William J. Durham

RyR1 S-Nitrosylation Underlies Environmental Heat Stroke and Sudden Death in Y522S RyR1 Knockin Mice

Mice with a malignant hyperthermia mutation (Y522S) in the ryanodine receptor (RyR1) display muscle contractures, rhabdomyolysis, and death in response to elevated environmental temperatures. We demonstrate that this mutation in RyR1 causes Ca(2+) leak, which drives increased generation of reactive nitrogen species (RNS). Subsequent S-nitrosylation of the mutant RyR1 increases its temperature sensitivity for activation, producing muscle contractures upon exposure to elevated temperatures. The Y522S mutation in humans is associated with central core disease. Many mitochondria in the muscle of heterozygous Y522S mice are swollen and misshapen. The mutant muscle displays decreased force production and increased mitochondrial lipid peroxidation with aging. Chronic treatment with N-acetylcysteine protects against mitochondrial oxidative damage and the decline in force generation. We propose a feed-forward cyclic mechanism that increases the temperature sensitivity of RyR1 activation and underlies heat stroke and sudden death. The cycle eventually produces a myopathy with damaged mitochondria.

Generation of Reactive Oxygen and Nitrogen Species in Contracting Skeletal Muscle

Since the early 1980s biologists have recognized that skeletal muscle generates free radicals. Of particular interest are two closely related redox cascades—reactive oxygen species (ROS) and nitric oxide (NO) derivatives. The ROS cascade is initiated by superoxide anion radicals derived from the mitochondrial electron transport chain, the membrane‐associated NAD(P)H oxidase complex, or other sources. NO is produced by two NO synthase isoforms constitutively expressed by muscle fibers. ROS and NO derivatives are produced continually and are detectable in both the cytosolic and extracellular compartments. Production increases during strenuous exercise. Both ROS and NO modulate contractile function. Under basal conditions, low levels of ROS enhance force production. Excessive ROS accumulation inhibits force, for example, during fatiguing exercise. NO inhibits skeletal muscle contraction, an effect that is partially mediated by cyclic GMP as a second messenger. With aging, redox modulation of muscle contraction may be altered by changes in the rates of ROS and NO production, the levels of endogenous antioxidants that buffer ROS and NO, and the sensitivities of regulatory proteins to ROS and NO action. The impact of aging on contractile regulation depends on the relative magnitude of these changes and their net effects on ROS and NO activities at the cellular level.

Heat- and anesthesia-induced malignant hyperthermia in an RyR1 knock-in mouse

Malignant hyperthermia (MH) is a life‐threatening disorder characterized by skeletal muscle rigidity and elevated body temperature in response to halogenated anesthetics such as isoflurane or halothane. Mutation of tyrosine 522 of RyR1 (the predominant skeletal muscle calcium release channel) to serine has been associated with human malignant hyperthermia. In the present study, mice created harboring this mutation were found to represent the first murine model of human malignant hyperthermia. Mice homozygous for the Y522S mutation exhibit skeletal defects and die during embryonic development or soon after birth. Heterozygous mice, which correspond to the human occurrence of this mutation, are MH susceptible, experiencing whole body contractions and elevated core temperatures in response to isoflurane exposure or heat stress. Skeletal muscles from heterozygous mice exhibit increased susceptibility to caffeine‐ and heat‐induced contractures in vitro. In addition, the heterozygous expression of the mutation results in enhanced RyR1 sensitivity to activation by temperature, caffeine, and voltage but not uncompensated sarcoplasmic reticulum calcium leak or store depletion. We conclude that the heterozygous expression of the Y522S mutation confers susceptibility to both heat‐ and anesthetic‐induced MH responses.

Age-related anabolic resistance after endurance-type exercise in healthy humans

Age‐related skeletal muscle loss is thought to stem from suboptimal nutrition and resistance to anabolic stimuli. Impaired microcirculatory (nutritive) blood flow may contribute to anabolic resistance by reducing delivery of amino acids to skeletal muscle. In this study, we employed contrast‐enhanced ultrasound, microdialysis sampling of skeletal muscle interstitium, and stable isotope methodology, to assess hemodynamic and metabolic responses of older individuals to endurance type (walking) exercise during controlled amino acid provision. We hypothesized that older individuals would exhibit reduced microcirculatory blood flow, interstitial amino acid concentrations, and amino acid transport when compared with younger controls. We report for the first time that aging induces anabolic resistance following endurance exercise, manifested as reduced (by ∼40%) efficiency of muscle protein synthesis. Despite lower (by ∼40–45%) microcirculatory flow in the older than in the younger participants, circulating and interstitial amino acid concentrations and phenylalanine transport into skeletal muscle were all equal or higher in older individuals than in the young, comprehensively refuting our hypothesis that amino acid availability limits postexercise anabolism in older individuals. Our data point to alternative mediators of age‐related anabolic resistance and importantly suggest correction of these impairments may reduce requirements for, and increase the efficacy of, dietary protein in older individuals. Durham, W. J., Casperson, S. L., Dillon, E. L., Keske, M. A., Paddon‐Jones, D., Sanford, A. P., Hickner, R. C., Grady, J. J., Sheffield‐Moore, M. Age‐related anabolic resistance after endurance‐type exercise in healthy humans. FASEB J. 24, 4117–4127 (2010). www.fasebj.org

Inflammatory burden and amino acid metabolism in cancer cachexia.

