Showing papers by "Donald M. Bers published in 1998"

Cardiac myocyte calcium transport in phospholamban knockout mouse: relaxation and endogenous CaMKII effects

Abstract: Increases in heart rate are accompanied by acceleration of relaxation. This effect is apparent at the single myocyte level and depends on sarcoplasmic reticulum (SR) Ca transport and Ca/calmodulin ...

Cytosolic and mitochondrial Ca2+ signals in patch clamped mammalian ventricular myocytes

Abstract: 1. Ventricular myocytes isolated from ferret or cat were loaded with the acetoxymethyl ester form of indo-1 (indo-1 AM) such that approximately 75% of cellular indo-1 was mitochondrial. The intramitochondrial indo-1 concentration was 0.5-2 mM. 2. Myocytes were also voltage clamped (membrane capacitance, Cm = 100 pF) and a typical wash-out time constant of cytosolic indo-1 by a patch pipette was found to be approximately 300 s. Depolarizations to +110 mV produced graded and progressive cellular Ca2+ load via Na(+)-Ca2+ exchange. 3. During these relatively slow Ca2+ transients, cell contraction (delta L) paralleled fluorescence ratio signals (R) such that delta L could be used as a bioassay of cytosolic [Ca2+] ([Ca2+]c), where [Ca2+]CL is the inferred signal which is delayed by approximately 200 ms from true [Ca2+]c. 4. In myocytes without Mn2+ quench, the kinetics of the total cellular indo-1 signal, delta R (including cytosolic and mitochondrial components), match delta L during stimulations at low basal [Ca2+]i. However, after progressive Ca2+ loading, delta R kinetics deviate from delta L dramatically. The deviation can be completely blocked by a potent mitochondrial Ca2+ uniport blocker, Ru360. 5. When cytosolic indo-1 is quenched by Mn2+, initial moderate stimulation triggers contractions (delta L), but no change in indo-1 signal, indicating both the absence of cytosolic Ca(2+)-sensitive indo-1 and unchanged mitochondrial [Ca2+] (delta [Ca2+]m). Subsequent stronger stimulation evoked larger delta L and also delta R. The threshold [Ca2+]c for mitochondrial Ca2+ uptake was 300-500 nM, similar to that without Mn2+ quench. 6. At high Ca2+ loads where delta [Ca2+]m is detected, the time course of [Ca2+]m was different from that of [Ca2+]c. Peak [Ca2+]m after stimulation has an approximately 1 s latency with respect to [Ca2+]c, and [Ca2+]m decline is extremely slow. 7. Upon a Ca2+ influx which increased [Ca2+]c by 0.4 microM and [Ca2+]m by 0.2 microM, total mitochondrial Ca2+ uptake was approximately 13 mumol (1 mitochondria)-1. 8. With Mn2+ quench of cytosolic indo-1, there was no mitochondrial uptake of Mn2+ until the point at which mitochondrial Ca2+ uptake became apparent. However, after mitochondrial Ca2+ uptake starts, mitochondria continually take up Mn2+ even during relaxation, when [Ca2+]c is low. 9. It is concluded that mitochondria in intact myocytes do not take up detectable amounts of Ca2+ during individual contractions, unless resting [Ca2+]c exceeds 300-500 nM. At high cell Ca2+ loads and [Ca2+]c, mitochondrial Ca2+ transients occur during the twitch, but with much slower kinetics than those of [Ca2+]c.

Ca flux, contractility, and excitation-contraction coupling in hypertrophic rat ventricular myocytes

Abstract: Left ventricular hypertrophy (∼40%) was induced in rats by banding of the abdominal aorta. After 16 wk, ventricular homogenates were prepared for biochemical measurements and ventricular myocytes w...

