REDISTRIBUTION OF PKC TO THE MITOCHONDRIA: COMPARING ... · Page 1 of 80 Introduction Background...

REDISTRIBUTION OF PKC TO THE MITOCHONDRIA: COMPARING MYOCARDIAL

ISCHEMIC AND PHARMACOLOGIC PRECONDITIONING

by

Steven Habbous

A thesis submitted in conformity with the requirements

for the degree of Master’s of Science Graduate Department of the Institute of Medical Science

University of Toronto

© Copyright by Steven Habbous 2010

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Redistribution of PKC to the Mitochondria: Comparing Myocardial Ischemic and Pharmacologic Preconditioning

Steven Habbous; Master’s of Science Convocation Date November 2010 Institute of Medical Science, University of Toronto Hospital for Sick Children, Toronto, Ontario

Abstract PKC plays a very important role in mediating the protection against

myocardial ischemia and reperfusion injury induced by ischemic preconditioning

(IPC) and pharmacologic preconditioning (PPC). The redistribution of PKC was

assessed by subcellular fractionation and western blotting in the Langendorff-

perfused rabbit heart. Either 5min ischemia or 5min administration of adenosine

A1 and/or A3 agonists, bradykinin, angiotensin II, and 1-opioid agonists resulted in

PKC redistribution from the cytosol to the mitochondria. This effect of IPC on

PKC redistribution was visible up to at least 30min of reperfusion, while that of

PPC was lost by 10min of drug washout, indicative of the transient nature of PKC

redistribution. PKC redistribution to mitochondria by IPC was also visualized

using immunogold electron microscopy. Thus, IPC and PPC caused PKC

redistribution from the cytosol to the mitochondria, which was longer-lasting in

IPC than in PPC.

Glossary of Acronyms

m – mitochondrial membrane potential[Ca2+]m – intramitochondrial Ca2+ concentration

V1-1 – PKC-specific inhibitor

V1-2 – PKC-specific inhibitor

RACK – PKC-specific activator

RACK – PKC-specific activator AMI – acute myocardial infarction AngII – angiotensin II APNEA – N6-2-(4-aminophenyl)ethyladenosine BK – bradykinin

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CCPA – 2-chloro-N6-cyclopentyladenosine Cl-IB-MECA – 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide

DADLE – 1-opioid agonist; [D-Ala2, D-Leu5]-enkephalinDAG – diacylglycerol dIPC – delayed ischemic preconditioning GA – glutaraldehyde GPCR – G-protein-coupled receptor IEM – immunoelectron microscopy IFM – interfibrillar mitochondria IPC – ischemic preconditioning IPost – ischemic postconditioning IR – ischemia/reperfusion MCU – mitochondrial calcium uniporter mitoKATP – mitochondrial potassium ATPase MPTP – mitochondrial permeability transition pore NCE – Na+-Ca2+ exchanger NHE – Na+-H+ exchanger NO – nitric oxide PF – paraformaldehyde PKC – protein kinase C PMA – phorbol-12-myristate-13-acetate PPC – pharmacologic preconditioning PPost – pharmacologic postconditioning RACK – receptor for activated C kinase RIPC – remote ischemic preconditioning RIPer – remote ischemic preconditioning RNS – reactive nitrogen species ROS – reactive oxygen species RyR – ryanodine receptor SSM – subsarcolemmal mitochondria TEM – transmission electron microscopy

Special Acknowledgements

This work was done in the laboratory of Dr. GJ Wilson under the co-supervision of Dr. RJ Diaz at the Hospital for Sick Children, Division of Cardiovascular Research. Thanks to Alina Hinek, Dr. A. Hinek, Dr. C. Caldarone, and Dr. John Coles (Hospital for Sick Children) for their insights throughout this study. Thanks to Aina Tilups and Yew Meng in the pathology department at the Hospital for Sick Children and to Robert Tempkin at Mt. Sinai Hospital for their assistance with electron microscopy.

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Contents Introduction ............................................................................................................................ 1

Background .................................................................................................................................. 1

Introduction to Ischemic Preconditioning (IPC) .......................................................................... 1

Disparities in Animal Models and Protocols of IPC Administration ............................................ 3

IPC in Pathological States and Tolerance to IPC .......................................................................... 3

Heterogeneity ......................................................................................................................... 4

Heterogeneity of the Heart ......................................................................................................... 4

Heterogeneity of Mitochondria .................................................................................................. 4

Figure 1: Hierarchy of Model Complexity in Preconditioning Experimentation ......................... 5

Ischemia/Reperfusion ............................................................................................................. 6

Energetics .................................................................................................................................... 6

Ionic Imbalance and Ca2+ Overload ............................................................................................. 7

Protein Kinase C (PKC) ............................................................................................................. 8

Table 1: Myocardial Protection Induced by Various Interventions and their Effects on PKC() . 9

RACKs ......................................................................................................................................... 12

Table 2: Summary of Published Studies of PKC Isoform Redistribution .................................... 13

Redistribution of PKC Isoforms ................................................................................................. 19

Preconditioning ..................................................................................................................... 19

Temporal Properties of PKC Redistribution .......................................................................... 21

PKC and PKC ........................................................................................................................... 21

Modulation of PKC Expression ................................................................................................ 22

PKC Substrates and End-Effectors ........................................................................................... 23

Formulation of Hypothesis .................................................................................................... 24

Materials and Methods ......................................................................................................... 24

Animals ...................................................................................................................................... 24

Assessment of Freezing on Tissue Morphology ........................................................................ 24

Figure 2: The Effect of Freezing on Ventricular Tissue Morphology ......................................... 25

Figure 3: Mitochondrial Fraction from Fresh and Frozen Tissue. ............................................. 26

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Subcellular Fractionation .......................................................................................................... 27

Bradford Protein Determination Assay ..................................................................................... 27

Western Blotting ....................................................................................................................... 27

Figure 4: The Effect of Freezing on PKC Redistribution........................................................... 28

Figure 5: Fractionation Pellets ................................................................................................... 29

Langendorff Perfusion ............................................................................................................... 30

Experimental Protocol ........................................................................................................... 30

Figure 6: Protocol Bars in Bar-Chart Format ............................................................................. 31

Microscopy ................................................................................................................................ 32

Sample Preparation for Immunogold .................................................................................... 32

Immunogold Labelling of PKC .............................................................................................. 32

Figure 7: Different Fixation Techniques on Snap-Frozen, Freeze-Substituted Ventricular Tissue

................................................................................................................................................... 33

Statistical Analysis ................................................................................................................. 34

Results ................................................................................................................................... 34

Fresh Tissue vs. Frozen Tissue ................................................................................................... 34

Fractionation ............................................................................................................................. 35

PKC Redistribution Following 5min Ischemia .......................................................................... 35

PKC Redistribution Following Pharmacological Preconditioning with 5min Infusion of Various

GPCR Agonists ........................................................................................................................... 35

Figure 8: The Distribution of Subcellular Markers Following Fractionation ............................. 36

Figure 9: PKC Dependence on Contamination ........................................................................ 37

Figure 10: Relative Density of PKC in Cytosol, Mitochondria, and Membranous Fractions in

Rabbit Ventricular Myocardium ................................................................................................ 38

Figure 11: Cytosolic and Mitochondrial PKC in the Langendorff-Perfused Rabbit Heart ...... 39

Figure 12: Cytosolic and Mitochondrial PKC in the Langendorff-Perfused Rabbit Heart

Following 5min Global Ischemia ............................................................................................... 40

Table 3: Pharmacological Preconditioning Agents Used .......................................................... 41

Figure 13a: PKC Redistribution to Mitochondria Following 5min Adenosine Agonist Drugs

with 2min Washout ................................................................................................................... 42

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Figure 13b: PKC Redistribution to Mitochondria Following 5min GPCR Agonists with 2min

Washout .................................................................................................................................... 43

Figure 14a: PKC Redistribution to Mitochondria Following 5min Adenosine Agonist Drugs

with 10min Washout ................................................................................................................ 44

Figure 14b: PKC Redistribution to Mitochondria Following 5min GPCR Agonists with 10min

Washout ................................................................................................................................... 45

Table 4: Statistics ....................................................................................................................... 46

Figure 15: Total PKC Levels Following 5min Ischemia ............................................................ 47

Immunogold Labeling of Ventricular Myocardium ................................................................... 48

Discussion ............................................................................................................................. 48

Model ........................................................................................................................................ 48

Figure 16: Perfusion-Fixed Ventricular MyocardiumFigure 17: Snap-Frozen Control and IPC

Ventricular Myocardium: Freeze-Substituted and Immunolabeled for PKC ............................ 49

Figure 17: Snap-Frozen Control and IPC Ventricular Myocardium: Freeze-Substituted and

Immunolabeled for PKC ........................................................................................................... 50

Figure 18: Density of gold particles ........................................................................................... 51

Fresh Tissue vs. Frozen Tissue ................................................................................................... 51

Fractionation ............................................................................................................................. 52

PKC Redistribution Following 5min Ischemia .......................................................................... 53

PKC Redistribution Following Pharmacological Preconditioning with 5min Infusion of Various

GPCR Agonists ........................................................................................................................... 54

Changes in Total PKC Levels Following Ischemic Stress .......................................................... 55

Microscopic Imaging of PKC at Mitochondria ......................................................................... 56

Novel Elements of This Work ................................................................................................. 58

Conclusions ........................................................................................................................... 58

Future Directions ................................................................................................................... 58

Limitations ............................................................................................................................ 60

Appendix ............................................................................................................................... 61

RACK Binding, Intramolecular Interactions, and Synthetic Activator/Inhibitor Peptides ..... 61

C1 Domain, Activation and Binding ....................................................................................... 62

C1 Domain Pseudosubstrate-C4 Substrate Binding Site Intramolecular Interaction ............ 62

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PKC Hinge Region ................................................................................................................... 63

PKC Catalytic Domain and Phosphorylation .......................................................................... 63

Phosphorylation of PKC ......................................................................................................... 63

References............................................................................................................................. 68

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Introduction

Background Insufficient oxygen delivery (hypoxia) or insufficient blood flow (ischemia) to the heart

contributes to the cardiovascular dysfunction that is the main cause of death in the Western

World. The heart is a strictly aerobic organ consuming copious amounts of oxygen at rest,

which is increased 4-5× upon exertion (O’Rourke B, 2005). Prolonged hypoxia/ischemia results

in cardiomyocyte death and is of great clinical importance. Myocardial ischemia occurs, for

example, during angina, acute myocardial infarction (AMI), and during cardiac surgery

[transplantation (reviewed in Ambros JR, 2007), coronary artery bypass grafting, percutaneous

coronary intervention, or abdominal aortic aneurysm repair]. In contrast to surgically-induced

ischemia, when and to what extent angina or AMI occurs is largely unpredictable. The current

standard of care is to remove the coronary artery obstructive agent (thrombus, atherosclerotic

plaque) by thrombolytic agents or angioplasty. The time between the onset of ischemia and

the restoration of blood flow (reperfusion) correlates with the extent of myocardial death or

post-infarct dysfunction. It is also evident that the very act of restoring blood flow to the organ

is itself associated with tissue injury, termed ‘reperfusion injury’. Ischemia/reperfusion (IR)

refers to injury resulting from the combination of both ischemia and reperfusion. Patients who

survive an IR incident sufficient to cause substantial cardiomyocyte necrosis often suffer from

post-infarct dysfunction, including impaired contractility and remodeling of the myocardium

with associated hypertrophy leading to heart failure. Different tissues, even within the same

organ, have varying degrees of resistance to ischemia (Asano G, 2003) with the cardiomyocytes

beginning to die after 20-30min without oxygen (Tapuria N, 2008; Moses MA, 2005).

Introduction to Ischemic Preconditioning (IPC) Increasing the resistance of the myocardium to ischemia has been a subject of extensive

research. It has been demonstrated in all mammalian animals studied, including humans, that

one or more cycles of brief ischemic insults followed by a brief reperfusion (5-10min) will

render the heart resistant to a prolonged ischemia (typically 30-90min) by reducing infarct size,

myocardial stunning, and arrhythmogenesis (Kiss A, 2008). This innate phenomenon of

cardioprotection is termed ischemic preconditioning (IPC). In 1986 the observation of IPC was

first published with a 75% reduction in infarct size in a canine model after four cycles of 5min

ischemia/5min reperfusion (Murry CE, 1986). IPC has two windows of protection: acute and

delayed. Acute IPC protects the myocardium from IR injury when the preconditioning IR occurs

a few minutes to 2-4h prior to the index (prolonged) ischemia (Liem DA, 2005), depending on

the species (Schwarz ER, 1998). Delayed/late IPC (dIPC), or the second window of protection

(SWOP), protects the myocardium starting about 24h after the preconditioning stimulus and

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lasts up to 72h (Moses MA, 2005) or even longer (Lott FD, 1996). dIPC is associated with the

synthesis of a host of proteins (Xuan YT, 2007; Wang GY, 2001; Kawata H, 2001; Kamota T,

2009; Joo JD, 2007; Arrington DD, 2008; Nayeem MA, 2004; Hochhauser E, 2004; Konishi H,

1997). All preconditioning techniques have an acute and a late phase of protection (Penna C,

2008; Wang GY, 2001; Baxter GF, 1997; Kristo G, 2004). In this regard it is interesting that

patients presenting with AMI have a higher likelihood of improved recovery and reduced post-

infarct dysfunction if preceded by angina 24h prior (Tomai F, 1999; Valen G, 2005). IPC is a very

powerful cardioprotective strategy, but it falls short of clinical applicability in AMI because 1)

the preconditioning ischemia must occur prior to the actual ischemic insult and, therefore, the

timing of the AMI must be known; 2) the resulting risk of stroke in already at-risk patients is

high (Ramzy D, 2006); and 3) patients with clinically silent chronic ischemia or an unknown

number of preceding angina attacks may not be protected or their condition even worsened by

IPC (Valen G, 2005; Liem DA, 2005).

The concept of ischemic postconditioning (IPost) has been shown efficacious in

protection against IR injury to a similar extent as IPC (Skychally A, 2009). IPost is identical to

IPC, except that the protective brief IR cycle(s) occur immediately following the index ischemia.

IPC and IPost have often yielded similar protection in animal studies, but IPost may not be as

protective in larger animals (Yang XM, 2010). IPost has the benefit over IPC such that it can be

initiated to patients already presenting with AMI. However the mechanical interventions

required for inducing myocardial ischemia in IPost pose similar risks to IPC that deter its clinical

use.

More recently discovered and more clinically applicable is remote ischemic

preconditioning (RIPC) and remote ischemic perconditioning (RIPer), which are analogous to IPC

and IPost, respectively, but require a tissue/organ other than the tissue of interest to be made

intermittently ischemic. The “perconditioning” of RIPer is not the same as postconditioning

because the protective remote conditioning cycles must be begun while the heart is ischemic,

not after myocardial reperfusion. Remote conditioning can be achieved by subjecting the

skeletal muscle of an upper or lower limb to repeated cycles of IR (typically 5min ischemia and

5min reperfusion; 5min I/5min R) by the minimally invasive procedure of inflating and deflating

a blood pressure cuff (Cheung MMH, 2006).

Efforts to elucidate the underlying mechanisms through which the protection of IPC is

achieved have elucidated pharmacological agents that can mimic the protection of IPC. This is

termed pharmacological preconditioning (PPC) and pharmacological postconditioning (PPost),

both of which are efficacious in many animal models. It is important to note that IPC, IPost,

RIPC and PPC are not reserved for the heart, but are also protective in many other organs and

tissues, including skeletal muscle (Hopper RA, 2000; Moses MA, 2005), intestinal epithelium

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(Cinel I, 2003), vascular endothelium (Inagaki K, 2003; Kharbanda RK, 2002), kidney (Joo JD,

2007), bowel (Ferencz A, 2010) and lung (Xia Z, 2003; Cheung MMH, 2006), all of which are

adversely effected by IR injury.

Disparities in Animal Models and Protocols of IPC Administration Various mammals have been employed in studies of preconditioning protection,

including humans (Hassouna A, 2004), non-human primates (Yang XM, 2010), pigs (Hopper RA,

2000), dogs (Murry CE, 1986; Gomez L, 2007), sheep (Xia Z, 2003), and more commonly rabbits,

rats, and mice. Not all IPC protocols are equally protective, and some of this variability is

attributable to the sex and age of the animal (Turcato S, 2006; Penna C, 2009; Welch WJ, 2008)

and the species under scrutiny. The number and duration of the preconditioning IR cycles

depend on the species. In swine, a 2-min cycle of IR was insufficient to confer protection,

whereas protection was achieved with a 3- or 10-min period of IR with the 10-min protocol

more protective than the 3-min (Schulz R, 1998). In the rabbit, protection against IR injury was

induced one cycle of 5min I/5min R (Omar BA 1991; Przklenk K, 2006) and one or two cycles of

5min I/10min R, but not by two cycles of 2min I/10min R (van Winkle DM, 1991). Once the

duration of an IPC stimulus is ascertained, the number of cycles may or may not provide

additional protection. Studies in rabbit heart have shown that three cycles of 5min I/10min R

was more protective than one cycle (Sandhu R, 1997), but four cycles did not reduce infarct size

any greater than one cycle did (Goto M, 1995). In the dog, one cycle of 5min I/10min R was as

effective as two, four, or twelve cycles. In cynomolgus monkeys, 2 cycles of 10min I/10min R

was very protective (Yang XM, 2010). There are a maximum number of cycles after which the

myocardial protection of repeated brief IR stimuli is lost (Zaugg M, 2004; Liem DA, 2005). In

rabbit myocardium one cycle of 5min I/10min R is protective but not when identical stimuli are

repeatedly given for 8h/d 3d prior to the index ischemia (Cohen MV, 1994). Since not all

species are protected equally by the same IPC or IPost protocol (reviewed in Skyschally A et al.,

2009), caution should be taken when extrapolating to humans.

