Calmodulin kinase modulates Ca2+ release in mouse skeletal muscle

Activation of the contractile machinery in skeletal muscle is initiated by the action-potential-induced release of Ca²⁺ from the sarcoplasmic reticulum (SR). Several proteins involved in SR Ca²⁺ release are affected by calmodulin kinase II (CaMKII)-induced phosphorylation in vitro , but the effect in the intact cell remains uncertain and is the focus of the present study. CaMKII inhibitory peptide or inactive control peptide was injected into single isolated fast-twitch fibres of mouse flexor digitorum brevis muscles, and the effect on free myoplasmic [Ca²⁺] ([Ca²⁺]_i) and force during different patterns of stimulation was measured. Injection of the inactive control peptide had no effect on any of the parameters measured. Conversely, injection of CaMKII inhibitory peptide decreased tetanic [Ca²⁺]_i by ≈25 %, but had no significant effect on the rate of SR Ca²⁺ uptake or the force-[Ca²⁺]_i relationship. Repeated tetanic stimulation resulted in increased tetanic [Ca²⁺]_i, and this increase was smaller after CaMKII inhibition. In conclusion, CaMKII-induced phosphorylation facilitates SR Ca²⁺ release in the basal state and during repeated contractions, providing a positive feedback between [Ca²⁺]_i and SR Ca²⁺ release.     

Minor sarcoplasmic reticulum membrane components that modulate excitation–contraction coupling in striated muscles

In striated muscle, activation of contraction is initiated by membrane depolarisation caused by an action potential, which triggers the release of Ca²⁺ stored in the sarcoplasmic reticulum by a process called excitation–contraction coupling. Excitation–contraction coupling occurs via a highly sophisticated supramolecular signalling complex at the junction between the sarcoplasmic reticulum and the transverse tubules. It is generally accepted that the core components of the excitation–contraction coupling machinery are the dihydropyridine receptors, ryanodine receptors and calsequestrin, which serve as voltage sensor, Ca²⁺ release channel, and Ca²⁺ storage protein, respectively. Nevertheless, a number of additional proteins have been shown to be essential both for the structural formation of the machinery involved in excitation–contraction coupling and for its fine tuning. In this review we discuss the functional role of minor sarcoplasmic reticulum protein components. The definition of their roles in excitation–contraction coupling is important in order to understand how mutations in genes involved in Ca²⁺ signalling cause neuromuscular disorders.     

Sarcoplasmic reticulum Ca2+ release and depletion fail to affect sarcolemmal ion channel activity in mouse skeletal muscle

In skeletal muscle, sarcoplasmic reticulum (SR) Ca²⁺ depletion is suspected to trigger a calcium entry across the plasma membrane and recent studies also suggest that the opening of channels spontaneously active at rest and possibly involved in Duchenne dystrophy may be regulated by SR Ca²⁺ depletion. Here we simultaneously used the cell-attached and whole-cell voltage-clamp techniques as well as intracellular Ca²⁺ measurements on single isolated mouse skeletal muscle fibres to unravel any possible change in membrane conductance that would depend upon SR Ca²⁺ release and/or SR Ca²⁺ depletion. Delayed rectifier K⁺ single channel activity was routinely detected during whole-cell depolarizing pulses. In addition the activity of channels carrying unitary inward currents of ∼1.5 pA at −80 mV was detected in 17 out of 127 and in 21 out of 59 patches in control and mdx dystrophic fibres, respectively. In both populations of fibres, large whole-cell depolarizing pulses did not reproducibly increase this channel activity. This was also true when, repeated application of the whole-cell pulses led to exhaustion of the Ca²⁺ transient. SR Ca²⁺ depletion produced by the SR Ca²⁺ pump inhibitor cyclopiazonic acid (CPA) also failed to induce any increase in the resting whole-cell conductance and in the inward single channel activity. Overall results indicate that voltage-activated SR Ca²⁺ release and/or SR Ca²⁺ depletion are not sufficient to activate the opening of channels carrying inward currents at negative voltages and challenge the physiological relevance of a store-operated membrane conductance in adult skeletal muscle.     

