Skeletal Muscle Triads Research Articles

Loss-of-function variants in JPH1 cause congenital myopathy with prominent facial and ocular involvement

BackgroundWeakness of facial, ocular and axial muscles is a common clinical presentation in congenital myopathies caused by pathogenic variants in genes encoding triad proteins. Abnormalities in triad structure and function...

Speg interactions that regulate the stability of excitation-contraction coupling protein complexes in triads and dyads

Here we show that striated muscle preferentially expressed protein kinase α (Spegα) maintains cardiac function in hearts with Spegβ deficiency. Speg is required for stability of excitation-contraction coupling (ECC) complexes and interacts with esterase D (Esd), Cardiomyopathy-Associated Protein 5 (Cmya5), and Fibronectin Type III and SPRY Domain Containing 2 (Fsd2) in cardiac and skeletal muscle. Mice with a sequence encoding a V5/HA tag inserted into the first exon of the Speg gene (HA-Speg mice) display a >90% decrease in Spegβ but Spegα is expressed at ~50% of normal levels. Mice deficient in both Spegα and Speg β (Speg KO mice) develop a severe dilated cardiomyopathy and muscle weakness and atrophy, but HA-Speg mice display mild muscle weakness with no cardiac involvement. Spegα in HA-Speg mice suppresses Ca2+ leak, proteolytic cleavage of Jph2, and disruption of transverse tubules. Despite it’s low levels, HA-Spegβ immunoprecipitation identified Esd, Cmya5 and Fsd2 as Spegβ binding partners that localize to triads and dyads to stabilize ECC complexes. This study suggests that Spegα and Spegβ display functional redundancy, identifies Esd, Cmya5 and Fsd2 as components of both cardiac dyads and skeletal muscle triads and lays the groundwork for the identification of new therapeutic targets for centronuclear myopathy.

STAC3 incorporation into skeletal muscle triads occurs independent of the dihydropyridine receptor.

Excitation‐contraction (EC) coupling in skeletal muscles operates through a physical interaction between the dihydropyridine receptor (DHPR), acting as a voltage sensor, and the ryanodine receptor (RyR1), acting as a calcium release channel. Recently, the adaptor protein SH3 and cysteine‐rich containing protein 3 (STAC3) has been identified as a myopathy disease gene and as an additional essential EC coupling component. STAC3 interacts with DHPR sequences including the critical EC coupling domain and has been proposed to function in linking the DHPR and RyR1. However, we and others demonstrated that incorporation of recombinant STAC3 into skeletal muscle triads critically depends only on the DHPR but not the RyR1. On the contrary, here, we provide evidence that endogenous STAC3 incorporates into triads in the absence of the DHPR in myotubes and muscle fibers of dysgenic mice. This finding demonstrates that STAC3 interacts with additional triad proteins and is consistent with its proposed role in directly or indirectly linking the DHPR with the RyR1.

STAC proteins associate to the IQ domain of CaV1.2 and inhibit calcium-dependent inactivation

The adaptor proteins STAC1, STAC2, and STAC3 represent a newly identified family of regulators of voltage-gated calcium channel (CaV) trafficking and function. The skeletal muscle isoform STAC3 is essential for excitation-contraction coupling and its mutation causes severe muscle disease. Recently, two distinct molecular domains in STAC3 were identified, necessary for its functional interaction with CaV1.1: the C1 domain, which recruits STAC proteins to the calcium channel complex in skeletal muscle triads, and the SH3-1 domain, involved in excitation-contraction coupling. These interaction sites are conserved in the three STAC proteins. However, the molecular domain in CaV1 channels interacting with the STAC C1 domain and the possible role of this interaction in neuronal CaV1 channels remained unknown. Using CaV1.2/2.1 chimeras expressed in dysgenic (CaV1.1-/-) myotubes, we identified the amino acids 1,641-1,668 in the C terminus of CaV1.2 as necessary for association of STAC proteins. This sequence contains the IQ domain and alanine mutagenesis revealed that the amino acids important for STAC association overlap with those making contacts with the C-lobe of calcium-calmodulin (Ca/CaM) and mediating calcium-dependent inactivation of CaV1.2. Indeed, patch-clamp analysis demonstrated that coexpression of either one of the three STAC proteins with CaV1.2 opposed calcium-dependent inactivation, although to different degrees, and that substitution of the CaV1.2 IQ domain with that of CaV2.1, which does not interact with STAC, abolished this effect. These results suggest that STAC proteins associate with the CaV1.2 C terminus at the IQ domain and thus inhibit calcium-dependent feedback regulation of CaV1.2 currents.