IPC in Pathological States and Tolerance to IPC The bulk of the myocardium consists of cardiomyocytes, which like any other cell type,

are subject to pro-survival and pro-death signals. Among the pro-death signals, reactive oxygen

species (ROS), reactive nitrogen species (RNS), and mitochondrial permeability transition pore

(MPTP) opening (Wang W, 2008; Brookes PS, 2004; Ryu SY, 2005; Sharov VG, 2007) are major

contributors. Under normal conditions, cardiomyocytes are better able to deal with these

stresses. However, under challenging circumstances, including diabetic cardiomyopathy

(Standen NB, 2006; Kristiansen SB, 2004) and hypertrophied myocardium (Miki T, 2003;

Hochhauser E, 2007), the ability of cardiomyocytes to withstand the insults of IR is greatly

reduced. In such cases, IPC may not be protective, but PPC may be beneficial if the agent used

bypasses the site of dysfunction. In the case of human diabetic myocardium (samples of right

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atrium), IPC failed to protect, as did multiple PPC protocols (phenylephrine, adenosine, mitoKATP

channel opener diazoxide), but PPC directed at PKC, presumably downstream of the three

agents that failed to protect, did protect (Standen NB, 2006). Myocardium in diabetes requires

more cycles of preconditioning IR in order to become protected (Carr RD, 2005), and IPC has

been shown to fail to protect remodeled myocardium two weeks following AMI by coronary

artery ligation (Miki T, 2000). Tolerance to adenosine-dependent IPC has been overcome by

PPC, which employs alternate signaling pathways (Liem DA, 2005). Since aged and diabetic

patients are among those who are particularly likely to require myocardial protection from IR

injury, PPC may be partially useful in this subset of individuals. Thus, precise molecular

pathways responsible for IPC, PPC, and RIPC protection merit study in order to capture the

greatest degree of protection while minimizing risk to the patient.

Heterogeneity

Heterogeneity of the Heart The heart is a heterogeneous organ that contains several different cell types:

cardiomyocytes, endothelial cells, neuronal cells, connective tissue cells, and blood cells. The

interplay of these various cell types should not be ignored, but may be excluded depending on

the model used (Figure 1). Although left ventricular tissue is most commonly used, ischemia

does not affect all ventricular myocardium to the same degree, with differences seen in the

subendocardium, mid-myocardium, and subepicardium (Asano G, 2003). Global ischemia of

30min duration in the buffer-perfused rat heart elicits different extents of necrosis throughout

the heart, often sparing the subepicardium (Pasdois P, 2008). In order to understand, at the

organ level, the effects of IR injury on the heart, one must look closer at the individual parts.

Since cardiomyocytes comprise the bulk of cardiac tissue and are essential for heart pumping

function, most attention has been placed on determining the response to ischemia at the level

of these cells (Figure 1). Furthermore, attention has been placed on the cardiomyocyte

mitochondria due to their important roles in regulating cardiomyocyte apoptosis and energy

production.

Heterogeneity of Mitochondria The mitochondrion plays a key role in cardiomyocyte physiology, and mitochondrial

dysfunction has a major impact on cell fate. This is especially true for cardiomyocytes because

of their high energy demand. In these cells, mitochondria take up ~30-40% of the cell volume,

generating >90% of the cell’s ATP (~30kg ATP/day) under aerobic conditions in order to support

2.5 billion contractions throughout the average human lifetime (Murphy E, 2007; Lemieux H,

2009; Barth E, 1992; Severs NJ, 1985). Not all mitochondria are equidistant from the cell

surface, giving rise to differences in the diffusive distance of O2 (and oxygen tension) among the

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Figure 1: Hierarchy of Model Complexity in Preconditioning Experimentation

Whole Animal (in vivo)

Neural stimulation intact Endothelium present

Humoral factors present

Isolated Heart (ex vivo)

Neuronal stimulation removed, but intracardiac neuronal tissue still present

Endothelium present Humoral factors absent

Individual Cardiomyocytes (in vitro)

Neural cells completely removed Endothelium completely removed

Humoral factors absent Cell-cell interactions abated

Isolated subcellular organelles (e.g. mitochondria)

All extramitochondrial signaling removed

Figure 1. Hierarchy of Model Complexity in Preconditioning Experimentation. A variety of model types are available for

preconditioning experiments. Simpler model systems sacrifice many signaling components, although may be advantageous in

that signaling stimuli can be added one at a time. The simpler systems become progressively more reductionistic, allowing

some observations not possible in more complex systems but lacking some potentially important factors operating in the

whole heart or whole animal.

Enzyme release, infarct size, animal survival, hemodynamic parameters can be measured, recovery of function after IR

Infarct size, ROS production, hemodynamic parameters can be measured (EF, LVDP, LVEDP, HR, CF, contractility), recovery of function after IR

Percent necrosis, ROS production, immunocytochemistry, morphology

Organelle rupture or swelling; intramitochondrial signaling

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mitochondria in an individual cardiomyocyte (Mik EG, 2009). The average oxygen tension in the

in vivo heart is 35mm Hg, but this is highly heterogeneous since 10% of mitochondria exist at

tensions between 0 and 10mm Hg (Mik EG, 2009), which is increased to 30% in the isolated

buffer-perfused heart. Mitochondrial microenvironments also depend on their proximity to

Ca2+ release channels (ryanodine receptors, RyR; inositol-3-phosphate receptors) of the SR,

which are exposed to much higher concentrations of Ca2+ than mitochondria further away

(Báthori G, 2006). The impact of mitochondrial microenvironments is further demonstrated by

the propagation of ROS production, Ca2+ overload, MPTP opening, and loss of mitochondrial

membrane potential (m) following localized photoexcitation and ROS release (Andreyev AY,

2005). Mitochondria act as energy producers, oxygen reducers, and calcium stores when

cytoplasmic calcium is high. The importance of mitochondrial function in IPC cannot be

overstated and is a subject of intensive research.

There are two major pools of cardiomyocyte mitochondria, differentiated by their

subcellular localization: the subsarcolemmal mitochondria (SSM) located beneath the

sarcolemma, and interfibrillar mitochondria (IFM), which are situated between myofibrils and

provide the energy for contraction (Palmer JW, 1977; Matlib MA, 1981). Most studies on

isolated mitochondria combine the mitochondria from the entire cell or look predominantly at

the subsarcolemmal pool. SSM and IFM have been separated from each other as early as 1977

(Palmer JW, 1977) and likely play differential roles in basal cardiac function, myocardial disease,

and IPC. In addition to their spatial differences, SSM and IFM differ in their morphology (cristae

are predominantly lamelliform in the former and tubular in the latter), biochemistry, rates of

respiration, and degrees of oxidative stress (Riva A, 2005; Williamson CL, 2010; Hoppel CL,

2009; Chen Q, 2008; Škárka L, 2002). IFM and SSM are differentially affected in hypoxia and

various cardiomyopathies (Duan J, 1989; Williamson CL, 2010), and are differentially adapted to

chronic exercise (Kavazis AN, 2009). Nonetheless, both pools exhibit similarities in

preconditioning (Quinlan CL, 2008). Functional differences are not necessarily attributed to

their morphological differences; proteomic differences appear more important to their

disparate performance (Duan J, 1989; Kavazis AN, 2009).

Ischemia/Reperfusion

Energetics The proton gradient across the inner mitochondrial membrane (IMM) is responsible for

the majority of the mitochondrial membrane potential necessary for mitochondrial function

(Škárka L, 2002). Loss of the m (depolarization) occurs in response to uncouplers or the

opening of pores in the IMM that allows ions to equilibrate across the membrane. When O2 is

not available (hypoxia/anoxia), the ETC becomes reduced, and the driving force for entry of

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NADH into the mitochondria from the cytosol is reduced and NAD+ is no longer generated.

Under such conditions, mammalian cells can use anaerobic metabolism, whereby ATP is

generated solely by glycolysis. The onset of anaerobic metabolism is rapid, beginning 8s into

ischemia, and a reduction in contraction is seen by 20s (O’Rourke B, 2005). Glycolysis alone

cannot supply the amount of ATP needed by the cardiomyocytes (Budas GR, 2007) and ionic

homeostasis is compromised. Indeed myocardial ATP levels decrease with the failing heart

(Lemieux H, 2009). Exogenously added ATP will not be beneficial because it is rapidly degraded

to adenosine, which will have effects on the vasculature (vasodilatation, inhibition of platelet

aggregation) and myocardium (negative inotropy and chronotropy) that are contraindicated in

most patients (Wang S, 2001). Ischemic acidosis is attributable to the production of lactate in

the process that regenerates NAD+. IPC and angiotensin II (AngII) pretreatment reduce this

acidosis during the index ischemia, but this is unlikely to be the sole basis of protection since

agents that do not ameliorate this acidosis still mimic IPC protection, including PKC activation

(Cross HR, 2002; Diaz RJ, 1997). Transient acidic conditions can nevertheless precondition

whole hearts akin to IPC (Li L, 2008). Ischemic tissue is characterized by a severely reduced ATP

supply (Murry CE, 1986), which has been shown to be reversed somewhat by IPC or in

transgenic mice expressing constitutively active PKC compared to controls (Cross HR, 2002).

Some clinical studies on IPC have shown success with respect to ATP preservation (76% higher

concentration in IPC vs. control) and CK-MB release, while others show no significant

difference, or even harm (Ramzy D, 2006).

Ionic Imbalance and Ca2+ Overload The increased [H+]C of ischemic acidosis is compensated for by extruding H+ into the

extracellular milieu via exchange for Na+ (Na+/H+ exchanger; NHE), resulting in [Na+]C overload.

This is exacerbated by the reduction in Na+/K+-ATPase activity under low ATP conditions, which

results in further intracellular Na+ loading. IPC preserves Na+/K+-ATPase activity during the early

phase of sustained IR (Saini HK, 2004). This cytosolic Na+ is exchanged for extracellular Ca2+

(Na+/Ca2+ exchanger; NCE), which in turn results in cytosolic calcium overload. Within minutes

of ischemia (Zaugg M, 2004), and more profoundly during reperfusion (Penna C, 2007), Ca2+

enters cardiomyocytes in toxic quantities, often associated with contractile rigor, mitochondrial

dysfunction, MPTP opening (Zhao ZQ, 2006) and cardiomyocyte death. The washout of H+ and

K+ (that build up during ischemia) upon reperfusion reduces the gradient for H+ efflux and

hence Ca2+ influx via NHE/NCE coupling (NHE is maximally active), resulting in further Ca2+

overload, concomitant with the rapid recovery of ATP (Zaugg M, 2004; Diaz RJ, 2004;

Ganitkevich V, 2006). At a high concentration of the PKC activator PMA (phorbol-12-myristate-

13-acetate), NHE was activated. Conversely, low concentrations lead to its inhibition and a

slower rate of recovery of intracellular pH upon reperfusion (Li L, 2008). The calcium

contracture that occurs may result in shearing of the cells and contraction band necrosis. Rigor

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contracture occurs at very low levels of ATP. Calcium overload also increases the osmolarity of

the cell, resulting in osmotic cell swelling (Zaugg M, 2004). Normally, the NCE operates to

extrude Ca2+ from the cells, but this function is reversed during IR in a voltage-dependent

manner (Hool LC, 2007). Cytosolic Ca2+ clearing is reduced during IR because SERCA2a

(sarcoendoplasmic reticulum Ca2+-ATPase) and ryanodine receptors (RyR) are modified by

nitrosylation and low ATP (Nadtochiy SM, 2009; Piper HM, 2006). Mitochondrial NCE and

mitochondrial calcium uniporters (MCUs) in the IMM have a very high affinity for Ca2+ (Kd <

2nM) and rapidly adjust cytosolic calcium levels (milliseconds), resulting in a rise in

intramitochondrial calcium concentration, [Ca2+]m, with mitochondrial calcium overload

(O’Rourke B, 2005; Paradies G, 2009; Škárka L, 2002; Brookes PS, 2004).

The ischemia-induced reduction in the ATP/ADP ratio opens mitochondrial potassium

ATPase (mitoKATP) channels, resulting in K+ influx into mitochondria. Thus, Ca2+ is not the only

ion that enters mitochondria. Mitochondrial Ca2+ entry (via MCUs) and K+ entry (via mitoKATP

channels) are both accompanied by an influx of PO43– to maintain electroneutrality and H2O to

maintain osmolarity. H2O influx results in mitochondrial swelling. IPC and many

preconditioning mimetics have been shown to prevent or reduce these effects associated with

the index ischemia, reducing necrosis (Gomez L, 2007; Penna C, 2006; Sun HY, 2005; Chen Q,

2002; Rodrigo GC, 2008; Pastukh V, 2005; Diaz RJ, 1999; Batthish M, 2002; Diaz RJ, 2003;

Juhaszova M, 2004). Reducing the m by uncouplers or potassium channel openers (KCOs)

will reduce the driving force (electropotential gradient) for Ca2+ and will reduce mitochondrial

calcium overload and ROS generation, resulting in IPC-like cardioprotection (Andrukhiv A, 2006;

Modrianský M, 2009). The effect of IPC to prevent the calcium-induced loss of diastolic

compliance is likely due to removal of calcium via SR-mediated uptake (via SERCA, which is

inhibited during IR injury), NCE, and/or Ca2+ pumps in the sarcolemma. (Chen Q, 2002).

Protein Kinase C (PKC) Almost every component mediating IR-induced cell injury appears to be modulated by

protein kinase C (PKC), including ion channels (K+, Ca2+, Cl–), ATP preservation, large pore

opening [MPTP, connexin-43 (Halestrap AP, 2006; Doble BW, 2000; Doble BW, 2001)], oxidative

stress (ROS producers/scavengers), and cell swelling, which is why much attention has been

placed on PKC activity ( isoform) in the setting of cardioprotection (Table 1). PKC is a family of

serine/threonine protein kinases found in a variety of cell types. Its role as a cardioprotective

mediator is well established (Table 1), since inhibition of PKC abrogates protection by IPC, IPost,

RPC, and PPC via many G-protein coupled receptor (GPCR) agonists (Ohnuma Y, 2002; Wang GY,

2001; Bell RM, 2005). Although adenosine, bradykinin (BK), and opioids all activate G-protein-

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Table 1: Myocardial Protection Induced by Various Interventions and their Effects on PKC()

Animal/Model Protection induced by

Protection Assessed by

Blocked by Protective Effector

Reference

Langendorff-perfused male Wistar rats

3× 3min I/5min R Recovery of hemodynamic parameters

1M chelerythrine

0.1M bisindolylmaleimide

PKC, Kawamura S, 1998

Primary newborn rat cardiomyocytes

0.5M RACK Viability and osmotic fragility

1M chelerythrine

3M GF109203X

PKC Dorn GW II, 1999

Transgenic mouse whole heart

RACK Ventricular function and geometry; cardiac hypertrophy

V1-2 PKC Wu G, 2000

Pig skeletal muscle flaps (latissimus dorsi)

IPC, OAG (0.1 mg/muscle), PMA (0.05 mg/muscle)

Infarction Chelerythrine (0.6 mg/muscle), polymyxin B (1.0 mg/muscle)

PKC (implied

PKC)

Hopper RA, 2000

Cultured adult rat cardiomyocytes and perfused rat hearts

RACK Cell death, CK release, infarct size

PKC Chen L, 2001

Male Sprague-Dawley rat ventricular cardiomyocytes

10nM PMA 24h prior

LDH release, percent necrosis

5M chelerythrine PKC Wang GY, 2001

Transgenic mice 10nM ganglioside GM-1

Recovery of LVDP, improved LVEDP, CK release

PKC knockout PKC Jin ZQ, 2002

Langendorff-perfused Japanese White male rabbits

Diazoxide Recovery of hemodynamic parameters (HR, LVDP, DF) and infarct size

Calphostin C PKC Ohnuma Y, 2002

Table 1: Myocardial Protection Induced by Various

Interventions and their Effects on PKC/PKC – (1/3)

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Whole male Wistar rat hearts

RPC Infarct size 5mg/kg i.v. chelerythrine PKC Wolfrum S, 2002

Transgenic mice PKC transgenic expression

Functional recovery, ATP preservation

PKC Cross HR, 2002

Isolated mitochondria from adult rabbit heart

400nM PMA MPTP opening PKC Korge P, 2002

Cultured adult rabbit cardiomyocytes

1M PMA Percent survival 5M chelerythrine PKC Batthish M, 2002

Cultured adult rabbit cardiomyocytes

IPC Cell swelling 5M chelerythrine PKC Diaz RJ, 2003

Langendorff-perfused rat hearts

RACK Hemodynamic parameters

PKC Inagaki K, 2003

Transgenic mice Active PKC Infarct size PKC Baines CP, 2003

Adult male mouse primary cardiomyocytes

Simulated short ischemia

LDH release, intact cells 1M chelerythrine PKC (implicates

PKC)

Nayeem MA, 2004

Human atrial biopsies IPC CK leakage, tissue viability

V1-2 PKC Hassouna A, 2004

HL-1 cells 500nM PMA,

0.25M RACK

Percentage dead cells 15M BIM, 0.5M V1-2 PKC Chaudary N, 2004

Infant rabbit hearts 1.0 U/mL erythropoietin

Recovery of LVDP 1M chelerythrine PKC (implicates

PKC)

Rafiee, 2005

Transgenic mice Antimycin A (ROS) Infarct size PKC knockout PKC Kabir AMN, 2006

Isolated rat heart IPost Infarct size, LDH release, recovery of LVDP

5M chelerythrine immediately at reperfusion or after IPost RI cycles

PKC Penna C, 2006

In vivo New Zealand White rabbit hearts

0.5g/kg/min PMA at

Infarct size 5mg/kg chelerythrine PKC Philipp S, 2006b



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reperfusion

Adult Wistar rat hearts

estrogen therapy,

10M 17-estradiol

Infarct size 2M chelerythrine PKC (implicates

PKC)

Sovershaev MA, 2006

Langendorff-perfused New Zealand White rabbits

IPC (5min I/10min R)

Infarct size 5M chelerythrine PKC (implicates

PKC)

Przyklenk K, 2006

H9c2 and neonatal rat cardiomyocytes

100M diazoxide, transfection with

active PKC

Percent cell death 10M V1-2 PKC Kim MY, 2006

Langendorff-perfused Sprague-Dawley rats

300nM DADLE (2×)

Infarct size 100nM V1-2, 200nM Calphostin C

PKC Miura T, 2007

Langendorff-perfused male C57/BL6 mice

chronic treatment with AT1-receptor blocker candesartan

Infarct size Chelerythrine PKC Lange SA, 2007

Transgenic mice fibroblast growth factor-2

Infarct size 1M bisindolylmaleimide PKC House SL, 2007

Isolated male Fischer 344 aged and adult male rat hearts

RACK Recovery of LVDP, infarct size

PKC Korzick DH, 2007

Langendorff-perfused New Zealand White rabbit hearts

cGMP analog CPT

(10M)

Infarct size 2.8M chelerythrine at reperfusion

PKC Kuno A, 2008

Langendorff-perfused male Sprague-Dawley rats

SNAP MPTP opening V1-2 PKC Costa ADT, 2008

Human atrial trabeculae

RACK Recovery of function PKC Sivaraman V, 2009

Table 1. Protection Induced by Various Interventions and their Effects on PKC/PKC. PKC() plays a major role in

preconditioning protection in various experimental models, induced by a variety of agents. Blocking PKC() appears to block

protection for IPC and all or almost all preconditioning pharmacological mimetics. A comprehensive review of PKC and

cardioprotection can be found in Inagaki K et al (2006).



