Spontaneous Ca2+ release from the sarcoplasmic reticulum limits Ca2+- dependent twitch potentiation in individual cardiac myocytes. A mechanism for maximum inotropy in the myocardium

We hypothesized that the occurrence of spontaneous Ca2+ release from the sarcoplasmic reticulum (SR), in diastole, might be a mechanism for the saturation of twitch potentiation common to a variety of inotropic perturbations that increase the total cell Ca. We used a videomicroscopic technique in single cardiac myocytes to quantify the amplitude of electrically stimulated twitches and to monitor the occurrence of the mechanical manifestation of spontaneous SR Ca2+ release, i.e., the spontaneous contractile wave. In rat myocytes exposed to increasing bathing [Ca2+] (Cao) from 0.25 to 10 mM, the Cao at which the peak twitch amplitude occurred in a given cell was not unique but varied with the rate of stimulation or the presence of drugs: in cells stimulated at 0.2 Hz in the absence of drugs, the maximum twitch amplitude occurred in 2 mM Cao; a brief exposure to 50 nM ryanodine before stimulation at 0.2 Hz shifted the Cao of the maximum twitch amplitude to 7 mM. In cells stimulated at 1 Hz in the absence of drugs, the maximum twitch amplitude occurred in 4 mM Cao; 1 microM isoproterenol shifted the Cao of the maximum twitch amplitude to 3 mM. Regardless of the drug or the stimulation frequency, the Cao at which the twitch amplitude saturated varied linearly with the Cao at which spontaneous Ca2+ release first occurred, and this relationship conformed to a line of identity (r = 0.90, p = less than 0.001, n = 25). The average peak twitch amplitude did not differ among these groups of cells. In other experiments, (a) the extent of rest potentiation of the twitch amplitude in rat myocytes was also limited by the occurrence of spontaneous Ca2+ release, and (b) in both rat and rabbit myocytes continuously stimulated in a given Cao, the twitch amplitude after the addition of ouabain saturated when spontaneous contractile waves first appeared between stimulated twitches. A mathematical model that incorporates this interaction between action potential-mediated SR Ca2+ release and the occurrence of spontaneous Ca2+ release in individual cells predicted the shape of the Cao-twitch relationship observed in other studies in intact muscle. Thus, the occurrence of spontaneous SR Ca2+ release is a plausible mechanism for the saturation of the inotropic response to Ca2+ in the intact myocardium.     

Single adult rabbit and rat cardiac myocytes retain the Ca2+- and species-dependent systolic and diastolic contractile properties of intact muscle

The systolic and diastolic properties of single myocytes and intact papillary muscles isolated from hearts of adult rats and rabbits were examined at 37 degrees C over a range of stimulation frequencies and bathing [Ca2+]o (Cao). In both rabbit myocytes and intact muscles bathed in 1 mM Cao, increasing the frequency of stimulation from 6 to 120 min-1 resulted in a positive staircase of twitch performance. During stimulation at 2 min-1, twitch performance also increased with increases in Cao up to 20 mM. In the absence of stimulation, both rabbit myocytes and muscles were completely quiescent in less than 15 mM Cao. Further increases in Cao caused the appearance of spontaneous asynchronous contractile waves in myocytes and in intact muscles caused scattered light intensity fluctuations (SLIF), which were previously demonstrated to be caused by Ca2+-dependent spontaneous contractile waves. In contrast to rabbit preparations, intact rat papillary muscles exhibited SLIF in 1.0 mM Cao. Two populations of rat myocytes were observed in 1 mM Cao: approximately 85% of unstimulated cells exhibited low-frequency (3-4 min-1) spontaneous contractile waves, whereas 15%, during a 1-min observation period, were quiescent. In a given Cao, the contractile wave frequency in myocytes and SLIF in intact muscles were constant for long periods of time. In both intact rat muscles and myocytes with spontaneous waves, in 1 mM Cao, increasing the frequency of stimulation from 6 to 120 min-1 resulted, on the average, in a 65% reduction in steady state twitch amplitude. Of the rat myocytes that did not manifest waves, some had a positive, some had a flat, and some had a negative staircase; the average steady state twitch amplitude of these cells during stimulation at 120 min-1 was 30% greater than that at 6 min-1. In contrast to rabbit preparations, twitch performance during stimulation at 2 min-1 saturated at 1.5 mM Cao in both intact rat muscles and in the myocytes with spontaneous waves. We conclude that the widely divergent, Ca2+-dependent systolic and diastolic properties of intact rat and rabbit cardiac muscle are retained with a high degree of fidelity in the majority of viable single myocytes isolated from the myocardium of these species, and that these myocytes are thus a valid model for studies of Ca2+-dependent excitation-contraction mechanisms in the heart.     

