Cardiovascular Research

Cardiovascular Research 1998 37(2):424-431; doi:10.1016/S0008-6363(97)00271-X
© 1998 by European Society of Cardiology

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Copyright © 1998, European Society of Cardiology

Contribution of reverse-mode sodium–calcium exchange to contractions in failing human left ventricular myocytes

Julian A Mattiello^a, Kenneth B Margulies^b, Valluvan Jeevanandam^c and Steven R Houser^a,*

^aDepartment of Physiology, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140, USA
^bDepartment of Cardiology, Temple University Hospital, 3401 North Broad Street, Philadelphia, PA 19140, USA
^cDepartment of Cardiothoracic Surgery, Temple University Hospital, 3401 North Broad Street, Philadelphia, PA 19140, USA

^* Corresponding author. Tel. (+1-215) 707-3278; Fax (+1-215) 707-4003; E-mail: srhouser@astro.ocis.temple.edu

Received 3 July 1997; accepted 15 October 1997

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: To examine the contribution of reverse mode sodium–calcium (Na–Ca) exchange to contractions in isolated left-ventricular myocytes from failing human heart. Methods: Low resistance patch pipettes were used to dialyze cells with Na-free or high-Na pipette solution ([Na]_pipette=0 and 20 mmol/L, respectively) to reduce or enhance Na–Ca exchange. Whole-cell membrane-potential, membrane-current and cell-shortening data were simultaneously acquired during whole-cell voltage clamp protocols. Thapsigargin (100 nmol/L) and nifedipine (1 µmol/L) were also used to inhibit sarcoplasmic reticulum (SR) Ca-ATPase and L-type Ca channels, respectively. Results: Two types of contractions were observed. Rapid phasic contractions were seen in both Na-free and high-Na cells. Slow tonic contractions were seen only in high-Na cells. Phasic contractions demonstrated bell-shaped voltage dependence over the voltage range that corresponds to the activity of the L-type Ca channel. Although the voltage dependence of phasic contractions were similar Na-free and high-Na cells, phasic contractions in high-Na cells were larger than phasic contractions in Na-free cells. Phasic contractions were sensitive to inhibition of SR Ca-ATPase and L-type Ca channels. Tonic contractions were not inhibited by either thapsigargin or nifedipine. In thapsigargin-treated high-Na cells, tonic contraction magnitude increased exponentially with test-potential. Conclusions: The increases in phasic contraction magnitude observed in high-Na cells compared to Na-free cells were most likely due to increased SR Ca loading resulting from increased reverse-mode Na–Ca exchange. Our results also suggest that tonic contractions in high-Na cells were mediated by Ca entry via reverse-mode Na–Ca exchange and were not the result of either SR Ca release or L-type Ca channel activity.

KEYWORDS Calcium influx; Contraction; Excitation-contraction coupling; Heart ventricle; Human myocyte; Sarcoplasmic reticulum; Sodium–calcium exchange

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Depolarization of the cell membrane during the ventricular action potential promotes calcium (Ca) influx by two major routes: through voltage dependent L-type Ca channels and through ‘reverse-mode’ electrogenic sodium–calcium exchange (Na–Ca exchange). Ca entry through the sarcolemma can activate contraction directly by elevating cytosolic Ca or can induce intracellular Ca release from the sarcoplasmic reticulum (SR) by a mechanism known as ‘Ca-induced Ca release’[1–3]. In most mammalian preparations, contraction is significantly altered when SR release is eliminated [4, 5]. For this reason, SR Ca is thought to be the primary source of Ca for activation of contractile proteins.

L-type Ca current is considered to be the principle trigger for SR Ca release in mammalian ventricular myocytes because the voltage dependence of SR Ca release follows the voltage dependence of Ca influx via L-type Ca channels [6–9]. However, a number of studies have shown that SR Ca release can be induced in the absence of L-type Ca current and that this release is dependent on either Na current or bulk cytoplasmic Na content [10–15]. These findings suggest that SR Ca release can also be triggered by Ca entry through reverse-mode Na–Ca exchange.

