TGF-β SIGNALLING IN SKELETAL MUSCLE ADAPTATION AND REGENERATION

TGF-β, myostatin and activin A signalling contributes to the progressive pathology of various muscle wasting disorders. Therefore, inhibition of TGF-β signalling may be a promising therapeutic approach to alleviate muscle pathologies and preserve muscle function. However, to date no successful therapeutic strategy based on TGF-β signalling inhibition has been developed yet. The aim of this thesis was to determine the role of TGF-β signalling via its type I receptors Acvr1b and Tgfbr1 in skeletal muscle physiology and regeneration. In chapter 2, we assessed time-dependent effects of TGF-β1 on gene expression in C2C12 myoblasts and myotubes. TGF-β1 induced Col1a1 expression was at least in part dependent on Ctgf and Fgf-2 expression. Knockdown by siRNA of Tgfbr1, but not Acvr1b, resulted in a reduction in fibrotic gene expression. These results indicate that, during muscle regeneration, TGF-β1 induces fibrosis via Tgfbr1 by stimulating the autocrine signalling of Ctgf and Fgf-2. TGF-β1 is well-known for its role in muscle fibrosis. However, TGF-β3 has been implicated to reduce scar formation and collagen production in skin and vocal mucosa. We hypothesised that TGF-β3 antagonises TGF-β1-induced fibrosis in muscle cells. In chapter 3 we examined the individual and combined effects of TGF-β1 and TGF-β3 on collagen expression in myoblasts and myotubes. After three days incubation with TGF-β1 and/or TGF-β3 myoblasts produce collagen in a dose independent manner. Collagen deposition was doubled in myotubes compared to myoblasts. As a consequence, TGF-β1 and/or TGF-β3 supplementation did not stimulate collagen production in myotubes any further. Taken together, these results indicate that in muscle cells, both TGF-β1 and TGF-β3 induce collagen production and TGF-β3 is ineffective to antagonize TGF-β1-induced collagen production. A myofibre specific conditional knockout mouse model was created to examine the individual and combined effects of Acvr1b and Tgfbr1 inhibition in vivo. In chapter 4 we show that 5 weeks of simultaneous knockout of Acvr1b and Tgfbr1 induces substantial muscle hypertrophy, while individual receptor knockout has no or modest effects on muscle mass. Hypertrophic effects are most substantial in type IIB myofibres, while myonuclear number per type IIB myofibre remains unaltered. Lack of both Acvr1b and Tgfbr1 increases the number of satellite cells per myofibre. During cardiotoxin-induced injury myogenic gene expression and CSA of regenerating myofibres are increased in muscles that lack both receptors, which may indicate enhanced regeneration. Gene expression of extracellular matrix (ECM) components is exclusively elevated in muscle with combined receptor knockout, both in intact muscle and after cardiotoxin induced injury. Taken together, Tgfbr1 and Acvr1b are synergistically involved in regulation of myofibre size and muscle regeneration, while myofibre specific receptor knockout increases rather than decreases ECM gene expression. In chapter 5 we tested effects of individual and simultaneous myofibre specific Acvr1b and Tgfbr1 receptor knockout for three months on force generating capacity and phenotype of both fast, glycolytic gastrocnemius medialis (GM) and slow, oxidative soleus muscle. Simultaneously blocking Acvr1b and Tgfbr1 induces substantial hypertrophy in GM and an increase in force generating capacity, but reduces specific force. In contrast, in soleus simultaneously blocking Acvr1b and Tgfbr1 induces moderate hypertrophy, accompanied by a similar increase in force generating capacity. In both muscle phenotypes lack of both Acvr1b and Tgfbr1 an increase in myofibre size is accompanied by an increase in mitochondrial enzyme activity, which indicates that these receptors are involved in both the adaptation of muscle size and oxidative capacity, which are usually mutually exclusive. Together these results indicate that lack of both Acvr1b and Tgfbr1 particularly has a beneficial effect on force generating capacity in slow, oxidative muscles and simultaneous receptor knockout may increase both muscle size and oxidative capacity.