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DOES SODIUM SALICYLATE TREATMENT ENHANCE HSP 72 EXPRESSION AND MYOCARDIAL PROTECTION?

Joel William Atance

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Community Health University of Toronto ,

0 Copyright by Joel William Atance (1998)

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Does sodium salicylate treatment enhance HSP 72 expression arid myocardial protection?

Master of Science, 1998. Joel Wiiam Atance, Graduate Department of Community

Health, University of Toronto.

It was of interest to determine whether an in vivo administration of salicylate,

combined with a mild heat shock, could potentiate the heat shock response, and confer

signiticant myocardial protection. To test this hypothesis, 25 male Sprague-Dawley rats

were divided into five groups (n=5 per group): 1) unstressed (control); 2) sodium

salicylate treated., 3) mildly heat-shocked; 4) mildly heat-shocked plus sodium salicylate

treated; and 5) severely heat shocked. Hearts were analyzed for hernodynamic performance

on a Langendorff apparatus. Following an ischemic episode, hearts from animals that were

severely heat shocked recovered a greater percentage of left ventricular developed pressure,

and rate of contraction and relaxation, compared to unstressed (control) animals. These

animals also showed a significantly greater accumulation of left ventricular HSP 72.

Animals that were mildly heat shocked, with or without sodium salicylate treatment, were

not conferred myocardial protection, and did not show significant increases in left

ventricular HSP 72 content. With this experimental design, it can be concluded that

sodium salicylate treatment does not potentiate the myocardial heat shock response in viva

F i t and foremost, I would like to thank my supervisor, Dr. Marius Locke, who a l l along displayed an admirable level of patience, while starting a lab from scratch, and supervising two graduate students, and occasional summer students, all of whom had veIy Little experience in the field. Marius, I can't think of one time when you were too busy to offer help, and I know your plate has been full for the last three years. For that I am very grateful. I would also like to thank Dr. Michael Plyley, and Dr. Nancy McKee, the other members of my advisory committee. Almost four years ago, I was a little uncertain of my future direction, and contemplating a career change. It was suggested I speak to Dr. Plyley. He had encouraging words for me, which gave me the confidence to undertake a fairly drastic change in academic disciplines. Thereafter, he was always willing to share his expertise on all types of academic issues. Dr. McKee has been instrumental in providing much needed lab space, and expert hands-on advice. Her generosity is much appreciated. Special thanks to Dr. Carol Rodgers, whose many courses I followed were always excellent, well-researched, and well-taught.

Thank you to my fellow students. My time at the U of T really got better as I moved along, and this was due in no small part to the many friends I made in the program. Thanks Robert for making an effort to unite graduate students spread out in the big city. Thanks also for pushing me to do OEP '98. It really helped out down the road. Thanks Cora for being a willing participant in aIl the little functions. Good luck Adrian in wrapping things up, and with future plans. Thanks to the office staff, especially Ruby, Tim, and Wenda, for courteous help, work hours, and the odd supplies.

You may never read this, but a sincere thanks to the many AC people I first befriended when I knew not a soul in this town. You made a difference when the going was tough.

Finally, thank you to my family, who don't know much about HSPs, but were there long before this all began, and without whom I wouldn't be typing this up.

*.

A B ~ T ~ A C T -------------------------------------------------.-----.----------------------------------------------------- 11

-.- AcmowLEDGMENTs ------.---------------*- **-.** * .*- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ----- ----- IJl

mumE OF CONTENTS -------------------------------------- .*------------------* *.* *---.--- *.***.**** -.---- iv

I X T OF TABLES ---.-----------------------------.-------------------------------------------------------------- vi

LIsT OF FIGURES --.----.-* **.**-** -*--------*.-.-------------- *.**.*.* ---------.- **.** +...--.- * * - * **..- --*.*--* vii ..-

LIST OF APPENDICES..* -----*------------- .--------------- .----- .------ . . --*------------------- .----------- VlLl

LIST OF -BRE-TIoNS. -- -- -- - - *-. *. - - -*. - - - -. * * * *. . - *. . - - - - - - - - * - - ** - --. . * -- * - * * - * . * - +.. . -- - - ix

TABLE

1 . Pre-ischemic absolute values for hernodynamic variables ---- .--------..-.+.------amiamiamiamiami 30

LIST OF FIGURES

FIGURE

Pre-heat stress (baseline) rectal temperatures are similar between groups- - --- - - 24

Mildly heat shocked, mildly heat shocked plus sodium salicylate treated animals, experienced heat shocks of similar duration ~ ~ ~ - - - ~ ~ ~ - ~ ~ a l a l a l a l a l a l a l a l a l a l a l a l a l a l 25

Mildly heat shocked, mildly heat shocked plus sodium salicylate treated animals, showed similar average T, during entire heat stress --------------- 26

Mildly heat shocked, mildly heat shocked plus sodium salicylate treated animals, showed similar average T, during 15 mio heat shock- -- -- - - --. 27

Graphical representation of left ventricular HSP 72 content following i=hemia-reperfusiontl - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - tl tl tl - - - - - - - - - - -. - - . -. . * * . + - * . - -. - - - - . . . * * * *a tl tl tl tl * tl tl . - - 45

vii

APPENDIX

ATF' AP ANOVA BSA sC T cm COX DNA DMF ddH20 g h i~ HSC HSE HSF HSP kD kg LVDP L mRNA

CLg

fi Cun mg min ml d . - l

rnm m g m m ~ g s-' mM nm NO NFDM NSAlD ID PGA, PGD, PGE, PGH +dP-dt -' -dP*dt " kdP*dt -' =r SDS

adenosine triphosphate alkaline p hosphatase analysis of variance bovine serum albumin degrees Celsius degrees Fahrenheit centimetre cyclooxygenase deoxyribonucleic acid N,N-dimethyl formamide double distilled de-ionized water gram hour(s) intraperitonealy heat shock cognate heat shock element heat shock factor heat shock protein kilodalton kilogram left ventricular developed pressure litre messenger ribonucleic acid micrograms microlitre micrometers milligram minu te(s) millilitre millilitres per minute millime= miltimetres of mercury millimetres of mercury per second millimolar nanometers nitric oxide non-fat dried milk powder non-steroidal anti-inflammatory drug one-dimensional prostaglandin A, prostaglandin 4 prostaglandin E, prostaglandin H synthase rate of contraction rate of relaxation rate of contraction and relaxation rectal temperature sodium dodecyl sulphate

SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis TBS tris-buffered saline Tr33S tris-buffered saline plus 0.05% Tween-20 U units v volt(s)

CHAPTER 1

1.1 Background

In response to elevated temperatures and other forms of protein damaging stresses,

both prokaryotic and eukaryotic cells temporarily suspend most gene transcription and

mRNA translation, while conspicuously increasing these processes in a select family of

genes (Lodish, 1995). These genes encode proteins which increase cell survival after

exposure to stress (for review see Morimoto et al., 1994). These proteins, originally

discovered in the salivary glands of fruit fly larvae subjected to temperature elevations,

were accordingly termed heat shock proteins (HSPs)(Ritossa, 1962; Tissieres et al., 1974).

Subsequent research has demonstrated the synthesis of HSPs to be a universal response to

a wide variety of protein damaging stresses, including hypoxia (Heacock and Sutherlmd,

1990), low pH (Whelan and Hightower, 1985), and in some cases, exercise (Locke et al.,

1990).

1.2 HSPs

The heat shock proteins are classified according to their molecular mass. Thus,

HSP 60, HSP 70, HSP 90, and HSP 110 are categorized as high molecular mass HSPs,

while those HSPs ranging in mass fiom 8 to 47 kD, are considered low molecular mass

HSPs (Mestril and Dillmann, 1995). HSP 70 is the most highly conserved HSP, both

within and between species (Hunt and Morimoto, 1985). Remarkably, 50% of the amino

acid sequence is conserved between E. coli and humans (Schlesinger, 1990). The term

'HSP 70' refers to one or more isofonns of the 70 kD family. Foremost among these are

the inducible isoform, HSP 72, and the cognate isoform, HSC 73, which can both be

elevated after exposure to various stresses (Locke, 1997). As well, both HSP 72, and

HSC 73, have been shown to be involved in various stages of protein synthesis

(Beckmann et al., 1990), transport (Chirico et al., 1988), and degradation (Chiang et al.,

1989). It is unclear why cells require both a cognate, and an inducible isoform of very

similar sequence, and function (Brown et al., 1993). However, HSC 73 possesses

intervening sequences not found in HSP 72 ( M a d et al,, 1994). It has been suggested

that the lack of intervening sequences in HSP 72 may facilitate the rapid transcription of

this protein during periods of stress (Hunt and Morimoto, 1985). In general, it is believed

that HSC 73 functions primarily during unstressed conditions, whereas HSP 72 is

synthesized inpponse to cellular demands during episodes of stress (Black and Subjeck,

1991).

1.3 Cellular function of HSP 70

In the cell, HSP 70 is thought to function as a protein chaperone. Using the

hydrolysis of ATP as an energy source, HSP 70 aids in the folding of newly synthesized

polypeptides (Ku et d., 1995), and the translocation of proteins across membranes

(Deshaies et al., 1988). HSP 70 has been shown to re-activate denatured proteins (often

the result of excessive heat or acidity) by restoring native conformation (for review see

Knowlton, 1995). This mechanism is thought to be critical for enhancing cell survival

during episodes of stress.

The heat shock response occurs rapidly and results in a robust induction of HSP

70. In fact, the heat shock gene is described as being in a constant state of readiness

(Mestril and Dilhqm, 1995), such that within 15 minutes of exposure to temperatures 3 to

5°C above normal, heat shock proteins axe preferentially synthesized (Lindquist and

Petersen, 1990). The exact means by which a cell 'senses' an increased temperature stress

still remains unknown. However, a model for the activation of the heat shock response has

been proposed (Abravaya et al., 1992).

1.4 HSP regulation

The promoter region of all heat shock genes contains a unique sequence termed the

heat shock element (HSE). In mammals, a constitutively expressed protein, d e d heat

shock factor (HSFl), binds to the HSE and mediates a stress-induced HSP induction. In

unstressed cells, the HSF is inactive, and is thought to be bound to HSP 70 (Abravaya et

al., 1992). Following stress, many proteins become denatured or unfold, and thus may

compete for HSP 70 binding. With a new pool of substrates available for binding, some

HSP 70 will release HSF. This allows the HSF to trirnerize to an active state, and

subsequently, to bind to the heat shock element, thus inducing HSP transcription.

Eventually, the amount of newly translated HSP 70 will outnumber the pool of denatured

proteins, and the excess HSP 70 is thought to rebind and deactivate HSF, preventing

further binding to the HSE,

1992).

1.5 Evidence for the

Thermotoleraflce is

temperatures. The synthesis

and effectively halting HSP 70 expression (Abravaya et al.,

role of HSP 70 in thermotolerance

described as the cell's ability to withstand

and degradation of HSPs precedes the acquisition

increased

and decay

of thermotolerance, suggesting a protective role for these proteins (Li and Mak, 1985).

After prior exposure to a brief, mild heat shock (-42OC), cells are able to withstand an

otherwise lethal heat shock ( - 4 5 O C ) (Li and Werb, 1982). This transient resistance to

elevated temperatures has been described as acquired thennotolerance (Henle and

Dethlefsen, 1978). Various experimental protocols have shown the importance of HSP 70

in conferring cellular thermotolerance. For example, when cultured fibroblasts were micro-

injected with antibodies to HSP 70, the cells were unable to survive even a brief incubation

at 45O C @abowol et al., 1988), suggesting that HSP 70 is indispensable for ceUula-r

survival during heat stress. In other studies, when cellular HSP 70 expression was

enhanced, a subsequent increase in thennotolerance was demonstrated. For example,

when an HSP 70 gene construct was placed under control of the p-actin promoter, and the

cells transfected (ensuring induction of HSPs at normal temperatures), the transfected cells

displayed a significantly greater degree of thermotolerance than non-transfected cells (Li et

al., 199 1; Angelidis et al., 199 1). In a similar experiment, myocytes were tramfected with

plasmids for various HSPs (under control of the p-actin promoter). Cells transfected with

an HSP 70 gene construct showed a significantly greater resistance to subsequent heat, or

ischemic stress (Heads et al., 1995). These results strongly suggest that HSP 70 can

confer protection to cells. Interestingly, whole body thermotolerance has also been

demonstrated in mice (L et al., 1983). A non-lethal preconditioning heat shock was

shown to protect C3H mice against thermal death during a subsequent, othenvise lethal,

hyperthermic episode, although the role of HSP 72 in the observed thermotolerance was

not investigated.

1.6 HSP 70 and myocardial protection

HSP 70 can be induced by several different types of stress (for review see

Morimoto et al., 1994). Accordingly, induction of HSPs by one stressor may confer

subsequent tolerance against another stressor. This phenomenon, known as cross-

tolerance, has- been demonstrated in several cases (for review see Yellon and Marber,

1994). Protection from ischemic stress through a prior heat stress is an example of cross-

tolerance that has been widely studied because of its potential for medical and general health

applications. In rat and rabbit models, whole body heat stress has been shown to result in

subsequent myocardial protection fiom ischemic insult (Currie et al., 1988; Karmazyn et

al., 1990; DonneJly et al., 1992; Hutter et al., 1994; Marber et al., 1994). Cunie et. al.

(1988) demonstrated that hearts from rats heated to 42°C for 15 min, 24 hours prior, had

better indices of hernodynamic function after an ischemic episode, than non-heat treated

controls. Creatine kinase release, a marker of cell injury, was significantly reduced in'

hearts from heat-shocked animals. Domeuy et al. (1992) observed a reduced infarct size in

hearts isolated from animals subjected to a 42OC heat shock for 20 min, 24 h prior. In

agreement with HSP 70 providing cell protection (Li and Werb, 1982; Li and Mak, 1985),

the cardiac tissue from heat treated animsls showed an elevated HSP 70 content.

