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Introduction
The osmolarity of body fluids is tightly controlled under normal physiological conditions. But, the volume of the cells,
including cardiomyocytes, can change under osmotic stress or in severe pathological states. For instance, after ischemia and
reperfusion insult, cardiomyocyte swelling
occurs[1]. The increase of the cell volume is also present in renal failure and after
excessive water ingestion[2]. In contrast, cell shrinkage is observed with
Na+/K+ pump inhibition and during
apoptosis[3,4]. Furthermore, it has been shown that the increased blood tonicity in streptozotocin-induced diabetic rats leads to shrinkage
of ventricular myocytes[5]. Many previous studies have indicated that both electrical and contractile properties of ventricular
myocytes are affected by osmotic
stress[1,5_9]. For example, it has been consistently found that L-type
Ca2+ currents, Ca2+ transients and contraction evoked by action potential are decreased by prolonged exposure to hyposmotic
medium[1,7].
It is well established that Ca2+ sparks are elementary events of
Ca2+ release mediated by ryanodine
receptors/Ca2+ release channels (RyRs) on the sarcoplasmic reticulum (SR) of muscle
cells[10]. It is generally accepted that the
Ca2+ transients evoked by action potential or depolarization are the spatial and temporal summation of many
Ca2+ sparks. In resting ventricular myocytes, spontaneous
Ca2+ sparks can be detected. However, the physiological significance of these spontaneous
events in cardiomyocytes is mostly unclear.
To our knowledge, there has been no study of the effect of osmotic stress on the spontaneous
Ca2+ sparks in cardiomyocytes. Investigating this problem will be helpful to understand whether and how osmotic stress modulates RyR
gating in cardiomyocytes. This study may also be relevant to further recognize the pathology of some diseases. For
instance, it has been observed that the Ca2+
spark frequency of cardiomyocytes
isolated from 5-week streptozo-tocin-induced
diabetic rats significantly increased with respect to aged-matched
control rats[11]. In addition,
Ca2+ spark and Ca2+ transients
exhibited a significantly prolonged duration in diabetic rats. An additional purpose of this study is that we have found that
the aggregation of isolated RyRs of rabbit skeletal muscle was modulated by the concentration of
K+ and Na+[12]. We wonder if such a modulation can be confirmed in intact myocytes through investigating the effect of osmotic stress on spontaneous
Ca2+ sparks.
Materials and methods
Cell isolation and permeabilization Rat ventricular myocytes were isolated as described
previously[13]. An adult Sprague-Dawley rat was anaesthetized by sodium pentobarbital (100 mg/kg, ip). The heart was quickly excised and perfused with
O2 saturated Ca2+-free Tyrode¡¯s solution at
37 °C for 5 min. The perfusion solution was then switched to Tyrode¡¯s solution containing 1mg/mL collagenase (type I),
0.1 mg/mL trypsin (type I) and 0.1 mmol/L
CaCl2, and the heart was perfused for another 20 min. Afterwards, the ventricle was
cut off, minced and filtered through nylon mesh. Isolated cells were washed with
Ca2+-free Tyrode¡¯s solution.
CaCl2 was then gradually added, and finally 1 mmol/L
Ca2+ concentration was achieved. Isolated myocytes were kept in the standard
physiological solution containing: NaCl 120 mmol/L, KCl 5.4 mmol/L,
MgCl2 1.2 mmol/L, CaCl2 1
mmol/L, HEPES 20 mmol/L, glucose 15 mmol/L, pH 7.4 (NaOH) at room temperature (22_24 °C) for 1
h before use.
Isolated myocytes were permeabilized with 0.01%
(w/v) saponin according to a modified method described in a previous
study[14]. After permeabilization, the myocytes were perfused with saponin-free internal solution composed of: potassium aspartate 100
mmol/L, KCl 20 mmol/L, ATP 3 mmol/L,
MgCl2
3.81 mmol/L, EGTA 0.5 mmol/L, CaCl2 0.1 mmol/L, phosphocreatine 10 mmol/L, creatine phosphokinase 5 U/mL, HEPES 10 mmol/L,
Fluo-4 potassium salt 0.04 mmol/L, and 8%
(w/v) dextran (MW 40 000); pH 7.2 (KOH). According to the computer program
WinMAXC 2.5 (Stanford University, Stanford, CA, USA), free
[Ca2+] and [Mg2+] in this solution were 43 nmol/L and 1 mmol/L,
respectively. The total Ca2+ and
Mg2+ necessary for obtaining different free
[Ca2+] were also calculated with this program.