STAC3 stably interacts through its C1 domain with CaV1.1 in skeletal muscle triads

The adaptor protein STAC3 is essential for skeletal muscle excitation-contraction (EC) coupling and a mutation in the STAC3 gene has been linked to a severe muscle disease, Native American myopathy (NAM). However the function of STAC3, its interaction partner, and the mode of interaction within the EC-coupling complex remained elusive. Here we demonstrate that STAC3 forms a stable interaction with the voltage-sensor of EC-coupling, CaV1.1, and that this interaction depends on a hitherto unidentified protein-protein binding pocket in the C1 domain of STAC3. While the NAM mutation does not affect the stability of the STAC3-CaV1.1 interaction, mutation of two crucial residues in the C1 binding pocket increases the turnover of STAC3 in skeletal muscle triads. Thus, the C1 domain of STAC3 is responsible for its stable incorporation into the CaV1.1 complex, whereas the SH3 domain containing the NAM mutation site may be involved in low-affinity functional interactions in EC-coupling.

Skeletal muscle triad junction ultrastructure by Focused-Ion-Beam milling of muscle and Cryo-Electron Tomography.

Cryo-electron tomography (cryo-ET) has emerged as perhaps the only practical technique for revealing nanometer-level three-dimensional structural details of subcellular macromolecular complexes in their native context, inside the cell. As currently practiced, the specimen should be 0.1-0.2 microns in thickness to achieve optimal resolution. Thus, application of cryo-ET to intact frozen (vitreous) tissues, such as skeletal muscle, requires that they be sectioned. Cryo-ultramicrotomy is notoriously difficult and artifact-prone when applied to frozen cells and tissue, but a new technique, focused ion beam milling (cryo-FIB), shows great promise for “thinning” frozen biological specimens. Here we describe our initial results in applying cryo-FIB and cryo-ET to triad junctions of skeletal muscle.

Skeletal muscle triad junction ultrastructure by Focused-Ion-Beam milling of muscle and Cryo-Electron Tomography

Skeletal muscle triad junction ultrastructure by Focused-Ion-Beam milling of muscle and Cryo-Electron Tomography

Unfolded Protein Response and Triad Formation in Skeletal Muscles of Catecholaminergic Polymorphic Ventricular Tachycardia Mouse / Odgovor Razvijenog Proteina I Formiranje Trijada U Skeletnim Mišićima Miševa Sa Kateholaminergičkom Polimorfnom Ventrikularnom Tahikardijom

Summary Isoform 2 of calsequestrin (CASQ2) is the main calcium-binding protein of sarcoplasmic reticulum (SR), expressed both in cardiac and in skeletal muscles. CASQ2 acts as an SR calcium (Ca2+) sensor and regulates SR Ca2+ release via interactions with triadin, junctin, and the ryanodine receptor. Various mutations of the csq2 gene lead to altered Ca2+ release and contractile dysfunction contributing to the development of arrhythmias and sudden cardiac death in young individuals affected by catecholaminergic polymorphic ventricular tachycardia (CPVT). Recently, a transgenic mouse carrying one of the identified CASQ2 point-mutations (R33Q) associated to CPVT was developed and a drastic reduction of the mutated protein was observed. Following a biomolecular approach, several analysis were performed using different antibody treatments in order to identify when the reduction of CASQ2 begins in skeletal muscles, unveil the mechanism involved in the reduction of CASQ2 in slow-twitch and fast twitch muscles and verify if other proteins are affected by the presence of the mutated protein. Mutated CASQ2 decreased soon after birth. Up-regulation of proteins associated to the unfolded protein response (UPR) was also observed. Important proteins in skeletal muscle triads formation were analyzed and increased protein levels were observed in adult knock-in CASQ2-R33Q/R33Q mice. Probably, R33Q mutation induced the decrease of CASQ2 by activation of the UPR and subsequently degradation through proteasome.

