Quantitative proteomic analysis of skeletal muscles from wild type and transgenic mice carrying recessive Ryr1 mutations linked to congenital myopathies

Skeletal muscle is a highly structured and differentiated tissue responsible for voluntary movement and metabolic regulation. Muscles however, are heterogeneous and depending on their location, speed of contraction, fatiguability and function, can be broadly subdivided into fast and slow twitch as well as subspecialized muscles, with each group expressing common as well as specific proteins. Congenital myopathies are a group of non-inflammatory non-dystrophic muscle diseases caused by mutations in a number of genes, leading to a weak muscle phenotype. In most cases specific muscles types are affected, with preferential involvement of fast twitch muscles as well as extraocular and facial muscles. Here we performed relative and absolute quantitative proteomic analysis of EDL, soleus and extraocular muscles from wild type and transgenic mice carrying compound heterozygous mutations in Ryr1 identified in a patient with a severe congenital myopathy. Our quantitative proteomic study shows that recessive Ryr1 mutations not only decrease the content of RyR1 protein in muscle, but also impact the content of many other proteins; in addition, we provide important insight into the pathological mechanism of congenital myopathies linked to mutations in other genes encoding components of the excitation contraction coupling molecular complex

HIV-1 capsid-cyclophilin interactions determine nuclear import pathway, integration targeting and replication efficiency.

Lentiviruses such as HIV-1 traverse nuclear pore complexes (NPC) and infect terminally differentiated non-dividing cells, but how they do this is unclear. The cytoplasmic NPC protein Nup358/RanBP2 was identified as an HIV-1 co-factor in previous studies. Here we report that HIV-1 capsid (CA) binds directly to the cyclophilin domain of Nup358/RanBP2. Fusion of the Nup358/RanBP2 cyclophilin (Cyp) domain to the tripartite motif of TRIM5 created a novel inhibitor of HIV-1 replication, consistent with an interaction in vivo. In contrast to CypA binding to HIV-1 CA, Nup358 binding is insensitive to inhibition with cyclosporine, allowing contributions from CypA and Nup358 to be distinguished. Inhibition of CypA reduced dependence on Nup358 and the nuclear basket protein Nup153, suggesting that CypA regulates the choice of the nuclear import machinery that is engaged by the virus. HIV-1 cyclophilin-binding mutants CA G89V and P90A favored integration in genomic regions with a higher density of transcription units and associated features than wild type virus. Integration preference of wild type virus in the presence of cyclosporine was similarly altered to regions of higher transcription density. In contrast, HIV-1 CA alterations in another patch on the capsid surface that render the virus less sensitive to Nup358 or TRN-SR2 depletion (CA N74D, N57A) resulted in integration in genomic regions sparse in transcription units. Both groups of CA mutants are impaired in replication in HeLa cells and human monocyte derived macrophages. Our findings link HIV-1 engagement of cyclophilins with both integration targeting and replication efficiency and provide insight into the conservation of viral cyclophilin recruitment

Store-operated calcium entry in skeletal muscle: what makes it different?

Current knowledge on store-operated Ca2+ entry (SOCE) regarding its localization, kinetics, and regulation is mostly derived from studies performed in non-excitable cells. After a long time of relative disinterest in skeletal muscle SOCE, this mechanism is now recognized as an essential contributor to muscle physiology, as highlighted by the muscle pathologies that are associated with mutations in the SOCE molecules STIM1 and Orai1. This review mainly focuses on the peculiar aspects of skeletal muscle SOCE that differentiate it from its counterpart found in non-excitable cells. This includes questions about SOCE localization and the movement of respective proteins in the highly organized skeletal muscle fibers, as well as the diversity of expressed STIM isoforms and their differential expression between muscle fiber types. The emerging evidence of a phasic SOCE, which is activated during EC coupling, and its physiological implication is described as well. The specific issues related to the use of SOCE modulators in skeletal muscles are discussed. This review highlights the complexity of SOCE activation and its regulation in skeletal muscle, with an emphasis on the most recent findings and the aim to reach a current picture of this mesmerizing phenomenon.</p

During post-natal human myogenesis, normal myotube size requires TRPC1- and TRPC4-mediated Ca²⁺ entry