Detection of global Ca2+ transients and
Ca2+ sparks Intact myocytes were loaded with 20 µmol/L Fluo-4 AM for 30 min at
room temperature. A glass coverslip attached by the cells was mounted as the bottom of a chamber, which was put on the
stage of an inverted microscope (Nikon Diaphot 300, Japan). Before taking records, the cells were superfused with standard
physiological solution for 30 min for de-esterification of Fluo-4 AM. For permeabilized cells, the step of loading dye was
omitted, and the dye Fluo-4 potassium salt was directly added into the bathing solution. Both global
Ca2+ transients and
Ca2+ sparks were observed by a laser scanning confocal microscope (MRC 1024, Bio-Rad, CA, USA) equipped with a ×60
oil-immersion objective (NA=
1.4). Fluo-4 was excited at a wavelength of 488 nm, and the fluorescence measured at >522 nm.
To monitor the global Ca2+ transients, the data of fluorescence intensity were collected at 1 Hz. Although the fluorescence
signals were not calibrated in terms of
Ca2+ concentra-tion, the baseline fluorescence was used as a rough estimate of
[Ca2+]i and the changes in fluorescence signal represented alterations in
[Ca2+]i. Caffeine-induced
Ca2+ transients (CaTs) were evoked by repetitive exposures to 10 mmol/L caffeine for approximately 20 s at an interval of 10 min. The amplitude of
F/F0 was used as an estimate of SR
Ca2+ content.
In order to record Ca2+ sparks, the fluorescence was recorded in either two-dimensional (X-Y) or line scan (X-T) mode. In
the latter case, each scan line consisted of 512 pixels, and the length of the line was 84 µm (512 pixels× 0.164 µm/pixel). The
scan line was oriented along the long axis of the myocyte, and the cell nuclei were avoided. A full image was obtained by
stacking 512 scan lines. It took approximately 1 s (2 ms/line). The image obtained by line scan mode was processed by IDL
software (Research Systems) and a modified spark detection
algorithm[15]. Ca2+ sparks were identified as local peak elevations
of fluorescent intensity (F), which were >3SD of the surrounding background levels
(F0). The measured parameters included
amplitude (F/F0), spatial full width at half maximum intensity (FWHM), full duration at half maximum intensity (FDHM), time
to peak intensity (RT), and time for decay to half-peak intensity (THR). The frequency of
Ca2+ sparks was represented by the number of
Ca2+ sparks in one image obtained by line scan mode, event·
S-1·84 µm-1.
To observe the effect of osmotic stress in intact ventricular myocytes, the collection of
Ca2+ sparks was started 10 min after changing the tonicity. The tonicity of standard physiological solution is referred to as ST. The tonicity in other
solutions used in this study was 1 T (isosmotic solu-tion), 0.6 T (hyposmotic solution) and 1.5 T or 2 T (hypero-smotic
solution). Their osmolalities were measured with an osmometer. The compositions of these solutions are represented in
Table 1.
Statistical analysis Statistical significance was determined by Student¡¯s
t-test. P<0.05 was considered to be statistically
significant.
Results
Effect of osmotic stress on spontaneous
Ca2+ sparks in intact myocytes Several effects of osmotic stress on
spontaneous Ca2+ sparks were found in this study. First, the frequency of spontaneous
Ca2+ sparks was increased and decreased by
hyper- and hypo-osmotic exposure, respec-tively. For instance, the frequency was increased from
1.80±0.25 event·S-1·84
µm-1 to 3.55±0.27
event·S-1·84
µm-1, when the tonicity was enhanced from ST to 1.5 T. In another series
of experiments it was found that the frequency in 1 T and 0.6 T were 5.11±0.52
event·S-1·84
µm-1 and 1.31±0.12
event·S-1·84
µm-1, respectively. The effects of osmotic challenge on the frequency were mostly reversible (Table 2).
Besides the frequency, the spatio-temporal properties of
Ca2+ sparks were also affected by osmotic stress.
Representative records are shown in Figure 1A. The summarized results are displayed in Figure 1B and Table 2. As shown in Figure 1Ba,
1.5 T caused a left shift of FWHM distribution and a right shift of the distribution of the temporal parameters (RT and THR),
respectively. The amplitude of Ca2+ sparks
(F/F0) was not affected (data not shown). The action of hyperosmotic exposure
on the temporal parameters was reversible, while the spatial parameter was irreversibly affected.
