Role of the sarcoplasmic reticulum in the modulation of rat cardiac action potential by stretch
Transcript of Role of the sarcoplasmic reticulum in the modulation of rat cardiac action potential by stretch
Role of the sarcoplasmic reticulum in the modulation of rat
cardiac action potential by stretch
C . H A N , 1 , 2 P . T A V I 1 and M . W E C K S T R OÈ M 1 , 2
1 Department of Physiology, Division of Biophysics, and Biocenter Oulu, University of Oulu, Oulu, Finland
2 Department of Physical Sciences, Division of Biophysics, and Biocenter Oulu, University of Oulu, Oulu, Finland
ABSTRACT
We have investigated the role of sarcoplasmic reticulum (SR) in the modulation on rat action
potentials by stretch. The action potentials were recorded intracellularly from rat atrial myocytes in an
isolated atrial preparation with small, physiological stretch produced by pressure (1±3 mmHg) inside
the atria. The SR function was inhibited by pharmacological interventions, either with ryanodine
(100 nmol L±1), thapsigargin (10 nmol L±1) or caffeine (1 mmol L±1). The duration of action potentials
was increased by stretch from 1 to 3 mmHg. The repolarization indices APD30% (P < 0.05), APD60%
(P < 0.01), and APD90% (P < 0.01) were all increased signi®cantly (n� 10). Ryanodine, thapsigargin,
and caffeine inhibited this prolongation, or even reversed the effect with repolarization indices
APD30% (P < 0.05) and APD60% (P < 0.05) which decreased in stretch with thapsigargin treatment.
As a conclusion, we suggest that the SR and the intracellular calcium balance play an important role in
the modulation of the shape of the rat atrial action potential during stretch.
Keywords caffeine, membrane potential, ryanodine, sarcoplasmic reticulum, thapsigargin.
Received 8 October 1998, accepted 24 June 1999
The effects of stretch on the shape of the action
potential (AP) of the myocytes are important
concerning, e.g. the arrhythmogenesis caused by stretch
(Tavi et al. 1996). In species with long AP plateau, such
as the guinea-pig and the rabbit, stretch reduces the
duration of the AP (Lerman et al. 1985, Dean & Lab
1990, White et al. 1993, Tung & Zou 1995, Wang et al.
1996). In contrast, stretch of cat papillary muscle
increases the duration of the APs at 80% repolarization
level (Allen 1977).
The stretch-sensitivity of the cardiac cells has
previously been explained on the basis of stretch-
activated ion channels (SACs) (Bustamante et al. 1991,
Sasaki et al. 1992, Kim 1992, 1993, Ruknudin et al.
19931 ) that would directly, by introducing an additional
conductance, change the shape of APs. Using the
so-called monophasic AP recordings from isolated
rabbit heart, combined with computer simulation that
included the SACs, Zabel et al. (1996) found that
stretch shortened the APs in early repolarization, but
increased the duration at late repolarization. However,
in cardiac myocytes the waveform of APs has also been
shown to be dependent on the size of the Ca2+ tran-
sients, via modulation in the Na+±Ca2+ exchange
current (Schouten & ter Keurs 19852 , DuBell et al.
1991). Interestingly, stretch increases the (systolic) Ca2+
transients after stretch (Allen & Kurihara 1982, Hongo
et al. 1995, 1996, Kentish & Wrzosek 1998, Tavi et al.
1998), suggesting that the sarcoplasmic reticulum (SR)
Ca2+ load is in¯uenced by stretch. This is because the
Ca2+ transients mainly develop from the Ca2+ released
from the SR (Delbridge et al. 1996, Bers 1997).
Supporting this, by using rapid cooling contractures,
Bluhm & Lew (1995) found that the Ca2+ content in
the SR is larger after stretch. Because the amount of
Ca2+ released is strongly dependent on the amount
stored in the SR (Janczewski et al. 1995), we postulated
that the SR should play an important role in the
modulation of cardiac APs in stretch. This hypothesis is
strongly supported by a recent study from our labora-
tory (Tavi et al. 1998), which suggested that in rat
atrium small, physiological stretch modulates the shape
of APs mainly via changes in the Na+/Ca2+ exchanger
current that are caused by increased calcium transients.
While this is in contrast to the proposed direct effects
of stretch-activated conductances on the APs in the
rabbit ventricle (Zabel et al. 1996), we investigated in
this study how the APs change during stretch when the
Correspondence: Prof. Matti WeckstroÈm MD PhD, Department of Physiology, University of Oulu, Kajaanintie 52 A, FIN-90220 Oulu, Finland.