Acidosis facilitates spontaneous sarcoplasmic reticulum Ca2+ release in rat myocardium

Previous studies have shown that acidosis increases myoplasmic [Ca2+] (Cai). We have investigated whether this facilitates spontaneous sarcoplasmic reticulum (SR) Ca2+ release and its functional sequelae. In unstimulated rat papillary muscles, exposure to an acid solution (produced by increasing the [CO2] of the perfusate from 5 to 20%) caused a rapid increase in the mean tissue Cai, as measured by the photoprotein aequorin. This was paralleled by an increase in spontaneous microscopic tissue motion caused by localized Ca2+ myofilament interactions, as monitored in fluctuations in the intensity of laser light scattered by the muscle. In regularly stimulated muscles, acidosis increased the size of the Ca2+ transient associated with each contraction and caused the appearance of Cai oscillations in the diastolic period. In unstimulated single myocytes, acidosis depolarized the resting membrane potential by approximately 5 mV and enhanced the frequency of spontaneous contractile waves. The small sarcolemmal depolarization associated with each contractile wave increased and occasionally initiated spontaneous action potentials. In regularly stimulated myocytes, acidosis caused de novo spontaneous contractile waves between twitches; these waves were associated with a decrease in the amplitude of the subsequent stimulated twitch. Ryanodine (2 microM) abolished all evidence of spontaneous Ca2+ release during acidosis, markedly reduced the acidosis-induced increase in aequorin light, and reduced resting tension. We conclude that acidosis increases the likelihood for the occurrence of spontaneous SR Ca2+ release, which can cause spontaneous action potentials, increase resting tension, and negatively affect twitch tension.     

Contribution of sarcolemmal sodium-calcium exchange and intracellular calcium release to force development in isolated canine ventricular muscle

The aim of this work was to determine the relationship between peak twitch amplitude and sarcoplasmic reticulum (SR) Ca2+ content during changes of stimulation frequency in isolated canine ventricle, and to estimate the extent to which these changes were dependent upon sarcolemmal Na(+)-Ca2+ exchange. In physiological [Na+]o, increased stimulation frequency in the 0.2-2-Hz range resulted in a positive inotropic effect characterized by an increase in peak twitch amplitude and a decrease in the duration of contraction, measured as changes in isometric force development or unloaded cell shortening in intact muscle and isolated single cells, respectively. Action potentials recorded from single cells indicated that the inotropic effect was associated with a progressive decrease of action potential duration and a marked reduction in average time spent by the cell near the resting potential during the stimulus train. The frequency-dependent increase of peak twitch force was correlated with an increase of Ca2+ uptake into and release from the SR. This was estimated indirectly using the phasic contractile response to rapid (less than 1 s) lowering of perfusate temperature from 37 degrees C to 0-2 degrees C and changes of twitch amplitude resulting from perturbations in the pattern of electrical stimulation. Lowering [Na+]o from 140 to 70 mM resulted in an increase of contractile strength, which was accompanied by a similar increase of apparent SR Ca2+ content, both of which could be abolished by exposure to ryanodine (1 x 10(-8) M), caffeine (3 x 10(-3) M), or nifedipine (2 x 10(-6) M). Increased stimulation frequency in 70 mM [Na+]o resulted in a negative contractile staircase, characterized by a graded decrease of peak isometric force development or unloaded cell shortening. SR Ca2+ content estimated under identical conditions remained unaltered. Rate constants derived from mechanical restitution studies implied that the depressant effect of increased stimulation frequency in 70 mM [Na+]o was not a consequence of a decreased rate of refilling of a releasable pool of Ca2+ within the cell. These results demonstrate that frequency-dependent changes of contractile strength and intracellular Ca2+ loading in 140 mM [Na+]o require the presence of a functional sarcolemmal Na(+)-Ca2+ exchange process. The possibility that the negative staircase in 70 mM [Na+]o is related to inhibition of Ca(2+)-induced release of Ca2+ from the SR by various cellular mechanisms is discussed.     

