Loss of endogenous estrogen alters mitochondrial metabolism and muscle clock-related protein Rbm20 in female mdx mice.

Female carriers of a Duchenne muscular dystrophy (DMD) gene mutation manifest exercise intolerance and metabolic anomalies that may be exacerbated following menopause due to the loss of estrogen, a known regulator of skeletal muscle function and metabolism. Here, we studied the impact of estrogen depletion (via ovariectomy) on exercise tolerance and muscle mitochondrial metabolism in female mdx mice and the potential of estrogen replacement therapy (using estradiol) to protect against functional and metabolic perturbations. We also investigated the effect of estrogen depletion, and replacement, on the skeletal muscle proteome through an untargeted proteomic approach with TMT-labelling. Our study confirms that loss of estrogen in female mdx mice reduces exercise capacity, tricarboxylic acid cycle intermediates, and citrate synthase activity but that these deficits are offset through estrogen replacement therapy. Furthermore, ovariectomy downregulated protein expression of RNA-binding motif factor 20 (Rbm20), a critical regulator of sarcomeric and muscle homeostasis gene splicing, which impacted pathways involving ribosomal and mitochondrial translation. Estrogen replacement modulated Rbm20 protein expression and promoted metabolic processes and the upregulation of proteins involved in mitochondrial dynamics and metabolism. Our data suggest that estrogen mitigates dystrophinopathic features in female mdx mice and that estrogen replacement may be a potential therapy for post-menopausal DMD carriers.

Effect of the mitochondrial transaminase (GOT2) on membrane potential-sensitive respiration in mitochondria of differentiated C2C12 muscle cells.

We previously showed that the transaminase inhibitor, aminooxyacetic acid, reduced respiration energized at complex II (succinate dehydrogenase, SDH) in mitochondria isolated from mouse hindlimb muscle. The effect required a reduction in membrane potential with resultant accumulation of oxaloacetate (OAA), a potent inhibitor of SDH. To specifically assess the effect of the mitochondrial transaminase, glutamic oxaloacetic transaminase (GOT2) on complex II respiration, and to determine the effect in intact cells as well as isolated mitochondria, we performed respiratory and metabolic studies in wildtype (WT) and CRISPR-generated GOT2 knockdown (KD) C2C12 myocytes. Intact cell respiration by GOT2KD cells versus WT was reduced by adding carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) to lower potential. In mitochondria of C2C12 KD cells, respiration at low potential generated by 1 µM FCCP and energized at complex II by 10 mM succinate + 0.5 mM glutamate (but not by complex I substrates) was reduced versus WT mitochondria. Although we could not detect OAA, metabolite data suggested that OAA inhibition of SDH may have contributed to the FCCP effect. C2C12 mitochondria differed from skeletal muscle mitochondria in that the effect of FCCP on complex II respiration was not evident with ADP addition. We also observed that C2C12 cells, unlike skeletal muscle, expressed glutamate dehydrogenase, which competes with GOT2 for glutamate metabolism. In summary, GOT2 KD reduced C2C12 respiration in intact cells at low potential. From differential substrate effects, this occurred largely at complex II. Moreover, C2C12 versus muscle mitochondria differ in complex II sensitivity to ADP and differ markedly in expression of glutamate dehydrogenase.NEW & NOTEWORTHY Impairment of the mitochondrial transaminase, GOT2, reduces complex II (succinate dehydrogenase, SDH)-energized respiration in C2C12 myocytes. This occurs only at low inner membrane potential and is consistent with inhibition of SDH. Incidentally, we observed that C2C12 mitochondria compared with muscle tissue mitochondria differ in sensitivity of complex II respiration to ADP and in the expression of glutamate dehydrogenase.

Effect of 2-aminoethoxydiphenyl borate on the functions of mouse skeletal muscle mitochondria.

