Molecular architecture of Ca2+ signaling control in muscle and heart cells

Ca2+ signaling in skeletal and cardiac muscles is a bi-directional process that involves cross-talk between signaling molecules in the sarcolemmal membrane and Ca2+ release machinery in the intracellular organelles. Maintenance of a junctional membrane structure between the sarcolemmal membrane and the sarcoplasmic reticulum (SR) provides a framework for the conversion of action potential arrived at the sarcolemma into release of Ca2+ from the SR, leading to activation of a variety of physiological processes. Activity-dependent changes in Ca2+ storage inside the SR provides a retrograde signal for the activation of store-operated Ca2+ channel (SOC) on the sarcolemmal membrane, which plays important roles in the maintenance of Ca2+ homeostasis in physiology and pathophysiology. Research progress during the last 30 years had advanced our understanding of the cellular and molecular mechanisms for the control of Ca2+ signaling in muscle and cardiovascular physiology. Here we summarize the functions of three key molecules that are located in the junctional membrane complex of skeletal and cardiac muscle cells: junctophilin as a "glue" that physiologically links the SR membrane to the sarcolemmal membrane for formation of the junctional membrane framework, mitsugumin29 as a muscle-specific synaptophysin family protein that contributes to maintain the coordinated Ca2+ signaling in skeletal muscle, and TRIC as a novel cation-selective channel located on the SR membrane that provides counter-ion current during the rapid process of Ca2+ release from the SR.     

Calsequestrin. Structure,function, and evolution

Calsequestrin is the major Ca²⁺ binding protein in the sarcoplasmic reticulum (SR), serves as the main Ca²⁺ storage and buffering protein and is an important regulator of Ca²⁺ release channels in both skeletal and cardiac muscle. It is anchored at the junctional SR membrane through interactions with membrane proteins and undergoes reversible polymerization with increasing Ca²⁺ concentration. Calsequestrin provides high local Ca²⁺ at the junctional SR and communicates changes in luminal Ca²⁺ concentration to Ca²⁺ release channels, thus it is an essential component of excitation-contraction coupling. Recent studies reveal new insights on calsequestrin trafficking, Ca²⁺ binding, protein evolution, protein-protein interactions, stress responses and the molecular basis of related human muscle disease, including catecholaminergic polymorphic ventricular tachycardia (CPVT). Here we provide a comprehensive overview of calsequestrin, with recent advances in structure, diverse functions, phylogenetic analysis, and its role in muscle physiology, stress responses and human pathology.     

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca²⁺ signaling is a fundamental physiological process in living cells. Ca²⁺ sparks are the elementary units of Ca²⁺ signaling in the striated muscle fibers that appear as highly localized Ca²⁺ release events mediated by ryanodine receptor (RyR) Ca²⁺ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca²⁺ sparks could provide information on the intracellular Ca²⁺ handling properties of healthy and diseased striated muscles. Although Ca²⁺ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.Detailed here is an experimental protocol for measuring Ca²⁺ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca²⁺ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca²⁺ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca²⁺ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP₃R) and RyR contributes to Ca²⁺ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca²⁺ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).     

Cytoplasmic Ca2+ does not inhibit the cardiac muscle sarcoplasmic reticulum ryanodine receptor Ca2+ channel,although Ca2+-induced Ca2+ inactivation of Ca2+ release is observed in native vesicles

