ER stress disturbs SR/ER-mitochondria Ca2+ transfer: Implications in Duchenne muscular dystrophy.

Besides its role in calcium (Ca2+) homeostasis, the sarco-endoplamic reticulum (SR/ER) controls protein folding and is tethered to mitochondria. Under pathophysiological conditions the unfolded protein response (UPR) is associated with disturbance in SR/ER-mitochondria crosstalk. Here, we investigated whether ER stress altered SR/ER-mitochondria links, Ca2+ handling and muscle damage in WT (Wild Type) and mdx mice, the murine model of Duchenne Muscular Dystrophy (DMD). In WT mice, the SR/ER-mitochondria links were decreased in isolated FDB muscle fibers after injection of ER stress activator tunicamycin (TM). Ca2+ imaging revealed an increase of cytosolic Ca2+ transient and a decrease of mitochondrial Ca2+ uptake. The force generating capacity of muscle dropped after TM. This impaired contractile function was accompanied by an increase in autophagy markers and calpain-1 activation. Conversely, ER stress inhibitors restored SR/ER-mitochondria links, mitochondrial Ca2+ uptake and improved diaphragm contractility in mdx mice. Our findings demonstrated that ER stress-altered SR/ER-mitochondria links, disturbed Ca2+ handling and muscle function in WT and mdx mice. Thus, ER stress may open up a prospect of new therapeutic targets in DMD.

Role of PGC-1α during acute exercise-induced autophagy and mitophagy in skeletal muscle.

Regular exercise leads to systemic metabolic benefits, which require remodeling of energy resources in skeletal muscle. During acute exercise, the increase in energy demands initiate mitochondrial biogenesis, orchestrated by the transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). Much less is known about the degradation of mitochondria following exercise, although new evidence implicates a cellular recycling mechanism, autophagy/mitophagy, in exercise-induced adaptations. How mitophagy is activated and what role PGC-1α plays in this process during exercise have yet to be evaluated. Thus we investigated autophagy/mitophagy in muscle immediately following an acute bout of exercise or 90 min following exercise in wild-type (WT) and PGC-1α knockout (KO) animals. Deletion of PGC-1α resulted in a 40% decrease in mitochondrial content, as well as a 25% decline in running performance, which was accompanied by severe acidosis in KO animals, indicating metabolic distress. Exercise induced significant increases in gene transcripts of various mitochondrial (e.g., cytochrome oxidase subunit IV and mitochondrial transcription factor A) and autophagy-related (e.g., p62 and light chain 3) genes in WT, but not KO, animals. Exercise also resulted in enhanced targeting of mitochondria for mitophagy, as well as increased autophagy and mitophagy flux, in WT animals. This effect was attenuated in the absence of PGC-1α. We also identified Niemann-Pick C1, a transmembrane protein involved in lysosomal lipid trafficking, as a target of PGC-1α that is induced with exercise. These results suggest that mitochondrial turnover is increased following exercise and that this effect is at least in part coordinated by PGC-1α.

AMPK activation stimulates autophagy and ameliorates muscular dystrophy in the mdx mouse diaphragm.

Duchenne muscular dystrophy (DMD) is characterized by myofiber death from apoptosis or necrosis, leading in many patients to fatal respiratory muscle weakness. Among other pathological features, DMD muscles show severely deranged metabolic gene regulation and mitochondrial dysfunction. Defective mitochondria not only cause energetic deficiency, but also play roles in promoting myofiber atrophy and injury via opening of the mitochondrial permeability transition pore. Autophagy is a bulk degradative mechanism that serves to augment energy production and eliminate defective mitochondria (mitophagy). We hypothesized that pharmacological activation of AMP-activated protein kinase (AMPK), a master metabolic sensor in cells and on-switch for the autophagy-mitophagy pathway, would be beneficial in the mdx mouse model of DMD. Treatment of mdx mice for 4 weeks with an established AMPK agonist, AICAR (5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside), potently triggered autophagy in the mdx diaphragm without inducing muscle fiber atrophy. In AICAR-treated mdx mice, the exaggerated sensitivity of mdx diaphragm mitochondria to calcium-induced permeability transition pore opening was restored to normal levels. There were associated improvements in mdx diaphragm histopathology and in maximal force-generating capacity, which were not linked to increased mitochondrial biogenesis or up-regulated utrophin expression. These findings suggest that agonists of AMPK and other inducers of the autophagy-mitophagy pathway can help to promote the elimination of defective mitochondria and may thus serve as useful therapeutic agents in DMD.