In general, there is a correlation between HSP 70 content and myocardial

protection. Hearts from rats exposed to a progressively higher temperature heat shock

express greater amounts of HSP 72, and are subsequently better protected against

myocardial ischemia (Hutter et al., 1994). Thus, a heat shock of 42OC is more effective at

inducing myocardial HSP 72, and reducing myocardial infarct size, than a heat shock of

40°C. Similarly, Kannazyn et al. (1990) showed that myocardial protection was related to

myocardial HSP 72 content. Animals were heat-shocked for 15 min at 42*C, and

myocardial protection assessed 24, 48, 96, and 192 h post-heat shock. Myocardial

protection was greater in animals heat-shocked 24 or 48 h prior, than in animals heat-

shocked 96, or 192 h prior. The degree of myocardial protection corresponded to

myocardial HSP 70 content, which after 48 h, decreased progressively over time. .

Though hypethermia and exercise are effective inducers of HSP 72, these stresses

are also known to alter the expression of other HSPs and antioxidant proteins (Trost et al.,

1998). As such, the correlation between HSP 70 accumulation and subsequent myocardial

protection cannot be interpreted as absolute proof of the protein's role in heart promtion.

The use of transgenic mice has proved advantageous in isolating the direct effect of HSP 72

on myocardial protection. Studies using these animals have provided strong evidence

implicating HSP 72 in myocardial protection (Plumier et d., 1995; Marber et al., 1995;

Radford et al., 1996). Isolated hearts from transgenic mice over-expressing human HSP

70 (under control of a 8-actin promoter), showed an improved functional recovery and

reduced cell& injury following ischemia, when compared to hearts fiom transgene

negative littermates (Plumier et al., 1995). Marber et al. (1995), using transgenic mice

overexpressing rat HSP 70, observed a reduced infarct size in post-ischemic hearts of

transgene positive animals. Similar results confirming the role of HSP 70 in conferring

myocardial protection, by reducing infarct size, were also demonstrated by Hutter et al.

(1996). Subsequent to global ischemia, transgenic mice hearts also displayed an enhanced

recovery of high energy phosphate stores, and a greater correction of metabolic acidosis

relative to hearts of non-transgenic Litter mates (Radford et al., 1996). Recently, rat hearts

transfected with the human HSP 70 gene, using a virus delivery system, also showed

better functional recovery than control hearts, when perfused using the Langendorff

isolated heart technique (Suzuki et al., 1997). Taken together, these experiments provide

strong support for HSP 70 playing a critical role in conferring myocardial protection.

An ischemic challenge to the myocardium actually presents two distinct periods of

stress, ischemia, and reperfusion. Each stress is defmed by a characteristic set of events

which are either the result of unavailability of oxygen (as a result of ischemia), or re-

introduction of oxygen (re-perfusion). During ischemia, irreversible injury in highly

oxidative myocardial tissue is thought to be associated with ATP depletion, cessation of

glycolysis, and inability to clear waste products (Jennings and Reimer, 198 1; Jennings and

Reimer, 1983). Ischemia causes disruption of the electron transport chain, disappearance

of glycogen particles, and eventual morphological damage to the cell which is evident in

sarcolernma disruption, distortion of 2-lines, and mitochondria swelling (Jennings and

Reimer, 198 1; Jennings and Reimer, 1983; Hammond et al., 1985; Hearse et al., 1977).

Despite the obvious dangers posed by ischemia, reperfusion also presents a unique

challenge, which initially exacerbates existing cell damage. Research has focussed on the

'oxygen paradox' which occurs during reperfusion. In effecf the rapid reestablishment of

normal oxygen tension following an ischemic period results in more tissue injury than if

oxygen levels are gradually restored. The restoration of molecular oxygen, which is

ultimately necessary for cell recovery from ischemia, is accompaaied by a heavy burst of

free radical production. However, free radicals such as the superoxide anion (SO,'), and the

hydroxyl radical (-OH) further damage the sarcolemma and mitochondria during

reperfusion, through lipid peroxidation (Guameri et al., 1980; Opie, 1989). Disruption of

the sarcolemma is believed to facilitate massive calcium influxes, which decrease the

contractile response of the myocardium, and possibly induce arrhythmias (Gao et al.. 1995;

Opie, 1989; Jennings et al., 1983). Thus, ultrastructural injuries originally caused by

ischemia are extended by re-perfision (Hearse et al., 1977). As well, recovery of ATP

stores and the restoration of normal metabolism may be delayed even after several days

following reperfusion (Reimer et al., 198 1 ).

Despite a wealth of evidence demonstrating the role of HSP 70 in myocardial

protection, littIe is known about the mechanisms involved. The role of k radicals in

causing cellular damage during re-perfusion was confirmed in several experiments in which

the introduction of anti-oxidants, such as catalase, or superoxide dismutase, conferred

myocardial protection @as et al., 1986; Myers et al., 1985; Otani et al., 1986). Karmazyn

et al- (1990) demonstrated that myocardial protection conferred by accumulation of HSP 72

may be mediated by catalase. It was suggested that HSP 72 may modulate the activity of

catalase (Karmazyn et d., 1990). This theory is concordant with suggestions that HSPs

exert their protective effect by stabilizing, or solubilizing damaged proteins, and preventing 3

heat-induced insoluble aggregates within the nucleus, andor the cytoplasm @ o ~ e l l y et

al., 1992; Nguyen et al., 1989). Furthermore, HSP 72 may protect and restore protein

synthesis to normal levels during re-perfusion (Trost et al., 1998).

1.7 The salicylates

In 1838, the Italian chemist Piria split salicin, the extract of willow bark, into a

sugar and an aromatic component, and through various oxidative processes converted the

latter into salicylic acid (Vane and Bottin, 1992). Some s i x t y years later, this compound

was acetylated by Hoffmann, and acetylsalicylic acid, or aspirin, was created wane and

Botting, 1992). Although aspirin and salicylic acid have similar modes of action, these

chemical relatives also possess some distinct properties, suggesting aspirin is not simply a

pro-drug for salicylate.

The most documented action of the salicylates is the prevention of prostaglandin

synthesis (Vane, 1971). Prostaglandins are cyclic fatty acids derived from precursor

arachadonic acids. Found in nearly all mammalian tissue, prostaglandins are lrnown to have

important physiologic and phannacologic activities (Murray et al., 1988). Originally

discovered for their role as initiators of smooth muscle contraction, prostaglandins are also

involved in the inflammatory response, and platelet aggregation. Thus, humans have

traditionally used aspirin to relieve pain and inflammation, or to reduce the risk of clot

formation, and subsequent strokes or myocardial infarctions.

The salicylates prevent prostaglandin synthesis by inhibiting the cyclo-oxygenase

activity of prostaglandin H synthase (PGH), one of two catalytic activities performed by

this enzyme in the conversion of arachadonic acids to prostaglandins (Ohki et al., 1979).

Aspirin irreversibly inactivates cyclo-oxygenase activity by transfening its acetyl group to

the PGH enzyme (Roth and Majerus, 1975). There is evidence that aspirin may also

inhibit cyclo-oxygenase activity by a mechanism independent of acetylation. Salicylate is

thought to prevent de novo synthesis of prostaglandin H synthase, thus inhibiting the

cyclo-oxygenase activity of this enzyme (Wu et al., 199 1). More recently, different forms

of the cyclooxygenase enzyme (PGH) have been identified, including COX-1, the

constitutive isoform, and COX-2 the inducible isoform. It has been suggested that the

mode of action of the salicylates and other non-steroidal anti-Mammatory drugs

(NSAIDS) may depend on their interaction with the different COX isofom (Frolich,

1997).

Apart fiom variations in their mode of action, aspirin and salicylic acid also appear

to differ in rate of plasma clearance. In rats administered either aspirin or salicylic acid, the

peak plasma concentration of salicylic acid OCCLKS within 1 h (Higgs et d., 1987). In either

case, salicylic acid concentration declines by half in approximately 6 h, but aspirin is

undetectable after 1 h (Eggs et al., 1987).

1.8 Salicylates and the heat shock response

The salicylates, though well known for relief of pain and idammation, are believed

to have additional properties. Human erythroleukemic cells treated with aspirin during, or

immediately after, heat shock, synthesized greater amounts of HSP 70, and for a longer

duration than non-treated cells (Amid et al., 1995). Mesalamine, a compound related to

sodium salicylate, increased the the& induction of HSP 72 in rat intestinal epithelial cells

(Burress et al., 1997). Conversely, Lee et al. (1995) concluded that when HeLa cells were

treated with indomethacin, combined with mild heat shock, the transcription of heat shock

genes was induced to greater levels than mild heat shock done. Indomethacin was reported

to lower the temperahxe threshold for the heat shock response, such that Hela cells heat-

shocked at 40°C in combination with indomethacin treatment, were capable of surviving a

subsequent 44S°C heat shock. This acquired thennotolerance was as effective as that

observed in cells previously heat-shocked at 41°C, but without indomethacin treatment (Lee

et al., 1995). gdomethacin has also been shown to enhance the heat induced expression of 1

HSP 70 in rat ghoma cells (Ito et al., 1996). Interestingly, in both yeast and Drosophila,

sodium salicylate activation of the HSF has been shown to prevent subsequent heat-

induced transcription of the heat shock gene (Winegarden et al.. 1996; Giardina et al.,

1995).

The various salicylates have been shown to exert their regulatory effect at the level

of the HSF (Jurivich et al., 1992; Jurivich et al., 1995; Amici et al., 1995). In the absence

of hyperthermic treatment, Jurivich et al. (1992) found that sodium salicylate treatment to

HeLa cells activated the HSF to its DNA binding state. This resulted in an HSF:HSE

binding to levels similar to that observed after a 42°C heat shock (Jurivich et al., 1992;

Jurivich et al., 1995). However, the increased HSF activation did not lead to a subsequent

increase in HSP 70 gene transcription. A number of different reasons have been given for

this observation, including the possibility that multiple HSF isoforrns may respond

uniquely to a given stress (Cotto et al., 1996). Thus, heat may activate one specific HSF

isofom, while sodium salicylate would presumably activate another. Alternatively,

salicylate may induce tkeonine phosphorylation of HSFl, while heat induces serine

phosphorylation of HSF1. Only the latter condition transactivates heat shock gene

expression (Jurivich et al., 1995).

Salicylate mediated enhancement of the heat shock response is not fully understood,

and in vivo research in the area is limited to one study. In rats treated with aspirin, HSP

70 mRNA induction was not increased in liver, lung, and kidney (Fawcett et al., 1997).

However, when aspirin was administered to animals one hour prior to a 30 min heat shock

in an ambient temperature of 37OC, a significantly greater induction of HSP 70 mRNA was

observed compared to animals that were heat-shocked alone. Based on Western blot

analyses, the authors also reported an elevation in HSP 70 content in the liver of animals

heat-stressed in combination with aspirin treatment. It was also reported that aspirin

enhanced the elevation of core body temperature in animals exposed to heat stress; mildly

heat-shocked animals treated with aspirin, reached significantly higher core temperatures 3

than animals that were only mildly heat-shocked. Thus, Fawcett et al. (1997) concluded

that the potentiation of heat induced HSP 70 expression was likely the result of an aspirin-

mediated elevation in core temperature. Based on this fmding, it remains unclear whether

salicylate can potentiate the heat shock response in vivo, without affecting animal core

temperatures.

1.9 Thesis objectives

The degree of myocardial protection conferred by a prior heat shock is strongly

associated with HSP 72 content (Kannazyn et al., 1990; Hutter et al., 1994). A heat

shock of 42OC confers significant myocardial protection, but also presents a severe stress to

the animal. Various salicylates have been shown to enhance the heat-induced expression of

HSP 70 in v i m . Aspirin has also been shown to potentiate the HSP 72 induction by a

mild heat shock in vivo, in rat liver, lung, and kidney. At present, it remains unknown

whether salicylates can directly affect HSP 72 induction in the rat heart during mild heat

shock in vivo. Furthermore, it is also unknown if any salicylate enhanced induction of

HSP 72 at lower heat shock temperatures, can confer an HSP 72 mediated myocardial

protection. Thus, the specific aims of this thesis are as follows:

1) to examine the effect of a common non-steroidal anti-inflammatory drug (sodium

salicylate) on myocardial HSP 72 accumulation in vivo.

2) to determine whether in vivo treatment with sodium salicylate, in combination with a

mild heat shock of 4WC, increases myocardial HSP 72 content to levels similar to those

observed following a severe heat shock of 42°C.

3) to determine whether in vivo treatment with sodium salicylate, in combination with a

mild heat shock of 40°C, results in myocardial protection similar to that observed following

a severe heat shock of 42°C.

CHAPTER 2

2.1 Animals,

AND METHODS

sodium salicylate and heat treatments

Adult, male, Sprague-Dawley rats (300-350 g; Charles River) were used in these

experiments. Animals were maintained on a 12 h dark/light cycle, housed in pairs at 2 1" C ,

50% xdative humidity, and were provided food and water ad libitum. Animals were

divided into five groups (n=5 per group): 1) control (unstressed), 2) sodium salicylate only

(400 mgokg'l), 3) mild heat shock (4U°C for 15 min), 4) mild heat shock ( W C for 15 min)

combined with sodium salicylate (400 mg-kg-'), and 5) severe heat shock (42OC for 15

min) (figure 1).

Sodium salicylate (salicyic acid, sodium salt; Sigma Chemical Company,

Mississauga, Ontario, Canada) was dissolved in H,O, and administered intraperitonealy

(ip) in a 0.5 ml volume. Preliminary results indicated a sodium salicylate dosage of 400

mg-kg-', in combination with a mild heat shock, was the minimum amount necessary for

detection of an increased HSP 72 response (appendix VT). For animals treated with'

sodium salicylate and also subjected to heat shock, sodium salicylate was administered one

hour prior to heat shock. For the purposes of this experiment, 'heat shock' refers to the 15 7

min period where T, was maintained at 40°C or 42OC, while 'heat stress' refers to the entire

period required to raise T, , and retum to baseline temperature. AU animals subjected to

heat shock were anesthetized with sodium pentobarbital (30 mg-kg-' ip), and baseline rectal

temperature (T,) was recorded. Animals subjected to total body heat stress were

subsequently placed on a heating pad that consisted of a Fisher Standard Isoelectric

Focusing System and Control Unit (Fisher Scientific, Nepean. Ontario, Canada) lined with

bench coat. The surface of the heating pad was maintained at 50" C until T, was within

Figure 1: Schematic illustration of methods

male Sprague-Dawley rats divided into 5 groups, n=5 per group

1 sodium salicylate injection1 400 mg-kge1, ip) P

I hour

I ltransfer of animal to heating pad 'I for I . 15 min heat . shock of: I

24 hour recovery

I isolation and perfusion of heart in Langendorff model I

I heart biopsy I

separation of proteins by one-dimensional SDS-PAGE

Western blot analysis of HSP 72/HSC 73 content

OS°C of the desired value for the 15 min heat shock. The pad temperature was then

adjusted to maintain a constant core temperature for 15 rnin. At the conclusion of the 15

min period, pad temperature was lowered to 20° C, and animal T, was allowed to return to

baseline. In the case of the mildly heat-shocked animals, and those mildly heat-shocked in

combination with sodium salicylate treatment, T, was raised to approximately 40°C.