Limited oxygen diffusion accelerates fatigue development in mouse skeletal muscle

Isolated whole skeletal muscles fatigue more rapidly than isolated single muscle fibres. We have now employed this difference to study mechanisms of skeletal muscle fatigue. Isolated whole soleus and extensor digitorum longus (EDL) muscles were fatigued by repeated tetanic stimulation while measuring force production. Neither application of 10 m m lactic acid nor increasing the [K⁺] of the bath solution from 5 to 10 m m had any significant effect on the rate of force decline during fatigue induced by repeated brief tetani. Soleus muscles fatigued slightly faster during continuous tetanic stimulation in 10 m m [K⁺]. Inhibition of mitochondrial respiration with cyanide resulted in a faster fatigue development in both soleus and EDL muscles. Single soleus muscle fibres were fatigued by repeated tetani while measuring force and myoplasmic free [Ca²⁺] ([Ca²⁺]_i). Under control conditions, the single fibres were substantially more fatigue resistant than the whole soleus muscles; tetanic force at the end of a series of 100 tetani was reduced by about 10% and 50%, respectively. However, in the presence of cyanide, fatigue developed at a similar rate in whole muscles and single fibres, and tetanic force at the end of fatiguing stimulation was reduced by ∼80%. The force decrease in the presence of cyanide was associated with a ∼50% decrease in tetanic [Ca²⁺]_i, compared with an increase of ∼20% without cyanide. In conclusion, lactic acid or [K⁺] has little impact on fatigue induced by repeated tetani, whereas hypoxia speeds up fatigue development and this is mainly due to an impaired Ca²⁺ release from the sarcoplasmic reticulum.     

A possible role of the junctional face protein JP-45 in modulating Ca2+ release in skeletal muscle

We investigated the functional role of JP-45, a recently discovered protein of the junctional face membrane (JFM) of skeletal muscle. For this purpose, we expressed JP-45 C-terminally tagged with the fluorescent protein DsRed2 by nuclear microinjection in myotubes derived from the C2C12 skeletal muscle cell line and performed whole-cell voltage-clamp experiments. We recorded in parallel cell membrane currents and Ca²⁺ signals using fura-2 during step depolarization. It was found that properties of the voltage-activated Ca²⁺ current were not significantly changed in JP-45–DsRed2-expressing C2C12 myotubes whereas the amplitude of depolarization-induced Ca²⁺ transient was decreased compared to control myotubes expressing only DsRed2. Converting Ca²⁺ transients to Ca²⁺ input flux using a model fit approach to quantify Ca²⁺ removal, the change could be attributed to an alteration in voltage-activated Ca²⁺ permeability rather than to altered removal properties or a lower Ca²⁺ content of the sarcoplasmic reticulum (SR). Determining non-linear capacitive currents revealed a reduction of Ca²⁺ permeability per voltage-sensor charge. The results may be explained by a modulatory effect of JP-45 related to its reported in vitro interaction with the dihydropyridine receptor and the SR Ca²⁺ binding protein calsequestrin (CSQ).     

Indirect coupling between Cav1.2 channels and ryanodine receptors to generate Ca2+ sparks in murine arterial smooth muscle cells

In arterial vascular smooth muscle cells (VSMCs), Ca²⁺ sparks stimulate nearby Ca²⁺-activated K⁺ (BK) channels that hyperpolarize the membrane and close L-type Ca²⁺ channels. We tested the contribution of L-type Ca_v1.2 channels to Ca²⁺ spark regulation in tibial and cerebral artery VSMCs using VSMC-specific Ca_v1.2 channel gene disruption in (SMAKO) mice and an approach based on Poisson statistical analysis of activation frequency and first latency of elementary events. Ca_v1.2 channel gene inactivation reduced Ca²⁺ spark frequency and amplitude by ∼50% and ∼80%, respectively. These effects were associated with lower global cytosolic Ca²⁺ levels and reduced sarcoplasmic reticulum (SR) Ca²⁺ load. Elevating cytosolic Ca²⁺ levels reversed the effects completely. The activation frequency and first latency of elementary events in both wild-type and SMAKO VSMCs weakly reflected the voltage dependency of L-type channels. This study provides evidence that local and tight coupling between the Ca_v1.2 channels and ryanodine receptors (RyRs) is not required to initiate Ca²⁺ sparks. Instead, Ca_v1.2 channels contribute to global cytosolic [Ca²⁺], which in turn influences luminal SR calcium and thus Ca²⁺ sparks.     