Single channel properties of cardiac and fast-twitch skeletal muscle sarcoplasmic reticulum (SR) release channels were compared in a planar bilayer by fusing SR membranes in a Cs⁺-conducting medium. We found that the pharmacology, Cs⁺ conductance and selectivity to monovalent and divalent cations of the two channels were similar. The cardiac SR channel exhibited multiple kinetic states. The open and closed lifetimes were not altered from a range of 10^–7 to 10^–3 M Ca²⁺, but the proportion of closed and open states shifted to shorter closings and openings, respectively.However, while the single channel activity of the skeletal SR channel was activated and inactivated by micromolar and millimolar Ca²⁺, respectively, the cardiac SR channel remained activated in the presence of high [Ca²⁺]. In correlation to these studies, [³H]ryanodine binding by the receptors of the two channel receptors was inhibited by high [Ca²⁺] in skeletal but not in cardiac membranes in the presence of adenine nucleotides. There is, however, a minor inhibition of [³H]ryanodine binding of cardiac SR at millimolar Ca²⁺ in the absence of adenine nucleotides.When Ca²⁺-induced Ca²⁺ release was examined from preloaded native SR vesicles, the release rates followed a normal biphasic curve, with Ca²⁺-induced inactivation at high [Ca²⁺] for both cardiac and skeletal SR. Our data suggest that the molecular basis of regulation of the SR Ca²⁺ release channel in cardiac and skeletal muscle is different, and that the cardiac SR channel isoform lacks a Ca²⁺-inactivated site.This work was supported by research grants from the National Institutes of Health HL13870 and AR38970, and the Texas Affiliate of the American Heart Association, 91A-188. M. Fill was the recipient of an NIH fellowship AR01834.     

Identification, Characterization and Partial Purification of a Thiol-protease Which Cleaves Specifically the Skeletal Muscle Ryanodine Receptor/Ca2+ Release Channel

A 94 kDa large subunit thiol-protease, as identified by anti-calpain antibodies, has been isolated from skeletal muscle junctional sarcoplasmic reticulum (SR). This protease cleaves specifically the skeletal muscle ryanodine receptor (RyR)/Ca²⁺ release channel at one site resulting in the 375 kDa and 150 kDa fragments. The 94 kDa thiol-protease degrades neither other SR proteins nor the ryanodine receptor of cardiac nor brain membranes. The partially purified 94 kDa protease, like the SR associated protease, had an optimal pH of about 7.0, was absolutely dependent on the presence of thiol reducing reagents, and was completely inhibited by HgCl₂, leupeptin and the specific calpain I inhibitor. However, while the SR membrane-associated protease requires Ca²⁺ at a submicromolar concentration, the isolated thiol-protease has lost the Ca²⁺ requirement. The 94 kDa thiol-protease had no effect on ryanodine binding but modified the channel activity of RyR reconstituted into planar lipid bilayer: in a time-dependent manner, the channel activity decreases and within several minutes the channel is converted into a subconducting state. The protease-modified channel activity is still Ca²⁺-dependent and ryanodine sensitive. This 94 kDa thiol-protease cross react with anti-calpain antibodies thus, may represent the novel large subunit of the skeletal muscle specific calpain p94. Received: 10 December 1996/Revised: 11 August 1997     

Dihydropyridine receptor-ryanodine receptor interactions in skeletal muscle excitation-contraction coupling

Much recent progress has been made in our understanding of the mechanism of sarcoplasmic reticulum Ca²⁺ release in skeletal muscle. Vertebrate skeletal muscle excitation-contraction (E-C) coupling is thought to occur by a mechanical coupling mechanism involving protein-protein interactions that lead to activation of the sarcoplasmic reticulum (SR) ryanodine receptor (RyR)/Ca²⁺ release channel by the voltage-sensing transverse (T–) tubule dihydropyridine receptor (DHPR)/Ca²⁺ channel. In a subsequent step, the released Ca²⁺ amplify SR Ca²⁺ release by activating release channels that are not linked to the DHPR. Experiments with mutant muscle cells have indicated that skeletal muscle specific DHPR and RyR isoforms are required for skeletal muscle E-C coupling. A direct functional and structural interaction between a DHPR-derived peptide and the RyR has been described. The interaction between the DHPR and RyR may be stabilized by other proteins such as triadin (a SR junctional protein) and modulated by phosphorylation of the DHPR.     

