Osteopontin ablation ameliorates muscular dystrophy by shifting macrophages to a pro-regenerative phenotype.

In the degenerative disease Duchenne muscular dystrophy, inflammatory cells enter muscles in response to repetitive muscle damage. Immune factors are required for muscle regeneration, but chronic inflammation creates a profibrotic milieu that exacerbates disease progression. Osteopontin (OPN) is an immunomodulator highly expressed in dystrophic muscles. Ablation of OPN correlates with reduced fibrosis and improved muscle strength as well as reduced natural killer T (NKT) cell counts. Here, we demonstrate that the improved dystrophic phenotype observed with OPN ablation does not result from reductions in NKT cells. OPN ablation skews macrophage polarization toward a pro-regenerative phenotype by reducing M1 and M2a and increasing M2c subsets. These changes are associated with increased expression of pro-regenerative factors insulin-like growth factor 1, leukemia inhibitory factor, and urokinase-type plasminogen activator. Furthermore, altered macrophage polarization correlated with increases in muscle weight and muscle fiber diameter, resulting in long-term improvements in muscle strength and function in mdx mice. These findings suggest that OPN ablation promotes muscle repair via macrophage secretion of pro-myogenic growth factors

Adhesion-contractile balance in myocyte differentiation

Tissue cells generally pull on their matrix attachments and balance a quasi-static contractility against adequate adhesion, but any correlation with and/or influence on phenotype are not yet understood. Here, we begin to demonstrate how differentiation state couples to actomyosin-based contractility through adhesion and substrate compliance. Myotubes are differentiated from myoblasts on collagen-patterned coverslips that allow linear fusion but prevent classic myotube branching. Postfusion, myotubes adhere to the micro-strips but lock into a stress fiber-rich state and do not differentiate significantly further. In contrast, myotubes grown on top of such cells do progress through differentiation, exhibiting actomyosin striations within one week. A compliant adhesion to these lower cells is suggested to couple to contractility and accommodate the reorganization needed for upper cell striation. Contractility is assessed in these adherent cells by mechanically detaching one end of the myotubes. All myotubes, whether striated or not, shorten with an exponential decay. The cell-on-cell myotubes relax more, which implies a greater contractile stress. The non-muscle myosin II inhibitor blebbistatin inhibits relaxation for either case. Myotubes in culture are thus clearly prestressed by myosin II, and this contractility couples to substrate compliance and ultimately influences actomyosin striation

Overexpression of SERCA1a in the \u3cem\u3emdx\u3c/em\u3e Diaphragm Reduces Susceptibility to Contraction-Induced Damage

Although the precise pathophysiological mechanism of muscle damage in dystrophin-deficient muscle remains disputed, calcium appears to be a critical mediator of the dystrophic process. Duchenne muscular dystrophy patients and mouse models of dystrophin deficiency exhibit extensive abnormalities of calcium homeostasis, which we hypothesized would be mitigated by increased expression of the sarcoplasmic reticulum calcium pump. Neonatal adeno-associated virus gene transfer of sarcoplasmic reticulum ATPase 1a to the mdx diaphragm decreased centrally located nuclei and resulted in reduced susceptibility to eccentric contraction-induced damage at 6 months of age. As the diaphragm is the mouse muscle most representative of human disease, these results provide impetus for further investigation of therapeutic strategies aimed at enhanced cytosolic calcium removal

Muscle-specific expression of insulin-like growth factor I counters muscle decline in mdx mice

Duchenne muscular dystrophy is an X-linked degenerative disorder of muscle caused by the absence of the protein dystrophin. A major consequence of muscular dystrophy is that the normal regenerative capacity of skeletal muscle cannot compensate for increased susceptibility to damage, leading to repetitive cycles of degeneration–regeneration and ultimately resulting in the replacement of muscle fibers with fibrotic tissue. Because insulin-like growth factor I (IGF-I) has been shown to enhance muscle regeneration and protein synthetic pathways, we asked whether high levels of muscle-specific expression of IGF-I in mdx muscle could preserve muscle function in the diseased state. In transgenic mdx mice expressing mIgf-I (mdx:mIgf+/+), we showed that muscle mass increased by at least 40% leading to similar increases in force generation in extensor digitorum longus muscles compared with those from mdx mice. Diaphragms of transgenic mdx:mIgf+/+ exhibited significant hypertrophy and hyperplasia at all ages observed. Furthermore, the IGF-I expression significantly reduced the amount of fibrosis normally observed in diaphragms from aged mdx mice. Decreased myonecrosis was also observed in diaphragms and quadriceps from mdx:mIgf+/+ mice when compared with age-matched mdx animals. Finally, signaling pathways associated with muscle regeneration and protection against apoptosis were significantly elevated. These results suggest that a combination of promoting muscle regenerative capacity and preventing muscle necrosis could be an effective treatment for the secondary symptoms caused by the primary loss of dystrophin

Functional muscle hypertrophy by increased insulin-like growth factor 1 does not require dysferlin.

