Ontogeny of Myocardial Excitation- Contraction Coupling · exclusive licence diowing the National...
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Ontogeny of Myocardial Excitation- Contraction Coupling Liqun Xu M.D., Nanjing Medical University, 1986 Thesis Submitted in Partial Fulfillrnent of the Requirements for the Degree of Master of Science in the School of Kinesiology O Liqun Xu 1999 SIMON FRASER UNIVERSITY March 1999 Al1 rights reserved. This work may not be reproduced in whole or in part, by photocopy or other means, without the permission of the author.
Transcript of Ontogeny of Myocardial Excitation- Contraction Coupling · exclusive licence diowing the National...
Ontogeny of Myocardial Excitation-
Contraction Coupling
Liqun Xu M.D., Nanjing Medical University, 1986
Thesis Submitted in Partial Fulfillrnent of the Requirements for the
Degree of Master of Science
in the School of
Kinesiology
O Liqun Xu 1999 SIMON FRASER UNIVERSITY
March 1999
Al1 rights reserved. This work may not be reproduced in whole or in part, by photocopy
or other means, without the permission of the author.
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Abstract
Excitation-contraction coupling of the neonatal heart differs from that of the adult which
is due in part to the lack of transverse (T) tubules and a sarcoplasmic reticulum (SR)
which is both sparse and less functional. We used caz'-dependent Ca" inactivation
kinetics of the sarcolemrnal L-type Ca2+ channel (DHPR) to probe the spatial relationship
behveen the DHPR and SR ca2+ release channel (RyR) dunng ontogeny. Single cardiac
myocytes from four age groups of rabbits (3, 6, 10, and 20 days old) were voltage
clamped in perforated patch configuration using Amphotericin B. We found that: 1) the
whole ce11 L-type ~ a ' + channel peak current density increased in parallel with the ce11
growth. but the voltage/current relationship was not changed by age; 2) L-type ca2+
channel ~a'+-dependent inactivation was significantly faster in 2Od group (p<0.002); 3)
this ~a"-dependent inactivation kinetics (r) were signi ficantl y increased (28%) by 5 mM
caffeine induced depletion of SR cal' in 2Od group @<0.0001); 4) the total releasable SR
~ a " content per ce11 increased significantly as a function of age. Our results suggest that
the number of functional DHPR increases with ontogeny, and that the spatial relationship
between the DHPR and the RyR is altered between the second and the third week of age,
allowing for "cross-talk" between these charnels.
Dedication
To my parents
Acknowledgements
1 am extremely grateful for the continuing guidance and support of my committee and
especially my supervisor, Glen Tibbits. 1 believe 1 was quite fortunate to work with such
an interested and motivated individual. He always gave me the freedom to develop my
own ideas. Working with him is both a privilege and a challenge. His insight,
enthusiasm, generous donation of time and financial support went into the work
presented here. 1 would like to thank Kenneth Spitzer who generously donated his lab
and led me into the door of the electrophysiological experiments, and Leif Hove-Medson
for his enthusiasm, tirne and patience in teaching me patch-clamping technique from
scratch. 1 thank Kerry Delaney and Eric Accili for their very helpful suggestions and
discussions about this study.
The support from al1 members of the lab has been most important for the successful
completion of this thesis. 1 am especially indebted to Haruyo Kashihara for not only her
technical assistance but also her caring throughout my time in this lab. 1 acknowledge al1
my colleagues in CMRL for helping out in many different capacities.
My sincere appreciation goes to my parents for their encouragement and my special
thanks to my family for putting up with my somewhat insane working hours. Many
thanks to Jingbo Huang who always keeps my spirit up.
1 am also indebted to the BC & Yukon Heart and Stroke Foundation for supporting theses
experiments.
Contents
. . Approval .......................................................................................................................... II
... Absrract ........................................................................................................................... iii
Dedication ....................................................................................................................... iv
........................................................................................................... Ac knowledgements v
Contents .......................................................................................................................... vi
List of Tables ................................................................................................................... x
List of Figures ................................................................................................................. xi
........................................................................................................... Introduction 1
Studies of mature myocardium ............................................................................ -4
2.1 Morphological studies of adult ventncular myocytes ............................... 4
................ 2.2 Major cellular elements reIated to E-C coupling in adult heart 5
......................................... 2.3 Excitation-Contraction coupling in adult heart 6
2.4 Cross-signaling of DHPR and RyR in adult myocytes ........................... 1 1
............................ 2.5 Electrophysiological studies of L-type caZf channels 12 t+ 2.5.1 Sarcolernmal Ca channels ........................................................ 12
2.5.2 Modulation of L-type Ica by cyclic nucleotides and
phosphorylation ......................................................................... 13
2.5.3 ~ a " dependent L-type ~ a " channel inactivation ....................... 14
3 Studies of the immature myocardium ..... .. .............................. ... ...................... 15
....................................... 3.1 MorphologicaI studies of the developing heart 15
3.1.1 Cell size ....................................................................................... 15
3.1 -2 Ce11 surface to volume ratio ..................................................... 16
3.1 -3 T-tubules and SR ......................................................................... 17
3.1.4 Myofibnls and mitochondria .................................................... . 18
3.1.5 Blood supply ............................................................................... 19
3.1 -6 Summary .................................................................................... 1 9
3.2 Biochemical studies of the developing heart .......................................... 22
3.2.1 DHPR .......................................................................................... 22 2+ 3.2.2 RyR and SR Ca ATPase ........................................................... 22
2+ 3 .2.3 ~a'/Ca exc hange ......................................... .... ................. -24
3 -3 ElectrophysiologicaI and ph ysiological studies of the developing heart 25
3.3.1 Resting membrane potential ....................................................... 25
3.3.2 Action potential ........................................................................... 26 2+ .................................................. ............... 3.3.3 L-typeCa channels ., 26
3 .3.4 Sarcoplasmic reticulum .............................................................. 28
3 -3 -5 ~ a ' / ~ a ' + exchange .................................................................... -30
....... 3.3.6 Cross-signaling of DHPR and RyR in the developing heart 32
................. 3 .3.7 Contractile force and [ca2'li of the developing heart 32
..................................................................................... 3 .3.8 Summary 34
7 Discussion ........................................................................................................... 82
........................................................................... 7.1 Perforated patch clamp 82
................................................... 7.2 Measurementofmyocytesurfacearea 83
.......................................................... 7.3 Tram-sarcolernmal ~ a " channels 85
............................................ 7.4 L-type ca2+ current and the curent density 85
.................................................................. .... 7.4.1 DHPR expression .. 88 2+ ................................................ 7.4.2 Functional L-type Ca channels 89
......................................... 7.4.3 Possible regulatory factor(s) changes 90
................................... 7.5 Voltage dependence of L-type ~ a ' + current .... . . 1 '+ ............................................................ 7.6 L-type Ca chamel inactivation 91
2+ 7.7 SR Ca content .................................................................... 97
....................................................................................................... 8 Conclusions 100
9 Bibliography ..................................................................................................... 103
List of Tables
Tabte 1 Summary of morphological changes o f neonatal myocytes .... .. .............. 20
Table 2 Protocols for ce11 isolation ....................................................................... 39
Table 3 Cornparison of extemal and intemal solutions .............. .. ....................... -60
Table 4 Summary o f developmental changes of cardiac myocytes ...................... 73
Table 5 Estimation of SR releasable ~ a " of four age groups ............................. 99
List of Figures
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Figure 1 1
Figure 12
................ Isolated single lefi ventricular rnyoçyte from an adult rat heart 5
............................ Schema of E-C coupling in the adult mammalian heart 7
"Fuzzy space" ........................................................................................ 9
Isolated single left ventricular myocyte from a
neonatal rabbit heart ................................................................................. 16
Specific DHP and ryanodine receptor binding and SERCA 2a
enzyme activity in the developing rabbit heart ................................. .. ..... 23
.............. Voltage protocol and capacitive current during the perforation 46
Voltage protocols for cornparing the efFect of SR ca2+ on
L-type Ica current inactivation ................................................................. 48
1 measurement ................................................................................. 51 cap
Voltage protocol for Ica 1-V relation and INa inactivation ........................ 54
Curve fitting of L-type Ica inactivation .......................... .... ............. 56
~ntegration of for SR caZt content measurement ............................. 58
Developmental change of capacitive currents ........................... ..... ......... 62
Figure 13
Figure 14
Figure 15
Figure 16
Figure 17
Figure 18
Figure 19
Figure 20
Figure 2 1
Figure 22
Figure 23
Figure 24
Figure 25
Developrnental change of C , ................................................................... 63
.... ........................................ Developmental change of the body weight .. 64
Voltage dependence of L-type Ic , ............................................... .. 66
................................................................................... Cadmium block Ic , 67
.................................... L-type b of the 3- and 6-days cardiac myocytes 6 9
................................ L-type Ica of the 10- and 20-days cardiac myocytes 70
L-type Ic, Current-voltage relation of four age groups ............................ 72
L-type Ica density of four age groups ............................................. 7 3
L-type Ica activation and inactivation ................................................... 75
................................................ CafTeine effect on L-type Ica inactivation 76
........................................... Caffeine effect on n of different age groups 78
............................................... Sampte recordings of L-type Ica and IN^^ 80
~ a + charge transported via NCX during caffeine application ................. 81
1. Introduction
During mamrnalian hem development the morphology, metabolism and mechanisms of
cardiac myocyte calcium regulation undergo significant changes [ 1 1,121, [3]. Because of
these changes, the immature heart has distinctive responses to various perturbations such
as pharmacological interventions and ischemidreperfusion injury when compared with
the mature heart. It is these unique responses that have made myocardial preservation a
challenging task for pediatric cardiac surgeons.
About 40% of children bom with congenital hem defect(s) die within the f in t year of
their lives unless treated surgically. Early correction of these defects is probably the only
way to increase survival into adulthood [4]. Today hypothermic cardioplegic arrest is
performed during pediatric cardiac surgery al1 over the world. This procedure is
necessary to allow a clear operative field during surgery and to ensure accurate correction.
However. this procedure also results in obligatory ischemia/reperfbsion injury during the
surgery. The ischemidreperfusion injury proves problematic for pediatric cardiac
surgery.
Many cl inical observations show that the overall mortality of pediatric open-hem surgery
is at least two folds higher than chat of adult surgery. Many experimental studies and
clinical observations find that this rate of surgical success is related to the age of the
patient [5] and the surgical protocols used. Most surgical protocols for children are
adapted from the adult cardiac surgery. This includes the composition of cardiaplegic
soiution. Special attention is paid to the concentrations of the ionized calcium and
magnesium, the control of temperature and the application dose of the cardiaplegic
solution 161, [7], [8]. These clinical settings are associated closely with our knowledge of
physiology, electrophysiology and biochemistry of the developing heart, which has also
been consistently improving over years. Decades of effort by numerous researchers and
pediatric surgeons have decreased the mortality rate of the open-heart surgery in
pediatrics. Mortality has been lowered from 57% to 17% in neonates (< 30 &y), fiom
25% to 4.8% in infants (1- to 12-month-old) and fiom 8.26% to 5.9% in children (1- to
12-year-old) [9 ] , [ 1 O], [4], [ 1 11, [ 121. However, the overall mortality rate for neonates
remains higher than that for adults (3%) [13].
In pediatric open-heart surgery, the most common cause of the death is acute
postoperative heart failure. The ventrïcular dystùnction results in the sudden
hemodynamic instability [14]. Although the aduh surgical protocols are effective in adult
myocardium preservation and pst-operative functional recovery, these protocols cannot
provide equally efficient myocardial protection in pediatric cardiac surgery. The
remaining questions are: 1 ) what are the mechanisms for better immature myocardial
preservation? 2) to what extent should pediatric surgeons adapt adult protocols when
they repair the congenital defects in immature hearts? In order to achieve the maximal
post-operative fûnctionaI recovery of immature hearts and to lower the rnortality and
morbidity of pediatric cardiac surgery more experimental and clinical research needs to be
done. The answers to these questions may lie in the structural and functional differences
between the developing heart and the mature hean. This is the basis for my studies.
2. Studies of mature
2.1 Morphological studies of adult ventricuar myocytes
Adul t mammalian ventricular myocytes tiom di fferen t species, inc luding human, s heep,
rabbit, rat, dog, cat, guinea pig and pig, share many common features. The single healthy
ventricular myocyte is quiescent, rectangular in shape and striated (Fig 1). Usually, the
ce11 has intercalated discs. It is about 100-1 50 p in length, 20 pm in width, and less
than 20 jm thick. The ceIl plasma membrane, sarcolemma (SL), invaginates into the ce11
at the position of the 2-disk to become transverse tubule (T-tubule) around myofibrils.
Within the myocyte, a closed intracellular membrane network (sarcoplasmic reticulum,
SR) around myofilamemts cornes ciose to T-tubules to form dyads. Large numbers of
myofibrils longitudinally distribute throughout the cell. These myofibrils occupy about
50-79% of the ce11 volume, depending on the species [ I S J , [16]. This regular array of
myofilaments gives the ventricular myocyte a striking striated appearance. The majority
of rnitochondria are arranged in rows adjacent to the sarcomeres.
Fig 1. Isolated single left ventricular myocyte fiom an adult rat heart.
2.2 Major cellular elemnts releted to €4 coupling in adult heert
Ultra-structural studies show that T-tubules and the terminal cisternae of SR corne close
to each other to form dyads. Dyads constitute two types of well-organized calcium
channels. One is the voltage dependent L-type ~ a ' + channel (also referred to as the
dihydropyridine receptor, DHPR) located in T tubules, an other is the SR ca2' release
channel found on SR membrane (also referred to as ryanodine receptor, RyR) [17].
Dyads are also observed between external sarcolemma (excluding T-tubules) and SR.
The number of the SL-SR dyads is much fewer than that found between T-tubules and
SR [18]. In contrast to skeletal muscle cells, there is no evidence of direct physical
connection between these two ~ a " channels in cardiac myocytes [19]. However, the
close spatial association of these two ~ a " channels is intimately reiated to their function.
This will be described in more detail later. The other ion transport devices in SL are
vo 1 tage-gated Na+ c bannel, ~ a ' / ~ a " exc hanger (NCX), SL ~ a ' + - A T P ~ S ~ (also named as
SL ~ a " - ~ u r n ~ ) and K* channels. in the SR membrane there is a large amount of SR
c ~ " - A T P ~ s ~ , also called SERCA 2a.
The force generation sites are myofilarnents. There are two types. They are the thin and
thick myofilaments. The thin and thick filaments are arranged in a repeating pattern
along the length of myofibrils. The thin myofilament is composed of at least five types of
proteins, including actin, tropomyosin, troponin C, troponin I and troponin T. The thick
myofilament is composed of protein myosin.
2.3 Excitation-contraction coupling in adult kar t
In mature marnmalian cardiac muscle, excitation-contraction coupling is generally
believed to be acting in a calcium-induced calcium reiease (CICR) fashion, which has
been characterized by rnany researchers. CICR has k e n shown both in skimed and
intact cardiac myocytes by Fabiato [20]; [2 11. Two separate groups, Morad et al. (1 990)
and Lederer et al. (1990) have demonstrated CICR via flash photolysis of caged cal+ in
the intact cardiac myocytes 1221, [23]. These accumulated data show that it is the
cytosolic free ~ a " ions rather than the trans-membrane voltage change that play a key
role in the release of ~ a " from SR. in contrast to cardiac muscle, a large body of
evidence shows that it is the trans-sarcolemmal charge movernent that gates ~ a " release
from the SR in skeletal muscle cells 1241, [Zj].
