Sarcoplasmic Reticulum Release Research Articles

Sarcoplasmic reticulum Ca2+ release channel complex and automatism: A matter of fine tuning

See article by Dirksen et al. [10] (pages 69–78) and Faber and Rudy [15] (pages 79–88) in this issue. In heart muscle, excitation contraction coupling (ECC) relies on proper release of Ca2+ from the sarcoplasmic reticulum (SR), which is accomplished by the activation of SR Ca2+ release channels (also known as ryanodine receptors, RyR) upon Ca2+ binding, a process termed calcium-induced calcium release [1]. The spontaneous activation of a cluster of RyRs produces local Ca2+ release seen under confocal fluorescence microscopy as minuscule flashes of light called Ca2+ sparks, the elementary Ca2+ release events [2]. SR release units are never totally quiescent. They keep on releasing Ca2+ randomly even during diastole, when sparks are rare in time and spatially separable [3]. During ECC, thousands of intracellular release events are synchronized by Ca2+ influx during the action potential, which causes both spatial and temporal overlap of elementary events in a way to produce a global intracellular transient increase in Ca2+ concentration ([Ca2+] i ) that initiates cell contraction. For relaxation to occur, [Ca2+] i must be reduced to the resting levels. This is accomplished by a number of transport systems that compete for Ca2+, mainly the SR Ca2+-ATPase and the Na+/Ca2+exchanger (NCX) [3]. The amount of Ca2+ released is strongly regulated by the SR Ca2+ content, both during systole [4] and diastole [5], so that release ceases at critically low SR loads, a condition in which a significant amount of Ca2+ (more than 40% of maximum load) can still be found in the organelle [4]. SR Ca2+ content, in turn, is regulated under normal physiological conditions by … *Centro de Engenharia Biomedica/UNICAMP, Caixa Postal 6040, 13084-971 Campinas, SP, Brazil. Tel.: +55 19 3521 9287; fax: +55 19 3289 3346. bassani{at}ceb.unicamp.br

HYPOXIC PRECONDITIONING INDUCES DELAYED CARDIOPROTECTION THROUGH P38 MAPK-MEDIATED CALRETICULIN UPREGULATION

The protective mechanisms of hypoxic preconditioning (HPC) involve the mitigation of cellular calcium overload in cardiomyocytes. The sarcoplasmic reticulum (SR) chaperone calreticulin (CRT) plays an important role in regulating calcium homeostasis and is upregulated by HPC. The goal of this study was to show whether the late cardioprotection of HPC is mediated by calreticulin upregulation and to demonstrate whether the calreticulin induction is mediated by p38 MAPK phosphorylation. Hypoxic preconditioning was induced by hypoxemic hypoxic exposure by a 24-h period of normoxic reoxygenation before undergoing LAD occlusion in rats or hypoxia/reoxygenation (H/R) in cardiomyocytes. Ca uptake and release of the SR vesicles was determined by use of Ca and the Millipore filtration technique. Western blotting analysis was used to detect calreticulin expression and activity of p38 MAPK. Hypoxic preconditioning induced calreticulin upregulation and attenuated H/R injury in neonatal cardiomyocytes and myocardial ischemia injury by increasing calcium uptake and reducing calcium release in SR. Hearts from the HPC group were more resistant to sustained ischemia and had much stronger phosphorylation of p38 MAPK than sham operation. Inhibition of p38 MAPK with SB202190 (a selective p38 MAPK inhibitor) abolished the calreticulin upregulation and cardioprotection by HPC. Hypoxic preconditioning upregulates calreticulin expression through a p38 MAPK signaling pathway and protects cardiomyocytes from H/R (and ischemia) injury.

Functional Significance of Na+/Ca2+ Exchangers Co‐localization with Ryanodine Receptors

Co-localization of Na+/Ca2+ exchangers (NCX) with ryanodine receptors (RyRs) is debated. We incorporate local NCX current in a biophysically detailed model of L-type Ca2+ channels (LCCs) and RyRs and study the effect of NCX on the regulation of Ca2+-induced Ca2+ release and the shape of the action potential. In canine ventricular cells, under pathological conditions, e.g., impaired LCCs, local NCXs become an enhancer of sarcoplasmic reticulum release. Under such conditions incorporation of local NCXs is critical to accurately capture mechanisms of excitation-contraction coupling.