Sarcoplasmic Reticulum Ca2+ Release Uses a Cascading Network of Intra-SR and Channel Countercurrents

In muscle, Ca²⁺ release from the sarcoplasmic reticulum (SR) into the cytosol is mediated through the ryanodine receptors (RyRs) and sustained by countercurrents that keep the SR membrane potential near 0 mV. Likewise, Ca²⁺ reuptake by the sarco/endoplasmic reticulum Ca²⁺ ATPase pump requires countercurrent. Although evidence has suggested that TRIC K⁺ channels and/or RyR K⁺ influx provide these countercurrents, the exact sources have not yet been determined. We used an equivalent circuit compartment model of a cardiac SR, the surrounding cytosol, and the dyadic cleft to probe the sources of countercurrent during a complete cardiac cycle. By removing and relocating TRIC K⁺ channels, as well as limiting when they are active, we explored the various possible sources of SR countercurrent under many conditions. Our simulations indicate that no single channel type is essential for countercurrent. Rather, a cascading network of countercurrents is present with anion fluxes within the SR redistributing charges throughout the full SR volume. This allows ion channels in the entire SR membrane, far from the Ca²⁺ fluxes through the RyRs in the junctional SR and sarco/endoplasmic reticulum Ca²⁺ ATPase pump in the nonjunctional SR, to mediate countercurrents that support Ca²⁺ release and reuptake. This multifactorial network of countercurrents allows Ca²⁺ release to be remarkably robust.     

Moment closure for local control models of calcium-induced calcium release in cardiac myocytes

In prior work, we introduced a probability density approach to modeling local control of Ca²⁺-induced Ca²⁺ release in cardiac myocytes, where we derived coupled advection-reaction equations for the time-dependent bivariate probability density of subsarcolemmal subspace and junctional sarcoplasmic reticulum (SR) [Ca²⁺] conditioned on Ca²⁺ release unit (CaRU) state. When coupled to ordinary differential equations (ODEs) for the bulk myoplasmic and network SR [Ca²⁺], a realistic but minimal model of cardiac excitation-contraction coupling was produced that avoids the computationally demanding task of resolving spatial aspects of global Ca²⁺ signaling, while accurately representing heterogeneous local Ca²⁺ signals in a population of diadic subspaces and junctional SR depletion domains. Here we introduce a computationally efficient method for simulating such whole cell models when the dynamics of subspace [Ca²⁺] are much faster than those of junctional SR [Ca²⁺]. The method begins with the derivation of a system of ODEs describing the time-evolution of the moments of the univariate probability density functions for junctional SR [Ca²⁺] jointly distributed with CaRU state. This open system of ODEs is then closed using an algebraic relationship that expresses the third moment of junctional SR [Ca²⁺] in terms of the first and second moments. In simulated voltage-clamp protocols using 12-state CaRUs that respond to the dynamics of both subspace and junctional SR [Ca²⁺], this moment-closure approach to simulating local control of excitation-contraction coupling produces high-gain Ca²⁺ release that is graded with changes in membrane potential, a phenomenon not exhibited by common pool models. Benchmark simulations indicate that the moment-closure approach is nearly 10,000-times more computationally efficient than corresponding Monte Carlo simulations while leading to nearly identical results. We conclude by applying the moment-closure approach to study the restitution of Ca²⁺-induced Ca²⁺ release during simulated two-pulse voltage-clamp protocols.     

Ca Alternans in a Cardiac Myocyte Model that Uses Moment Equations to Represent Heterogeneous Junctional SR Ca

Multiscale whole-cell models that accurately represent local control of Ca²⁺-induced Ca²⁺ release in cardiac myocytes can reproduce high-gain Ca²⁺ release that is graded with changes in membrane potential. Using a recently introduced formalism that represents heterogeneous local Ca²⁺ using moment equations, we present a model of cardiac myocyte Ca²⁺ cycling that exhibits alternating sarcoplasmic reticulum (SR) Ca²⁺ release when periodically stimulated by depolarizing voltage pulses. The model predicts that the distribution of junctional SR [Ca²⁺] across a large population of Ca²⁺ release units is distinct on alternating cycles. Load-release and release-uptake functions computed from this model give insight into how Ca²⁺ fluxes and stimulation frequency combine to determine the presence or absence of Ca²⁺ alternans. Our results show that the conditions for the onset of Ca²⁺ alternans cannot be explained solely by the steepness of the load-release function, but that changes in the release-uptake process also play an important role. We analyze the effect of the junctional SR refilling time constant on Ca²⁺ alternans and conclude that physiologically realistic models of defective Ca²⁺ cycling must represent the dynamics of heterogeneous junctional SR [Ca²⁺] without assuming rapid equilibration of junctional and network SR [Ca²⁺].     