Animals subjected to a severe heat shock had T, raised to approximately 42OC. In all cases,

rectal temperatures were maintained for 15 min, and subsequently returned to previously

determined baseline values. Two minutes prior, and throughout the entire heat stress (total

time rectal'temperature was above resting value), rectal temperature was measured

continuously using a Thermistor TSD 102C Tube Robe Transmistor (manufacturer reports

accuracy of M.0002"C) connected to a Biopac data acquisition system. Prior to each

individual heat shock, the Biopac software was calibrated for temperature by assigning

specific voltage values to two fixed temperatures within the physiological range observed in

the rat, 96OF and 107OF. A standard mercury thermometer (gradings of 0.2OF), and the

Tube Probe Transmistor were both immersed in a beaker of water. At watex temperatures

of 96OF, and 1079 (determined by visual inspection of the thermometer), the Thermistor

registered a specific voltage. This established a scale for the Biopac software to accurately

translate ensuing voltage measures into rectal temperatures. Following sodium dcylate

and/or heat shckk treatment, all animals were returned to their cages and allowed to recover

for 24 h. Twenty four hours after heat shock and/or sodium salicylate treatment, rats were

anesthetized with sodium pentobarbital (65 mg-kg-' ip) and injected with 1000 U of heparin

(Hepalean; Organon Teknika, Toronto, Ontario, Canada) via the tail vein, 10 min prior to

removal of the heart. Animals were sacrifiiced in a random manner. This ensured that no

group received emphasis at any point during experimentation, and furthermore, no group

was over-represented in a specific batch of animals.

2.2 Isolated heart preparation

2.2.1 Langendorff technique

Various Langendorff isolated and perfused heart systems exist and have been

described elsewhere (Langendofl, 1895; Neely et al., 1967; Neely et al., 1973); a constant

pressure, non-recirculating model was used for the present experiments (figure 2). This is

a retrograde perfusion method, i.e., the perfusate flows down the aorta, and not through

the left ventricle and out the aorta as blood does in v iva The inner chamber of a 2 L

water-jacketed reservoir (Radnoti Glass Technology, Inc., Monrovia, California, USA),

equipped with an oxygenating bubbler, was connected by 114 x 3/32 tygon tubing (Norton,

Akron, Ohio) to a three-way luer valve attached to a cannula hanging above a height-

adjustable heart chamber (Radnoti Glass Technology, Inc., Monrovia, California, USA).

A separate tygon tubing system (3/16 x 1/16) linked the water jacketed portions of the

buffer reservoir and the heart chamber with a pump (VWR 1 1 10; Preston Ind., Inc., Niles,

Illinois, USA) fixed to a 3 L pail. The pump warmed the water in the pail to 37°C and

ensured flow through the chamber outer jackets, thus maintaining the buffer at 37°C.

2.2.2 Langendorff preparation

Prior to each experiment, a Krebs-Henseleit buffer solution (Sigma Chemical

Company, Mississauga, Ontario, Canada) containing 4.7 mM KCI, 1.2 mM KH,PO,, 1.2

m M Mg,SO,, 118 mM NaCl, and 11 mM glucose, was freshly prepared. In order to

maintain ionic; and pH balance for proper function of the isolated heart, 2.0 mM CaCI, and

25 rnM NaHCO, were added to the Krebs-Henseleit buffer. The modified buffer solution

was filtered through a 0.8 micron filter (Gelman Scientific, Ann Arbor, Michigan, USA).

The buffer was placed in a 2 L water-jacketed reservoir and oxygenated with a 95% 045%

C 4 mixture, and maintained at 37OC. The reservoir was fastened to a stand 45 cm above

50 mmHg perfusion pressure flo

pacing I electrodes

Krebs-Henseleit

Mac computer

Figure 2. Langendorff apparatus. Shown is a retrograde perfusion, non-recirculating model (see text for further details). Buffer flow is indicated by the arrow.

the heart, a sufkient elevation to generate 50 mmHg perfusion pressure through the

cannula, thus maintaining the isolated heart.

2.2.3 Heart preparation

Anesthetized animals were placed on a dissecting board and hearts were exposed by

median sternotomy followed by cutting and retraction of the rib cage. The heart was

rapidly excised with a single cut across the arch of the aorta and the vena cava, and

immediately immersed in an icecold saline bath (0.9% NaCl). The heart was carefully

lifted from the saline using a pair of curved forceps, and the aorta was fitted on a cannula

suspended from a stand. The heart was secured to the cannula by tying the aorta to the

cannula using surgical thread. Excess tissue (lungs, fat) was removed. The heart was

gently flushed with ice cold saline from a 10 ml syringe attached to the opposite end of the

cannula. The cannulated heart was disconnected fkom the syringe and transferred to the

Langendorff apparatus. Immediately, perfusion of the heart was initiated. A water-fded,

balloon-tipped catheter (size 4; Radnoti Glass Technology, Inc., Monrovia, California,

USA) was inserted through the left atrium, into the left ventricle, and inflated to a volume

of 50 pl. Once inserted, hernodynamic measurements were started using MP 100

software. The pressure developed by each left ventricular contraction was measured

through ball- compression, and the resulting signal translated to the Biopac system by a

pressure transducer (COBE Labs Inc., Lakewood, Colorado, USA). Prior to each isolated

heart preparation, the Biopac software was calibrated for pressure by assigning a specific

voltage to two hxed pressure values within the range of left ventricular developed pressure

values (LVDP) observed in the isolated rat heart; 60 mmHg, and 180 mmHg. The rubber

tubing at the cuff end of an aneroid sphygmomanometer (gradings of 2 mmHg; Mabis

Healthcare Inc., Lake Forest, Illinois, USA) was cut, and tightly fitted over the pressure

transducer. The bulb of the shygmommometer was compressed to attain the desired

pressures of 60 m d g , and 180 mmHg (determined by visual inspection of the

shygmomanometer needle). At each pressure value, the tranducer registered a specific

voltage. This established a scale for the Biopac software to accurately translate ensuing

voltage measures into pressure readings. Rates of contraction and relaxation (kdP-dt 'I)

were calculated as the first derivative of the curve depicting left ventricular developed

pressure (LVDP), over time. Coronary flow, or the flow of perfusing buffer prior to

entering the cannulated heart was measured using a GiImont Instruments shielded

flowmeter. Flow values were manually recorded. Hearts were electrically paced at 320

beatsaid using plunge electrodes originating from an output channel on the Biopac

sys tern.

2.3.4 Langendorff protocol

The following protocol was used for the heart experiments: after a 45 min

equilibration period, the hearts were subjected to 45 min of complete, warm (37*C), global

ischemia by halting coronary perfusion and electrical pacing. This was achieved by closing

the 3-way luer valve above the heart cannula, further pinching off the tygon tubing with a

clip, and finally disconnecting one of the electrodes from the heart. Following 45 min of

global ischemia at 37OC, flow and pacing were restored, and the heart reperfused for 30

min. Data were recorded continuously throughout the protocol using the Biopac system

and Acknowledge MP lOO software. Hernodynamic indices (LVDP, *dP-dt - I , coronary

flow) were evaluated 5 min prior to ischemia, and at 0, 5, 10, 15, 20, 25, and 30 min of

reperfhion. Following reperfusion, the hearts were tdmmed of excess tissue (atria, great

vessels), and fiozen at -80°C.

2.4 Protein determination

Frozen portions near the apex of the left ventricle (40-60 mg) were placed in 13 x

100 mm disposable test tubes (Fisherbrand; Fisher Scientific, Nepean, Ontario, Canada)

containing 15 volumes of 600 mM NaCI, and 15 mM Tris (pH 7.3, and homogenized at

19

4OC using an Ultra-Turrax T8 grinder @(A Labortechnik, S taufen, Germany). Protein

concentrations were determined by the method of Lowry et al. (195 I), using bovine senun

albumin (BSA) as a standard. Five pL, of sample homogenate were added to 495 pL of

ddH,O in 13 x 100 mm tubes set-up in triplicate. Five ml of Lowry reagent (2 mVlOO ml

of 2% W/V CuS04-H20, 2 mVlOO ml of 4% w/v sodium tartrate in 96 ml of 3% w/v

NqCO, made in 100 mM NaOH) were added, vortexed, and allowed to react for a

minimum of 15 min. After 0.5 ml of phenol reagent (Anachemia, Mississauga, Ontario,

Canada) diluted 1:2 with d-0 was added,

for a minimum of 30 min. Absorbance was

samples were vortexed

measured at 660 nm in

and allowed to react

a Turner model 340

Spectrophotometer. A standard curve consisting of 10, 20, 40, 60, 80, and 100 pg of

BSA was constructed, and sample protein concentrations determined using a linear

regression equation.

2.5 One-dimensional separation of proteins

One-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis

(SDS-PAGE) was conducted according to the method described by L a e d (1970), using

a Bio-Rad mini-protean I1 gel electrophoresis system (Bio-Rad Laboratories, Mississauga,

Ontario, Canada). SDS-PAGE consisted of a 5- 15% polyacrylamide gradient separating

gel, and a 3% stacking gel. The glass plates were cleaned with 70% ethanol, and

assembled with 1.5 mm Teflon spacers in gel moulds, according to the manufacturer's

instructions. The separating gel was poured from equal amounts of 5% and 15%

acrylamide mixtures. A dualchambered mixer was placed on a stir plate, and tubing run

from the proximal chamber to the gel mould. The stopcock separating the two chambers

was initially closed, and the 5% mixture was added to the distal chamber. The stopcock

was opened very briefly to evacuate air between the two chambers. A small stir-bar was

added to the proximal chamber, followed by the 15% mixture, which immediately began

running into the gel mould. When the bottom of the mould was covered by the 15%

acrylamide, the stopcock was opened fully, and the acrylamide solutions mixed and drained

into the mould. When the pouring was complete, the separating gel was overlayed with

40-saturated butanol and allowed to polymerize for 30-45 min. The butanol was then

carefully rinsed off, a 10 well 1.5 mm Teflon comb was placed between the glass plates,

and a 3% stacking gel (pH 6.8) overlayed and allowed to polymerize for 30-45 min.

Protein samples were placed in 600 pl microtubes containing an equal volume of sample

buffer (0.5 M Tris (pH 6.8). 10% glycerol, 10% 2-f!-mercaptoethanol, 4% SDS, 0.05%

bromophenol blue), vortexed, and loaded into wells. SDS running buffer (192 mM

glycine, 25 mM Tris-Cl (pH 8.3), 0.1% SDS) was carefully added, and samples were

initially electrophoresed for 30 min at 70V, approximately until the loading dye front had

reached the separating gel, at which point the voltage was increased to 110V until the dye

front had reached the bottom. Human purified HSP 70, or bovine HSC 73 (product

#SPP-750, Stress-Gen, Victoria, British Columbia, Canada), were coelectrophoresed.

2.6 Protein transfer and immunoblotting

2.6.1 HSP 72

Following eelectrophoretic separation, proteins were transferred to nitrocellulose

membranes (0:22 jm thick, Bio-Rad Laboratories), as described by Towbin et al. (l979),

using the Bio-Rad mini-protean II gel transfer system. The gels were equilibrated in

transfer buffer (192 mM glycine, 25 mM Tris-C1 (pH 8.3), 0.1 % SDS and 20 % methanol)

for 10 min, removed and placed into a sandwich consisting of a Brillo pad, 3 pieces of

filter paper (Fisher Scientific, Nepean, Ontario, Canada), the nitrocellulose membrane, the

gel, three more pieces of filter paper, and a second Brillo pad. AU components of the

sandwich were immersed in transfer buffer prior to, and during, assembly. Two gel

sandwiches and an ice pack were placed in the gel transfer system. The proteins were

transferred to the nitrocellulose membrane at a constant 40 V for 4 h, with an ice pack

change at 2 h. Following protein transfer, the nitrocellulose membrane was blocked with

5% non-fat dried milk powder (NFDM) in Tris-buffered saline (TBS; 500 mM NaCl,

20mM Tris-C1 (pH 7.5)) for one hour, after which blots were washed twice, for 5 min

each time, in TTBS (TBS with 0.05 % Tween-20). The gels were stained to verify that

complete protein transfer had occurred. The blots were incubated overnight with a

polyclonal antibody 799 (1:2000 dilution in TTBS with 2% NFDM; a generous g& from

R.M. Tanguay, Lava1 University, Ste.-Foy, Quebec) specific for HSP 72. Following two

5 min washes in TTBS, the blots were immersed for 1 h in a solution of goat-anti-rabbit

IgG conjugated to alkaline phosphatase secondary antibody (BioRad, 1: 1000 dilution in

TI'BS with 2% NFDM). The blots were washed twice in ' I T B S , and once in TBS for 5

min each time, and immersed in a bicarbonate buffer (100mM Na&O,, 1 mM MgCl, (pH

9.8)) containing 3% w/v pnitro-blue-tetrazolium chloride ptoluidine salt in 70% N,N-

dimethyl-fonnamide (Dm and 1.5% wlv 5-bromo4chloro-3-indolyl phosphate in 100%

DMF. After development, blots were washed in d W 0 and allowed to dry. Immunoblots

were scanned using an AGFA Arcus 2 scanner, and HSP bands on the image were

quantified using Kodak 1D 1.0 image analysis software. Standard curves were constructed

to assure Linearity. The lane assignment for all blots run, whether showing HSP 72, or

HSC 73, was identical. On all blots, a control sample was run on the left most lane.