The mechanism by which reverse-mode exchange influences contraction in human heart cannot be easily predicted from animal studies because experiments in different species have produced significantly disparate results [16–18]. The principle route of Ca entry, as well as the degree of coupling of the transsarcolemmal Ca flux to SR Ca release and contraction, may be species dependent. Therefore, the respective roles of L-type Ca current, reverse-mode Na–Ca exchange, and the release of SR Ca in human heart must be directly determined.

Recent studies have demonstrated increased Na–Ca exchanger mRNA and protein levels in specimens from failing human heart, suggesting that the contribution of Na–Ca exchange to contraction may be even more significant in chronic heart failure than in normal human heart [19]. The purpose of the present study was to investigate the contribution of reverse-mode Na–Ca exchange to contraction in myocytes isolated from failing human left-ventricle. Our objective was to determine if Ca influx via reverse-mode exchange influences contraction in this cell population by inducing SR Ca release, directly elevating cytosolic Ca, and/or loading the SR.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Human myocyte isolation
The cells used in this study were isolated from failing hearts surgically removed from 15 human patients during the course of cardiac transplantation surgery. All protocols conform with the principles outlined in the Declaration of Helsinki. The method used to isolate myocytes was a modified version of previously published techniques [20–22]. In the operating room, cardioplegia solution was administered via a coronary catheter in vivo. Following arrest, the hearts were removed and placed in cold Ca-free modified Krebs–Henseleit (KH) solution containing (in mmol/L): glucose 12.5, KCl 5.4, lactic acid 1, MgSO₄ 1.2, NaCl 130, NaH₂PO₄ 1.2, NaHCO₃ 25, Na-pyruvate 2, and taurine 20 (pH 7.4 with NaOH). In the laboratory, a vessel on the surface of the left-ventricular free wall was cannulated. After the start of perfusion, the distribution of the cannulated vessel was excised from the remainder of the heart. All perfusion solutions were maintained at 37°C and equilibrated with 5% CO₂, 95% O₂. The constant flow perfusion rate was approximately 25 mL/min. KH solution was used to rinse plasma elements from the vasculature and to minimize extracellular Ca. The rinse-perfusate was discarded. After 30 min, the perfusion solution was changed to KH digestion solution that also contained CaCl₂ (50 µmol/L) collagenase (180 units/mL) and 2,3-butanedione monoxime (20 mmol/L). 2,3-Butanedione monoxime (BDM) has been shown to exert a protective effect on myocardium and to markedly increase the yield of rod-shaped myocytes following cell isolation protocols [20]. The digestion-perfusate was recirculated. Following 30 min of digestion, the solution was changed to a second non-recirculating KH rinse solution that contained CaCl₂ (200 µmol/L) and BDM (20 mmol/L). This second rinse was intended to clear the tissue of collagenase and to elevate extracellular Ca in the presence of BDM. The second rinse-perfusate was also discarded. After 10 min, the perfusion was stopped, and the tissue was bisected in the midmyocardial plane. From the cut surfaces of the midmyocardium, digested tissue was collected and suspended in the second KH rinse solution and filtered through nylon mesh (400 µm pore size) to remove large particulates from the isolated cells. Inspection of cells in solution by light microscopy revealed percentages of rod shaped cells that varied from 10 to 60%. The filtered cell suspension was centrifuged (25xg for 1 min) and resuspended in KH resuspension solution that contained CaCl₂ (200 µmol/L) and bovine serum albumin (1% w/v) without BDM. The isolated cells were washed twice in KH resuspension solution. The cells were stored at room temperature in KH resuspension solution continuously equilibrated with 5% CO₂, 95% O₂. Experiments were performed within 6 h of cell isolation.