Paradoxical SR Ca2+ release in guinea-pig cardiac myocytes after β-adrenergic stimulation revealed by two-photon photolysis of caged Ca2+

In heart muscle the amplification and shaping of Ca²⁺ signals governing contraction are orchestrated by recruiting a variable number of Ca²⁺ sparks. Sparks reflect Ca²⁺ release from the sarcoplasmic reticulum (SR) via Ca²⁺ release channels (ryanodine receptors, RyRs). RyRs are activated by Ca²⁺ influx via L-type Ca²⁺ channels with a specific probability that may depend on regulatory mechanisms (e.g. β-adrenergic stimulation) or diseased states (e.g. heart failure). Changes of RyR phosphorylation may be critical for both regulation and impaired function in disease. Using UV flash photolysis of caged Ca²⁺ and short applications of caffeine in guinea-pig ventricular myocytes, we found that Ca²⁺ release signals on the cellular level were largely governed by global SR content. During β-adrenergic stimulation resting myocytes exhibited smaller SR Ca²⁺ release signals when activated by photolysis (62.3% of control), resulting from reduced SR Ca²⁺ content under these conditions (58.6% of control). In contrast, local signals triggered with diffraction limited two-photon photolysis displayed the opposite behaviour, exhibiting a larger Ca²⁺ release (164% of control) despite reduced global and local SR Ca²⁺ content. This apparent paradox implies changes of RyR open probabilities after β-adrenergic stimulation, enhancing local regenerativity and reliability of Ca²⁺ signalling. Thus, our results underscore the importance of phosphorylation of RyRs (or of a related protein), as a regulatory physiological mechanism that may also provide new therapeutic avenues to recover impaired Ca²⁺ signalling during cardiac disease.     

Propagation in the transverse tubular system and voltage dependence of calcium release in normal and mdx mouse muscle fibres

Using a two-microelectrode voltage clamp technique, we investigated possible mechanisms underlying the impaired excitation–contraction coupling in skeletal muscle fibres of the mdx mouse, a model of the human disease Duchenne muscular dystrophy. We evaluated the role of the transverse tubular system (T-system) by using the potentiometric indicator di-8 ANEPPS, and that of the sarcoplasmic reticulum (SR) Ca²⁺ release by measuring Ca²⁺ transients with a low affinity indicator in the presence of high EGTA concentrations under voltage clamp conditions. We observed minimal differences in the T-system structure and the T-system electrical propagation was not different between normal and mdx mice. Whereas the maximum Ca²⁺ release elicited by voltage pulses was reduced by ∼67% in mdx fibres, in agreement with previous results obtained using AP stimulation, the voltage dependence of SR Ca²⁺ release was identical to that seen in normal fibres. Taken together, our data suggest that the intrinsic ability of the sarcoplasmic reticulum to release Ca²⁺ may be altered in the mdx mouse.     

Reactive oxygen species contribute to Ca2+ signals produced by osmotic stress in mouse skeletal muscle fibres

Ca²⁺ sparks, localized elevations in cytosolic [Ca²⁺], are rarely detected in intact adult mammalian skeletal muscle under physiological conditions. However, they have been observed in permeabilized cells and in intact fibres subjected to stresses, such as osmotic shock and strenuous exercise. Our previous studies indicated that an excess in cellular reactive oxygen species (ROS) generation over the ROS scavenging capabilities could be one of the up-stream causes of Ca²⁺ spark appearance in permeabilized muscle fibres. Here we tested whether the cytosolic ROS balance is compromised in intact skeletal muscle fibres that underwent osmotic shock and whether this misbalance contributes to unmasking Ca²⁺ sparks. Spontaneous Ca²⁺ sparks and the rate of ROS generation were assessed with single photon confocal microscopy and fluorescent indicators fluo-4, CM-H₂DCFDA and MitoSOX Red. Osmotic shock produced spontaneous Ca²⁺ sparks and a concomitant significant increase in ROS production. Preincubation of muscle cells with ROS scavengers (e.g. MnTBAP, Mn-cpx 3, TIRON) nearly eliminated Ca²⁺ sparks. In addition, inhibitors of NAD(P)H oxidase (DPI and apocynin) significantly reduced ROS production and suppressed the appearance of Ca²⁺ sparks. Taken together, the data suggest that ROS contribute to the abnormal Ca²⁺ spark activity in mammalian skeletal muscle subjected to osmotic stress and also indicate that NAD(P)H oxidase is a possible source of ROS. We propose that ROS-dependent Ca²⁺ sparks are an important component of adaptive/maladaptive muscle responses under various pathological conditions such as eccentric stretch, osmotic changes during ischaemia and reperfusion, and some muscle diseases.     