Effects of Na+/Ca2+ exchange induced by SR Ca2+ release on action potentials and afterdepolarizations in guinea pig ventricular myocytes

In cardiac cells, evoked Ca2+ releases or spontaneous Ca2+ waves activate the inward Na+/Ca2+ exchange current (INaCa), which may modulate membrane excitability and arrhythmogenesis. In this study, we examined changes in membrane potential due to INaCa elicited by sarcoplasmic reticulum (SR) Ca2+ release in guinea pig ventricular myocytes using whole cell current clamp, fluorescence, and confocal microscopy. Inhibition of INaCa by Na+-free, Li+-containing Tyrode solution reversibly abbreviated the action potential duration at 90% repolarization (APD90) by 50% and caused SR Ca2+ overload. APD90 was similarly abbreviated in myocytes exposed to the Na+/Ca2+ exchange inhibitor KB-R7943 (5 microM) or after inhibition of SR Ca2+ release with ryanodine (20 microM). In the absence of extracellular Na+, spontaneous SR Ca2+ releases caused minimal changes in resting membrane potential. After the myocytes were returned to Na+-containing solution, the potentiated intracellular Ca2+ concentration ([Ca2+]i) transients dramatically prolonged APD90 and [Ca2+]i oscillations caused delayed and early afterdepolarizations (DADs and EADs). Laser-flash photolysis of caged Ca2+ mimicked the effects of spontaneous [Ca2+]i oscillations, confirming that APD prolongation, DADs, and EADs could be ascribed to intracellular Ca2+ release. These results suggest that Na+/Ca2+ exchange is a major physiological determinant of APD and that INaCa activation by spontaneous SR Ca2+ release/oscillations, depending on the timing, can account for both DADs and EADs during SR Ca2+ overload.     

The C terminus (amino acids 75-94) and the linker region (amino acids 42-54) of the Ca2+-binding protein S100A1 differentially enhance sarcoplasmic Ca2+ release in murine skinned skeletal muscle fibers

S100A1, a Ca2+-binding protein of the EF-hand type, is most highly expressed in striated muscle and has previously been shown to interact with the skeletal muscle sarcoplasmic reticulum (SR) Ca2+ release channel/ryanodine receptor (RyR1) isoform. However, it was unclear whether S100A1/RyR1 interaction could modulate SR Ca2+ handling and contractile properties in skeletal muscle fibers. Since S100A1 protein is differentially expressed in fast- and slow-twitch skeletal muscle, we used saponin-skinned murine Musculus extensor digitorum longus (EDL) and Musculus soleus (Soleus) fibers to assess the impact of S100A1 protein on SR Ca2+ release and isometric twitch force in functionally intact permeabilized muscle fibers. S100A1 equally enhanced caffeine-induced SR Ca2+ release and Ca2+-induced isometric force transients in both muscle preparations in a dose-dependent manner. Introducing a synthetic S100A1 peptide model (devoid of EF-hand Ca2+-binding sites) allowed identification of the S100A1 C terminus (amino acids 75-94) and hinge region (amino acids 42-54) to differentially enhance SR Ca2+ release with a nearly 3-fold higher activity of the C terminus. These effects were exclusively based on enhanced SR Ca2+ release as S100A1 influenced neither SR Ca2+ uptake nor myofilament Ca2+ sensitivity/cooperativity in our experimental setting. In conclusion, our study shows for the first time that S100A1 augments contractile performance both of fast- and slow-twitch skeletal muscle fibers based on enhanced SR Ca2+ efflux at least mediated by the C terminus of S100A1 protein. Thus, our data suggest that S100A1 may serve as an endogenous enhancer of SR Ca2+ release and might therefore be of physiological relevance in the process of excitation-contraction coupling in skeletal muscle.     

Involvement of Ca2+ waves in excitation-contraction coupling of rat atrial cardiomyocytes

Two-dimensional and line-scan analyses of the early phase Ca2+ transients in rat cardiomyocytes were performed with a rapid-scanning laser confocal microscope and fluo-3 to elucidate the mechanism of activation of Ca2+ release from the sarcoplasmic reticulum in atrial myocytes which lack a well developed T-tubular network. On electrical stimulation of ventricular myocytes, Ca2+ concentration began to rise earliest at the Z-line level and became uniform throughout the cytoplasm within about 10 msec. In contrast, on stimulation of atrial myocytes, the earliest rise in Ca2+ occurred at the cell periphery and then spread to the cell interior; cytoplasmic Ca2+ became uniform after more than 30msec. The velocity of the propagation of rise in Ca2+ was 112 +/- 5.1 microm/sec (n = 10), which was similar to that of spontaneous Ca2+ waves observed in atrial and ventricular myocytes. No difference in frequency, amplitude and kinetics of spontaneous Ca2+ sparks was observed between the subsarcolemmal and central regions of atrial myocytes. Ryanodine concentration-dependently decreased the contractile force of isolated rat atrial and ventricular tissue preparations; the sensitivity was higher in atrial myocytes. The present study visualized the involvement of a propagated Ca2+-induced-Ca+ release mechanism in atrial but not ventricular myocytes. This difference may underlie some of the atrioventricular difference in response to physiological and pharmacological stimuli.     