This work examined the effect of 2-aminoethoxydiphenyl borate (2-APB) on the functioning of isolated mouse skeletal muscle mitochondria and modeled its putative interaction with mitochondrial proteins. We have shown that 2-APB is able to dose-dependently suppress mitochondrial respiration in state 3 and 3UDNP driven by substrates of complex I and II. This effect of 2-APB was accompanied by a slight dose-dependent decrease in mitochondrial membrane potential and appears to be due to inhibition of complex I and complex III of the electron transport chain (ETC) with IC50 values of 200 and 120 µM, respectively. The results of molecular docking identified putative 2-APB interaction sites in these ETC complexes. 2-APB was shown to dose-dependently inhibit both mitochondrial Ca2+ uptake and Ca2+ efflux, which seems to be caused by a decrease in the membrane potential of the organelles. We have found that 2-APB has no significant effect on mitochondrial calcium retention capacity. On the other hand, 2-APB exhibited antioxidant effect by reducing mitochondrial hydrogen peroxide production but without affecting superoxide generation. It is concluded that the effect of 2-APB on mitochondrial targets should be taken into account when interpreting the results of cell and in vivo experiments.

Impaired mitochondrial quality control in fibromyalgia: Mechanisms involved in skeletal muscle alteration.

Fibromyalgia (FMS) is a persistent syndrome marked by widespread musculoskeletal pain and behavioural symptoms. Given the hypothesis linking FMS aetiology to mitochondrial dysfunction and oxidative stress, we examined the biochemical correlation among these factors by studying specific proteins associated with mitochondrial homeostasis in muscle. Additionally, this study investigated the role of Boswellia serrata gum resin extract (BS), known for its various functions, including the potent induction of antioxidant enzymes, in determining protective or reparative mechanisms in the muscle cells. Sprague-Dawley rats were injected with reserpine to induce FMS. These animals exhibited moderate changes in hind limb skeletal muscles, experiencing mobility difficulties. Additionally, there were noteworthy morphological and ultrastructural alterations, along with the expression of myogenin, mitochondrial enzymes and oxidative stress markers in the gastrocnemius muscle. Interestingly, BS demonstrated a reduction in spontaneous motor activity difficulties. Moreover, BS showed a positive impact on musculoskeletal morphostructural aspects, as well as a decrease in oxidative stress and mitochondrial alterations. In particular, BS restored the mRNA expression of citrate synthase and cytochrome-c oxidase subunit II and the activity of electron transfer chain complexes. BS also influenced mitochondrial biogenesis, upregulating PGC-1α expression and the related transcription factors (Nrf1, Tfam, Nrf2, FOXO3a, SIRT3, GCLC, NQO1, SOD2 and GPx4), oxidative stress (lipid peroxidation, GSH levels and GSH-Px activity) and mitochondrial dynamics and function (Mnf2 expression and CoQ10 levels). Overall, this study underlined the key role of the mitochondrial alteration in FMS and that BS had a very high antioxidant effect in these organelles and also in the cells.

Mitochondrial hydrogen sulfide supplementation improves health in the C. elegans Duchenne muscular dystrophy model.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder characterized by progressive muscle degeneration and weakness due to mutations in the dystrophin gene. The symptoms of DMD share similarities with those of accelerated aging. Recently, hydrogen sulfide (H2S) supplementation has been suggested to modulate the effects of age-related decline in muscle function, and metabolic H2S deficiencies have been implicated in affecting muscle mass in conditions such as phenylketonuria. We therefore evaluated the use of sodium GYY4137 (NaGYY), a H2S-releasing molecule, as a possible approach for DMD treatment. Using the dys-1(eg33) Caenorhabditis elegans DMD model, we found that NaGYY treatment (100 µM) improved movement, strength, gait, and muscle mitochondrial structure, similar to the gold-standard therapeutic treatment, prednisone (370 µM). The health improvements of either treatment required the action of the kinase JNK-1, the transcription factor SKN-1, and the NAD-dependent deacetylase SIR-2.1. The transcription factor DAF-16 was required for the health benefits of NaGYY treatment, but not prednisone treatment. AP39 (100 pM), a mitochondria-targeted H2S compound, also improved movement and strength in the dys-1(eg33) model, further implying that these improvements are mitochondria-based. Additionally, we found a decline in total sulfide and H2S-producing enzymes in dystrophin/utrophin knockout mice. Overall, our results suggest that H2S deficit may contribute to DMD pathology, and rectifying/overcoming the deficit with H2S delivery compounds has potential as a therapeutic approach to DMD treatment.

An Amino Acid Mixture to Counteract Skeletal Muscle Atrophy: Impact on Mitochondrial Bioenergetics.

