Michael P. Siegel

Mitochondrial‐targeted peptide rapidly improves mitochondrial energetics and skeletal muscle performance in aged mice

Mitochondrial dysfunction plays a key pathogenic role in aging skeletal muscle resulting in significant healthcare costs in the developed world. However, there is no pharmacologic treatment to rapidly reverse mitochondrial deficits in the elderly. Here, we demonstrate that a single treatment with the mitochondrial‐targeted peptide SS‐31 restores in vivo mitochondrial energetics to young levels in aged mice after only one hour. Young (5 month old) and old (27 month old) mice were injected intraperitoneally with either saline or 3 mg kg−1 of SS‐31. Skeletal muscle mitochondrial energetics were measured in vivo one hour after injection using a unique combination of optical and 31P magnetic resonance spectroscopy. Age‐related declines in resting and maximal mitochondrial ATP production, coupling of oxidative phosphorylation (P/O), and cell energy state (PCr/ATP) were rapidly reversed after SS‐31 treatment, while SS‐31 had no observable effect on young muscle. These effects of SS‐31 on mitochondrial energetics in aged muscle were also associated with a more reduced glutathione redox status and lower mitochondrial H2O2 emission. Skeletal muscle of aged mice was more fatigue resistant in situ one hour after SS‐31 treatment, and eight days of SS‐31 treatment led to increased whole‐animal endurance capacity. These data demonstrate that SS‐31 represents a new strategy for reversing age‐related deficits in skeletal muscle with potential for translation into human use.

Defects in mitochondrial localization and ATP synthesis in the mdx mouse model of Duchenne muscular dystrophy are not alleviated by PDE5 inhibition

Given the crucial roles for mitochondria in ATP energy supply, Ca(2+) handling and cell death, mitochondrial dysfunction has long been suspected to be an important pathogenic feature in Duchenne muscular dystrophy (DMD). Despite this foresight, mitochondrial function in dystrophin-deficient muscles has remained poorly defined and unknown in vivo. Here, we used the mdx mouse model of DMD and non-invasive spectroscopy to determine the impact of dystrophin-deficiency on skeletal muscle mitochondrial localization and oxidative phosphorylation function in vivo. Mdx mitochondria exhibited significant uncoupling of oxidative phosphorylation (reduced P/O) and a reduction in maximal ATP synthesis capacity that together decreased intramuscular ATP levels. Uncoupling was not driven by increased UCP3 or ANT1 expression. Dystrophin was required to maintain subsarcolemmal mitochondria (SSM) pool density, implicating it in the spatial control of mitochondrial localization. Given that nitric oxide-cGMP pathways regulate mitochondria and that sildenafil-mediated phosphodiesterase 5 inhibition ameliorates dystrophic pathology, we tested whether sildenafils benefits result from decreased mitochondrial dysfunction in mdx mice. Unexpectedly, sildenafil treatment did not affect mitochondrial content or oxidative phosphorylation defects in mdx mice. Rather, PDE5 inhibition decreased resting levels of ATP, phosphocreatine and myoglobin, suggesting that sildenafil improves dystrophic pathology through other mechanisms. Overall, these data indicate that dystrophin-deficiency disrupts SSM localization, promotes mitochondrial inefficiency and restricts maximal mitochondrial ATP-generating capacity. Together these defects decrease intramuscular ATP and the ability of mdx muscle mitochondria to meet ATP demand. These findings further understanding of how mitochondrial bioenergetic dysfunction contributes to disease pathogenesis in dystrophin-deficient skeletal muscle in vivo.

Reduced coupling of oxidative phosphorylation In Vivo precedes electron transport chain defects due to mild oxidative stress in mice

Oxidative stress and mitochondrial function are at the core of many degenerative conditions. However, the interaction between oxidative stress and in vivo mitochondrial function is unclear. We used both pharmacological (2 week paraquat (PQ) treatment of wild type mice) and transgenic (mice lacking Cu, Zn-superoxide dismutase (SOD1−/−)) models to test the effect of oxidative stress on in vivo mitochondrial function in skeletal muscle. Magnetic resonance and optical spectroscopy were used to measure mitochondrial ATP and oxygen fluxes and cell energetic state. In both models of oxidative stress, coupling of oxidative phosphorylation was significantly lower (lower P/O) at rest in vivo in skeletal muscle and was dose-dependent in the PQ model. Despite this reduction in efficiency, in vivo mitochondrial phosphorylation capacity (ATPmax) was maintained in both models, and ex vivo mitochondrial respiration in permeabilized muscle fibers was unchanged following PQ treatment. In association with the reduced P/O, PQ treatment led to a dose-dependent reduction in PCr/ATP ratio and increased phosphorylation of AMPK. These results indicate that oxidative stress uncouples oxidative phosphorylation in vivo and results in energetic stress in the absence of defects in the mitochondrial electron transport chain.