Although increasing the frequency, 1 T had no influence on the spatio-temporal parameters of
Ca2+ sparks (Table 2). Reducing the tonicity from 1 T to 0.6 T evidently caused the changes of the spatio-temporal parameters, which were just
opposite to that induced by 1.5 T (Figure1Bb, Table 2). All of these effects of hyposmotic exposure were reversible.
Effect of osmotic stress on
[Ca2+]i and caffeine-induced
Ca2+ transients in intact myocytes To explore if these effects of
osmotic stress on spontaneous Ca2+ sparks result from the alterations of
[Ca2+]i and Ca2+ loading of the SR, the following
experiments were performed.
First, in consistence with the previous
studies[1,5_9], 1.5 T or 2 T caused an evident increase of resting
[Ca2+]i (Figure 2Aa), and this increase was eliminated in the absence of extracellular
Ca2+ (Figure 2Ab). It is indicated that this increase of
[Ca2+]i induced by hyperosmotic challenge is due to
Ca2+ influx. To see the effect of hyposmotic exposure, the myocytes were first
perfused with isosmotic solution (1 T) for 10 min and then with hyposmotic solution (0.6 T). Interestingly, it was observed
that an increase of [Ca2+]i occurred when extracellular solution was switched from ST to 1 T (Figure 2Ba). However, when the
extracellular solution was further changed to 0.6 T,
[Ca2+]i was returned to the baseline (Figure 2Ba). Similarly, the increase of
[Ca2+]i induced by 1 T was absent in
Ca2+ free extracellular solution (Figure 2Bb), indicating that it might be attributed to
enhanced Ca2+ entry or reduced
Ca2+ extrusion by
Na+-Ca2+
exchanger[1,7,16]. All of these effects of osmotic stress on
[Ca2+]i were reversible.
The effect of osmotic stress on CaTs was also investi-gated. The amplitude of the CaT was used as a rough estimate of
Ca2+ loading of the SR. Figure 3A shows representative records of the CaT under various conditions. The summarized results
displayed in Figure 3B illustrate that 1.5 T and 1 T significantly increased the CaT (124.5%±3.7% and 118.0%±3.4% of ST,
respectively). In contrast, 0.6 T evidently decreased the CaT (83.7%±3.5% of 1 T or 98.8%±
4.2% of ST).
Afterwards, the action of increasing external
Ca2+ ([Ca2+]o) was observed. In accordance with previous
studies[17], elevating
[Ca2+]o to 10 mmol/L from 1 mmol/L resulted in an increase in the resting fluorescence (Figure 3Ad) and greatly augmented
the CaT (135.4 %± 4.2% of ST). Correspondingly, the frequency of spontaneous
Ca2+ sparks significantly increased at 10
mmol/L [Ca2+]o (Table 2). However, raising
[Ca2+]o did not alter the spatio-temporal parameters (Figure 1Bc, Table 2).
Taken together, these results suggest that the change of the frequency induced by osmotic stress is attributed to the
alteration of the [Ca2+]i and
Ca2+ loading of SR, while other factor(s) are responsible for the change of the spatio-temporal
properties of Ca2+ sparks.
Effect of dextran, [Ca2+]i
and [K+]i on spontaneous
Ca2+ sparks in permeabilized myocytes
Dextran is an uncharged, inert and highly branched polymer that can increase the viscidity of the solution and hence reduce the diffusibility of solute. To
clarify the role of Ca2+ diffusion, the influence of dextran on the spatio-temporal properties of
Ca2+ sparks was examined. As a control, adding 16%
(w/v) dextran into external medium did not affect the spatio-temporal properties of
Ca2+ sparks in intact myocytes (Table 2). However, in permeabilized myocytes the spatio-temporal properties of
Ca2+ sparks were altered by dextran (Figure 4). In this study, 8% dextran was routinely used to prevent swelling of the permeabilized
cells. It was found that, removing dextran from the internal solution significantly changed the spatio-
temporal properties of Ca2+ sparks (Figure 4A,B). As shown in Figure 4B, the distribution of FWHM was evidently shifted to
the right, while distributions of RT and THR were shifted to the left. Increasing dextran from 8% up to 16% had an opposite
effect on these parameters (Figure 4B). The mean data are displayed in Table 3. The effect of dextran suggests the role of
Ca2+ diffusion in determining the spatio-temporal properties of
Ca2+ sparks.