Fig 2. Schema of E-C coupling in the adult mammalian heart [taken from Bers [15]]
Myocyte excitation starts with sarcolemmal depolarization that propagates along the T-
tubules into the interior of the ce11 to produce a small amount of ~ a " influx,
predorninatel y via voltagedependent L-type Ca2+ channels. This "trigger" ca2+ gates SR
~ a " release charnels to release a large amount of ~ a " from the SR. This is the weil-
known CICR phenomenon [20]. Both source of ~ a " , the trigger and the SR released
~ a " , cause an intracellular calcium ([ca2']i) transient [26], [27] (Fig 2) . When cytosolic
frer caL' concentration ( [~a"]~) increases, the calcium ions combine with troponin C
resulting in a conformational change of the thin filament. Subsequently, the thin and
thick myofilaments slide toward each other to generate a force inside the ce11 that results
in ceil contraction. Morad et al. (1995) quantified the eficacy of ~ a " influx either
through L-type ~ a " channels or via hJa*/ca2+ exchange (NCX) in uiggering SR ~ a "
release in adult rat heart. They found that the ~ a " channels were 20-160 times more
effective than the exchange [28] in rat ventncular cells [29]. Relaxation is initiated by
removal of ~ a " fiom the cytosol to the extracellular fluid via NCX and by
resequestrating Ca'+ into the SR via SERCA 2a [15]. As [ca2tJi declines to its resting
Ievel, the ce11 relaxation starts.
An alternative mechanism of CICR has been proposed. Several research groups
conducted experiments to explore the role of NCX in triggenng SR ~ a ' + release in
mamrnalian hearts [30], [3 11, (321. Using ventricular myocytes of adult guinea pig,
Leblanc and Hume (1990) demonstrated that in the absence of L-type ~ a " current,
elicitation of SR ~ a " release by depolarizing the ce11 membrane depended not only on
the ~ a ' entering through the SL ~ a ' channel but also on the extracellular Ca"
concentration [31]. Vites et al. (1996) present further evidence that it is the fast sodium
influx that triggen adult cat ventricular myocyte contraction when L-type ~ a " channels
are pharmacologically blocked by 10 FM nifedipine [30]. These studies irnply that CICR
may also be explained as a rapid ~ a ' influx through ~ a ' channels during the membrane
depolarization, which increases local Na' concentration in a restricted intracellular space
(SO callrd "fùzzy space") causing NCX to operate in reverse mode. That is, Ca" c m be
brought into the ce11 by this reverse mode ~ a + / c a ' + exchange to provide the "trigger"~a2+
to induce SR Ca" release (Fig 3). However, Sham et al. (1992) did not find similar
results in the adult rat ventricular myocytes and fûrther proved that only the trans-
membrane calcium current of the L-type Ca" channels induced SR ~ a " release under
9
physiological conditions [32]. The discrepancy between these results may be due to
species differences 1331. The physiological significance of ~ a ' / ~ a " exchanger-gated SR
~ a " release remains controversial in the adult mammalian hearts.
DHPR
Fig 3. The "Fuzzy space". Adapted fiom Lederer [34].
An interesting study by Santana et al. (1998) presents a new perspective of CICR [35].
They reponed that ~ a ' charnels could switch the ion selectivity to favor ~ a ' + influx into
the adult rat heart cells. They named this phenornenon as a slip-mode conductance of the
tetrodotoxin-sensitive Na' channel. The amount of ~ a ' + influx was great enough to
activate RyR to release SR ~ a " , which was observed as a 'Ca'+ spark". Howewr, the
conditions that allowed the slip-mode were either the activation of the P-adrenergic
receptor, or the protein kinase A, or the presence of nM concentrations of cardiotonic
steroids such as ouabain and digoxin [35].
Even though CICR is widely accepted as the predominant mechanism for ~ a " delivery in
excitation-contraction in the mammalian hem, the possibility of a contribution from a
voltage-gated Ca" release (VACR) mechanism cannot yet be excluded. Levi et al.
(1997) found that the SR ~ a " release could be elicited by depolarizing adult rabbit, rat
and guinea pig cardiac myocytes when the tram-membrane ca2+ currents were blocked by
5 rnM ~ i " . It should be noted that these experiments were conducted under specific
conditions 37OC and high concentration of 3', 5'-cyclic adenosine monophosphate
(CAMP, 50-100 FM). The magnitude of the VACR ïncreased proportionally with the
concentration of CAMP dialyzed into the ceIl through the pipette 1361.
In intact cardiac myocytes, intracellular calcium is tightly regulated on a beat-CO-beat
basis, no matter what kind of strategy the myocytes depend on to accomplish their
contraction and relaxation. This means that within each ka t , the amount of ~ a " going
into the cytoplasm during excitation must be removed from the cytoplasm to allow for
relaxation. Therefore. at the end of each beat, the intracellular Ca2' is kept in a steady
state by a combination of the efforts of the cellular ~ a " regdatory proteins.
2.4 Cross-signaling of OHPR and RyR in aduH myocjttes
The macroscopic behavior of CICR depends not only on the kinetics of sarcolemrnal and
SR ~ a " transporting proteins, but also on the spatial relationships arnong these proteins.
Especially, the distance between sarcolernma! L-type ~ a " chamel and the SR ~ a ' +
release channel is crucial in these spatial relationships [37]. In some species, the distance
between sarcolernmal ~ a + / ~ a ' + exchange and the SR Ca2+ release channel may be critical
for CICR [38], [34]. It is because the local [ca2']i has to be high enough to be the
intracellular signal to activate SR ~ a " release channels and to induce suficient SR ~ a "
release in order to induce ce11 contraction [39]. When Ca" ions enter the ce11 via L-type
ca2' channels during sarcolernmal depolarization, the local free ~ a " concentration
around the L-type ~ a " channel pores reach a much higher level than the global cytosolic
~ a " concentration within a restricted distance. A mathematical mode1 shows that the
local [ca2']i around the L-type Ca'+ channel pores drops dramatically within a couple of
micro-seconds and that the local [ca2?i decays rapidly with the distance from the ~ a "
chamel pores since the entering Ca" ions diffuse away from the channel pores with time
[27]. From indirect experimental evidence, Simon et al. (1985) has postulated that the
sarcolemmal L-type ~ a " channels are within about 10 to 20 nm of the SR caL' release
channels [37]. The space between these two ~a"channe1s has been referred to as the
''fuzzy space" (Fig 3) because of its unknown molecular anatomy. However, it is
generaliy believed that this space is a functionally restricted intracellular space that gives
the L-type ~ a " channel and maybe in some species the ~ a ' / ~ a " exchange a privileged
access to the calcium-sensing site of the SR ~ a " release channels [34]. Within this
restric ted space, ~ a " influx through L-type ~ a ' + channels signals SR ~ a ' + release
charnels to open and release SR ~ a " . This process is referred to as a "forward taik"
between these two types of ~ a " channels. On the other hand, the released SR ca2+ that
produces a very steep [~a"]i gradient in the restricted space can generate retrograde
signals that may modify the L-type ~ a " charnel inactivation kinetics. This process is
referred to as a "back talk" between these two ~ a " channels [40], [27]. The latter is also
known as the ca2+-dependent ca2+ channel inactivation. Sham et al. (1992) observed
both foms of talks, the "cross talk", between DHPR and RyR in adult rat cardiac cells,
but they did not have a similar observation between NCX and RyR in these cells [32].
2-5 EIectrophysiological studies of L-type CaZ* channels
2-5.1 Sarcolemmal& channels
There are two distinct types of Ca" channels in the sarcolemma of cardiac myocytes.
One is a 1,4-di hydropyridine @HP) sensitive and voltage-dependent ~ a " c hamei, whic h
is also known as the L-type ~ a " charnel. The L-type Ca" channels are characterized by
a high threshold for activation (membrane potential, E,, positive to -30 mV), large
conductance, and slow inactivation. The second type is a transient and DHP-insensitive
~ a " channel. This is also known as the T-type Ca" channel. The T-type Ca" channels
are characterized by a low threshold for activation (E, positive to 4 0 mV), small
conductance, and faster inactivation kinetics. The T-type ~ a " currents are observed
prominently in canine atrial and canine heart Purkinje cells [41], [42]. They aIso appear
in some aduit ventricular myocytes of guinea pig and rabbit heans as well 1431, [44]. The
physiological significance of the T-type Ica is not well understood. In contrast to the T-
type IC=, the L - t y p Ica is prevalent in ventricular myocytes in adult hearts [Ml , [29] and it
is also the major pathway of ~ a ' + influx to initiate CICR in adult cardiac myocytes. The
time and voltage dependence of L-type ca2' current inactivation cm be measured using
the whole-ce11 voltage clamp technique. In adult mammalian cardiac myocytes, the
maximum peak Ica is in the range of 1-2 nA (equivalent to a peak Ica density of 10-20
pNpF) and is reached around clamp potentials of O to + 1 O mV [45], [46].
2.5.2 Modulation of L-type b by cyclk nucleotides and phosphorylation
L-type ~ a " current is regulated by multi-factors or pathways and the magnitude of the Ica
is the result of the overall balance of those factors. A well-established and important
pathway is the P-adrenergic receptor - GTP binding protein (G,) - adenylate cyciase
system [47], [ 1 51, [48]. B-adrenergic agonists binding to P-adrenergic receptors activate a
G protein (Gs) which in turn activates adenyiate cyclase to produce cyclic AMP. Cyclic
AMP increases Ica density by increasing L-type ~ a " channel open probability dunng ce11
membrane depolarization via c AMP-dependent protein kinase A-induced L-type ~ a ' +
channel phosphorylation. L-type ~ a " channels may also be regulated directly by Gs
protein a subunit (G,). Within this receptor-Cis protein-CAMP system, the intracellular
CAMP level is also influenced by phosphodiesterase (PDE) which can accelerate CAMP
degradation. The protein kinase A activity which is directly associated with the ~ a "
channel phosphorylation can be inhibited by sub-micrornoiar [ ~ â " ] i . Furthermore, the
level of phosphorylation of L-type ~ a ' + channels can be altered by phosphatases that
dephosphorylate L-type ~ a ' + channels [49].
2.53 cfl-dependent L-type channel inactiv8tion
L-type cal' channels inactivate in response not only to the voltage but also to the
intracellular ~ a ' + transient which serves as an important negative feedback regulation
allowing for fine control of during E-C coupling [29], [SOI, [SI]. Single channel
studies suggest that the L-type ~ a " channel inactivation is achieved by reduction of L-
type ~ a " channel open probability as a result of the reduction of the channel open
frequency and charnel open tirne [52], [50]. ~ a " binding directly to an EF hand motif on
the ai sub-unit has been proposed for the ca2+-dependent L-type ~ a " channel
inactivation [50], [53], [54]. However, Bematchez et al. (1998) showed evidence against
this hypothesis [SS].
3. Studies of the immature myocardium
The immature heart faces many challenges afier birth, as it loses the placental circulation
and closes the foramen ovale and the ductus arteriosus. These changes accompanying
rapid animal growth after birth result in a constant increased volume and work load on the
newborn's heart.
3.1 Morpholog~cal studks of the developing heaH
3.1.1 CeIl size
In a mammalian hean, such as rabbit, dog, or cat, the neonatal cardiac myocyte is small in
size. In general, it is about 40-70 pm long and 3-8 pm in diameter. It has a smooth
cylindrical shape with tapered ends, and does not have intercalated discs in the first few
days. From the middle of the first week, more and more lefi ventricular myocytes appear
to have blunt ends and short intercalated discs along their borders (Fig 4).
Fig 4. Isolated single left ventricular myocytes from a neonatal rabbit hean (12-day-old).
3.1.2 CeIl surface to volume ratio
Immature mammalian cardiac myocytes are characterized by a high ce11 surface to volume
ratio (SN), lack of transverse (T) tubular system, and underdeveloped T-tubular SR
network in different species, including rat, rabbit, cat and canine [3], [56], [2], [l], [27],
[18]. In the rabbit heart, the S N is 1.2 pm-' in the 18 days pst-coitum fetal myocytes
(full term is 3 1 days) and it drops stightly to 1 .O 1 - 1 -05 pm-' at 24 days post-coitum and
remains stable up to 8 days post-parnim. Between 8 days and up to 3 months after birth,
the S N ratio declines to the adult level of 0.43 pf'. The surface area includes the T-
tubules. The fetal S N ratio is comparable to the arnphibian cardiac myocyte S N ratio,
which is 1.32 pm-' in frog [56]. The long and narrow shape of the young cardiac
myocytes contributes to the high surface-to-volume ratio of these cells, which favors the
extracelhlar ions to d i f i se into the cells. The decrease of this ratio with age may be due
to rapid ce11 growth in three dimensions, especially the increase of ce11 diameter. While
ce11 length increases 30% from day 8 after birth to mature, the ce11 diameter increases 2-3-
fold during this pet id [56].
3.1.3 T-tubules and SR
T-tubules are not observed until 8- 10 days of age, and it takes more than 30 days afier
birth to complete T-tubular maturation [57] in rabbit heart. The percentages of the
sarcolemma in T-tubules are 42% and 33% in adult rabbit and rat heart cell, respectively
[15]. However, the growing T-tubules are not suficient to counterbalance the declining
surface-to-volume ratio in mature myocytes. A rudimentary network of SR with
peripheral couplings at the sarcolemma was observed in neonatal riearts [2]. In rabbit
ventncular myocytes, the volume and the surface area of SR increase continually as the
ce11 grows and the volume fraction of SR increases 2-fold fkom day 1 to &y 14. From
day 13 on the SR volume of the ce11 is rnaintained at a constant level till adulthood [56].
Within the f int two weeks after birth, the SR surface area per myofibrillar volume
increases almost 2 to 3-fold in rabbit and rat myocytes [56], [58]. Using morphometrical
measurements of electron micrographs, Page et al. (1981) found that the total membrane
area (the area of SR in proximity with the SL) of the T-tubular-SR dyads was 4 to 6-fold
grrater than that of the extemal SL-SR dyads in adult rabbit and rat ventncular myocytes.
They also found that the external SL-SR dyadic density was low in late fetal myocytes,
and it rose to adult levels by the first day after birth in rabbit cardiac myocytes [18].
Starting from T-tubule formation at day 8-10 afier birth, the dyadic density in T-tubules
markedly approached adult values by the 1 2 ' ~ day of age. In adult rabbit and rat, the total
dyadic membrane of the ventricuIar myocyte is 2 1% and 48% of the T-tubular membrane,
respectively [ 1 51.
3.1.4 Myofibrils and mitochondria
Neonatal myocytes are also characterïzed by their sparse myofibrils and disorganized
myofilaments relative to the nuclei and the mitochondria 1561, [59]. Neonatal cells have
fewer and shorter myofibrils, and contain fewer sarcomeres than mature cells do. In
rabbit cardiac cells, the volume fraction of myofibril (VmfNceii) increases steadily by 15%
from its pre-neonatal value (0.346) up to the 8" day of age, then slowly increases towards
the adult value of 0.501, indicating that half of the intracellular space is occupied by
myofibriIs in adult cells. Similarly, the VrnfNceii increases from 37% to 45% within the
first 1 1 days of age in rat myoçytes. In fetus cardiac myocytes, the myofibriIs are located
under the sarcolemma with poor organization. With myocardium development,
myofibrils extend from their ends, fiom which the new sarcomeres forrn, and the
myofibrils are in apposition to each other along the ce11 longitudinal axis [56]. Nassar et
al. (1987) showed that myofibrils kept a shell-like distribution pattern under the
sarcolemma until age of 3 weeks while the two large oval nuclei surrounded by a large
aggregation of mitochondria occupied the central part of the ce11 almost from end-to-end
[59 ] . The total number and volume fraction of mitochondria (Vmi toNrn f or Vrnito/Vceii)
markedly increased with age. The VrnitoIVceii approached its adult value (0.309) within 8
days and the Vrn i toNmf reaches adult level of 0.50 - 0.65 within 3 days after birth. The
latter suggests that the myofibrils in mature myocytes may have better access to the
energy supplied by the rnitochondria in cornparison to immature cells [56].
3.1.5 BIood supply
Myocardial contractile function depends clearly on the blood supply of the myocardium
that may be defined as capillary density in the myocardium [58]. The fast postnatal
capillary proliferation in a growing rat heart was demonstrated by Anversa et al. [58].
These authors found that from day 1 to 11, both capillary surface areas per myocyte
surface area and capillary voIume per myocyte volume increase 2-3 fold, to the levels
comparable to that in the adult myocardium.
