Phosphoinositide substrates of myotubularin affect voltage-activated Ca²âº release in skeletal muscle.

Skeletal muscle excitation­contraction (E­C) coupling is altered in several models of phosphatidylinositol phosphate (PtdInsP) phosphatase deficiency and ryanodine receptor activity measured in vitro was reported to be affected by certain PtdInsPs, thus prompting investigation of the physiological role of PtdInsPs in E­C coupling. We measured intracellular Ca2+ transients in voltage-clamped mouse muscle fibres microinjected with a solution containing a PtdInsP substrate (PtdIns(3,5)P2 or PtdIns(3)P) or product (PtdIns(5)P or PtdIns) of the myotubularin phosphatase MTM1. No significant change was observed in the presence of either PtdIns(5)P or PtdIns but peak SR Ca2+ release was depressed by ~30% and 50% in fibres injected with PtdIns(3,5)P2 and PtdIns(3)P, respectively, with no concurrent alteration in the membrane current signals associated with the DHPR function as well as in the voltage dependence of Ca2+ release inactivation. In permeabilized muscle fibres, the frequency of spontaneous Ca2+ release events was depressed in the presence of the three tested phosphorylated forms of PtdInsP with PtdIns(3,5)P2 being the most effective, leading to an almost complete disappearance of Ca2+ release events. Results support the possibility that pathological accumulation of MTM1 substrates may acutely depress ryanodine receptor-mediated Ca2+ release. Overexpression of a mCherry-tagged form of MTM1 in muscle fibres revealed a striated pattern consistent with the triadic area. Ca2+ release remained although unaffected by MTM1 overexpression and was also unaffected by the PtdIns-3-kinase inhibitor LY2940002, suggesting that the 3-phosphorylated PtdIns lipids active on voltage-activated Ca2+ release are inherently maintained at a low level, inefficient on Ca2+ release in normal conditions.

Characterization of functional TRPV1 channels in the sarcoplasmic reticulum of mouse skeletal muscle.

TRPV1 represents a non-selective cation channel activated by capsaicin, acidosis and high temperature. In the central nervous system where TRPV1 is highly expressed, its physiological role in nociception is clearly identified. In skeletal muscle, TRPV1 appears implicated in energy metabolism and exercise endurance. However, how as a Ca(2+) channel, it contributes to intracellular calcium concentration ([Ca(2+)]i) maintenance and muscle contraction remains unknown. Here, as in rats, we report that TRPV1 is functionally expressed in mouse skeletal muscle. In contrast to earlier reports, our analysis show TRPV1 presence only at the sarcoplasmic reticulum (SR) membrane (preferably at the longitudinal part) in the proximity of SERCA1 pumps. Using intracellular Ca(2+) imaging, we directly accessed to the channel functionality in intact FDB mouse fibers. Capsaicin and resiniferatoxin, both agonists as well as high temperature (45°C) elicited an increase in [Ca(2+)]i. TRPV1-inhibition by capsazepine resulted in a strong inhibition of TRPV1-mediated functional responses and abolished channel activation. Blocking the SR release (with ryanodine or dantrolene) led to a reduced capsaicin-induced Ca(2+) elevation suggesting that TRPV1 may participate to a secondary SR Ca(2+) liberation of greater amplitude. In conclusion, our experiments point out that TRPV1 is a functional SR Ca(2+) leak channel and may crosstalk with RyR1 in adult mouse muscle fibers.

Ca2+ release in muscle fibers expressing R4892W and G4896V type 1 ryanodine receptor disease mutants.