To further confirm the role of
[Ca2+]i in modulating spontaneous
Ca2+ sparks, the influence of
[Ca2+]i was investigated. It
was revealed that raising
[Ca2+]i from 43 nmol/L to 75,
115 and 150 nmol/L increased the frequency from 3.19±0.14 to 6.13±0.34, 12.29±0.69 and 18.04±0.90
event·S-1·84
µm-1, respectively. Although the frequency was
[Ca2+]i-dependent, the spatio-temporal parameters of
Ca2+ sparks were not affected by moderate increase of
[Ca2+]i (data not shown).
In order to explore the role of ionic strength, spontaneous
Ca2+ sparks were recorded from permeablized myocytes
perfused with various concentrations of potassium aspartate ([K-Asp]) in the presence of 43 nmol/L
[Ca2+]i. It was observed that the frequency at 50, 100, 150 and or 200
mmol/L [K-Asp] was 2.95 ± 0.11, 3.20 ± 0.15, 2.31± 0.14 and 2.32 ±
0.13 event·S-1·84
µm-1, respectively. It is indicated that high [K-Asp] might slightly reduce the frequency. However, increasing [K-Asp] from
50 mmol/L to 100 mmol/L had no detectable influence on the distribution of FWHM, RT and THR. However, further raising
[K-Asp] up to 150 or 200 mmol/L slightly shifted these distribution curves to the left (Figure 4C).
Effect of osmotic stress and dextran on
Ca2+ waves The action on
Ca2+ waves of osmotic stress was observed in intact
myocytes. It is known that the propagating velocity of the
Ca2+ wave is proportional to the diffusion constant
of Ca2+[18]. Spontaneous
Ca2+ waves only appeared in a small proportion of the myocytes.
Figure 5A shows the representative images at different osmotic pressures. Summarized results are displayed in Figure 5B. It was observed that the velocity of
Ca2+ waves was significantly decreased and increased by hyper- and hypo-osmotic exposure, respectively. Interestingly, the
propagating velocity was not affected by 1 T (Figure 5B), although
[Ca2+]i and Ca2+ loading of the SR were increased (Figure 2B and
3Ab).
The effect of dextran on the propagating velocity of
Ca2+ waves was investigated with permeabilized myocytes.
Spontaneous Ca2+ waves could often be seen when
[Ca2+]i was elevated to 260 nmol/L (Figure 5C). The effect of dextran on the
velocity of Ca2+ waves is represented in Figure 5D. Dextran-induced decrease of
Ca2+ diffusion in permeabilized myocytes is
clearly shown.
Discussion
It was observed in this study that both the frequency and spatio-temporal properties of spontaneous
Ca2+ sparks in intact cells were affected by the tonicity of the extracellular solution. To understand the mechanisms underlying these effects, the
role of the following factors is discussed individually:
[Ca2+]i and SR
Ca2+ loading, ion strength, and
Ca2+ diffusion.
Role of [Ca2+]i and SR
Ca2+ loading In this study clear evidence was obtained that the alteration of
[Ca2+]i and Ca2+ loading
of the SR can account for the change of the frequency induced by osmotic stress but not for the change of the
spatio-temporal properties of spontaneous
Ca2+ sparks. First, exchanging 1T for ST or elevating
[Ca2+]o to 10 mmol/L increased
[Ca2+]i and SR
Ca2+ loading (Figure 2 and 3). These actions only increased the frequency but did not change the
spatio-temporal parameters of Ca2+ sparks in intact myocytes. Second, the frequency of
Ca2+ sparks in permeablized myocytes was
evidently increased by raising
[Ca2+]i from 43 nmol/L to 75 nmol/L, but the spatio-temporal parameters were not affected.
How are [Ca2+]i and SR
Ca2+ loading altered by osmotic stress? Several mechanisms may be involved. First, as the
alteration of [Ca2+]i induced by osmotic stress was dependent on extracellular
Ca2+, one mechanism is obviously
Ca2+ entry. Previous studies have shown that the
Ca2+ entry induced by osmotic stress are mediated by various channels, such as
L-type Ca2+ channels[1,7,19]
and stretch-activated channel[19,20]. Second, osmotic stress may change
[Ca2+]i and SR Ca2+
loading through altering cellular volume. Third, the alteration of
[Ca2+]i caused by the mechanisms just mentioned may be further
affected through Ca2+-induced Ca2+
release.