Skeletal muscle atrophy (SMA) is caused by a rise in muscle breakdown and a decline in protein synthesis, with a consequent loss of mass and function. This study characterized the effect of an amino acid mixture (AA) in models of SMA, focusing on mitochondria. C57/Bl6 mice underwent immobilization of one hindlimb (I) or cardiotoxin-induced muscle injury (C) and were compared with controls (CTRL). Mice were then administered AA in drinking water for 10 days and compared to a placebo group. With respect to CTRL, I and C reduced running time and distance, along with grip strength; however, the reduction was prevented by AA. Tibialis anterior (TA) muscles were used for histology and mitochondria isolation. I and C resulted in TA atrophy, characterized by a reduction in both wet weight and TA/body weight ratio and smaller myofibers than those of CTRL. Interestingly, these alterations were lightly observed in mice treated with AA. The mitochondrial yield from the TA of I and C mice was lower than that of CTRL but not in AA-treated mice. AA also preserved mitochondrial bioenergetics in TA muscle from I and C mice. To conclude, this study demonstrates that AA prevents loss of muscle mass and function in SMA by protecting mitochondria.

Reduction of Mitochondrial Calcium Overload via MKT077-Induced Inhibition of Glucose-Regulated Protein 75 Alleviates Skeletal Muscle Pathology in Dystrophin-Deficient mdx Mice.

Duchenne muscular dystrophy is secondarily accompanied by Ca2+ excess in muscle fibers. Part of the Ca2+ accumulates in the mitochondria, contributing to the development of mitochondrial dysfunction and degeneration of muscles. In this work, we assessed the effect of intraperitoneal administration of rhodacyanine MKT077 (5 mg/kg/day), which is able to suppress glucose-regulated protein 75 (GRP75)-mediated Ca2+ transfer from the sarcoplasmic reticulum (SR) to mitochondria, on the Ca2+ overload of skeletal muscle mitochondria in dystrophin-deficient mdx mice and the concomitant mitochondrial dysfunction contributing to muscle pathology. MKT077 prevented Ca2+ overload of quadriceps mitochondria in mdx mice, reduced the intensity of oxidative stress, and improved mitochondrial ultrastructure, but had no effect on impaired oxidative phosphorylation. MKT077 eliminated quadriceps calcification and reduced the intensity of muscle fiber degeneration, fibrosis level, and normalized grip strength in mdx mice. However, we noted a negative effect of MKT077 on wild-type mice, expressed as a decrease in the efficiency of mitochondrial oxidative phosphorylation, SR stress development, ultrastructural disturbances in the quadriceps, and a reduction in animal endurance in the wire-hanging test. This paper discusses the impact of MKT077 modulation of mitochondrial dysfunction on the development of skeletal muscle pathology in mdx mice.

Lipin1 deficiency causes sarcoplasmic reticulum stress and chaperone-responsive myopathy.

As a consequence of impaired glucose or fatty acid metabolism, bioenergetic stress in skeletal muscles may trigger myopathy and rhabdomyolysis. Genetic mutations causing loss of function of the LPIN1 gene frequently lead to severe rhabdomyolysis bouts in children, though the metabolic alterations and possible therapeutic interventions remain elusive. Here, we show that lipin1 deficiency in mouse skeletal muscles is sufficient to trigger myopathy. Strikingly, muscle fibers display strong accumulation of both neutral and phospholipids. The metabolic lipid imbalance can be traced to an altered fatty acid synthesis and fatty acid oxidation, accompanied by a defect in acyl chain elongation and desaturation. As an underlying cause, we reveal a severe sarcoplasmic reticulum (SR) stress, leading to the activation of the lipogenic SREBP1c/SREBP2 factors, the accumulation of the Fgf21 cytokine, and alterations of SR-mitochondria morphology. Importantly, pharmacological treatments with the chaperone TUDCA and the fatty acid oxidation activator bezafibrate improve muscle histology and strength of lipin1 mutants. Our data reveal that SR stress and alterations in SR-mitochondria contacts are contributing factors and potential intervention targets of the myopathy associated with lipin1 deficiency.

Insulin rapidly increases skeletal muscle mitochondrial ADP sensitivity in the absence of a high lipid environment.