The large and rapidly increasing number of potentially pathological mutants in the type 1 ryanodine receptor (RyR1) prompts the need to characterize their effects on voltage-activated sarcoplasmic reticulum (SR) Ca(2+) release in skeletal muscle. Here we evaluated the function of the R4892W and G4896V RyR1 mutants, both associated with central core disease (CCD) in humans, in myotubes and in adult muscle fibers. For both mutants expressed in RyR1-null (dyspedic) myotubes, voltage-gated Ca(2+) release was absent following homotypic expression and only partially restored following heterotypic expression with wild-type (WT) RyR1. In muscle fibers from adult WT mice, both mutants were expressed in restricted regions of the fibers with a pattern consistent with triadic localization. Voltage-clamp-activated confocal Ca(2+) signals showed that fiber regions endowed with G4896V-RyR1s exhibited an ∼30% reduction in the peak rate of SR Ca(2+) release, with no significant change in SR Ca(2+) content. Immunostaining revealed no associated change in the expression of either α1S subunit (Cav1.1) of the dihydropyridine receptor (DHPR) or type 1 sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA1), indicating that the reduced Ca(2+) release resulted from defective RyR1 function. Interestingly, in spite of robust localized junctional expression, the R4892W mutant did not affect SR Ca(2+) release in adult muscle fibers, consistent with a low functional penetrance of this particular CCD-associated mutant.

Defects in Ca2+ release associated with local expression of pathological ryanodine receptors in mouse muscle fibres.

Mutations of the gene encoding the type 1 ryanodine receptor (RyR1) are associated with skeletal muscle disorders including malignant hyperthermia susceptibility (MHS) and central core disease (CCD). We used in vivo expression of EGFP-RyR1 constructs in fully differentiated mouse muscle fibres to characterize the function of several RyR1 mutants. Wild-type and Y523S, R615C, R2163H and I4897T mutants of RyR1 were separately expressed and found to be present within restricted regions of fibres with a pattern consistent with triadic localization. Confocal measurements of voltage-clamp-activated myoplasmic Ca(2+) transients demonstrated alterations of sarcoplasmic reticulum (SR) Ca(2+) release spatially correlated with the presence of exogenous RyR1s. The Y523S, R615C and R2163H RyR1 MHS-related mutants were associated with enhanced peak Ca(2+) release for low and moderate levels of depolarization, whereas the I4897T CCD mutant produced a chronic reduction of peak SR Ca(2+) release. For example, peak Ca(2+) release in response to a depolarization to -20 mV in regions of fibres expressing Y523S and I4897T was 2.0 ± 0.3 (n = 9) and 0.46 ± 0.1 (n = 5) times the corresponding value in adjacent, non-expressing regions of the same fibre, respectively. Interestingly no significant change in the estimated total amount of Ca(2+) released at the end of large depolarizing pulses was observed for any of the mutant RyR1 channels. Overall, results are consistent with an 'inherent' increase in RyR1 sensitivity to activation by the voltage sensor for the MHS-related RyR1 mutants and a partial failure of voltage-gated release for the CCD-related I4897T mutant, that occur with no sign of change in SR Ca(2+) content. Furthermore, the results indicate that RyR1 channel density is tightly regulated even under the present conditions of forced exogenous expression.

In vivo expression of G-protein beta1gamma2 dimer in adult mouse skeletal muscle alters L-type calcium current and excitation-contraction coupling.

A number of G-protein-coupled receptors are expressed in skeletal muscle but their roles in muscle physiology and downstream effector systems remain poorly investigated. Here we explored the functional importance of the G-protein betagamma (Gbetagamma) signalling pathway on voltage-controlled Ca(2+) homeostasis in single isolated adult skeletal muscle fibres. A GFP-tagged Gbeta(1)gamma(2) dimer was expressed in vivo in mice muscle fibres. The GFP fluorescence pattern was consistent with a Gbeta(1)gamma(2) dimer localization in the transverse-tubule membrane. Membrane current and indo-1 fluorescence measurements performed under voltage-clamp conditions reveal a drastic reduction of both L-type Ca(2+) current density and of peak amplitude of the voltage-activated Ca(2+) transient in Gbeta(1)gamma(2)-expressing fibres. These effects were not observed upon expression of Gbeta(2)gamma(2), Gbeta(3)gamma(2) or Gbeta(4)gamma(2). Our data suggest that the G-protein beta(1)gamma(2) dimer may play an important regulatory role in skeletal muscle excitation-contraction coupling.

Transient receptor potential canonical type 1 (TRPC1) operates as a sarcoplasmic reticulum calcium leak channel in skeletal muscle.

