Muscle wasting is associated with a number of pathophysiologic conditions, including metabolic acidosis, diabetes, sepsis, and high angiotensin II levels. Under these conditions, activation of muscle protein degradation requires endogenous glucocorticoids. As the mechanism(s) underlying this dependence on glucocorticoids have not been identified, we analyzed the effects of glucocorticoids on muscle wasting in a mouse model of acute diabetes. Adrenalectomized, acutely diabetic mice given a physiologic dose of glucocorticoids exhibited decreased IRS-1–associated PI3K activity in muscle and progressive muscle atrophy. These responses were related to increased association of PI3K with the glucocorticoid receptor (GR). In mice with muscle-specific GR deletion (referred to as MGRKO mice), acute diabetes minimally suppressed IRS-1–associated PI3K activity in muscle and did not cause muscle atrophy. However, when a physiologic dose of glucocorticoids was given to mice with muscle-specific IR deletion, muscle protein degradation was accelerated. Fluorescence resonance energy transfer and an in vitro competition assay revealed that activated GRs competed for PI3K, reducing its association with IRS-1. Reexpression of WT GRs or those with a mutation in the nuclear localization signal in the muscle of MGRKO mice indicated that competition for PI3K was a prominent mechanism underlying reduced IRS-1–associated PI3K activity. This nongenomic influence of the GR contributes to activation of muscle protein degradation. We therefore conclude that stimulation of muscle proteolysis requires 2 events, increased glucocorticoid levels and impaired insulin signaling.
Zhaoyong Hu, Huiling Wang, In Hee Lee, Jie Du, William E. Mitch
Duchenne muscular dystrophy (DMD) is an X-linked, degenerative muscle disease that is exacerbated by secondary inflammation. Here, we characterized the immunological milieu of dystrophic muscle in mdx mice, a model of DMD, to identify potential therapeutic targets. We identified a specific subpopulation of cells expressing the Vβ8.1/8.2 TCR that is predominant among TCR-β+ T cells. These cells expressed high levels of osteopontin (OPN), a cytokine that promotes immune cell migration and survival. Elevated OPN levels correlated with the dystrophic process, since OPN was substantially elevated in the serum of mdx mice and muscle biopsies after disease onset. Muscle biopsies from individuals with DMD also had elevated OPN levels. To test the role of OPN in mdx muscle, mice lacking both OPN and dystrophin were generated and termed double-mutant mice (DMM mice). Reduced infiltration of NKT-like cells and neutrophils was observed in the muscle of DMM mice, supporting an immunomodulatory role for OPN in mdx muscle. Concomitantly, an increase in CD4+ and FoxP3+ Tregs was also observed in DMM muscle, which also showed reduced levels of TGF-β, a known fibrosis mediator. These inflammatory changes correlated with increased strength and reduced diaphragm and cardiac fibrosis. These studies suggest that OPN may be a promising therapeutic target for reducing inflammation and fibrosis in individuals with DMD.
Sylvia A. Vetrone, Encarnacion Montecino-Rodriguez, Elena Kudryashova, Irina Kramerova, Eric P. Hoffman, Scot D. Liu, M. Carrie Miceli, Melissa J. Spencer
Sarcolemma-associated neuronal NOS (nNOS) plays a critical role in normal muscle physiology. In Duchenne muscular dystrophy (DMD), the loss of sarcolemmal nNOS leads to functional ischemia and muscle damage; however, the mechanism of nNOS subcellular localization remains incompletely understood. According to the prevailing model, nNOS is recruited to the sarcolemma by syntrophin, and in DMD this localization is altered. Intriguingly, the presence of syntrophin on the membrane does not always restore sarcolemmal nNOS. Thus, we wished to determine whether dystrophin functions in subcellular localization of nNOS and which regions may be necessary. Using in vivo transfection of dystrophin deletion constructs, we show that sarcolemmal targeting of nNOS was dependent on the spectrin-like repeats 16 and 17 (R16/17) within the rod domain. Treatment of mdx mice (a DMD model) with R16/17-containing synthetic dystrophin genes effectively ameliorated histological muscle pathology and improved muscle strength as well as exercise performance. Furthermore, sarcolemma-targeted nNOS attenuated α-adrenergic vasoconstriction in contracting muscle and improved muscle perfusion during exercise as measured by Doppler and microsphere circulation. In summary, we have identified the dystrophin spectrin-like repeats 16 and 17 as a novel scaffold for nNOS sarcolemmal targeting. These data suggest that muscular dystrophy gene therapies based on R16/17-containing dystrophins may yield better clinical outcomes than the current therapies.