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coupled receptors (GPCRs), they do not follow identical intracellular signaling. There are >11

different PKC isoforms that are divided into three classes based on their general activation

requirements. The two most important isoforms for preconditioning are the novel PKCs and

, which are both activated by diacylglycerol (DAG) but not by Ca2+ (Ardehali H, 2006; Ping P,

1997). Different PKC splice variants have been detected (of PKC, , and ) that display both

tissue- and species-selectivity and differences in function (Pears C, 1991; Kofler K, 2002). Rabbit

ventricular myocardium contains most PKC isozymes (, , , , , , , , , , and ), and

among them only PKC and are translocated to the particulate fraction by ischemia (and not

by reperfusion), although the role of PKC in IPC is likely limited (Ping P, 1997; Wolfrum S,

2002; Hopper RA, 2000).

RACKsInactive PKC is predominantly located in the cytosol and is redistributed upon activation to

membranous destinations within the cell where its substrates lie. Thus PKC activation is often

measured by the ratio of its levels in the cytosol compared to some membranous

compartment, often the non-specific particulate fraction (Table 2). Upon stimulation by various

activators, binding to RACKs (Receptors for Activated C Kinases) facilitate PKC transport

throughout the cell by an unknown mechanism. PKC’s final destination depends on the specific

isozyme’s regulatory domain and the RACK to which it binds. PKCs bind RACKs in an isozyme-

specific manner, and only bind after initial activation. PKC specifically binds to the subunit of

the coatomer protein ('-COP) located at the Golgi that is essential for Golgi budding and

vesicular trafficking (Csukai M, 1997), known as RACK2. RACKs are highly selective for PKC

isoforms and are present in abundance (Schechtman D, 2004; Akita Y, 2002; Pass JM, 2001).

Mathematical modeling suggests PKC activation occurs in two steps (Schechtman D, 2004):

closed PKC in the cytosol open PKC in the cytosol

open PKC in the cytosol membrane-bound PKC

Once mature (tri-phosphorylated; see Appendix) and activated, PKC can bind to its RACK,

which transports it to some subcellular membranous location where it can be fully activated by

lipids (i.e. phosphatidylserine, DAG, cardiolipin). DAG and phorbol ester binding to PKC give the

enzyme a higher affinity for membranes (Ács P, 1997). Without initial activation, PKC may still

bind membranes, although with much less avidity and selectivity (Newton AC, 1995).

Movement is thought to occur via vesicular trafficking (slow), and perhaps by a much faster

vesicle-independent mechanism (Lehel C, 1996). Upon membrane binding, the regulatory

k2

k-1

k1

k-2

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Isoform Distribution to Activated by Inhibited by Model Method of Detection

Reference

, , but not

or Particulate

1M BK (high concentrations)

1-2d old rat ventricular myocytes

Fractionation and WB

Clerk A, 1996

Table 2: Summary of Published Studies of PKC Isoform Redistribution

Particulate LV stretch, 0.1-

10M AngII1M losartan Adult guinea pigs


Paul K, 1997

, , Particulate PMA NIH 3T3 fibroblasts Fractionation and WB

Ács P, 1997

Particulate Hypoxia Rat neonatal ventricular myocytes


Goldberg M, 1997

but

not, I,

II, , , , ,

,

Particulate Ischemia In vivi rabbit whole heart; ventricular


Ping P, 1997

, , , but

not Particulate IPC

Chelerythrine; bisindolylmaleimide

Rat whole heart Langendorff perfusion


Kawamura S, 1998

, , , but

not Particulate

Phorbol ester TPA

Bisindolylmaleimide II, stausporine, rottlerin

3Y1 rat fibroblast cell culture


Lu Z, 1998

, Nucleus H2O2 VSMCs IF (colocalization) Li PF, 1999

, , but not

Particulate UVB

Dominant-negative PKC mutants

Mouse epidermal JB6 cell culture


Chen N, 1999

, but not

or Particulate

RACK + 1nM PMA

Neonatal and adult rat cardiomyocytes


Dorn GW II, 1999

Perinuclear, cross-striations, cell-cell contact,

RACK, 1nM PMA, 100nM PMA

IF

perinuclear 100nM PMA

Particulate

Gq OE,

RACK, Gq

+RACK

Transgenic mouse ventricular tissue


Wu G, 2000

Table 2: Summary of Published Studies of PKC

Isoform Redistribution (1/6)

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Cross-striated elements and intercalated disks

RACK TGN V1-2 TGN TGN models with rat V1-2

or rat RACKIF

Mochly-Rosen D, 2000

, but not ,

, or Particulate

M phenylephrine,

100M R-PIA,

50M diCg (PKC

activator), 1M PMA

ChelerythrineAdult rat at ventricular myocytes


Lester JW, 2000

Cell-cell contacts FGF-2 Neonatal rat cardiomyocytes

IF Doble BW, 2000

, but not ,

I, II, ,

Not to sarcolemma/SR, but to particulate

IPC Pig latissimus dorsi skeletal muscle


Hopper RA, 2000

, but not

or Particulate

Metabolic inhibition,

10M U50 (-OR agonist)

Rat ventricular myocytes Fractionation and WB

Wang GY, 2001

10nM PMA

Particulate TGN PKC Heart tissue Fractionation and WB

Pass JM, 2001

but not Particulate

RACK

Adult (12-week-old) rat cardiac myocytes


Chen L, 2001 but not RACK

but not ,

, or

Nuclear but not Particulate

IPC 8 week old rats Fractionation and WB

Kawata H, 2001

Particulate

50M phenylephrine

Isolated adult rat cardiomyocytes in suspension (simulated ischemia by pellet)


Tsouka V, 2002 but not

10min and 20min ischemia



Table 2: Summary of Published Studies of PKC Isoform Redistribution (3/6)

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but not

or Particulate

Ganglioside GM-1

Langendorff-perfused mouse heart


Jin ZQ, 2002

, but not ,

, or Particulate

24h 1M -OR agonist U50,488H

Isolated rat ventricular myocytes


Zhou JJ, 2002

, but not ,

, or

15min 30M -OR agonist U50,488H

but not

or Particulate

IPC, ischemia, Dz

Male Japanese White rabbits Langendorff-perfused


Ohnuma Y, 2002

Mitochondria Ischemia

Particulate RPC MAO, bradykinin

HOE140 (B2 blocker), Partially by hexamethonium

Male rat Fractionation and WB

Wolfrum S, 2002

Particulate Global IPC Langendorff-perfused rabbit heart


Batthish M, 2002

Mitochondria TGN mice with constitutively

active PKC

Isolated mouse myocardial mitochondria

IF Baines CP, 2002

but not Particulate IPC and 10min ischemia

Hypertrophy, recovered with valsartan treatment

Male Japanese White rabbit heart tissue


Miki T, 2003

Mitochondria, nuclei, but not sarcolemma

IPC, APC Langendorff-perfused male rat heart

IF Uecker M, 2003

Sarcolemma, nuclei but not mitochondria

Plasma membrane GPCR stimulation via 10nM bombesin

V1-2 Swiss 3T3 cell culture

IF

Rey O, 2004 Particulate




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Mitochondria

PMA, 30M Dz, 200nM CCPA, hypoxic preconditioning, Hoe, 10nM DADLE, CsA, photoexcitation (ROS)

500M 5-HD, 1mM NAC

Neonatal and adult rat cardiomyocytes

Fractionation and WB, IF

Juhaszova M, 2004

Myofibrils Arachidonic acid, PMA

V1-2

Skinned rat cardiac myocytes

IF Huang X, 2004

Particulate IPC, OAG, ischemia

Colchicine, nocodazole

Male Japanese White Langendorff-perfused ventricular biopsies


Nakamura Y, 2004

Particulate 100nM PMA,

intramolecular mutation

Intramolecular mutation

CHO cells

Fractionation and WB Schechtman

D, 2004 Cell periphery IF

Particulate

300nM 20min CHA (A1AR agonist)

HL-1 (cardiac muscle cell line derived from the AT-1 mouse, atrial cardiomyocyte tumor lineage)


Chaudary N, 2004

PMA Neonatal mouse cardiomyocyte primary culture

but not t-tubular-like structures

2M CCPA,

PKC-GFP

overexpression DPCPX

Isolated rat ventricular myocytes

IF Miyazaki K, 2004

Cell surface

PMA Calphostin C (for

only)

Human myoblastic rhabdomyosarcoma (RD) cells

IF Sundberg C, 2004

but not

Particulate




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Particulate 1U/mL EPO 1M chelerythrine

Langendorff-perfused New Zealand White rabbits


Rafiee P, 2005

Plasma membrane

100nM PDBu (phorbol dibutyrate), 100nM PMA

Cultured HEK293 cells, adult rat ventricular myocytes IF Kang M, 2005

Nucleus

100nM PDBu (phorbol dibutyrate), 100nM PMA

Nucleus, cell membrane

100nM PMA Adult rat ventricular myocytes

t-tubular/z-lines V1 Adult rat ventricular myocytes

IF Robia SL, 2005

Particulate

0.1g/mL arachidonic acid

MPG Adult rat ventricular myocytes


Kabir AMN, 2006

PMA

Particulate 10M 17-estradiol (E2)

Chelerythrine Langendorff-perfused adult rat heart


Sovershaev MA, 2006

Particulate IPC Langendorff-perfused New Zealand White rabbit ventricular myocardium


Przyklenk K, 2006

, but not ,

I, , or

Particulate, Mitochondria

100M Dz, hypoxia

V1-2 but not 5-HD

H9c2, neonatal rat cardiomyocytes


Kim MY, 2006

Particulate IPC, DETA/NO eNOS–/– In vivo mouse heart Fractionation and WB

Xuan YT, 2007

Particulate

IPC

Langendorff-perfused adult mouse heart


Hund TJ, 2007 IPC + PKC- KO



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, but not ,

, or Particulate FGF2 TGN + IR

10-12 week-old Langendorff-perfused mouse heart


House SL, 2007

Sarcolemma, SR, nuclear/perinuclear, mitochondria, intercalated discs

FGF2 TGN + IR IF

Sarcolemma, SR, nuclear/perinuclear

Sarcolemma, SR, nuclear/perinuclear, mitochondria

Nuclear/perinuclear, SR, mitochondria

Mitochondria RACK Old rat Langendorff-perfused adult rat left ventricular tissue


Korzick DH, 2007

Particulate

500nM

truncated PKC C2 domain-like peptides + 3nM PMA

CHO cells, IP injection of mouse heart


Brandman R, 2007

NOT SEEN TO Mitochondria

IPC Male Wistar Langendorff-perfused rat hearts

Fractionation and wB

Clarke SJ, 2008

Nucleus Adenosine

Changing Ser729 to Ala729, microtubule depolymerizer nocodazole

H9c2 Cardiomyoblasts Fractionation and WB, IF

Xu TR, 2009

Table 2. Summary Published Studies of PKC Isoform Redistribution. Different PKC isoforms are translocated to different subcellular compartments.

Differences may be found among species, isoforms, stimulants, and tissues. Only a few previous studies have localized PKC to mitochondria, three

by western blotting (Ohnuma Y, 2002; Juhaszova M, 2004; Kim MY, 2006), but only one in rabbits (Ohnuma Y, 2002). WB = western blotting; IF =

immunofluorescence; OE = overexpession. The particulate fraction contains a mixture of various membranes, including mitochondria, sarcolemma,

sarcoendoplasmic reticulum, and sometimes nuclei.



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domain (see Appendix) of the enzyme is cleaved, leaving behind a constitutively active

fragment, termed PKM, that no longer requires cofactors for activation (Laher I, 2001; Basu A,

2002; Newton AC, 1995; Shimohata T, 2007). Therefore, even without activators PKC is

continuously being activated at basal levels, leading to background levels of the kinase in

various compartments.

Redistribution of PKC Isoforms

Inactive PKC is localized in the cardiomyocyte cytosol, Golgi, perinuclear region, cell-cell

contacts and intercalated disks, and in a striated pattern not seen with PKC (Robia SL, 2001; Li

PF, 1999; Kang M, 2005; Robia SL, 2005; Mochly-Rosen D, 2000; Tsouka V, 2002). Different

activators often cause redistribution of different PKCs to different subcellular locations and

differences also arise in various tissues of different species (Table 2) (Akita Y, 2008; Wolfrum S,

2002; Clerk A, 1996; Wang GY, 2001; Kang M, 2005). In response to DAG, PKC is recruited to

the plasma membrane, while arachidonic acid facilitates its translocation to Golgi networks

(Akita Y, 2002), and both of these second messengers are elevated during ischemia. PKC has

also been shown to translocate to nuclei after IPC (Kawata H, 2001) as well as z-lines after

activation with the phorbol ester PMA (Robia SL, 2005). PMA induces PKC accumulation in the

perinuclear region, intercalated disks, and in a longitudinal array indicative of mitochondrial

localization (Robia SL, 2001). Indeed it has been shown that PKC resides in mitochondria

under basal conditions and accumulates there upon ischemic stimulation (Table 2) (Baines CP,

2002; Baines CP, 2003; Sovershaev MA, 2006; Costa ADT, 2006; Jabůrek M, 2006). PKC has

been shown to bind several Golgi proteins, caveolae, and contractile filaments (Ping P, 2001;

Huang X, 2004). Binding to these various structures is reversible. PKC translocation is greater

and faster with PMA, followed by phenylephrine, and then the adenosine A1 receptor agonist R-

PIA (Lester JW, 2000; Tsouka V, 2002), all of which mimic the protection of IPC. More details on

the complex regulation of PKC are provided in the Appendix.

Preconditioning

As a mediator, PKC activity is required for the IPC protection during the subsequent

sustained ischemia and reperfusion (Wang GY, 2001; Zatta AJ, 2006, Hopper RA, 2000; Philipp S,

2006b; Hausenloy DJ, 2007) and its translocation to membranes increases with both the

number of cycles of short ischemia that constitute the IPC stimulus (Armstrong SC, 2004; Ping P,

1997) and the duration of the ischemic stimulus, increasing in the particulate fraction from

10min to 20min of ischemia (Tsouka V, 2002). Although a robust IPC stimulus (four cycles)

cannot be blocked by inhibiting other important mediators like ROS, chelerythrine (a PKC

blocker) completely abrogated four-cycle IPC protection (Bell RM, 2005). It appears that

several GPCR agonists, including bradykinin (Clerk A, 1996; Cohen MV, 2001; Oldenburg O,

2004; Philipp S, 2006a; Cohen MV, 2007), adenosine (Miyazaki K, 2004; Juhaszova M, 2004;

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Ohnuma Y, 2002), opioids (Zhou JJ, 2002; Zhang HY, 2002; Cohen MV, 2007; Miura T, 2007;

Philipp S, 2006a; Fryer RM, 2001b, Fryer RM, 2001c), and angiotensin (Chua S, 2009; Lester JW,

2000; Paul K, 1997) all converge on PKC downstream of activation of different receptors.

Additionally, the importance of PKC in RIPC has been documented. Myocardial IR injury

reduction by RIPC can be blocked by PKC inhibitors in situ in the rat (Weinbrenner C, 2002;

Wolfrum, 2002) and has been shown to redistribute PKC in the rabbit heart to the particulate

fraction (Shimizu M, 2009). RIPC cardioprotection has been demonstrated to require both

opioid and adenosine receptors (Surendra, 2009). Thus, in its effects on PKC redistribution in

the cardiomyocytes, it may be acting similar to PPC. The coronary effluent from preconditioned

rat hearts releases protective compounds that depend on PKC (Serejo FC, 2007). Consequently,

much attention has been focused on PKC’s role in cardioprotection and IR injury. GPCR

agonists are recognized by a variety of different tissues on which they can have substantial

effects, and so their administration to mimic IPC is contraindicated in most patients (Chiang

WC, 2007; Baxter GF, 1997; Wang S, 2001; Lester JW, 2000; Kristo G, 2004; Lasley RD, 1999; Ge

ZD, 2006).

Activating PKC either immediately before the index ischemia, or with a 10min washout

period in-between PKC administration and the index ischemia, confers protection against IR

injury (Sivaraman V, 2009). PKC can also be protective during reperfusion since a PPost

strategy of administering PMA at reperfusion was equally as protective (Liu Y, 2008).

Translocation of PKC itself is not enough for protection since kinase-inhibited PKC mutants,

although translocated sufficiently to membranes, do not mimic IPC protection (Cross HR, 2002).

Inhibition of translocation without altering catalytic competency (V1-2 treatment; see

Appendix) is similarly unable to mimic protection since PKC is unable to reach its substrates.

PKC translocation to the particulate fraction, and specifically to mitochondria, has been shown

with IPC, nitric oxide (NO) donor DETA/NO, mitoKATP channel opener diazoxide, PMA, hypoxic

preconditioning, 1-opioid agonist DADLE, MPTP inhibitor cyclosporine A, and photoexcitation

(presumably due to ROS since the ROS scavenger N-acetylcysteine blocked this effect)

(Juhaszova M, 2004; Xuan YT, 2007). ROS-induced preconditioning may proceed via specific

thiol oxidation that results in the loss of Zn2+ from the zinc-finger motif of PKC (Costa ADT,

2006, Ardehali H, 2006). Cells pretreated with H2O2 have elevated levels of activated tyrosine

phosphorylated PKC, , I, , , and (Konishi H, 1997). In human atrial tissue, IPC-induced

protection required PKC and , but not (Hassouna A, 2004). Atrial tissue is often used as a

source of human cardiac tissue because it is more readily available. Although human atrial

myocardium responds similar to animal ventricular myocardium to IPC (Hassouna A, 2004),

extrapolation of actual myocardial data to the whole heart warrants caution. The redistribution

of different PKCs is thought to occur via different mechanisms due to differences in their kinetic

properties.

of 80

Temporal Properties of PKC Redistribution

H2O2 treatment in vascular smooth muscle cells resulted in maximally active PKC at

2min, which was subsequently returned to baseline by 10min, without activation of PKC, , or

(Li PF, 1999). Upon stimulation by PMA in the GH3B6 (rat pituitary tumor) cell line, PKC, ,

, and were shown to translocate to membranes with different kinetics and required other

isoforms to remain active (Collazos A, 2006). Following treatment with thyrotropin-releasing

hormone in GH3B6 (pituitary) cells, PKCI was the first isoform to translocate to the plasma

membrane, followed 2s later by PKC to cell-cell contacts, then 3.5s later by PKC to cell-cell

contacts, which was followed immediately by PKC to the plasma membrane. PKC was active

the longest (15-20min) and PKCI, , and returned to the cytosol in less than 2min.