3.1.6 Summary
Myocardial growth is believed to be a physiological hypertrophie process. The data
above indicate that neonatal cardiac myocytes grow quickly. They rapidly accumulate the
contractile material, myofibrils and cellular organelles. Moreover, they assemble their
increasing organelles into a more mature cellular architecture to meet the increasing work
load of the hearts, It should be pointed out that physiological hypertrophy differs
distinctly from pathological hypertrophy caused by increasing work load (such as
hypertension) by the ratio of mitochondria to myofibrils. In the former, the mitochondria
growth rate is proportional to the myofibril growth rate. However, in the latter, the
mitoc hondria growth rate lags behind the myofibril growth rate.
Table 1 . Summary of the main morphological changes in developing and adult myocytes
[591, I6019 WI, I5819 Pl.
PARA MET ERS^ NEWBORN ADULT
S N ratio"
Ce11 diameter 5- 16+0.06 pm
3-8 pm (cat)
14.lf0.2 pm
5.13+0.08 pn (frog)
10-30 pm (cat)
Ce11 length 48I3 pm (-24d)
68+4 pm (+8d)
40-50 p (dog)
Nucleus located in ce11 center Located closely to ce11 SL
VnNcrii high IOW
vn 10-222 pn3 (rat)
SR spare, dyads of SL-SR increase, dyads of SL-SR & T-SR
~ a " uptake low Wh
VSR N c c l l d 0.0079~0.0006 0.0 13M.007
T-tubules no well developed
Capillary rudimentary
0.05 (rat)
0.06 (rat)
abundant
O. 1 1 (rat)
O. 17 (rat)
Table 1. Continued.
PARA MET ERS^ NEWBORN ADULT
Myofibrils low, poorly organized high, well organized
ATPase activity low
Vmf/Vcc~~ ' Iow high
37-46% (rat)
Vmf 334- 1200 pm3 (rat)
Mitochondria locate in ce11 center interspersed arnong myo f i brils
average diameter 0.56M.09 pm 0.73 t0.12 pm
Vrnird'Vcc~~ ' 10w high
VmitdVmt- ~ O W high
a ce11 surface to volume ratio.
nurnbers without notation are obtained from rabbit myocyte. C
V, is the volume of the nucleus and V,Ncell is the volume fiaction of the nucleus. d
VSR Ncrll is the volume fraction of the SR. e ratio of capillary luminal volume per myocyte volume.
ratio of capillary luminal surface per myocyte ce11 surface.
V,rN,,ii is the volume fraction of myofibril.
' VmiJV,,l~ is the volume fraction of mitochondna.
j VmjtJVmf is the ratio of mitochondrial to myofibrillar volume. H all features and nurnbers are fiom lefi ventricular myocytes fiom different species.
The word descriptions are applied to al1 mammalian cells mentioned in this table.
3.2 Biochemical studks of the developing kart
3.2.1 DHPR
A number of biochemical studies suggest that neonatal cardiac myocytes may have a
distinct cal' regdatory protein profile. Thomas [6 1 ] conducted a series of experiments
to determine the protein concentrations of DHPR and RyR, and the activity of SR ~ a " -
ATPase (SERCA) in 3-day, 6-day, IO-day and 20-day-old rabbit ventricles. Using crude
homogenates and specific ligand binding assay, it was found that there was no age-related
difference in the affinity and maximal specific DHP binding (B-) among the four
groups. The B,, of DHPR of the 3-day-old was the same as that of 20-day-old as shown
in Fig 5. Combining Thomas' results with Boucek et al.' study [62], in which it was
found that there was no marked developmental change of the B, of DHPR between 20-
day-old and adult rabbit hearts, we cannot see an age-related DHPR change in rabbit
myocardium aged from 3-day-old to adulthood. In contrast to the B,, of DHPR,
Brillante et al. (1994) found that the mRNA of DHPR increased postnatally, which might
suggest that the expression of this Ca" channel was up-regulated during myogenic
development [63]. The discrepancy of DHPR in protein and mRNA levels may require
funher work in order to define the meaning of the pre-translational change of this protein.
3.2.2 RyR and SR CB* ATmse
Biochemical data from different iaboratories show that the SR is not well developed in
newbom rabbit hearts [61], [Il , [Hl. The total amount of SR protein yielded from
newbom (2 to 5-day-old) rabbit hearts is 60% of the adult value and SR Ca2'-activated
ATPase activity in newbom hearts is also 60% of that in adult rabbit hearts [ I l .
Ontogenical increases of the B,, of RyR and SERCA activity were fourid only in the 20-
day-old ventricular homogenates of rabbits among the 3 to 20 days old age groups [61].
The B, of RyR increased nearly 30% frorn day 3 to day 20, while SERCA activity
tripled during the same penod (Fig 5).
O WPR = RYR
SERCA
Fig 5. Specific dihydropyridine and ryanodine receptor binding and SERCA activity in ventricular homogenates fiom different age groups of rabbits. The left ordinate is B,, of DHPR and RyR and the right ordinate is SERCA activity. The values
are in the form of means f SEM. * of RyR indicates significant @<0.01) difference fiom the other age groups. * of SERCA indicates significant @<O.OS) difference fiom the other age groups. Data are from Thomas' study with permission [6 11.
There were no statistical differences in 8, of RyR and SERCA activity found among 3-
day, 6-day and 10-day groups [6 11. Nayler et al. (1977) reported that ~ a " accumulation
and ATPase activity of SR-rich micmsomes changed at about the time of rabbit birth.
Microsornes prepared from fetal rabbit h e m muscle accumulated ~ a " at a comparatively
slow rate and the ability of the fetal microsornes to hydrolyze ATP was depressed relative
to the neonatal preparation [64].
3.2.3 /Va+/Caz* exchmge
In addition to the SR and SR-T tubular system changes in the developing heart,
sarcolemmal ~ a ' l ~ a " exchange also changes perinatally in protein and mRNA levels
[65]. [5 71. An exchanger immunoreactivity study demonstrates that in the developing
rabbit heart, both fetal (28-day gestation) and newbom (24-48 hours) sarcolemma have
2.5 times more ~ a + / c a ' + exchanger proteia in comparison with adult sarcolemma [65].
The distribution of ~ a + / C a " exchange in the cardiac myocytes in developing rabbit h e m
was investigated by Chen's group [57] using a monocolonal irnrnunolabelling technique.
These authors found that ~ a + / c a " exchanger distribution pattern changed during
ontogeny. In hearts from rabbits younger than 10-day-old, ~a+/ca '+ exchangers were
highly localized to the peripheral sarcolemma and appeared in the T-tubules during T-
tubule development- These authors also found that in terms of T-tubule development by
the age of one month, the myocytes were still immature in comparison with the adult
rabbit myocytes. These results are consistent with Boerth et al.' study of sarcolemmal
~ a ' / ~ a ' exchanger mRNA [66]. They found that in rabbit cardiac myocytes the mRNA
level peaked for fetuses that were 25 to 29 days old (full term is 3 1 days). During the first
week after birth (1-7 day of pst-partum), the ~ a + / ~ a ' + exchanger mRNA was 50% of the
peak value and at the end of the second week (14 days pst-partum) it decreased to 25%
of the peak value. The adult steady state mRNA level was about 1/6 of the peak value.
They also found that in rat myocytes the ~a 'Ka" exchanger mRNA change had the same
pattern as in rabbit heart cells. The highest level of the mRNA was at the first day of
birth. The mRNA level decreased to 30% at day 7, 75% at day 14 and 13% at adulthood.
Although the ~a'/ca'+ exchange mRNA peaks appear at different developmental days in
rabbit and rat, both peaks are near the time of birth and decline dramatically during the
first two weeks afier birth, gradually reaching adult levels. The postnatal decline of
exchanger protein and exchanger mRNA supports the idea that the ~a'lca'' exchanger
may play a relatively greater role in E-C coupling in neonatal myocytes in comparison
with mature myocytes.
3.3 Electrophysiological end physiologicel studks of the developing heart
3.3.1 Resting membrane potential
Developmental changes in the resting membrane potentials (RP) differ in various species.
In guinea pig cardiomyocytes there is no age-related RP change between 45 days post
conception as a fetus and 45 days post partum as an adult 1671. However in the rat
cardiac myocytes, the RP increases progressively fiom fetal to adult (-40 - -50 mV vs. -
75 - -85 mV) [68]. In rabbit cardiac myocytes, the RP increases slightly before birth (-68
mV at 21 days fetus vs. -73 mV at 28 days fetus) and stays stable at -75 mV after birth
( 1-5 days old) [69], [45]. The RP differences in species and developmental changes may
be due to the differences of the developmental changes in the types and number (density)
of various membrane ion channels such as outward and rectified potassium channels and
sodium channel in different species.
3.3.2 Action potential
Most of action potentials ( A h ) of mammalian cardiomyocytes have similar shape
including human, rabbit and guinea pig. The AP duration increases between neonatal and
adult period in human, guinea pig and rabbit myocytes. But the actual values of the
duration are reported differently by various investigators since the experimental
conditions differ from individual lab [70], [71], [67], [69]. In neonate rabbit and guinea
pig cardiomyocytes, the AP plateau phases are shorter as compared to the adult. The
upstroke velocity, amplitude (-105 mV in rabbit and -130 mV in guinea pig) and
overshoot (-30 mV in rabbit and -50 mV in guinea pig) of the APs have no significant
age-related difference [45], [67], [69]. As with the RP, the configuration of the APs
reflects the type, magnitude and kinetic properties of various membrane ion currents of
the cardiac myoçytes.
3.3.3 L-type Cd+ channel
Similar to what is in the mature heart, the L-type ca2+ c h a ~ e i is the prominent ca2+
channel in the immature cardiac myocytes in different species. While 9 1 % of adult rabbit
left ventncular cells have T-type ~ a ' + channels, only 39% of the neonatal myocytes (1 -5
27
days old) have the T-type ~ a " channels. The T-type ca2+ channel current is too small to
be significant in ternis of intracellular ~ a " regulation in immature cardiac myocytes [44].
To investigate the L-type ~ a " channel current (Ica) and its kinetics, whole-ce11 voltage-
clamp on isolated single cardiac myocytes of different species have k e n used by several
groups. Osaka and Joyner 1461 measured the absolute peak lc, and the peak current
density (the maximum peak current nomalized by the ce11 membrane capacitance) in
newborn (1 to 3-day-old) and adult rabbit ventricular cells. They found that the peak Ica
density was significantly higher in adult cells than in newborn cells, but there was no age-
related difference in the current-voltage relation behveen these two age groups. The time
to half inactivation was significantly longer in newbom cells than that in adult cells, and
this inactivation time difference could be eliminated when ~ a " was the charge carrier
through L-type ~ a " channels. This indicates that the inactivation is ~a"-dependant and
may have developmental changes- Wetzel et al. [72] expanded the study to fetal rabbit
myocytes (21 and 28-day of gestation). The results of Wetzel et al.' studies were in
partial agreement with that of Osaka et al. (1991). Their results indicated that the ~ a "
current density increased with age, suggesting that ~ a " channel density also increased in
the developing fetal heart [46]. Wetzel et al. (1993) did not observe the age-related time
course change of channel inactivation. But they showed that the L-type Ica was inhibited
by increase the frequency of stimulation, indicating that the time of ~ a " channel recovery
from inactivation was longer in younger cells than in adult cells. They also found that the
"window current" was significantly smaller in immature cells than in adult cetls. While
the steady-state activation curve (&) was the same among al1 age groups, the steady-state
inactivation curve (fa) was significantly shifted toward more positive potentials in mature
myocytes as compared with immature myocytes [66]- These results suggest that the L-
type Ica cootnbute to the relatively smaller ~ a " influx through ~ a " channels in the
developing rabbit heart compared with the mature hem. This concept is further
supported by a previous study by Wetzel et al. (199 1 ) on tension generation in newborn
( 1-7 day) rabbit ventricular papillary muscle and myocytes using the L-type caZ' channel
antagonist called diltiazem [45]. Diltiazem reduced action potential duration and
depressed the plateau phase, but it did not abolish the trans-sarcolemmal Ica completely in
the neonatal cells. While the trans-sarcolernrnal Ic, was reduced significantly by the drug,
there was no significant reduction in tension development. This indicates that the ~ a "
entering the neonatal myocyte via L-type ~ a ' + charnels may not be the only source of
extracellular ~ a " for E-C coupling [57]. During development, it appears that while the
DHPR protein density stays roughiy constant as described in biological studies, the
number of functional DHPR increases, as reflected in current density increase with age.
However, it should be noted that in al1 of the studies done on rabbits, the investigators
used a high concentration (1 0-14 mM) EGTA in the pipette, which may preferentially
enhance the L-type Ica currents in the mature heart [73], [45], [72], [29].
3.3.4 Sarcoplasmic reticulum
SR replates intracellular ~ a " during E-C coupling. caZ+ released from SR via CICR
and resequested into SR, results in the contraction and relaxation of the cells. RyR and
SR C ~ " - A T P ~ S ~ are major proteins for SR hnction. The reiiance of muscle contraction
on SR ~ a " is low in the neonatal rabbit heart in cornparison with the adult heart [15].
These results are consistent with the morphological and the biochemical findings that
include the underdeveloped SR, low RyR density and SERCA activity in the neonate
heans of various species as described at 3.2. L-type Ca" channel-gated SR Ca" release
exists in human atrial cardiac myocytes as early as 3 days after birth, and the rate of ~ a "
release increases with age (3 days versus 4 years), indicating the increase of efficiency of
~ a " signaling in the more mature heart [74]. Sirnilar observations were reported in
rabbit atrial and ventricular preparations. Seguchi et al. [75] observed that a few pM
ryanodine (an SR ~ a " release enhancer) induced a negative inoiropic effect in fetal, 3-
day, 7-day, and adult rabbit heart muscle strips, with the order of the magnitude k i n g
fetus, 3-day < 7-day < adult. Only in adult muscle did the presence of ryanodine result in
prolongation of the muscle relaxation, time to peak tension and higher resting tension. in
addition, 100 FM ryanodine (a RyR inhibitor) was more effective in attenuation of the
positive staircase in adult than in the neonate. These data suggest that the SR function
changes dramatically dunng perinatal myocardium development. ca2+ released fkom SR
via CICR increases with age in rabbit heart. SR ~ a ' + is the major source of contractile
~ a " in adult myocardium. This age-related SR-contraction dependency is also observed
in rat myocardium [76]. Myocardial relaxation was shortened by isoproterenol and
prolonged by ryanodine in the adult but not in the newborn, indicating that the contraction
of adult rat heart was highly dependent on SR ~ a " and therefore the neonatal heart might
be more dependent on trans-sarcolemmal ~ a ' + influx.
3.3.5 ~a?" exchange
During sarcolemmal depolarization, ~ a ' + enay via NCX in its reverse mode is favored,
which results in myocytes contraction. Repolarization favon ~ a " efflux to extracellular
space via NCX normal mode, which leads to ce11 relaxation [IS]. An increasing number
of studies on immature myocardial E-C coupling reveal that NCX may be the alternative
~ a " transport mechanism in the immature heart. This is because the amount of NCX
mRNA, NCX protein content and its rate of ~ a " uptake in SR vesicle preparation are
higher in the immature heart than in mature heart, as described previously (3.2) [77], [65].
Secondly, physioiogical studies of NCX provide more evidence to support this concept.
Artman et al. ( 1 995) found that the ~ a + K a ' + exchange current density was greatest at 1 -4
days and rapidly decreased within three weeks in rabbit heart [77]. Therefore, NCX is a
potential candidate for intracellular ~ a " regulation during E-C coupling in the immature
heart .
The direction and the amount of calcium ion transported by NCX depend upon the
membrane potential and ionic concentrations of ~ a ' and ~ a " on both sides of the cell
membrane [78], [l], [56] . Klitzner et al. (1988) used sucrose gap voltage clamp to
control membrane depolarization on the newborn and the adult rabbit ventricular muscles.