2.2 Whole-cell voltage clamp
An aliquot of cells was placed in a heated (37°Celsius) chamber on the stage of an inverted microscope (Zeiss Axiovert 10). Normal Tyrode bath solution contained (in mmol/L): glucose 10, HEPES 5, KCl 5.4, MgCl₂ 1.2, NaCl 150, Na-pyruvate 2 (pH 7.4 with NaOH). Voltage-clamp techniques were similar to those used previously in this laboratory [23]. Low resistance (1.5–4 M) glass patch pipettes were used to permit rapid dialysis of the cell interior. Fire-polished patch pipettes were filled with Na-free or high Na-solution that contained (in mmol/L): HEPES 20, KCl 130 or 110, MgCl₂ 5, K₂ATP 5, NaCl 0 or 20, respectively (pH 7.2 with KOH). Following gigaohm seal formation, whole cell voltage clamp protocols were initiated. Four pre-conditioning pulses to +10 mV (500 ms) were used to evenly load the SR with Ca prior to each test-step. The stimulation frequency was 0.5 Hertz. A holding potential of –50 mV was used to inactivate Na conductance. Test-step potentials increased by +10 mV with each successive episode. In experiments designed to examine the difference between Na-free and high-Na contractions, voltage clamp test-steps durations were equal to 500 ms. Longer 1,000 ms test-steps were used to examine the mechanism of slow-tonic contractions in Na-free and high-Na cells. No significant differences were observed in the rate or magnitude of contractions using the shorter test-step duration (data collected with these protocols were combined in Fig. 2A). Data were recorded during superfusion with control bath solution for each group of cells. An Axoclamp 2 voltage clamp (Axon Instruments) was used to generate membrane-potential and membrane-current signals. Cell shortening data was obtained with a CCD camera (Cohu) placed in the microscope eyepiece. Analog video edge detection (Cresent Electronics) was used to capture cell shortening data. Following amplification, analog signals were converted to digital data with a TL-1 analog to digital converter. Digital data were acquired on a 486-33 MHz personal computer with Clampex 5.5 (Axon Instruments). Data were analyzed with Clampfit 6.0.3 (Axon Instruments). Following acquisition of data in control bath solution, cells were treated with bath solution containing 100 nmol/L thapsigargin (a specific inhibitor of the SR Ca-ATPase) [24]. During thapsigargin treatment, depolarizing stimuli (1,000 ms voltage steps to +10 mV applied at 0.5 Hz) were used to cause contractions and to deplete SR Ca stores. After 2–4 min, new steady-state contractions were observed and the voltage-clamp protocol was repeated. Following thapsigargin treatment, cells were treated with both 100 nmol/L thapsigargin and 1 µmol/L nifedipine via the bath solution. Ca currents were observed during depolarizing stimuli (1,000 ms voltage steps to +10 mV applied at 0.5 Hz). When elimination of the Ca current and new steady-state contractions were observed, the voltage-clamp protocol was repeated. Four high-Na cells were treated with nifedipine alone. It is important to note that with the exception of nifedipine, no other specific ion channel antagonists were used to isolate currents. This limitation precluded the analysis of all but the dihydropyridine (DHP)-sensitive Ca channel current.

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Intracellular Na and voltage dependence of peak contraction. Panel A: These data illustrate the voltage dependence of the peak contraction magnitude in cells dialyzed with Na-free (open circles; n=11) and high-Na pipette solution (closed circles; n=24). Data are averages±S.E.M. Contractions in high-Na cells were significantly larger than contractions in Na-free cells at all potentials positive to –30 mV (* indicates P0.01; ** indicates P0.001). The voltage dependence of contraction was bell-shaped in Na-free and high-Na myocytes. At very positive potentials, tonic contractions in high-Na cells increased with membrane potential. Panel B: Whole cell current–voltage relationship (nifedipine sensitive current). These data illustrate the peak whole cell currents (normalized to one-hundred percent) ±S.E.M. observed in eight myocytes dialyzed with high-Na. The inward current demonstrates a bell-shaped voltage dependence which is characteristic of the L-type Ca channel activity. Identical results were obtained in Na-free myocytes.