Myogenesis involves expression of muscle-specific transcription factors such as myogenin and myocyte enhancer factor 2 (MEF2), and is essentially regulated by fluctuations of cytosolic Ca(2+) concentration. Recently we demonstrated that molecular players of store-operated Ca(2+) entry (SOCE), stromal interacting molecule (STIM) and Orai, were fundamental in the differentiation process of post-natal human myoblasts. Besides STIM and Orai proteins, the family of transient receptor potential canonical (TRPC) channels was shown to be part of SOCE in several cellular systems. In the present study, we investigated the role of TRPC channels in the human myogenesis process. We demonstrate, using an siRNA strategy or dominant negative TRPC overexpression, that TRPC1 and TRPC4 participate in SOCE, are necessary for MEF2 expression, and allow the fusion process to generate myotubes of normal size. Conversely, the overexpression of STIM1 with TRPC4 or TRPC1 increased SOCE, accelerated myoblast fusion, and produced hypertrophic myotubes. Interestingly, in cells depleted of TRPC1 or TRPC4, the normalization of SOCE by increasing the extracellular calcium concentration or by overexpressing STIM1 or Orai1 was not sufficient to restore normal fusion process. A normal differentiation occurred only when TRPC channel was re-expressed. These findings indicate that Ca(2+) entry mediated specifically by TRPC1 and TRPC4 allow the formation of normal-sized myotubes

Thapsigargin activates Ca²+ entry both by store-dependent, STIM1/Orai1-mediated, and store-independent, TRPC3/PLC/PKC-mediated pathways in human endothelial cells

The ER Ca²+ sensor STIM1 and the Ca²+ channel Orai1 are key players in store-operated Ca²+ entry (SOCE). In addition, channels from the TRPC family were also shown to be engaged during SOCE, while their precise implication remains controversial. In this study, we investigated the molecular players involved in SOCE triggered by the SERCA pump inhibitor thapsigargin in an endothelial cell line, the EA.hy926. siRNA directed against STIM1 or Orai1 reduced Ca²+ entry by about 50-60%, showing that a large part of the entry is independent from these proteins. Blocking the PLC or the PKC pathway completely abolished thapsigargin-induced Ca²+ entry in cells depleted from STIM1 and/or Orai1. The phorbol ester PMA or the DAG analog OAG restored the Ca²+ entry inhibited by PLC blockers, showing an involvement of PLC/PKC pathway in SOCE. Using pharmacological inhibitors or siRNA revealed that the PKCeta is required for Ca²+ entry, and pharmacological inhibition of the tyrosine kinase Src also reduced Ca²+ entry. TRPC3 silencing diminished the entry by 45%, while the double STIM1/TRPC3 invalidation reduced Ca²+ entry by more than 85%. Hence, in EA.hy926 cells, TG-induced Ca²+ entry results from the activation of the STIM1/Orai1 machinery, and from the activation of TRPC3

Distinct roles of NFATc1 and NFATc4 in human primary myoblast differentiation and in the maintenance of reserve cells

Ca2+ signaling plays a key role during human myoblast differentiation. Among Ca2+-sensitive pathways, calcineurin is essential for myoblast differentiation and muscle regeneration. Nuclear factor of activated T-cell (NFAT) transcription factors are the major calcineurin targets. We investigated the expression and the role of each NFAT gene during human primary myoblast differentiation. We found that three NFAT isoforms are present, NFATc1, NFATc3 and NFATc4. Importantly, while their mRNA expression increases during differentiation, NFATc1 is more highly expressed in myotubes, whilst NFATc4 is specifically maintained in reserve cells. NFATc3 is present in both cell types, although no specific role during myoblast differentiation was observed. Knockdown of either NFATc1 or NFATc4 affects the differentiation process similarly, by decreasing the expression of late differentiation markers, but impairs myotube formation differently. Whereas NFATc1 knockdown strongly reduced the number and the surface area of myotubes, NFATc4 knockdown increased the surface area of myotubes and reduced the pool of reserve cells. We conclude that NFAT genes have specific roles in myotube formation and in the maintenance of the reserve cell pool during human postnatal myogenesis

Nanopattern surface improves cultured human myotube maturation

Background: In vitro maturation of human primary myoblasts using 2D culture remains a challenging process and leads to immature fibers with poor internal organization and function. This would however represent a valuable system to study muscle physiology or pathophysiology from patient myoblasts, at a single cell level. Methods: Human primary myoblasts were cultured on 800 nm width striated surface between two layers of Matrigel, and in a media supplemented with an inhibitor of TGFreceptor. Gene expression, immunofluorescence and Ca2+ measurements upon electrical stimulations were performed at various time points during maturation to assess the organization and function of the myotubes. Results: We show that after 10 days in culture, myotubes display numerous functional acetylcholine receptor clusters and express the adult isoforms of myosin heavy chain and dihydropyridine receptor. In addition, the myotubes are internally well organized with striations of -actinin and STIM1, and occasionally ryanodine receptor 1. We also demonstrate that the myotubes present robust Ca2+ responses to repetitive electrical stimulations. Conclusion: The present method describes a fast and efficient system to obtain well matured and functional myotubes in 2D culture allowing thorough analysis of single cell Ca2+ signals