On the role of dysferlin in striated muscle: membrane repair, t-tubules and Ca2+ handling.

Dysferlin is a 237 kDa membrane-associated protein characterised by multiple C2 domains with a diverse role in skeletal and cardiac muscle physiology. Mutations in DYSF are known to cause various types of human muscular dystrophies, known collectively as dysferlinopathies, with some patients developing cardiomyopathy. A myriad of in vitro membrane repair studies suggest that dysferlin plays an integral role in the membrane repair complex in skeletal muscle. In comparison, less is known about dysferlin in the heart, but mounting evidence suggests that dysferlin's role is similar in both muscle types. Recent findings have shown that dysferlin regulates Ca2+ handling in striated muscle via multiple mechanisms and that this becomes more important in conditions of stress. Maintenance of the transverse (t)-tubule network and the tight coordination of excitation-contraction coupling are essential for muscle contractility. Dysferlin regulates the maintenance and repair of t-tubules, and it is suspected that dysferlin regulates t-tubules and sarcolemmal repair through a similar mechanism. This review focuses on the emerging complexity of dysferlin's activity in striated muscle. Such insights will progress our understanding of the proteins and pathways that regulate basic heart and skeletal muscle function and help guide research into striated muscle pathology, especially that which arises due to dysferlin dysfunction.

Changes of SERCA activity have only modest effects on sarcoplasmic reticulum Ca2+ content in rat ventricular myocytes.

Changes of the activity of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) affect the amplitude of the systolic Ca(2+) transient and thence cardiac contractility. This is thought to be due to alterations of SR Ca(2+) content. Recent work on mice in which the expression of SERCA is decreased found that a large reduction of SERCA expression resulted in a proportionately much smaller decrease of SR Ca(2+) content. The aim of the current work was to investigate the quantitative nature of the dependence of both the amplitude of the systolic Ca(2+) transient and SR Ca(2+) content on SERCA activity during acute partial inhibition of SERCA. Experiments were performed on rat ventricular myocytes. Brief application of thapsigargin (1 µm) resulted in a decrease of SERCA activity as measured from the rate of decay of the systolic Ca(2+) transient. This was accompanied by a decrease in the amplitude of the systolic Ca(2+) transient which was linearly related to that of SERCA activity. However, the fractional decrease in the SR Ca(2+) content was much less than that of SERCA activity. On average SR Ca(2+) content was proportional to SERCA activity raised to the 0.38 ± 0.07 power. This shallow dependence of SR content on SERCA activity arises because Ca(2+) release is a steep function of SR Ca(2+) content. In contrast SR Ca(2+) content was increased 4.59 ± 0.40 (n = 8)-fold by decreasing ryanodine receptor opening with tetracaine (1 mm). Therefore a modest decrease of SR Ca(2+) content results in a proportionately larger fall of Ca(2+) release from the SR which can balance a larger initiating decrease of SERCA. In conclusion, the shallow dependence of SR Ca(2+) content on SERCA activity is expected for a system in which small changes of SR Ca(2+) content produce larger effects on the amplitude of the systolic Ca(2+) transient.

Transverse tubules are a common feature in large mammalian atrial myocytes including human.

Transverse (t) tubules are surface membrane invaginations that are present in all mammalian cardiac ventricular cells. The apposition of L-type Ca(2+) channels on t tubules with the sarcoplasmic reticulum (SR) constitutes a "calcium release unit" and allows close coupling of excitation to the rise in systolic Ca(2+). T tubules are virtually absent in the atria of small mammals, and therefore Ca(2+) release from the SR occurs initially at the periphery of the cell and then propagates into the interior. Recent work has, however, shown the occurrence of t tubules in atrial myocytes from sheep. As in the ventricle, Ca(2+) release in these cells occurs simultaneously in central and peripheral regions. T tubules in both the atria and the ventricle are lost in disease, contributing to cellular dysfunction. The aim of this study was to determine if the occurrence of t tubules in the atrium is restricted to sheep or is a more general property of larger mammals including humans. In atrial tissue sections from human, horse, cow, and sheep, membranes were labeled using wheat germ agglutinin. As previously shown in sheep, extensive t-tubule networks were present in horse, cow, and human atrial myocytes. Analysis shows half the volume of the cell lies within 0.64 ± 0.03, 0.77 ± 0.03, 0.84 ± 0.03, and 1.56 ± 0.19 µm of t-tubule membrane in horse, cow, sheep, and human atrial myocytes, respectively. The presence of t tubules in the human atria may play an important role in determining the spatio-temporal properties of the systolic Ca(2+) transient and how this is perturbed in disease.

