Background Muscle wasting and weakness in Duchenne muscular dystrophy (DMD) causes severe locomotor limitations and early death due in part to respiratory muscle failure. Given that current clinical practice focuses on treating secondary complications in this genetic disease, there is a clear need to identify additional contributions in the aetiology of this myopathy for knowledge‐guided therapy development. Here, we address the unresolved question of whether the complex impairments observed in DMD are linked to elevated mitochondrial H 2 O 2 emission in conjunction with impaired oxidative phosphorylation. This study performed a systematic evaluation of the nature and degree of mitochondrial‐derived H 2 O 2 emission and mitochondrial oxidative dysfunction in a mouse model of DMD by designing in vitro bioenergetic assessments that attempt to mimic in vivo conditions known to be critical for the regulation of mitochondrial bioenergetics. Methods Mitochondrial bioenergetics were compared with functional and histopathological indices of myopathy early in DMD (4 weeks) in D2.B10‐DMD mdx /2J mice (D2. mdx )—a model that demonstrates severe muscle weakness. Adenosine diphosphate's (ADP's) central effect of attenuating H 2 O 2 emission while stimulating respiration was compared under two models of mitochondrial‐cytoplasmic phosphate exchange (creatine independent and dependent) in muscles that stained positive for membrane damage (diaphragm, quadriceps, and white gastrocnemius). Results Pathway‐specific analyses revealed that Complex I‐supported maximal H 2 O 2 emission was elevated concurrent with a reduced ability of ADP to attenuate emission during respiration in all three muscles (mH 2 O 2 : +17 to +197% in D2. mdx vs. wild type). This was associated with an impaired ability of ADP to stimulate respiration at sub‐maximal and maximal kinetics (−17 to −72% in D2. mdx vs. wild type), as well as a loss of creatine‐dependent mitochondrial phosphate shuttling in diaphragm and quadriceps. These changes largely occurred independent of mitochondrial density or abundance of respiratory chain complexes, except for quadriceps. This muscle was also the only one exhibiting decreased calcium retention capacity, which indicates increased sensitivity to calcium‐induced permeability transition pore opening. Increased H 2 O 2 emission was accompanied by a compensatory increase in total glutathione, while oxidative stress markers were unchanged. Mitochondrial b...

Key points Ninety‐eight per cent of patients with Duchenne muscular dystrophy (DMD) develop cardiomyopathy, with 40% developing heart failure. While increased propensity for mitochondrial induction of cell death has been observed in left ventricle, it remains unknown whether this is linked to impaired mitochondrial respiratory control and elevated H2O2 emission prior to the onset of cardiomyopathy. Classic mouse models of DMD demonstrate hyper‐regeneration in skeletal muscle which may mask mitochondrial abnormalities. Using a model with less regenerative capacity that is more akin to DMD patients, we observed elevated left ventricular mitochondrial H2O2 and impaired oxidative phosphorylation in the absence of cardiac remodelling or overt cardiac dysfunction at 4 weeks. These impairments were associated with dysfunctions at complex I, governance by ADP and creatine‐dependent phosphate shuttling, which results in a less efficient response to energy demands. Mitochondria may be a therapeutic target for the treatment of cardiomyopathy in DMD. Abstract In Duchenne muscular dystrophy (DMD), mitochondrial dysfunction is predicted as a response to numerous cellular stressors, yet the contribution of mitochondria to the onset of cardiomyopathy remains unknown. To resolve this uncertainty, we designed in vitro assessments of mitochondrial bioenergetics to model mitochondrial control parameters that influence cardiac function. Both left ventricular mitochondrial responsiveness to the central bioenergetic controller ADP and the ability of creatine to facilitate mitochondrial–cytoplasmic phosphate shuttling were assessed. These measurements were performed in D2.B10‐DMDmdx/2J mice – a model that demonstrates skeletal muscle atrophy and weakness due to limited regenerative capacities and cardiomyopathy more akin to people with DMD than classic models. At 4 weeks of age, there was no evidence of cardiac remodelling or cardiac dysfunction despite impairments in ADP‐stimulated respiration and ADP attenuation of H2O2 emission. These impairments were seen at both submaximal and maximal ADP concentrations despite no reductions in mitochondrial content markers. The ability of creatine to enhance ADP's control of mitochondrial bioenergetics was also impaired, suggesting an impairment in mitochondrial creatine kinase‐dependent phosphate shuttling. Susceptibly to permeability transition pore opening and the subsequent activation of cell death pathways remained unchanged. Mitochondrial H2O2 emission was elevated despite no change in markers of irreversible oxidative damage, suggesting alternative redox signalling mechanisms should be explored. These findings demonstrate that selective mitochondrial dysfunction precedes the onset of overt cardiomyopathy in D2.mdx mice, suggesting that improving mitochondrial bioenergetics by restoring ADP, creatine‐dependent phosphate shuttling and complex I should be considered for treating DMD patients.

