Summary Age-related loss of muscle mass and force (sarcopenia) contributes to disability and increased mortality. Ryanodine receptor 1 (RyR1) is the skeletal muscle sarcoplasmic reticulum calcium release channel required for muscle contraction. RyR1 from aged (24 months) rodents were oxidized, cysteine-nitrosylated, and depleted of the channel stabilizing subunit calstabin1, compared to RyR1 from younger (3–6 months) adults. This RyR1 channel complex remodeling resulted in “leaky” channels with increased open probability leading to intracellular calcium leak in skeletal muscle. Similarly, six-month old mice harboring leaky RyR1-S2844D mutant channels exhibited skeletal muscle defects comparable to 24-month old wild type mice. Treating aged mice with S107, stabilized binding of calstabin1 to RyR1, reduced intracellular calcium leak, decreased reactive oxygen species (ROS), and enhanced tetanic Ca2+ release, muscle specific force and exercise capacity. Taken together these data indicate that leaky RyR1 contribute to age-related loss of muscle function.

Cancer-associated muscle weakness is poorly understood and there is no effective treatment. Here, we find that seven different mouse models of human osteolytic bone metastases, representing breast, lung and prostate cancers, as well as multiple myeloma exhibited impaired muscle function, implicating a role for the tumor-bone microenvironment in cancer-associated muscle weakness. We found that TGF-β, released from the bone surface as a result of metastasis-induced bone destruction upregulated NADPH oxidase 4 (Nox4), resulting in elevated oxidization of skeletal muscle proteins, including the ryanodine receptor/calcium (Ca2+) release channel (RyR1). The oxidized RyR1 channels leaked Ca2+, resulting in lower intracellular signaling required for proper muscle contraction. We found that inhibiting RyR1 leak, TGF-β signaling, TGF-β release from bone or Nox4 all improved muscle function in mice with MDA-MB-231 bone metastases. Humans with breast cancer- or lung cancer-associated bone metastases also had oxidized skeletal muscle RyR1 that is not seen in normal muscle. Similarly, skeletal muscle weakness, higher levels of Nox4 protein and Nox4 binding to RyR1, and oxidation of RyR1 were present in a mouse model of Camurati-Engelmann disease, a non-malignant metabolic bone disorder associated with increased TGF-β activity. Thus, metastasis-induced TGF-β release from bone contributes to muscle weakness by decreasing Ca2+-induced muscle force production.

Age-related skeletal muscle dysfunction is a leading cause of morbidity that affects up to half the population aged 80 or greater. Here we tested the effects of increased mitochondrial antioxidant activity on age-dependent skeletal muscle dysfunction using transgenic mice with targeted overexpression of the human catalase gene to mitochondria (MCat mice). Aged MCat mice exhibited improved voluntary exercise, increased skeletal muscle specific force and tetanic Ca 2+ transients, decreased intracellular Ca 2+ leak and increased sarcoplasmic reticulum (SR) Ca 2+ load compared with age-matched wild type (WT) littermates. Furthermore, ryanodine receptor 1 (the sarcoplasmic reticulum Ca 2+ release channel required for skeletal muscle contraction; RyR1) from aged MCat mice was less oxidized, depleted of the channel stabilizing subunit, calstabin1, and displayed increased single channel open probability (P o ). Overall, these data indicate a direct role for mitochondrial free radicals in promoting the pathological intracellular Ca 2+ leak that underlies age-dependent loss of skeletal muscle function. This study harbors implications for the development of novel therapeutic strategies, including mitochondria-targeted antioxidants for treatment of mitochondrial myopathies and other healthspan-limiting disorders.aging | skeletal muscle | exercise capacity | muscle weakness | oxidation

Ventilator-induced diaphragmatic dysfunction (VIDD) refers to the diaphragm muscle weakness that occurs following prolonged controlled mechanical ventilation (MV). The presence of VIDD impedes recovery from respiratory failure. However, the pathophysiological mechanisms accounting for VIDD are still not fully understood. Here, we show in human subjects and a mouse model of VIDD that MV is associated with rapid remodeling of the sarcoplasmic reticulum (SR) Ca 2+ release channel/ryanodine receptor (RyR1) in the diaphragm. The RyR1 macromolecular complex was oxidized, S-nitrosylated, Ser-2844 phosphorylated, and depleted of the stabilizing subunit calstabin1, following MV. These posttranslational modifications of RyR1 were mediated by both oxidative stress mediated by MV and stimulation of adrenergic signaling resulting from the anesthesia. We demonstrate in the murine model that such abnormal resting SR Ca 2+ leak resulted in reduced contractile function and muscle fiber atrophy for longer duration of MV. Treatment with β-adrenergic antagonists or with S107, a small molecule drug that stabilizes the RyR1-calstabin1 interaction, prevented VIDD. Diaphragmatic dysfunction is common in MV patients and is a major cause of failure to wean patients from ventilator support. This study provides the first evidence to our knowledge of RyR1 alterations as a proximal mechanism underlying VIDD (i.e., loss of function, muscle atrophy) and identifies RyR1 as a potential target for therapeutic intervention.excitation-contraction coupling | beta adrenergic signaling | calcium | VIDD | skeletal muscle

BackgroundDisruption of the sarcolemma-associated dystrophin-glycoprotein complex underlies multiple forms of muscular dystrophy, including Duchenne muscular dystrophy and sarcoglycanopathies. A hallmark of these disorders is muscle weakness. In a murine model of Duchenne muscular dystrophy, mdx mice, cysteine-nitrosylation of the calcium release channel/ryanodine receptor type 1 (RyR1) on the skeletal muscle sarcoplasmic reticulum causes depletion of the stabilizing subunit calstabin1 (FKBP12) from the RyR1 macromolecular complex. This results in a sarcoplasmic reticular calcium leak via defective RyR1 channels. This pathological intracellular calcium leak contributes to reduced calcium release and decreased muscle force production. It is unknown whether RyR1 dysfunction occurs also in other muscular dystrophies.MethodsTo test this we used a murine model of Limb-Girdle muscular dystrophy, deficient in β-sarcoglycan (Sgcb−/−).ResultsSkeletal muscle RyR1 from Sgcb−/− deficient mice were oxidized, nitrosylated, and depleted of the stabilizing subunit calstabin1, which was associated with increased open probability of the RyR1 channels. Sgcb−/− deficient mice exhibited decreased muscle specific force and calcium transients, and displayed reduced exercise capacity. Treating Sgcb−/− mice with the RyR stabilizing compound S107 improved muscle specific force, calcium transients, and exercise capacity. We have previously reported similar findings in mdx mice, a murine model of Duchenne muscular dystrophy.ConclusionsOur data suggest that leaky RyR1 channels may underlie multiple forms of muscular dystrophy linked to mutations in genes encoding components of the dystrophin-glycoprotein complex. A common underlying abnormality in calcium handling indicates that pharmacological targeting of dysfunctional RyR1 could be a novel therapeutic approach to improve muscle function in Limb-Girdle and Duchenne muscular dystrophies.