Acta Physiol Scand 1999, 167, 111±117
Ó 1999 Scandinavian Physiological Society 111
SR is incapacitated by pharmacological interventions.
The main hypothesis was that with small, physiological
stretch the changes in calcium balance in the rat atrial
myocytes are more prominent than the direct effects of
any stretch-activated conductances on the shapes of the
APs. Thus, by incapacitating the SR, we should be able
to abolish the stretch-sensitivity of the APs.
MATERIALS AND METHODS
Animals, preparation and superfusion
Male Sprague±Dawley rats (n > 50) weighing 290±
400 g were used. The rats were decapitated, and their
hearts were rapidly removed and placed in oxygenated
cool (25 °C) buffer. The methods and the set-up used
to simulate the physiological stretch effect in rat left
atrium have been described in detail previously (Laine
et al. 1994, Tavi et al. 1996). Brie¯y, an X-branch
polyethylene adapter was inserted into the lumen of the
left auricle, and the tissue was placed in a constant
temperature (37 °C) organ bath. Another tube with
smaller diameter was inserted inside the adapter in
order to carry perfusate in¯ow into the lumen of the
auricle. The out¯ow from the lumen came from one
crossbranch of the X-cannula. The out¯ow tube was
connected to the chamber of a pressure generator
(WGA-200, Millar Instrument, USA), by which the
increased pressure could be generated into the lumen of
the atrium. The other crossbranch of the X-cannula
was connected to a pressure transducer (TCB 100,
Millar Instruments, USA), so that the pressure could be
monitored with an oscilloscope. In¯ow and out¯ow
(3 mL min±1) both to the auricle lumen and to the
organ bath (with constant temperature) were controlled
by a peristaltic pump (7553±85, Cole-Parmer Instru-
ment, USA).
Electrophysiological recordings
Intracellular APs were recorded at 37 °C with standard
glass microelectrodes with tip resistances of 70±
120 MW when ®lled with a solution 2 mol L±1
K-acetate and 5 mmol L±1 KCl (pH 7.0). To record
from moving (contracting and stretched) tissue, a
¯exible holder attachment for the microelectrodes was
used. The left atrial appendix was stimulated electrically
through bipolar Ag/AgCl electrodes placed in contact
with the auricle. Electrical stimulation (steps of 1-ms
duration, 50% over threshold voltage) was provided by
a stimulator (S44, Grass Instruments, USA). Membrane
potential signals were ampli®ed with an intracellular
ampli®er (Dagan 81001±1, Dagan, USA) and sampled
at 5 kHz with a 12-bit analogue-to-digital converter
(DT2821, Data Translation, USA). Data analysis was
done with MATLAB (The Math Natick, MA, USA) and
Origin (Microcal, USA) programs. The criteria for
accepted APs were: the resting potential at least
±70 mV and the overshoots of the APs at least 10 mV.
The APs were analysed with a MATLAB-based ana-
lysing program, and the durations determined were
APD15%, APD30%, APD60% and APD90% (where APD
means `action potential duration' and the following
number stands for the percentage of repolarization at
the determined time).
Experimental protocols
To study the physiological effects of stretch in the rat
atria all preparations were paced continually and were
left to stabilize in constant (diastolic) 1 mmHg pressure
level at least half an hour after preparation. Thereafter
the pressure inside of the atria was gradually but rapidly
increased from 1 to 3 mmHg. The pressure was held at
this level at least 10 min before recording. In phar-
macological interventions, ryanodine (100 nmol L±1),
thapsigargin (10 nmol L±1) and caffeine (1 mmol L±1)
were added to the normal superfusion solution at the
beginning and throughout the experiments. The
recordings were made not earlier than 30 min of
superfusion with a solution containing these agents, but
normally even later. The suf®cient depletion of the SR
was checked by monitoring the contraction of the atria,
which was abolished in all cases with all the pharma-
cological treatments used. The effectiveness of the SR
depletion may also be judged from the drastic effect
the treatments have on the shape of the APs (see
Table 2).
Solutions and agents
In all experiments we used a Krebs±Henseleit solution
of the following composition (as mmol L±1): 113.8
NaCl, 4.7 KCl, 17.6 NaHCO3, 1.2 KH2PO4, 1.1
MgSO4, 2.5 CaCl2, 11.0 glucose. Gassing this solution
with 95% O2 and 5% CO2 adjusted the pH to 7.4.
Ryanodine was obtained from Calbiochem (San Diego,
USA), thapsigargin from Alomone Labs (Jerusalem,
Israel), and caffeine from Sigma (USA).
