Nutraceutical and pharmaceutical cocktails did not preserve diaphragm muscle function or reduce muscle damage in D2-mdx mice.

NEW FINDINGS: What is the central question of this study? We previously demonstrated that quercetin transiently preserved respiratory function in dystrophin-deficient mice. To gain lasting therapeutic benefits, we tested quercetin in combination with nicotinamide riboside, lisinopril and prednisolone in the D2-mdx model. What is the main finding and its importance? We demonstrated that these quercetin-based cocktails did not preserve respiratory or diaphragmatic function or reduce histological damage after 7 months of treatment starting at 4 months of age. ABSTRACT: Duchenne muscular dystrophy is characterized by the absence of dystrophin protein and causes muscle weakness and muscle injury, culminating in respiratory failure and cardiomyopathy. Quercetin transiently improved respiratory function but failed to maintain long-term therapeutic benefits in mdx mice. In this study, we combined quercetin with nicotinamide riboside (NR), lisinopril and prednisolone to assess the efficacy of quercetin-based cocktails. We hypothesized that quercetin, NR and lisinopril independently would improve respiratory function and decrease diaphragmatic injury and when combined would have additive effects. To address this hypothesis, in vivo respiratory function, in vitro diaphragmatic function and histological injury were assessed in DBA (healthy), D2-mdx (dystrophic) and D2-mdx mice treated with combinations of quercetin, NR and lisinopril from 4 to 11 months of age. Respiratory function, assessed using whole-body plethysmography, was largely similar between healthy and dystrophin-deficient mice. Diaphragm specific tension was decreased by ∼50% in dystrophic mice compared with healthy mice (P < 0.05), but fatigue resistance was similar between groups. Contractile area was decreased by ∼10% (P < 0.05) and fibrotic area increased from 3.5% in healthy diaphragms to 27% (P < 0.05) in dystrophic diaphragms. Contrary to expectations, these functional and histological parameters of disease were not offset by any intervention. These data suggest that quercetin, NR and lisinopril, independently and in combination, did not prevent diaphragmatic injury or preserve respiratory function.

Autophagy in the heart is enhanced and independent of disease progression in mus musculus dystrophinopathy models.

BACKGROUND: Duchenne muscular dystrophy is a muscle wasting disease caused by dystrophin gene mutations resulting in dysfunctional dystrophin protein. Autophagy, a proteolytic process, is impaired in dystrophic skeletal muscle though little is known about the effect of dystrophin deficiency on autophagy in cardiac muscle. We hypothesized that with disease progression autophagy would become increasingly dysfunctional based upon indirect autophagic markers. METHODS: Markers of autophagy were measured by western blot in 7-week-old and 17-month-old control (C57) and dystrophic (mdx) hearts. RESULTS: Counter to our hypothesis, markers of autophagy were similar between groups. Given these surprising results, two independent experiments were conducted using 14-month-old mdx mice or 10-month-old mdx/Utrn± mice, a more severe model of Duchenne muscular dystrophy. Data from these animals suggest increased autophagosome degradation. CONCLUSION: Together these data suggest that autophagy is not impaired in the dystrophic myocardium as it is in dystrophic skeletal muscle and that disease progression and related injury is independent of autophagic dysfunction.

Autophagic dysfunction and autophagosome escape in the mdx mus musculus model of Duchenne muscular dystrophy.

AIM: Duchenne muscular dystrophy is caused by the absence of functional dystrophin protein and results in a host of secondary effects. Emerging evidence suggests that dystrophic pathology includes decreased pro-autophagic signalling and suppressed autophagic flux in skeletal muscle, but the relationship between autophagy and disease progression is unknown. The purpose of this investigation was to determine the extent to which basal autophagy changes with disease progression. We hypothesized that autophagy impairment would increase with advanced disease. METHODS: To test this hypothesis, 7-week-old and 17-month-old dystrophic diaphragms were compared to each other and age-matched controls. RESULTS: Changes in protein markers of autophagy indicate impaired autophagic stimulation through AMPK, however, robust pathway activation in dystrophic muscle, independent of disease severity. Relative protein abundance of p62, an inverse correlate of autophagic degradation, was dramatically elevated with disease regardless of age. Likewise, relative protein abundance of Lamp2, a lysosome marker, was decreased twofold at 17 months of age in dystrophic muscle and was confirmed, along with mislocalization, in histological samples, implicating lysosomal dysregulation in this process. In dystrophic muscle, autophagosome-sized p62-positive foci were observed in the extracellular space. Moreover, we found that autophagosomes were released from both healthy and dystrophic diaphragms into the extracellular environment, and the occurrence of autophagosome escape was more frequent in dystrophic muscle. CONCLUSION: These findings suggest autophagic dysfunction proceeds independent of disease progression and blunted degradation of autophagosomes is due in part to decreased lysosome abundance, and contributes to autophagosomal escape to the extracellular space.