Interestingly, PKC translocation was dependent on PKCI activity, but not of its own activity,

and similarly so for PKC, revealing a signal transduction cascade among different PKC isoforms

for activation/translocation (Collazos A, 2006). This may be extrapolated to the heart as well,

since PKC is required for PKC activity (see below), as well as PKC (Rey O, 2004). In the rat

heart, PKC returned to the cytosolic fraction 10min into reperfusion, while PKC and

remained in the particulate fraction even after 30min (Armstrong SC, 2004; Kawamura S, 1998).

In rabbit myocardium, PKC translocation to particulate fraction persisted up to 15min (Rafiee

P, 2005). An interesting correlation is the requirement of the continued activation of survival

kinases for at least an hour after the index ischemia for protection to be realized, including

adenosine receptor occupancy for at least 30min following the index ischemia (Solenkova NV,

2006; Cohen MV, 2008). Temporal activation of PKC to mitochondria has not been observed

comparing different cardioprotective stimulants.

PKC and PKC

Although both PKC and are activated by IPC and are translocated to the

mitochondria, they play opposing roles (Armstrong SC, 2004; Akita Y, 2008). In isolated

cardiomyocytes, ex vivo isolated hearts, and intact hearts in vivo, PKC was shown to be

protective, while PKC was deleterious (Chen L, 2001). However there is evidence that PKC

activation before ischemia may be required for protection, presumably by activating PKC

(Inagaki K, 2005; Sivaraman V, 2009; Miura T, 2007). Using isozyme-specific activators

(RACK, RACK) and inhibitors (V1-2, V1-1), this was shown to be the case (see Appendix).

Some studies have shown additive benefit of PKC activation (RACK) prior to ischemia plus

PKC inhibition (V1-1) at reperfusion (Inagaki K, 2003; Zatta AJ, 2006). This treatment not only

protected the myocardium by reducing infarct size, but also improved hemodynamic

parameters, reduced enzyme release, and reduced myocardial and endothelial dysfunction

(Table 1). PKC and expression promoted a positive inotropic response in addition to their

translocation in ventricular myocytes (Kang M, 2005). The actions of PKC and are recognized

to be antagonistic and likely regulate each other (Armstrong SC, 2004; Baines CP, 2002). In a

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NIH3T3 (mouse embryonic fibroblast) cell line PKC and promote and arrest cell growth,

respectively (Ács P, 1997; Bredel M, 1997). Thus, it should not be surprising that activation of

PKC and inhibition of PKC are each separately cardioprotective and together cardioprotective

(Korzick DH, 2007).

Modulation of PKC Expression

During fetal development, PKC promotes cell division in the heart (hyperplasia) when

cardiomyocyte precursors can still divide. Upon maturation, however, these cells become

amitotic and PKC overexpression induces cardiomyocyte hypertrophy, resulting in the extreme

in heart failure (Mochly-Rosen D, 2000). PKC-induced hypertrophied myocardium is refractory

to IPC and associated with GPCR desensitization (Wu G, 2000; Chen L, 2001; Mochly-Rosen D,

2000; Zhou JJ, 2002; Tomai F, 1999). By modulating the levels of PKC its role in IPC is further

supported (Tables 1 & 2). PKC knockout mice do not exhibit protection from myocardial IR

injury from IPC (Saurin AT, 2002), whereas its constitutive overexpression confers protection

with no change in basal contractility (Cross HR, 2002). PKC and its association with RACK2 in

the particulate fraction are elevated in transgenic mice expressing low levels of constitutively

active PKC (Pass JM, 2001). Transgenic mice with chronic inhibition of PKC (V1-2

overexpression) developed lethal dilated cardiomyopathy (Chen L, 2001; Pass JM, 2001).

Transgenic mice overexpressing V1-1, which will compete with PKC for RACK2, resulted in

death due to heart failure, depending on how much of the V1-1 peptide was present. Whereas

lower copy numbers of V1-1 were tolerated, high copy numbers led to early death from

insufficient myocardial growth (Mochly-Rosen D, 2000). Thus, too much or too little PKC

activity is detrimental to the heart, while PKC transgenic mice expressing low levels of

constitutively active PKC (Pass JM, 2001) or mice expressing a RACK construct are inherently

protected from IR injury (Edmondson RD, 2002; Dorn GW, 1999; Cross HR, 2002). The PKC-

specific activator (RACK) and inhibitor (V1-2) peptides mimic and block IPC and most PPC,

respectively, whether administered intravenously or intraperitoneally (Chen L, 2001; Pass JM,

2001; Wang GY, 2001; Brandman R, 2007; Dorn GW, 1999). Using these PKC-specific peptides,

it was confirmed that the isoform responsible for the protection of IPC was indeed PKC.

Studies employing isoform-specific peptides are more informative than those using non-specific

PKC activators (phorbol esters, dioctanolglycerol) or inhibitors (chelerythrine, calphostin C,

polymyxin B, staurosporin, bisindolylmaleimide, and H-7). Compounds like staurosporin,

bisindolylmaleimide, and H-7 are very strong inhibitors that compete for the ATP binding site in

many protein kinases, not just PKC. This makes this group of PKC inhibitors fairly non-selective.

A useful table of specific, non-specific, and class-specific PKC inhibitor compounds is presented

in a review by Laher et al (Laher I, 2001).

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PKC Substrates and End-Effectors

PKC has been shown to bind several of the proteins whose levels change under

conditions of myocardial IR, detected by PKC pull-down assays followed by mass spectrometry

to identify those proteins (Ping P, 2001; Edmondson RD, 2002; Zhang J, 2008). Although

deficient due to difficulties purifying membrane-bound proteins or the identification of rare

proteins, currently >93 different targets have been identified that interact with PKC

(Edmondson RD, 2002; Ping P, 2001). Of the 36 proteins first found to form complexes with

PKC in transgenic mice expressing constitutively active PKC, 24 of them undergo

posttranslational phosphorylation in the cardioprotected phenotype (Ping P, 2001), which is

highly suggestive of an important role for PKC since 23 of these 24 proteins contain putative

PKC phosphorylation sites. Of the 93 proteins found to associate with PKC, many are

structural proteins, stress-activated proteins (including various heat shock proteins), several

proteins known to play a role in the signaling of preconditioning protection (including the stress

kinases, nitric oxide synthase, tyrosine kinases, connexin 43), various transcription- and

translation-related proteins, as well as metabolic proteins (including enolase, glyceraldehyde-3-

phosphate dehydrogenase, isocitrate dehydrogenase, ATP synthase) (Ping P, 2001; Edmondson

RD, 2002). The metabolic enzymes associated with PKC, a few of which were found in the

mitochondria, likely play a role in ATP preservation (Cross HR, 2002) and improved glucose

metabolism (Mayr M, 2009).

The major end-effectors that may play a role in protecting cardiomyocytes from IR injury

include the putative mitoKATP channel (Ohnuma Y, 2002; Costa ADT, 2006; Fryer RM, 2001a,

Jabůrek M, 2006), the MPTP (Juhaszova M, 2004; Argaud L, 2004; Halestrap AP, 2004; Gomez L,

2007), modulation of ROS production (Ohnuma Y, 2002; Chen Q, 2008), and connexin-43

hemichannels (Ruiz-Meana M, 2008; Vetterlein F, 2006; Rodriguez-Sinovas A, 2006). PKC has

been shown to associate with the potential MPTP components voltage-dependent anion

channel (VDAC of the outer membrane), adenine nucleotide transporter (ANT1 and ANT2 of the

inner membrane), and hexokinase II in the transgenic PKC mouse heart (Baines CP, 2003;

Edmondson RD, 2002). The mitochondria marker prohibitin was shown to interact with PKC

(Ping P, 2001), so its use in assessing PKC movement to mitochondria in immunofluorescence

colocalization studies is reasonable. Unpublished immunofluorescence data from our lab has

shown elevated colocalization of PKC with prohibitin induced by 5min I/10min R and even

implicated the mitoKATP channels and ROS upstream of PKC. Since resolution of light

microscopy does not allow individual mitochondria to be resolved, electron microscopy was

employed here to visualize PKC in mitochondria.

of 80

Formulation of Hypothesis IPC and PPC with GPCR agonists protect the myocardium from prolonged IR injury, and

this protection is mediated through PKC. The redistribution of PKC within the myocardium

following brief ischemia or GPCR agonist administration may shed some light on which

subcellular organelle may play a role in this protection. Thus, it is feasible that PPC with the

GPCR agonists bradykinin, angiotensin II, 1-opioid DADLE, and adenosine A1 and/or A3

receptors will mimic the effects of PKC redistribution within the myocardium similar to IPC.

Since all of these agents have been shown to reduce myocardial IR injury, as does IPC, it is likely

that the resulting PKC redistribution should be spatiotemporally similar. Therefore the

experiments in this study set out to illustrate the following hypotheses: 1) the cardioprotective

GPCR agonists bradykinin, angiotensin II, adenosine A1/A3 (APNEA), adenosine A1 (CCPA),

adenosine A3 (Cl-IB-MECA), and 1-opioid (DADLE) mimic the effect of ischemia on PKC spatial

redistribution; and 2) activation of PKC by IPC and PPC will cause its redistribution with similar

kinetics.

Materials and Methods

Animals New Zealand White rabbits of either sex (approximately 3.5kg) were used. Animals

were treated under the ethical guidelines of the Animal Care Committee at the Hospital for Sick

Children.

Assessment of Freezing on Tissue Morphology Fresh ventricular tissue from the same rabbit heart was divided into three pieces in cold

saline. Piece 1 (Freeze-Clamped; FC) was squeezed in-between two pre-cooled metal plates in

N2(ℓ) prior to homogenization and fractionation. Piece 2 (5mm×4mm×4mm chunks) was

placed directly in N2(ℓ) (Snap-Frozen), and Piece 3 (Fresh) was homogenized and fractionated

immediately. To assess the morphology of the frozen tissue and the degree of mitochondrial

rupture, fresh, snap-frozen, and freeze-clamped tissue were cut into approximately 1-2mm3

pieces and immersed in 2.5% glutaraldehyde (GA) + 4% paraformaldehyde (PF), a standard

transmission electron microscopy (TEM) fixative, prior to embedding, sectioning, and imaging

as per standard TEM microscopy. Freezing artifact can be seen (Figure 2) in the freeze-clamped

and snap-frozen tissue when compared to freshly fixed tissue. Mitochondrial outer membranes

and cristae are distinguishable in all three methodologies. Following mitochondrial isolation

(see Fractionation below), mitochondria purified from fresh myocardium are better able to

withstand the homogenization and centrifugation steps of the fractionation procedure than

that from frozen myocardium (Figure 3). However, some preliminary data suggests that PKC

of 80

Figure 2: The Effect of Freezing on Ventricular Tissue Morphology

Figure 2. The Effect of Freezing on Ventricular Tissue Morphology. Fresh, snap-frozen, and freeze-clamped control ventricular tissue from the same heart were placed into 4% paraformaldehyde + 0.1% glutaraldehyde for TEM imaging. Mitochondria were consistently intact, and the outer and inner mitochondrial membrane can be discerned. Freezing artifact revealed as less dense cristae (). Third heart not shown; magnification 10,000×.

Heart 1 Heart 2

Fresh

Snap-Frozen

Freeze-

Clamped

of 80

Figure 3: Mitochondrial Fraction from Fresh and Frozen Tissue.

W

W

W

W

Fresh

Snap-Frozen

Freeze-

Clamped

W

W

W

W

W W

W

W

W W

W

Figure 3. Mitochondrial Fraction from Fresh and Frozen Tissue. Fractions were fixed in TEM fix following

homogenization and fractionation. Mitochondrial integrity is better preserved in the fresh tissue than from that of

the frozen tissue, as there are a greater number of whole mitochondria (W) and cristae ().

of 80

redistribution may still be occurring in myocardium freshly processed (Figure 4). Freeze-

clamping was chosen as the method of choice for preparing myocardial tissue in subsequent

experiments because 1) freezing artifact is not sufficient to rupture mitochondria, 2)

contamination among fractions is minimized (see Fractionation below), and 3) to avoid any

artificial redistribution of PKC that may occur during processing since freezing stops

biochemical processes that may influence the localization of PKC within the myocardium.

Subcellular Fractionation All homogenization and fractionation steps were performed on ice or otherwise at

4°C. Tissue samples were homogenized in a Polytron homogenizer in freshly prepared cold

fractionation buffer containing a cocktail of protease inhibitors (150mM NaCl, 50mM Tris pH

7.5, 5mM EDTA pH 7.5, 10mM EGTA pH 8, 10mM benzamidine, 50g/mL phenylmethylsulfonyl

fluoride, 10g/mL aprotinin, 10g/mL leupeptin, 10g/mL pepstatin A, 1mM sodium

orthovanadate in water). Homogenate was twice centrifuged at 1000g for 10min to pellet

nuclei, large cellular debris and whole cells. The supernatant (lysate) was spun at 10,000g for

30min to pellet the mitochondria (likely a high proportion of SSM), which resuspended in fresh

fractionation buffer, and pelleted again at 10,000g for 30min. The supernatant of the initial

10,000g spin was then spun at 30,000g for 30min to pellet any larger mitochondria and

small/medium-sized membrane fragments, which was discarded (optional step). This was

repeated at 46,000g for 30min. The supernatant was spun at 100,000g for 1hr, producing a

membranous pellet consisting predominantly of sarcolemma and sarcoendoplasmic reticulum,

as well as other membranes. The supernatant at the completion of this step was termed the

cytosolic fraction. Both sarcolemmal and mitochondrial pellets were rinsed with fractionation

buffer and resuspended in cold fractionation buffer + 0.5% v/v Triton X-100. These fractions

were subsequently sonicated on ice (20 pulses) and together with the cytosolic fractions stored

at -80°C until needed or western blotting performed immediately. Samples of homogenate,

1000g, 10,000g, 45,000g, and 100,000g pellets were stored in 2.5% glutaraldehyde and

observed with the transmission electron microscope (Figure 5).

Bradford Protein Determination Assay The Bradford assay was used to determine the protein concentration of all fractions,

lysates, and homogenates used. A standard curve was produced using 2, 4, 8, 12, 16, and

20g/mL bovine serum albumin (Sigma-Aldrich) in Bradford dye (1 part Bradford reagent

(Biorad) in 4 parts water) and absorbance detected at 595nm.

Western Blotting

The sample was mixed with water for a total volume of 10L. An additional 10L of

loading buffer (125mL Tris pH 6.5 (HCl), 0.2% glycerol v-v, 4% sodium-dodecyl sulphate, 5mM

of 80

0

0.5

1

1.5

2

2.5

3

3.5

1 2 3

Figure 4: The Effect of Freezing on PKC Redistribution

bromophenol blue, 4.6% -mercaptoethanol v-v, in water) was added, vortexed, and spun

down. Subfractions or homogenates/lysates were boiled for 5 or 7min, respectively, and

immediately placed on ice, spun, and loaded on 8-16% precast gels immersed in running buffer

(Invitrogen EC60452) with 10L protein standard (Fermentas SM0671). The gel was run at 80V

for 20min and then at 120V until the dye front reached the bottom of the gel. The blotting

procedure was conducted in cold transfer buffer (19mM Tris-HCl/Trizma, 192mM glycine, 20%

methanol). PVDF membranes (Millipore) were soaked in methanol for 15s, rinsed in water, and

both the membranes and gels were gently shaken in cold transfer buffer for 5min. Transfer of

Figure 4. The Effect of Freezing on PKC Redistribution. Cytosolic PKC (measured as

cyto/cyto+mito) levels were assessed in four control and one stressed heart processed fresh, snap-

frozen, and after freeze-clamping. Of the three methodologies, freeze-clamping represents the

native state of PKC most accurately, localized the highest in the cytosol. Freshly processed tissue

may retain enzyme activity longer and subcellular structure may still be preserved such that

redistribution of PKC can still occur while in cold saline. Fisher PLSD; mean ± SE.

Rat

io o

f R

ela

tive

Cyt

oso

lic P

KC

(Cyt

o/C

yto

+ M

ito

)p = 0.1590 p = 0.0281

p = 0.3399

Fresh Snap-Frozen Freeze-Clamped

n = 7 n = 5 n = 7

Cytosolic PKC Comparing Fresh, Snap-Frozen, and Freeze-

Clamped Ventricular Myocardium

of 80

Figure 5: Fractionation Pellets

Figure 5: Fractionation Pellets. (a) 3,000×; Initial

homogenization does not completely separate all

subcellular compartments; clusters of myofibrils (M)

with interfibrillar mitochondria (IFM) remain. (b)

3,000×; Following a 1000g spin, most of these clusters

have been removed, although some nuclei (N) may

remain. (c) 10,000×; at 10,000g, the mitochondrial

pellet is full of large vesicles and many mitochondria

(MI) are discernible. (d) 10,000×; the 45,000g pellet

consists of a plethora of vesicular structures, likely

including broken mitochondrial membranes as well as

other unidentified membranous compartments. (e)

20,000×; the 100,000g pellet is the putative

sarcolemmal/sarcoplasmic reticulum subfraction and

contains no discernible structures. Notice that the

particle size decreases as the spin speed increases.

a b

d e

c

Transmission Electron Microscopy of Subcellular Fractions

M

M

IFM

N

M

MI MI

MI

of 80

proteins from the gel to the membrane was performed in a transfer apparatus at 100V for

1hr. The membranes were quickly rinsed in water and submerged in blocking solution (5% w/v

Milk protein in wash buffer; TBS Blotto-A Santa Cruz Biotech) on a shaker for 1hr at room

temperature or at 4°C overnight. Excess blocking buffer was then washed off in washing buffer

(38mM Tris-HCl, 150mM NaCl pH 7.5) on a shaker for 10min at room temperature, repeated 3

times prior to incubation for 1h (or overnight) at room temperature with primary antibody.

Washes were repeated prior to incubation with 1:5000 secondary antibody (if necessary) and

washed as before. Mouse monoclonal anti-PKC antibody (BD Transduction Laboratories),

mouse monoclonal anti-prohibitin antibody (NeoMarkers), and mouse monoclonal anti-1–

integrin antibody (Abcam) were used at 1:1000 dilution; goat polyclonal anti-lactate

dehydrogenase antibody (Abcam) and mouse monoclonal anti-GAPDH antibody (Sigma) were

used at 1:30,000 dilution. Each membrane was overlaid with 1mL chemiluminescence

detection solution (Denvill Scientific Inc.) for 1min, blotted off, then immediately

detected. Densitometry was then performed with AlphaEase FC-40 software to determine the

relative intensity of the bands on the film with respect to the background.