 
2.3 Statistical analyses
In the experiments designed to examine the contribution of intracellular sodium to contractions (Fig. 3A), analysis of variance (ANOVA) for repeated measures with fixed effects was performed (two factor factorial design with repeated measures on voltage). Modified logarithmic transformation was used to stabilize variances and to improve normality. The groups were zero-Na and high-Na pipette solution. Multiple comparisons used F-tests adjusted for repeated measures design with a Bonferroni correction. Significance levels were measured at P0.05, 0.01 and 0.001.

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Inhibition of SR Ca-ATPase and L-type Ca current on phasic contractions. Panel A: This figure illustrates the effect of thapsigargin treatment on phasic contraction in a Na-free myocyte. Thapsigargin treatment almost totally abolished phasic contractions. Panel B: This figure illustrates the effect of nifedipine on phasic contractions in a single high-Na cell. Nifedipine inhibited phasic contractions. The line at 1 s on the time scale is included to indicate the duration of the voltage-clamp test-step which was initiated at time zero. The change in length observed (in microns) is shown on the ordinate.

 

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 The contribution of intracellular Na to contractions
Experiments were designed to characterize how the rate, the magnitude and the voltage dependence of contraction were influenced by intracellular Na. For these studies, two groups of cells were used: one group was dialyzed with Na-free pipette solution (0 mmol/L Na) and the other with high-Na pipette solution (20 mmol/L Na). The differences between these two groups are expected to demonstrate how intracellular Na, presumably via reverse-mode Na–Ca exchange, can alter contraction. Contractions elicited from cells dialyzed with Na-free pipette solution are expected to display small effects of reverse-mode Na–Ca exchange. In these Na-free cells, contraction magnitude is predicted to be proportional to the voltage-dependent L-type Ca current, the fractional release of SR Ca and the degree of SR Ca loading [25, 26]. In contrast, contractions elicited from cells dialyzed with high-Na solution are also expected to reflect the effect of high intracellular Na to generate a greater degree of Ca influx via reverse-mode exchange. In Na-loaded cells, contractions are expected to be increased by several possible mechanisms. Reverse-mode exchange mediated Ca influx may: (1) trigger SR Ca release; (2) elevate cytosolic Ca to cause direct activation of contractile proteins; (3) increase SR Ca content.

Fig. 1A illustrates contraction data from two cells, one cell dialyzed with Na-free pipette solution and one cell dialyzed with high-Na pipette solution (see legend to Fig. 1). In these cells, two types of contractions were observed: rapid phasic contractions and slow tonic contractions. Contractions in Na-free cells were phasic, while contractions in high-Na cells were phasic and/or tonic depending on the test-step command voltage. Phasic contractions were characterized by rapid onset following cell membrane depolarization, rapid rates of rise and times to peak (data not included). Phasic contractions were also characterized by relaxation that began during the depolarizing test-step. Tonic contractions, on the other hand, were characterized by slower onset following membrane depolarization and slower rates of rise, and slower times to peak (data not included). Furthermore, tonic contractions rose nearly monotonically during the duration of the voltage step and did not begin to relax until after the depolarizing test-step was terminated (see Fig. 1A).

View larger version (28K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Intracellular Na and the time course of contraction. Panel A: Each experiment consisted of 16 test-steps. Each test-step increased +10 mV over the preceding test-step. Only four contractions are shown in this figure. 0 mM Na and 20 mM Na correspond to the [Na] of the pipette filling solution (in mmol/L). The line at 1 second on the abscissa is included to indicate the duration of the voltage-clamp test-step which was initiated at time zero (test-step duration=1 s). The actual change in length observed (in microns) is shown on the ordinate. Panel B: Contractions at +20 and +100 mV are superimposed for the Na-free (left panel) and high-Na (right panel) cell. Only phasic contractions are seen in the Na-free cell. Phasic and tonic contractions are seen in the high-Na cell. The contraction at +100 mV in the high Na cell was typical of tonic contractions observed in these studies.

