Showing papers by "Donald M. Bers published in 1997"

Effects of [Ca2+]i, SR Ca2+ load, and rest on Ca2+ spark frequency in ventricular myocytes

Abstract: In heart, spontaneous local increases in cytosolic Ca2+ concentration ([Ca2+]i) called "Ca2+ sparks" may be fundamental events underlying both excitation-contraction coupling and resting Ca2+ leak from the sarcoplasmic reticulum (SR). In this study, resting Ca2+ sparks were analyzed in rabbit and rat ventricular myocytes with laser scanning confocal microscopy and the fluorescent Ca2+ indicator fluo 3. During the first 20 s of rest after regular electrical stimulation, both the frequency of Ca2+ sparks and SR Ca2+ content gradually decreased in rabbit. When rabbit SR Ca2+ content was decreased by reduction of stimulation rate. the initial resting spark frequency was also decreased, even though resting [Ca2+]i was unchanged. The rest-dependent decrease in spark frequency in rabbit cells was prevented by inhibition of Na+/Ca2+ exchange (which also prevents SR Ca2+ depletion during rest). These results suggest that elevation of SR Ca2+ content can increase Ca2+ spark frequency. In contrast to rabbit cells, 20 s of rest produced a gradual increase in spark frequency in rat cells, although SR Ca2+ content was constant and Ca2+ influx was completely prevented. This indicates that there is a time-dependent increase in spark probability during rest that is independent of [Ca2+]i or SR Ca2+. This effect was also apparent in rabbit cells when SR Ca2+ depletion was prevented by blocking Na+/Ca2+ exchange. Stimulation of Ca2+ extrusion via Na+/Ca2+ exchange in the rat (by Ca2+-free superfusion, which slowly depletes SR Ca2+ content) converted the normal rest-dependent increase in spark frequency to a decrease. The amplitude of individual Ca2+ sparks increased with increasing SR Ca2+ content. In the Ca2+-overloaded state, fusion of sparks or long-lasting localized increases of [Ca2+]i were observed with increased spark frequency. We conclude that the resting frequency of Ca2+ sparks can be independently affected by changes in SR Ca2+ content, [Ca2+]i, or rest period. The latter may reflect recovery of the SR Ca2+ release channels from inactivation or adaptation.

The effect of Ca2+–calmodulin‐dependent protein kinase II on cardiac excitation–contraction coupling in ferret ventricular myocytes

Abstract: 1. The effect of Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) on excitation-contraction coupling (E-C coupling) was studied in intact ferret cardiac myocytes using the selective inhibitor KN-93, KN-93 decreased steady-state (SS) twitch [Ca2+]i (by 51%), resting Ca2+ spark frequency (by 88%) and SS sarcoplasmic reticulum (SR) Ca2+ content evaluated by eaffeine application (by 37.5%). 2. Increasing extracellular Ca2+ concentration ([Ca2+]o) to 5 mM in KN-93 restored SR Ca2+ load and Ca2+ spark frequency towards that in control (2 mM Ca2+o), but SS twitch [Ca2+]i was still significantly depressed by KN-93. 3. KN-93 decreased Ca2+ transient amplitude of SS twitches much more strongly than the amplitude of post-rest (PR) twitches. In the control, the time constant (Tau) of [Ca2+]i decline of SS twitches was faster than that for PR twitches. This stimulation-dependent acceleration of [Ca2+]i decline was abolished by KN-93. 4. Voltage-clamp experiments demonstrated that KN-93 significantly inhibited sarcolemmal L-type Ca2+ current (ICa) during repetitive pulses by slowing recovery from inactivation. This may explain the preferential action of KN-93 to suppress SS vs. PR twitches. 5. In KN-93, even when both ICa and SR Ca2+ load were matched to the control levels by manipulation of conditioning voltage-clamp pulses, contraction and twitch Ca2+ transients were still both significantly depressed (to 39 and 49% of control, respectively). 6. Since KN-93 reduced SR Ca2+ release channel (RyR) activity during E-C coupling, even for matched SR Ca2+ load and trigger ICa, we infer that endogenous CaMKII is an important modulator of E-C coupling in intact cardiac myocytes. Effects of KN-93 on ICa and SS twitch [Ca2+]i decline also indicate that endogenous CaMKII may have stimulatory effects on ICa and SR Ca2+ uptake.

