Assessment of Cell Viability in Electrically Excitable Muscle Cells Through Intact Twitch Stimulation.

Muscle cells (i.e. skeletal muscle fibers) are fully viable and functional when their excitation-contraction (EC) coupling machinery is intact. This involves intact membrane integrity with polarized membrane, functional ion channels for action potential generation and conduction, an intact electro-chemical interface at the level of the fiber's triad, followed by sarcoplasmic reticulum Ca2+ release, and subsequent activation of the chemico-mechanical interface at the level of the contractile apparatus. The ultimate end result is then a visible twitch contraction upon a brief electrical pulse stimulation. For many biomedical studies involving single muscle cells, intact and viable myofibers are of utmost importance. Thus, a simple global screening method that involves a brief electrical stimulus applied to single muscle fibers and assessment of visible contraction would be of high value. In this chapter, we describe step-by-step protocols to (i) obtain intact single muscle fibers from freshly dissected muscle tissue using an enzymatic digestion procedure and (ii) provide a workflow for the assessment of twitch response of single fibers that can be ultimately classified as viable. For this, we have prepared a unique stimulation pen for which we provide the fabrication guide for do-it-yourself rapid prototyping to eliminate the need for expensive specialized commercial equipment.

Pathological conformations of disease mutant Ryanodine Receptors revealed by cryo-EM.

Ryanodine Receptors (RyRs) are massive channels that release Ca2+ from the endoplasmic and sarcoplasmic reticulum. Hundreds of mutations are linked to malignant hyperthermia (MH), myopathies, and arrhythmias. Here, we explore the first MH mutation identified in humans by providing cryo-EM snapshots of the pig homolog, R615C, showing that it affects an interface between three solenoid regions. We also show the impact of apo-calmodulin (apoCaM) and how it can induce opening by bending of the bridging solenoid, mediated by its N-terminal lobe. For R615C RyR1, apoCaM binding abolishes a pathological 'intermediate' conformation, distributing the population to a mixture of open and closed channels, both different from the structure without apoCaM. Comparisons show that the mutation primarily affects the closed state, inducing partial movements linked to channel activation. This shows that disease mutations can cause distinct pathological conformations of the RyR and facilitate channel opening by disrupting interactions between different solenoid regions.

Distinct mechanisms mediate pacemaker dysfunction associated with catecholaminergic polymorphic ventricular tachycardia mutations: Insights from computational modeling.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced ventricular arrhythmia associated with rhythm disturbance and impaired sinoatrial node cell (SANC) automaticity (pauses). Mutations associated with dysfunction of Ca2+-related mechanisms have been shown to be present in CPVT. These dysfunctions include impaired Ca2+ release from the ryanodine receptor (i.e., RyR2R4496C mutation) or binding to calsequestrin 2 (CASQ2). In SANC, Ca2+ signaling directly and indirectly mediates pacemaker function. We address here the following research questions: (i) what coupled-clock mechanisms and pathways mediate pacemaker mutations associated with CPVT in basal and in response to ß-adrenergic stimulation? (ii) Can different mechanisms lead to the same CPVT-related pacemaker pauses? (iii) Can the mutation-induced deteriorations in SANC function be reversed by drug intervention or gene manipulation? We used a numerical model of mice SANC that includes membrane and intracellular mechanisms and their interconnected signaling pathways. In the basal state of RyR2R4496C SANC, the model predicted that the Na+-Ca2+ exchanger current (INCX) and T-type Ca2+ current (ICaT) mediate between changes in Ca2+ signaling and SANC dysfunction. Under ß-adrenergic stimulation, changes in cAMP-PKA signaling and the sodium currents (INa), in addition to INCX and ICaT, mediate between changes in Ca2+ signaling and SANC automaticity pauses. Under basal conditions in Casq2-/-, the same mechanisms drove changes in Ca2+ signaling and subsequent pacemaker dysfunction. However, SANC automaticity pauses in response to ß-AR stimulation were mediated by ICaT and INa. Taken together, distinct mechanisms can lead to CPVT-associated SANC automaticity pauses. In addition, we predict that specifically increasing SANC cAMP-PKA activity by either a pharmacological agent (IBMX, a phosphodiesterase (PDE) inhibitor), gene manipulation (overexpression of adenylyl cyclase 1/8) or direct manipulation of the SERCA phosphorylation target through changes in gene expression, compensate for the impairment in SANC automaticity. These findings suggest new insights for understanding CPVT and its therapeutic approach.