Proceeding rightward, in successive adjacent lanes, samples were run from the sodium 3

salicylate group, the mild heat shock group, the mild heat shock plus sodium salicylate

group, and the severe heat shock group. On all blots, a common sample was run, in order

to allow for comparison of quantified bands between blots. Bands representing left

ventricular HSP 72 content for all animals were run on a total of 4 blots, each of which was

subsequently quantified. Each experiment was repeated twice.

2.6.2 HSC 73

The protocol for analysis of HSC 73 (the HSP 70 cognate isoform) foilowing

protein transfer was identical to the one described above for HSP 72, with the following

exception. Analysis of HSC 73 was performed by incubating the blots for 3 hours with an

AP-conjugated monoclonal antibody specific for HSC 73 (1:5000 dilution in ' I T B S with

2% blotto; catalog #SPA-8 ISAP, Stress-Gen, Victoria, British Columbia, Canada). Thus,

following incubation with this antibody, the blots were simply washed twice in 'ITBS, and

once in TBS for 5 min each time, and developed in the bicarbonate buffer, as previously

described for HSP 72.

2.7 Statistical analysis

InStat 2.01 was used to analyze all data. For all heat shock and hernodynamic

variables (see appendices p. 63 for a list of alI variables analyzed), a one-way ANOVA

was performed, followed by a Tukey's post-hoc test to determine where s i m c a n t

differences (pc0.05) existed between the twatment groups. Statistical analysis was

performed using data points obtained fiom all 25 animals, i.e., five treatment groups where

n=5 per group.(refer to appendices, p. 63, for all individual measurements).

Chapter 3

3.1 Heat Shock

Resting rectal temperatures CT, ) during unstressed conditions for animals in the

three heat shock treatment groups [mild heat shock (40°C), mild heat shock plus sodium

salicylate (40°C and 400 mg-kg-'), and severe heat shock (42OC)], prior to the 15 min heat

shock, were 37.0+0.3OC, 36.8M.1°C, and 36.9rt0.4OC, respectively (figure 3). Tr was

not significantly different between groups.

The total heat stress duration (total time rectal temperature was above resting value)

was not significantly different between mildly heat-shocked animals, and those subjected to

a mild heat shock in combination with sodium salicylate treatment (44.4fi.3 vs 45.5s. 1

min, figure 4). Conversely, animals subjected to a severe heat shock required an average

of 65.3f 1.8 min to raise Tr to 42°C for 15 min, and return T, to baseline. This was (by

design) a sigaiFicady longer heat stress period than that experienced by the two milder heat

shock groups @cO.OOl).

The average Tr of mildly heat-shocked animals, and mildly heat-shocked animals 7

treated with sodium salicylate, was not significantly different during the entire heat stress

(39.1f0.17 vs 39.W.O7OC, figure 5), and during the 15 rnin heat shock period

(40.lf0.02 vs 40. 1+0.02"C, figure 6). As expected, average Tr during the entire heat

stress (figure 5), and during the 15 min heat shock (figure 6), was significantly higher for

animals subjected to severe heat shock than for animals subjected to mild heat shock, or

mild heat shock plus sodium salicylate treatment (p4.001).

mild mild severe heat heat heat

shock shock shock PIUS

sodium salicy late

Figure 3. Re-heat stress (baseline) rectal temperatures are similar between groups. No significant differences in baseline T, were detected among the three groups. Data are expressed as meansfSE, n=5 per group.

Figure 4. Mildly heat shocked, mildly heat shocked plus sodium salicylate treated animals, experienced heat shocks of similar duration. Shown is the time required for animals from each group to reach desired heat shock T,, then return to pre-heat stress baseline Tr. Mildly heat shocked animals, and midly heat shocked animals treated with sodium salicylate, required a similar amount of time to reach 40°C and return to baseline. Predictably, severely heat shocked animals required significantly more time to reach 42"C, and return to baseline. *p<0.001 compared to mild heat shock and mild heat shock plus sodium salicylate groups. Data are expressed as meansSE, n=5 per g r o w

mild mild severe heat heat heat

shodc shock shock P I ~

sodium saiicy Iate

Figure 5.' Mildly heat shocked, mildly heat shocked plus sodium salicylate treated animals, showed similar average T, during entire heat stress. The severely heat shocked animals had a significantly higher average T, than animals from the other two groups for the total heat stress period, which includes the time to ascend and descend from the 15 min heat shock plateau. *p<0.001 compared with mild heat shock and miId heat shock plus sodium salicylate groups. Data are expressed as meansSE, n=5 per group.

mild mild severe heat heat heat shock shock shock

elus sodium salicy late

Figure 6. Mildly heat shocked, mildly heat shocked plus sodium salicylate treated animals, showed similar average T, during 15 min heat shock. Tr during the 15 minute heat shock was controlled using the isoelectric pad (see text). Thus, animals that were severely heat shocked maintained T, very close to 42OC, while Tr of animals that were mildly heat shocked, and those that were mildly heat shocked plus sodium salicylate treated, slightly surpassed 40°C, as desired. *p<0.001 compared with mild heat shock and mild heat shock plus sodium salicylate groups. Data are expressed as meanskSE, n=5 per group.

Animais subjected to a mild heat shock, and animals subjected to a mild heat shock

plus sodium salicylate treatment, reached a similar peak T, during the heat stress

(40.=.04 vs. 40.3M.02, respectively; figure 7). The peak T, of animals subjected to a

severe heat shock was significantly greater than the peak T of animals in all other groups

(pc0.00 1 ) . Thus, mildly heat-shocked animals, and mildly heat-s hocked plus sodium

salicylate treated animals, were subjected to very similar heat stresses, while severely heat-

shocked animals were subjected to a greater heat stress.

3.2 Coronary flow

The pre-ischemic absolute value for coronary flow (of perfusion buffer) in hearts of

unstressed animals was 6.2M.6 ml-mid (table 1). Prior to ischemia, hearts from

salicylate treated animals had a coronary flow of 5 .M. 2 ml- mid'. Re-ischemic flow in

hearts of mildly heat-shocked animals was 6. 1f 1.1 rnl-mid, while in hearts from animals

that were mildly heat-shocked plus sodium salicylate treated, flow was 5% 1.1 ml-min-' .

Finally, in hearts from animals subjected to severe heat shock, coronary flow was

5.8H.5 ml-mh-'. No simcant differences were detected between groups.

To allow for comparisons between the five groups, the post-ischemic recovery of

coronary flow during the 30 min reperfusion period was expressed as a percentage of

absolute pre-ischemic values (figure 8). Following this data normalization, hearts from

unstressed (control) animals recovered 78.3k5.65 of pre-ischemic coronary flow at 5 min

of reperfusion. The recovery of coronary flow in hearts from unstressed animals did not

change ~ i ~ c a n t l y during the remainder of reperfision.

Although there were no statistically sigruficant differences between groups, hearts

from the control, sodium salicylate, and mild heat shock plus sodium salicylate treated

animals regained less of their pre-ischemic flow than hearts from animals subjected to

severe heat shock, or hearts from animals subjected to mild heat shock. This trend was

mild mild severe heat heat heat

shock shock shock plus

sodium saiicylate

Figure 7. Mildly heat shocked, mildly heat shocked plus sodium salicylate treated animals, showed similar peak T, during heat stress. Shown is the peak Tr reached by each group, at some point during the 15 min heat shock. Animals that were severely heat shocked reached a significantly higher peak T, than mildly heat shocked, or mildly heat shocked plus sodium salicylate treated counterparts (*p<0.001). Data are expressed as meansfSE, n=5 per group.

unstressed (controi)

sodium salicylate

mild heat shock

d d heat

I shock plus sodium salicylate

severe heat 17

pre-ischemic rate of contraction I relaxation (+dP*dt 'I. (-dP-dt *I; ~ ~ H ~ - s - & s E ) ~ ~ H ~ - s - ~ * s E )

pre-ischemic left venbricular developed pressure amp; r n & S E )

Table 1. he-ischemic absolute values for hernodynamic variables are shown. Values were collected 5 min prior to ischemia Data are expressed as meanskSE.

10 io 3b L

reperfusion time (min)

control

q* sodium salicylate

,+ mild heat shock

mild heat shock ,+ plussodium

salicy late

-- severe heat shock

Figure 8. Post-ischemic recovery of coronary flow is unchanged by heat, or heat and sodium salicylate treatment. No significant differences were detected between groups. Data are expressed as a percentage of absolute pre-ischemic d u e s (meanskSE, n=5 per group).

most apparent after 10 min, and continued throughout the remainder of reperfusion. After

only 5 min of reperfusion, hearts fYom animals subjected to severe heat shock recovered

87.5&4.9% of pre-ischemic flow, and hearts from animals subjected to mild heat shock

recovered 83.2+4.7% of pre-ischemic flow values. After 5 min of reperfusion, hearts

from control, sodium salicylate, and mild heat shock plus sodium salicylate treated animals

recovered 78.3k5.68, 80.5&4.2%, and 74.7+4.5% of pre-ischemic flow, respectively.

Thus, at 5 min of reperfusion, no trend in recovery was immediately discernable, and there

was no indication of possible differences in degree of recovery. .However, after 10 min of

reperfusion, a trend was observed in the difference in flow recovery between groups. At

10 min, hearts from animals subjected to severe heat shock, and hearts from animals

subjected to mild heat shock, recovered over 80% of pre-ischemic coronary flow

(86.(W4.9% and 8 2.4k6.5, respectively). Conversely, hearts fkom unstressed (control)

animals, sodium salicylate treated animals, and mildly heat-shocked plus sodium salicylate

treated animals recovered less than 80% of pre-ischemic coronary flow (74.2H .3,

75.2B.3 and 74.4&4.7%, respectively). In all groups, there was fluctuation in the

percentage of absolute pre-ischemic coronary flow recovered during the reperfusion period.

Nevertheless, hearts from animals subjected to severe heat shock, and hearts from animals

subjected to mild heat shock, showed a trend towards a greater recovery of coronary flow

than hearts fkom all other groups.

In summary, the combinations of heat stress and/or sodium salicylate treatment

used here do not appear to confer any simcant advantage in a heart's ability to recover

coronary flow following ischemia.

3.3 Rate of contraction and relaxation

3.3.1 Rate of contraction

The pre-ischemic absolute value for rate of contraction, as indicated by +dP-dt -', in

hearts of unstressed animals was 1848k50.8 &gd (table 1). Prior to ischemia, hearts

fiom salicylate treated animals had a rate of contraction of 17793A40.2 mrnHg=s-'. h e -

ischemic +dP-dt in hearts of mildly heat-shocked animals was 1767S3.0 mm~g-s- ' ,

while in hearts fiom animals that were mildly heat-shocked plus sodium salicylate treated,

+dP-dt was 1498k109.3 mmHgd. Finally, in hearts from animals that were subjected

to severe heat shock, the pre-ischemic rate of contraction was 1637H40.9 rn~nHg-s-I. No

sigmficant differences were detected between groups.

When the post-ischemic recovery of +dP-dt -' was expressed as a percentage of

their respective absolute pre-ischemic values, hearts fiom control animals recovered

30.3&4.2% of their pre-ischemic +dl?& -' a . r 5 minutes of reperfusion (figure 9).

Thereafter, the recovery of +dP-dt -' in heats from control animals increased in a linear

manner. At 30 min of reperfusion, the recovery of rate of contraction in control hearts

reached 48.3&9.0% of pre-ischemic values.

As previously mentioned, hearts from unstressed (control) animals recovered

30.3&4.2% of pre-ischemic values at 5 min of reperfusion, while hearts from sodium

salicylate treated animals recovered 23.4&4.4%. Hearts from mildly heat-shocked animals

recovered 3 1 .=. 1 %, while those from mildly heat-shocked plus sodium salicylate treated

animals recovered 35.5s. 1%. Hearts from animals subjected to severe heat shock

recovered 24.5k4.295 of pre-ischemic +dP-dt -' values. At 5 min of reperfusion, there

were no significant differences in the recovery of +dP& " between groups

After 10 min, and throughout the remainder of reperfusion, differences were

observed in the recovery of +dP& between groups. Hearts from severely heat-shocked

animals, and those mildly heat-shocked plus sodium salicylate treated, recovered +dl?-dt '' to a greater extent than all other groups. This appeared to be the result of a more rapid rate

of +dP-dt " recovery. By 10 min, hearts fiom animals subjected to a severe heat shock

reached 54.7+10.3% of pre-ischemic +dP-dr -', more than twice the value observed at 5

min of repefision (24.5k4.2). In addition, hearts fiom mildly heat-shocked plus sodium

salicylate treated animals increased recovery of +dP-dt -' from 35.5&5.1% at 5 min, to

10 20 30

reperfusion time (min)

control

sodium salicylate

mild heat shock

miId heat shock plus sodium salicytate

severe heat shock

Figure 9. A 42°C heat shock, and a 40°C heat shock lus sodium salicylate enhanced ! post-ischemic recovery of rate of contraction (+dP-dr - ). Compared to hearts from unstressed (control) animals, hearts from severely heat shocked animals recovered a significantly greater percentage of re-ischemic +dP-dt -l (*p<0.05 after 15 rnin reperfusion). At 30 min of r e p e d i o n , +dP& was significantly greater in hearts from mildly heat shocked animals treated with sodium salicylate, than hearts from unstressed (control) animals (+pc0.05). Data are expressed as means+SE, n=5 per group.

50.5+9.3% at 10 min. Nonetheless, at LO min of reperfusion, the only si@cant

difference in recovery of +dP.dt " observed between groups was between hearts of animals

subjected to severe heat shock, which had recovered 54.7f 10.3% of +dP.dt 'I, and those

of sodium salicylate treated animals, which had recovered only 2 1.4--8% @<0.05).