Intracellular Ca2+ Increases the Mitochondrial NADH Concentration During Elevated Work in Intact Cardiac Muscle

Abstract: It is not clear how mitochondrial energy production is regulated in intact tissue when energy consumption suddenly changes. Whereas mitochondrial [NADH] ([NADH]m) may regulate cellular respiration rate and energetic state, it is not clear how [NADH]m itself is controlled during increased work in vivo. We have varied work and [Ca2+] in intact cardiac muscle while assessing [NADH]m using fluorescence spectroscopy. When increased work was accompanied by increasing average [Ca2+]c (by increasing [Ca2+]c or pacing frequency), [NADH]m initially fell and subsequently recovered to a new steady state level. Upon reduction of work, [NADH]m overshot and then returned to control levels. In contrast, when work was increased without increasing average [Ca2+]o (by increasing sarcomere length), [NADH]m fell similarly, but no recovery or overshoot was observed. This Ca(2+)-dependent recovery and overshoot may be attributed to Ca(2+)-dependent stimulation of mitochondrial dehydrogenases. We conclude that the immediate initial increase in respiration rate upon elevation of work is not activated by increased [NADH]m (since [NADH]m rapidly fell) or by [Ca2+]o (since work could also be increased at constant [Ca2+]c). However, during sustained high work, a Ca(2+)-dependent mechanism causes slow recovery of [NADH]m toward control values. This demonstrates a Ca(2+)-dependent feed-forward control mechanism of cellular energetics in cardiac muscle during increased work.

Downregulation of sarcoplasmic reticulum Ca(2+)-ATPase during progression of left ventricular hypertrophy.

Abstract: To determine whether reduced sarcoplasmic reticulum (SR) Ca(2+)-adenosinetriphosphatase (ATPase) (SERCA2) activity contributes to delayed myocardial relaxation during chronic left ventricular hypertrophy (LVH) progression, LVH was produced in rats by abdominal aortic coarctation. Systolic and diastolic functions were assessed in vivo 8 and 16 wk after surgery, and compositional alterations in LV myocardium [SERCA2 concentration, myosin heavy chain (MHC) isoenzymes, and tissue collagen] were correlated with the development of prolonged isovolumic relaxation and impaired cardiac performance over time. Myocardial relaxation was prolonged in 8-wk banded rats, despite normal isovolumic systolic function and LV end-diastolic pressure (LVEDP). No significant alterations in SERCA2 protein, beta-MHC, or fibrillar collagen levels were observed at this early time point. In contrast, LV SERCA2, beta-MHC, and fibrillar collagen concentrations were all significantly altered in 16-wk banded rats. These late compositional changes were associated with reduced cardiac performance, as manifested by a significant elevation in LVEDP (14 +/- 2 mmHg). The 34% decrease in SERCA2 protein was associated with reduced SR Ca2+ uptake and an even greater reduction (76%) in SERCA2 mRNA. SERCA2 mRNA levels were also significantly reduced to 43 +/- 10% of sham-operated rats 8 wk after banding, despite unchanged SERCA2 protein levels and normal SR Ca2+ uptake. These results argue against a significant contribution of SERCA2 downregulation to the subtle alterations in myocardial relaxation observed in compensated LVH. However, the early reduction in SERCA2 mRNA levels may serve as a molecular marker for impaired cardiac performance during the transition from compensated LVH to heart failure.

Cardiac myocyte volume, Ca2 fluxes, and sarcoplasmic reticulum loading in pressure-overload hypertrophy

Abstract: Alterations in cellular Ca2+ transport and excitation-contraction coupling may contribute to dysfunction in cardiac hypertrophy. Left ventricular myocytes were isolated from rat hearts after 15-18 wk of suprarenal abdominal aortic banding to evaluate the hypothesis that hypertrophy alters the relationship between Ca2+ current (ICa) and sarcoplasmic reticulum (SR) Ca2+ load during steady-state voltage-clamp depolarization. Mean arterial pressure (MAP) and heart weight-to-body weight ratio of banded (B) animals were significantly higher than in control or sham-operated animals (C). Isolated myocyte dimensions and volume increased in parallel with whole heart hypertrophy and elevation in MAP. However, the relationship between membrane surface area (measured by capacitance) and cell volume (measured by laser scanning confocal microscopy) was unaltered (C: 8.9 +/- 0.3; B: 8.5 +/- 0.4 pF/pl). No differences in the voltage dependence of ICa activation, steady-state inactivation, or recovery from inactivation were detected between C and B myocytes. Maximal ICa density for the two groups was also not different either under basal conditions (C: 4.28 +/- 0.98; B: 4.57 +/- 0.60 pA/pF) or in the presence of 1 microM isoproterenol (C: 16.6 +/- 2.3; B: 16.5 +/- 2.3 pA/pF). The fraction of Ca2+ released from the SR by a single twitch was 55.4 +/- 9.4% in C and 37.1 +/- 6.9% in B (not significantly different). Steady-state Ca2+ influx during a twitch was calculated in units of micromoles per liter of nonmitochondrial volume from the integral of ICa (C: 13.4 +/- 0.7 microM; B: 13.3 +/- 0.8 microM). The SR Ca2+ load was similarly calculated by integration of Na+/Ca2+ exchange current induced by rapid caffeine application (C: 140 +/- 9 microM; B: 169 +/- 18 microM). We conclude that significant cellular hypertrophy is associated with proportional increases in sarcolemmal ICa influx, SR Ca2+ loading, and the amount of SR Ca2+ released in this model of pressure overload.