Role of ionic strength Previous studies have indicated that the activity of RyRs and the interaction between RyRs are
modulated by ionic strength. For instance, with single-channel recording an increase of RyR activity has been shown with
increasing KCl concentration[21,22]. By photon correlation spectroscopy and atomic force microscopy we found that the
aggregation of isolated RyRs of rabbit skeletal muscle is decreased by raising KCl
concentration[12]. Accordingly, some properties of
Ca2+ sparks would be expected to be influenced by the alteration of ionic strength.
It was found in this study that the frequency of spontaneous
Ca2+ sparks in permeabilized myocytes was slightly
decreased by raising [K-Asp] from 100 mmol/L to 150 or 200 mmol/L. This change seems to be different from the results
obtained by single-channel
recording[21,22]. The exact reason for this discrepancy is unclear. It may be because the
Ca2+ spark is an event of
Ca2+ release from a cluster of RyRs.
In addition, an evident difference is noted when comparing the effect of changing ion strength in internal solutions of
permeabilized myocytes with the effect of changing the extracellular tonicity of intact myocytes. Raising [K-Asp] from 100
mmol/L to 200 mmol/L in permeabilized myocytes decreased THR by approximately 11%, whereas increasing the extracellular
tonicity from ST to 1.5 T in intact myocytes remarkably increased THR by 45% (Table 2). Moreover, decreasing [K-Asp] from
100 mmol/L to 50 mmol/L had no effect on the spatio-temporal parameters, while decreasing the extracellular tonicity from 1 T
to 0.6 T in intact myocytes increased FWHM by 4% and decreased THR by 20%. These results indicate that, if the alteration
of ionic strength induced by osmotic stress is responsible for the change of the spatial parameter, it could not account for the
change of the temporal parameters.
Role of Ca2+ diffusion Among the osmotic stress-induced changes of the spatio-temporal properties, the change of
temporal parameters, especially THR is most conspicuous. The main factors contributing to the decline of the local
[Ca2+]i include closure of RyR, pumping
Ca2+ back into the SR, Ca2+ diffusion and
Ca2+ binding to buffers[23]. Even though
Ca2+ uptake by the SR significantly influences the spatio-temporal parameters of
Ca2+ sparks, Ca2+ diffusion is proposed to be a
dominant process in local
[Ca2+]i decline during
Ca2+ sparks[23]. Hence, whether or not
Ca2+ diffusion during Ca2+ sparks is
altered by extracellular tonicity is questioned.
Previous observations have shown that transverse tubular-SR (T-SR) junction anatomy of amphibian skeletal muscle
fibers, including the T-SR distance, was affected by extracellular
tonicity[24,25]. The T-SR distance of about 16 nm was
increased and decreased by hypo- and hyper-osmotic exposure, respectively. Unfortunately, information about the effect of
the tonicity on the T-SR junction anatomy of ventricular myocytes is unavailable. If the effect in ventricular myocytes is
similar, the T-SR distance of ventricular myocytes would be increased and decreased by hypo- and hyper-osmotic exposure,
respectively. The T-SR distance is approximately 20 nm, while the spatial spread of
Ca2+ sparks is over one micron. Therefore,
the osmotic stress-induced alteration of the T-SR distance would affect
Ca2+ diffusion during Ca2+ sparks and in turn the
spatio-temporal para-meters.
Besides the T-SR distance, other mechanisms may be involved in altering
Ca2+ diffusion. For instance, accompanying cell
shrinkage or swelling, the alteration in the
Ca2+-binding proteins¡¯ concentration would occur. It potentially would influence
Ca2+ diffusion. In fact, our result showed that the propagating velocity of
Ca2+ waves was increased and decreased by hypo-
and hyper-osmotic exposure, respec-tively (Figure 5).
It was observed in this study that the propagating velocity of
Ca2+ wave in permeabilized myocytes was significantly
decreased by dextran (Figure 5). Furthermore, the FWHM of
Ca2+ sparks in permeabilized myocytes was significantly
reduced by dextran, whereas the temporal parameters, such as RT and THR, were remarkably increased (Figure 4B and Table
3). The effects of dextran on both
Ca2+ waves and Ca2+ sparks in permeabilized myocytes are the same as the effects of
hyperosmotic stress in intact myocytes. Therefore, it is likely that osmotic stress might affect
Ca2+ diffusion and hence change the spatio-temporal properties of
Ca2+ sparks in intact myocytes.