Ca2+-Induced Ca2+ Release through Localized Ca2+ Uncaging in Smooth Muscle

Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) occurs in smooth muscle as spontaneous SR Ca2+ release or Ca2+ sparks and, in some spiking tissues, as Ca2+ release that is triggered by the activation of sarcolemmal Ca2+ channels. Both processes display spatial localization in that release occurs at a higher frequency at specific subcellular regions. We have used two-photon flash photolysis (TPFP) of caged Ca2+ (DMNP-EDTA) in Fluo-4–loaded urinary bladder smooth muscle cells to determine the extent to which spatially localized increases in Ca2+ activate SR release and to further understand the molecular and biophysical processes underlying CICR. TPFP resulted in localized Ca2+ release in the form of Ca2+ sparks and Ca2+ waves that were distinguishable from increases in Ca2+ associated with Ca2+ uncaging, unequivocally demonstrating that Ca2+ release occurs subsequent to a localized rise in [Ca2+]i. TPFP-triggered Ca2+ release was not constrained to a few discharge regions but could be activated at all areas of the cell, with release usually occurring at or within several microns of the site of photolysis. As expected, the process of CICR was dominated by ryanodine receptor (RYR) activity, as ryanodine abolished individual Ca2+ sparks and evoked release with different threshold and kinetics in FKBP12.6-null cells. However, TPFP CICR was not completely inhibited by ryanodine; Ca2+ release with distinct kinetic features occurred with a higher TPFP threshold in the presence of ryanodine. This high threshold release was blocked by xestospongin C, and the pharmacological sensitivity and kinetics were consistent with CICR release at high local [Ca2+]i through inositol trisphosphate (InsP3) receptors (InsP3Rs). We conclude that CICR activated by localized Ca2+ release bears essential similarities to those observed by the activation of ICa (i.e., major dependence on the type 2 RYR), that the release is not spatially constrained to a few specific subcellular regions, and that Ca2+ release through InsP3R can occur at high local [Ca2+]i.

Caffeine administration results in greater tension development in previously fatigued canine muscle in situ

In isolated single skeletal myocytes undergoing long-term fatiguing contractions, caffeine (CAF) can result in nearly immediate restoration of generated tension to near-prefatigue levels by increasing Ca2+ release via activation of sarcoplasmic reticulum release channels. This study tested whether arterial CAF infusion (>5 mm) would cause a similar rapid restoration of tetanic isometric tension during contractions to fatigue in perfused canine hindlimb muscle in situ. Tetanic contractions were elicited by electrical stimulation (200 ms trains, 50 Hz, 1 contraction s(-1)), and biopsies were taken from the muscle at rest and during contractions: (1) following the onset of fatigue (tension approximately 60% of initial value); and (2) following CAF administration. Resting muscle ATP, PCr and lactate contents were 25.2 +/- 0.4, 76.9 +/- 3.3 and 14.4 +/- 3.3 mmol (kg dry weight)(-1), respectively. At fatigue, generated tetanic tension was 61.1 +/- 6.9% of initial contractions. There was a small but statistically significant recovery of tetanic tension (64.9 +/- 6.6% of initial value) with CAF infusion, after which the muscle showed incomplete relaxation. At fatigue, muscle ATP and PCr contents had fallen significantly (P < 0.05) to 18.1 +/- 1.1 and 18.9 +/- 2.1 mmol (kg dry weight)(-1), respectively, and lactate content had increased significantly to 27.7 +/- 5.4 mmol (kg dry weight)(-1). Following CAF, skeletal muscle ATP and PCr contents were significantly lower than corresponding fatigue values (15.0 +/- 1.3 and 10.9 +/- 2.2 mmol (kg dry weight)(-1), respectively), while lactate was unchanged (22.2 +/- 3.9 mmol (kg dry weight)(-1)). These results demonstrate that caffeine can result in a small, but statistically significant, recovery of isometric tension in fatigued canine hindlimb muscle in situ, although not nearly to the same degree as seen in isolated single muscle fibres. This suggests that, in this in situ isolated whole muscle model, alteration of Ca2+ metabolism is probably only one cause of fatigue.

Antisense strategies for treatment of heart failure.