Simultaneous assessment of calcium handling and contractility dynamics in isolated ventricular myocytes of a rat model of post-acute isoproterenol-induced cardiomyopathy.

Stress-induced cardiomyopathy (SIC) results from a profound catecholaminergic surge during strong emotional or physical stress. SIC is characterized by acute left ventricular apex hypokinesia, in the absence of coronary arteries occlusion, and can lead to arrhythmias and acute heart failure. Although, most SIC patients recover, the process could be slow, and recurrence or death may occur. Despite that the SIC common denominator is a large catecholamine discharge, the pathophysiological mechanism is incompletely understood. It is thought that catecholamines have direct cytotoxicity on apical ventricular myocytes (VM), which have the highest ß-adrenergic receptors density, and whose overstimulation might cause acute Ca2+ overload and oxidative stress, causing death in some VM and stunning others. Rodents receiving acute isoproterenol (ISO) overdose (OV) mimic SIC development, however, they have not been used to simultaneously assess Ca2+ handling and contractility status in isolated VM, which might explain ventricular hypokinesia. Therefore, treating rats with a single ISO-OV (67 mg/kg body weight), we sought out to characterize, with confocal imaging, Ca2+ and shortening dynamics in Fluo-4-loaded VM, during the early (1-5 days) and late post-acute phases (15 days). We found that ISO-OV VM showed contractile dysfunction; blunted shortening with slower force development and relaxation. These correlated with Ca2+ mishandling; blunted Ca2+ transient, with slower time to peak and SR Ca2+ recovery. SR Ca2+ content was low, nevertheless, diastolic Ca2+ sparks were more frequent, and their duration increased. Contractility and Ca2+ dysfunction aggravated or remained altered over time, explaining slow recovery. We conclude that diminished VM contractility is the main determinant of ISO-OV hypokinesia and is mostly related to Ca2+ mishandling.

Targeting pathological leak of ryanodine receptors: preclinical progress and the potential impact on treatments for cardiac arrhythmias and heart failure.

Introduction: Type-2 ryanodine receptor (RyR2) located on the sarcoplasmic reticulum initiate systolic Ca2+ transients within cardiomyocytes. Proper functioning of RyR2 is therefore crucial to the timing and force generated by cardiomyocytes within a healthy heart. Improper intracellular Ca2+ handing secondary to RyR2 dysfunction is associated with a variety of cardiac pathologies including catecholaminergic polymorphic ventricular tachycardia (CPVT), atrial fibrillation (AF), and heart failure (HF). Thus, RyR2 and its associated accessory proteins provide promising drug targets to scientists developing therapeutics for a variety of cardiac pathologies.Areas covered: In this article, we review the role of RyR2 in a variety of cardiac pathologies. We performed a literature search utilizing PubMed and MEDLINE as well as reviewed registries of trials from clinicaltrials.gov from 2010 to 2019 for novel therapeutic approaches that address the cellular mechanisms underlying CPVT, AF, and HF by specifically targeting defective RyR2 channels.Expert opinion: The negative impact of cardiac dysfunction on human health and medical economics are major motivating factors for establishing new and effective therapeutic approaches. Focusing on directly impacting the molecular mechanisms underlying defective Ca2+ handling by RyR2 in HF and arrhythmia has great potential to be translated into novel and innovative therapies.

Junctional membrane Ca2+ dynamics in human muscle fibers are altered by malignant hyperthermia causative RyR mutation.