Compared to all other groups at 10 min of reperfusion, the recovery of +dP-dt -' in hearts

fiom severely heat-shocked animals was not siplicantly different. Hearts from animals

that were mildly heat-shocked, mildly heat-shocked plus sodium salicylate treated, and

severely heat-shocked, showed a trend of an increasing recovery of +dP-dt throughout

reperfusion. In contrast, hearts fiom both unstressed, and salicylate treated animala

actually showed a decrease in recovery of idP-dt -' between minutes 5 and 10.

Subsequently, both groups exhibited continuing increases in recovery of +dP-dt " . Thus,

at 10 min of reperfusion, the significant difference observed in restoration of rate of

contraction between hearts fiom severely heat-shocked animals, and hearts from sodium

salicylate treated animals, was the result of a large increase in +dP& -' recovery in the

former group, and a small decrease in +dP-dt -' recovery in the latter group, between 5 and

10 minutes of reperfusion.

At 15 min of reperfusion, the recovery of rate of contraction in hearts from animals

subjected to severe heat shock reached 72Sf 10.9% of pre-ischemic values, and was

significantly greater than that observed in hearts of sodium salicylate treated animals

(23.6k3.3%, p<O.01), and also that observed in hearts from unstressed (control) animals

(39.1&6.5%, p<0.05). In addition, the recovery of +dP-dr for hearts from mildly heat-

shocked plus sodium salicylate treated animals was significantly greater than observed for

hearts from sodium salicylate treated animals (58.5f9.4% vs. 23.6*3.3%, pc0.05).

Hearts fiom animals in all groups increased their recovery of +dP/dt throughout the

remainder of reperfusion, and the significant differences were consistent for the rest of the

30 min period. In addition, +dP-dt -' recovery was sigruficantly greater in hearts from

severely heat-shocked animals compared to hearts from mildly heat-shocked animals, at 20

rnin of reperfbsion and thereafter (p4.05). Furthermore, after 30 min of reperhion,

hearts £iom mildly heat-shocked plus sodium salicylate treated animals, recovered a

significantly greater percentage of their pre-ischemic absolute +dP-dt - I , than hearts fkom

unstressed animals (75.8M.5% vs. 48.3B.0%, p<0.05). This difference was not

observed when comparing +dP& recovery of hearts fiom mildly heat-shocked animals,

to +dP& " recovery of hearts fiom unstressed animals.

A heat shock of 42OC for 15 min is capable of conferring myocardial protection, in

terms of recovery of rate of contraction, following an ischemic insult (figure 9). A milder

heat shock of 40°C for 15 min, in combination with sodium salicylate treatment,

significantly kproves +dP& -' recovery when compared to unstressed controls, but this is

only evident after 30 min of reperfusion. Conversely, a heat shock of 40°C for 15 min, in

the absence of sodium salicylate treatment, does not significantly improve +dP-dt -' recovery, compared to unstressed controls.

3.3.2 Rate of relaxation

The pre-ischemic absolute value for rate of relaxation, as indicated by -dP-dt -I, in

hearts of unstressed animals was 1162+68.8 mmHg-s" (table 1). Prior to ischemia, hearts

from salicylate treated animals had a rate of relaxation of 983k107.4 mm~g-s-' . Pre-

ischemic - d ~ & -' in hearts of mildly heat-shocked animals was lOO3H 1.4 mmHg-s",

while in hearts fkom animals that were mildly heat-shocked plus sodium salicylate treated,

-dP-dt -' was 91 1B7.6 mmHg-s-l. Finally, in hearts from animals that were subjected to

severe heat shock, the pre-ischemic absolute rate of relaxation was 1034k86.7 mmHgd.

No significant differences were detected between groups.

When the post-ischemic recovery of -dP& -' was expressed as a percentage of their

respective absolute pre-ischemic values, hearts from unstressed (control) animals recovered

35.6&5.0% of their pre-ischemic -dl?-dt '' after 5 min of reperfusion (figure 10).

Thereafter, hearts fkom unstressed animals recovered progressively more of their pre-

+ control

.I_Q1_ mild heat shock

mild heat shock plussodium salicylate * severe heat shock

reperfusion time (min)

Figure 10. A 42°C heat shock, and a 40°C heat shock plus sodium salicylate enhanced post-ischemic recovery of rate of relaxation (-dPdt -1). Compared to hearts fiom unstressed (controls) animals, hearts fiom severely heat shocked animals recovered a significantly greater percentage of pre-ischemic -dP& - I (*p<0.05 after 20 min reperfusion). At 30 rnin of reperfusion, -dP.dt -'is significantly greater in hearts from mildly heat shocked animals treated with sodium salicylate, than in hearts from unstressed (control) animals (+p<0.05). Data are expressed as meansfSE, n=5 per group.

ischemic -dP-dt -I. Following the 30 min reperfusion period, these hearts had recovered

53.4B.396 of their pre-ischemic -dP-dt -'. Considering all groups as a whole, the

recovery of rate of contraction differed somewhat fiom the recovery of rate of relaxation.

For example, at 5 min of reperfusion, no statistically significant differences existed

between groups in terms of both -dP& " and +dP-dr -'. After 10 min of reperfusion, there

was still no significant difference between groups in terms of recovery of rate of relaxation.

In contrast, at 10 min, the hearts from animals subjected to severe heat shock recovered

sipdicantly more of their pre-ischemic rate of contraction than hearts from sodium

salicylate treated animals. At 15 min of reperfusion, hearts fiom both severely heat-

shocked animals, and mildly heat-shocked plus sodium salicylate treated animals had

recovered significantly more of their pre-ischemic -dP.dt -' than hearts from sodium

salicylate treated animals (77.M 12.5% and 68 Ski 0.1 %, respectively vs. 3 1 .w .4%;

p<O.01 and p<0.05, respectively). This difference appears to be attributabIe to a slower

rate of recovery of -dP-dt -' in the hearts of sodium salicylate treated animals compared to

all other groups. Similarly, hearts from unstressed animals also showed a slower rate of

recovery during the middle portion of reperfusion. Thus, at 20 min, -dP.dt " recovery of

hearts fiom animals subjected to severe heat shock was significantly greater than that of

hearts from unstressed (control) animals. In the last 10 min of reperfusion, hearts fiom all

groups progressively increased their recovery of -dP-dt ". This was particularly evident in

hearts from sodium salicylate treated animals. As a result, at 25 and 30 min of reperfision,

there was no longer a significant difference in rate of relaxation recovery between hearts

from animals subjected to mild heat shock plus sodium salicylate treatment, and those fiom

animals only treated with sodium salicylate. Furthermore, at 30 min of reperfusion, there

was no sigruticant difference in -dP-dt -' recovery between hearts from severely heat-

shocked animals, and hearts from sodium salicylate treated animals. At the end of the 30

min reperfusion period, hearts fiom animals subjected to a severe heat shock, and those

fiom animals subjected to a mild heat shock plus sodium salicylate treatment, had recovered

a siacantly greater percentage of their pre-ischemic -dP-dt " than hearts fiom unstressed

animals (87.8k7.61 and 86.2+7.6%, respectively, vs. 53.4&9.3%, p<0.05).

A heat shock of 42°C for 15 min is capable of conferring myocardial protection, in

terms of recovery of rate of relaxation, following an ischemic insult (figure 10). A milder

heat shock of 40°C for 15 min, in combination with sodium salicylate treatment, also

significantly improves -dP-dt -' recovery, but this was only observed at the conclusion of

the 30 min reperfusion period. Conversely, a heat shock of W C for 15 min, without

sodium salicyate treatment, does not significantly improve -dP-dr " recovery, compared to

unstressed controls.

3.4 Left ventricular developed pressure

The pre-ischemic absolute value for left ventricular developed pressure (LVDP) in

hearts fkom unstressed animals was 85.7kl.7 mmHg (table 1). Prior to ischemia, hearts

from salicylate treated animals had an LVDP of 78.1S.2 mrnHg. Pre-ischemic LVDP in

hearts of mildly heat-shocked animals was 77.632.2 mmHg, while in hearts from animals

that were mildly heat-shocked plus sodium salicylate treated, LVDP was 67 .=.O rnmHg.

Finally, in hearts from animals that were subjected to severe heat shock, the pre-ischemic

absolute left ventricular developed pressure was 7426 .4 mrn Hg. No significant

differences were detected between groups.

When the post-ischemic recovery of LVDP was expressed as a percentage of their

respective absolute pre-ischemic values, hearts fkom unstressed (control) animals recovered

33.W4.7% of their pre-ischemic LVDP after 5 minutes of reperfusion (figure 1 1). After

10 min, recovery of left ventricular developed pressure had increased to 42.%7.2%. In

the middle portion of the reperfusion period, from 10 min to 20 min, the restoration of

LVDP plateaued (43.5+6.9% LVDP recovery at 20 min). Thereafter, these hearts showed

an increased recovery of pre-ischemic LVDP values. However, after 30 rnin of

control

* sodium salicylate - mild heat shock

mild heat shock --m- plus sodium

salicylate * severe heat shock

reperfusion time (min)

Figure 11. A 42OC heat shock enhanced post-ischemic recovery of left ventricular developed pressure (LVDP). In comparison to controls, recovery of LVDP is significantly greater in hearts from severely heat shocked animals after 15 min of reperfusion (*p<0.01). Hearts from animals in the mild heat shock plus sodium salicylate group did not exhibit significantly greater restoration of LVDP relative to hearts from unstressed (control) animals. Data are expressed as a percentage of absolute pre-ischemic d u e s (meanskSE, n=5 per group).

reperfusion, hearts fkom unstressed animals had regained only about half of their pre-

ischemic LVDP (5O.P+lO. 1 %). As previously mentioned, after 5 min of reperhsion,

hearts from unstressed animals recovered 33.01t4.795 of pre-ischemic LVDP, while hearts

from sodium salicylate treated animals, d d l y heat-shocked animals, mildly heat-shocked

plus sodium salicylate treated animals, and severely heat-shocked animals recovered

26.824.0%, 36.4rU.2, 39.1fi.4, and 25.W4.0 of their pre-ischemic LVDP, respectively.

At 5 min of reperfusion there were no significant differences in LVDP recovery between

groups. However, at 10 min of reperfusion, hearts fkom severely heat-shocked animals

recovered a significantly greater amount of pre-ischemic LVDP than those fiom sodium

salicylate treated animals (59.1H.6 vs. 23 .= .6%, p<0.05). The differences between

these two groups continued throughout the 30 minute reperfusion period. At 10 min of

reperfusion, the hearts of mildly heat-shocked plus sodium salicylate treated animals

recovered a similar percentage of LVDP when compared to hearts of severely heat-shocked

counterparts (53.2393% vs. 59.1B.6). However, recovery of LMlP in the hearts from

mildly heat-shocked plus sodium salicylate treated animals was not significantly different

from any other group at 10 min. Nevertheless, at this point and for the remainder of the

reperfusion period, a trend was observed where hearts fiom animals subjected to a mild

heat shock plus sodium salicylate treatment, recovered LVDP to a greater extent than hearts

fiom mildly heat-shocked animals, but to a lesser extent than hearts from severely heat-

shocked animals. After 15 min of reperfusion, the hearts fiom animals subjected to a

severe heat shock recovered LVDP to a significantly greater extent than hearts from

unstressed animals, this continued for the remainder of reperfusion, such that at 30 min,

values were 94.5k4.695 vs. 50.9&10.1% (pc0.05). In addition, the percentage of LVDP

recovered at 15 min of reperfusion was significantly greater in hearts from mildly heat-

shocked plus sodium salicylate treated animals than in hearts from animals treated only with

sodium salicylate (64.5k10.4 vs. 26.3&3.6%, p ~0.05). This observation was also made

at 20 minutes, but not thereafter. This difference existed when the rate of LVDP recovery

was increasing in the hearts of mildly heat-shocked plus sodium salicylate treated animals,

but decreasing in the hearts of sodium salicylate treated animals. LVDP recovery in the

hearts of sodiuk salicylate treated animals was low immediately upon reperfusion, and by

25 rnin reached values similar to those observed in unstressed animals.

In general, the recovery of LVDP during reperfusion was greatest in the hearts fkom

animals subjected to severe heat stress, followed by hearts from mildly heat-stressed plus

sodium salicylate treated animals, then in hearts from mildly heat-stressed animals only.

However, when compared to hearts fiom unstressed animals, only hearts fkom animals

subjected to severe heat shock differed significantly in terms of LVDP recovery. These

results demonstrate a heat shock of 42°C for 15 min is capable of conferring subsequent

myocardial protection. Other treatment forms u r n in this experiment did not confer

myocardial in terms of LVDP recovery.

3.5 HSP 70 content

3.51 ESP 72

To evaluate differences in HSP 72 content, portions (40-60 mg) of the left

ventricle, near the apex, were homogenized, total protein separated by SDS polyacryIamide

gel electrophoresis, and transferred to nitrocellulose membrane, as described in materials

and methods. A representative Western blot (figure 12A) shows HSP 72 content was

detectable in f l hearts examined. When purified human HSP 70 (catalog #SPP-755;

Stress-Gen, Victoria, British Columbia, Canada) was co-electrophoresed ( h e 6 ), and

used as a standard, it co-migrated to approximately the same position as proteins from

myocardial tissue, thus confirming the specificity of the HSP 72 antibody. HSP 72 content

was barely detectable in the heats of unstressed (control) animals (lane I). Conversely,

HSP 72 content was noticeably elevated in hearts fiom animals heat-shocked to 42O C

(lane 4 ). HSP 72 content in the hearts from animals in all other groups was similar to that

observed for controls, however, some trends were detected. Visual inspection suggested

HSP 72+

Figure 12. A) HSP 72 content is increased in left ventricle following heat shock to 42OC. A representative Western blot illustrates left ventricular HSP 72 content following heat and/or sodium salicylate treatment (100 pg protein loaded per lane). Portions of the left ventricle were homogenized, total protein separated by SDS polyacrylamide gel electrophoresis, and transferred to nitrocellulose membrane, as described in materials and methods. Only the heart of the animal heat shocked to 42°C has a discernably higher HSP 72 content (lane 4 ). Lane I : control; lane 2 : sodium salicylate only (400 mgkgL); lane 3 : mild heat shock (40°C); lane 4: severe heat shock (42°C); lane 5: mild heat shock (40°C) combined with sodium salicylate (400 mg-kg-I); lane 6 : human HSP 70 standard. B) HSC 73 content in left ventricle is unchanged following heat shock and/or sodium salicylate treatment. A representative Western blot illustrates left ventricular HSC 73 content following heat andlor sodium salicylate treatment (100 pg protein loaded per lane). Portions of the left ventricle were homogenized, total protein separated by SDS polyacrylamide gel electrophoresis, and transferred to nitrocellulose membrane, as described in materials and methods. Lane I: control; lane 2 : sodium s alicylate only (400 mg-kg-'); lane 3 : mild heat shock (40°C); lane 4: severe heat shocp (42°C); lane 5: mild heat shock (40°C) combined with sodium salicylate (400 mg-kg ).

myocardial HSP 72 content of sodium salicylate treated animals was less than that of

unstressed animals (compare [mte 2 to lrme I). Conversely, myocardial HSP 72 content

from mildly heat-stressed plus sodium salicylate treated animals was greater than

myocardial HSP 72 content in unstressed animals (compare Inne 5 to h e I).