AbstractIn the mammalian heart, intracellular Ca2+ movements are tightly regulated at various levels within the cell (1). The sarcoplasmic reticulum (SR) plays an important role in orchestrating the movement of calcium during each contraction and relaxation. Excitation leads to the opening of voltage-gated L-type Ca2+ channels, allowing the entry of a small amount of Ca2+ into the cell. Through a coupling mechanism between the L-type Ca2+ channel and the SR release channels (ryanodine receptors), a larger amount of Ca2+ is released, activating the myofilaments leading to contraction. During relaxation, Ca2+ is reaccumulated back into the SR by the SR Ca2+ adenosine triphosphatase (ATPase) pump (SERCA2a) and extruded extracellularly by the sarcolemmal Na+/Ca2+ exchanger. The contribution of each of these mechanisms for lowering cytosolic Ca2+ varies with species. In humans, approx 70% of the Ca2+ is removed by SERCA2a and approx 30% by the Na+/Ca2+ exchanger (1). The Ca2+ pumping activity of SERCA2a is influenced by phospholamban. In the unphosphorylated state, phospholamban inhibits the Ca2+-ATPase, whereas phosphorylation of phospholamban by cyclic adenosine monophosphate (cAMP)-dependent protein kinase and by Ca2+-calmodulin-dependent protein kinase reverses this inhibition (2). Ca2+ transients recorded from failing human myocardial cells or trabeculae reveal a significantly prolonged Ca2+ transient with an elevated end-diastolic intracellular Ca2+ (3,4).KeywordsSarcoplasmic ReticulumSarcoplasmic Reticulum CalciumHuman CardiomyocytesAdenoviral Gene TransferAntisense StrategyThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Attentuation of cardiac stunning by losartan in a cellular model of ischemia and reperfusion is accompanied by increased sarcoplasmic reticulum Ca2+ stores and prevention of cytosolic Ca2+ elevation.

This study investigates whether protective effects of an angiotensin II type 1 receptor antagonist (losartan) in ischemia and reperfusion are mediated by actions on Ca(2+) cycling. Effects of exposure to losartan (10 microM) in ischemia were evaluated in isolated guinea pig ventricular myocytes exposed to simulated ischemia and reperfusion at 37 degrees C. Field-stimulated myocytes were exposed to 30 min of simulated ischemia (hypoxia, acidosis, lactate, hyperkalemia, and glucose-free) and reperfusion with Tyrode's solution for 40 min. Cell shortening was measured with a video edge detector, and Ca(2+) concentration was measured with fura-2. Field-stimulated myocytes exhibited stunning in reperfusion, which was abolished in cells exposed to losartan. In microelectrode studies, losartan did not alter the responses of resting potentials or action potentials to ischemia and reperfusion. In the absence of losartan, diastolic Ca(2+) increased in ischemia, and Ca(2+) transients exhibited a rebound overshoot in early reperfusion. Losartan did not affect amplitudes of Ca(2+) transients in ischemia but prevented elevations in diastolic Ca(2+) in ischemia. Furthermore, losartan prevented the overshoot of Ca(2+) transients in early reperfusion and increased the magnitude of Ca(2+) transients in late reperfusion. Sarcoplasmic reticulum (SR) Ca(2+) stores, determined as Ca(2+) released by rapid application of 10 mM caffeine, were not altered in ischemia and reperfusion. However, losartan increased SR Ca(2+) stores in late reperfusion, even in cells that were not exposed to simulated ischemia. We conclude that losartan abolishes stunning in reperfusion by preserving normal diastolic Ca(2+) in ischemia and by increasing Ca(2+) transients through elevation of releasable SR Ca(2+).

Putting out the fire: what terminates calcium-induced calcium release in cardiac muscle?

