Biomedical subjects

D M Bers

Publications and source records attributed to D M Bers.

At least 109 records · Page 6Linked to original sources

PubMed1993

Twitch-dependent SR Ca accumulation and release in rabbit ventricular myocytes.

Using caffeine-induced contractures (Ccaf) and thapsigargin (TG), we estimated the fraction of sarcoplasmic reticulum (SR) Ca released at one twitch and also the number of twitches required to reload a Ca-depleted SR. Similar results were obtained for twitches or intracellular Ca (Cai) transient with the fluorescent indicator, indo 1. Sustained exposure to 10 mM caffeine completely depletes the SR of Ca in < 5 s (as assessed by a second Ccaf). After such Ca depletion, four to five twitches are necessary to reload the SR to the steady-state level (with a twitch constant, tau = 1.6 twitches). We also determined the time required for complete inhibition of the SR Ca-adenosinetriphosphatase (ATPase) by TG. After SR Ca depletion, 5 microM TG was applied for different periods of time before a train of "reloading" twitches. A TG exposure period of 90 s was sufficient to completely prevent Ccaf after these reloading twitches. When SR is Ca depleted, the twitch is larger in the presence of TG, indicating that the SR Ca-ATPase can limit the ability of Ca influx to activate contraction. To assess SR Ca released at one twitch in cells with normally Ca-loaded SR, 5 microM TG was applied for 90 s to prevent SR Ca reuptake. Then one or several twitches were activated (causing SR Ca release, but with reuptake completely blocked). After the twitch (or train), a Ccaf was used to assess remaining SR Ca.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Effects of thapsigargin and cyclopiazonic acid on twitch force and sarcoplasmic reticulum Ca2+ content of rabbit ventricular muscle.

Thapsigargin (TG) and cyclopiazonic acid (CPA) are reported to be specific high-affinity inhibitors of the sarcoplasmic reticulum (SR) Ca2+ pump in isolated membranes and cells, with TG causing complete pump inhibition at nanomolar concentrations. To evaluate the effectiveness of TG and CPA in small multicellular cardiac preparations, we used rapid cooling contractures (RCCs) to assess the SR Ca2+ load. In contrast to observations in single myocytes, TG caused remarkably slow and incomplete SR Ca2+ depletion in multicellular preparations. A 45-minute exposure to 500 microM TG at 30 degrees C and 0.5-Hz stimulation only decreased RCCs by 76 +/- 5% (and 100 microM CPA reduced RCCs by 59 +/- 10% [mean +/- SEM]). In contrast, 10 minutes with 20 mM caffeine completely abolished RCCs. This confirms that there was still a caffeine-sensitive pool of Ca2+ in the TG-treated muscle. The time constant of rest decay of RCCs was accelerated by both TG (from 83 +/- 18 to 26 +/- 6 seconds) and CPA (from 68 +/- 11 to 10 +/- 5 seconds). This might be expected since Ca2+ leaking from the SR during rest cannot be taken back up as efficiently, favoring Ca2+ extrusion by the sarcolemmal Na(+)-Ca2+ exchanger. TG and CPA decreased twitch force (by 44 +/- 7% and 40 +/- 11%, respectively) and increased twitch duration, presumably because of the SR effects. We conclude that complete blockade of SR Ca2+ uptake by TG or CPA in multicellular preparations cannot be assumed, even at high [TG] or [CPA], unless evaluated (eg, by RCC).

Animals↗

PubMed1993

Sarcoplasmic reticulum Ca2+ uptake and thapsigargin sensitivity in permeabilized rabbit and rat ventricular myocytes.