Targeted inhibition of sarcoplasmic reticulum CaMKII activity results in alterations of Ca2+ homeostasis and cardiac contractility

Transgenic (TG) mice expressing a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitory peptide targeted to the cardiac myocyte longitudinal sarcoplasmic reticulum (LSR) display reduced phospholamban phosphorylation at Thr17 and develop dilated myopathy when stressed by gestation and parturition (Ji Y, Li B, Reed TD, Lorenz JN, Kaetzel MA, and Dedman JR. J Biol Chem 278: 25063-25071, 2003). In the present study, these animals (TG) are evaluated for the effect of inhibition of sarcoplasmic reticulum (SR) CaMKII activity on the contractile characteristics and Ca2+ cycling of myocytes. Analysis of isolated work-performing hearts demonstrated moderate decreases in the maximal rates of contraction and relaxation (+/-dP/dt) in TG mice. The response of the TG hearts to increases in load is reduced. The TG hearts respond to isoproterenol (Iso) in a dose-dependent manner; the contractile properties were reduced in parallel to wild-type hearts. Assessment of isolated cardiomyocytes from TG mice revealed 40-47% decrease in the maximal rates of myocyte shortening and relengthening under both basal and Iso-stimulated conditions. Although twitch Ca2+ transient amplitudes were not significantly altered, the rate of twitch intracellular Ca2+ concentration decline was reduced by approximately 47% in TG myocytes, indicating decreased SR Ca2+ uptake function. Caffeine-induced Ca2+ transients indicated unaltered SR Ca2+ content and Na+/Ca2+ exchange function. Phosphorylation assays revealed an approximately 30% decrease in the phosphorylation of ryanodine receptor Ser2809. Iso stimulation increased the phosphorylation of both phospholamban Ser16 and the ryanodine receptor Ser2809 but not phospholamban Thr17 in TG mice. This study demonstrates that inhibition of SR CaMKII activity at the LSR results in alterations in cardiac contractility and Ca2+ handling in TG hearts.     

Comparison of Ca2+ release and uptake characteristics of the sarcoplasmic reticulum in isolated horse and rabbit cardiomyocytes

Both the cardiac action potential duration (APD) (0.6-1 s) and resting heart rate (30-40 beats/min) in the horse are significantly different from humans and smaller mammals, including the rabbit. This would be anticipated to have consequences for excitation-contraction (EC) coupling and require adaptation of the individual processes involved. The sarcoplasmic reticulum (SR) is one of the main components involved in EC coupling. This study examines and compares the activity of this organelle in the horse with that of the rabbit. In particular, the study focuses on SR Ca2+ release via the Ca2+ release channel/ryanodine receptor (RyR2) and Ca2+ uptake via the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pump. Isolated cardiomyocytes from both horse and rabbit hearts were permeabilized, bathed in a mock intracellular solution, and exposed to a specified [Ca2+]. Rabbit cardiomyocytes exposed to 260 nM [Ca2+] produced spontaneous Ca2+ release and propagated Ca2+ waves. Horse cells failed to produce Ca2+ waves; instead, only local release in the form of Ca2+ sparks was evident. However, at 550 nM [Ca2+], Ca2+ waves were produced in both species. Ca2+ waves were four times less frequent yet approximately 1.5 times greater in amplitude in the horse compared with the rabbit. Ca2+ wave velocity was comparable between the species. The reason for this disparity in Ca2+ wave characteristics is unknown. Separate measurements of oxalate-supported Ca2+ uptake into the SR suggest that both horse and rabbit cardiomyocytes have comparable levels SERCA activity. The possible reasons for the observed differences in SR Ca2+ release between the horse and rabbit are discussed.     

Temperature dependence of Ca2+ wave properties in cardiomyocytes: implications for the mechanism of autocatalytic Ca2+ release in wave propagation.