The majority of contractile calcium in cardiac muscle is released from stores in the sarcoplasmic reticulum (SR), by a process of calcium-induced calcium release (CICR) through ryanodine receptors. Because CICR is intrinsically self-reinforcing, the stability of and graded regulation of cardiac EC coupling appear paradoxical. It is now well established that this gradation results from the stochastic recruitment of varying numbers of elementary local release events, which may themselves be regenerative, and which can be directly observed as calcium sparks. Ryanodine receptors (RyRs) are clustered in dense lattices, and most calcium sparks are now believed to involve activation of multiple RyRs. This implies that local CICR is regenerative, requiring a mechanism to terminate it. It was initially assumed that this mechanism was inactivation of the RyR, but during the decade since the discovery of sparks, no sufficiently strong inactivation mechanism has been demonstrated in vitro and all empirically determined gating schemes for the RyR give unstable EC coupling in Monte Carlo simulations. We consider here possible release termination mechanisms. Stochastic attrition is the spontaneous decay of active clusters due to random channel closure; calculations show that it is much too slow unless assisted by another process. Calcium-dependent RyR inactivation involving third-party proteins remains a viable but speculative mechanism; current candidates include calmodulin and sorcin. Local depletion of SR release terminal calcium could terminate release, however calculations and measurements leave it uncertain whether a sufficient diffusion resistance exists within the SR to sustain such depletion. Depletion could be assisted by dependence of RyR activity on SR lumenal [Ca 2+]. There is substantial evidence for such lumenal activation, but it is not clear if it is a strong enough effect to account for the robust termination of sparks. The existence of direct interactions among clustered RyRs might account for the discrepancy between the inactivation properties of isolated RyRs and intact clusters. Such coupled gating remains controversial. Determining the mechanism of release termination is the outstanding unsolved problem of cardiac EC coupling, and will probably require extensive genetic manipulation of the EC coupling apparatus in its native environment to unravel the solution.

How many sodium ions does it take to turn an exchanger?

How many sodium ions does it take to turn an exchanger?

Activation of cardiac ryanodine receptors by cardiac glycosides.

This study investigated the effects of cardiac glycosides on single-channel activity of the cardiac sarcoplasmic reticulum (SR) Ca2+ release channels or ryanodine receptor (RyR2) channels and how this action might contribute to their inotropic and/or toxic actions. Heavy SR vesicles isolated from canine left ventricle were fused with artificial planar lipid bilayers to measure single RyR2 channel activity. Digoxin and actodigin increased single-channel activity at low concentrations normally associated with therapeutic plasma levels, yielding a 50% of maximal effect of approximately 0.2 nM for each agent. Channel activation by glycosides did not require MgATP and occurred only when digoxin was applied to the cytoplasmic side of the channel. Similar results were obtained in human RyR2 channels; however, neither the crude skeletal nor the purified cardiac channel was activated by glycosides. Channel activation was dependent on [Ca2+] on the luminal side of the bilayer with maximal stimulation occurring between 0.3 and 10 mM. Rat RyR2 channels were activated by digoxin only at 1 microM, consistent with the lower sensitivity to glycosides in rat heart. These results suggest a model in which RyR2 channel activation by digoxin occurs only when luminal [Ca2+] was increased above 300 microM (in the physiological range). Consequently, increasing SR load (by Na+ pump inhibition) serves to amplify SR release by promoting direct RyR2 channel activation via a luminal Ca2+-sensitive mechanism. This high-affinity effect of glycosides could contribute to increased SR Ca2+ release and might play a role in the inotropic and/or toxic actions of glycosides in vivo.

How can overexpression of Na+,Ca2+-exchanger compensate the negative inotropic effects of downregulated SERCA?

See article by Terracciano et al. [2] (pages 38–47) in this issue. The failing human ventricle suffers from two major problems: (1) During diastole, relaxation is retarded and remains eventually incomplete. (2) During systole, the force-frequency relation is blunted, i.e. an increase from 60 to 120 beats-per-min does not increase the contractile force as it is typical in non-failing tissue. Both problems have been linked to reduced expression and function of sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA) proteins. Studies in isolated human ventricular trabeculae [1] have shown that incomplete Ca2+ reuptake by SERCA can cause (1) a diastolic accumulation of Ca2+ ions in the cytosol which impairs diastolic relaxation, and (2) a reduction of releasable SR Ca2+ with the consequence of a reduced systolic Ca2+ activation of force and a blunted force-frequency relation. Since failing human myocardium was shown to overexpress the Na+/Ca2+-exchanger (mRNA and protein [1]), enhanced Ca2+ efflux by Na+/Ca2+-exchange has been suggested to partially compensate impaired diastolic Ca2+ removal. The paper of Terracciano et al. ([2] in this issue) confirms this view. In addition, it introduces a new idea: the enhanced expression and function of the Na+/Ca2+-exchanger may facilitate Ca2+ reuptake by SERCA and thereby compensate for the impaired SR Ca2+ load. Terraciano et al. [2,3] compared protein concentrations and functions between ventricular myocytes from transgenic mice (TR) that overexpress the Na+/Ca2+-exchanger and non-transgenic (non-TR) wild-type littermates. They find that the protein levels of the Na+/Ca2+-exchanger are approximately 2.4-fold elevated [3,4]) whilst the concentrations of other Ca2+ handling proteins such as SERCA, calsequestrin and phospholambam were not different. With this background, the authors can evaluate the consequences … * Tel.: +49-345-557-1886; fax: +49-345-557-4019 gerrit.isenberg{at}medizin.uni-halle.de