Ca2+ uptake by the sarcoplasmic reticulum (SR) and free [Ca2+] were measured simultaneously with indo 1 and a Ca(2+)-selective minielectrode in suspensions of permeabilized rabbit or rat ventricular myocytes (approximately 10 mg/mL protein). In the presence of 25 mumol/L ruthenium red and 10 mmol/L oxalate, the Km for Ca2+ uptake by the SR was approximately 250 nmol/L in rabbit and rat ventricular myocytes. The maximal Ca2+ uptake rate was 2.4 times higher in rat than in rabbit. Addition of 5 nmol thapsigargin (TG) per milligram cell protein abolished Ca2+ uptake completely in both species. The [TG] necessary for a half-maximal reduction of the uptake rate (K1/2) was 55 pmol/mg cell protein for rabbit and 390 pmol/mg cell protein for rat. Assuming that the number of pump sites is two times the concentration of TG necessary to inhibit half of the Ca2+ pump activity (ie, the TG affinity is very high), the density of pump sites is 7.7 mumol/kg wet wt for rabbit and 54.6 mumol/kg wet wt for rat. Despite a fivefold decrease of the Ca2+ uptake rate by a submaximal [TG], the permeabilized myocytes were still able to lower the free [Ca2+] to < 150 nmol/L from a peak value > 10 mumol/L. The relative inhibition of Ca2+ uptake by TG did not depend on the free [Ca2+]. Addition of more than 5 nmol TG per milligram cell protein abolished Ca2+ uptake by the SR completely in < 15 seconds and reduced the uptake rate by 95% in 5 seconds.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Citrate decreases Ca current and changes selectivity of cardiac Ca channels.

Previous studies using citrate as a low affinity Ca buffer have shown that this agent inhibits myocardial contractions and neurotransmitter release. Using several different low affinity Ca buffers we have demonstrated that these effects are unlikely to be attributable to Ca buffering per se. Here we demonstrate that citrate can directly inhibit Ca current (ICa). The decrease in ICa appears to be due to shifts in channel gating (due to surface charge effects) and also to a decrease in channel selectivity for Ca over monovalent ions.

Animals↗

PubMed1992

Indo-1 binding to protein in permeabilized ventricular myocytes alters its spectral and Ca binding properties.

We have examined the binding of the fluorescent Ca indicator indo-1 to cellular protein in permeabilized ventricular myocytes and also to soluble and particulate myocyte protein. Using either a filtration technique or equilibrium dialysis, and conditions similar to those in a cardiac myocyte patch clamped with 100 microM indo-1 in the patch pipette, we found that 72% of the total indo-1 was bound to myocyte protein at a protein concentration of 100 mg/ml. This corresponds to a binding of 3.8 +/- 0.5 nmol indo-1/mg protein. Separation of the myocyte protein into a soluble and a particulate fraction showed that 63% of the bound indo-1 was bound to soluble protein, corresponding to a binding of 3.22 +/- 0.99 nmol/mg, whereas 37% of the bound indo-1 was bound to particulate protein (0.85 +/- 0.14 nmol/mg) at a low [Ca] (pCa approximately 9). Binding of indo-1 in permeabilized myocytes was approximately 60% higher at a saturating Ca concentration (pCa = 3), than under Ca free conditions (1 mM EGTA). Simultaneous measurements of free [Ca] with a Ca selective electrode and indo-1 fluorescence showed that, the dissociation constant (Kd) for Ca was increased 4-5 fold in the presence of permeabilized myocytes as compared to the value obtained in vitro. In agreement with the binding experiments we estimate that the true Kd and the apparent Kd (using ratiometric measurements) for Ca binding to indo-1 are increased approximately four fold, at a myocyte protein concentration of 100 mg/ml.

Animals↗

PubMed1992

Mitochondrial and sarcolemmal Ca2+ transport reduce [Ca2+]i during caffeine contractures in rabbit cardiac myocytes.