Junctin and the histidine-rich Ca2+ binding protein: potential roles in heart failure and arrhythmogenesis

Contractile dysfunction and ventricular arrhythmias associated with heart failure have been attributed to aberrant sarcoplasmic reticulum (SR) Ca²⁺ cycling. The study of junctin (JCN) and histidine-rich Ca²⁺ binding protein (HRC) becomes of particular importance since these proteins have been shown to be critical regulators of Ca²⁺ cycling. Specifically, JCN is a SR membrane protein, which is part of the SR Ca²⁺ release quaternary structure that also includes the ryanodine receptor, triadin and calsequestrin. Functionally, JCN serves as a bridge between calsequestrin and the Ca²⁺ release channel, ryanodine receptor. HRC is a SR luminal Ca²⁺ binding protein known to associate with both triadin and the sarcoplasmic reticulum Ca²⁺-ATPase, and may thus mediate the crosstalk between SR Ca²⁺ uptake and release. Indeed, evidence from genetic models of JCN and HRC indicate that they are important in cardiophysiology as alterations in these proteins affect SR Ca²⁺ handling and cardiac function. In addition, downregulation of JCN and HRC may contribute to Ca²⁺ cycling perturbations manifest in the failing heart, where their protein levels are significantly reduced. This review examines the roles of JCN and HRC in SR Ca²⁺ cycling and their potential significance in heart failure.     

Lipid rafts, the sarcoplasmic reticulum and uterine calcium signalling: an integrated approach

The pathways involved in Ca²⁺ signalling in the uterus remain incompletely understood, impairing our ability to prevent preterm and difficult labours. In this review we focus on two elements in the pathway of Ca²⁺ signalling that have recently emerged as playing important roles: membrane lipid rafts and the sarcoplasmic reticulum. We examine the evidence for lipid rafts in the uterus and discuss their functional role. We suggest that the increases in cytosolic [Ca²⁺] and contractility that occur with raft disruption are due, at least in part, to effects on large conductance Ca²⁺-activated K⁺ (BK) channels that are localized to rafts. The role of the SR in contributing to subsarcolemmal cytosolic microdomains in uterus is evaluated, along with its interactions with ion channels on the plasma membrane. Thus, signalling microdomains play an important, but incompletely understood, role in the uterus, and integrating them into other Ca²⁺ signalling pathways is a challenge for further research. We suggest that the role of the SR changes in pregnancy, from promoting quiescence via BK channels or SR Ca²⁺ uptake, to promoting Ca²⁺ entry and contractility at term, and relate data on lipid rafts to clinical outcome in obese pregnant women.     

Discrete store-operated calcium influx into an intracellular compartment in rabbit arteriolar smooth muscle

This study tested the hypothesis that store-operated channels (SOCs) exist as a discrete population of Ca²⁺ channels activated by depletion of intracellular Ca²⁺ stores in cerebral arteriolar smooth muscle cells and explored their direct contractile function. Using the Ca²⁺ indicator fura-PE3 it was observed that depletion of sarcoplasmic reticulum (SR) Ca²⁺ by inhibition of SR Ca²⁺-ATPase (SERCA) led to sustained elevation of [Ca²⁺]_i that depended on extracellular Ca²⁺ and slightly enhanced Mn²⁺ entry. Enhanced background Ca²⁺ influx did not explain the raised [Ca²⁺]_i in response to SERCA inhibitors because it had marked gadolinium (Gd³⁺) sensitivity, which background pathways did not. Effects were not secondary to changes in membrane potential. Thus SR Ca²⁺ depletion activated SOCs. Strikingly, SOC-mediated Ca²⁺ influx did not evoke constriction of the arterioles, which were in a resting state. This was despite the fura-PE3-indicated [Ca²⁺]_i rise being greater than that evoked by 20 m m [K⁺]_o (which did cause constriction). Release of endothelial vasodilators did not explain the absence of SOC-mediated constriction, nor did a change in Ca²⁺ sensitivity of the contractile proteins. We suggest SOCs are a discrete subset of Ca²⁺ channels allowing Ca²⁺ influx into a 'non-contractile' compartment in cerebral arteriolar smooth muscle cells.     