Rapid onset of specific diaphragm weakness in a healthy murine model of ventilator-induced diaphragmatic dysfunction.

BACKGROUND: Controlled mechanical ventilation is associated with ventilator-induced diaphragmatic dysfunction, which impedes weaning from mechanical ventilation. To design future clinical trials in humans, a better understanding of the molecular mechanisms using knockout models, which exist only in the mouse, is needed. The aims of this study were to ascertain the feasibility of developing a murine model of ventilator-induced diaphragmatic dysfunction and to determine whether atrophy, sarcolemmal injury, and the main proteolysis systems are activated under these conditions. METHODS: Healthy adult male C57/BL6 mice were assigned to three groups: (1) mechanical ventilation with end-expiratory positive pressure of 2-4 cm H2O for 6 h (n=6), (2) spontaneous breathing with continuous positive airway pressure of 2-4 cm H2O for 6 h (n=6), and (3) controls with no specific intervention (n=6). Airway pressure and hemodynamic parameters were monitored. Upon euthanasia, arterial blood gases and isometric contractile properties of the diaphragm and extensor digitorum longus were evaluated. Histology and immunoblotting for the main proteolysis pathways were performed. RESULTS: Hemodynamic parameters and arterial blood gases were comparable between groups and within normal physiologic ranges. Diaphragmatic but not extensor digitorum longus force production declined in the mechanical ventilation group (maximal force decreased by approximately 40%) compared with the control and continuous positive airway pressure groups. No histologic difference was found between groups. In opposition with the calpains, caspase 3 was activated in the mechanical ventilation group. CONCLUSION: Controlled mechanical ventilation for 6 h in the mouse is associated with significant diaphragmatic but not limb muscle weakness without atrophy or sarcolemmal injury and activates proteolysis.

Chronic neuromuscular electrical stimulation improves muscle mass and insulin sensitivity in a mouse model.

Muscle wasting reduces functional capacity and increases cardiometabolic risk in chronic disease. Neuromuscular electrical stimulation (NMES) of the lower limb has been shown to reverse muscle wasting in these patients but its effect on cardiometabolic health is unclear. We investigated a mouse model of in-vivo non-invasive chronic NMES on muscle mass, insulin sensitivity and arterial blood pressure (BP). Twenty-three C57BL6 mice underwent unilateral NMES or sham training over 2.5 weeks while anesthetized by isoflurane. Lower limb muscle mass and the stimulated limb to non-stimulated limb muscle mass ratio were compared between groups (NMES vs. sham). Insulin sensitivity was assessed 48 h after training using an intraperitoneal insulin tolerance test (ITT) and BP was assessed before and after training using the tail-cuff technique. After training, muscle mass increased in NMES vs. sham (416 ± 6 vs. 397 ± 6 mg, p = 0.04) along with the ratio of muscle mass (+3 ± 1% vs. -1 ± 1% p = 0.04). Moreover, insulin sensitivity improved in NMES vs. sham (average blood glucose during ITT: 139.6 ± 8.5 vs. 161.9 ± 9.0 mg/dl blood, p = 0.01). BP was decreased in both groups, although it is likely that the effect of NMES on BP was dampened by repetitive anesthesia. The metabolic benefit of NMES training could be of great utility in patients with chronic disease. Moreover, the clinical-like mouse model of NMES is an effective tool to investigate the systemic effects of local muscle strengthening.

Effect of contractile activity on PGC-1α transcription in young and aged skeletal muscle.

