Mitochondrial oxidative stress impairs contractile function but paradoxically increases muscle mass via fibre branching.

BACKGROUND: Excess reactive oxygen species (ROS) and muscle weakness occur in parallel in multiple pathological conditions. However, the causative role of skeletal muscle mitochondrial ROS (mtROS) on neuromuscular junction (NMJ) morphology and function and muscle weakness has not been directly investigated. METHODS: We generated mice lacking skeletal muscle-specific manganese-superoxide dismutase (mSod2KO) to increase mtROS using a cre-Lox approach driven by human skeletal actin. We determined primary functional parameters of skeletal muscle mitochondrial function (respiration, ROS, and calcium retention capacity) using permeabilized muscle fibres and isolated muscle mitochondria. We assessed contractile properties of isolated skeletal muscle using in situ and in vitro preparations and whole lumbrical muscles to elucidate the mechanisms of contractile dysfunction. RESULTS: The mSod2KO mice, contrary to our prediction, exhibit a 10-15% increase in muscle mass associated with an ~50% increase in central nuclei and ~35% increase in branched fibres (P < 0.05). Despite the increase in muscle mass of gastrocnemius and quadriceps, in situ sciatic nerve-stimulated isometric maximum-specific force (N/cm2 ), force per cross-sectional area, is impaired by ~60% and associated with increased NMJ fragmentation and size by ~40% (P < 0.05). Intrinsic alterations of components of the contractile machinery show elevated markers of oxidative stress, for example, lipid peroxidation is increased by ~100%, oxidized glutathione is elevated by ~50%, and oxidative modifications of myofibrillar proteins are increased by ~30% (P < 0.05). We also find an approximate 20% decrease in the intracellular calcium transient that is associated with specific force deficit. Excess superoxide generation from the mitochondrial complexes causes a deficiency of succinate dehydrogenase and reduced complex-II-mediated respiration and adenosine triphosphate generation rates leading to severe exercise intolerance (~10 min vs. ~2 h in wild type, P < 0.05). CONCLUSIONS: Increased skeletal muscle mtROS is sufficient to elicit NMJ disruption and contractile abnormalities, but not muscle atrophy, suggesting new roles for mitochondrial oxidative stress in maintenance of muscle mass through increased fibre branching.

Oxidative stress-induced dysregulation of excitation-contraction coupling contributes to muscle weakness.

BACKGROUND: We have previously shown that the deletion of the superoxide scavenger, CuZn superoxide dismutase, in mice (Sod1-/- mice) results in increased oxidative stress and an accelerated loss of skeletal muscle mass and force that mirror the changes seen in old control mice. The goal of this study is to define the effect of oxidative stress and ageing on muscle weakness and the Excitation Contraction (EC) coupling machinery in age-matched adult (8-10 months) wild-type (WT) and Sod1-/- mice in comparison with old (25-28 months) WT mice. METHODS: In vitro contractile assays were used to measure muscle contractile parameters. The activity of the sarcoplasmic reticulum Ca2+ ATPase (SERCA) pump was measured using an NADH-linked enzyme assay. Immunoblotting and immunofluorescence techniques were used to measure protein expression, and real-time reverse transcription PCR was used to measure gene expression. RESULTS: The specific force generated by the extensor digitorum longus muscle was reduced in the Sod1-/- and old WT mice compared with young WT mice along with significant prolongation of time to peak force, increased half relaxation time, and disruption of intracellular calcium handling. The maximal activity of the SERCA calcium uptake pump was significantly reduced in gastrocnemius muscle from both old WT (≈14%) and adult Sod1-/- (≈33%) mice compared with young WT mice along with increased expression of sarcolipin, a known inhibitor of SERCA activity. Protein levels of the voltage sensor and calcium uptake channel proteins dihydropyridine receptor α1 and SERCA2 were significantly elevated (≈45% and ≈57%, respectively), while the ratio of calstabin, a channel stabilizing protein, to ryanodine receptor was significantly reduced (≈21%) in Sod1-/- mice compared with young WT mice. The changes in calcium handling were accompanied by substantially elevated levels of global protein carbonylation and lipid peroxidation. CONCLUSIONS: Our data suggest that the muscle weakness in Sod1-/- and old WT mice is in part driven by reactive oxygen species-mediated EC uncoupling and supports a role for reduced SERCA pump activity in compromised muscle function. The novel quantitative mechanistic data provided here can lead to potential therapeutic interventions of SERCA dysfunction for sarcopenia and muscle diseases.

Lifelong reduction in complex IV induces tissue-specific metabolic effects but does not reduce lifespan or healthspan in mice.

Loss of SURF1, a Complex IV assembly protein, was reported to increase lifespan in mice despite dramatically lower cytochrome oxidase (COX) activity. Consistent with this, our previous studies found advantageous changes in metabolism (reduced adiposity, increased insulin sensitivity, and mitochondrial biogenesis) in Surf1-/- mice. The lack of deleterious phenotypes in Surf1-/- mice is contrary to the hypothesis that mitochondrial dysfunction contributes to aging. We found only a modest (nonsignificant) extension of lifespan (7% median, 16% maximum) and no change in healthspan indices in Surf1-/- vs. Surf1+/+ mice despite substantial decreases in COX activity (22%-87% across tissues). Dietary restriction (DR) increased median lifespan in both Surf1+/+ and Surf1-/- mice (36% and 19%, respectively). We measured gene expression, metabolites, and targeted expression of key metabolic proteins in adipose tissue, liver, and brain in Surf1+/+ and Surf1-/- mice. Gene expression was differentially regulated in a tissue-specific manner. Many proteins and metabolites are downregulated in Surf1-/- adipose tissue and reversed by DR, while in brain, most metabolites that changed were elevated in Surf1-/- mice. Finally, mitochondrial unfolded protein response (UPRmt )-associated proteins were not uniformly altered by age or genotype, suggesting the UPRmt is not a key player in aging or in response to reduced COX activity. While the changes in gene expression and metabolism may represent compensatory responses to mitochondrial stress, the important outcome of this study is that lifespan and healthspan are not compromised in Surf1-/- mice, suggesting that not all mitochondrial deficiencies are a critical determinant of lifespan.