Microbiopsies of human skeletal muscle are increasingly adopted by physiologists for a variety of experimental assays given the reduced invasiveness of this procedure compared to the classic Bergstrom percutaneous biopsy technique. However, a recent report demonstrated lower mitochondrial respiration in saponin-permeabilized muscle fiber bundles (PmFB) prepared from microbiopsies vs. Bergstrom biopsies. We hypothesized that ADP-induced contraction (rigor) of smaller length microbiopsy PmFB causes a greater reduction in maximal respiration vs. Bergstrom, such that respiration could be increased by a myosin II ATPase-inhibitor (Blebbistatin; BLEB). Eleven males and females each received a 2 mm diameter percutaneous microbiopsy and a 5 mm diameter Bergstrom percutaneous biopsy in opposite legs. Glutamate/malate (5/0.5 mM)—supported respiration in microbiopsy PmFB was lower than Bergstrom at submaximal concentrations of ADP. 5 μM BLEB reduced this impairment such that there were no differences relative to Bergstrom ± BLEB. Surprisingly, pyruvate (5 mM)-supported respiration was not different between either biopsy technique ±BLEB, whereas BLEB increased succinate-supported respiration in Bergstrom only. H2O2 emission was lower in microbiopsy PmFB compared to Bergstrom PmFB in the presence of BLEB. Microbiopsies contained fewer type I fibers (37 vs. 47%) and more type IIX fibers (20 vs. 8%) compared to Bergstrom possibly due to sampling site depth and/or longitudinal location. These findings suggest that smaller diameter percutaneous biopsies yield lower glutamate-supported mitochondrial respiratory kinetics which is increased by preventing ADP-induced rigor with myosin inhibition. Microbiopsies of human skeletal muscle can be utilized for assessing mitochondrial respiratory kinetics in PmFB when assay conditions are supplemented with BLEB, but fiber type differences with this method should be considered.

Lipocalin-2 (Lcn2), a critical component of the innate immune response which binds siderophores and limits bacterial iron acquisition, can elicit spillover adverse proinflammatory effects. Here we show that holo-Lcn2 (Lcn2-siderophore-iron, 1:3:1) increases mitochondrial reactive oxygen species (ROS) generation and attenuates mitochondrial oxidative phosphorylation in adult rat primary cardiomyocytes in a manner blocked by -acetyl-cysteine or the mitochondria-specific antioxidant SkQ1. We further demonstrate using siderophores 2,3-DHBA (2,3-dihydroxybenzoic acid) and 2,5-DHBA that increased ROS and reduction in oxidative phosphorylation are direct effects of the siderophore component of holo-Lcn2 and not due to apo-Lcn2 alone. Extracellular apo-Lcn2 enhanced the potency of 2,3-DHBA and 2,5-DHBA to increase ROS production and decrease mitochondrial respiratory capacity, whereas intracellular apo-Lcn2 attenuated these effects. These actions of holo-Lcn2 required an intact plasma membrane and were decreased by inhibition of endocytosis. The hearts, but not serum, of Lcn2 knockout (LKO) mice contained lower levels of 2,5-DHBA compared with wild-type hearts. Furthermore, LKO mice were protected from ischemia/reperfusion-induced cardiac mitochondrial dysfunction. Our study identifies the siderophore moiety of holo-Lcn2 as a regulator of cardiomyocyte mitochondrial bioenergetics.

Contraction‐mediated lipolysis increases the association of lipid droplets and mitochondria, indicating an important role in the passage of fatty acids from lipid droplets to mitochondria in skeletal muscle. PLIN3 and PLIN5 are of particular interest to the lipid droplet–mitochondria interaction because PLIN3 is able to move about within cells and PLIN5 associates with skeletal muscle mitochondria. This study primarily investigated: 1) if PLIN3 is detected in skeletal muscle mitochondrial fraction; and 2) if mitochondrial protein content of PLIN3 and/or PLIN5 changes following stimulated contraction. A secondary aim was to determine if PLIN3 and PLIN5 associate and whether this changes following contraction. Male Long Evans rats (n = 21; age, 52 days; weight = 317 ± 6 g) underwent 30 min of hindlimb stimulation (10 msec impulses, 100 Hz/3 sec at 10–20 V; train duration 100 msec). Contraction induced a ~50% reduction in intramuscular lipid content measured by oil red‐O staining of red gastrocnemius muscle. Mitochondria were isolated from red gastrocnemius muscle by differential centrifugation and proteins were detected by western blotting. Mitochondrial PLIN5 content was ~1.6‐fold higher following 30 min of contraction and PLIN3 content was detected in the mitochondrial fraction, and unchanged following contraction. An association between PLIN3 and PLIN5 was observed and remained unaltered following contraction. PLIN5 may play a role in mitochondria during lipolysis, which is consistent with a role in facilitating/regulating mitochondrial fatty acid oxidation. PLIN3 and PLIN5 may be working together on the lipid droplet and mitochondria during contraction‐induced lipolysis.