We used the nanometer-wide tubules of the transverse tubular (t)-system of human skeletal muscle fibers as sensitive sensors for the quantitative monitoring of the Ca2+-handling properties in the narrow junctional cytoplasmic space sandwiched between the tubular membrane and the sarcoplasmic reticulum cisternae in single muscle fibers. The t-system sealed with a Ca2+-sensitive dye trapped in it is sensitive to changes in ryanodine receptor (RyR) Ca2+ leak, the store operated calcium entry flux, plasma membrane Ca pump, and sodium-calcium exchanger activities, thus making the sealed t-system a nanodomain Ca2+ sensor of Ca2+ dynamics in the junctional space. The sensor was used to assess the basal Ca2+-handling properties of human muscle fibers obtained by needle biopsy from control subjects and from people with a malignant hyperthermia (MH) causative RyR variant. Using this approach we show that the muscle fibers from MH-susceptible individuals display leakier RyRs and a greater capacity to extrude Ca2+ across the t-system membrane compared with fibers from controls. This study provides a quantitative way to assess the effect of RyR variants on junctional membrane Ca2+ handling under defined ionic conditions.

A model for cooperative gating of L-type Ca2+ channels and its effects on cardiac alternans dynamics.

In ventricular myocytes, membrane depolarization during the action potential (AP) causes synchronous activation of multiple L-type CaV1.2 channels (LTCCs), which trigger the release of calcium (Ca2+) from the sarcoplasmic reticulum (SR). This results in an increase in intracellular Ca2+ (Cai) that initiates contraction. During pulsus alternans, cardiac contraction is unstable, going from weak to strong in successive beats despite a constant heart rate. These cardiac alternans can be caused by the instability of membrane potential (Vm) due to steep AP duration (APD) restitution (Vm-driven alternans), instability of Cai cycling (Ca2+-driven alternans), or both, and may be modulated by functional coupling between clustered CaV1.2 (e.g. cooperative gating). Here, mathematical analysis and computational models were used to determine how changes in the strength of cooperative gating between LTCCs may impact membrane voltage and intracellular Ca2+ dynamics in the heart. We found that increasing the degree of coupling between LTCCs increases the amplitude of Ca2+ currents (ICaL) and prolongs AP duration (APD). Increased AP duration is known to promote cardiac alternans, a potentially arrhythmogenic substrate. In addition, our analysis shows that increasing the strength of cooperative activation of LTCCs makes the coupling of Ca2+ on the membrane voltage (Cai→Vm coupling) more positive and destabilizes the Vm-Cai dynamics for Vm-driven alternans and Cai-driven alternans, but not for quasiperiodic oscillation. These results suggest that cooperative gating of LTCCs may have a major impact on cardiac excitation-contraction coupling, not only by prolonging APD, but also by altering Cai→Vm coupling and potentially promoting cardiac arrhythmias.

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca2+ reservoir for contraction, which reuptakes intracellular Ca2+ during relaxation. The SR Ca2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa2+ reserve and diastolic Ca2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) to diastolic calcium removal was evaluated.

Quantitative assessment of passive electrical properties of the cardiac T-tubular system by FRAP microscopy.

Well-coordinated activation of all cardiomyocytes must occur on every heartbeat. At the cell level, a complex network of sarcolemmal invaginations, called the transverse-axial tubular system (TATS), propagates membrane potential changes to the cell core, ensuring synchronous and uniform excitation-contraction coupling. Although myocardial conduction of excitation has been widely described, the electrical properties of the TATS remain mostly unknown. Here, we exploit the formal analogy between diffusion and electrical conductivity to link the latter with the diffusional properties of TATS. Fluorescence recovery after photobleaching (FRAP) microscopy is used to probe the diffusion properties of TATS in isolated rat cardiomyocytes: A fluorescent dextran inside TATS lumen is photobleached, and signal recovery by diffusion of unbleached dextran from the extracellular space is monitored. We designed a mathematical model to correlate the time constant of fluorescence recovery with the apparent diffusion coefficient of the fluorescent molecules. Then, apparent diffusion is linked to electrical conductivity and used to evaluate the efficiency of the passive spread of membrane depolarization along TATS. The method is first validated in cells where most TATS elements are acutely detached by osmotic shock and then applied to probe TATS electrical conductivity in failing heart cells. We find that acute and pathological tubular remodeling significantly affect TATS electrical conductivity. This may explain the occurrence of defects in action potential propagation at the level of single T-tubules, recently observed in diseased cardiomyocytes.

Atrial-selective targeting of arrhythmogenic phase-3 early afterdepolarizations in human myocytes.