Altered Splicing of the BIN1 Muscle-Specific Exon in Humans and Dogs with Highly Progressive Centronuclear Myopathy

Amphiphysin 2, encoded by BIN1, is a key factor for membrane sensing and remodelling in different cell types. Homozygous BIN1 mutations in ubiquitously expressed exons are associated with autosomal recessive centronuclear myopathy (CNM), a mildly progressive muscle disorder typically showing abnormal nuclear centralization on biopsies. In addition, misregulation of BIN1 splicing partially accounts for the muscle defects in myotonic dystrophy (DM). However, the muscle-specific function of amphiphysin 2 and its pathogenicity in both muscle disorders are not well understood. In this study we identified and characterized the first mutation affecting the splicing of the muscle-specific BIN1 exon 11 in a consanguineous family with rapidly progressive and ultimately fatal centronuclear myopathy. In parallel, we discovered a mutation in the same BIN1 exon 11 acceptor splice site as the genetic cause of the canine Inherited Myopathy of Great Danes (IMGD). Analysis of RNA from patient muscle demonstrated complete skipping of exon 11 and BIN1 constructs without exon 11 were unable to promote membrane tubulation in differentiated myotubes. Comparative immunofluorescence and ultrastructural analyses of patient and canine biopsies revealed common structural defects, emphasizing the importance of amphiphysin 2 in membrane remodelling and maintenance of the skeletal muscle triad. Our data demonstrate that the alteration of the muscle-specific function of amphiphysin 2 is a common pathomechanism for centronuclear myopathy, myotonic dystrophy, and IMGD. The IMGD dog is the first faithful model for human BIN1-related CNM and represents a mammalian model available for preclinical trials of potential therapies.

Samaritan myopathy, an ultimately benign congenital myopathy, is caused by a RYR1 mutation

Congenital myopathies describe a group of inherited muscle disorders with neonatal or infantile onset typically associated with muscle weakness, respiratory involvement and delayed motor milestones. We previously reported a novel congenital myopathy in an inbred Samaritan family. All patients displayed severe neonatal hypotonia and respiratory distress, and unlike other congenital myopathies, a constantly improving health status. As clinical and pathological data did not point to preferential candidate genes, we performed exome sequencing complemented by linkage analysis to identify the mutation causing the benign Samaritan congenital myopathy. We identified the homozygous p.Tyr1088Cys mutation in RYR1, encoding the skeletal muscle ryanodine receptor. This sarcoplasmic reticulum calcium channel is a key regulator of excitation-contraction coupling (ECC). Western blot and immunohistofluorescence revealed a significant decrease of the RYR1 protein level and an abnormal organization of skeletal muscle triad markers as caveolin-3, dysferlin and amphiphysin 2. RYR1 mutations are associated with different myopathies and malignant hyperthermia susceptibility. The index patient had mild hyperthermia following anesthesia, indicating that the inbred Samaritan population might be a risk group for this disorder. Our results suggest an aberrant ECC as the primary cause of this disease, and broaden the clinical consequences of RYR1 defects.

Dynamics of Homologous and Heterologous Calcium Channel α1 and β Subunits in the Skeletal Muscle Triad

Skeletal muscle EC-coupling requires direct interactions of the dihydropyridine receptor (DHPR) with the ryanodine receptor (RyR1) and its organization in tetrads opposite RyR1. Accordingly, tetrad formation and skeletal muscle EC-coupling fail in myotubes expressing the cardiac α1C subunit or lacking the β1a subunit. This indicates that tissue-specific protein-protein interactions between α1S, β1a and RyR1 are important for the structure and function of the skeletal muscle EC-coupling apparatus. Here we apply fluorescence recovery after photobleaching (FRAP) in dysgenic (α1S-/-) myotubes reconstituted with GFP-tagged DHPR subunits to probe the importance of these interactions in stabilizing them in the triad. Consistent with its anchoring in the triad, the α1S subunit is essentially immobile. Surprisingly, also the cardiac α1C subunit, which does not interact with RyR1, is immobile. Thus, the II-III loop - RyR1 interaction is not the determing factor for stabilizing α1S in the triad. When coexpressed with α1S, the β1a subunit showed a higher fluorescence recovery rate than the α1 subunits, suggesting that it can be dynamically exchanged in the complex. Again, the recovery did not increase when β1a was coexpressed with α1C, indicating that its putative interaction with RyR1 does not contribute to the stability of β1a in the complex. Previously we showed that all βisoforms can associate with α1S and α1C in the triads. When the non-muscle β2a or β4b subunits were expressed with α1S in dysgenic myotubes their recovery after photobleaching was significantly higher than that of β1a, demonstrating that the homologous β subunit binds the channel complex with higher affinity than heterologous β subunits. Funding: FWF P20059, W1101, T443-B18.