To fuaher assess left ventricular HSP 72 content, quantification of bands

representing HSP 72 fiom Western blots was performed using 1-dimensional Kodak image

analysis software. In all cases, HSP 72 content was expressed as a percentage of the mean

value determined fiom the hearts of the unstressed (control) aniqals. Hearts from animals

subjected to severe heat shock showed a significant increase in HSP 72 content compared

to hearts f?om unstressed animals (Figure 13, p<0.001). No significant differexes were

observed in HSP 72 content between hearts from animals subjected to mild heat shock and

those from animals given the combined treatment of mild heat shock plus sodium salicylate.

Although left ventricular HSP 72 content was greater in the hearts from mildly heat-

shocked plus sodium salicylate treated animals, relative to hearts from unstressed animals,

no significant difference was detected. Hearts from sodium salicylate treated animals

showed a decreased HSP 72 content in comparison to unstressed animal hearts, but again,

this difference was not statistically significant These results demonstrate that a heat shock

of 42°C for 15 min is capable of sigmficantly increasing myocardial HSP 72 content.

However, a mild heat shock of 40°C for 15 min, alone or combined with sodium salicylate,

does not increase left ventricular HSP 72 content compared to unstressed animals.

3.5.2 HSC 73 content

To evaluate differences in HSC 73 content, portions (40-60 mg) of the left

ventricle, near the apex were homogenized, total protein separated by SDS polyacrylarnide

gel electrophoresis, and transferred to nitroceUulose membrane, as described in materials

and methods. A representative Western blot (figure 12B) shows HSC 73 content was

detectable in all hearts examined. While HSP 72 content was noticeably elevated in hearts

control sodium mild mild severe salicy late heat heat heat

shock shock shock PIUS

sodium salicy late

Figure 13. Graphical representation of left ventricular HSP 72 content following ischemia-reperfusion. HSP 72 content of the lefi ventricle was quantified using 1-dimensional image analysis software. Data are expressed as a percent of control. Left ventricular HSP 72 content in animals heat shocked to 42OC is significantly elevated compared to all other groups (*p<0.001). Data are expressed as means+SE, n=5 per group.

fkom severely heat-stressed animals, visual inspection suggested that the various treatments

did not affect left ventricular KSC 73 content. Thus, hearts fkom animals in ail groups

showed similar HSC 73 content.

To fuaher assess left ventnicdar HSC 73 content, quantification of bands

representing HSC 73 from Western blots was performed using 1-dimensional Kodak

image analysis software (figure 14). In all cases, HSC 73 content was expressed as a

percentage of .values determined for hearts from unstressed (control) animals. Various (r

combinations of heat andlor salicylate treatment did not affect the accumulation HSC 73 in

the left ventricle. HSC 73 is constitutively expressed, and generally detected following

stressed, or unstressed conditions. Thus, it was not surprising to observe no sigmficant

differences in myocardial HSC 73 content between groups.

r

- control sodium mild mild

salicylate heat heat shock shock

plus sodium salicy I ate

Severe heat

shock

Figure 14. Graphical represenration of left ventricular HSC 73 content following ischemia-repefision. HSC 73 content of the left ventricle was quantified using I-dimensional image analysis software. Data are expressed as a percent of control. Left ventricular HSC 73 content is srnilar in animals from all groups. Data are expressed as meanskSE, n=5 per group.

Chapter 4

The heat shock response is universal, and has been demonstrated in many

organisms (for review, see Morirnoto et al., 1994). Cells exposed to temperature

elevations, either in vitro or in vivo, respond by rapidly synthesizing HSP 72 and other

HSPs (Ritossa, 1962; Tissieres et d., 1974; Currie et al., 1982; Li et al., 1982; Barb et

aI. 1988; Blake et al., 1990). Heat-induced accumulation of HSP 72 has been shown to

confer subsequent myocardial protection (Currie et al., 1988; Kamxizyn et al., 1990;

Hutter et ai. 1994; Lofke et al., 1995). Recent evidence has suggested that various

salicylates potentiate the heat shock response in vitro (Amici et al., 1995; Ito et al., 1996;

Burress et al., 1997). However, only one study has examined this concept in vivo.

Fawcett et al. (1997) have reported that aspirin potentiated the heat shock response in rat

liver, lung, and kidney, but in that study, the myocardium was not examined.

Furthermore, Fawcett et al. (1997) did not determine whether potentiation of the heat shock

response provided any protective effect. Thus, it was of interest to propose two novel

questions. Firstly, does salicylate have an effect on the heat-induced HSP 72 accumulation

in the myocardium? And secondly, does any such accumulation of HSP 72 provide

myocardial protection?

In the present study, neither sodium salicylate treatmat alone, nor sodium

sulicylate treatment in combination with a mild heat shock of 409C for 1.5 min, were found

to increase lefr ventricular HSP 72 content signt~cuntly in rhe animals observed (figures

12A and 13). This result differs from that reported by Fawcett et al. (1997), who

concluded that aspirin potentiated the heat shock response in vivo. Reasons for this

discrepancy are not clear, but certainly, some rnethodologicaI differences between the two

studies need to be addressed.

First, in the present study, as in the research undertaken by Fawcett et al. (1 997),

the animals were treated with a 'salicylate' 1 h prior to the heat stress. However, in that

study, Fawcett et al. (1997) used aspirin (100 mg-kg1), whereas in the present study,

sodium salicylate was used (400 mg-kg-'). Thus, there were differences in both dosage

and in the type of salicylate used between the two studies. L

While .it is not clear whether the difference in dosage had an effect on the

potentiation of the heat shock response, it has been noted that sodium salicylate is

commonly administered in greater doses than aspirin (Vane and Botting, 1992). There are

differences in the chemical properties of aspirin and sodium salicylate which affect the

mode of action of the two drugs. In mammals, aspirin prevents prostaglandin production

by inhibiting cyclo-oxygenase through acetylation (Roth and Majerus, 1975). In contrast,

there is evidence that sodium salicylate stops prostaglandin production primarily through

the prevention of de novo synthesis of prostaglandin H synthase (Wu, 1991). It is unclear

whether the putative differences in the mode of action of these two drugs could affect

potentiation of the heat shock response. Furthermore, while sodium salicylate can be

dissolved and administered in an &O medium, aspirin is not easily dissolved in H,O, and

presumably for this reason, Fawcett et al. (1997) administered the aspirin via an ethanol

vehicle. However, it should be noted that ethanol itself has been shown to be an inducer of

HSPs (Li, 1983). Furthermore, it has been demonstrated that two or more stressors acting

in concert can have a synergistic effect on the expression of certain HSPs (Rodenhiser et

al., 1986; Hahn et al., 199 1). Thus, the administration of ethanol in combination with a

heat shock may produce a confounding induction of HSP 72.

Secondly, in comparing the present study to that of Fawcett et al. (1997), the

method of heat stress exposure should be closely evaluated. Fawcett et al. (1997) exposed

the animals to a constant, ambient temperature of 37OC for 30 min. ; this method allowed T,

to rise in an uncontrolled manner. In the present study, an adjustable thermal plate was

used to control the change in T, more precisely. In this way, a rise in T, above the desired

value was quickly corrected, thus ensuring that any observed potentiation of the heat shock

response by salicylate would not simply be the result of a higher T, Animals that were

mildly heat-stressed, and animals that were mildly heat-stressed in combination with

sodium salicylate treatment, required similar time periods to elevate rectal temperatures CT,)

to 400C for 15 min, and for T, to return to baseline Levels (figure 4). In addition, the

animals from both of these groups had identical average T, during the 15 min heat shock

(figure 6), and similar average T, during the entire heat stress (figure 5). This finding

stands in contrast to that reported by Fawcett et al. (1997), who observed a sigdicant

difference in core temperature between the mildly heat-shocked rats, and those mildly heat-

shocked in combination with aspirin treatment. In the Fawcett study, at the conchsion of

the 30 min heat shock period, the animals treated with aspirin reached an average core

temperature of 40.3"C, whereas the animals that experienced heat-shock without the

corresponding aspirin treatment reached an average core temperature of only 39.4OC. This

result would suggest that the aspirin potentiated a rise in core body temperature during heat

shock. In fact, Fawcett et al. (1997) concluded that aspirin enhanced the accumulation of

HSP 70 by mediating a rise in core body temperature. However, the accumulation of HSP

70 through a rise in core body temperature makes it difficult to isolate any independent

effect that aspirin may have had on HSP 70 induction. Indeed, a rise in core body

temperature alone is likely sufficient to enhance the heat shock response. Hutter et al.

(1994) demonstrated a correlation between core temperature during heat shock and

subsequent HSP accumulation. Following heat shocks of 40, 41, and 42"C, there was an

incremental elevation in myocardial HSP 72 (Hutter et al., 1994). Such a relationship

would imply that a significant increase in core body temperature, similar to the one

observed in the study by Fawcett et al. (1997), could in itself, result in a greater

accumulation of HSP 72.

Thirdly, as previously mentioned, Fawcett et al. (1997) observed a signifcant

elevation in core body temperature in the miIdly heat-shocked animals previously

administered aspirin, relative to counterparts that were only mildly heat-shocked. The

authors did not pursue this observation, giving no indication of how long core body

temperature remained elevated following the 30 min heat shock. It is possible that since

aspirin enhanced the rise in core temperature, the drug may have also prolonged the return

of core temperature to unstressed values. Thus, the animals would have been exposed to

an overall greater heat stress, which could possibly cause a greater induction of HSP 72.

Although a brief, but intense heat shock of 42°C for 15 min is known to elicit a robust

induction of HSP 72, heat shocks of lesser temperatures, but of longer durations have also

been shown to effectively induce HSP 72 (Blake et d., 1990; Kregel et al., 1995). Blake

et al. (1990) reported appreciable levels of HSP 70 mRNA in tissues of heat-shocked rats

whose core body temperature was less than 40°C. In the Blake study, animals were

exposed to a 90 min heat stress in which core body temperature remained less than 40°C.

Kregel et al. (1995) reported a four-fold elevation in both hepatic and myocardial HSP 72

content foilowing a heat shock of 4I0C for 30 min. Importantly, Kregel et al. (1995)

employed a heating system requiring approximately 45-55 min to raise the colonic

temperature of the rat to 41°C, and maintained this temperature for 30 rnin. Thus, in these

studies (Blake et al., 1990; Kregel et al., 1995), the animals were exposed to heat stresses

in which the core temperature of the animal remained elevated above the resting value for at

least 90 min. It is not clear how long animal core temperature remained elevated in the

study by Fawcett et al. (1997). However, it is likely that animals in the present study were

exposed to an overall shorter heat stress than those in the study by Fawcett et d. (1997). A

distinguishingefeature in the present study was the use of a heating pad which dowed a

rapid cooling of the animal following the 15 min heat shock, hence Limiting the duration of

the heat stress. Thus, the core temperature of the mildly heat-shocked animals, with or

without salicylate treatment, was elevated above resting values for only 45 min.

52

In the present study, the animalR treated with a mild heat shock plus sodium

salicylate showed a trend towards a higher peak T, than mildly heat-shocked only

counterparts, (40.3'C vs 40.Z°C; Figure 6). The observation of a higher peak T, in the

former group, albeit not statistically significant, may be noteworthy. Fawcen et al. (1997)

observed that aspirin potentiated the rise in core body temperature of animals at the end of a

30 min exposure to an elevated temperature. As mentioned previously, animals in the

study by Fawcett et al. (1997), subjected to a combination of heat stress and aspirin

treatment, exhibited an elevation in T, of approximately 1°C, compared to animals that were

only heat-strqsed (40.3OC vs 39.4"C, respectively). It should be reiterated that in that

study, animal T, was allowed to rise freely in response to an elevated temperature. In the

present study, the trend of a higher peak T, in animals that were mildly heat-shocked in

combination with sodium salicylate treatment occurred despite efforts to adjust plate

temperature, and keep T, of both groups as close to 4OT as possible. In effect, it seemed

more difficult to minimi;re the overshoot of T, in the animals that were subjected to mild

heat shock plus sodium salicylate katment after T, reached 40°C. In the present study, it is

unlikely that a dBerence of 0. 1°C in peak T, between animals in the two mild heat shock

groups carried any physiological significance, especially in terms of HSP 72 content.

Moreover, it .is debatable whether this trend in higher peak T, would be observed

repeatedly. Nevertheless, the present study may offer some support to the observation that

salicylate may enhance the rise in core body temperature during a mild heat shock, a s

reported by Fawcett et al. (1997).