Transient Receptor Potential Canonical Type 1 (TRPC1) Operates as a Sarcoplasmic Reticulum Calcium Leak Channel in Skeletal Muscle

Extensive studies performed in nonexcitable cells and expression systems have shown that type 1 transient receptor potential canonical (TRPC1) channels operate mainly in plasma membranes and open through phospholipase C-dependent processes, membrane stretch, or depletion of Ca²⁺ stores. In skeletal muscle, it is proposed that TRPC1 channels are involved in plasmalemmal Ca²⁺ influx and stimulated by store depletion or membrane stretch, but direct evidence for TRPC1 sarcolemmal channel activity is not available. We investigated here the functional role of TRPC1 using an overexpressing strategy in adult mouse muscle fibers. Immunostaining for endogenous TRPC1 revealed a striated expression pattern that matched sarcoplasmic reticulum (SR) Ca²⁺ pump immunolabeling. In cells expressing TRPC1-yellow fluorescent protein (YFP), the same pattern of expression was observed, compatible with a longitudinal SR localization. Resting electric properties, action potentials, and resting divalent cation influx were not altered in TRPC1-YFP-positive cells. Poisoning with the SR Ca²⁺ pump blocker cyclopiazonic acid elicited a contracture of the fiber at the level of the overexpression site in presence and absence of external Ca²⁺ which was not observed in control cells. Ca²⁺ measurements indicated that resting Ca²⁺ and the rate of Ca²⁺ increase induced by cyclopiazonic acid were higher in the TRPC1-YFP-positive zone than in the TRPC1-YFP-negative zone and control cells. Ca²⁺ transients evoked by 200-ms voltage clamp pulses decayed slower in TRPC1-YFP-positive cells. In contrast to previous hypotheses, these data demonstrate that TRPC1 operates as a SR Ca²⁺ leak channel in skeletal muscle.     

Mitochondria in cardiomyocyte Ca signaling

Ca²⁺ signaling is of vital importance to cardiac cell function and plays an important role in heart failure. It is based on sarcolemmal, sarcoplasmic reticulum and mitochondrial Ca²⁺ cycling. While the first two are well characterized, the latter remains unclear, controversial and technically challenging.In mammalian cardiac myocytes, Ca²⁺ influx through L-type calcium channels in the sarcolemmal membrane triggers Ca²⁺ release from the nearby junctional sarcoplasmic reticulum to produce Ca²⁺ sparks. When this triggering is synchronized by the cardiac action potential, a global [Ca²⁺]_i transient arises from coordinated Ca²⁺ release events. The ends of intermyofibrillar mitochondria are located within 20 nm of the junctional sarcoplasmic reticulum and thereby experience a high local [Ca²⁺] during the Ca²⁺ release process. Both local and global Ca²⁺ signals may thus influence calcium signaling in mitochondria and, reciprocally, mitochondria may contribute to the local control of calcium signaling. In addition to the intermyofibrillar mitochondria, morphologically distinct mitochondria are also located in the perinuclear and subsarcolemmal regions of the cardiomyocyte and thus experience a different local [Ca²⁺].Here we review the literature in regard to several issues of broad interest: (1) the ultrastructural basis for mitochondrion – sarcoplasmic reticulum cross-signaling; (2) mechanisms of sarcoplasmic reticulum signaling; (3) mitochondrial calcium signaling; and (4) the possible interplay of calcium signaling between the sarcoplasmic reticulum and adjacent mitochondria.Finally, this review discusses experimental findings and mathematical models of cardiac calcium signaling between the sarcoplasmic reticulum and mitochondria, identifies weaknesses in these models, and suggests strategies and approaches for future investigations.     

