Sarcoplasmic reticulum and excitation-contraction coupling at 20 and 10 °C in rainbow trout myocardium

Summary Isometric force and series membrane potential were recorded in isolated ventricular strips from rainbow trout at 20 and 10 °C. Preparations were electrically stimulated to contract at either 0.5 or 0.2 Hz. Single extrastimulations elicited a twitch force which diminished when the preceding diastole was shortened below the regular value. The stimulation following this extra stimulation evoked no potentiation of force. Apart from a marginal effect on the post extrasystolic force at 20 °C, ryanodine did not affect either of these responses or the steady-state force at 0.5 Hz. At 0.2 Hz the steady-state force was somewhat depressed by ryanodine at 20 but not at 10 °C. In contrast, extrastimulations preceded by diastoles of up to 1 h more than doubled extrasystolic force at 20 °C. This effect was removed by ryanodine. Both the potentiations and the effect of ryanodine were strongly reduced at 10 °C. Apparently, temperature acts on the release of Ca²⁺ from the sarcoplasmic reticulum, since Ca²⁺ seems to be taken up at both temperatures. Hence, at both 20 and 10 °C, the contractures evoked in a solution inhibiting sarcolemmal Ca²⁺ transfer and releasing Ca²⁺ from the sarcoplasmic reticulum were diminished by pretreatment with 15 mM caffeine. Action potential duration at 20 °C was less than half of that at 10 °C. At both temperatures it tended to be prolonged by periods of prolonged rest. No effect of ryanodine on action potential configuration was detected. The results suggest that trout myocardial sarcoplasmic reticulum, although powerful at unphysiologically low stimulation rates, does not partake in the beat-to-beat regulation of force at heart rates encountered in vivo.Abbreviations ESF extrasystolic force - SR sarcoplasmic reticulum - v _F maximal rate of force development - v _R maximal rate of relaxation - TPF time to peak force - TR _0.5 time for half relaxation - TTF duration of force development     

R4496C RyR2 mutation impairs atrial and ventricular contractility

Ryanodine receptor (RyR2) is the major Ca²⁺ channel of the cardiac sarcoplasmic reticulum (SR) and plays a crucial role in the generation of myocardial force. Changes in RyR2 gating properties and resulting increases in its open probability (P_o) are associated with Ca²⁺ leakage from the SR and arrhythmias; however, the effects of RyR2 dysfunction on myocardial contractility are unknown. Here, we investigated the possibility that a RyR2 mutation associated with catecholaminergic polymorphic ventricular tachycardia, R4496C, affects the contractile function of atrial and ventricular myocardium. We measured isometric twitch tension in left ventricular and atrial trabeculae from wild-type mice and heterozygous transgenic mice carrying the R4496C RyR2 mutation and found that twitch force was comparable under baseline conditions (30°C, 2 mM [Ca²⁺]_o, 1 Hz). However, the positive inotropic responses to high stimulation frequency, 0.1 µM isoproterenol, and 5 mM [Ca²⁺]_o were decreased in R4496C trabeculae, as was post-rest potentiation. We investigated the mechanisms underlying inotropic insufficiency in R4496C muscles in single ventricular myocytes. Under baseline conditions, the amplitude of the Ca²⁺ transient was normal, despite the reduced SR Ca²⁺ content. Under inotropic challenge, however, R4496C myocytes were unable to boost the amplitude of Ca²⁺ transients because they are incapable of properly increasing the amount of Ca²⁺ stored in the SR because of a larger SR Ca²⁺ leakage. Recovery of force in response to premature stimuli was faster in R4496C myocardium, despite the unchanged rates of recovery of L-type Ca²⁺ channel current (I_Ca-L) and SR Ca²⁺ content in single myocytes. A faster recovery from inactivation of the mutant R4496C channels could explain this behavior. In conclusion, changes in RyR2 channel gating associated with the R4496C mutation could be directly responsible for the alterations in both ventricular and atrial contractility. The increased RyR2 P_o and fractional Ca²⁺ release from the SR induced by the R4496C mutation preserves baseline contractility despite a slight decrease in SR Ca²⁺ content, but cannot compensate for the inability to increase SR Ca²⁺ content during inotropic challenge.     