Langendorff Perfusion New Zealand White rabbits (approximately 3.5kg) of either sex were anesthetized with

sodium pentobarbital. Anesthetics isoflurane, sevoflurane, propofol, and ketamine were

avoided because of their effects on the protection obtained from IPC (Uecker M, 2003; Zaugg

M, 2004; Zhao ZQ, 2006, Javadov SA, 2003; Müllenheim J, 2001). Hearts were excised following

thoracotomy and Langendorff-perfused in a retrograde and non-recirculating fashion at a

constant pressure of 100mm Hg with modified Krebs-Hanseleit buffer (118.5mM NaCl, 4.7mM

KCl, 1.2mM MgSO4, 2.5mM CaCl2, 24.8mM NaHCO3) bubbled with 95% O2/5% CO2 at 37°C.

Hemodynamic parameters were measured via insertion into the left ventricle of a balloon

connected to a pressure transducer. Diastolic pressure was set <10mmHg by adjusting the

volume of saline within the balloon. Hearts that underwent fibrillation or had a left ventricular

developed pressure less than 75mm Hg were excluded. Heart rate and coronary flow were

monitored.

Experimental Protocol

Several experimental groups were employed, summarized in Figure 6. Global ischemia

was attained by completely stopping perfusion while immersing the heart in a 37°C bath to

maintain temperature. Briefly, following 20min stabilization, IPC hearts were subject to 5min

global ischemia and various times of full reperfusion (Figure 6a) while pharmacologically

treated hearts were given constant doses of GPCR agonists followed by 2min or 10min drug

washout (Figure 6b). Control hearts were perfused for the entire duration without intervention

or with appropriate vehicle control (0.1% DMSO). N6-2-(4-aminophenyl)ethyladenosine

of 80

Figure 6: Protocol Bars in Bar-Chart Format

b

35'

150

'

60'

n = 9

n = 4

n = 3

n = 7

n = 5

n = 4

n = 3

n = 3

20'

35min Control

5min I/0min R

5min I/2min R

(IPC) 5min I/10min R

(IPC) 5min I/ 30min R

60min Control

5min I/120min R

150min Control

35min Control

DMSO

0.4M Bradykinin 2min WO

0.4MBradykinin 10min WO

20nM DADLE 2min WO

20nM DADLE 10min WO

0.1M APNEA 2min WO

0.1M APNEA 10min WO

0.2M CCPA 2min WO

0.2M CCPA 10min WO

50nM Cl-IB-MECA 2min WO

50nM Cl-IB-MECA 10min WO

0.1M AngII 2min WO

0.1M AngII 10min WO

35'

n = 2

n = 3

n = 3

n = 3

n = 9

n = 6

n = 3

n = 3

n = 3

n = 3

n = 3

n = 3

n = 4

n = 7

20'

Figure 6: Protocol Bars in Bar-Chart Format. (a) Ischemia-Reperfusion Protocols. Langendorff-perfused

rabbit hearts were subjected to 5min ischemia () followed by reperfusion (). For longer reperfusion

protocols an equally time-matched control was performed. (b) Drug Administration Protocols.

Langendorff-perfused rabbit hearts were subjected to 5min drug infusion () followed by 2 and 10min

washout (WO; ). DMSO (0.1%) was used as a vehicle control for DADLE, APNEA, CCPA, and Cl-IB-MECA.

Bradykinin and AngII were dissolved in water. All protocols begin with a 20min stabilization period.

Concentrations of drugs used were those shown to be cardioprotective (see text and Table 3).

a

of 80

(APNEA; 10nM; Rice PJ, 1996; Liu GS, 1994), 2-chloro-N6-cyclopentyladenosine (CCPA; 0.2M;

Batthish M, 2002; Liu GS, 1996; Rice PJ, 1996), 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-

methylcarboxamide (Cl-IB-MECA; 50nM; Lasley RD, 1999), and [D-Ala2, D-Leu5]-enkephalin

(DADLE; 20nM; Cohen MV, 2007) were dissolved in dimethylsulfoxide (DMSO). Bradykinin (BK;

0.4M; Oldenburg O, 2004; Krieg T, 2005; Krieg T, 2004; Cohen MV, 2007), and angiotensin II

(AngII; 0.1M; Diaz RJ, 1997) were dissolved in water. All drugs were obtained from Sigma

Aldrich. Upon completion of the experimental procedure (Figure 6), hearts were immediately

immersed in cold (4°C) saline (0.9% NaCl) and ventricular tissue was freeze-clamped in liquid

nitrogen, N2(ℓ), and stored at -80°C until required.

For assessment of total PKC levels before and after ischemia, large biopsies of the right

and left ventricles were taken after 25min stabilization, 5min global ischemia, and following

30min reperfusion. Each biopsy was further upstream from the last and snap-frozen

immediately upon excision. Tissue homogenates were measured for PKC on immunoblots

after loading the same protein per sample per lane, measured as micrograms of protein.

Microscopy

Sample Preparation for Immunogold Whole hearts were perfused with 4% paraformaldehyde (PF) + 0.1% glutaraldehyde

(GA), the standard immunoelectron microscopy (IEM) fix, fixing the heart within seconds. The

heart was incubated in IEM fix for 2h at room temperature, washed in 0.1M phosphate buffer

and stored in 0.2M sodium azide prior to cryosectioning, labeling, and counterstaining with

0.2% uranyl acetate in 2% methyl cellulose (1min in each). As an alternative, freeze-

substitution was performed in previously freeze-clamped tissue (the PKC distribution of which

was already assessed by western blotting). This tissue was thawed in various fixatives (2.5% GA

only, 4% PF + 0.1% GA, 2%PF + 0.01% GA, 6% PF only; Figure 7) then incubated in 2.3M sucrose

at 4°C overnight (optional). This was followed by plunge freezing in N2(ℓ) and placed in -80°C

0.5% uranyl acetate in 100% methanol for 72h (unfixed tissue began at the -80°C step). Tissues

were washed twice in -20°C 100% methanol for 2h, infiltrated with 1:1 methanol/Lowicryl

HM20 twice for 24h each, and twice in 100% Lowicryl for 24h each. Samples were placed in

fresh Lowicryl at -20°C for 48h under UV illumination and inverted for another 24h to allow

complete polymerization. Ultrathin sections were cut and dried onto formvar-coated nickel

grids and labelled.

Immunogold Labelling of PKCGrids were incubated in six changes of 1.5% glycine in 0.5% BSA/PBS 10min each. The

blocked grids were then incubated overnight at 4°C with either mouse monoclonal anti-N–

terminal PKC antibody (BD Transduction; 610086) or rabbit polyclonal anti-C–terminal PKC

of 80

Figure 7. Different Fixation Techniques on Snap-Frozen, Freeze-Substituted Ventricular Tissue

Figure 7. Different Fixation Techniques on Snap-Frozen, Freeze-Substituted Ventricular Tissue.

Incubated overnight in 1:1 N-terminal PKC mouse mAb as pilot. (a) 6% PF only; (b) 2% PF + 0.01%

GA; (c) 4% PF + 0.1% GA; (d) 2.5% GA only. Only unfixed (not shown) and 6% PF showed gold

particles. 6% PF has excellent structural preservation and more gold particles compared to the

other fixatives and was thus selected as the preparation of choice. Blocking was achieved with

0.15% glycine for the first two 10min changes only.

Chemical Fixation Techniques on Freeze-Clamped Ventricular Myocardium

ab

dc

of 80

antibody (Abcam; ab4140) in 0.5% BSA/PBS in various concentrations. This was followed by six

10min washes in 0.5% BSA/PBS and 1h with 1:20 GAM (or GAR) 10nm-gold-conjugated

secondary antibody at room temperature. To eliminate any unbound secondary antibody, grids

were washed 6 times in 0.5% BSA/PBS and 6 times in dH2O (5-10min each). Grids were allowed

to dry and then counterstained with 0.2% uranyl acetate and lead citrate (1min each) with

extensive washing in-between and after each stain.

Statistical AnalysisAll statistics was performed using StatView software. The Kolmogorov-Smirnov test for

normality was used to generate z-values to determine if the data were normally distributed.

Fisher’s PLSD (protected least significant difference) was the parametric test used to determine

differences across groups in the three-compartment analysis (cyto vs. mito vs. membrane) and

the two-compartment analyses (cyto vs. mito). In the case of measure the changes of PKC

redistribution with cyto and mito only, because cyto + mito is defined to equal 1, comparing

cytosolic PKC across groups is identical to using mitochondria (cytosol was used). A paired t-

test was used to assess the relative changes in PKC when prepared fresh, snap-frozen, or

freeze-clamped because samples came from the same heart. The t-test was used to assess the

difference of the number of gold particles/m2 in IPC from control. All variability was displayed

as a standard error (SE) of the mean.

Results

Fresh Tissue vs. Frozen Tissue The morphology of ventricular myocardium was analyzed by TEM following fixation in

2.5% glutaraldehyde + 4% paraformaldehyde (standard TEM fixation) prepared either fresh,

after snap-freezing, or by freeze-clamping. Freezing artifact in the form of ice-crystal damage

can be seen in the freeze-clamped and snap-frozen tissue when compared to freshly fixed

tissue (Figure 2). Mitochondrial outer membranes and cristae are distinguishable in all three

preparations. Following mitochondrial isolation (see Fractionation below), mitochondria

purified from fresh myocardium are better able to withstand the homogenization and

centrifugation steps of the fractionation procedure (more intact mitochondria) than that from

frozen myocardium (Figure 3). However, some preliminary data suggests that PKC

redistribution may still be occurring in myocardium processed (fractionated) without freezing

(Figure 4). Cytosolic PKC was measured in these samples and was highest in the freeze-

clamped and lowest in the freshly processed tissue. Under basal conditions, PKC has been

shown to be localized predominantly in the cytosol, which was the profile most closely matched

in the freeze-clamped myocardium. Freeze-clamping was chosen as the method of choice for

preparing myocardial tissue in subsequent experiments because 1) freezing artifact is not

of 80

sufficient to rupture mitochondria, 2) contamination among fractions is minimized (see

Fractionation below), and 3) to avoid any artificial redistribution of PKC that may occur during

processing since freezing stops biochemical processes that may influence the localization of

PKC within the myocardium.

Fractionation The mitochondrial marker prohibitin reveals a very dense band only in the

mitochondrial fraction, in conjunction with very little sarcolemmal marker 1-integrin and

cytosolic marker lactate dehydrogenase (LDH) (Figure 8). There was virtually zero prohibitin

found in the cytosolic compartment following the 100,000g spin. The relative level of

contaminants among fractions did not have any statistically significant correlation with the level

of PKC in any fraction (Figure 9). The cytosolic and mitochondrial fractions were the most

pure, being greatly enriched by their respective compartments with little contamination.

Fractionation steps were imaged by TEM, revealing vesicles of smaller sizes with subsequently

higher centrifugation steps (Figure 5). Mitochondria isolated from freshly fractionated tissue

had a better morphology than mitochondria obtained from tissue that was frozen (Figure 3), as

expected due to artifacts of freezing and subsequent thawing prior to TEM.

PKC Redistribution Following 5min Ischemia Compared to untreated hearts, 5min global ischemia followed by 10min reperfusion

resulted in the redistribution of PKC into the mitochondrial fraction with a concomitant

decline in the cytosolic pool. PKC in the sarcolemmal fraction, however, did not change

significantly, implying most of the PKC movement was from the cytosol to the mitochondria

only (Figure 10). Thus, the sarcolemmal fraction was not considered all subsequent analyses

which focused on redistribution of PKC between cytosol and mitochondria.

Under basal conditions in the Langendorff-perfused hearts, PKC was predominantly

localized to the cytosol with small amounts in the mitochondria. This held true for hearts

perfused for 35min up to 2.5h (Figure 11). 5min of ischemia caused a massive redistribution of

PKC to mitochondria, an effect that slowly returned to baseline throughout reperfusion (Figure

12). PKC was retained in the mitochondria up to at least 30min of reperfusion, after which the

cytosolic PKC level overtook that of the mitochondria normalizing at the basal level. By 2h of

reperfusion the mitochondrial/cytoplasmic distribution was indistinguishable from time-

matched control hearts.

PKC Redistribution Following Pharmacological Preconditioning with

5min Infusion of Various GPCR Agonists

PKC mitochondrial/cytoplasmic redistribution of the Langendorff-perfused rabbit heart

following global ischemia was compared to various pharmacological agents shown to mimic IPC

of 80

Figure 8. The Distribution of Subcellular Markers Following Fractionation

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Cyto Mito Mem

Distribution of Fraction-Specific Markers for All Hearts

Figure 8. The Distribution of Subcellular Markers Following Fractionation. (A) The cytosolic marker

lactate dehydrogenase (LDH) is enriched in the cytosolic fractions, as are prohibitin and 1-integrin in

the mitochondrial and membranous “Mem” fractions, respectively. There is some cross-

contamination of cytosol and mitochondria in the membranous fraction and cytosol and sarcolemma

in the mitochondrial fraction. All hearts were fractionated identically and each fraction is enriched by

their respective compartment. The membranous compartment represents the sarcolemmal/SR

compartment. Data represented as mean ± SE. (B) Representative western blots showing the

distribution of compartment-specific markers. LDH (1:10000), prohibitin (1:1000), 1-integrin (1:1000)

in Blotto milk.

Re

lati

ve D

ensi

ty

Cyto Mito

Mem

Cyto Mito

Mem

Cyto Mito Mem

Lactate Dehydrogenase Prohibitin -

Integrin

A

B

prohibitin LDH

1-integrin

of 80

Figure 9. PKC Dependence on Contamination

Cytosol Mitochondria Membrane

PKC vs LDH p = 0.6538 p = 0.225 p = 0.5699

PKC vs prohibitin p = 0.0483 p = 0.4241 p = 0.6875

PKC vs 1-integrin p = 0.1588 p = 0.7757 p = 0.8733

0

0.2

0.4

0.6

0.8

1

1.2

0 0.2 0.4 0.6 0.8

PKCε

Membranous PKCε

0

0.2

0.4

0.6

0.8

1

1.2

0 0.05 0.1 0.15 0.2

PKCε

Mitochondrial PKCε

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 0.1 0.2 0.3 0.4 0.5

PKCε

Cytosolic PKCε

prohibitin LDH

1-integrin

Figure 9. PKC Dependence on Contamination. Relative Density of prohibitin, LDH, and 1-integrin with respect to that of

PKC in the cytosolic, mitochondrial, and membranous fractions in control hearts. There is no correlation between PKC

density and the density of any subcellular marker, implying that any change in PKC distribution is not due to contamination

of any fraction, but to its movement from one compartment to another. The only exception is an apparent correlation

between PKC and prohibitin in the cytosolic fraction. However being virtually non-existent in the cytosol (Figure 10), this

correlation is meaningless. Correlation z-test 95% confidence interval upper and lower.

Re

lati

ve D

ensi

ty

Cyt

o +

Mit

o +

Me

m =

1)

of 80

p = 0.0032

p = 0.0086

p = 0.4653

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Redistribution of PKCε in Rabbit Ventricular Myocardium Following 5min Ischemia 10min Reperfusion

Figure 10. Relative Density of PKC in Cytosol, Mitochondria, and Membranous

Fractions in Rabbit Ventricular Myocardium

Figure 10. Relative Density of PKC in Cytosol, Mitochondria, and Membranous Fractions

in Rabbit Ventricular Myocardium. PKC in the membranous fraction (enriched with

sarcolemma and SER) does not change after 5min ischemia and 10min reperfusion, in

strong contrast to the decline in cytosol and rise in mitochondria. There was no change in

sarcolemmal PKC after 5min ischemia alone (not shown). All hearts subjected to total

35min perfusion protocols prior to snap-freezing and processing. Data are displayed as

mean ± SE. Significance assessed with Fisher’s PLSD. Representative immunoblots are

displayed (PKC; 1:1000) in Blotto milk.

Cyto Mito Mem

Control n = 7

Cyto Mito Mem

5min IPC 10min Rep n = 6

Re

lati

ve D

en

sity

(Cyt

o +

Mit

o +

Me

m =

1.0

)

of 80

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Cytosolic and Mitochondrial Distributation of the Langendorff-Perfused Rabbit Heart Without Intervention

Figure 11. Cytosolic and Mitochondrial PKC in

the Langendorff-Perfused Rabbit Heart

Cyto Mito

Control 35' n = 10

Cyto Mito

Control 60' n = 5

Cyto Mito

Control 150' n = 3

p = 0.3800 p = 0.2714

p = 0.7374

Figure 11. Cytosolic and Mitochondrial PKC in the Langendorff-Perfused Rabbit Heart.

Under basal conditions, PKC predominates in the cytosol with basal levels found in the

mitochondria. Despite the visual trend for mitochondrial PKC to rise with time, this is

not significant. Data are displayed as mean ± SE. Significance assessed with Fisher’s

PLSD. Representative immunoblots are displayed (PKC; 1:1000).

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Redistribution of PKCε Following 5min Global Ischemia in the Langendorff-Perfused Rabbit Heart

Figure 12. Cytosolic and

Mitochondrial PKC in the

Langendorff-Perfused Rabbit

Heart Following 5min Global

Ischemia

Cyto Mito

Control (weighted average)

Cyto Mito

IPC 2' Rep n = 4

Cyto Mito

IPC 30' Rep n = 5

Cyto Mito

IPC 0' Rep n = 4

Cyto Mito

IPC 10' Rep n = 7

Cyto Mito

IPC 2h Rep n = 3

Figure 12. Cytosolic and Mitochondrial PKC in the Langendorff-Perfused Rabbit Heart Following

5min Global Ischemia. Under ischemic conditions, PKC accumulates in the mitochondria within

5min and slowly reverts to basal conditions by 2h reperfusion. The control data is that from Figure

9, and the p-values are between time-matched groups. Data are displayed as mean ± SE.

Significance assessed with Fisher’s PLSD. Representative immunoblots are displayed (PKC;

1:1000).

p = 0.019 p = 0.024 p = 0.0006 p < 0.0001

p = 0.1067 p = 0.0047

p = 0.1512

p < 0.0001 p = 0.0003 p < 0.0001 p = 0.0205 p = 0.8131

p = 0.5956 p = 0.4389 p =0.05

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+ M

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(Table 3). 5min global ischemia followed by 2min of reperfusion resulted in mitochondrial PKC

accumulation indistinguishable from that of any of the GPCR agonists used (Figure 13). In

contrast to IPC, however, prolonging the washout period to 10min produced a PKC

redistribution which was not different than controls (Figure 14). There were no significant

differences between drugs (Table 4), such that all six GPCR agonists used (A1/A3, A1, A3,

bradykinin, AngII, 1-opioid) affected PKC redistribution similarly.