Digital imaging microscopy of fluo-3 fluorescence was used to study the velocity and shape of intracellular Ca2+ waves in isolated rat cardiomyocytes as a function of temperature. Decreasing the temperature from 37 to 17 degrees C reduced the longitudinal wave velocity by a factor of 1.8 and remarkably slowed the decay of [Ca2+]i in the trailing flank of a wave. Using image analysis, rise times, and half-maximum decay times of local Ca2+ transients, which characterize the processes of local Ca2+ release and removal, were determined as a function of temperature. Apparent activation energies for wave front propagation, local Ca2+ release, and local Ca2+ removal were derived from Arrhenius plots and amounted to -23, -28, and -46 kJ/mol, respectively. The high activation energy of Ca2+ removal, which arises from the activity of the sarcoplasmic reticulum (SR) Ca2+ ATPase, relative to those of longitudinal wave propagation and local Ca2+ release excludes the hypothetical mechanism of regenerative "spontaneous Ca2+ release," in which Ca2+ that has been taken up from the approaching wavefront triggers Ca2+ release at a luminal site of the SR. It is consistent, however, with the hypothesis that Ca2+ wave propagation is based on Ca(2+)-induced Ca2+ release where Ca2+ triggers release on the cytosolic face of the SR.     

Intracellular calcium transients and developed tension in rat heart muscle. A mechanism for the negative interval-strength relationship

The purposes of the present study were to determine (a) whether changes of intracellular [Ca2+] (Cai) can account for the decrease of developed tension observed in rat heart muscle when stimulation rate is increased, and (b) whether the effect of stimulation rate on Cai is altered in conditions in which the rate of repriming of the sarcoplasmic reticulum (SR) is altered, as when perfusate [Ca2+] (Cao) is increased, and in heart muscle from senescent animals. The photoprotein aequorin was used to monitor Cai in rat papillary muscles. In muscles from 6-mo-old rats, increasing the stimulation rate in the range 0.2-0.66 Hz led to parallel decreases of both the aequorin light transient and developed tension when Cao was 2 mM. When Cao was increased to 4 mM, changes in the stimulation rate had less effect on both the light transient and tension. At 8 mM Cao, changing the stimulation rate had no effect on either the light transient or developed tension. Papillary muscles from 24-mo-old rats, in which SR function is likely to be depressed, exhibited a prolonged Ca2+ transient and twitch. At a Cao of 4 or 8 mM, increasing the stimulation rate from 0.33 to 0.66 Hz still led to decreases in the size of the aequorin light transient and developed tension in these muscles. Developed tension and aequorin light responded to increases of Cao in the same way in both groups of muscles. We conclude that under the conditions of our experiments, developed tension is determined by Cai. The negative interval-strength relationship observed when Cao is in the physiological range can be accounted for by a time-dependent recycling of Ca2+ by the SR. The effects of increasing Cao and the age-related differences observed at high Cao can also be accounted for using this model.     

Evolution of cardiac calcium waves from stochastic calcium sparks

We present a model that provides a unified framework for studying Ca2+ sparks and Ca2+ waves in cardiac cells. The model is novel in combining 1) use of large currents (approximately 20 pA) through the Ca2+ release units (CRUs) of the sarcoplasmic reticulum (SR); 2) stochastic Ca2+ release (or firing) of CRUs; 3) discrete, asymmetric distribution of CRUs along the longitudinal (separation distance of 2 microm) and transverse (separated by 0.4-0.8 microm) directions of the cell; and 4) anisotropic diffusion of Ca2+ and fluorescent indicator to study the evolution of Ca2+ waves from Ca2+ sparks. The model mimics the important features of Ca2+ sparks and Ca2+ waves in terms of the spontaneous spark rate, the Ca2+ wave velocity, and the pattern of wave propagation. Importantly, these features are reproduced when using experimentally measured values for the CRU Ca2+ sensitivity (approximately 15 microM). Stochastic control of CRU firing is important because it imposes constraints on the Ca2+ sensitivity of the CRU. Even with moderate (approximately 5 microM) Ca2+ sensitivity the very high spontaneous spark rate triggers numerous Ca2+ waves. In contrast, a single Ca2+ wave with arbitrarily large velocity can exist in a deterministic model when the CRU Ca2+ sensitivity is sufficiently high. The combination of low CRU Ca2+ sensitivity (approximately 15 microM), high cytosolic Ca2+ buffering capacity, and the spatial separation of CRUs help control the inherent instability of SR Ca2+ release. This allows Ca2+ waves to form and propagate given a sufficiently large initiation region, but prevents a single spark or a small group of sparks from triggering a wave.     