Quantitative measurement of Ca²(+) in the sarcoplasmic reticulum lumen of mammalian skeletal muscle

Skeletal muscle stores Ca²⁺ in the sarcoplasmic reticulum (SR) and releases it to initiate contraction, but the concentration of luminal Ca²⁺ in the SR ([Ca²⁺]_SR) and the amount that is released by physiological or pharmacological stimulation has been difficult to measure. Here we present a novel, yet simple and direct, method that provides the first quantitative estimates of static content and dynamic changes in [Ca²⁺]_SR in mammalian skeletal muscle, to our knowledge. The method uses fluo-5N loaded into the SR of single, mammalian skeletal muscle cells (murine flexor digitorum brevis myofibers) and confocal imaging to detect and calibrate the signals. Using this method, we have determined that [Ca²⁺]_{SR, free} is 390 μM. 4-Chloro-m-cresol, an activator of the skeletal muscle ryanodine receptor, reduces [Ca²⁺]_{SR, free} to ∼8 μM, when values are corrected for background fluorescence from cytoplasmic pools of dye. Prolonged electrical stimulation (10 s) at 50 Hz releases 88% of the SR Ca²⁺ content, whereas stimulation at 1 Hz (10 s) releases only 20%. Our results lay the foundation for molecular modeling of the dynamics of luminal SR Ca²⁺ and for future studies of the role of SR Ca²⁺ in healthy and diseased mammalian muscle.     

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.     

Transport of Ca2+ from Sarcoplasmic Reticulum to Mitochondria in Rat Ventricular Myocytes

Studies with electron microscopy have shown that sarcoplasmic reticulum (SR) andmitochondria locate close to each other in cardiac muscle cells. We investigated the hypothesis thatthis proximity results in a transient exposure of mitochondrial Ca²⁺ uniporter (CaUP) to highconcentrations 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 aphysiological 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 thecaffeine-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 anincrease in [Ca²⁺]_m, which was related to the rate of [Ca²⁺]_c increase. Finally, electronmicroscopy 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) isvery short, ranging between approximately 37 and 270 nm. These results are consistent withthe idea that there is a preferential coupling of Ca²⁺ transport from SR to mitochondria incardiac muscle cells, because of their structural proximity.     

Enhanced ryanodine receptor-mediated calcium leak determines reduced sarcoplasmic reticulum calcium content in chronic canine heart failure

In this study, we investigated the role of elevated sarcoplasmic reticulum (SR) Ca²⁺ leak through ryanodine receptors (RyR2s) in heart failure (HF)-related abnormalities of intracellular Ca²⁺ handling, using a canine model of chronic HF. The cytosolic Ca²⁺ transients were reduced in amplitude and slowed in duration in HF myocytes compared with control, changes paralleled by a dramatic reduction in the total SR Ca²⁺ content. Direct measurements of [Ca²⁺]_SR in both intact and permeabilized cardiac myocytes demonstrated that SR luminal [Ca²⁺] is markedly lowered in HF, suggesting that alterations in Ca²⁺ transport rather than fractional SR volume reduction accounts for the diminished Ca²⁺ release capacity of SR in HF. SR Ca²⁺ ATPase (SERCA2)-mediated SR Ca²⁺ uptake rate was not significantly altered, and Na⁺/Ca²⁺ exchange activity was accelerated in HF myocytes. At the same time, SR Ca²⁺ leak, measured directly as a loss of [Ca²⁺]_SR after inhibition of SERCA2 by thapsigargin, was markedly enhanced in HF myocytes. Moreover, the reduced [Ca²⁺]_SR in HF myocytes could be nearly completely restored by the RyR2 channel blocker ruthenium red. The effects of HF on cytosolic and SR luminal Ca²⁺ signals could be reasonably well mimicked by the RyR2 channel agonist caffeine. Taken together, these results suggest that RyR2-mediated SR Ca²⁺ leak is a major factor in the abnormal intracellular Ca²⁺ handling that critically contributes to the reduced SR Ca²⁺ content of failing cardiomyocytes.     