1. Contraction and intracellular Ca2+ (Ca2+i) transients were measured in isolated rabbit ventricular myocytes during twitches and contractures induced by rapid application of 10 mM-caffeine. 2. The amplitude of caffeine-induced contractures and the accompanying Ca2+i transients were larger than during normal twitches and also declined more slowly. This may be because only a fraction of sarcoplasmic reticulum (SR) Ca2+ is released during a normal twitch, or because of a temporal overlap of SR Ca2+ release and uptake during the twitch. 3. When a caffeine contracture was initiated in Na(+)-free, Ca(2+)-free medium (to prevent sarcolemmal Na(+)-Ca2+ exchange) the contracture and Ca2+i transient were larger and decreased much more slowly. Thus, Ca2+ extrusion via Na(+)-Ca2+ exchange may limit the amplitude of caffeine-induced contractures. 4. Relaxation half-time (t1/2) for the twitch (0.17 +/- 0.03 s) was increased to 0.54 +/- 0.07 s for caffeine contractures in control solution and 8.8 +/- 1 s for caffeine-induced contractures in Na(+)-free, Ca(2+)-free solution. These results confirm that the SR Ca2+ pump and Na(+)-Ca2+ exchange are the predominant mechanisms for cytoplasmic Ca2+ removal during relaxation. However slower mechanisms can still reduce intracellular [Ca2+]. 5. Relaxation of caffeine contractures in Na(+)-free solution was further slowed when (a) mitochondrial Ca2+ uptake was inhibited with the oxidative phosphorylation uncoupler, FCCP (t1/2 = 19.7 +/- 3.2 s), or (b) the sarcolemmal Ca(2+)-ATPase pumping ability was depressed by a large transmembrane [Ca2+] gradient (t1/2 = 27.5 +/- 6.9 s). 6. When the four Ca2+ transport systems were simultaneously inhibited (i.e. SR Ca2+ pump, Na(+)-Ca2+ exchange, mitochondrial Ca2+ uptake and sarcolemmal Ca2+ pump), relaxation was practically abolished, but the cell could recover quickly when Na+ was reintroduced and caffeine removed. 7. We conclude that, under our experimental conditions, the sarcolemmal Ca2+ pump and mitochondria are approximately 37- and 50-fold slower than the Na(+)-Ca2+ exchange at removing Ca2+ from the cytoplasm. Additionally, the SR Ca2+ pump is about 3-4 times faster than Na(+)-Ca2+ exchange.

Animals↗

PubMed1992

Citrate alters Ca channel gating and selectivity in rabbit ventricular myocytes.

Addition of 10 mM citrate at constant free extracellular Ca concentration [( Ca]o; 2 mM) reduced contraction in rabbit ventricular muscle and isolated myocytes. We have recently shown that extracellular citrate decreases contraction and Ca current (ICa) in cardiac muscle by a direct effect on Ca channels rather than by Ca buffering per se [D. M. Bers, L. V. Hryshko, S. M. Harrison, and D. Dawson. Am. J. Physiol. 260 (Cell Physiol. 29): C900-C909, 1991]. Citrate rapidly depressed peak ICa and shifted both the peak ICa and the apparent reversal potential (Erev) to more negative potentials. When the impermeant cations, tetraethylammonium or N-methylglucamine were used instead of intracellular Cs, the citrate-induced shift in Erev was reduced or eliminated but depression of ICa was still observed. Thus citrate appears to alter the selectivity (PCa/PCs) of the Ca channel and reduce ICa. We also studied the effects of citrate on Na current through the Ca channel, observed when the divalent cation concentration is submicromolar. This current, termed INS for nonspecific, also exhibited leftward shifts in peak INS and smaller changes in Erev in the presence of citrate. However, neither peak INS nor single-channel conductance were affected by citrate. Thus the reduced PCa/PCs is due primarily to alteration of Ca permeation rather than monovalent cation permeation. Activation and inactivation curves for both ICa and INS were shifted toward more negative potentials by citrate. The shifts in gating and peak current to more negative membrane potentials would be consistent with a surface charge effect. The much larger shift in Erev for ICa (than for INS) is consistent with a reduction in Ca selectivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Quantitative analysis of regional variability in the distribution of transverse tubules in rabbit myocardium.

