Effects of a preferential myosin loss on Ca2+ activation of force generation in single human skeletal muscle fibres

Preferential loss of the motor protein myosin, as observed in patients with acute quadriplegic myopathy (AQM) or cancer cachexia, causes generalized muscle wasting, muscle weakness and a decrease in muscle fibre force normalized to cross-sectional area. It remains unclear, however, whether this myosin loss influences other important features of muscle fibre function, such as Ca²⁺ activation of the contractile proteins. To address this question, we have studied Ca²⁺ sensitivity of force generation using skinned muscle fibres from four patients with AQM or cancer cachexia and a preferential loss of myosin; and from seven healthy control individuals. Force and apparent rate constant of force redevelopment ( k _tr) were assessed in solutions with varying Ca²⁺ concentrations (pCa), allowing construction of relative force–pCa and k _tr–pCa relationships. Results showed a rightward shift of the relative force–pCa relationship and a leftward shift of the relative k _tr–pCa curve in muscle fibres with a preferential myosin loss. To improve the understanding of the mechanisms underlying these alterations, the relative stiffness–pCa relationship was evaluated. A rightward shift of this curve was observed, suggesting that the changes in the Ca²⁺ activation of force and k _tr were predominantly due to a decrease in the relative number of attached cross-bridges at different pCa values. Thus, a change in Ca²⁺ activation of the contractile apparatus in patients with preferential myosin loss is proposed as an additional factor contributing to the muscle function impairment in these patients.     

Defective regulation of contractile function in muscle fibres carrying an E41K β-tropomyosin mutation

A novel E41K β-tropomyosin (β-Tm) mutation, associated with congenital myopathy and muscle weakness, was recently identified in a woman and her daughter. In both patients, muscle weakness was coupled with muscle fibre atrophy. It remains unknown, however, whether the E41K β-Tm mutation directly affects regulation of muscle contraction, contributing to the muscle weakness. To address this question, we studied a broad range of contractile characteristics in skinned muscle fibres from the two patients and eight healthy controls. Results showed decreases (i) in speed of contraction at saturated Ca²⁺ concentration (apparent rate constant of force redevelopment ( k _tr) and unloaded shortening speed ( V ₀)); and (ii) in contraction sensitivity to Ca²⁺ concentration, in fibres from patients compared with controls, suggesting that the mutation has a negative effect on contractile function, contributing to the muscle weakness. To investigate whether these negative impacts are reversible, we exposed skinned muscle fibres to the Ca²⁺ sensitizer EMD 57033. In fibres from patients, 30 μ m of EMD 57033 (i) had no effect on speed of contraction ( k _tr and V ₀) at saturated Ca²⁺ concentration but (ii) increased Ca²⁺ sensitivity of contraction, suggesting a potential therapeutic approach in patients carrying the E41K β-Tm mutation.     

Calcium- and myosin-dependent changes in troponin structure during activation of heart muscle

Each heartbeat is triggered by a pulse of intracellular calcium ions which bind to troponin on the actin-containing thin filaments of heart muscle cells, initiating a change in filament structure that allows myosin to bind and generate force. We investigated the molecular mechanism of calcium regulation in demembranated trabeculae from rat ventricle using polarized fluorescence from probes on troponin C (TnC). Native TnC was replaced by double-cysteine mutants of human cardiac TnC with bifunctional rhodamine attached along either the C helix, adjacent to the regulatory Ca²⁺-binding site, or the E helix in the IT arm of the troponin complex. Changes in the orientation of both troponin helices had the same steep Ca²⁺ dependence as active force production, with a Hill coefficient ( n _H) close to 3, consistent with a single co-operative transition controlled by Ca²⁺ binding. Complete inhibition of active force by 25 μ m blebbistatin had very little effect on the Ca²⁺-dependent structural changes and in particular did not significantly reduce the value of n _H. Binding of rigor myosin heads to thin filaments following MgATP depletion in the absence of Ca²⁺ also changed the orientation of the C and E helices, and addition of Ca²⁺ in rigor produced further changes characterized by increased Ca²⁺ affinity but with n _H close to 1. These results show that, although myosin binding can switch on thin filaments in rigor conditions, it does not contribute significantly under physiological conditions. The physiological mechanism of co-operative Ca²⁺ regulation of cardiac contractility must therefore be intrinsic to the thin filaments.     