The Role of Dyadic Organization in Regulation of Sarcoplasmic Reticulum Ca Handling during Rest in Rabbit Ventricular Myocytes

The dyadic organization of ventricular myocytes ensures synchronized activation of sarcoplasmic reticulum (SR) Ca²⁺ release during systole. However, it remains obscure how the dyadic organization affects SR Ca²⁺ handling during diastole. By measuring intraluminal SR Ca²⁺ ([Ca²⁺]_SR) decline during rest in rabbit ventricular myocytes, we found that ∼76% of leaked SR Ca²⁺ is extruded from the cytosol and only ∼24% is pumped back into the SR. Thus, the majority of Ca²⁺ that leaks from the SR is removed from the cytosol before it can be sequestered back into the SR by the SR Ca²⁺-ATPase (SERCA). Detubulation decreased [Ca²⁺]_SR decline during rest, thus making the leaked SR Ca²⁺ more accessible for SERCA. These results suggest that Ca²⁺ extrusion systems are localized in T-tubules. Inhibition of Na⁺-Ca²⁺ exchanger (NCX) slowed [Ca²⁺]_SR decline during rest by threefold, however did not prevent it. Depolarization of mitochondrial membrane potential during NCX inhibition completely prevented the rest-dependent [Ca²⁺]_SR decline. Despite a significant SR Ca²⁺ leak, Ca²⁺ sparks were very rare events in control conditions. NCX inhibition or detubulation increased Ca²⁺ spark activity independent of SR Ca²⁺ load. Overall, these results indicate that during rest NCX effectively competes with SERCA for cytosolic Ca²⁺ that leaks from the SR. This can be explained if the majority of SR Ca²⁺ leak occurs through ryanodine receptors in the junctional SR that are located closely to NCX in the dyadic cleft. Such control of the dyadic [Ca²⁺] by NCX play a critical role in suppressing Ca²⁺ sparks during rest.     

The Role of Dyadic Organization in Regulation of Sarcoplasmic Reticulum Ca2+ Handling during Rest in Rabbit Ventricular Myocytes

The dyadic organization of ventricular myocytes ensures synchronized activation of sarcoplasmic reticulum (SR) Ca²⁺ release during systole. However, it remains obscure how the dyadic organization affects SR Ca²⁺ handling during diastole. By measuring intraluminal SR Ca²⁺ ([Ca²⁺]_SR) decline during rest in rabbit ventricular myocytes, we found that ∼76% of leaked SR Ca²⁺ is extruded from the cytosol and only ∼24% is pumped back into the SR. Thus, the majority of Ca²⁺ that leaks from the SR is removed from the cytosol before it can be sequestered back into the SR by the SR Ca²⁺-ATPase (SERCA). Detubulation decreased [Ca²⁺]_SR decline during rest, thus making the leaked SR Ca²⁺ more accessible for SERCA. These results suggest that Ca²⁺ extrusion systems are localized in T-tubules. Inhibition of Na⁺-Ca²⁺ exchanger (NCX) slowed [Ca²⁺]_SR decline during rest by threefold, however did not prevent it. Depolarization of mitochondrial membrane potential during NCX inhibition completely prevented the rest-dependent [Ca²⁺]_SR decline. Despite a significant SR Ca²⁺ leak, Ca²⁺ sparks were very rare events in control conditions. NCX inhibition or detubulation increased Ca²⁺ spark activity independent of SR Ca²⁺ load. Overall, these results indicate that during rest NCX effectively competes with SERCA for cytosolic Ca²⁺ that leaks from the SR. This can be explained if the majority of SR Ca²⁺ leak occurs through ryanodine receptors in the junctional SR that are located closely to NCX in the dyadic cleft. Such control of the dyadic [Ca²⁺] by NCX play a critical role in suppressing Ca²⁺ sparks during rest.     