 
For the purpose of comparison, phasic and tonic contractions observed in a single Na-free and a single high-Na cell are superimposed in Fig. 1B. The data in Fig. 1B demonstrate the results obtained in all other Na-free and high-Na cells. In Na-free cells, substantial tonic contractions were not observed. In high-Na cells, on the other hand, maximum tonic contractions were larger than phasic contractions. These results suggest that different cellular mechanisms are responsible for each type of contraction.

Fig. 2A demonstrates the voltage dependence of the average peak contraction magnitude from 11 Na-free cells and 24 high-Na cells (see figure legend). Fig. 2A demonstrates that, in both Na-free and high-Na cells, the voltage dependence of phasic contractions were bell-shaped. This contraction–voltage relationship was similar to the L-type Ca channel current–voltage relationship (Fig. 2B). Fig. 2A also demonstrates that the voltage dependence of tonic contractions in high-Na cells did not follow the voltage dependence of the Ca current. Instead, tonic contractions in high-Na cells increased in proportion to the depolarizing step potential. Contraction magnitudes were significantly greater in cells dialyzed with high-Na pipette solution at all potentials positive to –20 mV (see legend, Fig. 2A). The bell-shaped portion of the high-Na contraction–voltage relationship between –30 and +50 mV likely reflects increased SR Ca loading in the high-Na myocytes. The portion of this curve positive to +50 mV can be attributed to reverse-mode exchanger-mediated increases in either SR Ca release or cytosolic Ca content.

Phasic contractions are thought to result from SR Ca release. To test this idea Na-free myocytes were treated with thapsigargin, a specific inhibitor of the SR Ca-ATPase that can effectively deplete SR Ca stores [27]. A representative result is shown in Fig. 3A. In Na-free myocytes, phasic contractions were observed only under control conditions. Treatment with 100 nM thapsigargin almost totally eliminated these contractions. Similar results were obtained in three other myocytes. These results strongly support the idea that the phasic contractions in Na-free myocytes are the result of SR Ca release. Thapsigargin inhibited phasic contractions in high-Na cells in similar fashion (see below).

Ca current is thought to be the primary trigger for the SR Ca release that causes phasic contractions [8]. Ca influx from reverse mode Na/Ca exchange may also trigger SR Ca release [14]. Fig. 3B shows a representative example of the effects of nifedipine on the phasic contractions of a high-Na myocyte. In this experiment, nifedipine treatment eliminated the Ca current and abolished phasic contractions. Similar results were obtained in four other high-Na cells. These experiments indicate that both L-type Ca current and SR Ca release are required for phasic contractions in both Na-free and Na-loaded ventricular myocytes from failing human heart.

3.2 Reverse-mode Na–Ca exchange mediated contractions following inhibition of SR Ca reuptake with thapsigargin
Subsequent experiments examined by what mechanism high-pipette Na caused increased contractions. Following collection of control data, Na-free and high-Na myocytes were treated with thapsigargin. Comparison of contraction records from cells before and after treatment with thapsigargin should reveal the role of SR Ca release and reuptake in contractions mediated by reverse-mode exchange. Thapsigargin-treated cells were also exposed to nifedipine (see below). Representative phasic and tonic contractions from a thapsigargin-treated high-Na cell are shown in Fig. 4. The phasic contractions were almost totally abolished by thapsigargin in high-Na cells (Fig. 3A and Fig. 4). On the other hand, tonic contractions in high-Na cells were not inhibited by thapsigargin treatment (tonic contractions were not observed in Na-free cells). Average data illustrating the voltage dependence of contraction collected from 5 high-Na cells after thapsigargin treatment are shown in Fig. 5. These data and the data in Fig. 3A show that thapsigargin selectively inhibits phasic contractions in high-Na and Na-free cells. Thapsigargin treatment did not inhibit tonic contractions. In five out of five cells, phasic contractions in high-Na myocytes were almost totally eliminated by thapsigargin treatment (Fig. 4). In the same cohort, thapsigargin treatment did not diminish the magnitude of the maximal tonic contractions versus the control contractions. Our observation that tonic contraction magnitudes were not inhibited by thapsigargin treatment cells strongly suggests that tonic contractions do not involve SR calcium release.