In their experiments, the right ventricular papillary muscle of the newborn exhibited a
monotonically increasing tension response reaching a steady state level maintained
through out the duration of the depolarization controlled by the voltage clamp. However,
under the same conditions, the adult papillary muscle developed an early peak of tension
before relaxation to a sready state level, irnplying that SR ~ a " release resulting in the
papillary muscle contraction only occurred in adult heart [78]. Some earlier studies also
demonstrated that the myocardial contractility and relaxation depended on extracellular
~ a ' and ~ a ' + concentration [l], [56]. Hoener et al. (1 98 1 ) demonstrated that the low
extracellular ~ a ' ( ~ a ' ] , 7.2 mM) increased isometric tension of the right papillary
muscles of the fetal and newborn rabbit and slowed down myocardium relaxation, most
prominently in the 22-24 days old fetal hearts. This sensitivity of relaxation to low [Na'],
decreased with age up to 8 days after birth. In the û-day-old newborn, as in the adult
hean, relaxation was unaltered by a low wa'],, suggesting that the involvement of NCX
in myocardium E-C coupling, especially in fetus and newbom (< 7 days) rabbit hearts,
was greater than the more mature heart, and this involvement was progressively
substituted by postnatal developing SR [56]. In contrast to using low ma'],, Nakanishi et
al. (1984) raised va'], to 200 mM and observed that the high [Na+], decreased rabbit
myocardial contractility, and this negative inotropic effect was greater in the newbom
than in the adult rabbit h e m [79]. These early findings are consistent with the concept
that NCX is regulated by the concentrations o f ~ a ' and ~ a " across the ce11 membrane.
The early neonatal heart, therefore rely on NCX for contraction and relaxation more than
their mature counterparts due to the absence of a large L-type ca2+ curtent and
functionally mature SR in neonatal rabbit cardiac myocytes.
3.3.6 Cross-signalhg of DHPR and RyR in the developing heart
The cornerstone of the functional coupling of DHPR and RyR in myocardium is the close
spatial association of these two ~ a ' + channels, which is tightly related to the T-tubules
and SR structural maturation. Functional coupling of DHPR and RyR in cardiac E-C
coupling has been extensively studied in adult marnmalian hearts in different species [go],
[39], but there is very limited work done in immature myocardium. The presence of
CICR in human immature myocytes (3day to 4-year of age) has been reported by Hatem
et al. [74]. The age difference of L-type ~ a " inactivation has k e n studied in fetal, 1-5
days old and adult rabbit hearts by Wetzel et al. [72j and Osaka et al. [46].
3.3.7 Contractile force and [&JI of the developing heart
Physiological and electrophysiological differences between mature and immature
mammalian hearts are not unexpected, since the cellular structure and architecture are
different. The most predominant physiological differences are the lower contractility and
the slower rate of force generation in the immature heart 111, [79], 17 11. Jarmakani et al.
(1982) showed that the force of myocardial contraction per gram tissue in the newborn
rabbit was approximately one third that in the adult when extracellular ~ a ' + concentration
was 1.5 mM.
Myocardial contractile force is determined by the arnount of contractile proteins,
myofibrillar ATPase activity and the amount of ~ a " reaching the myofilaments. Several
studies have explored various cellular components to predict their physiological fbnction
in the immature hem. Myofibrîllar content progressively increases perinatally as
descri bed previousl y. A developmental increase in myo fibrillar ATPase ac tivity was
reportcd by Nakanishi et al [ I l . Intracellular ca2+ concentration is dependent on the ca2'
flux across the ce11 membrane, and the ca2+ released and uptaken by SR. Developmental
change in [ca2']i transient has not k e n well established yet. However, the magnitude of
[~a"]i might be speculated from many studies on the developing mammalian
myocardium. In the developing rabbit ventricular myocytes (1 -5 days old vs. adult), INcX
density increases 6-fold [77], L-type Ica density increases 2-3-fold [69], [46], ce11 surface
area increases 4-foid [77] and cell volume increases 3-fold [56]. Thus, the INCX and Ica
might provide a comparable size [~a' ']~ transient in neonatal and adult myocytes dunng
cellular depolarization, respectively. The assumption is that dunng the myocyte
depolarization the total arnount of ~ a " influx into the neonatal cell is from NCX and in
the adult ce11 is from L-type ~ a ' + channels. In addition to the ~ a " influx through SL, the
SR caL' release dunng E-C coupling affects [ca2+li transient as well. SR ca2'
contributes much less to the immature myocardiurn contraction compared to the mature
myocardium [75], [76]. Therefore, it seems logical to expect that the [ca2+]i transient is
lower in the neonate than in adult during cardiomyocyte contraction. However it should
be noted that linle is known about developmental effects on intracellular ~ a " buffering
capacity.
3.3.8 Summary
Myocardial excitation-contraction coupling results fiom the intracellular calcium
transient. In mature cardiac myocytes, the [~a' ']~ transient relies largely on the trans-
sarcolernrnal ~ a " influx to provide the activator ca2+ required to induce a large arnount
of ~ a " release fiom SR (CICR). CICR is a prevalent mechanisrn in mature myocardium
E-C coupling, depending on both T-tubules and SR structural and functional maturation.
This is absent in the fetal and newbom mammalian myocardium. Therefore, before the
T-SR system reaches its full maturation, immature hearts need alternative means to fulfill
their physiological fhct ion (pumping blood via contraction and relaxation cycles).
Despite the fact that there are a few studies presenting evidence of developmental
changes in Ica in rabbit ventricular cells, the ages of the rabbits in those studies are fetai,
neonatal (1-7 day old) and adult (1.5-3.0 kg) [al], [72], [16], [69]. There are no data
available, so far, to describe the L-type ~ a ' + current changes and to characterize the ca2+-
dependant L-type ~ a " channel inactivation changes between the neonatal and the adult
rabbit hearts. Our study is the first one to investigate postnatal development of Ic, and its
~a"-related inactivation in 3 to 20-day-old rabbit ventricular myocytes using the
perforated patch clamp technique. To expIore the time course of DHPR and RyR
functional coupling in the developing hem, SR ~ a " dependent L-type ca2+ chamel
inactivation ("back talk") was investigated in 3-20 days old rabbit ventricuiar myocytes.
4. Hypotheses and Objectives
4.1 Hypotheses
In the developing rabbit heart,
The spatial relationship between the L-type ~ a " channel and SR ca2+ release channel
is crucial for CICR,
The contribution of ~ a " flux through L-type ca2+ channels to ce11 contraction
increases.
Functional coupling of L-type ~ a " channels and SR ~ a " release channels should
occur after the development of T-tubules (NO days).
SR ~a"-dependent ~ a ' + channei inactivation, the "back talk" between the L-type
~ a " channel and SR ~ a ' + release channel, should occur from a certain age as the L-
type ~ a " channel and SR ~ a " release channel assume the adult spatial localization.
SR ~ a " content increases up until -20 days of age.
If these hypotheses are tme, then 1 should be able to detect the following postnatal
changes:
Increase in L-type ~ a " current density with age.
36
Appearance of ca2+ dependent L-type caZ' channel inactivation in the myocytes with
T-tubule development ( 1 0 days old).
Increase in SRca2'content withage.
Increase in SR releasable ~ a " removed by NCX.
4.2 Objectives
To characterize the following in the neonatal rabbit ventncular myocytes during ontogeny
( 1-2 1 days):
L-type Ca" current drnsity
L-type Ca" channel current and voltage relationships
L-type ~ a " channel inactivation kinetics (calcium-dependent inactivation)
SR ~ a " load
To investigate these, 1 observed the age differences in voltage-dependent L-type ~ a ' +
channel ~ a " current (Ica) and the kinetics of the inactivation of these charnels in
response to SR ca2' release witMwithout caffeine intervention. By using this
physiological approach, 1 hoped to gain insight into the functional and spatial maturity of
L-type ~ a " channels and SR ca2+ release charnels.
5. Material and Methods
The amphotencin B perforated patch clamp technique was used in single immature lefi
ventricular myocyte as descnbed in several papers [82], [83], [84]. Four age groups of
New Zealand White rabbit were obtained from a local farrn, including 3-day, 6-day, 10-
day and 20-day-old of either sex. The L-type ~ a " charnel ~ a " current, channel voltage-
current relationship and ~ a + / ~ a ~ + exchange current induced by caffeine depletion of SR
~ a " were measured. 50 cells from 10 rabbits (at least 5 cells per rabbit) were recorded
and analyzed for each of the four groups.
5.1 Ce11 preparation
5.1.1 Single ventricular myocyte isolation
The method of single ventricular rnyocyte enzymatic dissociation was adapted fiom Mitra
and Morad [Ml. Al1 solutions prepared with carefùl sterile procedures and aerated with
100% O2 before and during the isolation. New Zealand White rabbits aged 3-20 days old
of either sex were anesthetized by an intraperitoneal injection of sodium pentobarbital(60
rngikg body weight). For anticoagulation, heparin (sodium salt, 2700 uspikg body
weight, Sigma) was injected with pentobarbital simultaneously. The rabbit lost its pain
reflexes within several minutes. AAer thoracotomy, the heart was irnmediately excised
and kept in ice-chilled (4°C) dissecting solution for about 1 minute to arrest the heart,
The pericardium was removed first. The aorta was cannulated above the aortic valve and
coronary arterial blood was washed out by 5-10 ml cold Krafibriihe solution (KB
medium) [86] , which was free of calcium, sodium and ethylene glycol-bis (P-aminoethyl
ether)-N, N, N', N'- tetraacetic acid (EGTA). The heart was then mounted ont0 a
LangendortT apparatus and retrogradely perfùsed for 4 minutes with pre-warrned (37°C)
and oxygenated KI3 solution via a peristalic pump (MasterFlex Cole-Parmer Instrument
Company, Chicago). Then 5-8 minutes of enzymatic digestion (collagenase type II,
Worthington) at a flow rate of 1-4.6 ml/min at 34-3S°C was used (the flow rate, enzyme
concentrations, and the time of enzyme digestion were adjusted according to the age of
the animal) (Table 2). Finally, an EGTA containing KB solution was used to wash out
the enzymes. The heart was transferred to a Petri dish containing 50 ml EGTA-KB
solution and the left ventricle was dissected from the heart. The lefi ventricle was either
cut into very small pieces or gently teased with forceps to disperse individual myocytes.
The ce11 suspension was filtered through a 200 Fm coarse nylon mesh to get nd of tissue
chunks. The myocytes were incubated in KB solution with 0.5 mM EGTA at room
temperature for at least an hour to allow for recovery [87]. The cells were kept in Ki3
solution with 0.5 mM EGTA at 4OC before electropbysiological studies.
Table 2. Protocols for enzymatic dissociation of ventrïcular myocytes fiom rabbits of
different ages.
ISOLATIONS STEP 3 DAY 6DAYS IODAYS 20DAYS
(ml) 1 4 5 6 16
Collagenase (mglml) 2 1 6.5 0.5 0.5
Protease XIV (mg/ml) 0.2 O. 1 O. 1 O. 1
KB (ml) 5 8 1 O 20 - 25
(ml) 3 4 5 6 16
Perfusion speed (ml/min) 1 1.5 1.5 4.6
5.1.2 Myocyte evaluation criteria
Only myocytes meeting the following criteria were used for these experiments:
cylindrical and rectangular shaped with well-defined edges without sarcolemmal
blebbing
without any visible spontaneous contraction afier the myocytes settled down to the
bottorn of the plate in the experimental solution (2 mM CaCl-)
with clear striations and without obvious intracellular granulation
5.1.3 Solutions for ceIl isolation
Al1 the solutions were prepared with double de-ionized water passed through two sets of a
four-element purifier (Milli-RO and Milli-Q water system) and filtered with a 0.2 p n
filter (Nalge Company, NY, USA).
40
Dissecting solution (rrro CU*'). Solution contained (mM): 126 NaCl, 4.4 KCl, 5 MgCl?,
24 HEPES, 22 glucose, 20 taurine, 5 creatine, 5 Na-pyruvate, 1 NaH2P04, pH 7.4 (at
37'C) with NaOH.
KB solution wit/dwit/1out EGTA. Kraftbrùhe solution [87], [86] is a ~ a " - and Na*-free
solution. KB solution without EGTA was used to stop ~ a ' i ~ a " exchange during the
myocytes isolation but without affecting collagenase activity which was dependent on the
~ a " residue in the solution. The EGTA-containing KB solution prevented the myocyte
from hypemontracturing by inhibiting reverse mode ~ a * K a " exchange and allowed the
ce11 time to recover fiom the isolation intervention. KB solution contained (rnM): 10
taurine. 70 glutamic acid, 25 KCI, 10 KHzPOa, 22 dextrose, pH 7.3 with KOH at 22OC.
The osmolarity of Ki3 solution was about 245-260 rnOsmL EGTA-containing KB
solution was freshly prepared before ce11 isolation by adding 0.5 mM EGTA into the KB
solution.
External solution (bath solution). In order to block interfering current from K'
charnels, 20 mM CsCl was used in the extemai solution. In some control experiments,
0.01 mM TTX was also added into the bath solution. l T X ( I O FM) is sufficient to block
more than 98% of <he inward Na+ current in cardiac myocytes of neonatal rabbits [45].
The external solution contained (mM): 1 O7 NaCl, 10 HEPES, 20 CsCl, IO Glucose, 5 Na-
Pymvate, 1 MgC12, 2 CaClz, pH 7.4 with NaOH at room temperature. The osmolarity is
about 290-300 mOsm/L.
5.2 Amphotericin B perfomted patch prepamtion
To avoid dilution of cytosolic substances by the pipette filling solution, especially the
diffùsible intracellular factors involving cellular responses and intracellular ~ a " buffers,
the amphotericin B perforated patch whole ceIl recording technique was used. This
technique was first used by Hom and Marty [88]. This is based on the fact that
amphotericin B inserts into the ceil membrane to form channels that selectively allow
monovalent ions to pass through the ceIl membrane. In addition, amphotericin B
molecules do not pass through the membrane into the cell, but stay only in the membrane
patch within the pipette [89]. When there are many channels formed within the patched
membrane, the access resistance of the cell becomes low enough to allow voltage clamp
of the ce11 and to measure ionic currents of the whole ceIl [84]. Amphotericin B is light
sensitive and was handled with light protection through out the experiments.
5.2.1 Preparation of amphotericin B solution
Internai solution (pipette fiïling solution) It contained (mM): 1 1 0 CsCI, 5 MgATP, 1
MgCl?, 20 TEA, 0.025 EGTA, 10 HEPES, 5 Naz-phosphocreatine, adjusted the pH to 7.1
with CsOH. To maintain consistency of the pipette filling solution in al1 my expenments,
1 used the same intemal solution in the perforated cells as 1 used in the "routinely" patch-
clamped whole cells.
Amphotericin B stock solution. The arnphotericin B stock solution (100 mg/ml) in
dimethylsufoxide (DMSO) was prepared weekly and stored at -20°C in the dark [82],
[ S I . In a small centrifuge tube, 3 mg amphotericin B powder (Sigma) was well mixed
with 30 pl DMSO by rnicropipettor utill there were no visible particles on the wall of the
tube and sonicated for 1-2 minutes. Sometimes a couple of minutes of vortex were
needed for complete dissolution. During the experirnent, the stock solution was kept in a
container with ice.
Ampltutericin B working solution. The amphotericin B working solution (200-600
pç/ml) was prepared just prior to use and kept on ice no longer than 2 hours, since this
solution was overly saturated and amphotericin B would precipitate within 2 hours. If
this happened, the amphotericin B would very likely. be incapable of permeabilizing the
plasma membrane. Every 2 hours, a new working solution was made. 1 and 3 pl of stock
solution (100 mg/ml) was added to 499 and 497 pl pipette filling solution, respectively,
and vortexed well to give final arnphotericin B concentrations of 200 or 600 pg/ml,
respectiveiy, which were ready to fil1 pipettes.