Yi Lai, Gail D. Thomas, Yongping Yue, Hsiao T. Yang, Dejia Li, Chun Long, Luke Judge, Brian Bostick, Jeffrey S. Chamberlain, Ronald L. Terjung, Dongsheng Duan
The composition of skeletal muscle, in terms of the relative number of slow- and fast-twitch fibers, is tightly regulated to enable an organism to respond and adapt to changing physical demands. The phosphatase calcineurin and its downstream targets, transcription factors of the nuclear factor of activated T cells (NFAT) family, play a critical role in this process by promoting the formation of slow-twitch, oxidative fibers. Calcineurin binds to calsarcins, a family of striated muscle–specific proteins of the sarcomeric Z-disc. We show here that mice deficient in calsarcin-2, which is expressed exclusively by fast-twitch muscle and encoded by the myozenin 1 (Myoz1) gene, have substantially reduced body weight and fast-twitch muscle mass in the absence of an overt myopathic phenotype. Additionally, Myoz1 KO mice displayed markedly improved performance and enhanced running distances in exercise studies. Analysis of fiber type composition of calsarcin-2–deficient skeletal muscles showed a switch toward slow-twitch, oxidative fibers. Reporter assays in cultured myoblasts indicated an inhibitory role for calsarcin-2 on calcineurin, and Myoz1 KO mice exhibited both an excess of NFAT activity and an increase in expression of regulator of calcineurin 1-4 (RCAN1-4), indicating enhanced calcineurin signaling in vivo. Taken together, these results suggest that calsarcin-2 modulates exercise performance in vivo through regulation of calcineurin/NFAT activity and subsequent alteration of the fiber type composition of skeletal muscle.
Norbert Frey, Derk Frank, Stefanie Lippl, Christian Kuhn, Harald Kögler, Tomasa Barrientos, Claudia Rohr, Rainer Will, Oliver J. Müller, Hartmut Weiler, Rhonda Bassel-Duby, Hugo A. Katus, Eric N. Olson
Congenital myasthenias (CMs) arise from defects in neuromuscular junction–associated proteins. Deciphering the molecular bases of the CMs is required for therapy and illuminates structure-function relationships in these proteins. Here, we analyze the effects of a mutation in 1 of 4 homologous subunits in the AChR from a CM patient, a Leu to Pro mutation at position 42 of the δ subunit. The mutation is located in a region of contact between subunits required for rapid opening of the AChR channel and impedes the rate of channel opening. Substitutions of Gly, Lys, or Asp for δL42, or substitutions of Pro along the local protein chain, also slowed channel opening. Substitution of Pro for Leu in the ε subunit slowed opening, whereas this substitution had no effect in the β subunit and actually sped opening in the α subunit. Analyses of energetic coupling between residues at the subunit interface showed that δL42 is functionally linked to αT127, a key residue in the adjacent α subunit required for rapid channel opening. Thus, δL42 is part of an intersubunit network that enables ACh binding to rapidly open the AChR channel, which may be compromised in patients with CM.