Drug Compound Ligand Concentration References APNEA N6-2-(4-

aminophenyl)ethyladenosine

Adenosine A1 and A3 receptors

0.1M Diaz RJ, 1999; Batthish M, 2002; Rice PJ, 1996, Liu GS, 1994

CCPA 2-chloro-N6-cyclopentyladenosine

Adenosine A1 receptors

200nM Kristo G, 2004; Kilpatrick EL, 2001; Baxter GF, 1997; Bell RM, 2005; Batthish M, 2002; Liu GS, 1996

Cl-IB-MECA

2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide

Adenosine A3 receptors

50nM Kilpatrick EL, 2001; Lasley RD, 1999; Ge ZD, 2006; Peart J, 2003

Bradykinin Bradykinin acetate Bradykinin B1 and B2 receptors

0.4M Oldenburg O, 2004; Krieg T, 2005; Cohen MV, 2007; Weinbrenner C, 1996

DADLE [D-Ala2, D-Leu5]-enkephalin 1-opioid receptors 20nM Cohen MV, 2007

AngII Angiotensin II Angiotensin II AT1 and AT2 receptors

100nM Diaz RJ, 1997

Table 3: Pharmacological Preconditioning Agents Used. Concentrations used were shown to protect myocardium against IR injury. Those of the same concentration as the present study in the rabbit are in bold, while those with similar protocol (5min perfusion/10min washout) in rabbits are underlined. Kilpatrick et al. (2001) used the same concentration for 5min in the rabbit, but did not employ a washout.

Table 3: Pharmacological Preconditioning Agents Used

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PKCε Distribution After 5min Adenosine Receptor Agonist

Treatment and min Washout

Figure 13a. PKC Redistribution to

Mitochondria Following 5min

Adenosine Agonist Drugs with

2min Washout

Cyto Mito

DMSO n = 6

Cyto Mito

IPC 2'R n = 4

Cyto Mito

A1/A3

n = 3

Cyto Mito

A1

n = 3

Cyto Mito

A3

n = 3

Figure 13a. PKC Redistribution to Mitochondria Following 5min Adenosine Agonist Drugs with 2min

Washout. All adenosine agonists were dissolved in DMSO (0.1%), which had no effect on PKC distribution.

All drugs induced mitochondrial accumulation of PKC akin to IPC with 2min reperfusion. There is no

difference between control and DMSO vehicle control (not shown; p = 0.6646). Data are displayed as mean ±

SE. Significance assessed with Fisher’s PLSD. Representative immunoblots are displayed (PKC; 1:1000).

p = 0.0022 p = 0.002 p = 0.0829 p = 0.0405

p = 0.9799 p = 0.2097 p = 0.334

p = 0.2008 p = 0.3216

p = 0.7703

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PKCε Distribution After 5min Bradykinin, δ Opioid, and

AngII Agonist Treatment and min Washout

Figure 13b. PKC Redistribution to Mitochondria Following 5min GPCR Agonists with 2min Washout

Cyto Mito

Control n = 10

Cyto|Mito

DMSO n = 6

Cyto|Mito

IPC 2'R n = 4

Cyto|Mito

Bradykinin

n = 3

Cyto|Mito

DADLE

n = 3

Cyto|Mito

AngII

n = 7

Figure 13b. PKC Redistribution to Mitochondria Following 5min GPCR Agonists with 2min Washout. All

agents were dissolved in water, except for DADLE, which was dissolved in DMSO (0.1%). 5min 0.4M

bradykinin, 20nM DADLE (1-opioid), and 100nM angiotensin II with 2min washout all redistributed PKC to

mitochondria akin to IPC with 2min reperfusion. Data are displayed as mean ± SE. Significance assessed

with Fisher’s PLSD. Representative immunoblots are displayed (PKC; 1:1000).

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p = 0.6646 p = 0.0003 p = 0.0043 p = 0.0025 p < 0.0001

p = 0.0022 p = 0.0212 p = 0.0168 p = 0.0007

p = 0.4673 p = 0.3793 p = 0.7385

p = 0.6506

p = 0.9178 p = 0.5981

p = 0.4806

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1

PKCε Distribution After 5min Adenosine Receptor Agonist

Treatment and min WashoutFigure 14a. PKC Redistribution to

Mitochondria Following 5min

Adenosine Agonist Drugs

with 10min

Washout

Cyto Mito

DMSO n = 6

Cyto Mito

IPC 10'R n = 7

Cyto Mito

A1/A3

n = 4

Cyto Mito

A1

n = 3

Cyto Mito

A3

n = 3

Figure 14a. PKC Redistribution to Mitochondria Following 5min Adenosine Agonist Drugs with

10min Washout. All adenosine agonists were dissolved in DMSO (0.1%), which had no effect PKC

distribution. Unlike IPC, 10min washout was enough time for the PKC signal in the mitochondria

to reverse back into the cytosol. There is no difference between control and DMSO vehicle control

(not shown; p = 0.6646). Differences between the 2-min and 10-min washout groups for given

drugs are as follows: APNEA (p = 0.0004), CCPA (p = 0.1395), Cl-IB-MECA (p = 0.0774). Data are

displayed as mean ± SE. Significance assessed with Fisher’s PLSD. Representative immunoblots are

displayed (PKC; 1:1000).

p < 0.0001 p = 0.3373 p = 0.93 p = 0.9339

p < 0.0001 p = 0.0003 p = 0.0003

p = 0.349 p = 0.3512

p = 0.9965

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0.9

1

PKCε Distribution After 5min Bradykinin, δ Opioid, and

AngII Agonist Treatment and min Washout

Figure 14b. PKC Redistribution to Mitochondria Following 5min GPCR Agonists with 10min Washout

Cyto Mito

Control n = 10

Cyto Mito

DMSO n = 6

Cyto Mito

IPC 10'R n = 7

Cyto Mito

Bradykinin

n = 3

Cyto Mito

DADLE

n = 3

Cyto Mito

AngII

n = 4

Figure 14b. PKC Redistribution to Mitochondria Following 5min GPCR Agonists with 10min Washout. All

agents were dissolved in water, except for DADLE, which was dissolved in DMSO (0.1%). The effect of 5min

0.4M bradykinin, 20nM DADLE (1-opioid), and 100nM angiotensin II was lost with 10min washout, but retained

after 5min ischemia with 10min reperfusion. Differences between the 2-min and 10-min WO groups for given

drugs are as follows: bradykinin (p = 0.0046), DADLE (p = 0.0008), AngII (p = 0.0003). Data are displayed as mean

± SE. Significance assessed with Fisher’s PLSD. Representative immunoblots are displayed (PKC; 1:1000).

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.0)

p = 0.6646 p < 0.0001 p = 0.527 p = 0.2367 p = 0.9178

p < 0.0001 p = 0.3644 p = 0.1594 p = 0.6506

p < 0.0001 p < 0.0001 p < 0.0001

p = 0.6506

p = 0.6503 p = 0.3737

p = 0.3403

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Table 4: Statistics

Fischer’s PLSD Significance (p) Values for GPCR Agonists

IPC 10' Reperfusion

APNEA 10' WO

CCPA 10' WO

Cl-IB-MECA 10' WO

Bradykinin 10' WO

DADLE 10' WO

Angiotensin II 10' WO

IPC 10' Reperfusion <.0001 0.0003 0.0003 <.0001 <.0001 <.0001

APNEA 10' WO 0.349 0.3512 0.9622 0.6847 0.6018

CCPA 10' WO 0.9965 0.3737 0.1811 0.6301

Cl-IB-MECA 10' WO 0.376 0.1825 0.6334

Bradykinin 10' WO 0.6503 0.6374

DADLE 10' WO 0.3403


Fischer’s PLSD Significance (p) Values for GPCR Agonists

Table 4: Statistics. Statistical Analysis (p-values; = 0.05) for all pharmacologic agents including IPC with 2 or 10min reperfusion. Not all

comparisons were shown graphically, so the statistics are provided here, with statistical significance in red. The duration of the washout (WO) is

more important for differences PKC redistribution than the identity of the drug. However, for IPC this is not the case because there is no

difference between IPC with 2 or 10min of WO. On the contrary, between 2 and 10min of reperfusion there is little change in the PKC

redistribution, and no differences arise between IPC 2' reperfusion with any drug with 2' WO (left-most column of c),

a IPC 10' Reperfusion

APNEA 10' WO

CCPA 10' WO

Cl-IB-MECA 10' WO

Bradykinin 10' WO

DADLE 10' WO


IPC 10' Reperfusion <.0001 0.0003 0.0003 <.0001 <.0001 <.0001

APNEA 10' WO 0.349 0.3512 0.9622 0.6847 0.6018

CCPA 10' WO 0.9965 0.3737 0.1811 0.6301

Cl-IB-MECA 10' WO 0.376 0.1825 0.6334

Bradykinin 10' WO 0.6503 0.6374

DADLE 10' WO 0.3403


c IPC 10' Reperfusion

APNEA 10'WO

CCPA 10' WO

Cl-IB-MECA 10'

WO

Bradykinin 10' WO

DADLE 10' WO


IPC 2' Reperfusion 0.5756 0.0004 0.0073 0.0073 0.0005 0.0001 0.001

APNEA 2' WO 0.596 0.0004 0.0068 0.0068 0.0004 <.0001 0.0009

CCPA 2'WO 0.043 0.0173 0.1395 0.1384 0.0195 0.0058 0.041

Cl-IB-MECA 2' WO 0.091 0.008 0.0781 0.0774 0.0091 0.0025 0.0193

Bradykinin 2' WO 0.158 0.004 0.0462 0.0457 0.0046 0.0012 0.0098

DADLE 2' WO 0.094 0.003 0.0425 0.042 0.0035 0.0008 0.0075

Angiotensin II 2' WO 0.2505 0.0001 0.0045 0.0045 0.0002 <.0001 0.0003

b IPC 2' Reperfusion

APNEA 2' WO

CCPA 2' WO

Cl-IB-MECA 2' WO

Bradykinin 2' WO

DADLE 2' WO


IPC 2' Reperfusion 0.9799 0.2097 0.334 0.4673 0.3793 0.7385

APNEA 2' WO 0.2008 0.3216 0.4522 0.365 0.7162

CCPA 2' WO 0.7703 0.5942 0.6408 0.2488

Cl-IB-MECA 2' WO 0.8094 0.8771 0.4173

Bradykinin 2' WO 0.9178 0.5981

DADLE 2' WO 0.4806


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1.6

baseline 5min Ischemia 30min Reperfusion

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1.4

1.6

baseline 5min Ischemia 30min Reperfusion

To assess the possibility of PKC production or destruction causing mitochondrial or

cytoplasmic PKC decline, total levels of PKC in myocardial homogenates from both right and

left ventricles was measured at baseline, after 5min ischemia, and following 30min reperfusion.

Although the data were highly variable from heart to heart, total PKC levels do not appear to

change in either ventricle during 30min of reperfusion (Figure 15).

Figure 15. Total PKC Levels Following 5min Ischemia Crude PKC Levels Following 5min Ischemia

a b

p = 0.8398 p = 0.8542

p = 0.9583

p = 0.8730 p = 0.5229

p = 0.4284

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PKC

GAPDH

PKC

GAPDH

10g; Left Ventricle

10g; Right Ventricle

c

Figure 15. Total PKC Levels Following 5min Ischemia. Left (a) and right (b) ventricular

homogenates were assessed for total PKC. Although the extensive variability cannot be ignored,

PKC is neither created nor destroyed 30min after the 5min ischemic stimulus. Scheffe’s test; all data

are mean ± SE. (c) Representative immunoblots of homogenates. PKC (1:1000); GAPDH (1:10000).

Left Ventricle (n = 4) Right Ventricle (n = 4)

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Immunogold Labeling of Ventricular Myocardium Perfusion-fixation is the ideal method of preserving tissue because doing so rapidly

cross-links proteins in the entire perfused tissue bed by using the vasculature as a conduit.

Control and 5min I/10min R hearts were perfusion-fixed with IEM fix (4% PF + 0.1% GA) and

stored for 1h at room temperature. This resulted in immaculately preserved tissue (Figure 16)

but did not yield positive labeling. Consequently, freeze-clamped tissue was used because its

cytosolic and mitochondrial distributions of PKC were already determined by western blotting.

The best fixation technique (Figure 7) was 6% paraformaldehyde for 10min at room

temperature. By labeling overnight with 75% anti-C–terminal rabbit PKC polyclonal antibody,

gold particles were found in mitochondria in control and IPC hearts, but not in the negative

control (Figure 17). The number of gold particles per m2 of mitochondria increased in the IPC

(5min I/10min R) compared to control (Figure 18).

Discussion

Model IPC has been studied in a variety of experimental models (Ytrehus K, 2000) that vary in

complexity (Figure 1). New Zealand White rabbits were selected due to their similarities to

humans and mice (in which many of the most insightful experiments have been done, as cited

above) with respect to PKC being responsible for mediating myocardial protection from IR

injury.

The effect of circulating blood-borne factors and neuronal stimulation is removed by

excising the heart from the body and mounting it on a Langendorff apparatus for ex vivo

experiments. Although advantageous in many regards, caution must be taken when

extrapolating to the in vivo situation (Mik EG, 2009 and references therein). Some other

benefits of this model are the ease with which drugs can be administered at a constant

concentration in the perfusate and other manipulations made. Global ischemia is easily

attained by complete cessation of perfusate, or local ischemia by ligating one large artery as

performed in vivo. Perfusion pressure or flow can be modulated throughout the experiment

according to design, but one cannot forget that they are not the same. Constant perfusion

pressure preserves the heart’s innate ability to control local perfusion by vasoconstriction or

vasodilatation. This is important in a heterogeneous organ like the heart. IPost protection

differed in rat hearts perfused at constant flow than at constant pressure (Penna C, 2006;

Penna C, 2008), while there was no difference for IPC (Penna C, 2006). Constant perfusion

pressure was used in the present study. This model has been used by many experimenters and

is accepted as an ex vivo representation of the beating heart.

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Figure 16. Perfusion-Fixed Ventricular Myocardium

Electron Microscopy of Perfusion-Fixed

Ventricular Myocardium

a b

Figure 16. Perfusion-Fixed Ventricular Myocardium. a-b) TEM image of control (non-ischemic) ventricular

myocardium perfusion-fixed with IEM fixative and stored in universal fixative (2.5% GA + 4% PF). c-d) Ischemic

(no reperfusion) from the same preparation (IEM fix only; 5min I/10minR) was labeled with N-term PKC 1:10 1h

at RT. (a) 5,000× (b) 50,000×, (c) 20,000×, (d) 60,000×. Only a few gold particle clusters were detected ().

c d

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Figure 17: Snap-Frozen Control and IPC Ventricular Myocardium: Freeze-

Substituted and Immunolabeled for PKC

Figure 17: Snap-Frozen Control and IPC

Ventricular Myocardium: Freeze-

Substituted and Immunolabeled for PKC.

75% C-terminal 10nm gold was used to

probe PKC. (a) Control and (b) IPC samples

have many particles of gold () in and

around mitochondria, myofilaments, and

often concentrated in the z-lines. Only

mitochondrial gold was quantitated.

Blocking was in 1.5% glycine throughout the

entire labeling. (c) The negative control has

no gold, implying that the GAR secondary

antibody is not binding non-specifically.

Mitochondrial PKC by Immunogold Labelling

on Freeze-Clamped Myocardium

a b

c

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Figure 18: Density of gold particles

Fresh Tissue vs. Frozen Tissue Although enzyme activity is often lost upon freezing, many experimenters opt to freeze

tissue prior to processing (Nakamura Y, 2004; Pass JM, 2001; Wolfrum S, 2002). One major

concern for using frozen tissue is the emergence of ice crystals that can disrupt cell structure,

potentially causing mitochondrial rupture upon freezing and thawing, releasing their contents

into the surrounding cytosolic milieu. Frozen myocardium was not substantially inferior with

respect to maintaining mitochondrial structure, whether freeze-clamped or snap-frozen (Figure

2). Both inner and outer mitochondrial membranes remained intact as seen on TEM, although

ice crystal damage was often manifest as less dense cristae. If mitochondria were damaged

sufficiently that proteins leak out of the mitochondria and back into the cytosol, then it would

be more difficult to find significant PKC redistribution to mitochondria, which was indeed

obtained. Due to the speed at which freeze-clamping works, it is the preferred method of

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

# Gold Particles/μm² Mitochondria

Mitochondrial Gold Density: Control vs. IPC

Figure 18: Density of gold particles. Total number of gold particles per m2 of

mitochondria. IPC was induced by 5min I/10min R prior to freeze-clamping.

Mean ± SE. p = 0.0273. Two-tailed unpaired t-test ( = 0.05). N = 1 means

one rabbit heart was used, and one grid was scanned.

Control (N = 1)

18 pictures;

6.75 m2 mito/pic

IPC (N = 1)

27 pictures;

8.67 m2 mito/pic

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freezing non-cryoprotected myocardial tissue. PKC redistribution may also depend on the

manner in which tissue is stored prior to processing. Data suggests (Figure 4) that PKC

movement is dependent on the rapidity by which the tissue is frozen since plunging the heart in

cold saline does not rapidly stop all subcellular processes. Thus, the speed of freeze-clamping

not only preserves mitochondrial integrity reasonably well, but, more imporantly preserves the

subcellular localization of the protein of interest. When assessing a dynamic process like PKC

movement between cytosol and mitochondria, it is important to freeze-clamp the heart as

quickly as possible to avoid any changes during processing (Kawamura S, 1998). Removal of the

heart from the Langendorff apparatus, removal of extraventricular tissue in cold saline and

finally freeze-clamping took approximately 90s to complete. Thus, not only does freeze-

clamping provide convenience in batch-processing different samples simultaneously, but it

makes it possible to take a “snapshot” of the myocardium, avoiding changes in the distribution

of PKC.

