Mitochondrial creatine sensitivity is lost in the D2.mdx model of Duchenne muscular dystrophy and rescued by the mitochondrial-enhancing compound Olesoxime

Abstract

Duchenne muscular dystrophy (DMD) is associated with distinct mitochondrial stress responses. Here, we aimed to determine whether the prospective mitochondrial-enhancing compound Olesoxime prevents early-stage mitochondrial stress in limb and respiratory muscle from D2.mdx mice using a proof-of-concept short-term regimen spanning 10-28 days of age. As mitochondrial-cytoplasmic energy transfer occurs via ATP- or phosphocreatine-dependent phosphate shuttling, we assessed bioenergetics with or without creatine in vitro. We observed that disruptions in Complex I-supported respiration and H2O2 emission in D2.mdx quadriceps and diaphragm were amplified by creatine demonstrating mitochondrial creatine insensitivity manifests ubiquitously and early in this model. Olesoxime selectively rescued or maintained creatine sensitivity in both muscles, independent of the abundance of respiration-related mitochondrial proteins or mitochondrial creatine kinase cysteine oxidation in quadriceps. Mitochondrial calcium retention capacity and glutathione were altered in a muscle-specific manner in D2.mdx but were generally unchanged by Olesoxime. Treatment reduced serum creatine kinase (muscle damage) and preserved cage hang-time, microCT-based volumes of lean compartments including whole body, hindlimb and bone, recovery of diaphragm force after fatigue, and cross-sectional area of diaphragm type IIX fibre, but reduced type I fibres in quadriceps. Grip strength, voluntary wheel-running and fibrosis were unaltered by Olesoxime. In summary, locomotor and respiratory muscle mitochondrial creatine sensitivities are lost during early stages in D2.mdx mice but are preserved by short-term treatment with Olesoxime in association with specific indices of muscle quality suggesting early myopathy in this model is at least partially attributed to mitochondrial stress.

Disrupted cardiac bioenergetics linked to oxidized mitochondrial creatine kinase are rescued by the mitochondrial-targeting peptide SBT-20 in the D2.mdx model of Duchenne muscular dystrophy

“…We were unable to assess cardiac function in aged D2.mdx mice due to their qualitatively frail nature. Furthermore, while we did not see robust differences in grip strength compared to agematched wild type mice, we did note that the absolute values of grip strength in the 12 month old wild type mice are ~50% of the values we have reported previously in this strain at 4 weeks of age [7,16] suggesting an aging effect occurred in the control group. Nonetheless, the other parameters demonstrate a severe myopathy in the aged D2.mdx mice.…”

“…The greater abundance of cysteine oxidation on mtCK was consistent with higher rates of creatine-sensitive mH2O2 during oxidative phosphorylation and the more oxidized cellular environment (lower GSH:GSSG). mtCK oxidation may be unique to advanced stages of this disease, given we previously reported no differences in 4-week D2.mdx mice, at least in skeletal muscle [16]. Further studies could examine the precise form of thiol modification that occurs in aged D2.mdx mice, such as glutathionylation, considering its emerging role in linking redox signaling to metabolic control [20,21] and considering the shift in GSH:GSSG noted in the present study.…”

“…The increased creatine-dependent mH2O2 during oxidative phosphorylation in aged D2.mdx mice guided us to hypothesize that mtCK thiols would be more oxidized than in wild type given mtCK is a redox-sensitive protein (reviewed in [9]). We next immunoprecipitated mtCK from left ventricles and incubated the extract in a maleimide-tagged fluorophore that binds irreversibly to reduced cysteine thiols, with no affinity to oxidized thiols, as previously described ( [16][17][18], Supplemental Figure S3). This experiment demonstrated that mtCK is more oxidized in aged D2.mdx mice (Figure 3A).…”

Muscle weakness and mitochondrial stress occur before metastasis in a novel mouse model of ovarian cancer cachexia

“…25M, 100M and 500M ADP were selected to reflect creatine sensitivity as this is within the predicted range that is sensitive to the effects of mitochondrial creatine kinase (mtCK) [23,[46][47][48]. Maximal ADP-stimulated respiration was Another approach for evaluating changes in the relative control exerted by mitochondrial creatine kinase on ADP-stimulated respiration, or 'mitochondrial creatine sensitivity', is to calculate the ratio of respiration at submaximal ADP concentrations in both +Creatine/-Creatine conditions [16,23,28,46,49]. In the TA, creatine sensitivities were unchanged across time points indicating alterations in respiration were similar between both high energy phosphate shuttling systems (SFigure 4E).…”

Memory impairment in the D2.mdx mouse model of Duchenne muscular dystrophy is prevented by the adiponectin receptor agonist ALY688

“…Given that adiponectindeficient mice have impaired recognition memory (Bloemer et al, 2019), we hypothesized that a recently developed slow-release formulation (ALY688-SR) with improved pharmacokinetic properties enabling once-daily injection (Allysta Pharmaceutical, unpublished observations) would improve memory during early disease stages in young D2.mdx mice, in association with lower inflammation and enhanced mitochondrial metabolism. (Bellissimo et al, 2023;Coley et al, 2016;Hughes et al, 2020Hughes et al, , 2019Ramos et al, 2020). Mice were killed 20-24 h after the last dose.…”