Phosphorylation of Anchoring Protein by Calmodulin Protein Kinase Associated to the Sarcoplasmic Reticulum of Rabbit Fast-Twitch Muscle

Regulatory phosphorylation of phospholamban and of SR Ca2+-ATPase SERCA2a isoform by endogenous CaM-K II in slow-twitch skeletal and cardiac sarcoplasmic reticulum (SR) is well documented, but much less is known of the exact functional role of CaM K II in fast-twitch muscle SR. Recently, it was shown that RNA splicing of brain-specific α CaM K II, gives rise to a truncated protein (αKAP), consisting mainly of the association domain, serving to anchor CaM K II to SR membrane in rat skeletal muscle [Bayer, K.-U., et al. (1998) EMBO J. 19, 5598–5605]. In the present study, we searched for the presence of αKAP in sucrose-density purified SR membrane fractions from representative fast-twitch and slow-twitch limb muscles, both of the rabbit and the rat, using immunoblot techniques and antibody directed against the association domain of α CaM K II. Putative αKAP was immunodetected as a 23-kDa electrophoretic component on SDS–PAGE of the isolated SR from fast-twitch but not from slow-twitch muscle, and was further identified as a specific substrate of endogenous CaM K II, in the rabbit. Immunodetected, 32P-labeled, non-calmodulin binding protein, behaved as a single 23-kDa protein species under several electrophoretic conditions. The 23-kDa protein, with defined properties, was isolated as a complex with 60-kDa δ CaM K II isoform, by sucrose-density sedimentation analysis. Moreover, we show here that putative αKAP, in spite of its inability to bind CaM in ligand blot overlay, co-eluted with δ CaM K II from CaM-affinity columns. That raises the question of whether CaM K II-mediated phosphorylation of αKAP and triadin together might be involved in a molecular signaling pathway important for SR Ca2+-release in fast-twitch muscle SR.

Immunogold-labeled L-type Calcium Channels are Clustered in the Surface Plasma Membrane Overlying Junctional Sarcoplasmic Reticulum in Guinea-pig Myocytes—Implications for Excitation–contraction Coupling in Cardiac Muscle

Ca2+release through ryanodine receptors, located in the membrane of the junctional sarcoplasmic reticulum (SR), initiates contraction of cardiac muscle. Ca2+influx through plasma membrane L-type Ca2+channels is thought to be an important trigger for opening ryanodine receptors (««Ca2+-induced Ca2+-release»»). Optimal transmission of the transmembrane Ca2+influx signal to SR release is predicted to involve spatial juxtaposition of L-type Ca2+channels to the ryanodine receptors of the junctional SR. Although such spatial coupling has often been implicitly assumed, and data from immunofluorescence microscopy are consistent with its existence, the definitive demonstration of such a structural organization in mammalian tissue is lacking at the electron-microscopic level. To determine the spatial distribution of plasma membrane L-type Ca2+channels and their location in relation to underlying junctional SR, we applied two high-resolution immunogold-labeling techniques, label-fracture and cryothin-sectioning, combined with quantitative analysis, to guinea-pig ventricular myocytes. Label-fracture enabled visualization of colloidal gold-labeled L-type Ca2+channels in planar freeze-fracture electron-microscopic views of the plasma membrane. Mathematical analysis of the gold label distribution (by nearest-neighbor distance distribution and the radial distribution function) demonstrated genuine clustering of the labeled channels. Gold-labeled cryosections showed that labeled L-type Ca2+channels quantitatively predominated in domains of the plasma membrane overlying junctional SR. These findings provide an ultrastructural basis for functional coupling between L-type Ca2+channels and junctional SR and for excitation–contraction coupling in guinea-pig cardiac muscle.

Modeling short-term interval-force relations in cardiac muscle.