Mitochondria as all-round players of the calcium game

Although it has been known for over three decades that mitochondria are endowed with a complex array of Ca²⁺ transporters and that key enzymes of mitochondrial metabolism are regulated by Ca²⁺, the possibility that physiological stimuli that raise the [Ca²⁺] of the cytoplasm could trigger major mitochondrial Ca²⁺ uptake has long been considered unlikely, based on the low affinity of the mitochondrial transporters and the limited amplitude of the cytoplasmic [Ca²⁺] rises. The direct measurement of mitochondrial [Ca²⁺] with highly selective probes has led to a complete reversion of this view, by demonstrating that, after cell stimulation, the cytoplasmic Ca²⁺ signal is always paralleled by a much larger rise in [Ca²⁺] in the mitochondrial matrix. This observation has rejuvenated the study of mitochondrial Ca²⁺ transport and novel, unexpected results have altered long-standing dogmas in the field of calcium signalling. Here we focus on four main topics: (i) the current knowledge of the functional properties of the Ca²⁺ transporters and of the thermodynamic constraints under which they operate; (ii) the occurrence of mitochondrial Ca²⁺ uptake in living cells and the key role of local signalling routes between the mitochondria and the Ca²⁺ sources; (iii) the physiological consequences of Ca²⁺ transport for both mitochondrial function and the modulation of the cytoplasmic Ca²⁺ signal; and (iv) evidence that alterations of mitochondrial Ca²⁺ signalling may occur in pathophysiological conditions.     

Discrete influx events refill depleted Ca2+ stores in a chick retinal neuron

The depletion of ER Ca²⁺ stores, following the release of Ca²⁺ during intracellular signalling, triggers the Ca²⁺ entry across the plasma membrane known as store-operated calcium entry (SOCE). We show here that brief, local [Ca²⁺]_i increases (motes) in the thin dendrites of cultured retinal amacrine cells derived from chick embryos represent the Ca²⁺ entry events of SOCE and are initiated by sphingosine-1-phosphate (S1P), a sphingolipid with multiple cellular signalling roles. Externally applied S1P elicits motes but not through a G protein-coupled membrane receptor. The endogenous precursor to S1P, sphingosine, also elicits motes but its action is suppressed by dimethylsphingosine (DMS), an inhibitor of sphingosine phosphorylation. DMS also suppresses motes induced by store depletion and retards the refilling of depleted stores. These effects are reversed by exogenously applied S1P. In these neurons formation of S1P is a step in the SOCE pathway that promotes Ca²⁺ entry in the form of motes.     

Regenerative component of slow waves in the guinea-pig gastric antrum involves a delayed increase in [Ca2+]i and Cl− channels

Regenerative potentials were initiated by depolarizing short segments of single bundles of circular muscle isolated from the gastric antrum of guinea-pigs. When changes in [Ca²⁺]_i and membrane potential were recorded simultaneously, regenerative potentials were found to be associated with an increase in [Ca²⁺]_i, with the increase starting after a minimum latency of about 1 s. Although the increase in [Ca²⁺]_i was reduced by nifedipine, the amplitudes of the regenerative responses were little changed. Regenerative responses and associated changes in [Ca²⁺]_i were abolished by loading the preparations with the Ca²⁺ chelator MAPTA-AM. Regenerative potentials were abolished by 2-aminoethoxydiphenyl borate (2APB), an inhibitor of IP₃ induced Ca²⁺ release, by N -ethylamaleimide (NEM), an alkylating agent which blocks activation of G-proteins and were reduced in amplitude by two agents which block chloride (Cl⁻)-selective channels in many tissues. The observations suggest that membrane depolarization triggers IP₃ formation. This causes Ca²⁺ release from intracellular stores which activates Ca²⁺-dependent Cl⁻ channels.     