5.2.2 Preparation of amphotericin B perforated cardiac myocytes
Pipette pulfing procedure. Patch electrodes were made from thin-walled borosilicate
glass (Corning #7052) capillaries ( l.6Y 1.1 mm OD/ID, World Precision Instruments, FL)
using a vertical puller (Narishige Scientific Instrument, Tokyo, Japan). Pipette pulling
included two stages. in the first stage, 1 carefully clamped a capillary in the puller,
selected a relative high heating current (12.9 A), set a 10 mm space to stop the fa11 of the
pipette, and activated the puller to pull the capillary. Afier the coi1 filament completely
cooled down, the second stage of pulling was started. 1 selected a lower heating current
(7.9 A), removed the spacer, and allowed the puller to pull. During the two-stage pulling,
the coi1 was carefülly protected from air currents to get a consistent pipette resistance.
The diarneter of the pipette tip was about 1-2 pm and the pipette resistance ranged from
1-3 M R with the filling pipette solution. In order to form a bettrr seal. in genenl, the
higher resistance pipettes (2-3 MR) were used for the srnaller myocytes, especially, those
from 3 and 6-day-old rabbit hearts, since the diameters of these myocytes were small.
Pipette filfing procedure. In order to prevent amphotencin B from interfering with the
high resistance seal formation, the pipette tip was filled with an amphotericin B free
pipette solution first and then back filled with an amphotericin B containing pipette
soiution (amphotericin B working solution). I bnefly dipped the pipette tip into a small
beaker containing non-amphotericin B intemal solution. The filling distance of this
amphotericin B free pipette solution was proportional to the time of the dipping. Under
our experimental conditions, -1 second dipping could fil1 the piperte up to -200 pm. If
the tip contained too much amphotericin B free pipette solution, the time that it took the
amphotericin to d i f i se to the membrane patch was too long, sometimes longer than an
hour, which made the perforated patch an unattractive technique to use. Less than 1
second dipping gave the appropriate tilling distance to the pipette. Afier filling the tip,
the pipette was irnmediately back filled with amphotericin B working solution using a 1
ml tuberculin syringe up to 5- 10 mm of the pipette.
Seal formation
Isolated myocytes were plated ont0 a glass coverslip mounted on the stage of an
inverted microscope (Nikon, Tokyo) for about 5-10 minutes. The plate was
superfused with bath solution at a flow rate of 2-3 m h i n for about 5-10 minutes to
ivrish out the dead cells and ce11 debris. Then the superfusion rate was reduced to 1
mVmin and kept this rate of perfusion throughout the whole experiment.
The pipettes were filled as described above and mounted ont0 the headstage (CV-4,
headstage, Axon Instruments).
A motorized micromanipulator (MP-285, Sutter Instrument Co.) was used to quickly
insert the pipette into the bath solution and the junction potential was offset. The
pipette tip was moved near the center of the cell. As the pipette tip pressed against
the myocyte surface, the size of the test-pulse (t-test, a repeated test voltage pulse, 1
mV for 5 ms every 10 ms) became progressively smaller. To speed up the formation
of a giga-ohm (GR) seal, a negative voltage of -70 mV was applied to the pipette afier
applying a slight negative pressure by gentle suction on the interior of the pipette.
When the t-pulse was reduced to less than 10% of its original size, the negative
pressure was kept until the seal resistance was closed to GG!. Usually a seal was
established within 2 minutes. After compensation of pipette capacitance, the
capacitive current was recorded in response to a 10 mV hyperpolarizing pulse every
minute (Figure 6.). The voltage roto col was controlled bv a cornDuter with software
CLAMPEX 7. The ce11 capacitance (Cm) was deterrnined by observing the peak of
the capacitive current (lm,) while the ce11 was hyperpolarized by a step voltage pulse
from a holding potential of -70 to -80 mV for 8 ms every minute. ï h e series
resistance was monitored simultaneously. The capacitive current gradually became
bigger and faster over 20-40 minutes as more and more amphotericin B channels
formed in the patched membrane, and the series resistance decreased correspondingly
at the same time. When series resistance was stable at about 15-20 MR, the myocyte
was ready for the current measuring experiments.
-50 0 1 2 3 4 5 6 7
time (ms)
Fig 6 . The voltage protocol and the typical capacitive current change with time during amphotericin B perforation. Upper: the voltage protocol. Lower: the capacitive current traces recorded from the left véntncular myocyte of a 20- day-old rabbit (9841 7000) during the amphotericin B perforation. Each trace represents one event of the hyperpolanzation of the ce11 from -70 mV to 430 mV. From the bottom to the top, 21 traces (total) were obtained at 1 trace per minute after the GR seal formation. The inward capacitive current is expressed as an opposite direction.
5.3 EleclrophysiologicaI studks
Al1 the experiments were performed at room temperature (22-23°C). Data acquisition
and some data analysis were conducted by using software pCLAMP 7 that consisted of
CLAlMPEX 7 and CLAMPFIT 6. CLAMPEX 7 provided sequences of stimulation
(voltage protocols) to the testing myocytes via a Pentium computer, a DigiData anafog-to-
digital converter and an Axopatch-1 D amplifier (Axon Instruments, Inc., Foster City,
Calif.). Software CLAMPEX 7 was also used to acquire data in discrete episodes and
recorded data onto the computer hard disk. CLAMPFIT was used to analyze data. L-type
ca2' current and ~a'Ka'' exchange cunent were measured in amphotericin B perforated
single myocyte as descnbed in the "routine whole-celi" recording by Kato et al. [67],
[29] , [74], [90]. These membrane currents were filtered at 2 kHz using an eight-pole
Bessel filter built into the amplifier. The data was saved in the computer for later data
analysis.
5.3.1 Outline of voltage protocols
The voltage protocol that 1 used consists of four cornponents as shown in Figure 7. In the
first phase (ramp), a slow hyperpolarizing ramp was used to measure ce11 membrane
capacitance (C,). In the second phase (caff), 5 mM caffeine was used to empty the SR
~ a " to ensure al1 cells begin at the same stage. In the third phase (5 pre-pulses), 5
conditioning pulses were used to replenish the SR with ca2'. In the founh phase (caff),
the L-rype Ic, and its kinetics of inactivation were measured by depolarizing the ceIl tri
+10 mV for 400 ms fkom a holding potential of -40 mV. This 400 rns depolarization was
repeated once in each test cell. The first depolarization was conducted in the absence of
caffeine. The second depolarization was applied in the presence of 5 mM caffeine (- 3
seconds) in the bath solution to deplete SR ~ a " . Two L-type Ica were recorded and their
inactivation kinetics were compared later. During caffeine application, IN=.Y was elicited
and recorded.
ramP caff 5 pre-pulses 9
5 mM caff 9
Fig 7. Voltage protocols for Cm and L-type ~ a " current inactivation with caffeine
intervention,
5.3.2 Cm measuremnt
To estimate ceIl size (cell surface area) change with growth, cell membrane capacitance
was measured in each cell. n i e ce11 membrane capacitance (Cm) was determined by
measuring the ce11 steady-state capacitive cumnt (I,,) [69] while the ce11 was
hyperpolarized by a steady-rate (fiom -70 to -80 mV, -2 mV/ms) ramp voltage pulse for 5
ms as showed in Figure 8. During the measurements, the contribution of the time and
voltage dependent currents was minimized by using a brief and small hyperpolarizing
\-oltagci change. The neonatal cells are srnall, so are the steady-state capacitive currents
(is,,p). To estimate I,,, more accurately in small cells, -2 mV/ms ramp rate was used
instead of -1 mV/ms rate [69] to enlarge I,,, in the neonatal cells (Fig 8). To keep the
Cm measurements of the four groups consistent, -2 mV/ms ramp voltage was used in al1
age groups. Cm is obtained from:
c m - - dQ / dV
- - (dQ / dt) x (dt / dV) Equation (1 )
The ramp rate (dV/dt) was -2 mV/ms in our experiments. Therefore, dt/dV has a value of
4 . 5 ms/mV. The dQ/dt is the ce11 capacitive current (I,,). Hence, equation (1) becomes,
- c m - La, x ( -0 .9
when I,,, reaches a steady state,
- c m - - 1/2 I,,
It is assumed that the sarcolernma bas a specific capacitance of about 1 @/cm2 [91]. The
ce11 surface area was then determined by multiplying the measured Cm by 1 xl O % ~ ' / ~ F .
Series resistance compensation was not required to achieve voltage control. because of
the relatively small size of the cells and small L-type ca2+ currents (4 250 pA) in our
experiments. The voltage drop across the series resistance of 15-20 MR was within an
acceptable range (c 5 mV). In al1 experirnents, the whole ce11 L-type ~ a " currents
rncasurcd in each ce11 were normalized by Cm to correct for age-relaird ce11 size
differences.
1-P
time tms)
Fig 8. An example of the membrane capacitance O,,) measurement. A: the voltage protocol of the steady-rate ramp (-10 mV/Sms). B: the recording of the membrane capacitive current O,,) from a rabbit cardiomyocyte (14-day-old). C: the equation used to calculate Cm according to the I,, obtained from Lp recording.
5.3.3 Voltage &pendence of l-fype C 8 cumnt
To eliminate K+ currents, myocytes were superfbsed with a K'-free bath solution
containing CsCl (Table 3). To eliminate the Na' cumnt, either TTX was added into the
bath solution or the pre-pulse voltage protocol was used (Fig 9.). The left ventricular
myocytes nxre perforated by amphotericin B. Afier the access resistrince lowered than
15-20 M O and became stable, a 30-millisecond and 30 rnV pre-pulse was applied to the
ceIl from its holding potential of -70 mV to -40 mV. The pre-pulse elicited Na' current,
and i t inactivated Na' current dunng the L-type b measurements (A. in Fig 9). The fast
kinetic and large (- 1 -5 nA) inward current was the Na+ current.
The L-type ~ a " currents (Ica) were elicited by depolarking the ce11 from its holding
potential of -40 mV to the different test potentials up to +70 mV at 10 mV increments and
%second intervals as shown in Fig 9. The time of depolarization was set to 400 ms, since
Ic. almost completely inactivated within 400 ms of the depolarization. L-type b was
sampled at 10 kHz and recorded. The L-type ~ a " current was measured as the small and
low kinetic inward current after the capacitive current (the second upward spike) as
shown in Figure 9. The L-type Ica could be blocked by 100 pM CdC12 (Fig 16 in Result).
During our experiments, the La, was induced almost simultaneously by the
depolarization. The 6, reached the peak within 0.5 ms and 95% of the L, disappeared
within the first 2 ms of the depolarization. The L-type Ica had a slow kinetics compared
with the h, and reached their peaks at aboui 8-15 rns of the onset of the
depolarization at al1 testing potentials in Our experirnents. This voltage protocol was
based on the Ica current voltage relation in neonatal and adult rabbit myocyte as shown by
Wetzel et al. [45]. in that study, they showed that the voltage dependence of the ~ a "
current was not significantly different between neonatal and adult animals with the
maximum Ica occumng at about +10 mV.
time (second)
Fig 9. The voltage protocol for IN. inactivation and L-type Ica 1-V plot. A: the voltage protocol (voltage is expressed in mV). INa was dissected by the 30 ms pre- pulse. B: recorded IN, and Ica by using the voltage protocol in A. The data were recorded fkom a 20-day-old rabbit myocyte (cell-98427001). The first large and fast kinetic inward current is the INa obtained by depolarizing the ce11 to 4 0 mV fkom the holding of -70 mV. The small and slow kinetic inward current is the Ica. Three Ica traces were obtained at testing potentials of +10 mV (124 PA), -10 mV (42 PA) and -30 mV (8 PA), respectively. The peak Ica occurred in response to a potential of +l O mV.
5.3.4 Abasurement of L-type b inactivation
The inactivation of L-type b and its relation to SR ~ a " release were monitored by use of
400 ms clamp pulses fiom a holding potentiai of -40 mV to +10 mV before and after
depleting ~ a " from the SR with 5 mM caffeine. Caffeine was rapidly and briefly (< 3
seconds) applied into the bath solution. To optimize the Ica and SR ~ a " uptake, ftve 100
ms conditioning pulses from a holding potential of -70 mV to O mV were applied at 5-
second intervals to load SR ~ a " (Figure 7). After comparing the numben of the pre-
pulse (5 versus 10 and 15) and SR ~ a " loading, 5 pre-pulses were chosen. since more
pre-pulses did not induce more SR ~ a " loading. The time constants for the inactivation
of the L-type Ica were obtained by fitting the calcium current decay with a second order
exponential using the least squares method (CLAMPFIT curve-fitting algorith), since
the single exponential could not describe Ica inactivation well. The goodness of the curve
fitting of a single and two exponential equations is shown in Figure 10. Two components
of inactivation were estimated by fitting Ica by a two exponentially decaying function,
equation (2).
I ~ ~ ( ~ ) = % + a, ë*" + az e -tm Equation (2)
where TI is the fast component and T? is the slow cornponent of the Ica inactivation.
t irne (ms)
tirne (rns)
Fig 10. Curve fitting of the L-type Ica inactivation. b resulting fiorn depoiarizing the left ventricular myocyte of a 10-day-old rabbit fkom a holding potential of -40 rnV to +10 mV (cell-98505005). Upper: the ka decay is fitted with a
single exponential equation (r =29.1 ms). Lower panel: the Ic, decay is
fitted with a second order exponential equation, ~ 1 4 5 . 3 ms and ?2=99.5 ms. The light colored and white line are the fitting curves.
The slow component of the L-type inactivation time constant (r?) was expected to
become small afier a certain age of the rabbit cardiac myocytes. This fastened
inactivation time would be eliminated by SR Ca" depletion. The difference between the
rz and r?, (with and without SR ~ a " load) was used to indicate the presence of "back
talk" between RyR and DHPR during ontogeny.
5.3.5 Measurement of SR content
After the first depolarization (refer to the voltage protocol in Figure 7.), the ceil was
voltage clamped at a membrane potential of 4 0 mV, exposed to 5 mM caffeine resulting
in the total SR ~ a ' + content being released into the cytosol. The released ~ a " could not
be sequestered back the SR because caffeine was presented in the bath until the time of
the znd depolarization. The attendant increase in cytosolic Ca" shifted the reversal
potential of the ~ a ' l ~ a " exchanger (nomally - -30 mV) to more positive values and
increased the driving force for normal mode exchange. As a consequence an inward
~a'ica'' exchanger current occurred and Ca" released from SR was removed to
extracellular space by NCX. Since it is assumed that as each time NCX transports 3 Nac
ions into the cell, in exchange, it transports 1 ~ a ' + ion out of the cell, one net charge is
gained at each tum. Therefore, when NCX transports SR caZ' released by caffeine into
extracellular space, the total charge gained via NCX can be quantified by integrating the
(Fig 11). Correspondingly, at the sarne time, the total charge carried by extmded
~ a " via NCX (Qca) can be expressed as:
QCa(t) = -2 I INCX - dt Equation 3
where t is the duration of INCX afier the leak current subtraction
Thus, the arnount of the ~ a " removed from cytosol via NCX during caffeine application
couid be caiculated from QCa- Based on the measured cell membrane capacitance, the
ceIl surface area is obtained. From the ce11 surface to volume ratio and the density of the
cytosol. ce11 rnass can be estirnated. Therefore, the SR ~ a " content can be evaluated by
normalizing the amount of ~ a " removed by NCX for the ce11 mass [28].
tirne (ms)
Fig 1 1. Integrating IN^^ for SR ~ a " content measurement. A sample INCX trace fiom a 20 days old myocyte (cell98502012).
5.3.6 Data and s&tistical analysis
Values are reported in the form of mean f SEM unless otherwise indicated. Within each
age group, L-rype calcium channel ~ a ' + current inactivation time constants changed by
caffeine application (T, vs. ri, and ~2 vs. szc. c stands for caffeine) were analyzed by two-
tailed Student's t-test for paired samples, respectively. Among the four age groups, the
variables (5 ,, TI ,, TI, and T-,) of calcium channel inactivation were analyzed individually
by one-way completely randomized analysis of variance (ANOVA). Other
developmental variables measured in this study including ce11 membrane capacitance, Ica
density etc., were analyzed by one-way ANOVA as well. If there was a significant
difference among age groups the Newman-Keuls test for multiple comparisons was used
[W]. Differences were considered significant if p < 0.05.
5.3.7 Materials
Al1 chernicals were purchased fiom Sigma Chemical Co. unless otherwise noted.