Mitochondrial impairments are often noted in aged skeletal muscle. The transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is integral to maintaining mitochondria, and its expression declines in aged muscle. It remains unknown whether this is due to a transcriptional deficit during aging. Our study examined PGC-1α transcription in muscle from young and old F344BN rats. Using a rat PGC-1α promoter-reporter construct, we found that PGC-1α transcription was reduced by ∼65% in aged TA muscle, accompanied by decreases in PGC-1α mRNA and transcript stability. Altered expression patterns in PGC-1α transcription regulatory factors, including nuclear respiratory factor 2, upstream transcription factor 1, activating transcription factor 2, and yin yang 1, were noted in aged muscle. Acute contractile activity (CA) followed by recovery was employed to examine whether PGC-1α transcription could be activated in aged muscle similar to that observed in young muscle. AMPK and p38 signaling was attenuated in aged muscle. CA evoked an upregulation of PGC-1α transcription in both young and aged groups, whereas mRNAs encoding PGC-1α and cytochrome oxidase subunit IV were induced during the recovery period. Global DNA methylation, an inhibitory event for transcription, was enhanced in aged muscle, likely a result of elevated methyltransferase enzyme Dnmt3b in aged muscle. Successive bouts of CA for 7 days to evaluate longer-term consequences resulted in a rescue of PGC-1α and downstream mRNAs in aged muscle. Our data indicate that diminished mitochondria in aged muscle is due partly to a deficit in PGC-1α transcription, a result of attenuated upstream signaling. Contractile activity is an appropriate countermeasure to restore PGC-1α expression and mitochondrial content in aged muscle. NEW & NOTEWORTHY PGC-1α is a regulator of mitochondrial biogenesis in muscle. We demonstrate that PGC-1α expression is reduced in aging muscle due to decreases in transcriptional and posttranscriptional mechanisms. The transcriptional deficit is due to alterations in transcription factor expression, reduced signaling, and DNA methylation. Acute exercise can initiate signaling to reverse the transcriptional defect, restoring PGC-1α expression toward young values, suggesting a mechanism whereby aged muscle can respond to exercise for the promotion of mitochondrial biogenesis.

Contractile activity attenuates autophagy suppression and reverses mitochondrial defects in skeletal muscle cells.

Macroautophagy/autophagy is a survival mechanism that facilitates protein turnover in post-mitotic cells in a lysosomal-dependent process. Mitophagy is a selective form of autophagy, which arbitrates the selective recognition and targeting of aberrant mitochondria for degradation. Mitochondrial content in cells is the net balance of mitochondrial catabolism via mitophagy, and organelle biogenesis. Although the latter process has been well described, mitophagy in skeletal muscle is less understood, and it is currently unknown how these two opposing mechanisms converge during contractile activity. Here we show that chronic contractile activity (CCA) in muscle cells induced mitochondrial biogenesis and coordinately enhanced the expression of TFEB (transcription factor EB) and PPARGC1A/PGC-1α, master regulators of lysosome and mitochondrial biogenesis, respectively. CCA also enhanced the expression of PINK1 and the lysosomal protease CTSD (cathepsin D). Autophagy blockade with bafilomycin A1 (BafA) reduced mitochondrial state 3 and 4 respiration, increased ROS production and enhanced the accumulation of MAP1LC3B-II/LC3-II and SQSTM1/p62. CCA ameliorated this mitochondrial dysfunction during defective autophagy, increased PPARGC1A, normalized LC3-II levels and reversed mitochondrially-localized SQSTM1 toward control levels. NAC emulated the LC3-II reductions induced by contractile activity, signifying that a decrease in oxidative stress could represent a mechanism of autophagy normalization brought about by CCA. CCA enhances mitochondrial biogenesis and lysosomal activity, and normalizes autophagy flux during autophagy suppression, partly via ROS-dependent mechanisms. Thus, contractile activity represents a potential therapeutic intervention for diseases in which autophagy is inhibited, such as vacuolar myopathies in skeletal muscle, by establishing a healthy equilibrium of anabolic and catabolic pathways. ABBREVIATIONS: AMPK: AMP-activated protein kinase; BafA: bafilomycin A1; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; CCA: chronic contractile activity; COX4I1: cytochrome c oxidase subunit 4I1; DMEM: Dulbecco's modified Eagle's medium; GFP: green fluorescent protein; LSD: lysosomal storage diseases; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NAC: N-acetylcysteine; PPARGC1A: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; PINK1: PTEN induced putative kinase 1; ROS: reactive oxygen species; SOD2: superoxide dismutase 2, mitochondrial; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB.

High intensity aerobic exercise training improves chronic intermittent hypoxia-induced insulin resistance without basal autophagy modulation.

Chronic intermittent hypoxia (IH) associated with obstructive sleep apnea (OSA) is a major risk factor for cardiovascular and metabolic diseases (insulin resistance: IR). Autophagy is involved in the pathophysiology of IR and high intensity training (HIT) has recently emerged as a potential therapy. We aimed to confirm IH-induced IR in a tissue-dependent way and to explore the preventive effect of HIT on IR-induced by IH. Thirty Swiss 129 male mice were randomly assigned to Normoxia (N), Intermittent Hypoxia (IH: 21-5% FiO2, 30 s cycle, 8 h/day) or IH associated with high intensity training (IH HIT). After 8 days of HIT (2*24 min, 50 to 90% of Maximal Aerobic Speed or MAS on a treadmill) mice underwent 14 days IH or N. We found that IH induced IR, characterized by a greater glycemia, an impaired insulin sensitivity and lower AKT phosphorylation in adipose tissue and liver. Nevertheless, MAS and AKT phosphorylation were greater in muscle after IH. IH associated with HIT induced better systemic insulin sensitivity and AKT phosphorylation in liver. Autophagy markers were not altered in both conditions. These findings suggest that HIT could represent a preventive strategy to limit IH-induced IR without change of basal autophagy.