Xin-Ming Shen, Taku Fukuda, Kinji Ohno, Steven M. Sine, Andrew G. Engel
Hyperkalemic periodic paralysis (HyperKPP) produces myotonia and attacks of muscle weakness triggered by rest after exercise or by K+ ingestion. We introduced a missense substitution corresponding to a human familial HyperKPP mutation (Met1592Val) into the mouse gene encoding the skeletal muscle voltage-gated Na+ channel NaV1.4. Mice heterozygous for this mutation exhibited prominent myotonia at rest and muscle fiber-type switching to a more oxidative phenotype compared with controls. Isolated mutant extensor digitorum longus muscles were abnormally sensitive to the Na+/K+ pump inhibitor ouabain and exhibited age-dependent changes, including delayed relaxation and altered generation of tetanic force. Moreover, rapid and sustained weakness of isolated mutant muscles was induced when the extracellular K+ concentration was increased from 4 mM to 10 mM, a level observed in the muscle interstitium of humans during exercise. Mutant muscle recovered from stimulation-induced fatigue more slowly than did control muscle, and the extent of recovery was decreased in the presence of high extracellular K+ levels. These findings demonstrate that expression of the Met1592Val Na+ channel in mouse muscle is sufficient to produce important features of HyperKPP, including myotonia, K+-sensitive paralysis, and susceptibility to delayed weakness during recovery from fatigue.
Lawrence J. Hayward, Joanna S. Kim, Ming-Yang Lee, Hongru Zhou, Ji W. Kim, Kumudini Misra, Mohammad Salajegheh, Fen-fen Wu, Chie Matsuda, Valerie Reid, Didier Cros, Eric P. Hoffman, Jean-Marc Renaud, Stephen C. Cannon, Robert H. Brown Jr.
Numerous studies have suggested that muscle atrophy is accompanied by apoptotic loss of myonuclei and therefore recovery would require replenishment by muscle stem cells. We used in vivo time-lapse microscopy to observe the loss and replenishment of myonuclei in murine muscle fibers following induced muscle atrophy. To our surprise, imaging of single fibers for up to 28 days did not support the concept of nuclear loss during atrophy. Muscles were inactivated by denervation, nerve impulse block, or mechanical unloading. Nuclei were stained in vivo either acutely by intracellular injection of fluorescent oligonucleotides or in time-lapse studies after transfection with a plasmid encoding GFP with a nuclear localization signal. We observed no loss of myonuclei in fast- or slow-twitch muscle fibers despite a greater than 50% reduction in fiber cross-sectional area. TUNEL labeling of fragmented DNA on histological sections revealed high levels of apoptotic nuclei in inactive muscles. However, when costained for laminin and dystrophin, virtually none of the TUNEL-positive nuclei could be classified as myonuclei; apoptosis was confined to stromal and satellite cells. We conclude that disuse atrophy is not a degenerative process, but is rather a change in the balance between protein synthesis and proteolysis in a permanent cell syncytium.
Jo C. Bruusgaard, Kristian Gundersen
The intracellular signals that mediate skeletal muscle protein loss and functional deficits due to muscular disuse are just beginning to be elucidated. Previously we showed that the activity of an NF-κB–dependent reporter gene was markedly increased in unloaded muscles, and p50 and Bcl-3 proteins were implicated in this induction. In the present study, mice with a knockout of the p105/p50 (Nfkb1) gene are shown to be resistant to the decrease in soleus fiber cross-sectional area that results from 10 days of hindlimb unloading. Furthermore, the marked unloading-induced activation of the NF-κB reporter gene in soleus muscles from WT mice was completely abolished in soleus muscles from Nfkb1 knockout mice. Knockout of the B cell lymphoma 3 (Bcl3) gene also showed an inhibition of fiber atrophy and an abolition of NF-κB reporter activity. With unloading, fast fibers from WT mice atrophied to a greater extent than slow fibers. Resistance to atrophy in both strains of knockout mice was demonstrated clearly in fast fibers, while slow fibers from only the Bcl3–/– mice showed atrophy inhibition. The slow-to-fast shift in myosin isoform expression due to unloading was also abolished in both Nfkb1 and Bcl3 knockout mice. Like the soleus muscles, plantaris muscles from Nfkb1–/– and Bcl3–/– mice also showed inhibition of atrophy with unloading. Thus both the Nfkb1 and the Bcl3 genes are necessary for unloading-induced atrophy and the associated phenotype transition.
R. Bridge Hunter, Susan C. Kandarian