Table 3. Cornparison of extemal and intemal solutions (al1 concentrations are presented in mM) [29].
NaCl
CaCI:
CsCl
MgATP
MgCi2
glucose
TEA
EGTA
HEPES
Na-pyruvate
Na-phosphocreatine
PH 7.4 (with NaOH)
5
7.1 (with CsOH)
6. Results
Non-uniforrn appearance of rabbit ventricular myocytes sarnpled from the same hean was
very prevalent in the growing hearts, especially in the 10-day-old group. However, the
rabbit left ventricular myocytes used in our experiments were al1 clearly striated, but were
small and contained fewer sarcomeres compared to the adult ventricular myocytes (Figure
3 & Figure 1).
6.1 Rabbits and left ventricular myocyte growth
Rabbit left ventricular myocyte growth was evaluated by the ce11 surface area change.
Cell surface area was estimated from the cell membrane capacitance, which was
calciilated based on the measurernent of the cell steady-state capacitive current (I,,,) as
described in methods, since the biological membrane capacitance is assumed 1 p~lcmL.
The typical original recordings of the four age group ce11 capacitive cunents in response
to a - 10 mV (-2 mV/ms) ramp hyperpolarization fkom a holding potential of -70 mV to -
80 mV are shown in Figure 12. As expected, the ce11 steady-state capacitive current
increased up to 2-fold from day 3 to day 20. The mean membrane capacitance of the
myocyte doubled from 3-days (1 8.06 + 0.6 1 pF) to 20-days (3 5.6 i 1.3 pF), as shown in
Figure 13. The membrane capacitance of the myocytes gradually increased 22.8% and
39.4% by day 6 and day 10, respectively, and increased rapidly up to 96.9% by day 20,
compared to 3-day-old cells. The analysis of variance shows that age has a profound
effect on the rabbit left ventricular rnyocyte membrane capacitance (p = 6.0 x 10"~).
Xewmrin Ksuls test and Student's t-test indicate that the increase ce11 surface areas is
siçnificantly different between successive age groups @ = 1.99 x 10", 7.3 x lo4. and 6.2
x 1 O-' '. respectively).
-90 time (ms)
Fig 1 2. The representative recordings of whole-ce11 capacitive currents (I,,) fiom the
left ventricular myocytes of rabbits, aged 3-20 days. br was recorded in response to a rarnp pulse (-2 mV/ms) fiom a holding potential o f -70 mV to - 80 mV- I,,, is presented after leak current subtraction.
Fig 1 3. The left ventricular myocyte membrane capacitance change during postnatal
development of rabbit hearts. The values are in the form of mean f. SEM. N =
53 for each age group. Each *, ' and .denotes significant difference fiom the age group immediately before it.
Body weight increase with age is shown in Figure 14. 'fhe mean body weight increased
30.9% and 8 1.3% at day 6 and day 10, respectively, compared to 3-day-old rabbits. The
body weight increased markedly from àay 10 to day 20, and the rabbits more than
doubled their body weights during these 10 days. ANOVA indicates that age bas a
significant effect on the rabbit body weight. Newman Keuis test and Student's t-test
indicates that the increase of body weight is significant beniveen each successive age
P " P -
Fig 14. Rabbit body weight change with development. The values are in the form of
mean t SEM. N = 53 in each group. Each *, ' and .denotes significant difference fiom the age group immediately before it.
6.2 Voltage-dependent Cd+ curnnt
The voltage-dependent whole-ce11 ~ a " currents, Ica, were induced by depolanzing
arnphotericin B perforated isolated rabbit left ventrïcular myocytes with 2 mM ca2+ as the
permeable divalent cation in the bath solution (Table 2). The typical voltage and time
dependent L-type Ica is presented in Figure 15. Afier a 30 ms depolarization pre-pulse
fiom a holding potential of -70 mV to 4 0 mV, INa was inactivated as shown in Figure 9.
The L-type Ica was determined as the difference between the peak of the inward L-type
~ a " current and the current at the end of the depolarkation. Ica was recorded in a series
of 400 ms depolarizations fiom a holding potential of -40 mV to +70 mV with 10 mV
increments. ~ a " currents were activated at testing potentials more positive than -40 mV,
and the magnitude of Ica increased as the testing potentials became more positive than -
40 mV untiI + I O mV. As shown in Fig 15, the fast and large ouhvard capacitive current
appeared dmost simultaneously when depolarization started. and it reached its peak and
disappeared within 3 msec of the depolarization. The L-type Ic, reached its maximal
peak value when it was depolarized to +10 mV from its holding potential of 4 0 mV-
The maximal peak Ica in this myocyte was reached at 10 ms of the onset of the
depolarization, and inactivated to a steady state by 400 msec. When the myocyte was
depoIarized further positive than +IO mV, Ica started to decrease.
As observed in Fig 15, there were substantial outward background currents present during
the depolarization in al1 age groups. One of the control experiments showed chat when Ic,
was blocked by 100 PM cadmium (a known L-type ~ a " channel blocker), the
background currents were still present (Fig 16). In Fig 16, the large outward capacitive
current (Icap) appeared and reached its peak within the first 0.3 msec of the depolarization.
The fast inward IN, was elicited afier L, and the Is, was 99% inactivated within 6 msec
of the onset of the depolarization. There was no appreciable Ica detected in al1 different
testing potentials (trace b: +IO mV and trace c: +30 mV). Furthemore, the background
current increased when the ce11 was depolarized to the more positive potential (trace b vs.
c). The increase in background current was approximately linear to the increase in
66
voltage across al1 age groups (Fig17-18). The background current was almost constant
during the
time (ms)
Fig 15. Representative recordings of the whole-ce11 L-type ~ a " currents from the lefl
ventricular myocyte of a 6-day-old rabbit (cell-980714042). The holding
potential was 4 0 mV. The testing potentials were +IO, +20,0, +30 to +70 mV
starting from the position of the arrow. The peak Ica appeared at + I O mV. The
upward spikes at the begiming of the traces were capacitive currents.
depolarization as shown in Fig 16. To check the possibility that this outward background
current was caused by the ~ a + / ~ a " exchanger, 5 mM Ni+ was added into the ce11 bath
solution. The addition of Ni' partially blocked L-type Ica, but could only decrease the
outward current about 5-10 pA (data not shown). To correct for the background current
effect on the L-type Ica, the ka was determined as the difference between the peak and the
value at the end of the depolarkation.
tim (ms)
Fig 16. CdC12 blocks L-type Ica. Ica was blocked by 100 ph4 CdClz when the lefi ventricular myocyte of a IO-day-old rabbit was depolarized from a holding potential of -40 to the testing potentials of +10 mV (trace b) and +30 mV (trace c), respectively. Trace a: the ce11 was voltage-clamped at a potential of -40 mV.
The Ica and its voltage dependence for different aged myocytes were investigated while
controlling the temperature, pipette resistance and access resistance. Figure 17 illustrates
the voltage and time dependence of Ica fiom 3-day- and 6-day-old left ventricular
myocytes. Similarly, Figure 18 demonstrates the Ica fiom 1 O-day- and 20-day-old cells.
In Our experimental conditions, the inward L-type Ica was initially detected at -30 mV,
reached the peak values between 8 to 15 ms of the depolarization at al1 testing potentials,
and inactivation foIIowed. The maximum peak Ica was obtained at about +10 m V in al1
ages of myocytes within 8 to 9 msec of the onset of the depolarization and the magnitude
of the L-type Ic, increased with age (Fig 17 & 18). At the end of 100 ms depolarization,
IL-, neas almost inactivated completely at al1 tssting potentials in al1 four age groups. but
the inactivation time constants were not the same arnong age groups at the testing
potential of + 1 O mV.
time (ms)
B. 6day
time (ms)
Fig 17. Original Ica traces recorded from the left ventricular myocytes of 3-6 days old rabbits. The myocyte was depolarized fiom a holding potential o f -40 mV to
the testing potentials of - 10 and +10 mV. The Ica peaked at +10 mV.
time (ms)
tirne (ms)
Fig 18. Original Ica traces recorded from the lefi ventncular myocytes of 10-20 days old rabbits. The myocyte was depolarized from a holding potential of 4 0 mV to the testing potentials of - 1 O and + 10 mV. The Ica peaked at +10 mV.
The ~ a " current-voltage relationships (1-V) of the four age groups is displayed in Figure
19 using the same voltage protocol as described in Fig 9 (methods). The current-voltage
relations of al1 age groups show a similar characteristic "bel1 shape" with a maximum
peak current at around +10 mV, and the amplitudes of the Ic, continuously increasing
with age at potentials ranged from -10 to +40 mV. ANOVA indicated that there was a
significant age related increase @ c 0.01) in the magnitude of the maximum peak ~ a ' +
current. Student r-test indicated that the increase in maximum peak current from day 3 to
day 20 were significant in between each successive age group (al1 p < 0.01 for 3-day vs.
6-day. 6-day vs. 10-day, and IO-day vs. 20-&y). There was no significant voltage shift in
the 1-V reiation among the four age groups.
From 3-days to 20-days development, not only the maximum amplitude of the whole-ce11
L-type Ica increased with age (Figure 17 & Figure 18), but also the peak ca2* current
density increased at the same time. To eliminate the effect of myocyte growth during
development, the mean maximum peak Ica was normalized for the mean ce11 surface area
in each age g-roup. Ica density in pA/pF increased with age as shown in Figure 20.
During the first 3 weeks of life, the rabbit lefi ventricular myocyte Ica density drarnatically
increased from 17.9% to 42.3% and 68.5% from age of 6 to 1 O and 20 days, respectively.
The Ica density increased markedly by age 10 days and 20 days. Both increases were
significant @ < 0.01 as 6-day vs. 10-day, IO-day vs. 20-day). Although the Ic, density
increased from age of 3 days to 6 days, the change was not significant. Analysis of
variance suggests that age is a significant factor for Ica density increase.
voltage (mV)
Fig 19. The L-type ~ a " chamel current-voltage relations for four different age groups.
The values are in the form of mean k SEM. N = 44, 49, 47, 46 in the 36, 6d, 1 Od, and 20d group, respectively.
Ica rundown, a decrease in Ica with time after the myocyte is dialyzed with pipette
solution. is a prevalent phenornenon in the "routine" voltage clamp experiments. But in
Our amphotericin B perforated myocytes, it was rarely occurred during our data recording
period. The same amplitude of the L-type Ica could be recorded repeatedly up to 30 to 60
minutes, if the seal and the access resistance were stable.
c a CL Y
r Y .- V) E a O
0" -
Fig 20. The L-type ~ a " current density of four different age groups. Ica was elicited by depolarking the ce11 fiom a holding potential of -40 mV to +10 mV. The values are in the f o m of mean + SEM. N = 46 in each age group. * indicates a significant difference from 3-day and 6-day-old groups, and ' indicates a significant difference from the 1 O-day-old group.
Table 4. Summary of the developmental changes of surface area, Ic, density, charge density of Na', and body weight in single cardiac myocytes of four age groups.
3 d 6 d 10 d 20 d N
Peak 1, density 3.62k0.13 4.23+0.10 5-15kO-18 6.10i0.27 46
(pA/pF)
Cell surface area 18.06 + 0.61 22.17 + 0.63 25.20 + 0.68 35.60 + 1.30 53
( 1 0*cm')
Qx, (pC/pF) 0.4250.07 0.4540.05 0.66I0.03 0.61I0.05 53
Body weight (g) 74.57 k 1.45 97.64 t 4.34 135.2 + 2.29 308.10 + 7.10 46
6.3 CS dependent L-type Io inactivation
The effect of SR ~ a " release on L-type ~ a " current inactivation was examined by
exprrimentally manipulating the amount of ~ a " in SR. Five depolarization pre-pulses
from -70 mV to +10 mV were applied to each tested myocyte after the perforated patch
was obtained, by which SR was refilled with ~ a " . SR ~ a " was depleted by applying 5
mM caffeine into the ce11 bath solution. Afier the SR of each ce11 was fiiled with ~ a " by
the pre-puises, the myocyte was depolarized twice before and after caffeine application,
respectively, from its holding potential of 4 0 mV to +10 mV (refer to the protocol
shown in Figure 7). The change of Ica inactivation kinetics by changes of SR ~ a " release
were compared within each age group and among the four age groups (Figure 22).
The Ica inactivation rime constants (r) were determined by fitting the Ica inactivation
curves with a second order exponential function (refer to Methods), where two
components, fast (51) and slow (q), were obtained as shown in Figure 22.
t ime (ms)
Fig 21. The L-type Ica activation and inactivation. si and T? is the fast and slow Ica inactivation time constant, respectively. The Ica was elicited by depolarizing the left ventricular myocyte (20-day-old) from the hoiding potential of -40 mV to + 10 mV for 300 ms.
The fast inactivation time constant, si, is in a range of about 1 8-23 ms without caffeine
intervention and it is about 21-24 ms in the presence of caffeine (rl,) across al1 age
groups. Analysis of variance indicates that the fast inactivation time course has no
statistical difference with (ri,) or without (r i ) caffeine exposure arnong four age groups.
However, the Student's r-test suggests that in the presence of caffeine in the bath solution,
the fast inactivation time course (ri, vs. r i , p c 0.01) was significantly prolonged in 10
days group (Figure 22) and this change was not observed in the other three age groups.
Fig 22. L-type Ica inactivation time constants (T), for different age groups before and after caffeine application. ALI Ica was obtained by depolarizing the ce11 from a
holding potential of -40 mV to + 10 mV. The values are in the t o m of mean + SEM. N = 60, 64, 53, 66 for the 36, 6d, 10d and 20d group, respectively. ri
and sl, are the fast Ica inactivation time constants without and with caffeine
presence in the bath, respectively. sl and sz, are the slow Ica inactivation time
constants without and with caffeine in the bath, respectively. *: (T?,) is significantly different from 72 within the same age group. *: (Q) is
v significantly different fiom other age groups. : (TI,) is significantly different
from TI within the same age group.
The slow component of the inactivation (t?) is related to the ~a"-dependent Ica
inactivation [29]. As evident in Figure 22, only in the 20day group, is the Ica
inactivation tirne course significantly affected by SR ~ a " depletion @ < 0.000 1, T?, =
145.5 _+ 7.7 ms vs. Q = 113.6 I 4.4 ms). There is no significant caffeine effect on Ic,
slow inactivation kinetics (T? VS. n,) found among the other three age groups. When the
SR of four age groups refilled with ~ a " , the analysis of variance suggests that the Ica
inactivation time (tz) is significantly affected by the cell ages @ < 0.002, dotted bar).
ANOVA and Newman-Keuls test denote that the L-type ~ a ' + channel inactivation in the
20-day myocytes is significantly faster than that in 3-day, 6-day and 10-day-old cells (y <
0.0 1 ), while the sz of these three ages of cells are not different. In contrat to r?, 5 mM
caffeine eliminated the age-related difference of Ica inactivation kinetics, which means
that in the presence of 5 mM cafEeine, rt,s of al1 age groups are not significantly different
from each other. Again, only in 20-day-old myocytes, the slow component of Ica
inact ivarion time constant, rz, was markedly prolonged (28.2%) by SR caz' deplet ion,
and in other three groups, rr was only slightly increased by 5.7%, 6.0% and 5.4% for 3-
day, 6-day and 1 O-day group, respectively (Fig 23)-
Fig 23. The caffeine effect on the slow Ica inactivation time constant (rz) for different age groups. N = 60, 64, 53, 66 in 3d, 6d, 1 Od and 20d group, respectively.
6.4 Removal of [Ca], by ~ a ' / ~ e z * exchange
SR ca2' content of different ages of rabbit lefi ventricular myocytes was indirectly
assessed by a rapid application of caffeine. The ~ a " released from SR by cafEeine was
almost completely removed from the myocytes via NCX, since SR caL' re-uptake was
disabled in the presence of caffeine in the bath solution. When NCX removed the ca2'
ions out of the myocytes in exchange for the extracellular ~ a ' ions, it generated an
inward NCX current, as shown in Fig 24. Integration of caffeine-induced I N ~ X offered a
measurement of charges camed by ~ a ' transported via NCX during caffeine application.