The goal of the present investigation was to compare quantitatively the distribution of T-tubules between regions of the myocardium. The volume fraction and surface density of T-tubules in rabbit right atrial free wall, left atrial free wall, right ventricular free wall, left ventricular free wall, right ventricular papillary muscle, and left ventricular papillary muscle were measured using established, electron-microscopic, morphometric techniques. T-tubules were delineated using wheat-germ agglutinin conjugated to horseradish peroxidase as a tracer. No significant differences were found in the morphometric parameters between any two ventricular samples or between atrial samples. Furthermore, little difference between T-tubule volume fraction or surface density was found between individual animals for any given site. Both volume fraction and surface density of ventricular T-tubules were more than ten-times their values in atrial tissue (volume fraction: 3.43% +/- 0.35 vs. 0.20 +/- 0.09; surface density: 2.46 microns 2/microns 3 +/- 0.11 vs 0.10 +/- 0.04). Measurements show that there is greater variation of T-tubule volume fraction and surface density within atrial samples than within ventricular samples. This suggests greater inhomogeneity in T-tubule distribution in atrial myocardium than in ventricular myocardium. Morphometric data also indicate that the mean diameter of atrial T-tubules is greater than that of ventricular T-tubules while qualitative observations show that atrial T-tubules are distributed less regularly and have a larger longitudinal component to their organization than those in the ventricular myocardium.

Animals↗

PubMed1991

Diffusion around a cardiac calcium channel and the role of surface bound calcium.

The diffusion of Ca as it converges to the external mouth of a Ca channel is examined. Diffusional limitation on Ca ions entering Ca channels during current flow, cause local extracellular Ca depletions. Such extracellular Ca depletions have been reported in cardiac muscle. The cardiac sarcolemma has a large number of low-affinity Ca binding sites that can buffer these local Ca depletions. For a hemisphere of extracellular space (of radius less than 0.33 microns) centered on the external mouth of a Ca channel the amount of Ca bound at the membrane surface exceeds that which is free within the associated hemisphere. The ratio of bound Ca/free Ca increases as r decreases, such that the [Ca] nearest the Ca channel is the most strongly buffered by sarcolemmal bound Ca. It is demonstrated that Ca ions coming from these sarcolemmal Ca binding sites contribute quantitatively to the integrated Ca current. The electric field generated by the local depletion of Ca near the channel mouth has little impact on the extent of Ca depletion, but if an additional electric field exists at the mouth of the channel, Ca depletion can be significantly altered. Other low-affinity Ca binding sites in the interstitium may also contribute to the buffering of extracellular Ca. The complex geometry of the extracellular space in cardiac muscle (e.g., transverse tubules and restrictions of extracellular space between cells) increases both the predicted Ca depletions (in the absence of binding) and the bound/free ratio. Thus, the impact of this surface Ca binding is greatly increased. By considering arrays of Ca channels in transverse tubules or in parallel planes (e.g., membranes of neighboring cells), extracellular Ca depletions are predicted which agree with those measured experimentally. Membrane Ca binding may also be expected to buffer increases in [Ca] around the inner mouth of Ca channels. It is demonstrated that in the absence of other intracellular systems most of the Ca entering the cell via Ca channels might be expected to be bound to the inner sarcolemmal surface. It is concluded that surface Ca binding may have a substantial impact on the processes of extracellular Ca depletion (and intracellular Ca accumulation).

Animals↗

PubMed1991

Species differences and the role of sodium-calcium exchange in cardiac muscle relaxation.

During normal relaxation in rabbit, guinea-pig, and rat ventricular muscle, the Na-Ca exchange system competes with the SR Ca pump, with the former being responsible for about 20-30% of the Ca removal from the cytoplasm. Ca extrusion via Na-Ca exchange is Em-sensitive, whereas Ca uptake by the SR is not. Neither the sarcolemmal Ca-ATPase pump nor mitochondrial Ca uptake appear to contribute significantly to the decline of [Ca]i during relaxation. Furthermore, the diastolic efflux of Ca from cardiac muscle cells appears to be primarily attributable to Na-Ca exchange and not the sarcolemmal Ca-ATPase pump. In rabbit ventricle Ca entry via Na-Ca exchange is favored thermodynamically during much of a normal twitch contraction and Ca extrusion occurs primarily between beats. In rat ventricle Ca efflux via Na-Ca exchange occurs during the contraction and net Ca influx may occur between beats. This fundamental difference in Ca fluxes during the cardiac cycle in rat versus rabbit ventricle may be a simple consequence of the shorter action potential duration and higher aNai in rat ventricle (due to the effects of Em and [Na] and [Ca] gradients on Na-Ca exchange).

Animals↗

PubMed1991

Citrate decreases contraction and Ca current in cardiac muscle independent of its buffering action.