Purpose of reviewCancer cachexia is associated with marked alterations in skeletal muscle protein metabolism that lead to muscle wasting and, in some cases, death. The inflammatory response elicited by cancer is a likely, if not primary, mediator of these alterations. This review focuses on the possible relationship between inflammatory signaling and altered amino acid metabolism in cancer. Recent findingsLoss of skeletal muscle in cancer patients can potentially be due to anorexia and early satiety, reduced muscle protein synthesis, and/or increased muscle protein breakdown. Inflammation has been associated with each of these mechanisms. Effects on appetite appear to be mediated by the melanocortin system in the hypothalamus. Studies in animal models of cachexia suggest that modulation of orexigenic and anorexigenic pathways in this system may improve nutrient consumption. Inflammatory cytokines such as IL-6 and TNF-α are likely to contribute to the effects of inflammation on muscle protein metabolism through several pathways. SummaryLimited studies in humans suggest that targeted anti-inflammatory and nutritional interventions may ameliorate the net catabolic effect on skeletal muscle protein metabolism. Future studies of the precise mechanism of muscle protein loss, as well as novel or combination therapies to inhibit inflammation and promote anabolism, are warranted.

Progressive nuclear factor-κB activation resistant to inhibition by contraction and curcumin in mdx mice

Skeletal muscle of patients with Duchenne‐type muscular dystrophy and mdx mice exhibits elevated activity of the transcription factor NF‐κB (nuclear factor‐κB), which may play a role in muscle catabolism. We measured skeletal muscle NF‐κB activity in mdx mice at three ages (10 days, 4 weeks, and 8 weeks) to test the hypothesis that NF‐κB activity is elevated in an age‐dependent manner in these mice. In addition, we tested the hypothesis that NF‐κB activity could be reduced in mdx skeletal muscle by dietary supplementation with curcumin (1% w/v) or by fatiguing muscle contractions. We found that NF‐κB activity was elevated at 4 and 8 weeks of age but not at 10 days, and was resistant to inhibition by either fatiguing contractions or dietary curcumin. We conclude that NF‐κB activity is elevated in dystrophic skeletal muscle in an age‐related manner and is resistant to inhibition by physiological and pharmacological means. These findings are consistent with a role for NF‐κB activation in dystrophic muscle wasting but suggest that predicted interventions such as exercise or inhibitors of the early steps in the NF‐κ activation pathway may not be effective and that targeted research is needed to identify novel therapeutic strategies. Muscle Nerve, 2006

Effects of dietary curcumin or N-acetylcysteine on NF-κB activity and contractile performance in ambulatory and unloaded murine soleus

BackgroundUnloading of skeletal muscle causes atrophy and loss of contractile function. In part, this response is believed to be mediated by the transcription factor nuclear factor-kappa B (NF-κB). Both curcumin, a component of the spice turmeric, and N-acetylcysteine (NAC), an antioxidant, inhibit activation of NF-κB by inflammatory stimuli, albeit by different mechanisms. In the present study, we tested the hypothesis that dietary curcumin or NAC supplementation would inhibit unloading-induced NF-κB activity in skeletal muscle and thereby protect muscles against loss of mass and function caused by prolonged unloading.MethodsWe used hindlimb suspension to unload the hindlimb muscles of adult mice. Animals had free access to drinking water or drinking water supplemented with 1% NAC and to standard laboratory diet or diet supplemented with 1% curcumin. For 11 days, half the animals in each dietary group were suspended by the tail (unloaded) and half were allowed to ambulate freely.ResultsUnloading caused a 51–53% loss of soleus muscle weight and cross-sectional area relative to freely-ambulating controls. Unloading also decreased total force and force per cross-sectional area developed by soleus. Curcumin supplementation decreased NF-κB activity measured in peripheral tissues of ambulatory mice by gel shift analysis. In unloaded animals, curcumin supplementation did not inhibit NF-κB activity or blunt the loss of muscle mass in soleus. In contrast, NAC prevented the increase in NF-κB activity induced by unloading but did not prevent losses of muscle mass or function.ConclusionIn conclusion, neither dietary curcumin nor dietary NAC prevents unloading-induced skeletal muscle dysfunction and atrophy, although dietary NAC does prevent unloading induced NF-κB activation.