The action potential-evoked sarcoplasmic reticulum calcium release is impaired in mdx mouse muscle fibres

The mdx mouse, a model of the human disease Duchenne muscular dystrophy, has skeletal muscle fibres which display incompletely understood impaired contractile function. We explored the possibility that action potential-evoked Ca²⁺ release is altered in mdx fibres. Action potential-evoked Ca²⁺-dependent fluorescence transients were recorded, using both low and high affinity Ca²⁺ indicators, from enzymatically isolated fibres obtained from extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles of normal and mdx mice. Fibres were immobilized using either intracellular EGTA or N -benzyl- p -toluene sulphonamide, an inhibitor of the myosin II ATPase. We found that the amplitude of the action potential-evoked Ca²⁺ transients was significantly decreased in mdx mice with no measured difference in that of the surface action potential. In addition, Ca²⁺ transients recorded from mdx fibres in the absence of EGTA also displayed a marked prolongation of the slow decay phase. Model simulations of the action potential-evoked transients in the presence of high EGTA concentrations suggest that the reduction in the evoked sarcoplasmic reticulum Ca²⁺ release flux is responsible for the decrease in the peak of the Ca²⁺ transient in mdx fibres. Since the myoplasmic Ca²⁺ concentration is a critical regulator of muscle contraction, these results may help to explain the weakness observed in skeletal muscle fibres from mdx mice and, possibly, Duchenne muscular dystrophy patients.     

Spatial profiles of store-dependent calcium release in motoneurones of the nucleus hypoglossus from newborn mouse

Hypoglossal motoneurones (HMN) are selectively damaged in both human amyotrophic lateral sclerosis (ALS) and corresponding mouse models of this neurodegenerative disease, a process which has been linked to their low endogenous Ca²⁺ buffering capacity and an exceptional vulnerability to Ca²⁺-mediated excitotoxic events. In this report, we investigated local Ca²⁺ profiles in low buffered HMNs by utilizing multiphoton microscopy, CCD imaging and patch clamp recordings in slice preparations. Bath application of caffeine induced highly localized Ca²⁺ release events, which displayed an initial peak followed by a slow 'shoulder' lasting several seconds. Peak amplitudes were paralleled by Ca²⁺-activated, apamin-sensitive K⁺ currents ( I _KCa), demonstrating a functional link between Ca²⁺ stores and HMN excitability. The potential involvement of mitochondria was investigated by bath application of CCCP, which collapses the electrochemical potential across the inner mitochondrial membrane. CCCP reduced peak amplitudes of caffeine responses and consequently I _KCa, indicating that functionally intact mitochondria were critical for store-dependent modulation of HMN excitability. Taken together, our results indicate localized Ca²⁺ release profiles in HMNs, where low buffering capacities enhance the role of Ca²⁺-regulating organelles as local determinants of [Ca²⁺]_i. This might expose HMN to exceptional risks during pathophysiological organelle disruptions and other ALS-related, cellular disturbances.     

Modulation of Ca2+ release and Ca2+ oscillations in HeLa cells and fibroblasts by mitochondrial Ca2+ uniporter stimulation

The recent availability of activators of the mitochondrial Ca²⁺ uniporter allows direct testing of the influence of mitochondrial Ca²⁺ uptake on the overall Ca²⁺ homeostasis of the cell. We show here that activation of mitochondrial Ca²⁺ uptake by 4,4',4"-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) or kaempferol stimulates histamine-induced Ca²⁺ release from the endoplasmic reticulum (ER) and that this effect is enhanced if the mitochondrial Na⁺–Ca²⁺ exchanger is simultaneously inhibited with CGP37157. This suggests that both Ca²⁺ uptake and release from mitochondria control the ability of local Ca²⁺ microdomains to produce feedback inhibition of inositol 1,4,5-trisphosphate receptors (InsP₃Rs). In addition, the ability of mitochondria to control Ca²⁺ release from the ER allows them to modulate cytosolic Ca²⁺ oscillations. In histamine stimulated HeLa cells and human fibroblasts, both PPT and kaempferol initially stimulated and later inhibited oscillations, although kaempferol usually induced a more prolonged period of stimulation. Both compounds were also able to induce the generation of Ca²⁺ oscillations in previously silent fibroblasts. Our data suggest that cytosolic Ca²⁺ oscillations are exquisitely sensitive to the rates of mitochondrial Ca²⁺ uptake and release, which precisely control the size of the local Ca²⁺ microdomains around InsP₃Rs and thus the ability to produce feedback activation or inhibition of Ca²⁺ release.     