Mechanistic insights into store-operated Ca2+ entry during excitation-contraction coupling in skeletal muscle

Skeletal muscle fibres support store-operated Ca²⁺-entry (SOCE) across the t-tubular membrane upon exhaustive depletion of Ca²⁺ from the sarcoplasmic reticulum (SR). Recently we demonstrated the presence of a novel mode of SOCE activated under conditions of maintained [Ca²⁺]_SR. This phasic SOCE manifested in a fast and transient manner in synchrony with excitation contraction (EC)-coupling mediated SR Ca²⁺-release (Communications Biology 1:31, doi: https://doi.org/10.1038/s42003-018-0033-7). Stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel 1 (ORAI1), positioned at the SR and t-system membranes, respectively, are the considered molecular correlate of SOCE. The evidence suggests that at the triads, where the terminal cisternae of the SR sandwich a t-tubule, STIM1 and ORAI1 proteins pre-position to allow for enhanced SOCE transduction.Here we show that phasic SOCE is not only shaped by global [Ca²⁺]_SR but provide evidence for a local activation within nanodomains at the terminal cisternae of the SR. This feature may allow SOCE to modulate [Ca²⁺]_SR during EC coupling. We define SOCE to occur on the same timescale as EC coupling and determine the temporal coherence of SOCE activation to SR Ca²⁺ release. We derive a delay of 0.3 ms reflecting diffusive Ca²⁺-equilibration at the luminal ryanodine receptor 1 (RyR1) channel mouth upon SR Ca²⁺-release. Numerical simulations of Ca²⁺-calsequestrin binding estimates a characteristic diffusion length and confines an upper limit for the spatial distance between STIM1 and RyR1. Experimental evidence for a 4- fold change in t-system Ca²⁺-permeability upon prolonged electrical stimulation in conjunction with numerical simulations of Ca²⁺-STIM1 binding suggests a Ca²⁺ dissociation constant of STIM1 below 0.35 mM. Our results show that phasic SOCE is intimately linked with RyR opening and closing, with only μs delays, because [Ca²⁺] in the terminal cisternae is just above the threshold for Ca²⁺ dissociation from STIM1 under physiological resting conditions.This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.     

Molecular characterization and functional properties of cardiomyocytes derived from human inducible pluripotent stem cells

In view of the therapeutic potential of cardiomyocytes derived from induced pluripotent stem (iPS) cells (iPS‐derived cardiomyocytes), in the present study we investigated in iPS‐derived cardiomyocytes, the functional properties related to [Ca²⁺]_i handling and contraction, the contribution of the sarcoplasmic reticulum (SR) Ca²⁺ release to contraction and the b‐adrenergic inotropic responsiveness. The two iPS clones investigated here were generated through infection of human foreskin fibroblasts (HFF) with retroviruses containing the four human genes: OCT4, Sox2, Klf4 and C‐Myc. Our major findings showed that iPS‐derived cardiomyocytes: (i) express cardiac specific RNA and proteins; (ii) exhibit negative force–frequency relations and mild (compared to adult) post‐rest potentiation; (iii) respond to ryanodine and caffeine, albeit less than adult cardiomyocytes, and express the SR‐Ca²⁺ handling proteins ryanodine receptor and calsequestrin. Hence, this study demonstrates that in our cardiomyocytes clones differentiated from HFF‐derived iPS, the functional properties related to excitation–contraction coupling, resemble in part those of adult cardiomyocytes.     

Effects of temperature and calcium availability on cardiac contractility in Synbranchus marmoratus, a neotropical teleost

An isometric muscle preparation was used to investigate the importance of the ventricular sarcoplasmic reticulum (SR) and extracellular Ca²⁺ (1.25 up to 11.25 mM) to force generation at 25 °C (acclimation temperature), 15 and 35 °C. The post-rest tension and force–frequency relationship were conducted with and without 10 μM ryanodine in the bathing medium. Increments in extracellular Ca²⁺ resulted in increases in twitch force development only at 35 °C. A significant post-rest potentiation was recorded for the control preparations at 25 °C (100% to 119.8 ± 4.1%). However, this post-rest potentiation was inhibited by ryanodine only at 25 °C (100% to 97.6 ± 1.5%). At 35 °C, force remained unchanged in the control preparations, but a significant post-rest decay was recorded in the presence of ryanodine (100% to 76.6 ± 4.6%) while at 15 °C, ryanodine was not able to preventing the post-rest potentiation observed in the control preparations. The increases in the imposed contraction frequency caused a decline of the force at 25 and 35 °C and ryanodine decreased significantly peak tension at both temperatures. The findings suggest a high or medium calcium turnover, possibly related to the presence of a functional SR, whose functionality is diminished when temperature is decreased.     