Extensive studies performed in nonexcitable cells and expression systems have shown that type 1 transient receptor potential canonical (TRPC1) channels operate mainly in plasma membranes and open through phospholipase C-dependent processes, membrane stretch, or depletion of Ca(2+) stores. In skeletal muscle, it is proposed that TRPC1 channels are involved in plasmalemmal Ca(2+) influx and stimulated by store depletion or membrane stretch, but direct evidence for TRPC1 sarcolemmal channel activity is not available. We investigated here the functional role of TRPC1 using an overexpressing strategy in adult mouse muscle fibers. Immunostaining for endogenous TRPC1 revealed a striated expression pattern that matched sarcoplasmic reticulum (SR) Ca(2+) pump immunolabeling. In cells expressing TRPC1-yellow fluorescent protein (YFP), the same pattern of expression was observed, compatible with a longitudinal SR localization. Resting electric properties, action potentials, and resting divalent cation influx were not altered in TRPC1-YFP-positive cells. Poisoning with the SR Ca(2+) pump blocker cyclopiazonic acid elicited a contracture of the fiber at the level of the overexpression site in presence and absence of external Ca(2+) which was not observed in control cells. Ca(2+) measurements indicated that resting Ca(2+) and the rate of Ca(2+) increase induced by cyclopiazonic acid were higher in the TRPC1-YFP-positive zone than in the TRPC1-YFP-negative zone and control cells. Ca(2+) transients evoked by 200-ms voltage clamp pulses decayed slower in TRPC1-YFP-positive cells. In contrast to previous hypotheses, these data demonstrate that TRPC1 operates as a SR Ca(2+) leak channel in skeletal muscle.

Evaluation of remodeling in left and right ventricular myocytes from heterozygous (mRen2)27 transgenic rats.

Cardiac remodeling was assessed both in the pressure-overloaded left ventricle and in the normotensive right ventricle of hypertensive transgenic rats (mRen2)27 (TGR27). The present study combined histology, electrophysiology, molecular biology and biochemistry techniques. A significant increase in action potential (AP) duration was recorded both in right and left ventricular myocytes wheareas only in the latter ones were hypertrophic. The increase in AP duration is mainly supported by the reduction of the transient outward K current (I(to)) density since no significant modification was observed for the L-type calcium current (I(Ca,L)), the sodium-calcium exchange current (I(NCX)), the delayed rectifier current (I(K)) and the inward rectifier current (I(K1)). The lower amplitude of I(to) current was associated with a lower Kv4.3 protein expression both in right and left ventricles while Kv4.3 mRNA levels was decreased only in left ventricle. Thus, a differential ventricular remodeling takes place in the TGR27 model. The possible cause of electrical remodeling in right ventricular myocytes of TGR27 is discussed.

Expression of the muscular dystrophy-associated caveolin-3(P104L) mutant in adult mouse skeletal muscle specifically alters the Ca(2+) channel function of the dihydropyridine receptor.

Caveolins are plasma-membrane-associated proteins potentially involved in a variety of signalling pathways. Different mutations in CAV3, the gene encoding for the muscle-specific isoform caveolin-3 (Cav-3), lead to muscle diseases, but the underlying molecular mechanisms remain largely unknown. Here, we explored the functional consequences of a Cav-3 mutation (P104L) inducing the 1C type limb-girdle muscular dystrophy (LGMD 1C) in human on intracellular Ca(2+) regulation of adult skeletal muscle fibres. A YFP-tagged human Cav-3(P104L) mutant was expressed in vivo in muscle fibres from mouse. Western blot analysis revealed that expression of this mutant led to an approximately 80% drop of the level of endogenous Cav-3. The L-type Ca(2+) current density was found largely reduced in fibres expressing the Cav-3(P104L) mutant, with no change in the voltage dependence of activation and inactivation. Interestingly, the maximal density of intramembrane charge movement was unaltered in the Cav-3(P104L)-expressing fibres, suggesting no change in the total amount of functional voltage-sensing dihydropyridine receptors (DHPRs). Also, there was no obvious alteration in the properties of voltage-activated Ca(2+) transients in the Cav-3(P104L)-expressing fibres. Although the actual role of the Ca(2+) channel function of the DHPR is not clearly established in adult skeletal muscle, its specific alteration by the Cav-3(P104L) mutant suggests that it may be involved in the physiopathology of LGMD 1C.

Spontaneous and voltage-activated Ca2+ release in adult mouse skeletal muscle fibres expressing the type 3 ryanodine receptor.