IntroductionDysferlin loss-of-function mutations cause muscular dystrophy, accompanied by impaired membrane repair and muscle weakness. Growth promoting strategies including insulin-like growth factor 1 (IGF-1) could provide benefit but may cause strength loss or be ineffective. The objective of this study was to determine whether locally increased IGF-1 promotes functional muscle hypertrophy in dysferlin-null (Dysf-/- ) mice.MethodsMuscle-specific transgenic expression and postnatal viral delivery of Igf1 were used in Dysf-/- and control mice. Increased IGF-1 levels were confirmed by enzyme-linked immunosorbent assay. Testing for skeletal muscle mass and function was performed in male and female mice.ResultsMuscle hypertrophy occurred in response to increased IGF-1 in mice with and without dysferlin. Male mice showed a more robust response compared with females. Increased IGF-1 did not cause loss of force per cross-sectional area in Dysf-/- muscles.DiscussionWe conclude that increased local IGF-1 promotes functional hypertrophy when dysferlin is absent and reestablishes IGF-1 as a potential therapeutic for dysferlinopathies

Myotubes differentiate optimally on substrates with tissue-like stiffness : pathological implications for soft or stiff microenvironments

Contractile myocytes provide a test of the hypothesis that cells sense their mechanical as well as molecular microenvironment, altering expression, organization, and/or morphology accordingly. Here, myoblasts were cultured on collagen strips attached to glass or polymer gels of varied elasticity. Subsequent fusion into myotubes occurs independent of substrate flexibility. However, myosin/actin striations emerge later only on gels with stiffness typical of normal muscle (passive Young\u27s modulus, E ~12 kPa). On glass and much softer or stiffer gels, including gels emulating stiff dystrophic muscle, cells do not striate. In addition, myotubes grown on top of a compliant bottom layer of glass-attached myotubes (but not softer fibroblasts) will striate, whereas the bottom cells will only assemble stress fibers and vinculin-rich adhesions. Unlike sarcomere formation, adhesion strength increases monotonically versus substrate stiffness with strongest adhesion on glass. These findings have major implications for in vivo introduction of stem cells into diseased or damaged striated muscle of altered mechanical composition

Systemic Myostatin Inhibition via Liver-Targeted Gene Transfer in Normal and Dystrophic Mice

Background: Myostatin inhibition is a promising therapeutic strategy to maintain muscle mass in a variety of disorders, including the muscular dystrophies, cachexia, and sarcopenia. Previously described approaches to blocking myostatin signaling include injection delivery of inhibitory propeptide domain or neutralizing antibodies. Methodology/Principal Findings: Here we describe a unique method of myostatin inhibition utilizing recombinant adenoassociated virus to overexpress a secretable dominant negative myostatin exclusively in the liver of mice. Systemic myostatin inhibition led to increased skeletal muscle mass and strength in control C57 Bl/6 mice and in the dystrophindeficient mdx model of Duchenne muscular dystrophy. The mdx soleus, a mouse muscle more representative of human fiber type composition, demonstrated the most profound improvement in force production and a shift toward faster myosin-heavy chain isoforms. Unexpectedly, the 11-month-old mdx diaphragm was not rescued by long-term myostatin inhibition. Further, mdx mice treated for 11 months exhibited cardiac hypertrophy and impaired function in an inhibitor dose–dependent manner. Conclusions/Significance: Liver-targeted gene transfer of a myostatin inhibitor is a valuable tool for preclinical investigation of myostatin blockade and provides novel insights into the long-term effects and shortcomings of myostatin inhibition o

Osteopontin ablation ameliorates muscular dystrophy by shifting macrophages to a proregenerative phenotype

In the degenerative disease Duchenne muscular dystrophy, inflammatory cells enter muscles in response to repetitive muscle damage. Immune factors are required for muscle regeneration, but chronic inflammation creates a profibrotic milieu that exacerbates disease progression. Osteopontin (OPN) is an immunomodulator highly expressed in dystrophic muscles. Ablation of OPN correlates with reduced fibrosis and improved muscle strength as well as reduced natural killer T (NKT) cell counts. Here, we demonstrate that the improved dystrophic phenotype observed with OPN ablation does not result from reductions in NKT cells. OPN ablation skews macrophage polarization toward a pro-regenerative phenotype by reducing M1 and M2a and increasing M2c subsets. These changes are associated with increased expression of pro-regenerative factors insulin-like growth factor 1, leukemia inhibitory factor, and urokinase-type plasminogen activator. Furthermore, altered macrophage polarization correlated with increases in muscle weight and muscle fiber diameter, resulting in long-term improvements in muscle strength and function in mdx mice. These findings suggest that OPN ablation promotes muscle repair via macrophage secretion of pro-myogenic growth factors