Reductions in mitochondrial function have been proposed to cause insulin resistance, however the possibility that impairments in insulin signaling negatively affects mitochondrial bioenergetics has received little attention. Therefore, we tested the hypothesis that insulin could rapidly improve mitochondrial ADP sensitivity, a key process linked to oxidative phosphorylation and redox balance, and if this phenomenon would be lost following high-fat diet (HFD)-induced insulin resistance. Insulin acutely (60 min post I.P.) increased submaximal (100-1000 µM ADP) mitochondrial respiration ∼2-fold without altering maximal (>1000 µM ADP) respiration, suggesting insulin rapidly improves mitochondrial bioenergetics. The consumption of HFD impaired submaximal ADP-supported respiration ∼50%, however, despite the induction of insulin resistance, the ability of acute insulin to stimulate ADP sensitivity and increase submaximal respiration persisted. While these data suggest that insulin mitigates HFD-induced impairments in mitochondrial bioenergetics, the presence of a high intracellular lipid environment reflective of an HFD (i.e. presence of palmitoyl-CoA) completely prevented the beneficial effects of insulin. Altogether, these data show that while insulin rapidly stimulates mitochondrial bioenergetics through an improvement in ADP sensitivity, this phenomenon is possibly lost following HFD due to the presence of intracellular lipids.

Breast cancer-associated skeletal muscle mitochondrial dysfunction and lipid accumulation is reversed by PPARG.

The peroxisome proliferator-activated receptors (PPARs) have been previously implicated in the pathophysiology of skeletal muscle dysfunction in women with breast cancer (BC) and animal models of BC. This study investigated alterations induced in skeletal muscle by BC-derived factors in an in vitro conditioned media (CM) system and tested the hypothesis that BC cells secrete a factor that represses PPAR-γ (PPARG) expression and its transcriptional activity, leading to downregulation of PPARG target genes involved in mitochondrial function and other metabolic pathways. We found that BC-derived factors repress PPAR-mediated transcriptional activity without altering protein expression of PPARG. Furthermore, we show that BC-derived factors induce significant alterations in skeletal muscle mitochondrial function and lipid accumulation, which are rescued with exogenous expression of PPARG. The PPARG agonist drug rosiglitazone was able to rescue BC-induced lipid accumulation but did not rescue effects of BC-derived factors on PPAR-mediated transcription or mitochondrial function. These data suggest that BC-derived factors alter lipid accumulation and mitochondrial function via different mechanisms that are both related to PPARG signaling, with mitochondrial dysfunction likely being altered via repression of PPAR-mediated transcription, and lipid accumulation being altered via transcription-independent functions of PPARG.

Species-specific metabolic responses of songbird, shorebird, and murine cultured myotubes to n-3 polyunsaturated fatty acids.

Migratory birds may benefit from diets rich in polyunsaturated fatty acids (PUFAs) that could improve exercise performance. Previous investigations suggest that different types of birds may respond differently to PUFA. We established muscle myocyte cell culture models from muscle satellite cells of a migratory passerine songbird (yellow-rumped warbler, Setophaga coronata coronata) and a nonpasserine shorebird (sanderling, Calidris alba). We differentiated and treated avian myotubes and immortalized murine C2C12 myotubes with n-3 PUFA docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and with monounsaturated oleic acid (OA) to compare effects on aerobic performance, metabolic enzyme activities, key fatty acid (FA) transporters, and expression of peroxisome proliferator-activated receptors (PPARs). Sanderling and C2C12 myotubes increased expression of PPARs with n-3 PUFA treatments, whereas expression was unchanged in yellow-rumped warblers. Both sanderlings and yellow-rumped warblers increased expression of fatty acid transporters, whereas C2C12 cells decreased expression following n-3 PUFA treatments. Only yellow-rumped warbler myotubes increased expression of some metabolic enzymes, whereas the sanderling and C2C12 cells were unchanged. PUFA supplementation in C2C12 myotubes increased mitochondrial respiratory chain efficiency, whereas sanderlings increased proton leak-associated respiration and maximal respiration (measurements were not made in warblers). This research indicates that songbirds and shorebirds respond differently to n-3 PUFA and provides support for the hypothesis that n-3 PUFA increase the aerobic capacity of migrant shorebird muscle, which may improve overall endurance flight performance.

Time-to-treatment window and cross-sex potential of ß2-adrenergic receptor-induced mitochondrial biogenesis-mediated recovery after spinal cord injury.