Role of other factors Besides the possible mechanisms mentioned above, other factors, such as SR
Ca2+-ATPase, Ca2+ release kinetics of RyR and control of RyR by dihydro-pyridine receptor (DHPR) control, may be involved.
In this study we used mag-fluo-4, a low affinity
Ca2+ indicator (Kd =22 µmol/L), to directly measure the change of free
Ca2+ concentration in the SR lumen. It was found that, following 10 mmol/L caffeine exposure, the rate of
Ca2+ uptake back to the SR was not affected by osmotic stress (data not shown). In addition, it has been shown previously that, by completely
blocking the SR Ca2+-ATPase with thapsigargin, the time constant of
[Ca2+]i decline (t) and FWHM of
Ca2+ sparks increased 36% and 42%, respectively. On the contrary, stimulating the SR
Ca2+-ATPase by isoprenaline, the t and FWHM decreased
by 33% and 18%, respective-
ly[23]. It was revealed in the present study that, increasing the tonicity from ST to 1.5 T remarkably enhanced THR by 45% and
decreased FWHM by 13%, while decreasing the tonicity from 1 T to 0.6 T decreased THR by 20% and enhanced FWHM by
4%. Because the changes of the spatio-temporal properties of
Ca2+ sparks induced by altering the tonicity or activity of the
SR ATPase are qualitatively different, the role of the SR
Ca2+-ATPase may be excluded.
The RT of Ca2+ sparks may provide an estimate of
Ca2+ release duration[26]. We have observed that the RT of
Ca2+ sparks was increased by 29% and decreased by 10% at 1.5 T and 0.6 T, respectively. As the change in
Ca2+ diffusion may also alter the RT, it is still uncertain whether or not osmotic stress affects the gating of RyR.
The previous study on the effect of osmotic stress on
Ca2+ sparks in mice skeletal muscle fibers showed a robust and
transient appearance of Ca2+ sparks when the fibers were returned to normal
solution after being briefly exposed to hypotonic
solution[27]. The initiation of
Ca2+ sparks is proposed to result from osmotic shock-induced change of RyR gating controlled
by DHPR. Differing from skeletal muscle fibers, the effect of osmotic stress on
Ca2+ sparks in ventricular myocytes
maintained for an hour after osmotic challenge. Although the contact between RyR and DHPR in cardiac myocytes is not so close
as in skeletal muscle fibers, the alteration in the T-SR distance may change DHPR-RyR coupling and hence
Ca2+ sparks frequency.
Functional consequence of osmotic stress-induced change
of Ca2+ sparks In cardiac myocytes, the electrical
depolarization of the surface membrane leads to the influx of
Ca2+ and then the Ca2+ release from the SR. As a result, the contraction of
cardiomyocytes is evoked. Therefore, any fault in
Ca2+ handling may influence the contractile func-
tion[28_31]. This study showed that in ventricular myocytes
[Ca2+]i , Ca2+ loading in the SR and the frequency of spontaneous
Ca2+ sparks enhanced with increasing the tonicity of extracellular solution. Moreover, the spatio-temporal properties of
spontaneous Ca2+ spark and the propagating velocity of
Ca2+ wave were also affected. These alterations induced by
hyperosmotic stress may affect the contractile properties in various ways. For example, compared with that under normal or
isosmotic condition, the time course of the contraction will be prolonged because of slower propagating of
Ca2+ and slower decline of local
[Ca2+]i. As a result of the increased
[Ca2+]i, the relaxation may be incomplete. Opposite changes would be
expected under hyposmotic stress. In fact, it has been reported that the time course (including the time to peak shortening
and the time to half relaxation) of myocyte shortening in response to electrical stimulation decreases and increases by
prolonged hypo- and hyper-osmotic exposures,
respectively[1,5,7]. Besides, the alterations of
[Ca2+]i and the frequency of
spontaneous Ca2+ sparks induced by osmotic stress may affect the pathways of the intracellular signal transduction. For
instance, Ca2+ sparks in myocytes are able to yield mitochodrial
Ca2+ uptake called Ca2+ marks, and in turn affect the cellular
metabolism[32].
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