The concentration of free Ca(2+) in the sarcoplasmic reticulum of frog cut twitch skeletal muscle fibers estimated with tetramethylmurexide

One aim of this article was to determine the resting concentration of free Ca²⁺ in the sarcoplasmic reticulum (SR) of frog cut skeletal muscle fibers ([Ca²⁺]_SR,R) using the calcium absorbance indicator dye tetramethylmurexide (TMX). Another was to determine the ratio of [Ca²⁺]_SR,R to TMX's apparent dissociation constant for Ca²⁺ (K_app) in order to establish the capability of monitoring [Ca²⁺]_SR(t) during SR Ca²⁺ release – a signal needed to determine the Ca²⁺ permeability of the SR. To reveal the properties of TMX in the SR, the surface membrane was rapidly permeabilized with saponin to rapidly dissipate myoplasmic TMX. Results indicated that the concentration of Ca-free TMX in the SR was 2.8-fold greater than that in the myoplasm apparently due to binding of TMX to sites in the SR. Taking into account that such binding might influence K_app as well as a dependence of K_app on TMX concentration, the results indicate an average [Ca²⁺]_SR,R ranging from 0.43 to 1.70 mM. The ratio [Ca²⁺]_SR,R/K_app averaged 0.256, a relatively low value which should not depend on factors influencing K_app. As a result, the time course of [Ca²⁺]_SR(t) in response to electrical stimulation is well determined by, and approximately linearly related to, the active TMX absorbance signal.     

Importance of the sarcoplasmic reticulum and adrenergic stimulation on the cardiac contractility of the neotropical teleost <Emphasis Type="Italic">Synbranchus marmoratus</Emphasis> under different thermal conditions

Experiments were carried out to investigate the heart rate of Synbranchus marmoratus after changing the temperature of the water contained in the experimental chamber of the acclimated fish (from 25 to 35°C and from 25 to 15°C). Then, an isometric cardiac muscle preparation was used to test the relative importance of Ca²⁺ released from the sarcoplasmic reticulum and Ca²⁺ influx across the sarcolemma for the cardiac performance under different thermal conditions: 25°C (acclimation temperature), 15 and 35°C. Adrenaline and ryanodine were used to modulate the Ca²⁺ flux through the sarcolemma and the sarcoplasmic reticulum, respectively. Ryanodine reduced the peak tension by approximately 47% at 25°C, and by 53% at 35°C; however, it had no effect at 15°C. A high adrenaline concentration was able to ameliorate the negative effects of ryanodine. Despite increasing the peak tension, adrenaline increased the times necessary for contraction and relaxation. We conclude that the sarcoplasmic reticulum is active in contributing Ca²⁺ to the development of tension at physiological contraction frequencies. The adrenaline-stimulated Ca²⁺ influx is able to increase the peak tension, even after addition of ryanodine, at physiologically relevant temperatures and pacing frequencies.     

Some disturbances in the function of ryanodine receptors of spontaneously hypertensive rat cardiomyocytes detected using 4-chloro<Emphasis Type="Italic">m</Emphasis>-cresol

The rate of Ca²⁺ release from the sarcoplasmic reticulum in response to the activation of ryanodine receptors with 4-chlorom-cresol in the cardiomyocytes of three rat strains—spontaneously hypertensive (SHR), normotensive (WKY), and Wistar rats—during 5 weeks of their growth and development was studied aiming to detect the functional differences in the operation of these receptors at various stages of arterial hypertension. In response to 4-chlorom-cresol, a drastic increase in the rates of [Ca²⁺]_i accumulation in SHR myocytes after 17 days of development is recorded versus a decrease in the rates of Ca²⁺ efflux from the sarcoplasmic reticulum of Wistar and WKY rat cardiomyocytes. A correlation between this phenomenon and a genetic defect of ryanodine receptors in SHR rats seems rather unlikely, as the newborn WKY and SHR rats exposed to 4-chlorom-cresol at a concentration range of 0.5–2.0 mM did not display any differences in the rate of sarcoplasmic reticulum Ca²⁺ release. On the other hand, it is possible that the pathological changes in the function of ryanodine receptors manifest themselves later in the ontogenesis. The connection of this phenomenon with an increase in the role of ryanodine receptors in the excitation-contraction coupling in muscle cells and an increase in the calpain expression in SHR rats (absent in the WKY rats) by the age of 3 weeks is discussed. It is assumed that the cleavage of ryanodine receptor subunits by calpain can noticeably intensify the sarcoplasmic reticulum Ca²⁺ release after activation of these receptors without influencing the receptor binding characteristics.     