The regulation of mitochondrial transcription factor A (Tfam) expression during skeletal muscle cell differentiation.

The ATP demand required for muscle development is accommodated by elevations in mitochondrial biogenesis, through the co-ordinated activities of the nuclear and mitochondrial genomes. The most important transcriptional activator of the mitochondrial genome is mitochondrial transcription factor A (Tfam); however, the regulation of Tfam expression during muscle differentiation is not known. Thus, we measured Tfam mRNA levels, mRNA stability, protein expression and localization and Tfam transcription during the progression of muscle differentiation. Parallel 2-fold increases in Tfam protein and mRNA were observed, corresponding with 2-3-fold increases in mitochondrial content. Transcriptional activity of a 2051 bp promoter increased during this differentiation period and this was accompanied by a 3-fold greater Tfam mRNA stabilization. Interestingly, truncations of the promoter at 1706 bp, 978 bp and 393 bp promoter all exhibited 2-3-fold higher transcriptional activity than the 2051 bp construct, indicating the presence of negative regulatory elements within the distal 350 bp of the promoter. Activation of AMP kinase augmented Tfam transcription within the proximal promoter, suggesting the presence of binding sites for transcription factors that are responsive to cellular energy state. During differentiation, the accumulating Tfam protein was progressively distributed to the mitochondrial matrix where it augmented the expression of mtDNA and COX (cytochrome c oxidase) subunit I, an mtDNA gene product. Our data suggest that, during muscle differentiation, Tfam protein levels are regulated by the availability of Tfam mRNA, which is controlled by both transcription and mRNA stability. Changes in energy state and Tfam localization also affect Tfam expression and action in differentiating myotubes.

Exercise and the Regulation of Mitochondrial Turnover.

Exercise is a well-known stimulus for the expansion of the mitochondrial pool within skeletal muscle. Mitochondria have a remarkable ability to remodel their networks and can respond to an array of signaling stimuli following contractile activity to adapt to the metabolic demands of the tissue, synthesizing proteins to expand the mitochondrial reticulum. In addition, when they become dysfunctional, these organelles can be recycled by a specialized intracellular system. The signals regulating this mitochondrial life cycle of synthesis and degradation during exercise are still an area of great research interest. As mitochondrial turnover has valuable consequences in physical performance, in addition to metabolic health, disease, and aging, consideration of the signals which control this cycle is vital. This review focuses on the regulation of mitochondrial turnover in skeletal muscle and summarizes our current understanding of the impact that exercise has in modulating this process.

Combined Strategies for Maintaining Skeletal Muscle Mass and Function in Aging: Myostatin Inactivation and AICAR-Associated Oxidative Metabolism Induction.

Myostatin (mstn) blockade, resulting in muscle hypertrophy, is a promising therapy to counteract age-related muscle loss. However, oxidative and mitochondrial deficit observed in young mice with myostatin inhibition could be detrimental with aging. The aim of this study was (a) to bring original data on metabolic and mitochondrial consequences of mstn inhibition in old mice, and (b) to examine whether 4-weeks of AICAR treatment, a pharmacological compound known to upregulate oxidative metabolism, may be useful to improve exercise capacity and mitochondrial deficit of 20-months mstn KO versus wild-type (WT) mice. Our results show that despite the enlarged muscle mass, the oxidative and mitochondrial deficit associated with reduced endurance running capacity is maintained in old mstn KO mice but not worsened by aging. Importantly, AICAR treatment induced a significant beneficial effect on running limit time only in old mstn KO mice, with a marked increase in PGC-1α expression and slight beneficial effects on mitochondrial function. We showed that AICAR effects were autophagy-independent. This study underlines the relevance of aged muscle remodelling by complementary approaches that impact both muscle mass and function, and suggest that mstn inhibition and aerobic metabolism activators should be co-developed for delaying age-related deficits in skeletal muscle.