Predicting Local SR Ca Dynamics during Ca Wave Propagation in Ventricular Myocytes

Of the many ongoing controversies regarding the workings of the sarcoplasmic reticulum (SR) in cardiac myocytes, two unresolved and interconnected topics are 1), mechanisms of calcium (Ca²⁺) wave propagation, and 2), speed of Ca²⁺ diffusion within the SR. Ca²⁺ waves are initiated when a spontaneous local SR Ca²⁺ release event triggers additional release from neighboring clusters of SR release channels (ryanodine receptors (RyRs)). A lack of consensus regarding the effective Ca²⁺ diffusion constant in the SR (D_Ca,SR) severely complicates our understanding of whether dynamic local changes in SR [Ca²⁺] can influence wave propagation. To address this problem, we have implemented a computational model of cytosolic and SR [Ca²⁺] during Ca²⁺ waves. Simulations have investigated how dynamic local changes in SR [Ca²⁺] are influenced by 1), D_Ca,SR; 2), the distance between RyR clusters; 3), partial inhibition or stimulation of SR Ca²⁺ pumps; 4), SR Ca²⁺ pump dependence on cytosolic [Ca²⁺]; and 5), the rate of transfer between network and junctional SR. Of these factors, D_Ca,SR is the primary determinant of how release from one RyR cluster alters SR [Ca²⁺] in nearby regions. Specifically, our results show that local increases in SR [Ca²⁺] ahead of the wave can potentially facilitate Ca²⁺ wave propagation, but only if SR diffusion is relatively slow. These simulations help to delineate what changes in [Ca²⁺] are possible during SR Ca²⁺release, and they broaden our understanding of the regulatory role played by dynamic changes in [Ca²⁺]_SR.     

Mechanisms of excitation-contraction coupling and calcium liberation in striated muscles of vertebrates

In vertebrate skeletal muscle, the main part of excitation-contraction coupling occurs at the level of the triad, where membranes of T-system and of junctional SR are facing each other. From place to place, the junctional gap is bridged by "feet" structures which include the SR Ca2+ channel. Half of them are closely apposed to tubular intramembranous structures assumed to be DHP-sensitive voltage-sensors which are similar to tubular Ca2+ channels and act by controlling Ca2+ release from SR. During a twitch, the release of Ca2+ activator from SR is controlled both by voltage-sensors via the feet structures and by a tubular Na+ current via a Na+-induced Ca2+ release mechanism. During long-duration mechanical responses, additional mechanisms are involved: a Ca2+-induced Ca2+ release which can be activated by ICa; the release of Ca2+ from membrane, controlled by the operation of a Na+/Ca2+ exchanger and/or new arrangements of surface membrane charges. An IP3-mediated Ca2+ release could be involved too. All these mechanisms can be regulated by intracellular biochemical or ionic processes.     

Characterization of junctional and longitudinal sarcoplasmic reticulum from heart muscle

Longitudinal tubules and junctional sarcoplasmic reticulum (SR) were prepared from heart muscle microsomes by Ca2+-phosphate loading followed by sucrose density gradient centrifugation. The longitudinal SR had a high Ca2+ loading rate (0.93 +/- 0.08 mumol.mg-1.min) which was unchanged by addition of ruthenium red. Junctional SR had a low Ca2+ loading rate (0.16 +/- 0.02 mumol.mg-1.min) which was enhanced about 5-fold by ruthenium red. Junctional SR had feet structures observed by electron microscopy and a high molecular weight protein with Mr of 340,000, whereas longitudinal SR was essentially devoid of both. Thus, these subfractions have similar characteristics to longitudinal and junctional terminal cisternae of SR from fast twitch skeletal muscle. Ryanodine binding was localized to junctional cardiac SR as determined by [3H]ryanodine binding. Scatchard analysis of the binding data showed two types of binding (high affinity, Kd approximately 7.9 nM; low affinity, Kd approximately 1 microM), contrasting with skeletal junctional terminal cisternae where only one site with Kd of approximately 50 nM was observed. The ruthenium red enhancement of Ca2+ loading rate in junctional cardiac SR was blocked by pretreatment with low concentrations of ryanodine as reported for junctional terminal cisternae of skeletal muscle SR. The Ca2+ loading rate of junctional cardiac SR was enhanced by preincubation with high concentrations of ryanodine. The apparent inhibition constant (Ki approximately 7 nM) and stimulation constant (Km approximately 1.1 microM) for ryanodine on junctional SR corresponded to the Kd for high affinity binding (Kd approximately 7.9 nM) and low affinity binding (Kd approximately 1.1 microM), respectively. These results suggest that high affinity ryanodine binding locks the Ca2+ release channels in the open state and that low affinity binding closes the Ca2+ release channels of the junctional cardiac SR. The characteristics of the Ca2+ release channels of junctional cardiac SR appear to be similar to that of skeletal muscle SR, but the Ca2+ release channels of cardiac SR are more sensitive to ryanodine.     