New factors contributing to dynamic calcium regulation in the skeletal muscle triad-a crowded place.

Skeletal muscle is a highly organized tissue that has to be optimized for fast signalling events conveying electrical excitation to contractile response. The site of electro-chemico-mechanical coupling is the skeletal muscle triad where two membrane systems, the extracellular t-tubules and the intracellular sarcoplasmic reticulum, come into very close contact. Structure fits function here and the signalling proteins DHPR and RyR1 were the first to be discovered to bridge this gap in a conformational coupling arrangement. Since then, however, new proteins and more signalling cascades have been identified just in the last decade, adding more diversity and fine tuning to the regulation of excitation-contraction coupling (ECC) and control over Ca2+ store content. The concept of Ca2+ entry into working skeletal muscle has become attractive again with the experimental evidence summarized in this review. Store-operated Ca2+ entry (SOCE), excitation-coupled Ca2+ entry (ECCE), action-potential-activated Ca2+ current (APACC), and retrograde EC-coupling (ECC) are new concepts additional to the conventional orthograde ECC; they have provided fascinating new insights into muscle physiology. In this review, we discuss the discovery of these pathways, their potential roles, and the signalling proteins involved that show that the triad may become a crowded place in time.

Does the voltage‐gated calcium channel α2δ‐1 subunit play a dual function in skeletal muscle?

The dihydropyridine receptor (DHPR), a high voltage-activated calcium (Ca2+) channel, represents a key element in the excitation–contraction (EC) coupling machinery of muscle cells. In skeletal muscle, the DHPR fulfils two known functions: (i) it controls the ryanodine receptor activity, which makes it commonly referred to as the voltage-sensor for EC coupling and (ii) it supports an L-type voltage-activated Ca2+ current. The DHPR is a macromolecular complex composed of the Cav1.1 pore-forming subunit, surrounded by β1a, γ1 and α2δ-1 auxiliary subunits. Whereas knock-out animals have clearly demonstrated the essential role of Cav1.1 and β1a subunits in EC coupling and in the control of the Ca2+ conductance, the functional importance of α2δ-1 in skeletal muscle remains largely elusive, despite the lethal phenotype of α2δ-1-null embryos. Interestingly, down-regulation of the α2δ-1 protein in the BC3H1 muscle cell line with siRNA only resulted in acceleration of Ca2+ current activation kinetics with no effect on both EC coupling and Cav1.1 trafficking (Obermair et al. 2005). However, it is hard to understand how alteration of Ca2+ current kinetics could be the cause of the lethal phenotype observed in α2δ-1-null mice embryos; instead α2δ-1 is most likely to be involved in other cell functions independent of the DHPR activity. Previous observations suggested such a function: (i) α2δ-1 appears early during muscle development, at a period when the Cav1.1 subunit is not yet expressed, and (ii) sequence analysis of α2δ-1 revealed the presence of consensus domains, known to be involved in cell adhesion and protein–protein interactions (see Fig. 1 for structural organization of the α2δ-1 subunit). In a recent issue of The Journal of Physiology, Garcia et al. raised the possibility that α2δ-1 could mediate the interaction of muscle cells with the extracellular matrix (Garcia et al. 2008). Using immunohistochemistry and a siRNA knock-down strategy, the authors provided new insights into the functional involvement of α2δ-1 in skeletal muscle cells.