Altered excitation-contraction coupling with skeletal muscle specific FKBP12 deficiency

The immunophilin FKBP12 binds the skeletal muscle Ca2+ release channel or ryanodine receptor (RyR1), but the functional consequences of this interaction are not known. In this study, we have generated skeletal muscle specific FKBP12‐deficient mice to investigate the role of FKBP12 in skeletal muscle. Primary myotubes from these mice show no obvious change in either Ca2+ stores or resting Ca2+ levels but display decreased voltage‐gated intracellular Ca2+ release and increased L‐type Ca2+ currents. Consistent with the decreased voltage‐gated Ca2+ release, maximal tetanic force production is decreased and the force frequency curves are shifted to the right in extensor digitorum longus (EDL) muscles of the mutant mice. In contrast, there is no decrease in maximal tetanic force production in the mutant diaphragm or soleus muscle. The force frequency curve is shifted to the left in the FKBP12‐deficient diaphragm muscle compared with controls. No changes in myosin heavy chain (MHC) phenotype are observed in EDL or soleus muscle of the FKBP12‐deficient mice, but diaphragm muscle displays an increased ratio of slow to fast MHC isoforms. Also, calcineurin levels are increased in the diaphragm of the mutant mice but not in the soleus or EDL. In summary, FKBP12 deficiency alters both orthograde and retrograde coupling between the L‐type Ca2+ channel and RyR1 and the consequences of these changes depend on muscle type and activity. In highly used muscles such as the diaphragm, adaptation to the loss of FKBP12 occurs, possibly due to the increased Ca2+ influx.

TIMP3: a physiological regulator of adult myogenesis

Myogenic differentiation in adult muscle is normally suppressed and can be activated by myogenic cues in a subset of activated satellite cells. The switch mechanism that turns myogenesis on and off is not defined. In the present study, we demonstrate that tissue inhibitor of metalloproteinase 3 (TIMP3), the endogenous inhibitor of TNFα-converting enzyme (TACE), acts as an on–off switch for myogenic differentiation by regulating autocrine TNFα release. We observed that constitutively expressed TIMP3 is transiently downregulated in the satellite cells of regenerating mouse hindlimb muscles and differentiating C2C12 myoblasts. In C2C12 myoblasts, perturbing TIMP3 downregulation by overexpressing TIMP3 blocks TNFα release, p38 MAPK activation, myogenic gene expression and myotube formation. TNFα supplementation at a physiological concentration rescues myoblast differentiation. Similarly, in the regenerating soleus, overexpression of TIMP3 impairs release of TNFα and myogenic gene expression, and delays the formation of new fibers. In addition, downregulation of TIMP3 is mediated by the myogenesis-promoting microRNA miR-206. Thus, TIMP3 is a physiological regulator of myogenic differentiation.

A Randomized Pilot Study of Monthly Cycled Testosterone Replacement or Continuous Testosterone Replacement Versus Placebo in Older Men

CONTEXT Cycling androgens has been reported by athletes to improve physical performance by enhancing muscle mass and strength, a paradigm that has not been studied, and may have clinical value in older men being treated with testosterone. OBJECTIVE We investigated the efficacy of a monthly cycled testosterone regimen that uses half the testosterone dose as the current standard of care continuous therapy on body composition and muscle strength in older men. DESIGN, SETTING, AND PATIENTS Twenty-four community-dwelling older men 70 ± 2 yr of age with total testosterone levels below 500 ng/dl were randomized at the Institute for Translational Sciences-Clinical Research Center into a 5-month double-blind placebo-controlled trial. INTERVENTION Subjects were dosed weekly for 5 months, receiving continuous testosterone (TE, n = 8; 100 mg testosterone enanthate, im injection), monthly cycled testosterone (MO, n = 8; alternating months of testosterone and placebo), or placebo (PL, n = 8). MAIN OUTCOME MEASURES Main outcomes included body composition by dual-energy x-ray absorptiometry and upper and lower body muscle strength. Secondary outcomes included body weight, serum hormones, and mixed-muscle protein fractional synthesis rate (FSR). RESULTS Total lean body mass was increased and percent fat was reduced after 5 months in TE and MO (P < 0.05). Upper body muscle strength increased in TE, and lower body muscle strength increased in TE and MO (P < 0.05). FSR increased in TE and MO (P < 0.05) but not in PL. CONCLUSIONS Cycled testosterone improved body composition and increased muscle strength compared with placebo and increased FSR similarly to continuous testosterone.