Differences in intracellular calcium homeostasis between atrial and ventricular myocytes.

The role that Ca(2+) plays in ventricular excitation contraction coupling is well defined and much is known about the marked differences in the spatiotemporal properties of the systolic Ca(2+) transient between atrial and ventricular myocytes. However, to date there has been no systematic appraisal of the Ca(2+) homeostatic mechanisms employed by atrial cells and how these compare to the ventricle. In the present study we sought to determine the fractional contributions made to the systolic Ca(2+) transient and the decay of [Ca(2+)](i) by the sarcoplasmic reticulum and sarcolemmal mechanisms. Experiments were performed on single myocytes isolated from the atria and ventricles of the rat. Intracellular Ca(2+) concentration, membrane currents, SR Ca(2+) content and cellular Ca(2+) buffering capacity were measured at 23 degrees C. Atrial cells had smaller systolic Ca(2+) transients (251+/-39 vs. 376+/-41 nmol x L(-1)) that decayed more rapidly (7.4+/-0.6 vs. 5.45+/-0.3 s(-1)). This was due primarily to an increased rate of SR mediated Ca(2+) uptake (k(SR), 6.88+/-0.6 vs. 4.57+/-0.3 s(-1)). SR Ca(2+) content was 289% greater and Ca(2+) buffering capacity was increased approximately 3-fold in atrial cells (B(max) 371.9+/-32.4 vs. 121.8+/-8 micromol x L(-1), all differences P<0.05). The fractional release of Ca(2+) from the SR was greater in atrial cells, although the gain of excitation contraction coupling was the same in both cell types. In summary our data demonstrate fundamental differences in Ca(2+) homeostasis between atrial and ventricular cells and we speculate that the increased SR Ca(2+) content may be significant in determining the increased prevalence of arrhythmias in the atria.

What role does modulation of the ryanodine receptor play in cardiac inotropy and arrhythmogenesis?

In this article we review the role of the Ryanodine Receptor (RyR) in cardiac inotropy and arrhythmogenesis. Most of the calcium that activates cardiac contraction comes from the sarcoplasmic reticulum (SR) from where it is released through the RyR. The amplitude of the systolic Ca transient depends steeply on the SR Ca content and it is therefore important that SR content be regulated. This regulation occurs via changes of SR Ca content affecting systolic Ca and thence sarcolemmal Ca fluxes. In the steady state, the cardiac myocyte must be in Ca flux balance on each beat and this has implications for understanding even simple inotropic manoeuvres. The main part of the review considers the effects of modulating the RyR on systolic Ca. Potentiation of RyR opening produces an increase of the amplitude of the Ca transient but this effect disappears within a few beats because the increased sarcolemmal efflux of Ca decreases SR Ca content. We conclude that it is therefore unlikely that potentiation of the RyR by phosphorylation plays a dominant role in the actions of positive inotropic agents such as beta-adrenergic stimulation. Some cardiac arrhythmias result from release of Ca from the SR in the form of waves. This is best known to occur when the SR is overloaded with calcium. Mutations in the RyR also produce cardiac arrhythmias attributed to Ca waves due to leaky RyRs and a similar leak has been suggested to contribute to arrhythmias in heart failure. We show that, due to compensatory changes of SR Ca content, simply making the RyR leaky does not produce Ca waves in the steady state and that SR Ca content is critical in determining whether Ca waves occur.

What is the purpose of the large sarcolemmal calcium flux on each heartbeat?