Functional consequence of mutation in rat cardiac troponin T is affected differently by myosin heavy chain isoforms

Cardiac troponin T (cTnT) is an essential component of the thin filament regulatory unit (RU) that regulates Ca²⁺ activation of tension in the heart muscle. Because there is coupling between the RU and myosin crossbridges, the functional outcome of cardiomyopathy-related mutations in cTnT may be modified by the type of myosin heavy chain (MHC) isoform. Ca²⁺ activation of tension and ATPase activity were measured in muscle fibres from normal rat hearts containing α-MHC isoform and propylthiouracil (PTU)-treated rat hearts containing β-MHC isoform. Muscle fibres from normal and PTU-treated rat hearts were reconstituted with two different mutations in rat cTnT; the deletion of Glu162 (cTnT_E162DEL) and the deletion of Lys211 (cTnT_K211DEL). α-MHC and β-MHC isoforms had contrasting impact on tension-dependent ATP consumption (tension cost) in cTnT_E162DEL and cTnT_K211DEL reconstituted muscle fibres. Significant increases in tension cost in α-MHC-containing muscle fibres corresponded to 17% ( P < 0.01) and 23% ( P < 0.001) when reconstituted with cTnT_E162DEL and cTnT_K211DEL, respectively. In contrast, tension cost decreased when these two cTnT mutants were reconstituted in muscle fibres containing β-MHC; by approximately 24% ( P < 0.05) when reconstituted with cTnT_E162DEL and by approximately 17% ( P = 0.09) when reconstituted with cTnT_K211DEL. Such differences in tension cost were substantiated by the mechano-dynamic analysis of cTnT mutant reconstituted muscle fibres from normal and PTU-treated rat hearts. Our observation demonstrates that qualitative changes in MHC isoform alters the nature of cardiac myofilament dysfunction induced by mutations in cTnT.     

Protein kinase C and A sites on troponin I regulate myofilament Ca2+ sensitivity and ATPase activity in the mouse myocardium

Cardiac troponin I (cTnI) is a phosphoprotein subunit of the troponin-tropomyosin complex that is thought to inhibit cardiac muscle contraction during diastole. To investigate the contributions of cTnI phosphorylation to cardiac regulation, transgenic mice were created with the phosphorylation sites of cTnI mutated to alanine. Activation of protein kinase C (PKC) by perfusion of hearts with phorbol-12-myristate-13-acetate (PMA) or endothelin-1 (ET-1) inhibited the maximum ATPase rate by up to 25 % and increased the Ca²⁺ sensitivity of ATPase activity and of isometric tension by up to 0.15 pCa units. PKC activation no longer altered cTnI phosphorylation, depressed ATPase rates or enhanced myofilament Ca²⁺ sensitivity in transgenic mice expressing cTnI that could not be phosphorylated on serines^43/45 and threonine¹⁴⁴ (PKC sites). Modest changes in myosin regulatory light chain phosphorylation occurred in all mouse lines, but increases in myofilament Ca²⁺ sensitivity required the presence of phosphorylatable cTnI. For comparison, the β-adrenergic agonist isoproterenol caused a 38 % increase in maximum ATPase rate and a 0.12 pCa unit decrease in myofilament Ca²⁺ sensitivity. These β-adrenergic effects were absent in transgenic mice expressing cTnI that could not be phosphorylated on serines^23/24 (protein kinase A, PKA, sites). Overall, the results indicate that PKC and PKA exert opposing effects on actomyosin function by phosphorylating cTnI on distinct sites. A primary role of PKC phosphorylation of cTnI may be to reduce the requirements of the contractile apparatus for both Ca²⁺ and ATP, thereby promoting efficient ATP utilisation during contraction.     

Differential effects of a green tea-derived polyphenol (−)-epigallocatechin-3-gallate on the acidosis-induced decrease in the Ca2+ sensitivity of cardiac and skeletal muscle

(-)-Epigallocatechin-3-gallate (EGCg), a green tea-derived polyphenol, has received much attention as a protective agent against cardiovascular diseases. In this study, we determined its effects on the acidosis-induced change in the Ca(2+) sensitivity of myofilaments in myofibrils prepared from porcine ventricular myocardium and chicken pectoral muscle. EGCg (0.1 mM) significantly inhibited the decrease caused by lowering the pH from 7.0 to 6.0 in the Ca(2+) sensitivity of myofibrillar ATPase activity in cardiac muscle, but not in skeletal muscle. Studies on recombinant mouse cardiac troponin C (cTnC) and chicken fast skeletal troponin C (sTnC) using circular dichroism and intrinsic and extrinsic fluorescence spectroscopy showed that EGCg bound to cTnC with a dissociation constant of approximately 3-4 muM, but did not bind to sTnC. By presumably binding to the cTnC C-lobe, EGCg decreased Ca(2+) binding to cTnC and overcame the depressant effect of protons on the Ca(2+) sensitivity of the cardiac contractile response. To demonstrate isoform-specific effects of the action of EGCg, the pH sensitivity of the Ca(2+) response was examined in cardiac myofibrils in which endogenous cTnC was replaced with exogenous sTnC or cTnC and in skeletal myofibrils in which the endogenous sTn complex was replaced with whole cardiac Tn complex (cTn). The results suggest that the binding of EGCg to the cardiac isoform-specific TnC or Tn complex alters the effect of pH on myofilament Ca(2+) sensitivity in striated muscle.     