Impaired adaptability of in vivo mitochondrial energetics to acute oxidative insult in aged skeletal muscle

Periods of elevated reactive oxygen species (ROS) production are a normal part of mitochondrial physiology. However, little is known about age-related changes in the mitochondrial response to elevated ROS in vivo. Significantly, ROS-induced uncoupling of oxidative phosphorylation has received attention as a negative feedback mechanism to reduce mitochondrial superoxide production. Here we use a novel in vivo spectroscopy system to test the hypothesis that ROS-induced uncoupling is diminished in aged mitochondria. This system simultaneously acquires (31)P magnetic resonance and near-infrared optical spectra to non-invasively measure phosphometabolite and O(2) concentrations in mouse skeletal muscle. Using low dose paraquat to elevate intracellular ROS we assess in vivo mitochondrial function in young, middle aged, and old mice. Oxidative phosphorylation was uncoupled to the same degree in response to ROS at each age, but this uncoupling was associated with loss of phosphorylation capacity and total ATP in old mice only. Using mice lacking UCP3 we demonstrate that this in vivo uncoupling is independent of this putative uncoupler of skeletal muscle mitochondria. These data indicate that ROS-induced uncoupling persists throughout life, but that oxidative stress leads to mitochondrial deficits and loss of ATP in aged organisms that may contribute to impaired function and degeneration.

Nitric Oxide Regulates Skeletal Muscle Fatigue, Fiber Type, Microtubule Organization, and Mitochondrial ATP Synthesis Efficiency Through cGMP-Dependent Mechanisms

AIM Skeletal muscle nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathways are impaired in Duchenne and Becker muscular dystrophy partly because of reduced nNOSμ and soluble guanylate cyclase (GC) activity. However, GC function and the consequences of reduced GC activity in skeletal muscle are unknown. In this study, we explore the functions of GC and NO-cGMP signaling in skeletal muscle. RESULTS GC1, but not GC2, expression was higher in oxidative than glycolytic muscles. GC1 was found in a complex with nNOSμ and targeted to nNOS compartments at the Golgi complex and neuromuscular junction. Baseline GC activity and GC agonist responsiveness was reduced in the absence of nNOS. Structural analyses revealed aberrant microtubule directionality in GC1-/- muscle. Functional analyses of GC1-/- muscles revealed reduced fatigue resistance and postexercise force recovery that were not due to shifts in type IIA-IIX fiber balance. Force deficits in GC1-/- muscles were also not driven by defects in resting mitochondrial adenosine triphosphate (ATP) synthesis. However, increasing muscle cGMP with sildenafil decreased ATP synthesis efficiency and capacity, without impacting mitochondrial content or ultrastructure. INNOVATION GC may represent a new target for alleviating muscle fatigue and that NO-cGMP signaling may play important roles in muscle structure, contractility, and bioenergetics. CONCLUSIONS These findings suggest that GC activity is nNOS dependent and that muscle-specific control of GC expression and differential GC targeting may facilitate NO-cGMP signaling diversity. They suggest that nNOS regulates muscle fiber type, microtubule organization, fatigability, and postexercise force recovery partly through GC1 and suggest that NO-cGMP pathways may modulate mitochondrial ATP synthesis efficiency. Antioxid. Redox Signal. 26, 966-985.

Soluble guanylyl cyclase regulates skeletal muscle fiber type plasticity, fatigue resistance and whole body insulin resistance

Pathogenic defects in NO-cGMP signaling drive skeletal muscle dysfunction in Duchenne muscular dystrophy (DMD). These defects arise from decreased nNOS and guanylyl cyclase (GC) activity and loss of spatial control of NO production. Therapeutics such as sildenafil that amplify NO-cGMP signaling reduce skeletal muscle dysfunction in DMD patients and mouse models of DMD making GC and cGMP attractive therapeutic targets. However, GC and cGMP functions in skeletal muscle are poorly defined hindering therapy development. To remove this barrier, we investigated the functions of GC in skeletal muscle. We report that α1β1 soluble GC (sGC) is the primary cGMP source in skeletal muscle. α1β1 sGC expression was greater in oxidative muscles suggesting a greater cGMP synthesis capacity and muscle-specific differences in sGC function. Interestingly, sGC activity exhibited partial nNOS dependence. Analyses of sGC subcellular localization revealed a pool of α1β1 at the cis-Golgi complex in muscle cells. α1β1 and α2β1 sGC localized to the microvasculature. Muscles lacking a functional α1 sGC subunit (α1β1 sGC deficient) exhibited reduced fatigue resistance and normal hypertrophic growth. Surprisingly, α1β1 sGC deficiency had no impact on mitochondrial content suggesting that mitochondrial density may not be as tightly regulated by cGMP as previously thought. α1β1 sGC deficiency had a modest effect on mitochondrial ATP synthesis. Also, loss of α1β1 sGC triggered a type IIA to type IIX fiber shift. Although this shift was unlikely to significantly enhance fatigability, it may impact insulin metabolism because type IIX fiber content positively correlates with insulin insensitivity. Indeed, α1β1 sGC null mice exhibited gender-specific defects in whole body insulin sensitivity consistent with reports of gender-specific effects of cGMP on metabolism but normal glucose tolerance indicating compensatory changes in glucose metabolism. In summary, these findings argue that NO signaling through sGC plays important roles in muscle fatigue and fiber type specification and suggest sGC as a target for mitigating skeletal muscle fatigue in DMD. These data also suggest that disruption of NO-cGMP signaling may contribute to the poorly understood metabolic defects in DMD patients. By showing that reductions in cGMP synthesis promote muscle fatigue, type IIX content and insulin resistance, these data suggest new insights as to how decreases in cGMP may contribute to the development of metabolic dysfunction and disease, particularly over time. Importantly, these results suggest sGC as a potential target for mitigating insulin resistance.