BACKGROUND: We have previously shown that non-equilibrium Na(+) current (INa) reactivation drives isoproterenol-induced phase-3 early afterdepolarizations (EADs) in mouse ventricular myocytes. In these cells, EAD initiation occurs secondary to potentiated sarcoplasmic reticulum Ca(2+) release and enhanced Na(+)/Ca(2+) exchange (NCX). This can be abolished by tetrodotoxin-blockade of INa, but not ranolazine, which selectively inhibits ventricular late INa. AIM: Since repolarization of human atrial myocytes is similar to mouse ventricular myocytes in that it is relatively rapid and potently modulated by Ca(2+), we investigated whether similar mechanisms can evoke EADs in human atrium. Indeed, phase-3 EADs have been shown to re-initiate atrial fibrillation (AF) during autonomic stimulation, which is a well-recognized initiator of AF. METHODS: We integrated a Markov model of INa gating in our human atrial myocyte model. To simulate experimental results, we rapidly paced this cell model at 10Hz in the presence of 0.1µM acetylcholine and 1µM isoproterenol, and assessed EAD occurrence upon return to sinus rhythm (1Hz). RESULTS: Cellular Ca(2+) loading during fast pacing results in a transient period of hypercontractility after return to sinus rhythm. Here, fast repolarization and enhanced NCX facilitate INa reactivation via the canonical gating mode (i.e., not late INa burst mode), which drives EAD initiation. Simulating ranolazine administration reduces atrial peak INa and leads to faster repolarization, during which INa fails to reactivate and EADs are prevented. CONCLUSIONS: Non-equilibrium INa reactivation can critically contribute to arrhythmias, specifically in human atrial myocytes. Ranolazine might be beneficial in this context by blocking peak (not late) atrial INa.

Calcium homeostasis in a local/global whole cell model of permeabilized ventricular myocytes with a Langevin description of stochastic calcium release.

Population density approaches to modeling local control of Ca(2+)-induced Ca(2+) release in cardiac myocytes can be used to construct minimal whole cell models that accurately represent heterogeneous local Ca(2+) signals. Unfortunately, the computational complexity of such "local/global" whole cell models scales with the number of Ca(2+) release unit (CaRU) states, which is a rapidly increasing function of the number of ryanodine receptors (RyRs) per CaRU. Here we present an alternative approach based on a Langevin description of the collective gating of RyRs coupled by local Ca(2+) concentration ([Ca(2+)]). The computational efficiency of this approach no longer depends on the number of RyRs per CaRU. When the RyR model is minimal, Langevin equations may be replaced by a single Fokker-Planck equation, yielding an extremely compact and efficient local/global whole cell model that reproduces and helps interpret recent experiments that investigate Ca(2+) homeostasis in permeabilized ventricular myocytes. Our calculations show that elevated myoplasmic [Ca(2+)] promotes elevated network sarcoplasmic reticulum (SR) [Ca(2+)] via SR Ca(2+)-ATPase-mediated Ca(2+) uptake. However, elevated myoplasmic [Ca(2+)] may also activate RyRs and promote stochastic SR Ca(2+) release, which can in turn decrease SR [Ca(2+)]. Increasing myoplasmic [Ca(2+)] results in an exponential increase in spark-mediated release and a linear increase in nonspark-mediated release, consistent with recent experiments. The model exhibits two steady-state release fluxes for the same network SR [Ca(2+)] depending on whether myoplasmic [Ca(2+)] is low or high. In the later case, spontaneous release decreases SR [Ca(2+)] in a manner that maintains robust Ca(2+) sparks.

Assessment of calcium sparks in intact skeletal muscle fibers.

Maintaining homeostatic Ca(2+) signaling is a fundamental physiological process in living cells. Ca(2+) sparks are the elementary units of Ca(2+) signaling in the striated muscle fibers that appear as highly localized Ca(2+) release events mediated by ryanodine receptor (RyR) Ca(2+) release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca(2+) sparks could provide information on the intracellular Ca(2+) handling properties of healthy and diseased striated muscles. Although Ca(2+) 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 Ca(2+) sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca(2+) 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 Ca(2+) sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca(2+) 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 Ca(2+) spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca(2+) 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).