In cardiac muscle, although most of the calcium that activates contraction comes from the sarcoplasmic reticulum (SR), a significant fraction (up to 30%, depending on the species) enters from outside the cell and is then pumped out at the end of systole. Although some of this calcium influx is required to trigger calcium release from the SR, the bulk serves to reload the cell (and thence the SR) with calcium to replace the calcium that is pumped out of the cell. An alternative strategy would be for the heart to have a much smaller calcium influx balancing a smaller efflux. We demonstrate that this would result in a slowing of inotropic responses due to changes of SR calcium content. We conclude that the large sarcolemmal calcium fluxes facilitate rapid changes of contractility.

The mechanism and significance of the slow changes of ventricular action potential duration following a change of heart rate.

This article reviews the effects of changes of heart rate on the ventricular action potential duration. These can be divided into short term (fractions of a second), resulting from the kinetics of recovery of membrane currents, through to long term (up to days), resulting from changes of protein expression. We concentrate on the medium-term changes (time course of the order of 100 s). These medium-term changes occur in isolated tissues and in the intact human heart. They may protect against cardiac arrhythmias. Finally, we discuss the cellular mechanisms responsible for these changes.

From the ryanodine receptor to cardiac arrhythmias.

Cardiac contraction is activated by an increase of intracellular calcium concentration ([Ca(2+)](i)), most of which comes from the sarcoplasmic reticulum (SR) where it is released, via the ryanodine receptor (RyR), in response to Ca(2+) entering the cell on the L-type Ca(2+) current. This phenomenon is termed Ca(2+)-induced Ca(2+) release (CICR). However, under certain circumstances, the SR can become overloaded with Ca(2+) and once a threshold SR Ca(2+) content is reached Ca(2+) is released spontaneously. Such spontaneous Ca(2+) release from the SR propagates as a Ca(2+) wave by CICR. Some of the Ca(2+) released during a wave is removed from the cell on the electrogenic Na - Ca exchanger resulting in depolarization. This is the cellular mechanism producing delayed afterdepolarizations and is common to those arrhythmias produced by digitalis toxicity and right ventricular outflow tract tachycardia. More recently it has been suggested that arrhythmogenic Ca(2+) waves can also occur if the properties of the RyR are altered, resulting in increase of RyR open probability, for example by phosphorylation. However, in this review experimental evidence will be presented to support the view that such arrhythmias still require a threshold SR Ca(2+) content to be exceeded and that this threshold is decreased by increasing RyR open probability.

Nanoindentation of histological specimens: Mapping the elastic properties of soft tissues.

Although alterations in the gross mechanical properties of dynamic and compliant tissues have a major impact on human health and morbidity, there are no well-established techniques to characterize the micromechanical properties of tissues such as blood vessels and lungs. We have used nanoindentation to spatially map the micromechanical properties of 5-mum-thick sections of ferret aorta and vena cava and to relate these mechanical properties to the histological distribution of fluorescent elastic fibers. To decouple the effect of the glass substrate on our analysis of the nanoindentation data, we have used the extended Oliver and Pharr method. The elastic modulus of the aorta decreased progressively from 35 MPa in the adventitial (outermost) layer to 8 MPa at the intimal (innermost) layer. In contrast, the vena cava was relatively stiff, with an elastic modulus >30 MPa in both the extracellular matrix-rich adventitial and intimal regions of the vessel. The central, highly cellularized, medial layer of the vena cava, however, had an invariant elastic modulus of ~20 MPa. In extracellular matrix-rich regions of the tissue, the elastic modulus, as determined by nanoindentation, was inversely correlated with elastic fiber density. Thus, we show it is possible to distinguish and spatially resolve differences in the micromechanical properties of large arteries and veins, which are related to the tissue microstructure.

Reducing ryanodine receptor open probability as a means to abolish spontaneous Ca2+ release and increase Ca2+ transient amplitude in adult ventricular myocytes.