It is not clear how the salicylates could mediate a heat-induced rise in core body

temperature. Though well known for their antipyretic properties, the salicylates are thought

to have little effect in the afebrile state (Vane et al., 1992). In response to pyrogens derived

from invading organisms, animais produce interleukin- 1, which stimulates arachidonic acid

metabolism, and eventual prostaglandin production. Through complex thermoregulatory

mechanisms, the prostaglandins increase body temperature. Accordingly, the antipyretic

effect of salicylate is achieved by inhibiting prostaglandin synthesis. Studies in which

animals were heat-stressed have shown that the prostaglandins are not involved in normal

body temperature regulation (Vane et ai., 1992). Thus, salicylate enhancement of heat-

induced elevations in body temperahue are probably not mediated by the drug's effect on

prostaglandins. Moreover, if the prostaglandins were involved in the temperature

elevations caused by the imposed heat stress, it is reasonable to assume that salicylate

would act to oppose the rise in core body temperature. It is possible that the salicylates

exert a direct effect on the animal's temperat regulation centers, such as the

hypothalamus, the medulla oblongata, or the spinal cord. If salicylate were to affect the

ability of any of these centers to control heat loss, then animal core temperature may rise

faster than normal. Thus, an animal treated with salicylate in combination with a mild heat

shock, may experience a greater rise in core body temperahlre than a mildly heat-shocked

only counterpart.

The adxninistration of salicylate to exercising animals may be helpful in gaining

insight into the drug's effect on core body temperature. In an exercise modeI, animal core

temperature would presumably rise freely, as in the study by Fawcett et al. (1997). It

would be of interest to determine whether those animals treated with salicylate prior to

exercise would attain a higher core temperature than untreated animals. In addition, would

higher core temperatures lead to an increased accumulation of HSP 72?

Fawcett et al. (1997) provided evidence that aspirin potentiated the heat-induced

accumulation of HSP 72 mRNA in rat liver, lung, and kidney. Nevertheless, the authors

only documented an increase of the protein in the liver. This was perhaps surprising, given

that the hepatic tissue in question was harvested only 30 min after heat shock. Currie et al.

(1982) demonstrated that HSP 72 synthesis is not detectable immediately following heat

shock, nor within 30 min post-exposure, in several tissues, including liver. In any case,

observations regarding HSP 72 accumulation in Liver should not be extended to the

myocardium, since there is evidence that the heat shock response is tissue specific. Blake

et al. (1990) found discordance of in vivo expression of HSP 72 among different tissues.

The authors noted that HSP 70 mRNA accumulated in a time dependent manner in liver. In

this tissue, HSP 70 mRNA accumulation was several fold higher at 6 h post-heat shock

than at one hour post-heat shock. Hanagan et al. (1995) demonstrated that the liver was

sensitive to the rate of heating. The authors showed that a high rate of heating during heat

shock could induce greater hepatic HSP 72 induction than a lower rate of heating,

irrespective of-total heat load. More importantly, Hanagan et al. (1995) suggested that the

liver may be a tissue susceptible to early thermal damage. Thus, it is possible that a whole

body heat stress of a given intensity would elicit HSP induction more vigorously in the

liver than in the heart. Taken together, these results indicate that interpretation of the heat

shock response should be k t e d to the specific tissue under investigation. Accordingly,

this reasoning could partially explain why Fawcett et al. (1997) observed salicylate

potentiation of HSP 72 induction in the liver, while in the present study, salicylate did not

potentiate HSP 72 induction in the myocardium.

In the present study, animal core temperature was precisely controlled during a heat

shock of 40°C; ensuring that sodium salicylate had no effect on core temperature. Thus, it

was demonstrated that in the absence of a temperature elevation, salicylate did not potentiate

HSP 72 induction during a mild heat shock Accordingly, it is suggested that aspirin

potentiation of the heat shock response observed by Fawcett et al. (1997) was solely the

result of the drug's enhancement of the rise in core temperature during heat shock It is not

known why the actions of salicylate observed in vitro were not observed in vivo. The

evidence supporting an in vitro potentiation of the heat shock response by the salicylates

must be carefully interpreted, as the type of cell line studied, and salicylate type and dose

used, as well as timing of administration, all appear to be of importance. Aspirin,

mesalamine, ahd indomethacin, administered either during or after a heat shock, were al l

found to potentiate the heat shock response in various human cell lines (Amici et al., 1995;

Lee et al., 1995; Buress et al., 1997). Taken together, these results provide encouraging

evidence that various non-steroidal anti-inflammstory drugs (NSAIDs) can potentiate the

heat shock response in vitro. However, it is possible that in vivo, in the presence of

complex physiological systems, the effects of salicylate are quite different, and cannot be

compared to those observed in isolated cell systems.

While the studies discussed above have reported that the various salicylates enhance

the heat shock response, others have suggested that these drugs may only partially activate

the heat shock response (Jurivich et al., 1992; Jurivich et al., 1995). Jurivich et al. (1992)

found that sodium salicylate activated the heat shock fztor (HSF) to its DNA binding state

in HeLa cells. However, the increased activation of HSF did not lead to a subsequent

elevation in tripscription of the HSP 70 gene. In the present study, HSF activation was

not examined. However, Fawcett et al. (1997), in their work on the aspirin-mediated in

vivo potentiation of the heat shock response, did not observe any effect of the drug on

HSF:HSE binding. There is some evidence that salicylate activation of HSF inhibits

subsequent heat-induced HSP 70 gene transcription in both Drosophih and yeast

Winegarden et al., 1996; Giardina et al., 1995). However, both the study by Fawcett et

al. (1997), and the present study, did not fmd evidence indicating salicylate inhibits heat-

induced transcription of the HSP 70 gene.

In the present study, and in the study conducted by Fawcett et al., salicylate was

administered htraperitonedy. It is unknown whether orai administration could have

potentiated a heat shock response. I . man, plasma concentrations of aspirin rise rapidly,

and peak 20 min after oral ingestion (Rowland et al., 1972). Plasma salicylate levels reach

a peak approximately 1 h following ingestion (Rowland et al., 1972). Similarly, and as

mentioned previously, peak plasma salicylate levels were observed in the rat approximately

1 h post-oral ingestion (Higgs et al., 1987). It seems reasonable to assume that

intrapentoned administration of salicylate would accelerate the appearance of the drug in

the plasma. Consequently, it appears unlikely that oral administration of salicylate, one

hour prior to heat shock, would be favourable in potentiating the heat shock response,

when compared to intraperitoneal administration.

A possibility not explored in the present study, nor in the study by Fawcett et aI.

(1997), is that saiicylate requires a longer time period in vivo to affect the HSP 70 gene. A

rapid appearance of the drug in plasma might not necessarily lead to an immediate effect on

the HSP 70 gene. For example, it may be insightful to observe HSP 72 accumulation in

the myocardium following whole body heat shocks at 6, 12, and 24 h post-salicylate

administration. It is also unknown how chronic use of aspirin may affect the heat shock

response. There is evidence showing that distribution of salicylate in vivo is tissue-

dependent, with high concentrations occuring in the liver, and the kidney ( B m e , 1974;

B m e et d., 1976). Thus, salicylate may not accumulate preferentially in the myocardium.

In the present study, animals treated with sodium salicylate only showed a trend

towards a decreased left ventricular HSP 72 content compared to controls (figure 13). It is

unclear whether a similar trend existed in the tissues examined by Fawcett et al. (1997).

Visual inspection of their blots suggested reduced levels of HSP 70 and HSP 70 rnRNA in

the liver of rats breated with aspirin only, but a lack of quantification makes reasonable

observations difficult. It is tempting to speculate about possible explanations for the

decreased left ventricular HSP 72 content observed in the animals that were treated with

sodium salicylate. Sodium salicylate may have an indirect effect on the synthesis of HSP

72. The anti-proliferative prostaglandins PGA, and P G 4 have been implicated in the

induction of HSP 72 (Amici et al., 1992; Holbrook et al., 1992). It is possible that the

inhibition of these prostaglandins by salicylate could result in a decreased synthesis of the

protein. Recently, it was reported that following heat shock, a sharp increase in nitric

oxide (NO) precedes, and is necessary for HSP 70 accumdation in rat heart (Malyshev et

al., 1995). Interestingly, additional research has demonstrated that both aspirin and

sodium saticylate inhibit the production of NO in murine macrophage cell Lines (Kepka-

Lenhart et d., 1996). Furthermore, Farivar et al. (1996) reported that salicylate is a

transcriptional inhibitor of nitric oxide synthase in cardiac fibroblasts. This finding was

confirmed in murine macrophage cells (Amin et al., 1995). Thus, it is possible that even in

the absence of heat shock, attenuation of NO synthesis by salicylate may adversely affect

HSP 72 expression.

In mammals, an episode of whole body heat stress, and attendant accumulation of

HSP 70, has been shown to confer subsequent myocardial protection. This has been

demonstrated successfixlly by several researchers using various models (Cunie et al., 1988;

Karmazyn et al., 1990; Donnelly et al., 1992; Hutter et aI., 1994; Locke et al., 1995). In

the present study, it was of interest to determine a) whether in vivo administration of

sodium salicyIate could potentiate the heat shock response, and b) if so, would the

response be sufficient to confer myocardial protection following a mild heat shock of 40°C.

This question had not previously been examined.

Hearts from mildly heat-shocked plus sodium salicylate treated animals showed a

significantly improved recovery of the rates of contraction and relaxation compared to

control hearts;but only after 30 min of reperfusion (figure 8, A and B). However, hearts

fkom mildly heat-shocked plus sodium salicylate treated animals did not show a

significantly greater post-ischemic recovery of LVDP at any time during reperfusion

compared to their control counterparts (figure 9). Animals exposed to a 15 min heat shock

at 42"C, 24 h prior were conferred myocardial protection. The hearts from these animals

recovered a significantly greater percentage of their pre-ischemic left ventricular developed

pressure (LVDP), and the rate of contraction and relaxation (kdP-dt-'), than hearts from the

unstressed (control) animals. These results were in agreement with others reported

previously (Currie et al., 1988; Karmazyn et al., 1990; Locke et al., 1995; Lncke et al.,

1996). The hearts isolated from rats exposed to 15 min of 42OC hyperthermia, 24 h prior,

and subjected to complete global ischemia, showed an improved recovery of contractile

function, and reduced indices of reperfkion injury, as evidenced by a decreased creatine

kinase efflux (Currie et al., 1988). The hearts fiom rats exposed to whole body

hyperthermia of 42OC for 20 min, 24 h prior, showed a signif~cant reduction in infarct size,

and a greater degree of myocardial salvage (Domelly et al., 1992). Similarly, the stress of

3 consecutive days of treadmiU running has been shown to confer myocardial protection

equal to that provided by a single heat shock of 4Z0C for 15 min W k e et al., 1995).

Studies showing a significant myocardial recovery from an ischemic episode have

reported a significant elevation of myocardial HSP 70 follohhg a brief, but severe,

hyperthennic stress (Currie et al., 1988; Karmazyn et al., 1990; Domelly et al., 1992;

Hutter et al., 1994). Using two-dimensional gels, Currie et al. detected a 7 1 kD protein in

hearts from rak that were subjected to a heat shock of 42OC for 15 min, 24 h prior, and

subsequently conferred myocardial protection. The protein was undetectable in hearts from

unstressed animals (Currie et al., 1988). Karmazyn et al. (1990) subjected rats to a 42°C

heat shock for 15 min, and examined myocardial HSP 70 content 24, 48, 96, and 192 h

post-heat shock On a two-dimensional gel, a protein of 7 1 k D was easily detectable at the

four time intervals, but its intensity diminished in a time dependent manner, following a

peak at 24 h post-heat shock. Again, this protein was undetectable in hearts fkom

unstressed controls. Hutter et al. (1994) demonstrated a direct correlation between HSP 70

accumulation and myocardial protection by observing infarct size in hearts fkom animals

exposed to a heat shock of either 40-41, or 42OC. The hearts fiom animals heat-shocked to

41 or 42OC had a sigmflcantly higher HSP 72 content than hearts from the unstressed

controls, and conversely, showed a marked reduction in infarct size. In the present study,

the animals heat-shocked for 15 min at 42"C, and allowed to recover for 24 h, showed a

significant elevation in left ventricular HSP 72 content, compared to the unstressed

controls, and compared to animals that were mildly heat-shocked, with or without salicylate

treatment (figures 12A and 13). The left ventricular HSP 72 content in hearts fiom

severely heat-stressed animals (42OC for 15 min) was elevated approximately five-fold

relative to hearts fiom the unstressed animals (figures 12A and 13), and accordingly, the

hearts from the severely heat-stressed animals were conferred protection, in terms of LVDP

and 2dPd.t -'. These findings are in agreement with results of other studies in which HSP

72 content was quantified following heat shock. In rats heat-shocked for 20 min at 42"C,

24 h prior, an approximate four-fold increase in myocardial HSP 72, above unstressed

levels, was observed (Huner et al., 1994).

Previous studies have shown low levels of myocardial HSP 72 are insufficient to

confer myocardial protection (Domelly et d., 1992; Hutter et al., 1994; Locke et al.,

1995). The hearts of animals heat-shocked to 40°C for 15 or 20 min, 24 h prior, did not

show a significant elevation of HSP 72, and accordingly were not protected (Hutter et al.,

1994; Locke et al., 1995). Similarly, rats exposed to a 20 min ischemic pre-treatment

showed a modest elevation in HSP 72 content 24 h later, and were not conferred

myocardial protection (Do~e l ly et id., 1992). In the present study, a mild heat shock of

40°C for 15 min, with or without sodium salicylate treatment, did not sigmficantly increase

left ventricular HSP 72 content above the levels observed in the unstressed controls

(figures 12A and 13). Not surprisingly, these hearts were not conferred any significant

myocardial protection in terms of LVDP. Hearts from animals that were mildly heat-

shocked plus sodium salicylate treated were conferred myocardial protection in terms of

+dP-dt 'l, but only at 30 min of reperfusion.

In comparison to controls, the hearts fkoq the animals treated with sodium

salicylate only initially showed a decreased recovery of LVDP and +dP-dt -' during reperfusion than was observed subsequently (figures 7 & 8). It is tempting to speculate on

possible explanations for this trend, which although noticeable, was not statistically

signifcant. The impaired recovery of LVDP and &dP& -' observed in the hearts from the

sodium salicylate treated animals, at the onset of reperfusion, may be explained by a lower

HSP 72 content relative to the unstressed animals. However, such reasoning does not

account for the eventual improvement in myocardial function observed in these hearts

(figures 7 & 8).

organism 24 h

Some effects of aspirin are known to be

beyond administration. For instance,

long lasting,

it has been

and could affect the

shown that aspirin

irreversibly inactivates some enzymes, such as cyclo-oxygenase in platelets (Pedersen and

FitzGerald, 1984), and glutamate decarboxylase (Gould et al., 1965). In the former

scenario, platelet function is altered for severd days (Vane and Botting, 1992).