After adjusting for ceIl growth with age, the net charges camied by Na' ions per unit of
ceil membrane capacitance is 0.42, 0.44,0.66 and O.6lpC/pF for 3-day, 6-day, 10-day and
20-day-old cell, respectively (Fig 25). The normalized ~ a ' charges in 10-day and 20-day
groups are significantly higher than that in the 3-day-old and 6-day-old groups. Newman
keuls test indicates that this Na' charge density is not statistically different between either
the 3-day-old and 6-day-old groups, or the 10-day-old and the 20-day-old groups (both p
> 0.05). In contrast, the Student's t-test indicates that the ~ a * charge density significantly
increased from day 6 to day 1 O @ = 0.001 1 ). This indicates that the net amount of ~ a '
charges per unit of ce11 surface membrane transported during caffeine application has
been critically changed from day 6 to day 10. The total amount of net ~ a ' charges that
cross the ceIl membrane induced by caffeine was increased with age. They were 7.59,
1 0.06, 1 6.22 and 1 9.9 1 pC for 3-day, 6-day, 10-day and 20-day group, respectively. The
amount of ~ a " removed from the myocyte via NCX with caffeine application was
calculated (in pmoVkg wet weight) according to the 3: 1 ratio of Na' and ~ a " transponed
by NCX at each turn. However, the calculations were based on several assumptions. It
was assumed that the lefi ventricular myocyte surface to volume ratio was 1.05, 1.05,
0.95 and 0.75 for 3-day, 6-day, 1 Oday and 20-day-old myocytes, respectively [56], and
that the density of the cytosol was 1.07g/ml [15]. According to these calculations, the
amount of caL' released from SR and removed from the ceIl by NCX with caffeine
application was 41.62, 46.24, 59.45 and 38.75 pmol/kg wet wt for 3-day, 6-&y, 10-day
and 20-day group, respectively.
caffeine
time (sec)
Fig 24. Ica and INcx recorded from a ventricular myocyte of a 6-day-old rabbit (cell- 9850 1055). The 1' 4, was elicited by depolarizing the ce11 to +10 mV from a holding potential of -40 mV after 5 pre-pulses in absence of caffeine. ïNcx was induced by applying 5 mM caffeine to the ce11 bath solution with the ce11 voltage clarnped at 4 0 mV. The znd Ica was elicited by depolarizing the ce11 to +10 mV from a holding potential of -40 mV with 5 mM caffeine. Currents were leak subtracted.
Fig 25. The net Na' charge transported via PJa'/ca2' exchange during caffeine application. The values are in the form of mean k SEM. N = 46 for each age group. * and ' indicate a significant difference from the 3-day and the 6-day groups.
7. Discussion
The present study focused on the postnatal (3-day- to 30-day-old) development of the
fûnctional coupling of L-type ~ a " channel and SR ~ a " release channel in rabbit lefi
ventricular myocytes. The major findings of this shidy are: 1) an age-related increase in
peak current density of the voltage-dependent L-type ~ a " channels; 2) the presence of
DHPR and RyR hnctional coupling in the left ventncular myocytes of 20-day-old rabbit
hearts; 3) L-type ~ a ' + channel inactivation time constant changed by SR ~ a ' + in 20-day-
old ventricular cells, but not in 3-day to 1 O-day-old heart cells.
7.1 Perforated patch clamp
In the whole-ce11 recording configuration, myocytes are likely to lose their intracellular
diffusible factors, including caZ' buffers through dialysis into the pipette. This can
account for, at least in part, the ~ a " current rundown which is almost invariably observed
during whole-ce11 ~ a " current measurement experiments. The advantage of using
amphotericin B perforated patch clamp is to keep the intact intracellular buffer system
and to prevent ka rundown. In Our experiments Ic, was fairly stable and could be
repeatedly recorded from the same ce11 for a long period of time (up to 60 minutes). Ica
run down rarely occurred if the seal and access resistance was stable. Arnphotericin B,
600 pglml, was used in the most of the 6 to 20-day-old myocytes. This concentration was
higher than the concentration used by other researchers (240 &ml) [83], [82]. However,
in our pilot studies, we did not find significant concentration-dependent difference in Ic,
and Ica inactivation over the range of 200-600 pg/ml within the same age group (data not
shown). For the ventricular myocytes from the 3-day-old rabbits, 200-300 pg/ml
amphotericin B was used to perforate the membrane. The high concentration of the dmg
induced ce11 contraction that sometimes worsened the seal or caused the drug leak into the
cells. When leakage occurred, the amplitudes of IN, and Ica were reduced dramaticall y
and the signal-noise ratio dropped markedly. This response was similar to what we
observed when the patch was purposely broke to allow amphotericin B to get into the ceIl
by giving a negative pressure on the patch. The disadvantage of using a low
concentration of arnphotericin B in our experiments is the increased waiting t h e to
obtain an access resistance Iower than 15-20 M R (> 1 hour). Even though we used 6 0
pg/ml to perforate the cells, Our perforating time (30-40 minutes) was still much longer
than those reported by others: 5- 10 minutes by Rae [83], 10- 15 minutes by Bassani 1821
and 5-30 minutes by Zhou [84]. The time difference may be caused by the different
geometry of the pipette tips used for current measurement [83].
7.2 Measurement of myocyte surface are8
Myocyte surface areas, to serve as an index of the ce11 growth, were calculated fiom
cellular capacitive current (capacitance) measurernents in Our study. The ceIl surface area
showed a small increase from 3 days to 10 days, and a large increase from 10 days to 20
days, reflecting the ventricular myocyte growing Pace after birth. Our measurernent of
ceIl surface areas are in reasonable agreement with the values of neonatal rabbit hem cell
surface areas reported by other researchers, approximately 22-25 x 104 cm' for 2-5 days
cells [69], [45]. The ceIl surface a x a increased non-linearly during the heart
development from 3 days to 20 days. These changes are not unexpected since cell length
increases 42% and ceIl diameter shows no change until 8 days post partum. 8-days after
birth, cell diameter increases dramatically (up to 2-3 fold) [56]. Cell diarneter change has
a profound effect on the surface area of the cell. The appearance of T-tubules can
partially account for the increase in ce11 membrane capacitance, since in adult cells T-
tubule can be up to 33-42% of the total cell membrane [15]. In the 20 days old cells, the
capacitance increased 96.6% comparing to the 3 days old cells, which indicates that the
ceIl membrane capacitance increase is due to the increase of ceIl surface area except the
T-tubule development. Our results are also supported by Hoeter's and Vasson's studies
[56], in which they used stereological analysis to evaluate morphological development in
fetal, newbom and adult rabbit hearts. They interpreted their data as follows: during the
later fetal and the first postnatal week, the heart grew mainly by myocyte proliferation.
However, starting from the second week, heart growth was characterized by marked
cellular hypertrophy.
7.3 Trans-sarcoIemmal c$' channels
In the Ica and the Ica inactivation kinetics cornparison experiments, a holding potential of
1 0 mV was used to inactivate T-type Ca" channels, even though the T-type ~ a "
channels are much less prevalent in immature rabbit ventricular myocytes (1-5 days old)
compared to adult cells [44]. If L-type ~ a " channel ion selectivity changes postnatally is
not clear so far. In our control experiments, 100 pM cadmium could completely block
the assumed L-type ~ a ' + current in al1 age groups.
7.4 L-type Ca2* current and the curmnt density
Although there are a few studies presenting evidence of developmental changes in Ica in
rabbit ventricular cells, the ages of the rabbits in those studies are fetal, neonatal (1-5 day
old) and adult (1.5-3.0 kg) [81], [72], [46], [69]. There are no data availabte, so far, to
describe the L-type Ca2+ current changes between the neonatal and the adult rabbit hearts.
Our study is the first one to investigate postnatal development of Ica in 3 to 20-day-old
rabbit ventricular myocytes using the perforated patch clamp technique.
During L-type Ica measurement, the outward background currents were corrected by
determining the Ic, as the difference between the Ica peak and the value at the end of the
400 ms depolarization. Therefore, the values of the Ica in the Ica measurement are
equivalent to the data with leak subtraction and the background current correction.
Our results show that the amplitude of the elicited maximum L-type Ic, increases
postnatally (Figure 16- 18). In developing rnyocytes, the increased ce11 size may partially
account for the increase of the whole ce11 Ica. However, after norrnalizing Io for myocyte
surface area, Ica density in 20-day- (6.10 pA/pF) and 10-day-old cells (5.15 pNpF) were
significantly higher than chat in 6-day-old (4.2 pNpF) cells. Within the fint week (3-day-
old vs. 6-day-old) Ic, density did not change signîficantIy (3.8 vs. 4.2 pA/pF) (Table 4)-
In Our study, we found that L-type Ica density increased fiom 6-day to 20day. which
parallels other investigators' results. These studies compared neonatal (1-7 day) and
adult ventricular myocyte L-type Ica in rabbit or guinea pig hearts. They found that in
both species, Ica and Ica density of the adult cells were significantly larger than those of
the neonatal cells [46], [45], [69], [67]. However, Vornanen et al. found that the Ica
density remained fairly constant in developing rat ventricular myocytes [93]. The
disparity of those results may be due to species differences. The smaller IG and the lower
Ica density in neonatal venuicular myocytes indicate that the L-type ~ a " chamel may
play a less significant role in generating the ceIl action potential and in raising
intracell ular ~ a " to produce force compared to mature rabbit ventricular cells.
Our mean values of the maximum Ica (- 50 to 90 pA in 3-day and 6-day, respectively) are
smaller than those reported by other investigators (1 14-150 pA by Wetzel et al. [45] and
Huynh et al. [69], in 1-5 days old venvicular cells of rabbits). However, afier adjusting
for the ce11 size change, Our peak Ica density (3.6-4.2 pA/pF in 3- to 6-day-old cells) is
close to the low end of the range of the peak Ica density reported by these investigators
(3.3-5.1 pA/pF by Wetzel et al. and 5.6 pA/pF by Osaka et al. in 1-5 days old cells). The
different values of the Ica or the peak Ica density of the same age of rabbits may be due to
the different expenmental conditions used, Ica decreased by 40% by just lowenng the
experimental temperature fiom 36C0 to roorn temperature as reported by Osaka et al.
[46]. After adjusting for temperature, our Ica would be the same as Osaka's.
Furthennore, both Wetzel's and Huynh's groups used 14 mM EGTA in their pipette
solutions to dialyze the rnyocytes and 10 mM ~ a " as charge carriers in the ce11 bath
solutions as they recorded the Ica in a whole-ce11 configurations. High concentrations of
the ~ a " chelator (EGTA) might keep the intra-cellular ~ a " activity at a relatively low
level ( 100- 150 nM) [29]- Thus, Ica might be enlarged by eliminating the direct and the
indirect ~ a " related L-type ca2' channel inhibition [29], [94]. High extra-cellular ca2+
would generate a relatively large driving force for ~ a ' + passing through the L-type ~ a "
charnels when the channels were assumed to be hlly opened at +10 mV in the rabbit
ventncular cells of the different age groups.
During our L-type ~ a 2 ' current measurement, we used a holding potential of 4 0 mV in
al1 cells of al1 age groups to eliminate the T-type ~ a " current. However, we cannot
exclude the T-type ~ a ' * current contamination completely, since at 4 0 mV, T-type ~ a "
channels may not be completely inactivated [15]. There is no data available on voltage
dependency of T-type ~ a ' + current in rnammalian neonatal cardiac myocytes. According
to the studies in adult cells, the degree of T-type ~ a ' + current contamination in our snidies
would be low. It is because the T-type ~ a ' + channel is much less prevalent in the
neonatal rabbit cardiac myoçytes compared to adult cells and the residual activating T-
type ~ a " current is small at 4 0 mV [44], [ 151.
Despite the different experimental conditions used by different investigators, al1 studies
of the Ica in rabbit h e m agree that the Ica and the peak 4, density increase postnatally
throughout heart development. This implies a few possible changes occur during cardiac
myocyte growth, which include: 1) L-type ~ a " channel expression may increase after
birth: 2) the functional L-type ~ a " charnels may increase postnatally. This may be due
to in changes of the channel intrinsic property, such as increasing single channel
conductance andor channel open probability; 3) L-type ~ a ' + channel regulatory factods)
may exhibit developmental changes, therefore to affect the Ica density.
7.4.1 DHPR expmssion
Brillantes et al. 1631 found that the mRNA of DHPR increased posmatally in the rabbit
heart. However, this does not necessarily mean that the DHPR protein increase during
that same period of time. Several specific ligand-binding studies in neonatal and adult
rabbit hearts do not suppon the DHPR expression change hypothesis. Boucek et al. used
enrkhed isolated sarcolemmal vesicles to estimate the arnount of the DHPR in 2-3 weeks
old rabbit hearts. They found that the numbers of the L-type ca2+ channels identified as
the B, for DHP-binding and that the dissociation constant had no developmental change
[63]. However, Boucek et al. might have underestimated the numbers of DHPR because
of T-tubular membrane loss during the sample preparation. Thomas fiom Our lab avoided
this problem by using crude homogenates of whole rabbit hearts. To be consistent with
other studies in our lab, Thomas used the same age groups of rabbits as we did, and she
found the similar results as Boucek et al.'. This suggests that the expression of al subunit
of the L-type ~ a " channel may not exhibit developmenta! changes in density in rabbit
ventricular myocytes. It is worthwhile to note that the speciiic ligand-binding assay can
only test the numbers of the ai subunit in the myocytes, since the dihydropyridine-
binding site of the channel is on the a, subunit. The specific binding results do not
necessarily reflect the numbers of the fÙlIy fünctioning channels in the hearts, since the
other four subunits (az, B, y and 6) of this type of ~ a " charnel have regulatory effects on
DHPR function [95], [96]. To date, these regulatory roles are still under intensive bio-
molecular and electrophysiological investigation.
7.4.2 Functional L-type Cd+ channels
Single channel studies demonstrate that cloned cardiac al subunits expressed in Chinese
hamster fibroblast cells exhibit four distinct levels of conductance (22.7, 14.3,6.2 and 3.2
pS). The coexpression of f3 subunits significantly increases the open probability (Po) in
al1 four Ievels of conductance without changing the conductance [97]. This is consistent
with a previous study by Wakamon et al. [98]. ïhey expressed cloned human heart L-
type ~ a " channel ai subunit in Xenopus oocytes and found that I== activation and
inactivation were much slower than that of the native cells. The presence of the B subunit
could accelerate the Ica kinetics by increasing Po without changing the channel open time
and conductance. Therefore, the P subunit could increase Ica amplitude. There is no
information available about the developmental change of the cardiac DHPR B subunit.
But Brillantes et al. reported that the rabbit skeletat muscle DHPR P subunit mRNA
reached adult values on the first day of birth [63], suggesting that these subunits of the
~ a " channel may have developmental changes. Thus the following questions need to be
addressed: first, is there an age-specific cardiac a, subunit conductance in the developing
heart? second, if so, does the p subunit itself undergo developmental changes? third, if
the p subunit does change, how does this change affect L-type Ica in the developing hean?
7.4.3 Possibk regulatory factor(s) changes
Within the P adrenergic receptor-G,-adenylate cyclase regulatory system as described in
section 2.5.2, any enzyme activity change(s) in this system that affects the intracellular
CAMP levels or channel phosphorylation will affect the Ica of the cardiac myocytes.
Therefore. even though the L-type ca2+ channel intrinsic properties (the basal level of L-
type caL' charnel hinctions) rnay be the sarne in al1 age cells, the I=- densities of the cells
of different ages could still possibly be different. There is evidence that both PDE and
phosphatase may change with age in rabbit hearts. The most important PDE isozyme for
regulation of Ica in rabbit myocytes changes from PDE IV (CAMP specific PDE) to PDE
III (cGMP-inhibited PDE) during the postnatal period [49] and the PDE i l1 activity
increases fivefold during rabbi t ventricular myocyte maturation [99]. Phosphatase
activity in newbom rabbit heart cells is greater than that in the adult heart cells [100].