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Inhibition of SR Ca-ATPase on phasic and tonic contractions in high-Na cells. The effects of thapsigargin on phasic contraction at +20 mV and tonic contractions at +100 mV are shown. Thapsigargin almost eliminated the phasic contraction but had no major effect on the tonic contraction. Identical results were obtained in four other high-Na myocytes.

 

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Inhibition of SR Ca-ATPase voltage dependence of peak contraction. These data illustrate the voltage dependence of the tonic contraction magnitude in high-Na cells recorded following treatment with thapsigargin (n=5). The figure also contains a plot of the intracellular Ca concentration ([Ca]i) predicted by the thermodynamics of the exchanger reaction under the conditions of this study (see text). The voltage dependence of the magnitude of tonic contractions in thapsigargin-treated high-Na cells closely follows the voltage dependence of the predicted [Ca]i with a small deviation in the region corresponding to the region of the Ca current (see Fig. 6).

 
In thapsigargin-treated high-Na cells, small residual contractions were occasionally observed in the voltage range that corresponded to large Ca currents (Fig. 2B). Fig. 6 illustrates that these small contractions were eliminated when thapsigargin-treated myocytes were also exposed to the Ca channel blocker nifedipine (similar results were observed in eight other cells). Nifedipine did not significantly diminish tonic contractions in thapsigargin-treated myocytes (data not shown). As has been previously suggested, our data indicate that Ca current can cause small contractions when SR Ca uptake is inhibited with thapsigargin [5].

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Elimination of residual small thapsigargin resistant contractions with nifedipine in high-Na cell.

 
If tonic contractions are determined by exchanger-mediated elevations of cytosolic Ca, then the voltage dependence of the magnitude of these contractions should follow the voltage dependence of the steady-state intracellular Ca concentration ([Ca]_i) predicted by the thermodynamics of the Na–Ca exchange reaction: [28]

The relationship between the predicted [Ca]_i and the membrane voltage is shown in Fig. 5B where all of the variables in the above equation were assigned values from the experimental conditions (E_m=test-step potential, [Na]_o=152 mmol/L, [Na]_i=20 mmol/L, [Ca]_o=1 mmol/L, [Ca]_i=dependent variable in mmol/L, and T=310 K). The similarity between the shape of the predicted [Ca]_i–voltage relationship and the shape of the tonic contraction–voltage relationship in thapsigargin-treated (Fig. 5B) high-Na cells strongly suggests that tonic contractions are mediated by reverse-mode exchange.

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
These studies were specifically designed to determine the contribution of Na–Ca exchange-mediated Ca influx to excitation-contraction processes in isolated myocytes from the myocardium of failing human left ventricle, however, our results are also relevant to the controversy surrounding the role of Na–Ca exchange-mediated Ca influx in general. The debate over whether Ca influx via reverse-mode exchange acts to influence contraction by a direct mechanism (through increasing cytosolic Ca) or by an indirect mechanism (by increasing SR Ca release and/or storage) is unresolved. Animal studies have fueled the controversy, in large part, because they have provided varied results [15–17]. Recent molecular studies have indicated that Na–Ca exchanger message and protein levels are increased in failing human heart, suggesting that the contribution of the Na–Ca exchanger to Ca homeostasis and to contraction may be increased [18]. An additional purpose of this study was to investigate the mechanism of the contribution of Ca influx via reverse-mode exchange to contractions in failing human myocytes. Experiments were designed to accentuate or to diminish reverse-mode exchanger activity and to eliminate Ca current and/or SR Ca release. It is important to note that this study did not intend to determine if the magnitude of Na–Ca exchange mediated contributions are altered in failing versus normal myocytes.