Statistics
All results are given as mean � SE. The statistical tests
were done using either SigmaStat (USA) or Origin
(Microcal, USA) software. ANOVA, followed by
Student±Newman±Keuls test, or the student's t-test,
where appropriate, were used to evaluate the statistical
signi®cance of the results. P < 0.05 was accepted as the
level of signi®cance.
Sarcoplasmic reticulum and stretch � C Han et al. Acta Physiol Scand 1999, 167, 111±117
112 Ó 1999 Scandinavian Physiological Society
RESULTS
Effects of stretch on the action potentials
In the present study we investigated the changes in the
electrophysiological properties of myocytes in the
isolated rat left atrium when they were subjected to
small (physiological) stretch from (diastolic) intra-atrial
pressure of 1±3 mmHg. Stretch modulated the shape of
the APs after 10 min of stretch. As shown in Fig. 1 and
Table 1, stretch signi®cantly prolonged all phases of
repolarization, except the earliest one. When compared
with control (pressure level 1 mmHg), moderate stretch
(3 mmHg) increased the APD30% (n� 10, P < 0.05),
the APD60% (n� 10, P < 0.01) and the APD90%
(n� 10, P < 0.01). There was no signi®cant change in
the resting potentials or the amplitudes of the APs.
Effects of stretch after SR depletion
To study the role of the SR in the stretch-induced
changes in the shape of the APs, we treated the atria
with pharmacological agents known to interfere with
the intracellular Ca2+ balance in a way that reduces the
effects of calcium release from the SR by emptying the
calcium stores. Firstly, we wanted to know the effects
of these drugs on the APs at control pressure
(1 mmHg). The drugs we used were ryanodine, an
agent that locks the calcium release channels (the
so-called ryanodine receptors, Bers et al. 1987, Lewar-
towski et al. 1990) into a semi-open state in the
concentration used (100 nmol L±1) thereby depleting
the calcium stores, thapsigargin, an inhibitor of the SR
calcium-ATPase that is required to replenish the stores
(Thastrup et al. 1990, Kirby et al. 1992), or caffeine that
increases the calcium leakage from SR via the ryano-
dine-sensitive channels (Chapman and LeÂoty 1976,
Smith et al. 1988). Figure 2 shows that after 10 min of
treatment, ryanodine, thapsigargin and caffeine all
signi®cantly prolonged the duration of the APs at
control pressure of 1 mmHg (see Table 2 for statistics).
Ryanodine had a more prominent effect on the early
than late repolarization phase, but thapsigargin's effect
was almost the same in early and late repolarization.
Low concentration of caffeine (1 mmol L±1) prolongs
moderately all repolarization phases, in a similar
manner as does ryanodine and thapsigargin. High
concentration of caffeine (10 mmol L±1) mainly effects
the early repolarization and was thus not used in the
further analysis (data not shown), because it can be
assumed that these additional effects are caused by
other, unspeci®c effects of the drug. We may conclude
in this part of the study that the intact function of SR
calcium release has a signi®cant contribution to the
shape of the APs, and the inhibition of the release by
depletion of the calcium stores, however, achieved,
prolongs the repolarization. These results are inde-
pendent of whether the SR was fully depleted or not,
and in this context a depletion that is able to inhibit the
contraction of the muscle, which was achieved in all
preparations treated (see Methods), is effective enough.
Second, experiments were conducted to ®nd out the
effects of stretch on the shape of the AP under
conditions where the SR calcium release in incapaci-
tated. Figure 3 and Table 3. show, respectively, the
averaged APs and the repolarization indices from these
experiments. When small stretch (again from 1 to
3 mmHg) is applied, the effects of the pharmacological
interventions persist. However, as apparent from the
®gures and from the repolarization indices, we did not
®nd signi®cant prolongation of the APs by stretch
under those conditions. The conclusion from this
second set of experiments is that when SR calcium
release is non-functional, the sensitivity of the APs to
stretch is abolished. In addition, it has to be noted that
Table 1 Effect of stretch on the voltage and duration parameters of
rat atrial action potential
1 mmHg (n = 10) 3 mmHg (n = 10)
Resting Potential (mV) )76.2 � 0.6 )75.6 � 0.8
Amplitude (mV) 91.7 � 0.8 89.6 � 1.0
Overshoot (mV) 15.5 � 0.8 14.0 � 1.0
APD10% (ms) 5.2 � 0.5 7.0 � 0.4
APD15% (ms) 6.6 � 0.6 8.9 � 0.5
APD30% (ms) 10.4 � 0.6 14.0 � 0.7*
APD60% (ms) 20.2 � 1.6 29.8 � 1.3**
APD90% (ms) 53.7 � 4.2 73.4 � 2.2**
*P < 0.05; **P < 0.01 (t-test).