Fractionation

Redistribution of PKC has been shown among several subcellular compartments after

activation, with the majority of experimenters looking at the particulate fraction (see Table 2),

which includes a plethora of membranous organelles, not limited to mitochondria, SER,

sarcolemma, peroxisomes, myofibrils, and sometimes nuclei. Although subcellular

fractionation can be used to isolate mitochondria and cytosol, one can never completely avoid

contamination by other organelles (Foster B, 2008). Although LDH is found in much greater

quantities in the cytosolic fraction, the weaker band in the sarcolemmal fraction is likely due to

inadequate washing, but any LDH in the mitochondrial fraction (Figure 8) may be attributable to

a mitochondrial pool of LDH (Brooks GA, 1999). Although some contamination is unavoidable,

it is minimized since the mitochondrial fraction was resuspended, repelleted, and rinsed. The

effect of contamination on PKC distribution among the fractions was negligible because PKC

levels within a fraction did not change with varying levels of any of the three suborganellar

markers used (Figure 9). Also, since the fractionation protocol was followed identically for all

hearts, the amount of contamination of any fraction should be similar among all groups. With

respect to purity there was no difference between freezing and fresh processing (not shown),

but the morphology of the isolated mitochondria was superior in fresh tissue than frozen

(Figure 3). There were a greater number of damaged mitochondria in the frozen fractionated

tissue, presumably due to ice crystal formation compromising the membrane’s integrity and

ability to withstand the 10,000g spins. Nevertheless, intact mitochondria were not necessary

since differences of PKC distribution were still seen across groups. Assuming there was some

leakage of PKC back into the cytosol due to freeze-thaw injury, this would make it harder to

achieve significant changes in redistribution of the kinase from cytosol to mitochondria, which

were clearly achieved following 5min ischemic stress. Moreover, the cytosolic fraction (Figure

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8) had no prohibitin marker, indicative of an absence of mitochondria in this fraction. On the

other hand, the mitochondrial fraction had very little cytosolic marker lactate dehydrogense.

Cross-contamination of the fractions was adequately minimized, and significant changes in

PKC were still seen.

PKC Redistribution Following 5min Ischemia

In unstimulated control hearts, PKC existed at basal levels in mitochondria,

sarcolemma, and cytosol (Figure 10 and 11). 5min global ischemia followed by 10min

reperfusion in the rabbit heart is enough to confer cardioprotection (Schulz R, 1998; Przyklenk

K, 2006; Liem DA, 2005; Omar BA, 1991; van Winkle DM, 1991; Sandhu R, 1997; Cohen MV,

1994; Tracey WR, 1997; Weinbrenner C, 1996). The nuclear pool of PKC has been shown to

increase after IPC (Table 2), but this fraction has not been examined in the present experiments

due to its limited role in acute protection. The results shown in Figure 10 implicate PKC

movement from the cytosol to the mitochondria and not the sarcolemma/SR. This is in

corroboration with data obtained in pig skeletal muscle (Hopper RA, 2000) where the decline in

cytosolic PKC was not accompanied by a rise in the sarcolemmal/SR-enriched fraction, but was

seen in that study for the particulate fraction in which mitochondria and nuclei were included.

Thus, in studies that quantitated PKC movement to particulate the PKC redistribution can

likely be attributed predominantly to mitochondrial accumulation (provided nuclei have been

removed), which applies to most of the studies reporting on the particulate fraction. Since the

sarcolemma exhibits no change using western blotting techniques, this compartment was

removed from subsequent analyses. This, however, does not preclude some PKC movement

to the sarcolemma/SR, which has been shown in studies using alternative techniques (Table 2).

The SR network in cardiomyocytes is very extensive, but only accounts for up to 3.2% of the

volume of myocardial tissue and that of the sarcolemma is no greater than 1% (Severs NJ,

1985). Thus, even a two-fold rise in PKC content in the sarcolemma or SR would represent a

small change compared to a roughly two-fold rise in mitochondrial PKC, which comprises 30%

of the volume of the rabbit myocardium and an even greater percentage in rodents (Barth E,

1992; Severs NJ, 1985). Similarly, the nuclei comprise 2.5% of the volume of the rat

cardiomyocyte and likely represent a small portion of the PKC pool. The majority of the

cardiomyocyte volume is occupied by myofibrils (60%), which are largely removed in the 1000g

spin (Palmer JW, 1977; Figure 5).

Under basal conditions, despite the length of ex vivo perfusion, PKC predominates in

the cytosol, with basal levels in the mitochondria (Figure 10 and 11). Following 5min of global

ischemia, the cytosolic/mitochondrial PKC ratio is completely inverted, such that mitochondria

become the dominant pool of the kinase (Figure 12). Upon subsequent reperfusion,

mitochondrial PKC rapidly reverts back to the cytosol, at a rate that decreases with the length

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of reperfusion. By 30min of reperfusion, PKC in the mitochondria and cytosol are nearly

identical, as PKC in the cytosol once again begins to overtake that in the mitochondria. By

120min of reperfusion, the distribution of the kinase is nearly indistinguishable from control,

indicating the transient effects of ischemia on PKC redistribution. This suggests the importance

of PKC activation because IPC induced protection against myocardial IR injury in the rabbit 10,

30, and 60min into reperfusion, but not after 120min (van Winkle DM, 1991). Not surprisingly,

ischemia is insufficient to confer protection by itself, but requires a period of reperfusion to

provide time for intracellular signaling to provide for protection from a subsequent longer

period of ischemia (the index ischemia). Following the index ischemia, PKC activity is also

required, since reperfusing with PKC blockers can abrogate IPC protection (Inagaki K, 2003;

Zatta AJ, 2006; Hausenloy DJ, 2007). Many of the changes necessary for IPC protection must

therefore be occurring during reperfusion. The mechanism facilitating PKC movement

throughout the cell is currently unknown, but is postulated to involve microtubules (Quinlan CL,

2008; Nakamura Y, 2004; Xu TR, 2009) and perhaps a caveolin-coated signaling vesicle, termed

a signalosome (Quinlan CL, 2008; Jiao J, 2008).

PKC Redistribution Following Pharmacological Preconditioning with

5min Infusion of Various GPCR Agonists Adenosine, bradykinin, and opioids are the main contributors of protection in the in situ

heart (Critz SD, 2005). Blocking adenosine, bradykinin, or opioid receptors raises the threshold

for IPC protection, which is not the case for angiotensin II, endothelin-1, or norepinephrine

receptor blockade, suggesting a limited role of these latter agents in mediating IPC (Cohen MV,

2008; Miki T, 2000; Schulz R, 1998; Critz SD, 2005; Liem DA, 2005; Pears J, 2003). Bradykinin,

opioids and angiotensin II likely play a smaller role than adenosine in mediating IPC since their

abundance in the isolated heart is relatively low (Cohen MV, 2008; Qin Q, 2002; Downey JM,

2007; Miki T, 2000). Upon their release, the protective substances (i.e. adenosine) likely act

through autocrine and/or paracrine mechanisms by binding to their respective receptors and

culminating in a cardioprotected phenotype. PPC can protect similar to IPC if the

pharmacologic agent is administered for only 5min immediately prior to the index ischemia,

that is, no washout is required (Kilpatrick EL, 2001) presumably because the myocardium is

normoxic and has sufficient metabolic energy to complete the protective signaling cascade.

Pharmacological agents were administered to the Langendorff-perfused rabbit heart at doses

that induced protection in the rabbit (Table 3). All these GPCR agonists caused signaling that

converges on PKC, and in fact have been shown to promote PKC movement. All of the

pharmacological agents used (CCPA, Cl-IB-MECA, APNEA, bradykinin, angiotensin II, DADLE)

resulted in mitochondrial accumulation of PKC, seen 2min following washout of the drug

(Figure 13). The effects of each of these drugs on PKC redistribution between cytosol and

mitochondria were indistinguishable from that of IPC with 2min reperfusion. However, these

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effects on PKC’s redistribution disappeared by 10min of drug washout (Figure 14), while IPC

resulted in PKC retention at mitochondria at least 30min after the stimulus has been removed.

This indicates that IPC produces a more persistent activation of PKC although there is no

evidence that this increases the extent of protection compared to PPC. It has been shown that

0.4M bradykinin administered for 5min followed by 10min washout (identical to the

procedure used here) prior to prolonged IR injury resulted in reduced infarction (Krieg T, 2005;

Oldenburg O, 2004; Cohen MV, 2001; Cohen MV, 2007), despite data from the present study

indicating that PKC reverted back by 10min reperfusion to its basal, primarily cytosolic

localization. This may implicate PKC as putative trigger of protection, not just a mediator.

IPC is recognized to activate multiple GPCR signaling pathways of protection (Yang X,

2010). Whereas pharmacological treatment only activates one specific receptor, IPC activates

multiple receptors. Perhaps this culminates in longer mitochondrial PKC retention. It is also

likely that the duration of a GPCR agonist, unlike a direct PKC activator, would require a much

longer administration than IPC for a given amount of PKC redistribution (Tsouka V, 2002). It is

possible that the disparities between IPC and PPC may lie in the chemical and physical

manifestations of rendering the heart ischemic. Unlike anoxic or hypoxic preconditioning or

PPC, IPC is associated with a complete cessation of perfusion, and there is a buildup of

metabolites that cannot be washed away until reperfusion. In addition, the reperfusion

following the preconditioning ischemia may alter the cardiomyocyte’s environment, leading to

myofibrillar stretching (AngII release) that may persistently activate PKC well after reperfusion

has been initiated (Pisarenko OI, 1993; Browe DM, 2004; Stawowy P, 2005; Paul K, 1997). In

fact, reperfusion injury may be ameliorated by a gradual reperfusion protocol, which is as

protective as IPost and IPC, as shown in the brain (Gao X, 2008). It has been demonstrated that

movement of PKC to the particulate fraction increases with the number of preconditioning

cycles (Ping P, 1997) as well as the duration of the ischemia (Tsouka V, 2002). This, however,

does not preclude the role of reperfusion in providing excess stimuli that prevent PKC

returning to the cytoplasm from mitochondria.

Changes in Total PKC Levels Following Ischemic Stress

In order to investigate whether the decline in mitochondrial PKC is due to its

degradation or production, biopsies of the left and right ventricles were taken before IPC, after

5min global ischemia, and following 30min of reperfusion, each piece taken upstream from the

last. This approach eliminates rabbit-to-rabbit variability in endogenous levels of PKC, but

leaves region-to-region variability unaccounted for. Despite extensive variability among

samples taken from different regions of the same heart, PKC levels did not appear to change

from baseline after 30min of reperfusion, suggesting PKC is neither produced nor destroyed

(Figure 15). The expression of a variety of proteins is upregulated following an ischemic

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stimulus, resulting in dIPC. The upregulation of some proteins (e.g. SOD) that arise from

existing precursors has been shown as early as 40min into reperfusion (Thornton J, 1990 and

references within). If PKC were newly synthesized, it would be expected to appear first in the

cytosol after release, like any other newly synthesized protein, from the Golgi apparatus (see

Appendix). Any new PKC synthesis would, thus, reduce the ratio of mitochondrial PKC to

cytosolic PKC, giving the impression that mitochondrial PKC was returning towards baseline.

Accumulation of PKC in the particulate fraction upon treatment with PMA or hypoxia was not

due to degradation in the cytosol since total PKC levels did not change (Sovershaev MA, 2006;

Schechtman D, 2004; Goldberg M, 1997). Neither degradation products (smaller fragments)

nor ubiquitinated PKC intermediates (larger fragments; Lu Z, 1998) were seen in the present

experiments in any fraction or homogenate, precluding PKC destruction as a potential

confouner. Ubiquitination of PKC, , and isoforms in 3Y1 rat fibroblasts has been shown

after activation of PKC with phorbol ester (Lu Z, 1998), which results in their degradation

(Poulin B, 2009). Thus, in some environments, PKC activity is responsible for its own

degradation.

Acute protection is not due to protein synthesis since the presence of translation and

transcription inhibitors did not block protection in an in vivo rabbit model after 3h of

reperfusion (Thornton J, 1990). Moreover, the change in mitochondrial-to-cytosolic PKC levels

from 2min to 10min drug washout or from 0min to 2min reperfusion (Figure 12) occurs over

too short a time frame to allow for synthesis of new PKC. The decline in mitochondrial PKC is

likely attributable to the return of mitochondrial-bound PKC to the cytosol rather than its

cytosolic production or mitochondrial destruction. Rat neonatal cardiomyocytes subjected to

hypoxia (Goldberg M, 1997) and PMA-treated PKC transgenic and wild-type mice (Schetchman

D, 2004) resulted in PKC redistribution to the particulate fraction and were not associated with

a change in its abundance.

Microscopic Imaging of PKC at MitochondriaOptimization of immunogold labeling is subject to manipulation with respect to the

duration of the fixation, temperature of the fixation, concentration of individual fixatives, and

combination of individual fixatives. The immunogold labeling depends on the strength of the

antibody and the degree to which the fixative affects antibody binding. In the case of PKC a

fixative solution of minimal concentration of glutaraldehyde was desired to maximize signal.

Myocardium that was freeze-clamped and previously homogenized, fractionated, and

subjected to western western blotting were freeze-substituted (embedded in Lowicryl HM20 at

-80 to -20°C), sectioned, and labeled for electron microscopy. Samples from previously frozen

tissue were chosen in lieu of perfusion-fixation not only because signaling was negative (Figure

16), but because cytosolic/mitochondrial PKC redistribution can be measured from western

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blotting of the same sample, but not the reverse. Five different fixation techniques were

compared over a range of paraformaldehyde (PF) and glutaraldehyde (GA) concentrations:

2.5% GA, 4% GA + 0.1% PF, 2% PF + 0.01% GA, and 6% PF (Figure 7). Although some studies

with certain antibodies (not anti-PKC) have found success using up to 6% GA + 2% PF for 20min

(Salnikov V, 2009), all of the aforementioned techniques were incubated at room temperature

for only 10min. Of the different fixation techniques used, 6% PF alone appeared to provide

identical if not superior fixation compared to any of the fixation protocols employing

glutaraldehyde. Thus grids from this preparation and unfixed samples were used to optimize

labeling and a new polyclonal anti-C–terminal rabbit antibody was selected. This antibody was

chosen because a polyclonal antibody has a higher likelihood of binding PKC if not all available

epitopes are masked. 1:10 or 1:1 dilutions (overnight) of this antibody did not result in enough

staining while 100% primary Ab produced too much background staining (not shown). Thus, a

3:1 dilution was used (Figure 17), which resulted in adequate and clean labeling with no

background staining. Quantitation revealed an increase in the number of gold particles per m2

of mitochondria (Figure 18). PKC was found to be localized to mitochondria under basal

conditions and increased significantly following 5min of ischemia, in agreement with the

western blot observations.

Only two other studies to my knowledge have employed immunogold with PKC in

mitochondria. Hearts treated with PMA were perfusion-fixed (4% PF + 0.05% GA) had PKC

labeled with gold particles in and around mitochondria in intact cardiomyocytes (Juhaszova M,

2004). This was not quantitated, a control group was not provided, and a heart preconditioned

with ischemia was not assessed. More recently, PKC was demonstrated with gold in isolated

mitochondria (fixed with 4% PF + 0.025% GA), particularly the inner mitochondrial membrane

(Budas GR, 2010). These data were quantitated as the number of gold particles per

mitochondria, which does not account for variations in the size of different mitochondria when

sampling. Isolated mitochondria were used instead of whole tissue, as in the present study.

Additionally, the rise in gold particles in the IR group (Budas GR, 2010) compared to control was

not due to a preconditioning stimulus: the Langendorff-perfused rat hearts were subject to

35min global ischemia and 15min reperfusion, far beyond the prototypical 5min ischemia for a

rat heart to elicit protection. The shortcoming on the immunogold data (Budas GR, 2010) were

avoided here, as the counts and statistics were performed on the number of gold particles per

mitochondria area (corrected for differences in mitochondrial size or plane of sectioning

through any mitochondrion), with reference to a control group, and were performed in whole

tissue instead of isolated mitochondria.

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Novel Elements of This Work PKC translocation to the particulate fraction of cardiomyocytes in IPC is well

established. However, evidence of PKC translocation to myocardial mitochondria has only

been published by three groups (Baines, 2002; Juhaszova 2008; Ohnuma, 2002) in mice, rats

and rabbits, respectively. The Baines study was limited to stimulation from constitutively active

PKC, not IPC. Halestrap has expressed skepticism that PKC redistribution to mitochondria has

been reliably demonstrated, referring to technical problems with obtaining pure isolates of the

mitochondrial fraction uncontaminated by other membranes (Clarke, 2008). However, his work

in rats failed to show PKC redistribution to mitochondria, much less to the particulate fraction,

which has been well documented (Table 2). Because of the contamination issue, it is important

to establish PKC redistribution to mitochondria by an assay independent of mitochondrial

isolation in the intact cardiomyocyte under conditions of IPC.

My supervisor’s group has been able to show this using immunofluorescence

colocalization of PKC with prohibitin, a selective marker for mitochondria, but this light

microscopic technique cannot resolve individual mitochondria. In the present study, that

limitation has been overcome by immunogold electron microscopy. Through this technique,

the redistribution of PKC to mitochondria has been verified independent of contamination

issues.

PKC redistribution to the mitochondria has previously received very limited attention

under conditions of PPC (Ohnuma Y, 2002 with diazoxide; Juhaszova M, 2004 with diazoxide,

CCPA, DADLE). These studies did not compare the kinetics of the PKC between PPC and IPC.

Conclusions Ischemia of 5min duration caused a redistribution of PKC from the cytosol to the

mitochondria, which was slowly reversed throughout reperfusion. Administration of GPCR

agonists adenosine A1/A3 (APNEA), adenosine A1 (CCPA), adenosine A3 (Cl-IB-MECA), bradykinin,

angiotensin II, and 1-opioid (DADLE) for 5min also resulted in PKC redistribution from the

cytosol to the mitochondria. PKC was retained in the mitochondria much longer following IPC

than following PPC. Thus, although IPC and PPC exhibit similar spatial effects on PKC

redistribution, temporal differences do exist.

Future Directions The findings of this study pave the way for many lines of potential experimental work.

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1) There are several potential effectors of preconditioning in the mitochondria since this

organelle plays an important role in mediating apoptosis and ATP production. Some of these

effectors, namely, the putative mitoKATP channel and the MPTP, have not been resolved. The

very existence of this mitoKATP channel is debated (Das M, 2003). Of the putative mitoKATP

channel, antibodies to sulfonylurea receptors are available, although this protein is only

expected to be part of the channel if mitoKATP is similar to the sarcolemmal KATP channel.