Ca transport during contraction and relaxation in mammalian ventricular muscle.

Abstract: During relaxation of cardiac muscle four Ca transport systems can compete to remove Ca from the myoplasm. These are 1) the SR Ca-ATPase, 2) the sarcolemmal Na/Ca exchange, 3) the sarcolemmal Ca-ATPase, and 4) the mitochondrial Ca uniporter. Isolated ventricular myocytes loaded with the intracellular fluorescent Ca indicator indo-1 were used to study [Ca]i decline during relaxation. By selective inhibition of the various Ca transporters above the dynamic interaction of these systems during relaxation was evaluated. Quantitatively the SR Ca-ATPase and Na/Ca exchange are clearly the most important (accounting for > 95% of Ca removal). However, the balance of Ca fluxes between these systems vary in a species dependent manner. For example, the SR is much more strongly dominant in rat ventricular myocytes, where ~ 92% of Ca removal is via SR Ca-ATPase and only 7% via Na/Ca exchange during a twitch. In other species (rabbit, ferret, cat, and guinea-pig) the balance is more in the range of 70–75% SR Ca-ATPase and 25–30% Na/Ca exchange. Ferret ventricular myocytes also exhibit a unusually strong sarcolemmal Ca-ATPase. During the normal steady state cardiac contraction-relaxation cycle the same amount of Ca must leave the cell as enters over a cardiac cycle. This implies that 25–30% of the Ca required to activate contraction must enter the cell at each cardiac cycle. Experiments using voltage clamp to measure both Ca current and Na/Ca exchange current demonstrate that this amount of Ca may be supplied by the L-type Ca current.

Myosin heavy chain gene expression in neonatal rat heart cells: Effects of [Ca2+](i) and contractile activity

Abstract: To determine if mechanical signals or alterations in intracellular Ca2+ concentration ([Ca2+]i) affect myosin heavy chain (MHC) gene expression in spontaneously beating, neonatal rat ventricular myocytes, contractile activity was inhibited with verapamil, KCl, or 2,3-butanedione monoxime (BDM), and their acute and chronic effects on myocyte shortening, [Ca2+]i, and MHC gene expression were examined. Despite their differing effects on [Ca2+]i, verapamil, KCl, and BDM all inhibited contractile activity and markedly downregulated beta-MHC mRNA levels to 24 +/- 5, 21 +/- 7, and 6 +/- 2% of contracting cells, respectively. In contrast, these inhibitors of contraction upregulated alpha-MHC mRNA levels to 163 +/- 19, 156 +/- 7, and 198 +/- 20% of contracting cells, respectively. Transient transfection with a rat beta-MHC promoter-luciferase expression plasmid demonstrated that all inhibitors of contraction significantly decreased beta-MHC promoter activity. Paradoxically, contractile arrest also inhibited alpha-MHC promoter activity, suggesting that increased alpha-MHC mRNA levels resulted from posttranscriptional mechanisms. Actinomycin D mRNA stability assays indicated that alpha-MHC mRNA half-life was prolonged in noncontracting cells (33 h) compared with contracting myocytes (14 h). Contraction-dependent alterations in MHC gene expression were not dependent on release of angiotensin II or other growth factors into the culture medium. Thus intrinsic mechanical signals rather than alterations in [Ca2+]i regulate alpha-MHC and beta-MHC gene expression by both transcriptional and posttranscriptional mechanisms.

Diastolic Sr Ca Efflux In Atrial Pacemaker Cells And Ca-overloaded Myocytes.