The physiological properties and role of the type 3 ryanodine receptor (RyR3), a calcium release channel expressed in a wide variety of cell types, remain mysterious. We forced, in vivo, the expression of RyR3 in adult mouse skeletal muscle fibres using a GFP-RyR3 DNA construct. GFP fluorescence was found within spatially restricted regions of muscle fibres where it exhibited a sarcomere-related banded pattern consistent with a localization within or near the junctional sarcoplasmic reticulum membrane. Immunostaining confirmed the presence of RyR3 together with RyR1 within the GFP-positive areas. In approximately 90% of RyR3-positive fibres microinjected with the calcium indicator fluo-3, we detected repetitive spontaneous transient elevations of intracellular Ca2+ that persisted when fibres were voltage-clamped at -80 mV. These Ca2+ transients remained essentially confined to the RyR3 expression region. They ranged from wide local events to propagating Ca2+ waves and were in some cases associated with local contractile activity. When voltage-clamp depolarizations were applied while fluo-3 or rhod-2 fluorescence was measured within the RyR3-expressing region, no voltage-evoked 'spark-like' elementary Ca2+ release event could be detected. Still global voltage-activated Ca2+ release exhibited a prominent early peak within the RyR3-expressing regions. Measurements were also taken from muscles fibres expressing a GFP-RyR1 construct; positive fibres also yielded a local banded pattern of GFP fluorescence but exhibited no spontaneous Ca2+ release. Results demonstrate that RyR3 is a very potent source of voltage-independent Ca2+ release activity. Conversely we find no evidence that it could contribute to the production of discrete voltage-activated Ca2+ release events in differentiated mammalian skeletal muscle.

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 Ca(2+) channel in mouse primary cultured myotubes and fetal skeletal muscle fibres. Confocal immunomiscrocopy indicated a colocalization of Cav-3 and Ca(v)1.1, the pore-forming subunit of the L-type Ca(2+) 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(v)1.1 mean labelling intensity. Our results suggest that Cav-3 is involved in L-type Ca(2+) channel membrane function and localization in skeletal muscle cells and that an alteration of L-type Ca(2+) channels could be involved in the physiopathological mechanisms of caveolinopathies.

Microarray analysis of knockout mice identifies cyclin D2 as a possible mediator for the action of thyroid hormone during the postnatal development of the cerebellum.

Thyroid hormone is a major regulator of postnatal brain development, but the precise molecular mechanisms underlying its action in this organ remain poorly understood. We used microarray analysis to identify new target genes in brain. Thyroid hormone treatment of hypothyroid Pax8(-/-) knockout mice, which lack thyroid follicular cells, had a very limited global effect on brain transcripts. This analysis mainly identified cyclin D2 as a new thyroid hormone target gene in the cerebellum of hypothyroid mice. Thyroid hormone receptor (TRalpha and/or TRbeta) knockout mice studies provided further genetic evidence that cyclin D2 is likely to mediate the antiapoptotic effect exerted by thyroid hormone on the cerebellum external granular layer neuroblasts but that this transcriptional activation is not directly exerted by the thyroid hormone receptors.

Persistence of oligodendrocyte precursor cells and altered myelination in optic nerve associated to retina degeneration in mice devoid of all thyroid hormone receptors.

Thyroid hormone (3,5,3'-triiodo-l-thyronine or T3) exerts a pleiotropic activity during central nervous system development. Hypothyroidism during the fetal and postnatal life results in an irreversible mental retardation syndrome. At the cellular level, T3 is known to act on neuronal and glial lineages and to control cell proliferation, apoptosis, migration, and differentiation. Oligodendrocyte precursor cells (OPC) found at birth in the optic nerves are self-renewing cells that normally differentiate during the first 3 weeks of rodent postnatal life into postmitotic myelinating oligodendrocytes. In vitro, the addition of T3 to OPC is sufficient to trigger their terminal differentiation. The present analysis of T3 receptor knockout mice reveals that the absence of all T3 receptor results in the persistence of OPC proliferation in adult optic nerves, in a default in myelination, and sometimes in the degeneration of the retinal ganglion neurons. Thus, T3 signaling is necessary in vivo to promote the complete differentiation of OPC.