Control of Maximum Sarcoplasmic Reticulum Ca Load in Intact Ferret Ventricular Myocytes : Effects of Thapsigargin and Isoproterenol

Abstract: In steady state, the Ca content of the sarcoplasmic reticulum (SR) of cardiac myocytes is determined by a balance among influx and efflux pathways. The SR Ca content may be limited mainly by the ATP-supplied chemical potential that is inherent in the gradient between SR and cytosol. That is, forward Ca pumping from cytosol to SR may be opposed by energetically conservative reverse pumping dependent on intra-SR free [Ca]. On the other hand, SR Ca loading may be limited by dissipative pathways (pump slippage and/or pump-independent leak). To assess how SR Ca content is limited, we loaded voltage-clamped ferret ventricular myocytes cumulatively with known amounts of Ca via L-type Ca channels (ICa), using Na-free solutions to prevent Na/Ca exchange. We then measured the maximal resulting caffeine-released SR Ca content under control conditions, as well as when SR Ca pumping was accelerated by isoproterenol (1 μM) or slowed by thapsigargin (0.2–0.4 μM). Under control conditions, SR Ca content reached a limit of 137 μmol·liter cytosol−1 (nonmitochondrial volume) when measured by integrating caffeine-induced Na/Ca exchange currents (∫INaCaXdt) and of 119 μmol·liter cytosol−1 when measured using fluorescence signals dependent on changes in cytosolic free Ca ([Ca]i). When Ca-ATPase pumping rate was slowed 39% by thapsigargin, the maximal SR Ca content decreased by 5 (∫INaCaXdt method) or 23% (fluorescence method); when pumping rate was increased 74% by isoproterenol, SR Ca content increased by 10% (fluorescence method) or 20% (∫INaCaXdt method). The relative stability of the SR Ca load suggests that dissipative losses have only a minor influence in setting the SR Ca content. Indeed, it appears that the SR Ca pump in intact cells can generate a [Ca] gradient approaching the thermodynamic limit.

Effects of left ventricular hypertrophy on force and Ca2+ handling in isolated rat myocardium.

Abstract: To study the effect of left ventricular (LV) hypertrophy on force and Ca2+handling in isolated rat myocardium, LV hypertrophy was induced in rats by banding of the abdominal aorta. After 16 wk, art...

Bay K 8644 Increases Resting Ca2+ Spark Frequency in Ferret Ventricular Myocytes Independent of Ca Influx Contrast With Caffeine and Ryanodine Effects

Abstract: Bay K 8644, an L-type Ca2+ channel agonist, was shown previously to increase resting sarcoplasmic reticulum (SR) Ca2+ loss and convert post-rest potentiation to decay in dog and ferret ventricular muscle. Here, the effects of Bay K 8644 on local SR Ca2+ release events (Ca2+ sparks) were measured in isolated ferret ventricular myocytes, using laser scanning confocal microscopy and the fluorescent Ca2+ indicator fluo-3. The spark frequency under control conditions was fairly constant during 20 s of rest after interruption of electrical stimulation. Bay K 8644 (100 nmol/L) increased the spark frequency by 466+/-90% of control at constant SR Ca2+ load but did not change the spatial and temporal characteristics of individual sparks. The increase in spark frequency was maintained throughout the period of rest. The increase in Ca2+ spark frequency induced by Bay K 8644 was not affected by superfusion with Ca2+-free solution (with 10 mmol/L EGTA) but was suppressed by the addition of 10 micromol/L nifedipine (which by itself did not alter resting Ca2+ spark frequency). This suggests that the effect of Bay K 8644 on Ca2+ sparks is mediated by the sarcolemmal dihydropyridine receptor but is also independent of Ca2+ influx. Low concentrations of caffeine (0.5 mmol/L) increased both the average frequency and duration of sparks. Ryanodine (50 nmol/L) increased the spark frequency and also induced long-lasting Ca2+ signals. This may indicate long-lasting openings of SR Ca2+ release channels and a lack of local SR Ca2+ depletion. In lipid bilayers, Bay K 8644 had no effect on either single-channel current amplitude or open probability of the cardiac ryanodine receptor. It is concluded that Bay K 8644 activates SR Ca2+ release at rest, independent of Ca2+ influx and perhaps through a functional linkage between the sarcolemmal dihydropyridine receptor and the SR ryanodine receptor. In contrast, caffeine and ryanodine modulate Ca2+ sparks by a direct action on the SR Ca2+ release channels.