Calcium-dependent facilitation and graded deactivation of store-operated calcium entry in fetal skeletal muscle

Activation of store-operated Ca²⁺ entry (SOCE) into the cytoplasm requires retrograde signaling from the intracellular Ca²⁺ release machinery, a process that involves an intimate interaction between protein components on the intracellular and cell surface membranes. The cellular machinery that governs the Ca²⁺ movement in muscle cells is developmentally regulated, reflecting maturation of the junctional membrane structure as well as coordinated expression of related Ca²⁺ signaling molecules. Here we demonstrate the existence of SOCE in freshly isolated skeletal muscle cells obtained from embryonic days 15 and 16 of the mouse embryo, a critical stage of muscle development. SOCE in the fetal muscle deactivates incrementally with the uptake of Ca²⁺ into the sarcoplasmic reticulum (SR). A novel Ca²⁺-dependent facilitation of SOCE is observed in cells transiently exposed to high cytosolic Ca²⁺. Our data suggest that cytosolic Ca²⁺ can facilitate SOCE whereas SR luminal Ca²⁺ can deactivate SOCE in the fetal skeletal muscle. This cooperative mechanism of SOCE regulation by Ca²⁺ ions not only enables tight control of SOCE by the SR membrane, but also provides an efficient mechanism of extracellular Ca²⁺ entry in response to physiological demand. Such Ca²⁺ signaling mechanism would likely contribute to contraction and development of the fetal skeletal muscle.     

Sarcoplasmic reticulum calsequestrins: Structural and functional properties

Calsequestrin is the major Ca²⁺-binding protein localized in the terminal cisternae of the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle cells. Calsequestrin has been purified and cloned from both skeletal and cardiac muscle in mammalian, amphibian, and avian species. Two different calsequestrin gene products namely cardiac and fast have been identified. Fast and cardiac calsequestrin isoforms have a highly acidic amino acid composition. The amino acid composition of the cardiac form is very similar to the skeletal form except for the carboxyl terminal region of the protein which possess variable length of acidic residues and two phosphorylation sites. Circular dichroism and NMR studies have shown that calsequestrin increases its -helical content and the intrinsic fluorescence upon binding of Ca²⁺. Calsequestrin binds Ca²⁺ with high-capacity and with moderate affinity and it functions as a Ca²⁺ storage protein in the lumen of the SR. Calsequestrin has been found to be associated with the Ca²⁺ release channel protein complex of the SR through protein-protein interactions. The human and rabbit fast calsequestrin genes have been cloned. The fast gene is skeletal muscle specific and transcribed at different rates in fast and slow skeletal muscle but not in cardiac muscle. We have recently cloned the rabbit cardiac calsequestrin gene. Heart expresses exclusively the cardiac calsquestrin gene. This gene is also expressed in slow skeletal muscle. No change in calsequestrin mRNA expression has been detected in animal models of cardiac hypertrophy and in failing human heart.     

Calcium signaling in insulin action on striated muscle

Striated muscles (skeletal and cardiac) are major physiological targets of insulin and this hormone triggers complex signaling pathways regulating cell growth and energy metabolism. Insulin increases glucose uptake into muscle cells by stimulating glucose transporter (GLUT4) translocation from intracellular compartments to the cell surface. The canonical insulin-triggered signaling cascade controlling this process is constituted by well-mapped tyrosine, lipid and serine/threonine phosphorylation reactions. In parallel to these signals, recent findings reveal insulin-dependent Ca²⁺ mobilization in skeletal muscle cells and cardiomyocytes. Specifically, insulin activates the sarco-endoplasmic reticulum (SER) channels that release Ca²⁺ into the cytosol i.e., the Ryanodine Receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP₃R). In skeletal muscle cells, a rapid, insulin-triggered Ca²⁺ release occurs through RyR, that is brought about upon S-glutathionylation of cysteine residues in the channel by reactive oxygen species (ROS) produced by the early activation of the NADPH oxidase (NOX2). In cardiomyocytes insulin induces a fast and transient increase in cytoplasmic [Ca²⁺]_i trough L-type Ca²⁺ channels activation. In both cell types, a relatively slower Ca²⁺ release also occurs through IP₃R activation, and is required for GLUT4 translocation and glucose uptake. The insulin-dependent Ca²⁺ released from IP₃R of skeletal muscle also promotes mitochondrial Ca²⁺ uptake. We review here these actions of insulin on intracellular Ca²⁺ channel activation and their impact on GLUT4 traffic in muscle cells, as well as other implications of insulin-dependent Ca²⁺ release from the SER.     