This study employs two modeling approaches to investigate short-term interval-force relations. The first approach is to develop a low-order, discrete-time model of excitation-contraction coupling to determine which parameter combinations produce the degree of postextrasystolic potentiation seen experimentally. Potentiation is found to increase 1) for low recirculation fraction, 2) for high releasable fraction, i.e., the maximum fraction of Ca(2+) released from the sarcoplasmic reticulum (SR) given full restitution, and 3) for strong negative feedback of the SR release on sarcolemmal Ca(2+) influx. The second modeling approach is to develop a more detailed single ventricular cell model that simulates action potentials, Ca(2+)-handling mechanisms, and isometric force generation by the myofilaments. A slow transition from the adapted state of the ryanodine receptor produces a gradual recovery of the SR release and restitution behavior. For potentiation, a small extrasystolic release leaves more Ca(2+) in the SR but also increases the SR loading by two mechanisms: 1) less Ca(2+)-induced inactivation of L-type channels and 2) reduction of action potential height by residual activation of the time-dependent delayed rectifier K(+) current, which increases Ca(2+) influx. The cooperativity of the myofilaments amplifies the relatively small changes in the Ca(2+) transient amplitude to produce larger changes in isometric force. These findings suggest that short-term interval-force relations result mainly from the interplay of the ryanodine receptor adaptation and the SR Ca(2+) loading, with additional contributions from membrane currents and myofilament activation.

Transport of Ca2+ from sarcoplasmic reticulum to mitochondria in rat ventricular myocytes.

Studies with electron microscopy have shown that sarcoplasmic reticulum (SR) and mitochondria locate close to each other in cardiac muscle cells. We investigated the hypothesis that this proximity results in a transient exposure of mitochondrial Ca2+ uniporter (CaUP) to high concentrations of Ca2+ following Ca2+ release from the SR and thus an influx of Ca2+ into mitochondria. Single ventricular myocytes of rat were skinned by exposing them to a physiological solution containing saponin (0.2 mg/ml). Cytosolic Ca2+ concentration ([Ca2+]c) and mitochondrial Ca2+ concentration ([Ca2+]m) were measured with fura-2 and rhod2, respectively. Application of caffeine (10 mM) induced a concomitant increase in [Ca2+]c and [Ca2+]m. Ruthenium red, at concentrations that block CaUP but not SR release, diminished the caffeine-induced increase in [Ca2+]m but not [Ca2+]c. In the presence of 1 mM BAPTA, a Ca2+ chelator, the caffeine-induced increase in [Ca2+]m was reduced substantially less than [Ca2+]c. Moreover, inhibition of SR Ca2+ pump with two different concentrations of thapsigargin caused an increase in [Ca2+]m, which was related to the rate of [Ca2+]c increase. Finally, electron microscopy showed that sites of junctions between SR and T tubules from which Ca2+ is released, or Ca2+ release units, CRUs, are preferentially located in close proximity to mitochondria. The distance between individual SR Ca2+ release channels (feet or ryanodine receptors) is very short, ranging between approximately 37 and 270 nm. These results are consistent with the idea that there is a preferential coupling of Ca2+ transport from SR to mitochondria in cardiac muscle cells, because of their structural proximity.

Intracellular calcium during fatigue of cane toad skeletal muscle in the absence of glucose.

Mechanisms of fatigue were studied in single muscle fibres of the cane toad (Bufo marinus) in which force, intracellular calcium ([Ca2+]i), [Mg2+]i, glycogen and the rapidly releasable Ca2+ from the sarcoplasmic reticulum (SR) were measured. Fatigue was produced by repeated tetani continued until force had fallen to 50%. Two patterns of fatigue in the absence of glucose were studied. In the first fatigue run force fell to 50% in 8-10 min. Fatigue runs were then repeated until force fell to 50% in < 3 min in the final fatigue run. Addition of extracellular glucose after the final fatigue run prolonged a subsequent fatigue run. In the first fatigue run peak tetanic [Ca2+]i initially increased and then declined and at the time when force had fallen to 50% tetanic [Ca2+]i was 54+/-5% of initial value. In the final fatigue run force and peak tetanic [Ca2+]i declined more rapidly but to the same level as in first fatigue runs. At the end of the first fatigue run, the rapidly releasable SR Ca2+ store fell to 46+/-6% of the pre-fatigue value. At the end of the final fatigue run the rapidly releasable SR Ca2+ store was 109+/-16% of the pre-fatigue value. In unstimulated fibres the nonwashable glycogen content was 176+/-30 mmol glycosyl units/l fibre. After one fatigue run the glycogen content was 117+/-17 mmol glycosyl units/l fibre; at the end of the final fatigue run the glycogen content was reduced to 85+/-9 mmol glycosyl units/l fibre. [Mg2+]i did not change significantly at the end of fatigue in either the first or the final fatigue run suggesting that globally-averaged ATP does not decline substantially in either pattern of fatigue. These results suggest that different mechanisms are involved in the decline of tetanic [Ca2+]i in first compared to final fatigue runs. The SR Ca2+ store is reduced in first fatigue runs; this is not the case for the final fatigue run which is associated with a decline in glycogen and possibly related to either a non-metabolic effect of glycogen or a spatially-localised metabolic decline.