Subcellular properties of [Ca2+](i) transients in phospholamban- deficient mouse ventricular cells

Abstract: The regulatory protein phospholamban exerts a physiological inhibitory effect on the sarcoplasmic reticulum (SR) Ca2+ pump that is relieved with phosphorylation. We have studied the subcellular pro...

Regulation of Mitochondrial [NADH] by Cytosolic [Ca2+] and Work in Trabeculae From Hypertrophic and Normal Rat Hearts

Abstract: Pressure overload hypertrophy has previously been shown to reduce contractility but paradoxically to increase O2 consumption rates at a given force. Because O2 consumption rates are related to mitochondrial [NADH] ([NADH]m), we tested the hypothesis that with hypertrophy, control of [NADH]m may be altered. Left ventricular trabeculae were isolated from banded and control rat hearts, and fluorescence spectroscopy was used to monitor [NADH]m and cytosolic [Ca2+] ([Ca2+]c). The hearts from banded rats developed hypertrophy (heart-to-body weight ratio increased from 4.1+/-0.1 to 4.9+/-0.1 mg/g) and hypertension (systolic arterial pressure increased from 117+/-4 to 175+/-5 mm Hg). Muscle workload was increased by stepwise increases in pacing frequency (up to 2 Hz). After increased work, [NADH]m fell and then slowly recovered toward control levels. When work was decreased, [NADH]m overshot control values and then slowly returned. The Ca2+-independent initial fall was larger for trabeculae from rats with hypertrophied hearts than from control rats (eg, 17+/-2% versus 11+/-1% when work was increased by increasing the frequency from 0.25 to 1 Hz). At 1 Hz, average [Ca2+]c was approximately 280 nmol/L, and the Ca2+-dependent [NADH]m recovery was larger for trabeculae from rats with hypertrophied hearts (17+/-4% versus 10+/-2%) despite similar average [Ca2+]c. At steady state after Ca2+-dependent recovery, there was no difference in [NADH]m (fall of 1+/-2% versus 1+/-1%). Furthermore, the Ca2+-dependent overshoot was larger for trabeculae from hypertrophied than from control hearts (increase of 14+/-2% versus 9+/-2% when frequency was decreased from 1 to 0.25 Hz). We conclude that (1) there is initially a larger imbalance in NADH production versus consumption rate in hypertrophy (because NADH fell more) and (2) the Ca2+-dependent recovery mechanism is enhanced in hypertrophy (because NADH recovered and overshot more), thus compensating for the larger imbalance.

Factors That Control Sarcoplasmic Reticulum Calcium Release in Intact Ventricular Myocytesa

Abstract: Much has been discovered studying sarcoplasmic reticulum (SR) Ca release channels in SR vesicles and lipid bilayers. We have focused on how SR Ca release is regulated in intact mammalian ventricular myocytes, using fluorescent Ca indicators, voltage clamp, and confocal microscopy. Three major factors appear to contribute to the probability of spontaneous localized SR Ca release events (or Ca “sparks”) in resting myocytes: (1) cytosolic [Ca], (2) SR Ca content, and (3) time after previous activity (i.e., recovery from adapted or inactivated state). These same three factors function during excitation-contraction (E-C) coupling and can explain rest potentiation of twitches, increased fractional SR Ca release at higher SR Ca loads, and Ca overload. Since SR Ca release is sensitive to both ICa and SR Ca load, we have controlled (and measured) these parameters. At constant SR Ca load and ICA in intact cells we have found that SR Ca release is increased by Ca-calmodulin-dependent protein kinase (CaMKII) and FK506 (which may interfere with the interaction between the Ca release channel and the FK binding protein) and is reduced by the Ca channel agonist Bay K 8644, CaMKII inhibitors, and during ventricular hypertrophy. Thus the regulation of the SR Ca release channel in the intact cell is an important factor in cellular cardiac function.

Coordinated feet and the dance of ryanodine receptors.

Abstract: When a muscle contracts, it does so all at once and suddenly. Marx et al. (p. 818) show that one of the coordinated events underlying this simultaneous tensing is the release of calcium into the cytoplasm of the muscle cell. In their Perspective, Bers and Fill describe how ryanodine receptors, which release the calcium, act together to instantly raise the amount of calcium in the cytoplasm to a very high level, triggering a strong contraction of the muscle.