Extracellular Ca (Cao) depletions that occur during cardiac muscle contractions are indicative of net Ca entry. Buffering Cao concentration ([Ca]o) with citrate can limit the magnitude of these Cao depletions [e.g., Shattock and Bers. Am. J. Physiol. 256 (Cell Physiol. 25): C813-C822, 1989] which theoretically would allow more Ca entry and consequently greater force at the same free [Ca]o. However, Shimoni and Ginsburg [Am. J. Physiol. 252 (Cell Physiol. 21): C248-C252, 1987] have shown that citrate inhibits cardiac contractions and suggested that this was due to its Ca-buffering action (i.e., dissipating a local elevation of [Ca] at the outer sarcolemmal surface and thereby decreasing Ca influx). To examine the effects of Ca buffering per se, we compared the effects of four low-affinity Ca buffers [citrate, nitrilotriacetic acid (NTA), dipicolinic acid (DPA), and acetamidoiminodiacetic acid (ADA)] on several cardiac preparations. In Mg-free medium with 2 mM free Ca (measured using murexide), citrate, DPA, and ADA (10 mM) decreased the force of twitch contractions in rabbit ventricle to 76 +/- 2, 60 +/- 2, and 85 +/- 2%, respectively, but 10 mM NTA increased force slightly to 105 +/- 2%. No simple correlation was observed between the Ca affinity of the buffer and its effect on tension. These effects were not due to changes in sarcoplasmic reticulum (SR) Ca loading because rapid cooling contractures were not affected and similar results were observed in the presence of caffeine or ryanodine. The depressant effects of citrate and ADA on tension were greater at pH 5.5-6 and ADA had no effect at pH 8.5. Thus the depressant effect is stronger with more protonated forms of citrate and ADA, which are also poorer Ca buffers. Citrate (but not NTA) decreased Ca current in whole cell voltage clamp and shifted the current-voltage relationship and reversal potential to more negative potentials. Citrate decreased Ca current more effectively at higher citrate and lower Ca concentrations. We conclude that citrate (and some other weak Ca buffers) may directly decrease Ca current and contraction in a manner independent of Ca buffering ability.

Action Potentials↗

PubMed1991

Dihydropyridine receptors are primarily functional L-type calcium channels in rabbit ventricular myocytes.

We measured [3H]PN200-110 binding and patch-clamp currents in rabbit ventricular myocytes to determine if there is a disparity between the density of dihydropyridine-specific receptors and functional L-type calcium channels, as has been reported for skeletal muscle. The dihydropyridine receptor density was 74.7 +/- 4.2 fmol/mg protein (mean +/- SEM, Kd = 1.73 +/- 0.29 nM, n = 6) in ventricular homogenates and 147 +/- 6 fmol/mg protein (Kd = 1.15 +/- 0.16 nM, n = 4) in myocytes. Ventricular homogenates contained 121 +/- 9 mg protein/g wet wt (n = 7). These values were used to calculate a dihydropyridine receptor density of 12.9 dihydropyridine sites/micron2 for ventricular homogenates and 14.8 dihydropyridine sites/micron2 for myocytes. The number of functional L-type calcium channels (N) was calculated from measurements of whole-cell current (I), single-channel current (i), and open probability (po), where N = I/(i x po). We measured sodium current through calcium channels (I(ns)) to avoid calcium-induced inactivation. Whole-cell (I(ns)) and single-channel (i(ns) and po) measurements were obtained under similar ionic conditions at a test potential of -20 mV. In six cells, the peak I(ns) was approximately 105 pA/pF. The single-channel conductance was 40.8 +/- 2.6 pS (n = 12), and i(ns) at -20 mV was 1.96 pA. The mean po at -20 mV was 0.030 +/- 0.002 in 16 patches in which only a single channel was evident. The calculated density of functional L-type calcium channels was approximately 18 channels/micron2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Ca regulation in cardiac muscle.