The aim of this work was to investigate whether it is possible to remove arrhythmogenic Ca2+ release from the sarcoplasmic reticulum that occurs in calcium overload without compromising normal systolic release. Exposure of rat ventricular myocytes to isoproterenol (1 micromol/L) resulted in an increased amplitude of the systolic Ca2+ transient and the appearance of waves of diastolic Ca2+ release. Application of tetracaine (25 to 50 micromol/L) decreased the frequency or abolished the diastolic Ca2+ release. This was accompanied by an increase in the amplitude of the systolic Ca2+ transient. Cellular Ca2+ flux balance was investigated by integrating Ca2+ entry (on the L-type Ca2+ current) and efflux (on Na-Ca2+ exchange). Isoproterenol increased Ca2+ influx but failed to increase Ca2+ efflux during systole (because of the abbreviation of the duration of the Ca2+ transient). To match this increased influx the bulk of Ca2+ efflux occurred via Na-Ca2+ exchange during a diastolic Ca2+ wave. Subsequent application of tetracaine increased systolic Ca2+ efflux and abolished the diastolic efflux. The increase of systolic efflux in tetracaine resulted from both increased amplitude and duration of the systolic Ca2+ transient. In the presence of isoproterenol, those Ca2+ transients preceded by diastolic release were smaller than those where no diastolic release had occurred. When tetracaine was added, the amplitude of the Ca2+ transient was similar to those in isoproterenol with no diastolic release and larger than those preceded by diastolic release. We conclude that tetracaine increases the amplitude of the systolic Ca2+ transient by removing the inhibitory effect of diastolic Ca2+ release.

Analysis of cellular calcium fluxes in cardiac muscle to understand calcium homeostasis in the heart.

Central to controlling intracellular calcium concentration ([Ca(2+)](i)) are a number of Ca(2+) transporters and channels with the L-type Ca(2+) channel, Na(+)-Ca(2+) exchanger and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) being of particular note in the heart. This review concentrates on the regulation of [Ca(2+)](i) in cardiac muscle and the homeostatic mechanisms employed to ensure that the heart can operate under steady-state conditions on a beat by beat basis. To this end we discuss the relative importance of various sources and sinks of Ca(2+) responsible for initiating contraction and relaxation in cardiac myocytes and how these can be manipulated to regulate the Ca(2+) content of the major Ca(2+) store, the sarcoplasmic reticulum (SR). We will present a simple feedback system detailing how such control can be achieved and highlight how small perturbations to the steady-state operation of the feedback loop can be both beneficial physiologically and underlie changes in systolic Ca(2+) in ageing and heart disease. In addition to manipulating the amplitude of the normal systolic Ca(2+) transient, the tight regulation of SR Ca(2+) content is also required to prevent the abnormal, spontaneous or diastolic release of Ca(2+) from the SR. Such diastolic events are a major factor contributing to the genesis of cardiac arrhythmias in disease situations and in recently identified familial mutations in the SR Ca(2+) release channel (ryanodine receptor, RyR). How such diastolic release arises and potential mechanisms for controlling this will be discussed.

Na/Ca exchange: regulator of intracellular calcium and source of arrhythmias in the heart.

The major effect of Na/Ca exchange (NCX) on the systolic Ca transient is secondary to its effect on the Ca content of the sarcoplasmic reticulum (SR). SR Ca content is controlled by a mechanism in which an increase of SR Ca produces an increase in the amplitude of the systolic Ca transient. This, in turn, increases Ca efflux on NCX as well as decreasing entry on the L-type current resulting in a decrease of both cell and SR Ca content. This control mechanism also changes the response to other maneuvers that affect excitation-contraction coupling. For example, potentiating the opening of the SR Ca release channel (ryanodine receptor, RyR) with caffeine produces an immediate increase in the amplitude of the systolic Ca transient. However, this increases efflux of Ca from the cell on NCX and then decreases SR Ca content until a new steady state is reached. Changing the activity of NCX (by decreasing external Na) changes the level of SR Ca reached by this mechanism. If the cell and SR are overloaded with Ca then Ca waves appear during diastole. These waves activate the electrogenic NCX and thereby produce arrhythmogenic-delayed afterdepolarizations. A major challenge is how to remove this arrhythmogenic Ca release without compromising the normal systolic release. We have found that application of tetracaine to decrease RyR opening can abolish diastolic release while simultaneously potentiating the systolic release.