Differential Ca(2+) sensitivity of skeletal and cardiac muscle ryanodine receptors in the presence of calmodulin

Calmodulin (CaM) activates the skeletal muscle ryanodine receptorCa²⁺ release channel (RyR1) in the presence of nanomolarCa²⁺ concentrations. However, the role of CaM activation inthe mechanisms that control Ca²⁺ release from thesarcoplasmic reticulum (SR) in skeletal muscle and in the heart remainsunclear. In media that contained 100 nM Ca²⁺, the rate of⁴⁵Ca²⁺ release from porcine skeletal muscle SRvesicles was increased approximately threefold in the presence of CaM(1 µM). In contrast, cardiac SR vesicle⁴⁵Ca²⁺ release was unaffected by CaM,suggesting that CaM activated the skeletal RyR1 but not the cardiacRyR2 channel isoform. The activation of RyR1 by CaM was associated withan approximately sixfold increase in the Ca²⁺ sensitivityof [³H]ryanodine binding to skeletal muscle SR, whereasthe Ca²⁺ sensitivity of cardiac SR[³H]ryanodine binding was similar in the absence andpresence of CaM. Cross-linking experiments identified both RyR1 andRyR2 as predominant CaM binding proteins in skeletal and cardiac SR,respectively, and [³⁵S]CaM binding determinations furtherindicated comparable CaM binding to the two isoforms in the presence ofmicromolar Ca²⁺. In nanomolar Ca²⁺, however,the affinity and stoichiometry of RyR2 [³⁵S]CaM bindingwas reduced compared with that of RyR1. Together, our results indicatethat CaM activates RyR1 by increasing the Ca²⁺ sensitivityof the channel, and further suggest differences in CaM's functionalinteractions with the RyR1 and RyR2 isoforms that may potentiallycontribute to differences in the Ca²⁺ dependence of channelactivation in skeletal and cardiac muscle.

     

Inactivation of the cardiac ryanodine receptor calcium release channel by nitric oxide

We have recently reported [Mészáros L.G., Minarovic I., Zahradníková A. Inhibition of the skeletal muscle ryanodine receptor calcium release channel by nitric oxide. FEBS Lett 1996; 380: 49–52] that nitric oxide (NO) reduces the activity of the skeletal muscle ryanodine receptor Ca²⁺ release channel (RyRC), a principal component of the excitation-contraction coupling machinery in striated muscles. Since (i) as shown here, we have obtained evidence which indicates that the NO synthase (eNOS) of cardiac muscle origin co-purified with RyRC-containing sarcoplasmic reticulum (SR) fractions; and (ii) the effects of NO donors on the release channel, as well as on cardiac function, appear somewhat contradictory, we have made an attempt to investigate the response of the cardiac RyRC to NO that is generated in situ from L-arginine in the NOS reaction. We found that L-arginine-derived NO inactivates Ca²⁺ release from cardiac SR and reduces the steady-state activity (i.e. open probability) of single RyRCs fused into a planar lipid bilayer. This reduction was prevented by NOS inhibitors and the NO quencher hemoglobin and was reversed by 2-mercaptoethanol. We thus conclude that: (i) in isolated SR preparations, it is possible to assess the effects of NO that is generated from L-arginine in the NOS reaction; and (ii) cardiac RyRc responds to NO in a manner which is identical to that we have previously found with the skeletal channel. These findings suggest that the direct modulation of the RyRC by NO is a signaling mechanism which likely participates in earlier demonstrated NO-induced myocardial contractility changes.     