Contraction in cardiac muscle is activated by a combination of Ca influx via sarcolemmal Ca channels and release of Ca from the sarcoplasmic reticulum (SR). The release of Ca from the SR appears to be triggered by Ca influx via a Ca-induced Ca-release mechanism. The amount of Ca influx and Ca released from the SR vary with conditions and with different cardiac muscle preparations. In contrast to the Ca-induced Ca-release process in cardiac muscle, excitation-contraction coupling in skeletal muscle appears to be via a more direct coupling between sarcolemmal voltage sensors and the Ca release (sometimes referred to as depolarization-induced Ca-release). During cardiac relaxation Ca is removed from the cytoplasm by the SR Ca-pump and the sarcolemmal Na/Ca exchange (with the former being somewhat more dominant). Ca extrusion from the cell during both relaxation and diastole is predominantly via Na/Ca exchange, with the sarcolemmal Ca ATPase pump contributing very little. Thus, sarcolemmal Na/Ca exchange appears to be critically involved in the overall regulation of cardiac cellular Ca content.

Animals↗

PubMed1990

Functional interconversion of rest decay and ryanodine effects in rabbit and rat ventricle depends on Na/Ca exchange.

Rapid cooling contractures were used to assess changes in sarcoplasmic reticulum (SR) Ca content in isolated rabbit and rat ventricular muscle during rest, with altered transsarcolemmal [Na] and [Ca] gradients and in the presence and absence of 100 nM ryanodine. In rabbit there is normally a rest-duration dependent decline in SR Ca content (rest decay), whereas in rat there is a short-term increase in SR Ca content (rest potentiation) and little evidence of rest decay. Ryanodine greatly accelerates the rate of rest decay in rabbit, depleting the SR of Ca in approximately 1 s, whereas in rat, ryanodine does not appear to drain the SR even after a 10 min rest. Elevation of intracellular Na activity in rabbit (by Na-pump inhibition) to a level similar to that measured in control rat during rest (Shattock and Bers, Am. F. Physiol., 256: C813-C822, 1989) makes rest-dependent changes of SR Ca content in these two tissues similar. The rest decay in rabbit in the presence of ryanodine is also markedly slowed after Na-pump inhibition. In rat, reduction of [Ca]0 allows rest decay to occur (+/- ryanodine), but this rest decay can be largely prevented by simultaneous reduction of [Na]o (to maintain [Na]3/[ Ca] constant) which serves to keep the thermodynamic driving force on a 3:1 Na/Ca exchange constant. We conclude that the process of rest decay and rest potentiation in both rabbit and rat ventricle depends on the sarcolemmal Na/Ca exchange. Furthermore, these species can be functionally interconverted by manipulation of the [Na] and [Ca] gradients. The ability of ryanodine to deplete the SR of Ca also depends critically on other transport systems (particularly Na/Ca exchange) to remove Ca from the cytoplasm.

Animals↗

PubMed1990

Excitation-contraction coupling in heart cells. Roles of the sodium-calcium exchange, the calcium current, and the sarcoplasmic reticulum.

Experimental data do not support the idea that excitation-contraction coupling in heart muscle can be explained by a simple calcium-induced calcium release mechanism alone or a simple voltage-dependent calcium release mechanism alone. Our data on excitation-contraction coupling combined with the results of others suggest the need to either develop more complex and more sophisticated single-mechanism models, or to establish dual-control models.

Animals↗

PubMed1990

Temperature dependence of myofilament Ca sensitivity of rat, guinea pig, and frog ventricular muscle.

Cooling the superfusate of intact ventricular muscle, from 30 degrees C to below 4 degrees C in less than 2 s, leads to contractures thought to reflect the amount of Ca available for release from the sarcoplasmic reticulum (SR). On rewarming, tension transiently increases in guinea pig and rat ventricular muscle. It has been proposed that this rewarming tension spike reflects changes in myofilament Ca sensitivity and maximum Ca-activated force (Cmax) associated with rewarming. There are differences in intracellular Ca regulation among cardiac muscle preparations. Some characteristics of rapid-cooling contractures (e.g., the magnitude of the rewarming spike) also differ between species. Therefore, the Ca sensitivity of skinned ventricular muscle from the rat, guinea pig, and frog was determined at 29 (22 degrees C for frog ventricular preparations), 8, and 1 degrees C. The results show that cooling rat and guinea pig ventricular muscle from 29 to 1 degrees C shifts the pCa vs. tension relationship toward higher [Ca2+] by 0.65 and 0.55 pCa units, respectively. Cooling to 1 degrees C also reduced Cmax to 3.3 and 7.8% of that at 29 degrees C in rat and guinea pig ventricular muscle, respectively. Similar results were found for frog ventricular muscle, in which cooling from 22 to 1 degrees C reduced Ca sensitivity by 0.6 pCa units and Cmax to 45.7% of its value at 22 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin Cytoskeleton↗