Antibodies to potential components of the MPTP are commercially available, namely the

voltage-dependent anion channel (VDAC), adenine nucleotide transporter (ANT), and

cyclophilin-D. The VDAC has been claimed to be a target of PKC in the constitutively active

PKC transgenic mouse heart (Baines CP, 2003; Edmondson RD, 2002), but there is no published

evidence of this in either the mouse or the rabbit under conditions of acute IPC. The availability

of these antibodies makes it possible to ascertain whether PKC interacts with any of these

components by co-immunoprecipitation or immunofluorescence colocalization experiments. In

co-immunoprecipitation experiments, agarose beads conjugated to antibodies against PKC,

sulfonylurea receptor proteins, VDAC, ANT, and cyclophilin-D can be used to bind their

respective antigens in mitochondrial fractions obtained from control and preconditioned

myocardium. Once these antibodies bind their antigens, they can be subject to western

blotting and probed for all of the aforementioned proteins to assess which are associated with

which, a technique well-known to our lab. In the case of colocalization, immunohistochemistry

can be performed on tissue sections, where fluorophore-conjugated antibodies to the

aforementioned antigens can bind, allowing the visualization by fluorescence of the proteins of

interest. Colocalization studies have been performed with PKC and the mitochondrial marker

prohibitin in our lab in the past, linking the mitoKATP channel, ROS production, and PKC

redistribution (unpublished data). Several of the pharmacologic agents used in this study have

been shown to produce ROS in cardiomyocytes, namely angiotensin II (Cheng TH, 2004) as well

as bradykinin and DADLE (Cohen MV, 2007), assessed with ROS-sensitive fluorescent dyes (all

used at the same concentration as in this study to assess PKC redistribution).

The proteomic shotgun approach (Ping P, 2001; Edmondson RD, 2002) has revealed a

few, but not many, mitochondrial proteins associated with PKC in the constitutively active

PKC transgenic mouse. Since the samples that were measured in these studies by mass

spectrometry were done on tissue lysates, these mitochondrial proteins may have been

detected largely because of their abundance. Employing a similar approach using isolated

mitochondria from preconditioned cardiomyocytes might resolve a greater number of proteins

associated with PKC that would have not been detected due to their paucity in lysates.

Instead of a transgenic animal with constitutively active PKC, this can be done after pulling

down PKC from the mitochondrial fraction, that is, specific targets do not need to be identified

in advance. A more general approach would be to look at the entire mitochondrial fractions

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with or without IPC looking at mass spectrometry. This should reveal an increase in PKC,

among other compounds, in mitochondria after IPC and should provide insights into other

mediators of the preconditioning signaling cascade acting at the mitochondrion.

2) While IPC is due to activation of several different GPCRs, specific GPCR agonists

activate a single specific receptor, and so the effects of IPC and PPC on the kinetics of PKC

redistribution to mitochondria may differ. Administration of a cocktail of GPCR agonists may

mimic IPC’s effects on PKC redistribution more closely than any one, or two, pharmacological

agents administered alone. This would be expected to manifest as prolonged mitochondrial

PKC retention following the drug cocktail. Data from a single experiment our lab has done

using a cocktail of all six GPCR agonists with 10min washout resulted in PKC redistribution to

mitochondria in-between that of control and IPC.

3) PKC’s importance in IPC has been well documented, but whether the myocardium

can still be protected by activators or inhibitors of the aforementioned mitochondrial end

effectors with the absence of PKC has not been documented. Messenger RNA silencing of

PKC and attempting to precondition cardiomyocytes using various pharmacological agents that

open the putative mitoKATP channel (e.g. diazoxide), inhibit MPTP opening (e.g. cyclosporine A),

or other mediators of protection may clarify their protective roles downstream of PKC. This

would be more reliable than knockout animals since compensatory changes in proteins

associated with the gene knockout would be avoided.

4) The memory of PPC protection has not been adequately documented compared to

that of IPC, the protection of which was documented after 10, 30, and 60min of reperfusion,

but lost by 120min (van Winkle DM, 1991). Protection against myocardial IR injury at different

time points following 5min ischemia or 5min GPCR agonist treatment would be helpful to

compare between IPC and PPC in regard to the memory effect.

5) PKC is redistributed to the particulate fraction in RIPC (Shimizu, 2009) and RIPC

cardioprotection can be prevented by blocking either opioid or adenosine receptors (Surendra,

2009). Thus, one might expect the PKC redistribution to the mitochondria to be similar to PPC.

It would be interesting to examine the effect of RIPC in such PKC redistribution and its

dependence on opioid, adenosine, and potentially other GPCR receptors.

Limitations Unlike research animals, the typical types of patients who would require protection

from IPC or PPC are older, obese, smokers, diabetic, or have various complications.

Unfortunately, these conditions may prevent or even worsen the potentially therapeutic effects

of IPC or PPC. Thus, although experimental evidence has shown substantial benefit reducing IR

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injury on healthy young laboratory animals, the extrapolation to humans may not be realistic in

practice.

Appendix

RACK Binding, Intramolecular Interactions, and Synthetic

Activator/Inhibitor Peptides

The primary structure of PKC can shed light on its functional properties (Figure A1).

PKC, like all other PKCs, consist of two domains: a 20-40kDa N-terminal regulatory domain and

a 45kDa C-terminal catalytic domain that are separated by a hinge region (Newton AC, 1995).

PKC consists of five variable domains (V1-5) interspersed by four conserved domains (C1-4),

with differences appearing for different PKC classes. The C2 domain of cPKCs (conventional

PKCs) is responsible for the Ca2+-dependency of this class of PKCs. The Ca2+-independent nPKCs

(novel PKCs) have a C2-like domain located at the very N-terminus making PKC and PKC (both

are novel PKCs) unresponsive to elevations in Ca2+ (Kofler K, 2002). aPKCs (atypical PKCs) do

not have a conventional C2 domain, and only have one of the two zinc fingers that conventional

and novel PKCs have. Immediately distal to the C2-like domain in nPKCs is the V1 domain,

which is very important for the regulation of the protein. This domain contains the RACK

binding site (aa 14-21 in PKC) that binds RACK2 which facilitates the movement of PKC

throughout the cell upon activation. The interaction between the RACK binding site on PKC and

the RACK is prevented by an amino acid sequence, also in V1, termed the pseudo-RACK

(RACK) because it resembles the site on the RACK that will dock the RACK binding site of PKC

(Figure A2). This interaction is imperative to keep PKC inactive under non-stimulatory

conditions. The sequence of the RACK (residues 85-92; HDAPIGYD) in PKC shares ~75%

homology with a sequence in RACK2 (residues 285-292; NNVALGYD). Changing the sequence of

the endogenous RACK within PKC itself to one that more closely resembles that of RACK2,

which has a higher affinity for the RACK binding site, will stabilize the closed conformation by

making competition of the RACK binding site for RACK2 more unfavourable (Schechtman D,

2004; Brandman R, 2007). In fact, the tighter the association of RACK with the RACK binding

site, the longer it will take to open the enzyme and result in activation/translocation because it

would be harder for RACK2 to outcompete the RACK. It is based on this reasoning that the

isozyme-specific PKC inhibitory peptides were created. For PKC, this is the V1-2 peptide, which

corresponds to the RACK-binding site, will outcompete the endogenous RACK binding site for

RACK2, and prevent PKC from binding RACK2 thus inhibiting PKC translocation (Figure A3); this

is classical competitive inhibition. Using the same rationale, isozyme-specific activator peptides

have also been synthesized. RACK is an octapeptide that corresponds to the endogenous

RACK sequence of PKC (Chen L, 2001; Brandman R, 2007; Dorn GW, 1999) and will bind to it

once PKC is open, thereby stabilizing the open conformation. Both V1-2 and RACK are

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derived from the C2-like region of PKC (V1), implying the importance of this domain in

regulating PKC isozymes (Brandman R, 2007). Similarly, RACK and V1-1 have been

synthesized to specifically activate and inhibit, respectively, PKC (Chen L, 2001). These

activator and inhibitory peptides are designed specifically for each individual isoform and will

not directly affect other PKC isozymes (Dorn GW, 1999; Mochly-Rosen D, 2000).

C1 Domain, Activation and BindingC terminal to the V1 domain in nPKC is the C1 domain, which unlike in aPKCs, is

repeated twice in tandem in cPKCs and nPKCs. The C1 domain is responsible for the

binding/activation of phospholipids, diacylglycerol (DAG), phosphatidylserine (PS), and phorbol

ester via a highly conserved cysteine-rich region. PKC without the C1 domain binds

membranes will less affinity than its full-length counterpart, presumably because the C1

domain gives PKC a hydrophobic surface with which to interact with membranes more avidly

(Robia SL, 2001). Phorbol esters work in a similar fashion by acting as a hydrophobic switch

(Ács P, 1997). Phorbol esters are more potent PKC activators than DAG because they are not

easily metabolized and therefore have a longer half-life (Newton AC, 1995). The C1 domain

also contains the putative RAS binding site as well as an actin-binding motif (aa 223-228 in

PKC) (Akita Y, 2008; Xu TR, 2009; Huang X, 2004). Although PKC binding to F-actin is

promoted by arachidonic acid or PMA, activation is not necessary for F-actin binding, which is

the case for several of PKC’s binding partners (Huang X, 2004).

C1 Domain Pseudosubstrate-C4 Substrate Binding Site Intramolecular

Interaction An important aspect of the C1 domain is its function as a pseudo-substrate site, which

interacts in the tertiary structure with the C4 domain (substrate binding site and ATP transfer

site). Similar to the RACK/RACK binding site interaction, the basic pseudosubstrate region

exerts an autoinhibitory action on the substrate binding site. Thus, until activated/opened by

activators like DAG or PS, the substrate binding site will remain closed (bound to the

pseudosubstrate), and its kinase activity blocked. Proteolysis of the pseudosubstrate, and

therefore activation of PKC, may occur upon activation of the enzyme, independently of

membrane binding (Newton AC, 1995). Upon translocation to an anionic lipid-based

compartment (e.g. sarcolemma), the phospholipid (e.g. PS) releases the autoinhibitory pseudo-

substrate from the substrate binding site, allowing PKC access to its substrates at that location

within the cell (Cenni V, 2002). PKC is inactive in the cytosol (Rey O, 2004) and elimination of

the pseudo-substrate site results in a constitutively active PKC that is found in the particulate

fraction (Schechtman D, 2004). It is indeed the C1 and C2 domains of the regulatory domain

that dictate its subcellular localization by determining which factors can bind and influence the

enzyme’s conformation. Notably, differences among PKC isozymes do exist for binding these

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factors and the subcellular redistributions that may result (Áca P, 1997). The importance of the

regulatory domain of PKC was shown after this domain was conjugated to the PKC catalytic

domain. This chimeric construct did not show properties of PKC, presumably because the

PKC regulatory domain prevented the PKC catalytic activity from being exposed to its

substrates (in a different subcellular location) (Pears C, 1991).

PKC Hinge Region

The C1 domain in nPKCs (of which PKC is one example) is followed by the V2 and V3

domains. V3 serves as a hinge region that separates the regulatory (C1/C2/V1/V2) from the

catalytic (C3/C4/V4/V5) halves of PKC. Upon activation, PKC becomes labile to proteolysis at

the hinge by Ca2+-dependent proteases (chymotrypsin, calpain), resulting in the subsequent

release of the regulatory domain from the catalytic domain. The effect of this is the removal of

any inhibition from the regulatory domain, leaving behind a constitutively active catalytic

fragment, termed PKM (because it is found in the membrane, or particulate, fraction) (Laher I,

2001; Basu A, 2002; Newton AC, 1995; Shimohata T, 2007). The pseudosubstrate region is

protected from proteolysis when PKC is not catalytically active and its removal or disruption (via

an antibody directed to this site) leads to activation (Newton AC, 1995). Cleavage of certain

PKCs may lead to apoptosis (PKC, ), or prevention of apoptosis (PKC) (Basu A, 2002),

highlighting the contrasting roles of different PKC isozymes.

PKC Catalytic Domain and PhosphorylationThe catalytic domain is largely conserved among PKCs but the most C-terminal residues

of V5 are the more variable. This catalytic core contains the substrate binding site, ATP binding

site, as well as the ATP transfer site. C4 and V5 of the catalytic domain together contain three

motifs with specific residues that must be phosphorylated for the enzyme to be catalytically

competent. These phosphorylations are accomplished upon translation of the protein,

otherwise it misfolds and is subsequently degraded (Sossin WS, 2007). Upon synthesis, PKC is

released from the Golgi as an inactive precursor, not yet capable of phosphorylating substrates.

Upon its phosphorylation at three fairly conserved residues in the catalytic domain (Cenni V,

2002; Ács P, 1997), PKC becomes catalytically competent, but not yet active (not translocated).

It is not completely understood how all three of these posttranslational events take place, but

the final two may be due to autophosphorylation from the PKC itself (cis-autophosphorylation)

or by a second PKC (trans-autophosphorylation). Phosphorylation not only renders the

enzyme more soluble, but the catalytic domain also plays a role in the final destination of PKC

upon activation with phorbol esters (Ács P, 1997).

Phosphorylation of PKC Although highly conserved, different PKCs have three phosphorylation sites at different

residues. Only those for PKC will be described here. The first phosphorylation (and the rate-

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limiting one) occurs in the activation-loop motif (Thr566) of C4, resulting in a conformational

change in PKC, allowing the subsequent phosphorylation events to occur (Cenni V, 2002).

These sites are the turn motif (Thr710) and hydrophobic motif (Ser729) in human PKC, both of

which are located in the enzyme’s most variable domain, V5 (Zhu Y, 2006). Ser729

phosphorylation is important for the association of PKC with actin since mutation of this site

blocks this interaction (Xu TR, 2009). Truncation mutants of PKC have revealed that the C-

terminal segment following Ser729 is necessary for catalytic activity, even though PDK1

(phosphoinoside-dependent kinase-1) can still dock onto PKC without the entire V5 region

(Zhu Y, 2006). DAG, a potent activator of PKC, cannot bind PKC and activate it until all three

phosphorylation sites are phosphorylated. It appears that phosphorylation of PKC precedes its

translocation via RACK2 binding. Fully or partially dephosphorylated PKC localize near the

centrosome/Golgi region of the cell, and the phosphorylation events appear to occur in this

region (Akita Y, 2002). PKC will not bind its RACK until phosphorylated and activated

(Schechtman D, 2004). Since RACK2 is a Golgi-derived membrane protein involved in vesicular

trafficking, the phosphorylated (catalytically competent) PKC can then be recruited to its

destination. It is unknown what triggers initial PKC opening, but may be due to its

phosphorylation or oxidation (Konishi H, 1997). The importance of PDK1 activity in

preconditioning is fortified by the inability to precondition mice with hypomorphic expression

(~80% reduction) of PDK1 (Budas GR, 2007). In contrast to PKC, phosphorylation of PKC’s

activation loop (Thr505) is not required to activate the enzyme, since PKC is already catalytically

competent, but this posttranslational modification would nevertheless alter the enzyme’s

properties (Rybin VO, 2007). PKC phosphorylation may be partially modulated by PKC, and

somewhat by PKC activity. Evidence suggests that PKC either changes PKC’s substrate

selectivity, or inhibits the enzyme. PKC, but not PKC, I, II, or , was shown to be

translocated to nuclei in H9c2 cardiomyoblast cell line (where it may serve as a transcriptional

regulator), and this was associated with dephosphorylation at Ser729 (Xu TR, 2009; Ueker M,

2003). Thus effects of phosphorylation on PKC enzymology may be more complex than

originally thought. A similar observation of Ser729 dephosphorylation of PKC after

translocation to plasma membrane was seen in fibroblasts. Thus dephosphorylation of PKC

may lock it in place by inhibiting its RACK-binding and subsequent redistribution. PKC can

phosphorylate and therefore modulate the activity and/or subcellular distribution of other

kinases, which is the case for PKD. PKC has to be active (membrane-bound) for it to

phosphorylate PKD, while knockdown of PKC expression reversed PKD activation (Rey O,

2004). Thus, the complex nature by which different PKCs are regulated allow modulation by

various activators or inhibitors that are altered during ischemic stress.

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Thr710

Turn motif

C1 C1 C3 V3 V4 V5 C4 V2 V1

zinc finger motif cystein-rich DAG/PS/phorbol

ester/RAS binding site pseudo-substrate

ATP binding site PDK binding site

Hinge; cleaved by trypsin or calpain I/II

737 1

phosphate transfer site substrate binding site

RACK binding site (14-21); V1-2

RACK (85-92)

Regulatory domain Catalytic domain Hinge

Thr566

Activation loop

~142

N C

actin binding site

Homologous to C2 of

cPKCs

C2 in cPKC

Figure A1. PKC Primary Structure. PKC, like all novel PKCs, has no Ca2+-binding domain and is therefore not activated by Ca2+

like conventional PKCs are (i.e. PKC). The zinc-finger motif is altered upon oxidation/nitrosylation, changing the properties of

PKC. See Appendix for more detail and references within.

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Regulatory domain

Catalytic domain

RACK-binding site

14-21

-

Substrate

RACK amino acids 85-

92 Substrate-binding

site

N

C

Opening via activation (conformational change)

RACK

RACK-binding site

14-21

-

Substrate

Substrate binding site

now open Translocation to membranous

compartment

Figure A2: PKC Binding to RACK2. PKC initially in the closed conformation is induced to open by an unknown mechanism, presumably due to

cofactor binding. For PKC (not activated by Ca2+), this may be achieved by oxidation or nitrosylation. Upon opening, the RACK-binding site is free to bind RACK2, which allows its translocation to various subcellular membranous compartments, including the sarcolemma, sarcoendoplasmic reticulum, mitochondria, and nuclei (Table 1). Once there, lipids in the membrane (phosphatidylserine, cardiolipin, diacylglycerol) may fully open the enzyme, stabilizing its open conformation and revealing its substrate binding site. The hinge region is exposed to proteolytic cleavage, releasing the regulatory domain leaving behind a constitutively active catalytic domain, PKM. See Appendix for more detail and references within.

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TRANSLOCATION INHIBITED Cannot bind to RACK2 (RACK binding site blocked)

TRANSLOCATION ACTIVATED Can easily bind to RACK2 (RACK binding site exposed)

Regulatory domain

Catalytic domain

N

C

Opening via activation (conformational change)

Substrate binding site

now open

V1-2

RACK

Figure A3. PKC Activation and Inhibition with RACK and V1-2. V1-2 looks like RACK2 and so competitively blocks the RACK

binding site, preventing PKC from translocating to its final destination where its substrates lie. Thus, although the enzyme may be

active, it does not reach its substrates and is functionally inactive. RACK on the other hand binds the pseudo-RACK within PKC

that normally keeps the enzyme closed. Thus, once PKC is primed to open, RACK may bind, thereby stabilizing the active

conformation of PKC, increasing its propensity to activation and enhances the rate at which translocation can be initiated. See Appendix for further detail and references therein.

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REDISTRIBUTION OF PKC TO THE MITOCHONDRIA: COMPARING ... · Page 1 of 80 Introduction Background...

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