Effects of a R133W β-tropomyosin mutation on regulation of muscle contraction in single human muscle fibres

A novel R133W β-tropomyosin (β-Tm) mutation, associated with muscle weakness and distal limb deformities, has recently been identified in a woman and her daughter. The muscle weakness was not accompanied by progressive muscle wasting or histopathological abnormalities in tibialis anterior muscle biopsy specimens. The aim of the present study was to explore the mechanisms underlying the impaired muscle function in patients with the β-Tm mutation. Maximum force normalized to fibre cross-sectional area (specific force, SF), maximum velocity of unloaded shortening ( V ₀), apparent rate constant of force redevelopment ( k _tr) and force–pCa relationship were evaluated in single chemically skinned muscle fibres from the two patients carrying the β-Tm mutation and from healthy control subjects. Significant differences in regulation of muscle contraction were observed in the type I fibres: a lower SF ( P < 0.05) and k _tr ( P < 0.01), and a faster V ₀ ( P < 0.05). The force–pCa relationship did not differ between patient and control fibres, indicating an unaltered Ca²⁺ activation of contractile proteins. Collectively, these results indicate a slower cross-bridge attachment rate and a faster detachment rate caused by the R133W β-Tm mutation. It is suggested that the R133W β-Tm mutation induces alteration in myosin–actin kinetics causing a reduced number of myosin molecules in the strong actin-binding state, resulting in overall muscle weakness in the absence of muscle wasting.     

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.     

Spatiotemporal patterning of IP3-mediated Ca2+ signals in Xenopus oocytes by Ca2+-binding proteins

Ca²⁺-binding proteins (CaBPs) are expressed in a highly specific manner across many different cell types, yet the physiological basis underlying their selective distribution patterns remains unclear. We used confocal line-scan microscopy together with photo-release of IP₃ in Xenopus oocytes to investigate the actions of mobile cytosolic CaBPs on the spatiotemporal properties of IP₃-evoked Ca²⁺ signals. Parvalbumin (PV), a CaBP with slow Ca²⁺-binding kinetics, shortened the duration of IP₃-evoked Ca²⁺ signals and 'balkanized' global responses into discrete localized events (puffs). In contrast, calretinin (CR), a presumed fast buffer, prolonged Ca²⁺ responses and promoted 'globalization' of spatially uniform Ca²⁺ signals at high [IP₃]. Oocytes loaded with CR or PV showed Ca²⁺ puffs following photolysis flashes that were subthreshold in controls, and the spatiotemporal properties of these localized events were differentially modulated by PV and CR. In comparison to results we previously obtained with exogenous Ca²⁺ buffers, PV closely mimicked the actions of the slow buffer EGTA, whereas CR showed important differences from the fast buffer BAPTA. Most notably, puffs were never observed after loading BAPTA, and this exogenous buffer did not show the marked sensitization of IP₃ action evident with CR. The ability of Ca²⁺ buffers and CaBPs with differing kinetics to fine-tune both global and local intracellular Ca²⁺ signals is likely to have significant physiological implications.     

Properties of Orai1 mediated store-operated current depend on the expression levels of STIM1 and Orai1 proteins

Two cellular proteins, stromal interaction molecule 1 (STIM1) and Orai1, are recently discovered essential components of the Ca²⁺ release activated Ca²⁺ (CRAC) channel. Orai1 polypeptides form the pore of the CRAC channel, while STIM1 plays the role of the endoplasmic reticulum Ca²⁺ sensor required for activation of CRAC current ( I _CRAC) by store depletion. It is not known, however, if the role of STIM1 is limited exclusively to Ca²⁺ sensing, or whether interaction between Orai1 and STIM1, either direct or indirect, also defines the properties of I _CRAC. In this study we investigated how the relative expression levels of ectopic Orai1 and STIM1 affect the properties of I _CRAC. The results show that cells expressing low Orai1 : STIM1 ratios produce I _CRAC with strong fast Ca²⁺-dependent inactivation, while cells expressing high Orai1 : STIM1 ratios produce I _CRAC with strong activation at negative potentials. Moreover, the expression ratio of Orai1 and STIM1 affects Ca²⁺, Ba²⁺ and Sr²⁺ conductance, but has no effect on the current in the absence of divalent cations. The results suggest that several key properties of Ca²⁺ channels formed by Orai1 depend on its interaction with STIM1, and that the stoichiometry of this interaction may vary depending on the relative expression levels of these proteins.     