Constitutive beta2-adrenergic signalling enhances sarcoplasmic reticulum Ca2+ cycling to augment contraction in mouse heart.

1. Transgenic overexpression of the beta2-adrenergic receptor (beta2AR) in mouse heart augments baseline cardiac function in a ligand-independent manner, due to the presence of spontaneously active beta2AR (beta2AR*). This study aims to elucidate the mechanism of beta2AR*-mediated modulation of cardiac excitation-contraction (EC) coupling. 2. Confocal imaging was used to analyse Ca2+ sparks and spatially resolve Ca2+ transients in single ventricular myocytes from transgenic (TG4) and non-transgenic (NTG) littermates. Whole-cell voltage- and current-clamp techniques were used to record L-type Ca2+ currents (ICa) and action potentials, respectively. 3. In the absence of any beta2AR ligand, TG4 myocytes had greater contraction amplitudes, larger Ca2+ transients and faster relaxation times than did NTG cells. 4. The action potentials of TG4 and NTG myocytes were similar, except for a prolonged end-stage repolarization in TG4 cells; the ICa density and kinetics were nearly identical. The relationship between peak Ca2+ and contraction, which reflects myofilament Ca2+ sensitivity, was similar. 5. In TG4 cells, the frequency of Ca2+ sparks (spontaneous or evoked at -40 mV) was 2-7 times greater, despite the absence of change in the resting Ca2+, sarcoplasmic reticulum (SR) Ca2+ content, and ICa. Individual sparks were brighter, broader and lasted longer, leading to a 2.3-fold greater signal mass. Thus, changes in both spark frequency and size underlie the greater Ca2+ transient in TG4 cells. 6. The inverse agonist ICI 118,551 (ICI, 5 x 10-7 M), which blocks spontaneous beta2AR activation, reversed the aforementioned beta2AR* effects on cardiac EC coupling without affecting the sarcolemmal ICa. However, ICI failed to detect significant constitutive beta2AR activity in NTG cells. 7. We conclude that beta2AR*-mediated signalling enhances SR release channel activity and Ca2+-induced Ca2+ release in TG4 cardiac myocytes, and that beta2AR* enhances EC coupling by reinforcing SR Ca2+ cycling (release and reuptake), but bypassing the sarcolemmal ICa.

The role of calcium stores in fatigue of isolated single muscle fibres from the cane toad.

1. Intracellular calcium ([Ca2+]i) and tension were measured from single muscle fibres dissected from the cane toad (Bufo marinus). The amount of Ca2+ which could be released from the sarcoplasmic reticulum (SR) was estimated by brief (approximately 20 s) exposures to 4-chloro-m-cresol (4-CmC) or caffeine. 2. Muscle fatigue was produced by repeated tetani at 4 s or shorter intervals and continued until tension had fallen to 50% of the control. The intracellular free calcium concentration during a tetanus (tetanic [Ca2+]i) first increased and then steadily declined to 43+/-2% of control by the time tension had fallen to 50%. Over the period of fatigue the rapidly releasable Ca2+ from the SR fell to 46+/-6% of control. Tension and tetanic [Ca2+]i recovered to 93+/-3% and 100+/-4% of the control values after 20 min of rest. Over the same period rapidly releasable SR Ca2+ recovered to 98+/-12%. 3. When a similar number of tetani (200) were repeated at longer intervals (10 s), fibres showed only a small reduction in tension (to 85+/-1%) and tetanic [Ca2+]i did not change significantly. Under these conditions the rapidly releasable SR Ca2+ did not change significantly. 4. The recovery of rapidly releasable SR Ca2+ after fatigue was unaffected by removal of extracellular calcium but did not occur when oxidative phosphorylation was inhibited with cyanide. 5. These results suggest that an important cause of the decline of tetanic [Ca2+]i during fatigue is an equivalent decline in the amount of rapidly releasable SR Ca2+. The results show that the decline of rapidly releasable SR Ca2+ is related to a metabolic consequence of fatigue and are consistent with the hypothesis that Ca2+ precipitates with phosphate in the SR during fatigue.

