We identified two critical events that

We identified two critical events that might be associated with pathomechanism collagen VI deficiency in our mice. The first event is a non-cell-autonomous effect from the collagen VI deficiency, causing the dysregulation in the number of myofibers in a muscle. In normal control mice, myofibers increase in number during neonatal growth period until 3weeks of age. During this period is also the time when skeletal muscles have reached reconstitution of extracellular matrix, in parallel with muscle growth, before building up robust and endurable contractile structures in mature muscle. In our model, total number of myonuclei within myofibers in a whole section was not decreased (Fig. 4c) suggesting that the cells, which involved in muscle construction (myogenic cells), were not affected. We also observed the decreased number of membrane indentation within myofibers in Col6a1GT/GT muscles in 2weeks of age (Fig. 4d). These findings clearly suggest that the dysregulation in the myofiber number in collagen VI deficient muscle may be not due to a reduction of the number of myogenic cells, but due to a defect in myofiber splitting/branching which would then lead to an increment of myofiber number. We demonstrated that the IGF-1 signaling pathway is insufficiently activated, leading to lower serum IGF-1 levels and impaired activation of downstream signaling. It has been only reported that Grb10 is a determinant factor for the number of myofibers in a muscle (Holt et al., 2012). Grb10 is an adaptor protein that links the receptor tyrosine kinase to downstream signal mediators on IGF-1 signaling, and negatively regulates the IGF signaling by enhancing the internalization and degradation of the receptor kinase (Vecchione et al., 2003). Our finding of the insufficient activation of IGF-1 signaling pathway in Col6a1GT/GT muscle during perinatal development may suggest that the impairment of growth factor-mediated signal transduction leads to defects in myofiber growth and splitting resulted in fewer myofiber numbers. The second event is a cell-autonomous influence by skeletal muscle-resident MPCs in promoting PF-3758309 fibrosis. PDGFRα-positive MPCs produce fibrosis and ectopic fat tissue at the late stage of muscular dystrophy (Uezumi et al., 2010, 2011), can be a source of extracellular matrix proteins in skeletal muscles (Ito et al., 2013), and can provide the environment “normal muscle niche” for myofibers (Joe et al., 2010). In this study, we characterized PDGFRα-positive MPCs as a source of collagen VI which may be comparable to the fibroblastic cells identified as an origin of collagen VI in human muscles and also as being positive for some fibroblastic markers, vimentin and FAP, fibroblast activation protein (data not shown). In both Col6a1GT/GT mice and human UCMD muscles, we observed that there is a lot of MPCs with elongated morphology; moreover, the MPCs in Col6a1GT/GT mice can rapidly proliferate and contribute to fibrosis in response to CTXi on muscle necrosis. Spontaneous activation of MPC in collagen VI deficiency remains to be clarified, because we did not find alteration of IL-4 expression in muscles in Col6a1GT/GT mice. It has been suggested that the MPCs are suppressed by the physical interaction with muscle fibers (Uezumi et al., 2011); it may be conceivable that collagen VI can act as a scaffold to allow interaction between MPCs and myofibers and therefor help maintain normal muscle environment by MPC suppression. The interaction between satellite cells and MPCs, which are also identified as Tcf4+ muscle connective tissue fibroblasts (Murphy et al., 2011), is crucial for muscle regeneration. When muscle necrosis ensues, MPCs lose the interaction with myofibers, hence are activated. In a response to injury, MPCs undergo several steps to initiate a cascade of repair: MPCs phagocytose the necrotic debris and secrete cytokines (IL-6, IGF-1) and ECM proteins; then MPCs assist satellite cells to promote correct myogenic regeneration; after myofiber regeneration, MPCs become inactivated as the amount of connective tissue is diminished (Heredia et al., 2013). During muscle regeneration, ablation of Tcf4+ fibroblasts leads to premature satellite cell differentiation, leading to the formation of early regenerating myofibers with smaller diameter at the point of completion of muscle regeneration (Murphy et al., 2011). In contrast, the Col6a1GT/GT mice show a remarkable increase in MPCs on 1day after muscle injury and a persistence of muscle regeneration as large and multiple nucleated myofibers are seen. The number of proliferating satellite cells in Col6a1GT/GT muscles, however, was similar to that of normal control. Thus in the absence of collagen VI, the signal (from the myofiber) to suppress MPCs is decreased during muscle necrosis, while during myofiber regeneration, the MPC signals needed to promote induction of satellite cell differentiation are dysregulated. In addition to defects in muscle regeneration, MPCs also contribute to the fibrotic cell population.