Depressed β-adrenergic inotropic responsiveness and intracellular calcium handling abnormalities in Duchenne Muscular Dystrophy patients’ induced pluripotent stem cell–derived cardiomyocytes

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is an X-linked disease affecting male and rarely adult heterozygous females, resulting in death by the late 20s to early 30s. Previous studies reported depressed left ventricular function in DMD patients which may result from deranged intracellular Ca²⁺-handling. To decipher the mechanism(s) underlying the depressed LV function, we tested the hypothesis that iPSC-CMs generated from DMD patients feature blunted positive inotropic response to β-adrenergic stimulation. To test the hypothesis, [Ca²⁺]_i transients and contractions were recorded from healthy and DMD-CMs. While in healthy CMs (HC) isoproterenol caused a prominent positive inotropic effect, DMD-CMs displayed a blunted inotropic response. Next, we tested the functionality of the sarcoplasmic reticulum (SR) by measuring caffeine-induced Ca²⁺ release. In contrast to HC, DMD-CMs exhibited reduced caffeine-induced Ca²⁺ signal amplitude and recovery time. In support of the depleted SR Ca²⁺ stores hypothesis, in DMD-CMs the negative inotropic effects of ryanodine and cyclopiazonic acid were smaller than in HC. RNA-seq analyses demonstrated that in DMD CMs the RNA-expression levels of specific subunits of the L-type calcium channel, the β1-adrenergic receptor (ADRβ1) and adenylate cyclase were down-regulated by 3.5-, 2.8- and 3-fold, respectively, which collectively contribute to the depressed β-adrenergic responsiveness.     

Mitochondrial Ca2+-Handling in Fast Skeletal Muscle Fibers from Wild Type and Calsequestrin-Null Mice

Mitochondrial calcium handling and its relation with calcium released from sarcoplasmic reticulum (SR) in muscle tissue are subject of lively debate. In this study we aimed to clarify how the SR determines mitochondrial calcium handling using dCASQ-null mice which lack both isoforms of the major Ca²⁺-binding protein inside SR, calsequestrin. Mitochondrial free Ca²⁺-concentration ([Ca²⁺]_mito) was determined by means of a genetically targeted ratiometric FRET-based probe. Electron microscopy revealed a highly significant increase in intermyofibrillar mitochondria (+55%) and augmented coupling (+12%) between Ca²⁺ release units of the SR and mitochondria in dCASQ-null vs. WT fibers. Significant differences in the baseline [Ca²⁺]_mito were observed between quiescent WT and dCASQ-null fibers, but not in the resting cytosolic Ca²⁺ concentration. The rise in [Ca²⁺]_mito during electrical stimulation occurred in 20−30 ms, while the decline during and after stimulation was governed by 4 rate constants of approximately 40, 1.6, 0.2 and 0.03 s⁻¹. Accordingly, frequency-dependent increase in [Ca²⁺]_mito occurred during sustained contractions. In dCASQ-null fibers the increases in [Ca²⁺]_mito were less pronounced than in WT fibers and even lower when extracellular calcium was removed. The amplitude and duration of [Ca²⁺]_mito transients were increased by inhibition of mitochondrial Na⁺/Ca²⁺ exchanger (mNCX). These results provide direct evidence for fast Ca²⁺ accumulation inside the mitochondria, involvement of the mNCX in mitochondrial Ca²⁺-handling and a dependence of mitochondrial Ca²⁺-handling on intracellular (SR) and external Ca²⁺ stores in fast skeletal muscle fibers. dCASQ-null mice represent a model for malignant hyperthermia. The differences in structure and in mitochondrial function observed relative to WT may represent compensatory mechanisms for the disease-related reduction of calcium storage capacity of the SR and/or SR Ca²⁺-leakage.     