Interaction between the sarcoplasmic reticulum and mitochondria in the control of contractile calcium in guinea-pig atria--does it exist?

The inhibitory action of caffeine on calcium (Ca2+) release from the sarcoplasmic reticulum (SR) and interference with mitochondrial (Ca2+) fluxes by a mitochondrial uncoupler protonfore CCCP were utilized to define a calcium pool responsible for potentiation of post-rest twitch tension in guinea-pig atria. The Ca2+ fluxes were assessed by means of 45Ca2+. Caffeine and CCCP when applied separately did not affect post-rat 45Ca2+ content. Yet, when they were applied together it was markedly reduced to the resting level. It is concluded that a possible source of contractile Ca2+ may be located in mitochondria and an eventual shift of Ca2+ between mitochondria and the SR seems to be a plausible assumption.     

CaMKII-dependent reactivation of SR Ca(2+) uptake and contractile recovery during intracellular acidosis

In hearts, intracellular acidosis disturbs contractile performance by decreasing myofibrillar Ca(2+) response, but contraction recovers at prolonged acidosis. We examined the mechanism and physiological implication of the contractile recovery during acidosis in rat ventricular myocytes. During the initial 4 min of acidosis, the twitch cell shortening decreased from 2.3 +/- 0.3% of diastolic length to 0.2 +/- 0.1% (means +/- SE, P < 0.05, n = 14), but in nine of these cells, contractile function spontaneously recovered to 1.5 +/- 0.3% at 10 min (P < 0.05 vs. that at 4 min). During the depression phase, both the diastolic intracellular Ca(2+) concentration ([Ca(2+)](i)) and Ca(2+) transient (CaT) amplitude increased, and the twitch [Ca(2+)](i) decline prolonged significantly (P < 0.05). In the cells that recovered, a further increase in CaT amplitude and a reacceleration of twitch [Ca(2+)](i) decline were observed. The increase in diastolic [Ca(2+)](i) was less extensive than the increase in the cells that did not recover (n = 5). Blockade of sarcoplasmic reticulum (SR) function by ryanodine (10 microM) and thapsigargin (1 microM) or a selective inhibitor of Ca(2+)-calmodulin kinase II, 2-[N- (2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methyl benzylamine (1 microM) completely abolished the reacceleration of twitch [Ca(2+)](i) decline and almost eliminated the contractile recovery. We concluded that during prolonged acidosis, Ca(2+)-calmodulin kinase II-dependent reactivation of SR Ca(2+) uptake could increase SR Ca(2+) content and CaT amplitude. This recovery can compensate for the decreased myofibrillar Ca(2+) response, but may also cause Ca(2+) overload after returning to physiological pH(i).     

Overexpression of Na+/Ca2+ exchanger alters contractility and SR Ca2+ content in adult rat myocytes

The functional consequences of overexpression of rat heart Na+/Ca2+ exchanger (NCX1) were investigated in adult rat myocytes in primary culture. When maintained under continued electrical field stimulation conditions, cultured adult rat myocytes retained normal contractile function compared with freshly isolated myocytes for at least 48 h. Infection of myocytes by adenovirus expressing green fluorescent protein (GFP) resulted in >95% infection as ascertained by GFP fluorescence, but contraction amplitude at 6-, 24-, and 48-h postinfection was not affected. When they were examined 48 h after infection, myocytes infected by adenovirus expressing both GFP and NCX1 had similar cell sizes but exhibited significantly altered contraction amplitudes and intracellular Ca2+ concentration ([Ca2+]i) transients, and lower resting and diastolic [Ca2+]i when compared with myocytes infected by the adenovirus expressing GFP alone. The effects of NCX1 overexpression on sarcoplasmic reticulum (SR) Ca2+ content depended on extracellular Ca2+ concentration ([Ca2+]o), with a decrease at low [Ca2+]o and an increase at high [Ca2+]o. The half-times for [Ca2+]i transient decline were similar, suggesting little to no changes in SR Ca2+-ATPase activity. Western blots demonstrated a significant (P < or = 0.02) threefold increase in NCX1 but no changes in SR Ca2+-ATPase and calsequestrin abundance in myocytes 48 h after infection by adenovirus expressing both GFP and NCX1 compared with those infected by adenovirus expressing GFP alone. We conclude that overexpression of NCX1 in adult rat myocytes incubated at high [Ca2+]o resulted in enhanced Ca2+ influx via reverse NCX1 function, as evidenced by greater SR Ca2+ content, larger twitch, and [Ca2+]i transient amplitudes. Forward NCX1 function was also increased, as indicated by lower resting and diastolic [Ca2+]i.     