Mitochondrial dysfunction is a well-characterized consequence of spinal cord injury (SCI). We previously reported that treatment with the FDA-approved ß2-adrenergic receptor agonist formoterol beginning 8 h post-SCI induces mitochondrial biogenesis (MB) and improves body composition and locomotor recovery in female mice. To determine the time-to-treatment window of formoterol, female mice were subjected to 80 kdyn contusion SCI and daily administration of vehicle or formoterol (0.3 mg/kg) beginning 24 h after injury. This delayed treatment paradigm improved body composition in female mice by 21 DPI, returning body weight to pre-surgery weight and restoring gastrocnemius mass to sham levels; however, there was no effect on locomotor recovery, as measured by the Basso-Mouse Scale (BMS), or lesion volume. To assess the cross-sex potential of formoterol, injured male mice were treated with vehicle or formoterol (0.3 or 1.0 mg/kg) beginning 8 h after SCI. Formoterol also improved body composition post-SCI in male mice, restoring body weight and muscle mass regardless of dose. Interestingly, however, improved BMS scores and decreased lesion volume was observed only in male mice treated with 0.3 mg/kg. Additionally, 0.3 mg/kg formoterol induced MB in the gastrocnemius and injured spinal cord, as evidenced by increased MB protein expression and mitochondrial number. These data indicate that formoterol treatment improves recovery post-SCI in both male and female mice in a dose- and initiation time-dependent manner. Furthermore, formoterol-induced functional recovery post-SCI is not directly associated with peripheral effects, such as muscle mass and body weight.

Extramyocellular interleukin-6 influences skeletal muscle mitochondrial physiology through canonical JAK/STAT signaling pathways.

Interleukin-6 (IL-6) is a pleiotropic cytokine that has been shown to be produced acutely by skeletal muscle in response to exercise, yet chronically elevated with obesity and aging. The mechanisms by which IL-6 influences skeletal muscle mitochondria acutely and chronically are unclear. To better understand the influence of extramyocellular IL-6 on skeletal muscle mitochondrial physiology, we treated differentiated myotubes with exogenous IL-6 to evaluate the dose- and duration-dependent effects of IL-6 on salient aspects of mitochondrial biology and the role of canonical IL-6 signaling in muscle cells. Acute exposure of myotubes to IL-6 increased the mitochondrial reactive oxygen species (mtROS) production and oxygen consumption rates (JO2 ) in a manner that was dependent on activation of the JAK/STAT pathway. Furthermore, STAT3 activation by IL-6 was partly attenuated by MitoQ, a mitochondrial-targeted antioxidant, suggesting that mtROS potentiates STAT3 signaling in skeletal muscle in response to IL-6 exposure. In concert with effects on mitochondrial physiology, acute IL-6 exposure induced several mitochondrial adaptations, consistent with the stress-induced mitochondrial hyperfusion. Exposure of myotubes to chronically elevated IL-6 further increased mtROS with eventual loss of respiratory capacity. These data provide new evidence supporting the interplay between cytokine signaling and mitochondrial physiology in skeletal muscle.

Colchicine treatment impairs skeletal muscle mitochondrial function and insulin sensitivity in an age-specific manner.

The aim of the study was to investigate the impact of autophagy inhibition on skeletal muscle mitochondrial function and glucose homeostasis in young and aged mice. The transcriptional co-activator PGC-1α regulates muscle oxidative phenotype which has been shown to be linked with basal autophagic capacity. Therefore, young and aged inducible muscle-specific PGC-1α knockout (iMKO) mice and littermate lox/lox controls were used in three separate experiments performed after either saline or colchicine injections on two consecutive days: (1) Euthanization in the basal state obtaining skeletal muscle for mitochondrial respirometry, (2) whole body glucose tolerance test, and (3) in vivo insulin-stimulated 2-deoxyglucose (2-DG) uptake into skeletal muscle. Muscle PGC-1α was not required for maintaining basal autophagy flux, regardless of age. Colchicine-induced inhibition of autophagy was associated with impairments of skeletal muscle mitochondrial function, including reduced ADP sensitivity and altered mitochondrial redox balance in both young and aged mice. Colchicine treatment reduced the glucose tolerance in aged, but not young mice, and similarly in iMKO and lox/lox mice. Colchicine reduced insulin-stimulated 2-DG uptake in soleus muscle in aged mice, independently of PGC-1α, and without affecting insulin-regulated phosphorylation of proximal or distal mediators of insulin signaling. In conclusion, the results indicate that autophagy regulates the mitochondrial ADP sensitivity and redox balance as well as whole body glucose tolerance and skeletal muscle insulin sensitivity in aged mice, with no additional effects of inducible PGC-1α deletion.