Ca2+ Release through Ryanodine Receptors in the Sarcoplasmic Reticulum and Ca2+ Sequestration by the Mitochondria in Smooth Muscle Cells

The contribution of the Ca²⁺-induced Ca²⁺ release (CICR) mechanism in excitation-contraction (E-C) coupling and the tightness of the coupling between Ca²⁺ influx and Ca²⁺ release are still controversial in smooth muscle cells (SMC). In SMC isolated from the guinea-pig vas deferens or urinary bladder, a depolarizing stimulus initially induced spot-like increases in the intracellular Ca²⁺ concentration ([Ca²⁺] _i ), called “Ca²⁺ hot spots,” at several superficial areas in the cell. When a weak stimulus (a small or a short depolarizing step) was applied, only a few Ca²⁺ hot spots appeared transiently in the superficial area but did not spread into other regions, to trigger global [Ca²⁺] _i rise. Such depolarization-evoked local Ca2+ transients were distinctive from spontaneous Ca²⁺ sparks, since the former were susceptible to Ca²⁺ blockers, ryanodine, and inhibitors of the Ca²⁺ pump in the sarcoplasmic reticulum (SR), suggesting pivotal roles of Ca²⁺ influx through voltage-dependent Ca²⁺ channels (VDCC) and Ca²⁺ release from the SR through ryanodine receptors (RyR) for the activation of Ca²⁺ spots. Frequently discharging Ca²⁺ spark sites (FDS) under resting conditions were located exactly in the same areas as Ca²⁺ hot spots evoked by depolarization, indicating the existence of distinct local junction sites for tight coupling between VDCC in the plasmalemma and RyR in the SR. Co-localization of clusters of RyR and large-conductance Ca²⁺-activated K⁺ (BK) channels was also suggested. The fast and tight coupling for CICR in these junctional sites was triggered also by an action potential, whereas a slower spread of Ca²⁺ wave to the whole-cell areas suggests the loose coupling in propagating CICR to other cell areas. It can therefore be postulated that CICR may occur in two steps upon depolarization; the initial CICR in distinct junctional sites shows tight coupling between Ca²⁺ influx and release, and the following CICR may propagate slow Ca²⁺ waves to other areas. Ryanodine receptors form a multiprotein complex with molecules such as calsequestrin, junctin, triadin, junctophilins, and FK506-binding proteins, which directly or indirectly regulate the RyR activity and the tight coupling. Moreover, an evoked Ca²⁺ spot may enhance Ca²⁺ uptake by neighboring mitochondria and their ATP production to increase energy supply to the Ca²⁺ pump of the SR in the microdomain.     

Potentiation in mouse lumbrical muscle without myosin light chain phosphorylation: Is resting calcium responsible?

The increase in isometric twitch force observed in fast-twitch rodent muscles during or after activity, known universally as potentiation, is normally associated with myosin regulatory light chain (RLC) phosphorylation. Interestingly, fast muscles from mice devoid of detectable skeletal myosin light chain kinase (skMLCK) retain a reduced ability to potentiate twitch force, indicating the presence of a secondary origin for this characteristic feature of the fast muscle phenotype. The purpose of this study was to assess changes in intracellular cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) after a potentiating stimulus in mouse lumbrical muscle (37°C). Lumbricals were loaded with the Ca²⁺-sensitive fluorescent indicators fura-2 or furaptra to detect changes in resting and peak, respectively, intracellular Ca²⁺ levels caused by 2.5 s of 20-Hz stimulation. Although this protocol produced an immediate increase in twitch force of 17 ± 3% (all data are n = 10) (P < 0.01), this potentiation dissipated quickly and was absent 30 s afterward. Fura-2 fluorescence signals at rest were increased by 11.1 ± 1.3% (P < 0.01) during potentiation, indicating a significant increase in resting [Ca²⁺]_i. Interestingly, furaptra signals showed no change to either the amplitude or the duration of the intracellular Ca²⁺ transients (ICTs) that triggered potentiated twitches during this time (P < 0.50). Immunofluorescence work showed that 77% of lumbrical fibers expressed myosin heavy chain isoform IIx and/or IIb, but with low expression of skMLCK and high expression of myosin phosphatase targeting subunit 2. As a result, lumbrical muscles displayed no detectable RLC phosphorylation either at rest or after stimulation. We conclude that stimulation-induced elevations in resting [Ca²⁺]_i, in the absence of change in the ICT, are responsible for a small-magnitude, short-lived potentiation of isometric twitch force. If operative in other fast-twitch muscles, this mechanism may complement the potentiating influence of myosin RLC phosphorylation.     