Figure 1 Effect of stretch on action potentials of rat atrial myocytes.
Small, physiological stretch (3 mmHg, dotted line) increases the
duration of action potentials of rat atrium when compared with the
control (1 mmHg, solid line). The resting potentials of the recordings
were set to be the same to facilitate comparison of AP shape. The
®gure shows two individual action potentials, for statistics, see
Table 1.
Ó 1999 Scandinavian Physiological Society 113
Acta Physiol Scand 1999, 167, 111±117 C Han et al. � Sarcoplasmic reticulum and stretch
in case of thapsigargin treatment the APD30% and
APD60%, and in the case of ryanodine, the APD30%,
values are actually decreased (repolarization is acceler-
ated) by stretch (for statistics, see Table 3).
DISCUSSION
The main ®nding from this study was that the duration
of rat atrial AP was prolonged during small, physio-
logical stretch, and that this change was abolished by
pharmacological treatments that deplete the intracel-
lular calcium stores and thereby render the stores non-
functional. An immediate conclusion from this is that
stretch of the myocytes is mediated, at least in the case
of the rat left atrium, via a change in the intracellular
calcium balance into changes in the shape of APs.
When the SR function is incapacitated, stretch is not
able to change the APs. This ®nding points to mech-
anisms that are able to modulate the intracellular
calcium transients during stretch, possibly by changing
the amount of Ca2+ in the stores.
Stretch-induced changes in the action potential
Many investigations have found that the duration of the
cardiac AP is reduced after stretching the cardiac
muscle (Nakagawa et al. 1988, Dean & Lab 1990, White
et al. 1993, Tung & Zou 1995, Wang et al. 1996). Both
rapid and gradual stretch is also able to trigger
arrhythmias owing to stretch-induced depolarizations
(Stacy et al. 1992, Franz et al. 1992). The change in
membrane voltage in response to stretch suggests that
stretch was able to activate a conductance change in the
plasma membrane. Based on these observations,
changes in the APs during stretch have been explained
by activation of stretch-activated channel in the plas-
malemma (Hansen et al. 1991). Using MAP-recordings,
Zabel et al. (1996) found that the duration of the early
repolarization phase decreased and the late repolariza-
tion phase increased after stretch in the guinea-pig
ventricle. They were able to mimic this observation by
introducing the stretch-activated channels (SACs) into a
mathematical model in computer simulations.
Following this logic, the introduction of a signi®cant
calcium conductance into the membrane should lead to
an increase in stored calcium. In fact, using the rapid
cooling method, Bluhm & Lew (19953 ) showed that the
amount of Ca2+ in the SR is increased by stretch.
Mechanisms of stretch-sensitivity
The present work emphasizes the importance of the
intracellular calcium balance in response to stretch, in
contrast to direct modulation of the APs by stretch-
activated ionic channels. One possibility is that stretch
causes an increase in the SR calcium stores. Many
Table 2 Change of the duration of the action potentials by phar-
macological treatments (ryanodine at 100 nmol L)1, thapsigargin at
10 nmol L)1 and caffein at 1 mmol L)1) incapacitating the SR
Control
(n = 10)
Ryanodine
(n = 10)
Thapsigargin
(n = 10)
Caffeine
(n = 10)
APD30% (ms) 10.4 � 0.9 21.1 � 1.2** 15.9 � 0.7** 12.5 � 0.2
APD60% (ms) 20.2 � 1.6 33.2 � 1.6** 30.4 � 1** 24.7 � 0.9
APD90% (ms) 53.7 � 4 59.7 � 2.6* 67.6 � 2.6* 62.8 � 1.4*
*P < 0.05; **P < 0.01 (ANOVA).
Figure 2 Effect of (a) ryanodine
(100 nmol L±1), (b) thapsigargin
(10 nmol L±1), and (c) caffeine (1 mmol L±1)
(dotted lines) on the action potential of the
rat atrium at the low stretch level (1 mmHg)
compared with untreated controls (contin-
uous lines). All these pharmacological
interventions prolong the duration of action
potential of rat atrial myocytes. For statis-
tics, see Table 2.
114 Ó 1999 Scandinavian Physiological Society
Sarcoplasmic reticulum and stretch � C Han et al. Acta Physiol Scand 1999, 167, 111±117
experiments have shown that the Ca2+ transients
increase during stretch (Allen & Kurihara 1982, Hongo
et al. 1995, 1996, Kentish & Wrzosek 1998). Allen et al.