Calcium alternans in a couplon network model of ventricular myocytes: role of sarcoplasmic reticulum load.

Intracellular calcium (Ca) alternans in cardiac myocytes have been shown in many experimental studies, and the mechanisms remain incompletely understood. We recently developed a "3R theory" that links Ca sparks to whole cell Ca alternans through three critical properties: randomness of Ca sparks; recruitment of a Ca spark by neighboring Ca sparks; and refractoriness of Ca release units. In this study, we used computer simulation of a physiologically detailed mathematical model of a ventricular myocyte couplon network to study how sarcoplasmic reticulum (SR) Ca load and other physiological parameters, such as ryanodine receptor sensitivity, SR uptake rate, Na-Ca exchange strength, and Ca buffer levels affect Ca alternans in the context of 3R theory. We developed a method to calculate the parameters used in the 3R theory (i.e., the primary spark rate and the recruitment rate) from the physiologically detailed Ca cycling model and paced the model periodically to elicit Ca alternans. We show that alternans only occurs for an intermediate range of the SR Ca load, and the underlying mechanism can be explained via its effects on the 3Rs. Furthermore, we show that altering the physiological parameters not only directly changes the 3Rs but also alters the SR Ca load, having an indirect effect on the 3Rs as well. Therefore, our present study links the SR Ca load and other physiological parameters to whole cell Ca alternans through the framework of the 3R theory, providing a general mechanistic understanding of Ca alternans in ventricular myocytes.

Malignant hyperthermia susceptibility arising from altered resting coupling between the skeletal muscle L-type Ca2+ channel and the type 1 ryanodine receptor.

Malignant hyperthermia (MH) susceptibility is a dominantly inherited disorder in which volatile anesthetics trigger aberrant Ca(2+) release in skeletal muscle and a potentially fatal rise in perioperative body temperature. Mutations causing MH susceptibility have been identified in two proteins critical for excitation-contraction (EC) coupling, the type 1 ryanodine receptor (RyR1) and Ca(V)1.1, the principal subunit of the L-type Ca(2+) channel. All of the mutations that have been characterized previously augment EC coupling and/or increase the rate of L-type Ca(2+) entry. The Ca(V)1.1 mutation R174W associated with MH susceptibility occurs at the innermost basic residue of the IS4 voltage-sensing helix, a residue conserved among all Ca(V) channels [Carpenter D, et al. (2009) BMC Med Genet 10:104-115.]. To define the functional consequences of this mutation, we expressed it in dysgenic (Ca(V)1.1 null) myotubes. Unlike previously described MH-linked mutations in Ca(V)1.1, R174W ablated the L-type current and had no effect on EC coupling. Nonetheless, R174W increased sensitivity of Ca(2+) release to caffeine (used for MH diagnostic in vitro testing) and to volatile anesthetics. Moreover, in Ca(V)1.1 R174W-expressing myotubes, resting myoplasmic Ca(2+) levels were elevated, and sarcoplasmic reticulum (SR) stores were partially depleted, compared with myotubes expressing wild-type Ca(V)1.1. Our results indicate that Ca(V)1.1 functions not only to activate RyR1 during EC coupling, but also to suppress resting RyR1-mediated Ca(2+) leak from the SR, and that perturbation of Ca(V)1.1 negative regulation of RyR1 leak identifies a unique mechanism that can sensitize muscle cells to MH triggers.

Identification of biomarkers of meat tenderisation and its use for early classification of Asturian beef into fast and late tenderising meat.