Ca2+ sensitivity of regulated cardiac thin filament sliding does not depend on myosin isoform

Myosin heavy chain (MHC) isoforms in vertebrate striated muscles are distinguished functionally by differences in chemomechanical kinetics. These kinetic differences may influence the cross-bridge-dependent co-operativity of thin filament Ca²⁺ activation. To determine whether Ca²⁺ sensitivity of unloaded thin filament sliding depends upon MHC isoform kinetics, we performed in vitro motility assays with rabbit skeletal heavy meromyosin (rsHMM) or porcine cardiac myosin (pcMyosin). Regulated thin filaments were reconstituted with recombinant human cardiac troponin (rhcTn) and α-tropomyosin (rhcTm) expressed in Escherichia coli . All three subunits of rhcTn were coexpressed as a functional complex using a novel construct with a glutathione S-transferase (GST) affinity tag at the N-terminus of human cardiac troponin T (hcTnT) and an intervening tobacco etch virus (TEV) protease site that allows purification of rhcTn without denaturation, and removal of the GST tag without proteolysis of rhcTn subunits. Use of this highly purified rhcTn in our motility studies resulted in a clear definition of the regulated motility profile for both fast and slow MHC isoforms. Maximum sliding speed (pCa 5) of regulated thin filaments was roughly fivefold faster with rsHMM compared with pcMyosin, although speed was increased by 1.6- to 1.9-fold for regulated over unregulated actin with both MHC isoforms. The Ca²⁺ sensitivity of regulated thin filament sliding speed was unaffected by MHC isoform. Our motility results suggest that the cellular changes in isoform expression that result in regulation of myosin kinetics can occur independently of changes that influence thin filament Ca²⁺ sensitivity.     

Buffer Kinetics Shape the Spatiotemporal Patterns of IP3-Evoked Ca2+ Signals

Ca²⁺ liberation through inositol 1,4,5-trisphosphate receptors (IP₃Rs) plays a universal role in cell regulation, and specificity of cell signalling is achieved through the spatiotemporal patterning of Ca²⁺ signals. IP₃Rs display Ca²⁺-induced Ca²⁺ release (CICR), but are grouped in clusters so that regenerative Ca²⁺ signals may remain localized to individual clusters, or propagate globally between clusters by successive cycles of Ca²⁺ diffusion and CICR. We used confocal microscopy and photoreleased IP₃ in Xenopus oocytes to study how these properties are modulated by mobile cytosolic Ca²⁺ buffers. EGTA (a buffer with slow 'on-rate') speeded Ca²⁺ signals and 'balkanized' Ca²⁺ waves by dissociating them into local signals. In contrast, BAPTA (a fast buffer with similar affinity) slowed Ca²⁺ responses and promoted 'globalization' of spatially uniform Ca²⁺ signals. These actions are likely to arise through differential effects on Ca²⁺ feedback within and between IP₃R clusters, because Ca²⁺ signals evoked by influx through voltage-gated channels were little affected. We propose that cell-specific expression of Ca²⁺-binding proteins with distinct kinetics may shape the time course and spatial distribution of IP₃-evoked Ca²⁺ signals for specific physiological roles.     

Reactive oxygen species reduce myofibrillar Ca2+ sensitivity in fatiguing mouse skeletal muscle at 37°C

The mechanisms of muscle fatigue were studied in small muscle bundles and single fibres isolated from the flexor digitorum brevis of the mouse. Fatigue caused by repeated isometric tetani was accelerated at body temperature (37°C) when compared to room temperature (22°C). The membrane-permeant reactive oxygen species (ROS) scavenger, Tiron (5 m m ), had no effect on the rate of fatigue at 22°C but slowed the rate of fatigue at 37°C to that observed at 22°C. Single fibres were microinjected with indo-1 to measure intracellular calcium. In the accelerated fatigue at 37°C the tetanic [Ca²⁺]_i did not change significantly and the decline of maximum Ca²⁺-activated force was similar to that observed at 22°C. The cause of the greater rate of fatigue at 37°C was a large fall in myofibrillar Ca²⁺ sensitivity. In the presence of Tiron, the large fall in Ca²⁺ sensitivity was abolished and the usual decline in tetanic [Ca²⁺]_i was observed. This study confirms the importance of ROS in fatigue at 37°C and shows that the mechanism of action of ROS is a decline in myofibrillar Ca²⁺ sensitivity.     