Furthermore, salicylate in rat plasma is thought to have a half-life of approximately 6h

(Higgs et al., 1987). Clearly, certain effects of salicylate could persist for 24 h or longer, L

or perhaps until washout by reperfusion in the isolated heart. In a model of local ischemia

using isolated-rabbit hearts, aspirin was shown to be proarrhythmic (a proarrhythmia is a

drug-aggravated, or drug-induced, cardiac arrhythmia) (Dhein et al., 1997). Thus, hearts

from animals given sodium salicylate may be susceptible to electrophysiological side

effects. Aspirin is an inhibitor of certain vasodilators, such as the prostaglandins PGE, and

PGD, (Feinrnan et al, ; Vane and Botting, 1992), and an antagonist of the powerfd

vasodilator, bradykinin (Vane and Botting, 1992). Initial constriction of critical heart

vessels during reperfusion may explain a slowed recovery of contractile function.

Winegarden et al. (1996) proposed that lowered cellular ATP levels were the result of

sodium salicylate intedering with oxidative respiration. A reduced energy state in the cell

could explain the impaired contractility at the start of reperfusion in the h e m from the

animals treated with sodium salicylate. Finally, the role of the salicylates as modifiers of

transcriptional activity in certain genes has been documented and discussed above. Recent

research has indicated that the constitutively expressed HSP 25 is located adjacent to the

myofiblils in the heart, and thus, is thought to play a part in contractile function (Hoch et

al., 1996). In an isolated rat heart model, the expression of heme oxygenase, an

antioxidant protein, was found to be stimulated by reperfision, a period of consequential

fke radical production (M-aulik et al., 1996). A salicylate-mediated impairment in the

synthesis of either of these two proteins could impair myocardial function. In summary, it

is possible that some of the aforementioned effects could be deleterious to the contractile

function of the heart, which may initially be compromised during reperfusion, until the

aspirin is washed out. The dysfunction observed during early reperfusion in the hearts of

the animals treated with sodium salicylate, and hypothesized to be a result of the drug, was

not observed in the hearts of the animals treated with sodium salicylate in combination with

mild heat shock. It is possible that a heat shock, given concurrently with salicylate

treatment, may have a stabilizing effect on myocardial function.

Conclusion C

Animals subjected to a heat shock of 42°C- as brief as 15 min, are conferred

myocardial protection, as evidenced by a significant post-ischemic recovery of both left

ventricular developed pressure, and rates of contraction and relaxation. This protective

effect appears'to be mediated, at least in part, by an increased expression of the heat

inducible isofom of the HSP 70 family, HSP 72. In the present study, administration of

sodium salicylate, either alone, or in combination with a mild heat shock of 40°C for 15

min, did not potentiate the induction of HSP 72 in vivo. The HSP 72 content in the h e m

from mildly heat-shocked animals, whether treated or untreated with sodium salicylate, was

similar to that observed in the hearts from unstressed controls. Furthermore, the animals

that were mildly heat-shocked plus sodium salicylate treated were not coderred myocardial

protection. This result was not surprising given the strong association between HSP 72

accumulation and myocardial protection. It is not clear whether past results obtained in

vitro, and showing a potentiation of the heat shock response by various salicylates, can be

reproduced in v i v a Prior to the present study, only one other group had attempted to

demonstrate that a salicylate could potentiate the heat shock response in vivo (Fawcett et

al., 1997). In the presence of aspirin, the heat-induced accumulation of HSP 72 was

enhanced; however, it was concluded that this was the result of a rise in core body

temperature, somehow caused by aspirin (Fawcett et al., 1997). Similarly, the present

study was unable to show that sodium salicylate potentiated the heat shock response in vivo

by some direct action on the HSP 72 gene. The actions of salicylate in the complex

environment of a multiceuular organism are not fully understood. More research will be

needed to fully elucidate the action of this drug in viva

I APPENDIX I I Selected temperature, and heat stress duration values for all animals subjected to heat shock

animal unstressed duration ave temp (heat ave temp (heat temp(OC) (-1 stress) ( T I shock) ("C)

. mhs 1 36.0 53.89 38.6 40.1 mhs2 36.8 43.47 39.0 40.1

I

mhs 5 37 -9 37.76 39.5 1 40.1 mhss 1 37.0 45.26 39.0 1 40.2 mhss 2 37.0 40.76 39.1 40.1 mhss 3 36.6 47.44 39.0 40.2 mhss 4 36.3 52.20 38.8 40.1 mhss 5 37.1 41.57 39.2 40.1 sb 1 37.0 59.6 1 40.2 42.1 shs 2 3 5 -4 68.64 39.8 42.1 shs 3 37.4 69.36 40.4 42.0 shs 4 37.0 64.37 40.2 42.1 shs 5 37.5 64.58 40.4 42.0

mhs: mildly heat shocked animal rnhss: mildly heat shocked and sodium salicyIate treated animal shs: severely heat shocked animal

APPENDIX I1

Absolute LVDP values (mmHG), for al l animals, at end of equilibration period (40 min), and thoughout reperhion

ss: sodium salicylate treated animal mhs: mildly heat shocked animal mhss: mildly heat shocked and sodium salicylate treated animal shs: severely heat shocked animal

20 min

33.1 35.0 23.8 32.9 63.4 18.0 31-5 21.8'- 28.0 29.3 34.2 32.3 51.0 51.2 58.3 48.1 49.9 68.2 35.8- 28.9 56.5 82.2 82.9 38.4 48.4

animal

control 1

10 min 32.5

25 min

32.5 43.3 19.4 46.6 64.0 27.4 36.7 45.9 33.0 32.0 43.6 51.7 57.8 41.2 62.4 50.1 49.1 74.8 41.4 38.2 62.8 82.2 82.9 44.7 56.6

control 2 control 3 control 4 control 5 ss 1 ss 2 ss 3 ss 4 ss 5 mhs 1 mhs 2 mhs 3 mhs4 mhs 5 mhss 1 mhss 2 mhss 3 mhss 4 mhss 5 shs 1 shs 2 shs 3 shs 4 shs 5

40 min equilibration

87 -7

15 min

32.2

30 rnin

31.1 47.1 17.1 59-5 63.3 30-1 40.4 45.0 37.7 34.6 57.5 61.5 59.0 40.9 62.8 49.3 50.6 79.7 45.6 43.8 62.8 82.2 82.9 52.8 67.2

5min reperfusion

14.7 84.2 84.5 81.1 91.1

34.4 25.1 31.5 62.2

34.1 23.9 31.5 36.9

12.2 26.4 15.5 22.6 25.8 29.3 28.8 37.9 39.8 53.9' 47.2 54.1 65.8 30.8 21.7 47.9 82.2 82.0 33.3 40.1

38.2 22.3 29.8 62.7 10.8 24.7 12.6 21.2 24.2 29.3 15.5 36.3 38.3 43.3 40.1 52.0 42.4 26.0 18.2 31.7 73.0 56.5 31.6 27.1

69.2' 67 -6 67.5 95.1 91.3 72.4 77.7 75.4 76.9 85.8 57 .O 67.2 8 1 .O' 77.5 56.7 62.8 82.2 82.9 55.1 87.9

13.8 28.5 14.3 25.7 21.5 26.3 22.7 35.5 29.5 26.4 33.7 22.3. 33.3' 23.7 17.8 8.5

18.9 20.5 21.4 21.9

APPENDIX III

Absolute K O W values (d-mid'), for all animals, at end of equilibration period (40 min), and thoughout reperfusion

animal 1 40 min 1 5 min I 1 0 I15 120 125 I30 equilibration reperfusion m h min 1 min min rnin

control 1 6.0 5 .4 5.6 5.6 1 5.8 5.6 5.4 I I I I 1 I I

control 2 I 5.6 1 3.11 3.11 3.11 3.11 2.91 2.9 I I 1 I I I I

control 3 I 4.8 1 2.01 1-61 1.81 2.01 1.81 1.3 I 1

control 4 6.6 3.3 2.3 3.1 3.3 3.5 3.5 control 5 8.1 6.6 6.0 6.8 6.4 6.0 6.0 ss 1 4.6 3.6 3.3 3.8 3.8 3.8 3.6 ss 2 5.2 3.1 3.1 3.6 3.6 4.0 3.8 ss 3 4.4 3.6 2.4 2.3 3.3 2.5 2.3

I

ss 5 5.6 2.5 2.3 2.3 2.4 2.5 2.5 mhs1 4.4 2.9 3.3 3.8 4.0 4.0 3.8 mhs 2 5.6 5.2 5.6 5.4 5.2 4.8 4.8 mhs 3 4.8 4.0 3.8 4.2 4.2 4.0 3.6 mhs4 5.6 3.3 3 . 1 3.6 4.0 4.0 4.2 mhs5 10.4 5.8 4.6 4.8 5.2 5.2 5.2 mhss 1 I 4.6 2.9 2.9 3.3 3.3 3.3 3.1 mhss 2 1 9.9 5.2 5.6 6.8 7.0 6.8 6.8 . I

mhss 3 5.2 4.0' 4.0 5.0 5.2 5.2 4.8 mhss 4 6.4 3.8 3.6 3.6 3.6 3.6 3.5 mhss 5 3.5 1.3 1.2 1.4 1.5 1.5 1.4 sfis 1 7.6 6.8 6.4 6.8 6.8 6.4 6.4 shs 2 4.8 4.8 1 4.8 4.8 4.8 4.8 4.8

k

shs 3 5.6 4.8 4.6 5.0 5.2- 5.2 5.2 shs 4 5 -6 3.3 3.3 3.6 3.6 3.6 3.6

I I I 1 - -

I I

shs 5 1 5.6 1 3.31 3.11 3.31 3.31 3.51 3.5

b.

ss: sodium salicylate treated animal mhs: mildly heat shocked animal mhss: mildly heat shocked and sodium salicylate treated animal shs: severely heat shocked animal

I APPENDIX IV

Absolute +dP-dr -' values (mmHG-s-I), for all animals, at end of equilibration period (40 min), and thoughout reperfusion

ss: sodium salicylate treated animal mhs: mildly heat shocked animal mhss: mildly heat shocked and sodium salicylate treated animal shs: severely heat shocked animal

animal

control 1 control 2 control 3 control 4 control 5 ss 1 ss 2 ss 3 ss 4 ss 5 mhs I

40 min. equilibration

1700.2 1842.1 1857.6 1823.5 2018.3 16 16.5 1500.7 1543.4

5 min reperhion

270.8 721.8 503.9 572.7 760.6 255.6 603.3 263.5

285.5 684.4 804-1 875-8 842.1

1040.0 920.3 558.8 361.1

480.8 676.9 601.2 536.0 676.7 482.3 656.3 506.0 308.4 166.1 601.0 399.4 398.8 423.5

mhs2 mhs 3 mhs4 mhs5 mhss 1 mhss 2 mhss 3 mhss 4 mhss 5 shs 1 shs 2 shs 3 shs 4 s h 5

10 min

579.3 714.7 503.9 610.4 317.9 240.6 527.9 233.4

2165.2 2069.8 1549.7

- 1803.0 1789.9 1750.9 1940.3 1263.1 1386.5 18 10.4 1706.7 1324.0 1449.5 1795.5 1861.2 1 173.9 1905.7

401-2 483-4 586.8

548.4 714.5 826.6

1072.1 962.4

1047.4 1259.8 611.7 466.4

514.8 415.4 51 1.6

15 min

571.7 654.1 518.9 617.9

1302.9 278.2 520.4 278.6

634.2 1563.0 1168.0 647.1 468.9

25 min 20 min

594.3 646.6 556.5 617.9 1333

383-5 610.9 399.0

454.2 ' 559.0

1322.2 1256.C 796.5

1283.4 1045.1 949.4

1719.9 966.6

1008.1

586.0 1000.3 901.8

1155.1 1037.6 934.4

1463.4 725.0 586.8

1019.2 1795.5 1725.6 692.3 771.3

30 min

560.2 589.2

1449.5 1795.5 1861.2 1106.2

651.0 679.8

1051.8 1158.2 841.6

1291.0 1030.1 91 1.8

1606.8 883.6 850.1

1230.6 1795.5 1861.2 767.5

601.8

726.8 702.5

1419.3 1795.5 1861.2 903.0

594.3 87-9.7 473.8 926.8

1355.6 586.5 731.5 948.7

960.4

579.2 962-4 398.f

1205.f 1363.1 669.2 852.2 963.7

1173.6 677.1

1141.9

872.6

1436.8

APPENDIX V

Absolute -@I& '' values (mmHGd), for a l l animals, at end of equilibration period (40 min), and thoughout reperfusion I

animal l40rnin 15min I 1Ominl 15 min i2Omin 125 min equilibration reperhion

control 1 1324.0 233.2 473.9 526-6 541.6 534.1

ss: sodium salicylate treated animal mhs: mildly heat shocked animal mhss: mildly heat shocked and sodium salicylate treated animal shs: severely heat shocked animal

APPENDIX VI

Determination of sodium salicylate dosage

Myocardial HSP 72 content 24 hours after treatment with I S min heat shock (hs) plus sodium salicylate. Total protein content from left ventricle was separated by SDS-PAGE, transferred to nitrocellulose, and reacted with HSP 72-specific antibody. Shown is a western blot. Lane 1, control (unstressed); lane 2, mild hs (40°C); lane 3, severe hs (42°C); lanes 4 & 5, mild hs (40°C) plus 200 mg+kgl sodium salicylate; lanes 6 & 7, mild hs (40°C) plus 300 mg.kg1sodiurn salicylate; lanes 8 & 9, mild h: (40°C) plus 400 mg-kg1 sodium salicylate; lane 10, mild hs (40°C) plus 100 mg-kg sodium salicylate. When compared to lane 3 (severe heat shock), only lanes 8 & 9 show a similar elevation in HSP 72 content. Therefore, on the basis of this preliminary data, a sodium salicylate dosage of 400 mg-kglwas chosen for hrther study.

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