Ca2+ Overload and Sarcoplasmic Reticulum Instability in tric-a Null Skeletal Muscle

The sarcoplasmic reticulum (SR) of skeletal muscle contains K⁺, Cl⁻, and H⁺ channels may facilitate charge neutralization during Ca²⁺ release. Our recent studies have identified trimeric intracellular cation (TRIC) channels on SR as an essential counter-ion permeability pathway associated with rapid Ca²⁺ release from intracellular stores. Skeletal muscle contains TRIC-A and TRIC-B isoforms as predominant and minor components, respectively. Here we test the physiological function of TRIC-A in skeletal muscle. Biochemical assay revealed abundant expression of TRIC-A relative to the skeletal muscle ryanodine receptor with a molar ratio of TRIC-A/ryanodine receptor ∼5:1. Electron microscopy with the tric-a^−/− skeletal muscle showed Ca²⁺ overload inside the SR with frequent formation of Ca²⁺ deposits compared with the wild type muscle. This elevated SR Ca²⁺ pool in the tric-a^−/− muscle could be released by caffeine, whereas the elemental Ca²⁺ release events, e.g. osmotic stress-induced Ca²⁺ spark activities, were significantly reduced likely reflecting compromised counter-ion movement across the SR. Ex vivo physiological test identified the appearance of “alternan” behavior with isolated tric-a^−/− skeletal muscle, i.e. transient and drastic increase in contractile force appeared within the decreasing force profile during repetitive fatigue stimulation. Inhibition of SR/endoplasmic reticulum Ca^{2+ ATPase} function could lead to aggravation of the stress-induced alternans in the tric-a^−/− muscle. Our data suggests that absence of TRIC-A may lead to Ca²⁺ overload in SR, which in combination with the reduced counter-ion movement may lead to instability of Ca²⁺ movement across the SR membrane. The observed alternan behavior with the tric-a^−/− muscle may reflect a skeletal muscle version of store overload-induced Ca²⁺ release that has been reported in the cardiac muscle under stress conditions.     

The unraveling architecture of the junctional sarcoplasmic reticulum

The sarcoplasmic reticulum (SR) of skeletal muscle controls the contraction-relaxation cycle by raising and lowering the myoplasmic free-Ca²⁺ concentration. The coupling between excitation, i.e., depolarization of sarcolemma and transvers tubule (TT) and Ca²⁺ release from the terminal cisternae (TC) of SR takes place at the triad. The triad junction is formed by a specialized region of the TC, the junctional SR, and the TT. The molecular architecture and protein composition of the junctional SR are under active investigation. Since the junctional SR plays a central role in excitation-contraction coupling and Ca²⁺ release, some of its protein constituents are directly involved in these processes. The biochemical evidence supporting this contention is reviewed in this article.     

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.     

Unique isoform-specific properties of calsequestrin in the heart and skeletal muscle

Calcium signaling in myocytes is dependent on the cardiac ryanodine receptor (RyR2) calcium release channel and the calcium buffering protein in the sarcoplasmic reticulum, cardiac calsequestrin (CSQ2). The overall properties of CSQ2 and its regulation of RyR2 have not been explored in detail or directly compared with skeletal CSQ1 and its regulation of the skeletal RyR1, with physiological ionic strength and Ca²⁺ concentrations. We find that there are major differences between the two isoforms under these physiological conditions. Ca²⁺ binding to CSQ2 is 50% lower than to CSQ1. Only ～30% of CSQ2 is bound to cardiac junctional face membrane (JFM), compared with ～70% of CSQ1 and the ratio of CSQ2 to RyR2 is only 50% of the CSQ1/RyR1 ratio. Chemical crosslinking shows that CSQ2 is mostly monomer/dimer, while CSQ1 is mostly polymerized. In single channel lipid bilayer experiments, CSQ2 monomers and/or dimers increase the open probability of both RyR1 and RyR2 channels, while CSQ1 polymers decrease the activity of RyR1. We speculate that CSQ2 facilitates high rates of Ca²⁺ release through RyR2 during systole, while CSQ1 curtails RyR1 opening in response to a single action potential to maintain Ca²⁺ and allow repeated Ca²⁺ release and graded activation with increased stimulation frequency.