A mechanism distinct from the L-type Ca current or Na-Ca exchange contributes to Ca entry in rat ventricular myocytes.

The aim of this paper was to characterize the pathways that allow Ca(2+) ions to enter the cell at rest. Under control conditions depolarization produced an increase of intracellular Ca concentration ([Ca(2+)](i)) that increased with depolarization up to about 0 mV and then declined. During prolonged depolarization the increase of [Ca(2+)](i) decayed. This increase of [Ca(2+)](i) was inhibited by nifedipine and the calculated rate of entry of Ca increased on depolarization and then declined with a similar time course to the inactivation of the L-type Ca current. We conclude that this component of change of [Ca(2+)](i) is due to the L-type Ca current. If intracellular Na was elevated then only part of the change of [Ca(2+)](i) was inhibited by nifedipine. The nifedipine-insensitive component increased monotonically with depolarization and showed no relaxation on prolonged depolarization. This component appears to result from Na-Ca exchange (NCX). When the L-type current and NCX were both inhibited (nifedipine and Na-free solution) then depolarization decreased and hyperpolarization increased [Ca(2+)](i). These changes of [Ca(2+)](i) were unaffected by modifiers of B-type Ca channels such as chlorpromazine and AlF(3) but were abolished by gadolinium ions. We conclude that, in addition to L-type Ca channels and NCX, there is another pathway for entry of Ca(2+) into the ventricular myocyte but this is distinct from the previously reported B-type channel.

Coordinated control of cell Ca(2+) loading and triggered release from the sarcoplasmic reticulum underlies the rapid inotropic response to increased L-type Ca(2+) current.

The aim of this study was to investigate how sarcoplasmic reticulum (SR) Ca(2+) content and systolic Ca(2+) are controlled when Ca(2+) entry into the cell is varied. Experiments were performed on voltage-clamped rat and ferret ventricular myocytes loaded with fluo-3 to measure intracellular Ca(2+) concentration ([Ca(2+)](i)). Increasing external Ca(2+) concentration ([Ca(2+)](o)) from 1 to 2 mmol/L increased the amplitude of the systolic Ca(2+) transient with no effect on SR Ca(2+) content. This constancy of SR content is shown to result because the larger Ca(2+) transient activates a larger Ca(2+) efflux from the cell that balances the increased influx. Decreasing [Ca(2+)](o) to 0.2 mmol/L decreased systolic Ca(2+) but produced a small increase of SR Ca(2+) content. This increase of SR Ca(2+) content is due to a decreased release of Ca(2+) from the SR resulting in decreased loss of Ca(2+) from the cell. An increase of [Ca(2+)](o) has two effects: (1) increasing the fraction of SR Ca(2+) content, which is released on depolarization and (2) increasing Ca(2+) entry into the cell. The results of this study show that the combination of these effects results in rapid changes in the amplitude of the systolic Ca(2+) transient. In support of this, the changes of amplitude of the transient occur more quickly following changes of [Ca(2+)](o) than following refilling of the SR after depletion with caffeine. We conclude that the coordinated control of increased Ca(2+) entry and greater fractional release of Ca(2+) is an important factor in regulating excitation-contraction coupling.

Integrative analysis of calcium cycling in cardiac muscle.

The control of intracellular calcium is central to regulation of contractile force in cardiac muscle. This review illustrates how analysis of the control of calcium requires an integrated approach in which several systems are considered. Thus, the calcium content of the sarcoplasmic reticulum (SR) is a major determinant of the amount of Ca(2+) released from the SR and the amplitude of the Ca(2+) transient. The amplitude of the transient, in turn, controls Ca(2+) fluxes across the sarcolemma and thence SR content. This control of SR content influences the response to maneuvers that modify, for example, the properties of the SR Ca(2+) release channel or ryanodine receptor. Specifically, modulation of the open probability of the ryanodine receptor produces only transient effects on the Ca(2+) transient as a result of changes of SR content. These interactions between various Ca(2+) fluxes are modified by the Ca(2+) buffering properties of the cell. Finally, we predict that, under some conditions, the above interactions can result in instability (such as alternans) rather than ordered control of contractility.