An antagonist of cADP-ribose inhibits arrhythmogenic oscillations of intracellular Ca2+ in heart cells.

Oscillations of Ca2+ in heart cells are a major underlying cause of important cardiac arrhythmias, and it is known that Ca2+-induced release of Ca2+ from intracellular stores (the sarcoplasmic reticulum) is fundamental to the generation of such oscillations. There is now evidence that cADP-ribose may be an endogenous regulator of the Ca2+ release channel of the sarcoplasmic reticulum (the ryanodine receptor), raising the possibility that cADP-ribose may influence arrhythmogenic mechanisms in the heart. 8-Amino-cADP-ribose, an antagonist of cADP-ribose, suppressed oscillatory activity associated with overloading of intracellular Ca2+ stores in cardiac myocytes exposed to high doses of the beta-adrenoreceptor agonist isoproterenol or the Na+/K+-ATPase inhibitor ouabain. The oscillations suppressed by 8-amino-cADP-ribose included intracellular Ca2+ waves, spontaneous action potentials, after-depolarizations, and transient inward currents. Another antagonist of cADP-ribose, 8-bromo-cADP-ribose, was also effective in suppressing isoproterenol-induced oscillatory activity. Furthermore, in the presence of ouabain under conditions in which there was no arrhythmogenesis, exogenous cADP-ribose was found to be capable of triggering spontaneous contractile and electrical activity. Because enzymatic machinery for regulating the cytosolic cADP-ribose concentration is present within the cell, we propose that 8-amino-cADP-ribose and 8-bromo-cADP-ribose suppress cytosolic Ca2+ oscillations by antagonism of endogenous cADP-ribose, which sensitizes the Ca2+ release channels of the sarcoplasmic reticulum to Ca2+.     

Mechanistic basis of differences in Ca2+ -handling properties of sarcoplasmic reticulum in right and left ventricles of normal rat myocardium

This study investigated Ca2+ -cycling properties of sarcoplasmic reticulum (SR) in right ventricle (RV) and left ventricle (LV) of normal rat myocardium. Intracellular Ca2+ transients and contractile function were monitored in freshly isolated myocytes from RV and LV. SR in RV displayed nearly fourfold lower rates of ATP-energized Ca2+ uptake in vitro than SR of LV. The Ca2+ concentration required for half-maximal activation of Ca2+ transport was nearly twofold higher in SR of RV. The lower Ca2+ -sequestering activity of SR in RV was accompanied by a matching decrement in Ca2+ -induced phosphoenzyme formation during the catalytic cycle of the Ca2+ -pumping ATPase (SERCA2). Western immunoblot analysis showed that protein levels of Ca2+ -ATPase and its inhibitor phospholamban (PLN) were only approximately 15% lower in SR of RV than in SR of LV. Coimmunoprecipitation experiments revealed that PLN-bound, functionally inert Ca2+ -ATPase molecules in SR of RV greatly exceed (> 50%) that in SR of LV. Endogenous Ca2+/calmodulin-dependent protein kinase-mediated phosphorylation of SR substrates did not abolish the huge disparity in SR Ca2+ pump function between RV and LV. Intracellular Ca2+ transients, evoked by electrical field stimulation, were significantly prolonged in RV myocytes compared with LV myocytes, mainly because of slow decay of intracellular Ca2+ concentration. The slow decay of intracellular Ca2+ concentration in RV and consequent decrease in the speed of RV relaxation may promote temporal synchrony of the end of diastole in RV and LV. The preponderance of functionally silent SR Ca2+ pumps in RV reflects a higher diastolic reserve required to protect and maintain RV function in the face of a sudden rise in afterload or resistance in the pulmonary circulation.