Direct measurement of SR release flux by tracking 'Ca2+ spikes' in rat cardiac myocytes.

1. Ca2+ release flux across the sarcoplasmic reticulum (SR) during cardiac excitation-contraction coupling was investigated using a novel fluorescence method. Under whole-cell voltage-clamp conditions, rat ventricular myocytes were dialysed with a high concentration of EGTA (4.0 mM, 150 nM free Ca2+), to minimize the residence time of released Ca2+ in the cytoplasm, and a low-affinity, fast Ca2+ indicator, Oregon Green 488 BAPTA-5N (OG-5N; 1.0 mM, Kd approximately 31 microM), to optimize the detection of localized high [Ca2+] in release site microdomains. Confocal microscopy was employed to resolve intracellular [Ca2+] at high spatial and temporal resolution. 2. Analytical and numerical analyses indicated that, under conditions of high EGTA concentration, the free [Ca2+] change is the sum of two terms: one major term proportional to the SR release flux/Ca2+ influx, and the other reflecting the running integral of the released Ca2+. 3. Indeed, the OG-5N transients in EGTA-containing cells consisted of a prominent spike followed by a small pedestal. The OG-5N spike closely resembled the first derivative (d[Ca2+]/dt) of the conventional Ca2+ transient (with no EGTA), and mimicked the model-derived SR Ca2+ release function reported previously. In SR Ca2+-depleted cells, the OG-5N transient also closely followed the waveform of L-type Ca2+ current (ICa). Using ICa as a known source of Ca2+ influx, SR flux can be calibrated in vivo by a linear extrapolation of the ICa-elicited OG-5N signal. 4. The OG-5N image signal was localized to discrete release sites at the Z-line level of sarcomeres, indicating that the local OG-5N spike arises from 'Ca2+ spikes' at transverse (T) tubule-SR junctions (due to the imbalance between calcium ions entering the cytosol and the buffer molecules). 5. Both peak SR release flux and total amount of released Ca2+ exhibited a bell-shaped voltage dependence. The temporal pattern of SR release also varied with membrane voltage: Ca2+ release was most synchronized and produced maximal peak release flux (4.2 mM s-1) at 0 mV; in contrast, maximal total release occurred at -20 mV (71 versus 61 microM at 0 mV), but the localized release signals were partially asynchronous. Since the maximal conventional [Ca2+] transient and contraction were elicited at 0 mV, it appears that not only the amount of Ca2+ released, but also the synchronization among release sites affects the whole-cell Ca2+ transient and the Ca2+-myofilament interaction.

Transient mitochondrial depolarizations reflect focal sarcoplasmic reticular calcium release in single rat cardiomyocytes.

Digital imaging of mitochondrial potential in single rat cardiomyocytes revealed transient depolarizations of mitochondria discretely localized within the cell, a phenomenon that we shall call "flicker." These events were usually highly localized and could be restricted to single mitochondria, but they could also be more widely distributed within the cell. Contractile waves, either spontaneous or in response to depolarization with 50 mM K+, were associated with propagating waves of mitochondrial depolarization, suggesting that propagating calcium waves are associated with mitochondrial calcium uptake and consequent depolarization. Here we demonstrate that the mitochondrial flicker was directly related to the focal release of calcium from sarcoplasmic reticular (SR) calcium stores and consequent uptake of calcium by local mitochondria. Thus, the events were dramatically reduced by (a) depletion of SR calcium stores after long-term incubation in EGTA or thapsigargin (500 nM); (b) buffering intracellular calcium using BAPTA-AM loading; (c) blockade of SR calcium release with ryanodine (30 microM); and (d) blockade of mitochondrial calcium uptake by microinjection of diaminopentane pentammine cobalt (DAPPAC), a novel inhibitor of the mitochondrial calcium uniporter. These observations demonstrate that focal SR calcium release results in calcium microdomains sufficient to promote local mitochondrial calcium uptake, suggesting a tight coupling of calcium signaling between SR release sites and nearby mitochondria.