Conformation-dependent Stability of Junctophilin 1 (JP1) and Ryanodine Receptor Type 1 (RyR1) Channel Complex Is Mediated by Their Hyper-reactive Thiols

Junctophilin 1 (JP1), a 72-kDa protein localized at the skeletal muscle triad, is essential for stabilizing the close apposition of T-tubule and sarcoplasmic reticulum membranes to form junctions. In this study we report that rapid and selective labeling of hyper-reactive thiols found in both JP1 and ryanodine receptor type 1 (RyR1) with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin, a fluorescent thiol-reactive probe, proceeded 12-fold faster under conditions that minimize RyR1 gating (e.g. 10 mM Mg2+) compared with conditions that promote high channel activity (e.g. 100 microM Ca2+, 10 mM caffeine, 5 mM ATP). The reactivity of these thiol groups was very sensitive to oxidation by naphthoquinone, H2O2, NO, or O2, all known modulators of the RyR1 channel complex. Using preparative SDS-PAGE, in-gel tryptic digestion, high pressure liquid chromatography, and mass spectrometry-based peptide sequencing, we identified 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin-thioether adducts on three cysteine residues of JP1 (101, 402, and 627); the remaining five cysteines of JP1 were unlabeled. Co-immunoprecipitation experiments demonstrated a physical interaction between JP1 and RyR1 that, like thiol reactivity, was sensitive to RyR1 conformation and chemical status of the hyper-reactive cysteines of JP1 and RyR1. These findings support a model in which JP1 interacts with the RyR1 channel complex in a conformationally sensitive manner and may contribute integral redox-sensing properties through reactive sulfhydryl chemistry.

In Hot Pursuit

In Hot Pursuit

Triadins Are Not Triad-specific Proteins: TWO NEW SKELETAL MUSCLE TRIADINS POSSIBLY INVOLVED IN THE ARCHITECTURE OF SARCOPLASMIC RETICULUM

We have cloned two new triadin isoforms from rat skeletal muscle, Trisk 49 and Trisk 32, which were named according to their theoretical molecular masses (49 and 32 kDa, respectively). Specific antibodies directed against each protein were produced to characterize both new triadins. Both are expressed in adult rat skeletal muscle, and their expression in slow twitch muscle is lower than that in fast twitch muscle. Using double immunofluorescent labeling, the localization of these two triadins was studied in comparison to well-characterized proteins such as ryanodine receptor, calsequestrin, desmin, Ca(2+)-ATPase, and titin. None of these two triadins are localized within the rat skeletal muscle triad. Both are instead found in different parts of the longitudinal sarcoplasmic reticulum. We attempted to identify partners for each isoform: neither is associated with ryanodine receptor; Trisk 49 could be associated with titin or another sarcomeric protein; and Trisk 32 could be associated with IP(3) receptor. These results open further fields of research concerning the functions of these two proteins; in particular, they could be involved in the set up and maintenance of a precise sarcoplasmic reticulum structure.

Electron Tomography of Frozen-Hydrated Isolated Triad Junctions

Cryoelectron microscopy and tomography have been applied for the first time to isolated, frozen-hydrated skeletal muscle triad junctions (triads) and terminal cisternae (TC) vesicles derived from sarcoplasmic reticulum. Isolated triads were selected on the basis of their appearance as two spherical TC vesicles attached to opposite sides of a flattened vesicle derived from a transverse tubule (TT). Foot structures (ryanodine receptors) were resolved within the gap between the TC vesicles and TT vesicles, and some residual ordering of the receptors into arrays was apparent. Organized dense layers, apparently containing the calcium-binding protein calsequestrin, were found in the lumen of TC vesicles underlying the foot structures. The lamellar regions did not directly contact the sarcoplasmic reticulum membrane, thereby creating an ≈5-nm-thick zone that potentially constitutes a subcompartment for achieving locally elevated [Ca 2+ ] in the immediate vicinity of the Ca 2+-conducting ryanodine receptors. The lumen of the TT vesicles contained globular mass densities of unknown origin, some of which form cross-bridges that may be responsible for the flattened appearance of the transverse tubules when viewed in cross-section. The spatial relationships among the TT membrane, ryanodine receptors, and calsequestrin-containing assemblage are revealed under conditions that do not use dehydration, heavy-metal staining, or chemical fixation, thus exemplifying the potential of cryoelectron microscopy and tomography to reveal structural detail of complex subcellular structures.