Effects of Mg2+ and SR luminal Ca2+ on caffeine-induced Ca2+ release in skeletal muscle from humans susceptible to malignant hyperthermia

Regulation of the ryanodine receptor (RYR) by Mg²⁺ and SR luminal Ca²⁺ was studied in mechanically skinned malignant hyperthermia susceptible (MHS) and non-susceptible (MHN) fibres from human vastus medialis. Preparations were perfused with solutions mimicking the intracellular milieu and changes in [Ca²⁺] were detected using fura-2 fluorescence. At 1 m m cytosolic Mg²⁺, MHS fibres had a higher sensitivity to caffeine (2-40 m m ) than MHN fibres. The inhibitory effect of Mg²⁺ on caffeine-induced Ca²⁺ release was studied by increasing [Mg²⁺] of the solution containing 40 m m caffeine. Increasing [Mg²⁺] from 1 to 3 m m reduced the amplitude of the caffeine-induced Ca²⁺ transient by 77 ± 7.4 % ( n = 8) in MHN fibres. However, the caffeine-induced Ca²⁺ transient decreased by only 24 ± 8.1 % ( n = 9) in MHS fibres. In MHN fibres, reducing the Ca²⁺ loading period from 4 to 1 min (at 1 m m Mg²⁺) decreased the fraction of the total sarcoplasmic reticulum (SR) Ca²⁺ content released in response to 40 m m caffeine by 90.4 ± 6.2 % ( n = 6). However, in MHS fibres the response was reduced by only 31.2 ± 17.4 % ( n = 6) under similar conditions. These results suggest that human malignant hyperthermia (MH) is associated with reduced inhibition of the RYR by (i) cytosolic Mg²⁺ and (ii) SR Ca²⁺ depletion. Both of these effects may contribute to increased sensitivity of the RYR to caffeine and volatile anaesthetics.     

Voltage-controlled Ca2+ release and entry flux in isolated adult muscle fibres of the mouse

The voltage-activated fluxes of Ca²⁺ from the sarcoplasmic reticulum (SR) and from the extracellular space were studied in skeletal muscle fibres of adult mice. Single fibres of the interosseus muscle were enzymatically isolated and voltage clamped using a two-electrode technique. The fibres were perfused from the current-passing micropipette with a solution containing 15 m m EGTA and 0.2 m m of either fura-2 or the faster, lower affinity indicator fura-FF. Electrical recordings in parallel with the fluorescence measurements allowed the estimation of intramembrane gating charge movements and transmembrane Ca²⁺ inward current exhibiting half-maximal activation at −7.60 ± 1.29 and 3.0 ± 1.44 mV, respectively. The rate of Ca²⁺ release from the SR was calculated after fitting the relaxation phases of fluorescence ratio signals with a kinetic model to quantify overall Ca²⁺ removal. Results obtained with the two indicators were similar. Ca²⁺ release was 2–3 orders of magnitude larger than the flux carried by the L-type Ca²⁺ current. At maximal depolarization (+50 mV), release flux peaked at about 3 ms after the onset of the voltage pulse and then decayed in two distinct phases. The slower phase, most likely resulting from SR depletion, indicated a decrease in lumenal Ca²⁺ content by about 80% within 100 ms. Unlike in frog fibres, the kinetics of the rapid phase of decay showed no dependence on the filling state of the SR and the results provide little evidence for a substantial increase of SR permeability on depletion. The approach described here promises insight into excitation–contraction coupling in future studies of genetically altered mice.