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.     

Calcium oscillations in interstitial cells of the rabbit urethra

Measurements were made (using fast confocal microscopy) of intracellular Ca²⁺ levels in fluo-4 loaded interstitial cells isolated from the rabbit urethra. These cells exhibited regular Ca²⁺ oscillations which were associated with spontaneous transient inward currents recorded under voltage clamp. Interference with d - myo -inositol 1,4,5-trisphosphate (IP₃) induced Ca²⁺ release using 100 μ m 2-aminoethoxydiphenyl borate, and the phospholipase C (PLC) inhibitors 2-nitro-4-carboxyphenyl N , N -diphenylcarbamate and U73122 decreased the amplitude of spontaneous oscillations but did not abolish them. However, oscillations were abolished when ryanodine receptors were blocked with tetracaine or ryanodine. Oscillations ceased in the absence of external Ca²⁺, and frequency was directly proportional to the external Ca²⁺ concentration. Frequency of Ca²⁺ oscillation was reduced by SKF-96365, but not by nifedipine. Lanthanum and cadmium completely blocked oscillations. These results suggest that Ca²⁺ oscillations in isolated rabbit urethral interstitial cells are initiated by Ca²⁺ release from ryanodine-sensitive intracellular stores, that oscillation frequency is very sensitive to the external Ca²⁺ concentration and that conversion of the primary oscillation to a propagated Ca²⁺ wave depends upon IP₃-induced Ca²⁺ release.     

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.     

Modelling and measuring electromechanical coupling in the rat heart

Tension-dependent binding of Ca²⁺ to troponin C in the cardiac myocyte has been shown to play an important role in the regulation of Ca²⁺ and the activation of tension development. The significance of this regulatory mechanism is quantified experimentally by the quantity of Ca²⁺ released following a rapid change in the muscle length. Using a computational, coupled, electromechanics cell model, we have confirmed that the tension dependence of Ca²⁺ binding to troponin C, rather than cross-bridge kinetics or the rate of Ca²⁺ uptake by the sarcoplasmic reticulum, determines the quantity of Ca²⁺ released following a length step. This cell model has been successfully applied in a continuum model of the papillary muscle to analyse experimental data, suggesting the tension-dependent binding of Ca²⁺ to troponin C as the likely pathway through which the effects of localized impaired tension generation alter the Ca²⁺ transient. These experimental results are qualitatively reproduced using a three-dimensional coupled electromechanics model. Furthermore, the model predicts that changes in the Ca²⁺ transient in the viable myocardium surrounding the impaired region are amplified in the absence of tension-dependent binding of Ca²⁺ to troponin C.     

Loss of caveolin-3 induced by the dystrophy-associated P104L mutation impairs L-type calcium channel function in mouse skeletal muscle cells

Caveolins are membrane scaffolding proteins that associate with and regulate a variety of signalling proteins, including ion channels. A deficiency in caveolin-3 (Cav-3), the major striated muscle isoform, is responsible for skeletal muscle disorders, such as limb-girdle muscular dystrophy 1C (LGMD 1C). The molecular mechanisms leading to the muscle wasting that characterizes this pathology are poorly understood. Here we show that a loss of Cav-3 induced by the expression of the LGMD 1C-associated mutant P104L (Cav-3^P104L) provokes a reduction by half of the maximal conductance of the voltage-dependent L-type Ca²⁺ channel in mouse primary cultured myotubes and fetal skeletal muscle fibres. Confocal immunomiscrocopy indicated a colocalization of Cav-3 and Ca_v1.1, the pore-forming subunit of the L-type Ca²⁺ channel, at the surface membrane and in the developing T-tubule network in control myotubes and fetal fibres. In myotubes expressing Cav-3^P104L, the loss of Cav-3 was accompanied by a 66% reduction in Ca_v1.1 mean labelling intensity. Our results suggest that Cav-3 is involved in L-type Ca²⁺ channel membrane function and localization in skeletal muscle cells and that an alteration of L-type Ca²⁺ channels could be involved in the physiopathological mechanisms of caveolinopathies.     

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.