Ca2+-mobility in the sarcoplasmic reticulum of ventricular myocytes is low

The sarcoplasmic reticulum (SR) in ventricular myocytes contains releasable Ca²⁺ for activating cellular contraction. Recent measurements of intra-SR (luminal) Ca²⁺ suggest a high diffusive Ca²⁺-mobility constant (D_CaSR). This could help spatially to unify SR Ca²⁺-content ([Ca²⁺]_SRT) and standardize Ca²⁺-release throughout the cell. But measurements of localized depletions of luminal Ca²⁺ (Ca²⁺-blinks), associated with local Ca²⁺-release (Ca²⁺-sparks), suggest D_CaSR may actually be low. Here we describe a novel method for measuring D_CaSR. Using a cytoplasmic Ca²⁺-fluorophore, we estimate regional [Ca²⁺]_SRT from localized, caffeine-induced SR Ca²⁺-release. Caffeine microperfusion of one end of a guinea pig or rat myocyte diffusively empties the whole SR at a rate indicating D_CaSR is 8-9 μm²/s, up to tenfold lower than previous estimates. Ignoring background SR Ca²⁺-leakage in our measurement protocol produces an artifactually high D_CaSR (>40 μm²/s), which may also explain the previous high values. Diffusion-reaction modeling suggests that a low D_CaSR would be sufficient to support local SR Ca²⁺-signaling within sarcomeres during excitation-contraction coupling. Low D_CaSR also implies that [Ca²⁺]_SRT may readily become spatially nonuniform, particularly under pathological conditions of spatially nonuniform Ca²⁺-release. Local control of luminal Ca²⁺, imposed by low D_CaSR, may complement the well-established local control of SR Ca²⁺-release by Ca²⁺-channel/ryanodine receptor couplons.     

Characterization of Functional TRPV1 Channels in the Sarcoplasmic Reticulum of Mouse Skeletal Muscle

TRPV1 represents a non-selective cation channel activated by capsaicin, acidosis and high temperature. In the central nervous system where TRPV1 is highly expressed, its physiological role in nociception is clearly identified. In skeletal muscle, TRPV1 appears implicated in energy metabolism and exercise endurance. However, how as a Ca²⁺ channel, it contributes to intracellular calcium concentration ([Ca²⁺]_i) maintenance and muscle contraction remains unknown. Here, as in rats, we report that TRPV1 is functionally expressed in mouse skeletal muscle. In contrast to earlier reports, our analysis show TRPV1 presence only at the sarcoplasmic reticulum (SR) membrane (preferably at the longitudinal part) in the proximity of SERCA1 pumps. Using intracellular Ca²⁺ imaging, we directly accessed to the channel functionality in intact FDB mouse fibers. Capsaicin and resiniferatoxin, both agonists as well as high temperature (45°C) elicited an increase in [Ca²⁺]_i. TRPV1-inhibition by capsazepine resulted in a strong inhibition of TRPV1-mediated functional responses and abolished channel activation. Blocking the SR release (with ryanodine or dantrolene) led to a reduced capsaicin-induced Ca²⁺ elevation suggesting that TRPV1 may participate to a secondary SR Ca²⁺ liberation of greater amplitude. In conclusion, our experiments point out that TRPV1 is a functional SR Ca²⁺ leak channel and may crosstalk with RyR1 in adult mouse muscle fibers.