Frequency-modulated atomic force microscopy localises viscoelastic remodelling in the ageing sheep aorta.

Age-related aortic stiffening is associated with cardiovascular diseases such as heart failure. The mechanical functions of the main structural components of the aorta, such as collagen and elastin, are determined in part by their organisation at the micrometer length scale. With age and disease both components undergo aberrant remodelling, hence, there is a need for accurate characterisation of the biomechanical properties at this length scale. In this study we used a frequency-modulated atomic force microscopy (FM-AFM) technique on a model of ageing in female sheep aorta (young: ~18 months, old: >8 years) to measure the micromechanical properties of the medial layer of the ascending aorta. The novelty of our FM-AFM method, operated at 30kHz, is that it is non-contact and can be performed on a conventional AFM using the ×³cantilever tune' mode, with a spatial (areal) resolution of around 1.6µm(2). We found significant changes in the elastic and viscoelastic properties within the medial lamellar unit (elastic lamellae and adjacent inter-lamellar space) with age. In particular, there was an increase in elastic modulus (Young; geometric mean (geometric SD)=42.9 (2.26)kPa, Old=113.9 (2.57)kPa, P<0.0001), G' and G″ (storage and loss modulus respectively) (Young; G'=14.3 (2.26)kPa, Old G'=38.0 (2.57)kPa, P<0.0001; Young; G″=14.5 (2.56)kPa, Old G″=32.8 (2.52)kPa, P<0.0001). The trends observed in the elastic properties with FM-AFM matched those we have previously found using scanning acoustic microscopy (SAM). The utility of the FM-AFM method is that it does not require custom AFM hardware and can be used to simultaneously determine the elastic and viscoelastic behaviour of a biological sample.

The control of sarcoplasmic reticulum Ca content in cardiac muscle.

Most of the calcium that activates contraction in the heart comes from the sarcoplasmic reticulum (SR) and it is therefore essential to control the SR Ca content. SR Ca content reflects the balance between uptake (via the SR Ca-ATPase, SERCA) and release, largely via the ryanodine receptor (RyR). Unwanted changes of SR Ca are prevented because, for example, an increase of SR Ca content increases the amplitude of the systolic Ca transient and this, in turn, results in increased loss of Ca from and decreased Ca entry into the cell thereby restoring cell and SR Ca towards control levels. We discuss the parameters that affect the steady level of SR Ca and how these may change in heart failure. Finally, we discuss disordered Ca regulation with particular emphasis on the condition of alternans where successive heartbeats alternate in amplitude. This behaviour can be explained by excessive feedback gain in the processes controlling SR Ca.

Enhanced sarcolemmal Ca2+ efflux reduces sarcoplasmic reticulum Ca2+ content and systolic Ca2+ in cardiac hypertrophy.