Mitochondrial division inhibitor 1 (mdivi-1) increases oxidative capacity and contractile stress generated by engineered skeletal muscle.

In skeletal muscle fibers, mitochondria are densely packed adjacent to myofibrils because adenosine triphosphate (ATP) is needed to fuel sarcomere shortening. However, despite this close physical and biochemical relationship, the effects of mitochondrial dynamics on skeletal muscle contractility are poorly understood. In this study, we analyzed the effects of Mitochondrial Division Inhibitor 1 (mdivi-1), an inhibitor of mitochondrial fission, on the structure and function of both mitochondria and myofibrils in skeletal muscle tissues engineered on micromolded gelatin hydrogels. Treatment with mdivi-1 did not alter myotube morphology, but did increase the mitochondrial turbidity and oxidative capacity, consistent with reduced mitochondrial fission. Mdivi-1 also significantly increased basal, twitch, and tetanus stresses, as measured using the Muscular Thin Film (MTF) assay. Finally, mdivi-1 increased sarcomere length, potentially due to mdivi-1-induced changes in mitochondrial volume and compression of myofibrils. Together, these results suggest that mdivi-1 increases contractile stress generation, which may be caused by an increase in maximal respiration and/or sarcomere length due to increased volume of individual mitochondria. These data reinforce that mitochondria have both biochemical and biomechanical roles in skeletal muscle and that mitochondrial dynamics can be manipulated to alter muscle contractility.

Givinostat as metabolic enhancer reverting mitochondrial biogenesis deficit in Duchenne Muscular Dystrophy.

Duchenne Muscular Dystrophy (DMD) is a rare disorder characterized by progressive muscle wasting, weakness, and premature death. Remarkable progress has been made in genetic approaches, restoring dystrophin, or its function. However, the targeting of secondary pathological mechanisms, such as increasing muscle blood flow or stopping fibrosis, remains important to improve the therapeutic benefits, that depend on tackling both the genetic disease and the downstream consequences. Mitochondrial dysfunctions are one of the earliest deficits in DMD, arise from multiple cellular stressors and result in less than 50% of ATP content in dystrophic muscles. Here we establish that there are two temporally distinct phases of mitochondrial damage with depletion of mitochondrial mass at early stages and an accumulation of dysfunctional mitochondria at later stages, leading to a different oxidative fibers pattern, in young and adult mdx mice. We also observe a progressive mitochondrial biogenesis impairment associated with increased deacetylation of peroxisome proliferator-activated receptor-gamma coactivator 1 α (PGC-1α) promoter. Such histone deacetylation is inhibited by givinostat that positively modifies the epigenetic profile of PGC-1α promoter, sustaining mitochondrial biogenesis and oxidative fiber type switch. We, therefore, demonstrate that givinostat exerts relevant effects at mitochondrial level, acting as a metabolic remodeling agent capable of efficiently promoting mitochondrial biogenesis in dystrophic muscle.

Mutations in the intellectual disability gene Ube2a cause neuronal dysfunction and impair parkin-dependent mitophagy.

The prevalence of intellectual disability is around 3%; however, the etiology of the disease remains unclear in most cases. We identified a series of patients with X-linked intellectual disability presenting mutations in the Rad6a (Ube2a) gene, which encodes for an E2 ubiquitin-conjugating enzyme. Drosophila deficient for dRad6 display defective synaptic function as a consequence of mitochondrial failure. Similarly, mouse mRad6a (Ube2a) knockout and patient-derived hRad6a (Ube2a) mutant cells show defective mitochondria. Using in vitro and in vivo ubiquitination assays, we show that RAD6A acts as an E2 ubiquitin-conjugating enzyme that, in combination with an E3 ubiquitin ligase such as Parkin, ubiquitinates mitochondrial proteins to facilitate the clearance of dysfunctional mitochondria in cells. Hence, we identify RAD6A as a regulator of Parkin-dependent mitophagy and establish a critical role for RAD6A in maintaining neuronal function.

Alisporivir Treatment Alleviates Mitochondrial Dysfunction in the Skeletal Muscles of C57BL/6NCrl Mice with High-Fat Diet/Streptozotocin-Induced Diabetes Mellitus.