Our major findings are: (1) Phasic contractions had a bell-shaped voltage dependence in both Na-free and high-Na cells and were eliminated by blocking either Ca current or SR Ca release. (2) The magnitude of both phasic and tonic contractions were larger in myocytes dialyzed with high-Na solution compared to Na-free solution. (3) Tonic contractions in high-Na myocytes were insensitive to thapsigargin and to nifedipine and increased with membrane voltage in a manner that was well fit by the energetics of the Na–Ca exchanger.

4.1 Phasic contractions
Phasic contractions were marked by a rapid rising phase and relaxation which began before the termination of the voltage clamp test-step (Fig. 1). The bell-shaped voltage dependence (Fig. 2A) was similar to the whole cell DHP-sensitive Ca current (Fig. 2B). Our results also showed that phasic contractions were largely eliminated when thapsigargin (Fig. 3A and Fig. 5) caused SR Ca stores to become diminished [23, 26]and by nifedipine (Fig. 3B). L-type Ca current is known to trigger contraction in cardiac myocytes by inducing Ca release from the SR [1, 2]and our results strongly support that Ca current induced SR Ca release is the primary mechanism for phasic contractions in failing human myocytes.

Phasic contractions in high-Na myocytes were significantly larger than phasic contractions in Na-free myocytes (Fig. 2A). Increased reverse-mode Na–Ca exchange activity could account for this difference by increasing SR Ca loading and/or by combining with the L-type Ca current to produce a larger trigger for SR Ca release. The fact that nifedipine blocked phasic contractions in high-Na myocytes suggests that Ca current is still the primary trigger in these cells. Our results suggest that exchanger-mediated contributions to phasic contractions in failing human ventricular myocardium are the result of increased SR Ca loading and not the result of increased triggering of SR Ca release.

4.2 Tonic contractions
The kinetics and the voltage dependence of tonic contractions were different than the kinetics and the voltage dependence of phasic contractions (Figs. 1 and 2). Tonic contractions were very small or absent in myocytes dialyzed with Na-free solutions. In high-Na cells, maximal tonic contractions were greater than maximal phasic contractions. In thapsigargin treated high-Na cells, our experimental results indicated that tonic contractions varied with membrane voltage in the same fashion as the predicted steady-state intracellular [Ca] (Fig. 5B).

Tonic contractions could result from a direct elevation of cytosolic free Ca by reverse-mode exchange activity with or without a contribution from the SR [29–31]. If slow SR Ca release is involved in tonic contractions, then inhibition of SR function should reduce tonic contraction magnitude. In our experiments, inhibition of SR Ca uptake and depletion of SR Ca stores with thapsigargin did not diminish tonic contraction magnitude. This suggests that under control conditions, SR Ca release is not responsible for tonic contractions. That tonic contraction magnitudes were increased in thapsigargin treated high-Na cells indicates that the SR may accumulate Ca that enters the cytoplasm via reverse-mode exchange. Our findings are strong evidence that Ca entry via reverse-mode Na–Ca exchange is the primary mechanism for Ca entry during tonic contractions. It is important to note, that these experiments were designed to elucidate the mechanism of exchanger mediated contributions to contractions and that large tonic contractions were observed with long duration voltage clamp steps at very positive potentials outside the physiologically relevant voltage range. Our results suggest that Ca influx via reverse-mode Na–Ca exchange is unlikely to play a direct role in E-C coupling in failing human ventricular myocytes independent of its contribution to SR Ca loading.

In summary, the results of the present study suggest that Ca influx via reverse-mode Na–Ca exchange contributes to contractions in failing human ventricular myocytes by loading the SR. Moreover, at very positive potentials reverse mode exchange can cause contraction directly. However, under our conditions, it does not appear that Ca influx via reverse-mode exchange induces SR Ca release.

Time for primary review 36 days.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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