Myostatin inhibition promotes fast fibre hypertrophy but causes loss of AMP-activated protein kinase signalling and poor exercise tolerance in a model of limb-girdle muscular dystrophy R1/2A.

KEY POINTS: Limb-girdle muscular dystrophy R1 (LGMD R1) is caused by mutations in the CAPN3 gene and is characterized by progressive muscle loss, impaired mitochondrial function and reductions in the slow oxidative gene expression programme. Myostatin is a negative regulator of muscle growth, and its inhibition improves the phenotype in several muscle wasting disorders. The effect of genetic and pharmacological inhibition of myostatin signalling on the disease phenotype in a mouse model of LGMD R1 (CAPN3 knockout mouse-C3KO) was studied. Inhibition of myostatin signalling in C3KO muscles resulted in significant muscle hypertrophy; however, there were no improvements in muscle strength and exacerbation of exercise intolerance concomitant with further reduction of muscle oxidative capacity was observed. Inhibition of myostatin signalling is unlikely to be a valid therapeutic strategy for LGMD R1. ABSTRACT: Limb-girdle muscular dystrophy R1 (LGMD R1) is caused by mutations in the CAPN3 gene and is characterized by progressive muscle loss, impaired mitochondrial function and reductions in the slow oxidative gene expression programme. There are currently no therapies available to patients. We sought to determine if induction of muscle growth, through myostatin inhibition, represents a viable therapeutic strategy for this disease. Myostatin is a negative regulator of muscle growth, and its inhibition improves the phenotype in several muscle wasting disorders. However, the effect of myostatin depends on the genetic and pathophysiological context and may not be efficacious in all contexts. We found that genetic inhibition of myostatin through overexpression of follistatin (an endogenous inhibitor of myostatin) in our LGMD R1 model (C3KO) resulted in 1.5- to 2-fold increase of muscle mass for the majority of limb muscles. However, muscle strength was not improved and exercise intolerance was exacerbated. Pharmacological inhibition of myostatin, using an anti-myostatin antibody, resulted in statistically significant increases in muscle mass; however, functional testing did not reveal changes in muscle strength nor endurance in treated C3KO mice. Histochemical and biochemical evaluation of follistatin overexpressing mice revealed a reduction in the percentage of oxidative fibres and decreased activation of AMP-activated protein kinase signalling in transgenics compared to C3KO muscles. Our data suggest that muscle hypertrophy, induced by myostatin inhibition, leads to loss of oxidative capacity, which further compromises metabolically impaired C3KO muscles and thus is unlikely to be a valid strategy for treatment of LGMD R1.

Spp1 (osteopontin) promotes TGFß processing in fibroblasts of dystrophin-deficient muscles through matrix metalloproteinases.

Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding dystrophin. Prior work has shown that DMD progression can vary, depending on the genetic makeup of the patient. Several modifier alleles have been identified including LTBP4 and SPP1. We previously showed that Spp1 exacerbates the DMD phenotype in the mdx mouse model by promoting fibrosis and by skewing macrophage polarization. Here, we studied the mechanisms involved in Spp1's promotion of fibrosis by using both isolated fibroblasts and genetically modified mice. We found that Spp1 upregulates collagen expression in mdx fibroblasts by enhancing TGFß signaling. Spp1's effects on TGFß signaling are through induction of MMP9 expression. MMP9 is a protease that can release active TGFß ligand from its latent complex. In support for activation of this pathway in our model, we showed that treatment of mdx fibroblasts with MMP9 inhibitor led to accumulation of the TGFß latent complex, decreased levels of active TGFß and reduced collagen expression. Correspondingly, we found reduced active TGFß in Spp1-/-mdxB10 and Mmp9-/-mdxB10 muscles in vivo. Taken together with previous observations of reduced fibrosis in both models, these data suggest that Spp1 acts upstream of TGFß to promote fibrosis in mdx muscles. We found that in the context of constitutively upregulated TGFß signaling (such as in the mdxD2 model), ablation of Spp1 has very little effect on fibrosis. Finally, we performed proof-of-concept studies showing that postnatal pharmacological inhibition of Spp1 reduces fibrosis and improves muscle function in mdx mice.