Furthemore, postnatal increases in the basal level of adenylyl cyclase activity were found
in the developing ventricular cells in rat [ l O I ] . Thus, the possible reasons for the
postnatal increase of Ica density may rely on hypotheses 2) and 3) as listed above.
7.5 Voltage dependence of the L-type CaZ* current
In present study, although the maximum elicited peak Ica of ventricular myocytes (Figure
16) kept increasing with age, the current-voltage relations (I-V plot) were uniform and
"bel1 shape" in the four age groups. In addition, the maximum peak currents in the four
groups were induced around the same potential, at about +10 mV. This finding is
consistent with previous studies in rabbit [46], [69] and guinea pig [67] veniricular
myocytes. In these studies, the L-type caZ' currents in adult cells were significantly
larger than those in neonatal cells, but the potentials (W10 mV) to elicit the maximum
peak Ica exhibited no age difference, which suggested that there was no current shifiing
during cardiac development. These similaritics in L-type ~ a " current and voltage
relations in al1 age groups indicate that the voltage gating property of the L-type ~ a "
channels do not change from day 3 to day 20, perhaps to adult as well.
7.6 L-type Cah chennel inactivation
According to the goodness-of-fit that is depicted in Figure 10, we chose two exponential
functions to quanti@ the whole ce11 L-type ~ a " current inactivation in each of the four
age groups. The two time constants (si and n) were different by a factor of 5, suggesting
that there were two different factors to affect the channel inactivation. In Our
experiments, the fast inactivation time constants (r ,) of the four age groups were similar
(about 19-23 ms without caffeine) and were unaffected by caffeine application (about 19-
24 ms with caffeine), except in the IO-day group. This indicates that in general, the fast
inactivation does not change with age and is not SR ~ a " related. According to statistical
test the fast time constant (ri) was significantly prolonged by caffeine but only in 1 O-days
group (18.8 vs. 23.4 ms), it is dificult to interpret it as an age related efTect. It may
represent the difficulty in choosing the same experimental conditions as an optimal
condition for al1 age groups. Vornaneon et al. (1996) had similar observations in the
developing rat heart [93]. However, Sham et al. (1995) showed that in adult rat cell, the
fast inactivation was also siowed down by caffeine 1291. The reason for these different
observations of fast inactivation in response to caffeine is not clear. If this fast
component of the inactivation is not completely [ca2']i-dependent, it may be, at least,
partiaIIy voltage-related.
In contrat to TI, the slow inactivation component, rl, did not show changes fiom day 3 to
day 1 O (about 130- 140 ms), but it substantially decreased to -1 10 ms at day 20. This time
course difference could be abolished by 5 mM caffeine, suggesting that the slow
inactivation is profoundly affected by the caZf released from SR. Our finding is in
agreement with the results of Tseng [102], Sham [29] and Masaki [5 1 1. They have also
reported that caffeine or ryanodine can prolong the Ica decay time course in adult canine,
rat and transgenic mouse (overexpression of phospholamban) ventricular myocytes. In
addition to activating RyR causing release of SR ~ a " , caffeine is also a
phosphodiesterase inhibitor that can increase intracellular CAMP. Therefore, it can
increase Ica by enhancing L-type ~ a " channel phosphorylation. A larger Ica will
accelerate L-type ~ a " channel inactivation [29]. To test the possibility that Our rz change
was a result of an enlarged Ica by caffeine-phosphodiesterase inhibitory effect, we used 30
mM 3-is0 buty l- 1 -methyl-xanthine (IBMX), a nonselective PDE inhibitor, as the caffeine
substitute to investigate the sr change. Under the similar experimental conditions, using
the sarne protocol as shown in Figure 7, we did not observe IBMX-related tz change in
any of the age groups (data not shown). However, when we applied 30 pM IBMX into
the bath solution for more than - 3 seconds, Ica kept increasing markedly with the
decreasing tl (data not shown).
Our results can be explained by morphological studies of the developing hem. These
studies demonstrate that in the mammalian hem, immature ventricular cells have an
under developed T-tubular-SR system as described previously. This is further supported
by irnmunolabelling studies of DHPR and RyR in adult and neonatal rabbit hearts by
using double labeling immunofluorescence and laser scaming confocal microscopy. in
adult rabbit ventricular cells, Car1 et al. (1995) found a complete punctuate bands spaced
at -2 Fm intervals along the whole length of the muscle fibers, indicating the close spatial
association of DHPR and RyR at T-tubules [103]. From our lab, Farhad Sedarat found
that the time course of the formation the close spatial localization of DHPR and RyR tit
well to the time course of the T-tubule maturation in rabbit ventricular cetls. He observed
that the CO-localization of DHPR and RyR originates from discrete spots along the
sarcolemma before age of 8 days to even distribution along the developing T-tubules
throughout the most of the space of the whole ce11 at age of 20 days [104].
DHPRs and RyRs not only establish a close spatial association but also exhibit functional
coupling in adult mamrnalian cardiac myocytes [29]. Thus, it seems logical that SR ~ a "
could shape the time course of the L-type ~ a " channel inactivation of the myocytes from
20-day-old rabbit hearts, since the DHPR and RyR become spatially CO-localized at this
developrnental stage. The SR ~ a " did not affect the time course of the L-type ~ a "
channel inactivation in the myocytes younger than 20-&YS, suggesting that the DHPR and
RyR were not spatially close and functionally coupled before the age of 20 days. Before
T-tubule development (< 8-1 0 days old), DHPRs are distributed in the sarcolernma where
they can associate with RyRs. The limitation of the spatial resolution of confocal images
of the irnrnunolabelling still places the "spatial CO-localization" of DHPRs and RyRs
wi thin a speculative distance. The undetectable ca2+ -dependent ~ a " c hannel inactivation
in 3 to 10 days old rabbit myocytes may suggest that at these myocardium developmental
stages DHPRs and RyRs are not in the positions within the "microdomain" [27] or T a "
release unit" [105]. The Ta2 ' release unit" is proposed to be exist in adult cardiac
myocytes [38], [105]. Therefore, the absence of T-tubules results in a different cellular
architecture in neonatal cells from the adult cells. The functional ontogenic change is
secondary to the structural ontogenic change between DHPRs and RyRs. More studies
are necessary to clariS, the spatial [ca2']i changes in the developing heart without T-
tubules and the non-uniformities in [ca2']i is highly expected.
Our results contradict the studies of Kato et al. and Wetzel et al. They did not identiw a
drvelopmental change of the ~ a " dependent ~ a ' + channel inactivation in either rabbit or
guinea pig ventricular myoçytes (-70-80 ms in rabbit and -40-50 ms in guinea pig) [67],
[72]. Furthemore, in Wetzel's study, the L-type ~ a " channel inactivation in rabbit
ventricular cells were faster (-80 ms in fetus, neonate and adult cells) than that we found
(- 120 ms in 20-day and -140 ms in 3-day-, 6-day- & 10-day-old cells). The discrepancy
of the results from different research groups is not well understood. Intracellular buffers
rnay also change this inactivation time course by directly affecting the [ca2*]i. The
"routine" whole-ce11 voltage clamp technique and the 10-14 m M EGTA in the interna1
solutions used by other researchers would also change the intra-myocyte ca2+ buffer
system compared to Our perforated patch clarnping without EGTA dialysis into the cell.
Of course, Our experiments were based on an assumption that the intracellular ~ a " buffer
capacity was relatively constant during day 3 to day 20.
There were consistent outward background currents during Our Ica recording. Using 100
Fm cadmium to block Ica, these currents showed a linear increase with the voltage
increase to more positive direction with the reversal potentials of -50 rnV in four age
groups. They were about 2540 pA when cells were depolarized to +10 mV from a
holding potential of 4 0 mV. The time dependence of these currents was small. Thus
they would affect the amplitude of the Ica but not the inactivation kinetics of the Ica very
rnuch. Ni' (5 mM) could only decreased -30% of these background currents, indicating
that the INCX might contribute to a part of these background currents. The INCX activation
time course might overlap to the Ica inactivation time therefore to interfere with the
estimation of the Ica inactivation. However, the outward currents did not show a
significant difference among the age groups, these currents affected the Ica kinetics would
in a same manner among four age groups. As a result of this, the absolute values of the
time constants of Ica inactivation of four age groups would be still comparable among the
four age groups. The increase in [Cali rnay also activate other channels to result in non-
specific cation currents.
1 t should be noted that Ca2'-dependent ca2+ charnel inactivation is subject not only to the
~ a " released from the SR, but also to the ~ a " influx through the L-type ~ a " channel
[29], [106]. AAer caffeine depleted SR ca2', there was still substantial inactivation
present. This residual inactivation may be related to the ~ a " influx through L-type ca2+
channels andor NCX [107]. Both neonatal and adult rabbit cardiac myocytes posses
~a"-dependent ~ a " channel inactivation, which was demonstrated by Toshiyuki et al.
using ~ a " as the charge carrier to prolong the Ca2+ charnel inactivation [46]. If SR ca2+
does not affect Ica inactivation in the neonatal cells as we present in this study, the trans-
sarcolemmal ~ a " influx could be responsible for this ~a"-dependent Ica inactivation in
thesr immature cells. The molecular mechanisms of the ~a"-dependent ~ a " channel
inactivation are under intensive study, especially the function of the a,, sub-unit EF-hand
binding site. However the results of these studies present an inconsistent picture of ~a" -
dependent Ica inactivation [41], [52], [50], [53].
7.7 SR ce2' content
Rapid application of caffeine induces SR Ca" release. The released ~ a ' + quickly
combines to cytosolic ~ a " buffers and at the same t h e , Ca" is extruded out of the ce11
by ~ a ' / ~ a " exchange. In the presence of caffeine in the bath solution, which makes SR
extremely Ieaky, the ~ a " released from the buffers is not be able to be sequestrated back
to the SR for storage, and is transported continuously to the extracellular space by NCX.
Therefore. the amount of ~ a " transported out of the ce11 by NCX can be used as an index
to indirectly evaluate the SR releasable ~ a " [ 151. According to the measured IN^^, we
estirnated that the SR releasable ~ a " was about 40-60 pmolkg wet W. in four age rabbit
ventricular myocytes (Table S), if it was assumed that 100% ce11 capacitance measured
represented surface membrane. If the T-tubular membrane was assumed to be 33% of the
total sarcolemma of the 20-&y-myocyte and sarcolernmal caveolae was assumed to
attribute -1 0% of the total sarcolemrna in four age myocytes [15], the SR Ca" content
was calculated to be 45-80 pmoVkg wet wt (Table 5) . Our calculation is consistent with
previous findings in other adult mamrnalian ventricular cells such as rat [15], 100-200
pmolkg wet wt. The calculation is based on a few assumptions including the ce11 surface
to volume ratio of some of the age groups and the intracellular buffer condition, etc.
(Table 5). Since the released Ca" quickly equilibrated with the intracellular buffers the
precise value of the SR content cannot be determined. Therefore, the accuracy of the
value may be at a range of * 50% [Ml. The similar arnount of SR ~ a " content per cell
kg wet wt. in the different age groups does not mean that the total SR content is the same
in these four age myocytes, since the volume of the SR almost has been doubled during
the ventricular myoçyte growth (Table 1). But this number does mean that after
normalized for the ce11 mass, the SR of a neonatal rabbit ventricular myocyte may have a
sirnilar ~ a " storage capacity to that of a more mature ce11 (3-days vs. 20-days). But the
stored ~ a ' + requires certain mechanisms to be released. One important difference
between immature and mature cardiac myocytes is the degree of involvement of the SR
~ a " in intracellular Ca" regulation and in cardiac myocyte excitation-contraction
coupling, both of which are based on the formation of functional coupling between the
DHPR and the RyR. In another words, the ontogeny of E-C coupling relies critically on
the spatial arrangement of L-type Ca" charnel and SR ca2+ release channel.
Table 5 . Estimation o f SR releasable ~ a " of four age groups.
3 days 6 days 10 days 20 days
Icap (PA) 37.08 44.24 50.18 69.66
Surface area 1854 2212 2509 3483
Assumed surface/volume (cim-9"
Volume 1765 2106 2641 4975
Weight (ng) 1.89 2.25 2.83 5.32
Net ~ a ' influx via NCX (1 O-" pmol)
SR releasabie ~ a " (pmoVkg wet wt) 4 1.62 46.24 59.47 38.75
Assurned T tubule (% of S L ) ~ O O 2 O 33-42
Assumed caveolae (% o f S L ) ~ 10 10 10 10
Adjusted SR releasable ~ a " (pmolkg wet wt)'
" [561 WI
C Afier the adjustment of assumed T tubule and caveolae of each ce11 of each age group.
8. Conclusions
In mature mammalian cardiac myocytes, the refined cellular DHPR-RyR architecture
enables these two ~ a " charnels to be functional coupled. Therefore, DHPR and RyR are
the central players of the excitation-contraction coupling process. In contrast, the DHPR
and RyR of the immature mamrnalian cardiac myocytes do not have this delicate
structural association because of the less deveioped SR and T-tubules. This alters the
d e s of these two ~ a " charnels in the ce11 E-C coupling. Using an electrophysiological
approach, 1 have investigated the ontogeny of the functional coupling of the L-type ~ a "
channels and the SR ~ a " release charmels in the left ventricular myocytes of rabbits,
aged 3-20 days. In this study, 1 have observed the following for the development of the 3-
20 days old hearts of rabbits:
The cardiac myocytes grow quickly. The most obvious morphological change was
the change of ce11 appearance from the long cytindrical to the wider and more
rectangular shape. The ce11 surface area dramatically increased 22.8%, 39.4% and
96.9% at by the age of 6, 1 O and 20 days, respectively.
The L-type Ica of the lefi ventricular myocytes of rabbits increased from -60 pA to
-2 10 pA during the fint 3 weeks afier binh. The L-type Ica density of these single
cardiac myocytes slightly increased 17.9% during the first week (3-6 days) afier birth
and significantly increased 42.3% and 68.5% by the age OC 10 days and 20 days,
respectively .
The current and voltage relations of the L-type Ic, of al1 age groups had a similar
characteristic "bel1 shape" and the maximum peak Ica occurred around +10 mV in
four different age groups. This indicates that the gating property of L-type ~ a "
channels has no developrnental differences.
The L-type ~ a " channel inactivation (t2) increased significantly in 20-day-old cardiac
myocytes ( 1 3 8.9f 7.8 ms of 3 days vs. 1 1 3 -6k4.4 ms for 20 days, p< 0.002) but had no
age-related difference arnong 3-10 day old age groups. Furthermore, this faster
inactivation of L-type Ica in 20-day group couId be eliminated by 5 mM caffeine
induced SR ~ a " depletion (145.5k7.7 ms with 5 mM caffeine, p < 0.0001),
suggesting that the faster inactivation in 20 days old myocytes are SR Ca2+ dependent.
The total releasable SR ~ a " significantly increased in the lefl ventricular myocytes of
3-20 days old rabbits (7.9-20.6 x IO-' ho l ) . However, the releasable SR Ca"
content was aImost the same in four different age groups in tems of the ce11 mass.
I t is suggested from these data that the left ventricular myocytes of the rabbit undergo not
only rnorphological changes but also functional change during the first 20-days of life.
The age-related increased peak Ica may be attributed mainly to the increase in the number
of functional L-type ~ a ' + channels while the voltage dependence of this channel does not
change during the development under Our experirnental conditions. The developmental
change of the kinetics of Ica decay in 20 days old myocytes dernonstrated that DHPR and
RyR were functionally coupled ("back talk") at this stage, but not in the myocytes from
younger nbbits. This suggests that at the age of 20 days, these two ~ a " charnels have a
close spatial retationship as they do in the mature mammalian cardiac myocytes.
Although the SR ~ a " content expressed as per ce11 wet weight has no significant age-
related difference, the SR ca2' may not contribute to the same extent to the ce11
excitation-contraction as it does in the adult celf. As a result of this that DHPR and RyR
are neither structurally nor functionally mature in the ventricular myocytes of the rabbits
younger than 20-day-old. Therefore, the mechanism(s) of E-C coupling in the immature
myocardium may change with deveIopment-
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