Cooperation of two-domain Ca(2+) channel fragments in triad targeting and restoration of excitation- contraction coupling in skeletal muscle.

The specific incorporation of the skeletal muscle voltage-dependent Ca(2+) channel in the triad is a prerequisite of normal excitation-contraction (EC) coupling. Sequences involved in membrane expression and in targeting of Ca(2+) channels into skeletal muscle triads have been described in different regions of the alpha(1S) subunit. Here we studied the targeting properties of two-domain alpha(1S) fragments, green fluorescent protein (GFP)-I x II (1-670) and III x IV (691-1873) expressed alone or in combination in dysgenic (alpha(1S)-null) myotubes. Immunofluorescence analysis showed that GFP-I x II or III x IV expressed separately were not targeted into triads. In contrast, on coexpression the two alpha(1S) fragments were colocalized with one another and with the ryanodine receptor in the triads. Coexpression of GFP-I x II and III x IV also fully restored Ca(2+) currents and depolarization-induced Ca(2+) transients, despite the severed connection between the two channel halves and the absence of amino acids 671-690 from either alpha(1S) fragment. Thus, triad targeting, like the rescue of function, requires the cooperation and coassembly of the two complementary channel fragments. Transferring the C terminus of alpha(1S) to the N-terminal two-domain fragment (GFP-I x II x tail), or transferring the I-II connecting loop containing the beta interaction domain to the C-terminal fragment (III x IV x beta in) did not improve the targeting properties of the individually expressed two-domain channel fragments. Thus, the cooperation of GFP-I.II and III.IV in targeting cannot be explained solely by a sequential action of the beta subunit by means of the I-II loop in releasing the channel from the sarcoplasmic reticulum and of the C terminus in triad targeting.

Deficiency of triad formation in developing skeletal muscle cells lacking junctophilin type 1

Junctophilins (JP-1, JP-2, and JP-3) are transmembrane proteins expressed in the junctional membrane complexes in excitable cells. Both JP-1 and JP-2 are co-expressed in the triads of skeletal muscle, but only JP-2 is expressed in cardiac muscle. We analyzed the roles played by JP-1 and JP-2 in triad formation in skeletal muscle by comparing developing skeletal muscles in wild-type and JP-1-knockout (KO) mice (both before and after birth). In the skeletal muscles of embryos, most of the couplings between sarcoplasmic reticulum (SR) and transverse tubule (T-tubule) were diads, with triads being very scarce. The number of triads increased markedly after birth in wild-type mice. However, there was no increase in the number of triads in the neonates of JP-1-KO mice, and they died within 1 day after birth. JP-2 expression was constant before and after birth, while expression of JP-1 increased with birth. Quantitative and morphological differences were not seen between wild-type and JP-1-KO mice in the formation of diads in the period just before the JP-1-KO mice died. The SR swelled and developed large vacuoles in skeletal muscle cells just before the JP-1-KO mice died. The present results strongly suggest that JP-1 and JP-2 play important roles in the formation of triads and diads, respectively, during the development of skeletal muscle in mouse.

Asymmetric Arrangement of Auxiliary Subunits of Skeletal Muscle Voltage-Gated L-Type Ca2+ Channel

Highly purified L-type Ca2+ channel complexes containing all five subunits (α1, α2, β, γ, and δ) and complexes of α1-β subunits were obtained from skeletal muscle triad membranes by three-step purification and by 1% Triton X-100 treatment, respectively. Their structures and the subunit arrangements were analyzed by electron microscopy. Projection images of negatively stained Ca2+ channels and α1-β complexes were aligned, classified and averaged. The α1-β complex showed a hollow trapezoid shape of 12 nm height. In top view, four asymmetric domains surrounded a central depression predicted to form the channel pore. The complete Ca2+ channel complex exhibited the cylindrical shape of 20 nm in height binding a spherical domain on one edge. Further image analysis of higher complexes of the Ca2+ channel using a monoclonal antibody against the β subunit showed that the α1-β complex forms the non-decorated side of the cylinder, which can traverse the membrane from outside the cell to the cytoplasm. Based on these results, we propose that the Ca2+ channel exhibits an asymmetric arrangement of auxiliary subunits.