PubMed1990

Modification of temperature dependence of myofilament Ca sensitivity by troponin C replacement.

The Ca sensitivity of chemically skinned right ventricular trabeculae from the rat heart was determined at 22 and 8 degrees C. Endogenous troponin C (TnC) was then extracted with EDTA and replaced with either bovine cardiac TnC or rabbit fast-twitch skeletal TnC. The temperature dependence of myofilament Ca sensitivity was then reevaluated. Cooling native cardiac tissue from 22 to 8 degrees C reduced the pCa (-log10 [Ca2+]), generating half-maximal tension (K1/2) from 5.20 +/- 0.07 to 4.89 +/- 0.08 (SD, n = 14), and also reduced maximum Ca-activated force to 33 +/- 6% of its value at 22 degrees C. After extraction of endogenous TnC and reconstitution with cardiac TnC, cooling from 22 to 8 degrees C caused a similar shift in mean K1/2 from 4.93 +/- 0.08 to 4.69 +/- 0.06 (n = 7). When skeletal TnC was reconstituted into TnC-extracted ventricular fibers, cooling from 22 to 8 degrees C led to a much smaller mean shift in K1/2 from 4.88 +/- 0.07 to 4.78 +/- 0.04 (n = 7). The results show that the magnitude of the cooling-induced shift in myofilament Ca sensitivity observed in the native state (or after reconstitution with cardiac TnC) is significantly reduced if the fiber is reconstituted with skeletal TnC (P less than 0.001). This indicates that the temperature dependence of myofilament Ca sensitivity of cardiac muscle can be modified by incorporation of skeletal TnC. Thus Ca binding to TnC plays an important role in determining the temperature dependence of myofilament Ca sensitivity.

Actin Cytoskeleton↗

PubMed1990

Intracellular Ca transients in rat cardiac myocytes: role of Na-Ca exchange in excitation-contraction coupling.

Membrane current and intracellular Ca concentration ([Ca]i) transients were recorded from isolated rat ventricular myocytes under voltage-clamp control. The cells were dialyzed by the patch pipette solution, which contained the fluorescent Ca indicator indo-1 and 0.5 mM Na. Under these experimental conditions, Ca entry via Na-Ca exchange did not appear to be appreciable even in the absence of extracellular Na. Increasing the duration of voltage-clamp pulses from 5 to 80 ms produced [Ca]i transients of increasing amplitude, while the peak Ca current was not changed. This duration dependence of the [Ca]i transient was most demonstrable at more negative test potentials (e.g., -20 to -30 mV) and was not qualitatively modified by Na-free solutions. This latter result indicates that Ca extrusion by Na-Ca exchange is not responsible for the smaller [Ca]i transients observed when the membrane is repolarized after very brief depolarizations. Although the peak Ca current was not changed by increasing pulse duration, the integrated Ca current was increased. These observations are consistent with a Ca-release mechanism in cardiac excitation-contraction coupling in which 1) the Ca-release process can be modulated by membrane potential or 2) the Ca entering the cell via Ca channels has a preferential access [compared with Ca from the sarcoplasmic reticulum (SR)] to the site(s) that control SR Ca release. The role of Na-Ca exchange in the decline of [Ca]i during relaxation was also explored. Removal of extracellular Na (Nao) resulted in 20% slowing of the decline in [Ca]i during relaxation. From this, we conclude that the Na-Ca exchange competes with SR to remove Ca from the cytoplasm and that under our control conditions the exchanger may account for 20% of this decline. The Nao dependence of relaxation was reduced at more positive membrane potentials and increased by SR Ca loading.

Animals↗