BACKGROUND: The objective of this work was to study the post-mortem evolution of potential biomarkers (µ-calpain activity and proteolytic profile) of meat tenderisation in bovine longissimus dorsi (LD) muscle from several biotypes coming from two beef breeds ('Asturiana de los Valles' and 'Asturiana de la Montaña') and showing different levels of muscular hypertrophy (mh/mh, mh/+, + /+). RESULTS: LD samples were taken at 2, 12, 24 and 48 h and 3, 7, 14 and 21 days post-mortem. The presence of muscular hypertrophy produced a faster rate of pH decline, faster exhaustion of µ-calpain activity and earlier occurrence of proteolytic changes. Changes in the electrophoretic pattern of some peptides from sarcoplasmic (glyceraldehyde-3-phosphate dehydrogenase) and myofibrillar (troponin T and troponin I) muscle extracts within the first 24 h significantly correlated with meat toughness and allowed accurate discrimination of meat products into two groups: (1) fast tenderising meat, coming from mh-biotypes, and (2) late tenderising meat, from normal (+/+) biotypes. CONCLUSION: Early monitoring (within 24 h after slaughter) of selected biomarkers in LD muscle allowed accurate prediction of ultimate meat toughness and could be used in the meat industry as a tool for early classification of beef into fast and late tenderising meat.

Perturbation analysis of spontaneous action potential initiation by stochastic ion channels.

A stochastic interpretation of spontaneous action potential initiation is developed for the Morris-Lecar equations. Initiation of a spontaneous action potential can be interpreted as the escape from one of the wells of a double well potential, and we develop an asymptotic approximation of the mean exit time using a recently developed quasistationary perturbation method. Using the fact that the activating ionic channel's random openings and closings are fast relative to other processes, we derive an accurate estimate for the mean time to fire an action potential (MFT), which is valid for a below-threshold applied current. Previous studies have found that for above-threshold applied current, where there is only a single stable fixed point, a diffusion approximation can be used. We also explore why different diffusion approximation techniques fail to estimate the MFT.

Passive Ca(2+) overload in H9c2 cardiac myoblasts: assessment of cellular damage and cytosolic Ca(2+) transients.

Increase of resting Ca(2+) levels and amplitude of vasopressin-induced Ca(2+) transients were observed when cells in serum-free medium were exposed to 5mM Ca(2+) for 2h. Small effect on cell viability was also observed. A rapid cytotoxic effect was developed in the presence of 10mM Ca(2+) and absence of serum. However, cells exposed to 10mM Ca(2+) in the presence of serum were protected from damage for at least 2days. Resting Ca(2+) levels and cytosolic Ca(2+) transients in serum-containing medium with 10mM Ca(2+) displayed lower increases and a tendency to recover control values. When serum was absent, cells preincubated with 10mM Ca(2+) were more sensitive to thapsigargin-induced damage than cells preincubated with lower Ca(2+). The sensitivity was similar when serum was present. Tolerance to high Ca(2+) in the presence of serum was linked to potentiation of the mitochondrial Ca(2+) entry to decrease the sarcoplasmic reticulum Ca(2+) overload.

Increased store-operated Ca2+ entry in skeletal muscle with reduced calsequestrin-1 expression.

Store-operated Ca(2+) entry (SOCE) contributes to Ca(2+) handling in normal skeletal muscle function, as well as the progression of muscular dystrophy and sarcopenia, yet the mechanisms underlying the change in SOCE in these states remain unclear. Previously we showed that calsequestrin-1 (CSQ1) participated in retrograde regulation of SOCE in cultured skeletal myotubes. In this study, we used small-hairpin RNA to determine whether knockdown of CSQ1 in adult mouse skeletal muscle can influence SOCE activity and muscle function. Small-hairpin RNA against CSQ1 was introduced into flexor digitorum brevis muscles using electroporation. Transfected fibers were isolated for SOCE measurements using the Mn(2+) fluorescence-quenching method. At room temperature, the SOCE induced by submaximal depletion of the SR Ca(2+) store was significantly enhanced in CSQ1-knockdown muscle fibers. When temperature of the bathing solution was increased to 39 degrees C, CSQ1-knockdown muscle fibers displayed a significant increase in Ca(2+) permeability across the surface membrane likely via the SOCE pathway, and a corresponding elevation in cytosolic Ca(2+) as compared to control fibers. Preincubation with azumolene, an analog of dantrolene used for the treatment of malignant hyperthermia (MH), suppressed the elevated SOCE in CSQ1-knockdown fibers. Because the CSQ1-knockout mice develop similar MH phenotypes, this inhibitory effect of azumolene on SOCE suggests that elevated extracellular Ca(2+) entry in skeletal muscle may be a key factor for the pathophysiological changes in intracellular Ca(2+) signaling in MH.