(19884 ) suggested that the Ca2+ transient augmentation
is caused by activation of cation-permeable stretch-
activated ion channels during stretch. By rising diastolic
[Ca2+] this would lead to an increase of the SR Ca2+
content (Frampton et al. 1991). Stretch-activated chan-
nels are found in various tissues, including the
mammalian heart (Bustamante et al. 1991, Sasaki et al.
1992, Kim 1992, 1993, Ruknudin et al. 1993). It has
been suggested that the activation of a cationic
conductance, large enough to change the membrane
voltage signi®cantly, would be responsible for the
introduction of calcium into the cell and into the SR
(Zabel et al. 1996). This idea is opposed by the fact that
diastolic [Ca2+]i seems to remain unaltered during
stretch (Hongo et al. 1996, Kentish & Wrzosek 1998,
Tavi et al. 1998). Also, in the present study, we did not
®nd any change in the resting potentials of the cells
during stretch, which should have been prominent with
a large stretch-activated conductance (Table 1.). Our
results suggest that stretch-induced changes in the rat
atrial AP are mediated by SR Ca2+ loading. This would
clearly require some additional Ca2+ in¯ux during
stretch. However, this Ca2+ in¯ux can be mediated
either by SA-channel activation or by some other
(unknown) mechanism. The results are supported by
recent ®ndings combining experimental results and a
mathematical modelling (Tavi et al. 1998).
Species differences in the mechanisms
In species other than the rat, and also in parts of the
heart other than the atrium, the calcium balance is likely
to be somewhat different (cf. Bers 1993). In species
with long APs the direct effect of SAC-activation may
be more prominent, because the calcium causing the
contraction of the myocyte is more weighted in favour
of the outside source (Bers 1997). Thus the effect of
intracellular calcium stores is probably much smaller
that in the rat atrium we have used. However,
therelationship between the calcium transients and
theshape of the APs is fairly complex. Depending on
the amount of calcium in the stores, the changes in the
size of the transient can plausibly be suggested to
modulate the repolarization either way. If the stores are
well loaded (or, if the released calcium plays a major
Figure 3 Effect of (a) ryanodine
(100 nmol L±1), (b) thapsigargin
(10 nmol L±1) and (c) caffeine
(1 mmol L±1) on the stretch-
induced changes in the action
potentials in the rat atrial
myocytes. Dotted lines show
representative APs with stretch
caused by pressure of 3 mmHg
and the continuous lines show
control APs at 1 mmHg. For
statistics, see Table 3
Table 3 The effect of stretch on the
duration of the action potentials when
the SR is incapacitated (ryanodine at
100 nmol L)1, thapsigargin at
10 nmol L)1 and caffeine at
1 mmol L)1)
Ryanodine Thapsigargin Caffeine
1 mmHg 3 mmHg 1 mmHg 3 mmHg 1 mmHg 3 mmHg
APD30% (ms) 21.1 � 1.2 20.8 � 0.9* 15.9 � 0.7 13.9 � 0.3* 12.5 � 0.4 13.8 � 0.2
APD60% (ms) 33.2 � 1.6 33.2 � 1.1 30.4 � 1 27.5 � 0.7* 24.7 � 0.9 27.4 � 0.8
APD90% (ms) 59.7 � 2.6 57.9 � 1.8 67.6 � 2.6 65 � 2.4 62.8 � 1.4 66.5 � 1.3
*P < 0.05 with ANOVA.
Ó 1999 Scandinavian Physiological Society 115
Acta Physiol Scand 1999, 167, 111±117 C Han et al. � Sarcoplasmic reticulum and stretch
role), the inward current via the Na+±Ca2+ exchanger is
dominating. Conversely, if the stores are depleted,
the effects of the changes in the calcium transients via
the L-type calcium current are more prominent. This is
supported by our ®nding that in SR-depleted situations
stretch is able to accelerate the repolarization (Table 3.),
an effect which is opposite to that found in the
untreated preparations.
In conclusion, this study showed that the duration
of APs in rat atrial myocytes is prolonged by stretch,
and that this effect can be inhibited by pharmacological
agents that deplete the SR calcium stores. This is
consistent with the hypothesis that in rat atrial
myocytes the effects of stretch on AP shape are
mediated by a change in the calcium balance.
The authors are grateful for the help of Prof. Heikki Ruskoaho during
this work, to Anneli Rautio and Eero Kouvalainen for technical
assistance. The work was supported by the Academy of Finland
(M.W.) and Orion-Farmos Scienti®c Foundation (P.T.).
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