Abstract: Evidence has shown that the sarcoplasmic reticulum (SR) of cardiac cells releases Ca not only during excitation-contraction coupling but also during diastole, albeit at a much lower rate. This diastolic SR Ca release (leak) has also been implicated in the generation of spontaneous depolarization in latent atrial pacemaker cells of the cat right atrium. In the present work, we sought to measure Ca transients in pacemaker and nonpacemaker cells of the cat using the fluorescent Ca indicator indo 1. Atrial latent pacemaker cells develop a slow Ca transient when rested in the presence of both Na- and Ca-free solution and thapsigargin [used to inhibit Na/Ca exchange and SR Ca adenosinetriphosphatase (Ca-ATPase), respectively]. This increase in cytosolic Ca concentration ([Ca]i) is probably caused by the rate of SR Ca leak exceeding the capacity of the remaining Ca transport systems (e.g., sarcolemmal Ca-ATPase and mitochondrial Ca uptake). However, neither cat sinoatrial (SA) node cells nor myocytes from cat atrium or ventricle exhibited a similar increase in [Ca]i during the same protocol. This indicates that SR Ca leak in these cells occurred at a rate low enough to be within the capacity of the slow Ca transporters, as observed previously in rabbit ventricular myocytes. When atrial and ventricular myocytes were stimulated at higher frequencies, sufficient to markedly increase diastolic and systolic [Ca]i and approach Ca overload (and spontaneous activity), they responded to inhibition of SR Ca-ATPase and Na/Ca exchange with a slow Ca transient similar to that normally observed in atrial latent pacemaker cells. Furthermore, the SR Ca depletion by thapsigargin did not affect spontaneous activity of SA node cells, but it prevented or slowed pacemaker activity in the atrial latent pacemaker cells. These findings suggest that enhanced diastolic SR Ca efflux contributes significantly to the generation of spontaneous activity in atrial subsidiary pacemakers under normal conditions and in Ca-overloaded myocytes but not in SA node cells.

Diastolic SR Ca efflux in atrial pacemaker cells and Ca-overloaded myocytes

Abstract: Evidence has shown that the sarcoplasmic reticulum (SR) of cardiac cells releases Ca not only during excitation-contraction coupling but also during diastole, albeit at a much lower rate. This diastolic SR Ca release (leak) has also been implicated in the generation of spontaneous depolarization in latent atrial pacemaker cells of the cat right atrium. In the present work, we sought to measure Ca transients in pacemaker and nonpacemaker cells of the cat using the fluorescent Ca indicator indo 1. Atrial latent pacemaker cells develop a slow Ca transient when rested in the presence of both Na- and Ca-free solution and thapsigargin [used to inhibit Na/Ca exchange and SR Ca adenosinetriphosphatase (Ca-ATPase), respectively]. This increase in cytosolic Ca concentration ([Ca]i) is probably caused by the rate of SR Ca leak exceeding the capacity of the remaining Ca transport systems (e.g., sarcolemmal Ca-ATPase and mitochondrial Ca uptake). However, neither cat sinoatrial (SA) node cells nor myocytes from cat atrium or ventricle exhibited a similar increase in [Ca]i during the same protocol. This indicates that SR Ca leak in these cells occurred at a rate low enough to be within the capacity of the slow Ca transporters, as observed previously in rabbit ventricular myocytes. When atrial and ventricular myocytes were stimulated at higher frequencies, sufficient to markedly increase diastolic and systolic [Ca]i and approach Ca overload (and spontaneous activity), they responded to inhibition of SR Ca-ATPase and Na/Ca exchange with a slow Ca transient similar to that normally observed in atrial latent pacemaker cells. Furthermore, the SR Ca depletion by thapsigargin did not affect spontaneous activity of SA node cells, but it prevented or slowed pacemaker activity in the atrial latent pacemaker cells. These findings suggest that enhanced diastolic SR Ca efflux contributes significantly to the generation of spontaneous activity in atrial subsidiary pacemakers under normal conditions and in Ca-overloaded myocytes but not in SA node cells.

BayK 8644 increases resting calcium spark frequency in ferret ventricular myocytes.

Abstract: BayK 8644, an L-type Ca2+ channel agonist, has been shown to increase sarcoplasmic reticulum (SR) Ca2+ release in dog and ferret ventricular muscle. The visualization of local increase in cytosolic Ca2+ concentration, called the "Ca2+ spark", has enabled us to investigate the elementary events of the BayK-induced Ca2+ release from the SR. In this study, the effects of BayK on twitch Ca2+ transients and Ca2+ sparks were examined in ferret ventricular myocytes with laser scanning confocal microscopy and a fluorescent calcium indicator, fluo 3. BayK converted the post rest potentiation of twitch Ca2+ transients to decay. The Ca2+ spark frequency under control conditions was fairly constant during 20 s of rest after interruption of electrical stimulation. BayK (100 nM) increased the spark frequency by 465.7 +/- 90.3% of control but did not change the spatial and temporal characteristics of the individual sparks. The increase in spark frequency by BayK was not affected by perfusion with Ca(2+)-free solution, but was suppressed by the addition of nifedipine (10 microM), suggesting that the BayK effects on Ca2+ sparks were mediated by the sarcolemmal dihydropyridine (DHP) receptor, but.were independent of Ca2+ influx. These findings suggest that BayK activates SR Ca2+ release at rest through a putative linkage between the sarcolemmal DHP receptor and the SR ryanodine receptor.