Spontaneous Ca2+ sparks and Ca2+ homeostasis in a minimal model of permeabilized ventricular myocytes.

Many issues remain unresolved concerning how local, subcellular Ca(2+) signals interact with bulk cellular concentrations to maintain homeostasis in health and disease. To aid in the interpretation of data obtained in quiescent ventricular myocytes, we present here a minimal whole cell model that accounts for both localized (subcellular) and global (cellular) aspects of Ca(2+) signaling. Using a minimal formulation of the distribution of local [Ca(2+)] associated with a large number of Ca(2+)-release sites, the model simulates both random spontaneous Ca(2+) sparks and the changes in myoplasmic and sarcoplasmic reticulum (SR) [Ca(2+)] that result from the balance between stochastic release and reuptake into the SR. Ca(2+)-release sites are composed of clusters of two-state ryanodine receptors (RyRs) that exhibit activation by local cytosolic [Ca(2+)] but no inactivation or regulation by luminal Ca(2+). Decreasing RyR open probability in the model causes a decrease in aggregate release flux and an increase in SR [Ca(2+)], regardless of whether RyR inhibition is mediated by a decrease in RyR open dwell time or an increase in RyR closed dwell time. The same balance of stochastic release and reuptake can be achieved, however, by either high-frequency/short-duration or low-frequency/long-duration Ca(2+) sparks. The results are well correlated with recent experimental observations using pharmacological RyR inhibitors and clarify those aspects of the release-reuptake balance that are inherent to the coupling between local and global Ca(2+) signals and those aspects that depend on molecular-level details. The model of Ca(2+) sparks and homeostasis presented here can be a useful tool for understanding changes in cardiac Ca(2+ )release resulting from drugs, mutations, or acquired diseases.

Sevoflurane modulation of Ca2+ regulation in skeletal muscle sarcoplasmic reticulum vesicles from young and mature rabbits.

INTRODUCTION: Developmental differences in splice variants of the two key sarcoplasmic reticulum (SR) calcium regulatory proteins, ryanodine (RyR1), and sarcoendoplasmic reticulum calcium pump (SERCA1) have been linked to various neuromuscular disorders, but not malignant hyperthermia (MH). However, it is unclear whether an age-related difference in volatile anesthetic-mediated SR calcium function exists that could add to our current understanding of the clinical presentation of MH syndrome and provide insight into molecular mechanisms for general anesthesia that may have other physiologic and/or pathophysiologic significance. Therefore, the effects of sevoflurane on intracellular calcium regulation in isolated SR membrane vesicles from the skeletal muscle of healthy young rabbits were compared to their adult counterpart using an established in vitro model with the assumption that exposure to sevoflurane would elicit a weaker response in the young SR. METHODS: Through dual wavelength spectroscopy of Ca(2+): Arsenazo III difference absorbance, the effects of sevoflurane on SR Ca(2+) uptake rate and release in heavy and light fraction SR membrane vesicles isolated from the white muscle of anesthetized, postweaned (age = 6 weeks, n = 5) and adult (age = 6 months, n = 5) male New Zealand rabbits were examined. RESULTS: The adult group showed a 50% increase in Ca(2+) uptake rate from control at both subclinical and clinically relevant anesthetic concentrations, whereas in the SR from the younger animals, Ca(2+) uptake rate was not altered by any concentration of sevoflurane. The sensitivity of both the low and high affinity Ca(2+)-binding sites on RyR1 was increased by sevoflurane to the same extent in the SR vesicles from the young and mature adult rabbits. Interestingly, a greater potency of sevoflurane for the high affinity-binding site was identified, and this was independent of age. CONCLUSIONS: These findings suggest that the sensitivity of the SR to sevoflurane-mediated Ca(2+) uptake may be increased with maturity, while an analogous developmental effect on RyR1 is less probable. Nonetheless, this study shows for the first time that a potent inhalational agent such as sevoflurane can influence the high affinity SR calcium-binding site by lowering the extraluminal concentration of calcium necessary to trigger calcium release. While this may not be of consequence when inhaled anesthetics are administered to normal children or adults, it may have life-threatening consequences in carriers of RyR1 mutations.