OBJECTIVE: Recent work has identified reductions in the systolic Ca(2+) transient in cardiac disease states. The aim of the present study was to identify the mechanisms responsible for perturbations of intracellular calcium homeostasis in isolated cardiac myocytes and determine if such changes can quantitatively explain the reduced systolic Ca(2+) transient. METHODS: Left ventricular hypertrophy (LVH) was induced by aortic coarctation in adult ferrets. Changes in intracellular Ca(2+) regulation, sarcolemmal Ca(2+) fluxes and SR function were measured in single left ventricular cardiac myocytes. RESULTS: Cardiac hypertrophy was associated with a 29% increase in action potential duration (APD(90)); a 48% reduction in the amplitude of and 19% slowing in the rate of decay of the systolic Ca(2+) transient; a 20% decrease in SR Ca(2+) content and a 36% increase in inward Na(+)-Ca(2+) exchange current for a given change in [Ca(2+)](i) (all P<0.05). Peak L-type Ca(2+) current density, integrated Ca(2+) influx and SERCA2a protein levels remained unchanged in hypertrophy. By determining the relationship between SR Ca(2+) content and systolic Ca(2+), the reduction in SR Ca(2+) content quantitatively explained the smaller systolic Ca(2+) transient. The reduced SR Ca(2+) content also accounted for a smaller fractional release of Ca(2+) from the SR and lower gain of excitation contraction coupling in cardiac hypertrophy. The increased sarcolemmal-mediated Ca(2+) efflux was sufficient to explain the reduction in SR Ca(2+) content. CONCLUSIONS: The findings indicate that the primary mechanism underlying the smaller systolic Ca(2+) transient amplitude in cardiac hypertrophy is decreased SR Ca(2+) content occurring as a consequence of reduced SR Ca(2+)-ATPase-mediated Ca(2+) uptake and increased sarcolemmal-mediated Ca(2+) efflux from the cell. The increased Na(+)-Ca(2+) exchange-mediated current for a given change in intracellular Ca(2+) concentration provides a mechanism for the development of arrhythmias in the face of a reduced SR Ca(2+) load in cardiac hypertrophy.

Stimulation of Ca-induced Ca release only transiently increases the systolic Ca transient: measurements of Ca fluxes and sarcoplasmic reticulum Ca.

OBJECTIVE: To investigate the effects of stimulating calcium induced Ca release with low concentrations (100-200 microM) of caffeine and, in particular, to study the cellular mechanisms responsible for the transient responses found previously. METHODS: Experiments were performed on isolated rat ventricular myocytes. Intracellular calcium concentration ([Ca2+]i) was measured with Indo-1, the cells were voltage-clamped with the perforated patch technique and sarcoplasmic reticulum (s.r.) Ca content was estimated from the integral of the caffeine-evoked current. RESULTS: The systolic Ca transient produced by the first depolarization in the presence of caffeine was larger than the control. Over the next few pulses the magnitude of the Ca transient returned to control levels despite the maintained presence of caffeine. The s.r. Ca content was decreased by 9% after one pulse in caffeine and by 21% after several pulses in caffeine. The first pulse in the low concentration of caffeine was followed by an enhanced inward (Na-Ca exchange) current tail indicating increased efflux of calcium from the cell. The extra loss of calcium calculated from the tail current agreed quantitatively with that from the change of s.r. Ca content. CONCLUSIONS: These results show that stimulating calcium induced calcium release produces only a transient increase of the systolic Ca transient. This is due to the larger Ca transient decreasing the s.r. Ca content. It is concluded that any agent whose sole mode of action is stimulation of calcium-induced calcium release will not produce a maintained inotropic effect. The consequences of this for the effects of other modulators of calcium induced calcium release are discussed.

The control of Ca release from the cardiac sarcoplasmic reticulum: regulation versus autoregulation.

This review discusses the mechanism and regulation of Ca release from the cardiac sarcoplasmic reticulum. Ca is released through the Ca release channel or ryanodine receptor (RyR) by the process of calcium-induced Ca release (CICR). The trigger for this release is the L-type Ca current with a small contribution from Ca entry on the Na-Ca exchange. Recent work has shown that CICR is controlled at the level of small, local domains consisting of one or a small number of L-type Ca channels and associated RyRs. Ca efflux from the s.r. in one such unit is seen as a 'spark' and the properties of these sparks produce controlled Ca release from the s.r. A major factor controlling the amount of Ca released from the s.r. and therefore the magnitude of the systolic Ca transient is its Ca content. The Ca content depends on both the properties of the s.r. and the cytoplasmic Ca concentration. Changes of s.r. Ca content and the Ca released affect the sarcolemmal Ca and Na-Ca exchange currents and this acts to control cell Ca loading and the s.r. Ca content. The opening probability of the RyR can be regulated by various physiological mediators as well as pharmacological compounds. However, it is shown that, due to compensatory changes of s.r. Ca, modifiers of the RyR only produce transient effects on systolic Ca. We conclude that, although the RyR can be regulated, of much greater importance to the control of Ca efflux from the s.r. are effects due to changes of s.r. Ca content.