Diabetes mellitus is a systemic metabolic disorder associated with mitochondrial dysfunction, with mitochondrial permeability transition (MPT) pore opening being recognized as one of its pathogenic mechanisms. Alisporivir has been recently identified as a non-immunosuppressive analogue of the MPT pore blocker cyclosporin A and has broad therapeutic potential. The purpose of the present work was to study the effect of alisporivir (2.5 mg/kg/day i.p.) on the ultrastructure and functions of the skeletal muscle mitochondria of mice with diabetes mellitus induced by a high-fat diet combined with streptozotocin injections. The glucose tolerance tests indicated that alisporivir increased the rate of glucose utilization in diabetic mice. An electron microscopy analysis showed that alisporivir prevented diabetes-induced changes in the ultrastructure and content of the mitochondria in myocytes. In diabetes, the ADP-stimulated respiration, respiratory control, and ADP/O ratios and the level of ATP synthase in the mitochondria decreased, whereas alisporivir treatment restored these indicators. Alisporivir eliminated diabetes-induced increases in mitochondrial lipid peroxidation products. Diabetic mice showed decreased mRNA levels of Atp5f1a, Ant1, and Ppif and increased levels of Ant2 in the skeletal muscles. The skeletal muscle mitochondria of diabetic animals were sensitized to the MPT pore opening. Alisporivir normalized the expression level of Ant2 and mitochondrial susceptibility to the MPT pore opening. In parallel, the levels of Mfn2 and Drp1 also returned to control values, suggesting a normalization of mitochondrial dynamics. These findings suggest that the targeting of the MPT pore opening by alisporivir is a therapeutic approach to prevent the development of mitochondrial dysfunction and associated oxidative stress in the skeletal muscles in diabetes.

Exercise, but Not Metformin Prevents Loss of Muscle Function Due to Doxorubicin in Mice Using an In Situ Method.

Though effective in treating various types of cancer, the chemotherapeutic doxorubicin (DOX) is associated with skeletal muscle wasting and fatigue. The purpose of this study was to assess muscle function in situ following DOX administration in mice. Furthermore, pre-treatments with exercise (EX) or metformin (MET) were used in an attempt to preserve muscle function following DOX. Mice were assigned to the following groups: control, DOX, DOX + EX, or DOX + MET, and were given a single injection of DOX (15 mg/kg) or saline 3 days prior to sacrifice. Preceding the DOX injection, DOX + EX mice performed 60 min/day of running for 5 days, while DOX + MET mice received 5 daily oral doses of 500 mg/kg MET. Gastrocnemius-plantaris-soleus complex function was assessed in situ via direct stimulation of the sciatic nerve. DOX treatment increased time to half-relaxation following contractions, indicating impaired recovery (p < 0.05). Interestingly, EX prevented any increase in half-relaxation time, while MET did not. An impaired relaxation rate was associated with a reduction in SERCA1 protein content (p = 0.07) and AMPK phosphorylation (p < 0.05). There were no differences between groups in force production or mitochondrial respiration. These results suggest that EX, but not MET may be an effective strategy for the prevention of muscle fatigue following DOX administration in mice.

Alisporivir Improves Mitochondrial Function in Skeletal Muscle of mdx Mice but Suppresses Mitochondrial Dynamics and Biogenesis.

Mitigation of calcium-dependent destruction of skeletal muscle mitochondria is considered as a promising adjunctive therapy in Duchenne muscular dystrophy (DMD). In this work, we study the effect of intraperitoneal administration of a non-immunosuppressive inhibitor of calcium-dependent mitochondrial permeability transition (MPT) pore alisporivir on the state of skeletal muscles and the functioning of mitochondria in dystrophin-deficient mdx mice. We show that treatment with alisporivir reduces inflammation and improves muscle function in mdx mice. These effects of alisporivir were associated with an improvement in the ultrastructure of mitochondria, normalization of respiration and oxidative phosphorylation, and a decrease in lipid peroxidation, due to suppression of MPT pore opening and an improvement in calcium homeostasis. The action of alisporivir was associated with suppression of the activity of cyclophilin D and a decrease in its expression in skeletal muscles. This was observed in both mdx mice and wild-type animals. At the same time, alisporivir suppressed mitochondrial biogenesis, assessed by the expression of Ppargc1a, and altered the dynamics of organelles, inhibiting both DRP1-mediated fission and MFN2-associated fusion of mitochondria. The article discusses the effects of alisporivir administration and cyclophilin D inhibition on mitochondrial reprogramming and networking in DMD and the consequences of this therapy on skeletal muscle health.