Tine Borum Billeskov

BACKGROUND: During aging, there is a functional decline in the pool of muscle stem cells (MuSCs) that influences the functional and regenerative capacity of skeletal muscle. Preclinical evidence has suggested that nicotinamide riboside (NR) and pterostilbene (PT) can improve muscle regeneration, e.g., by increasing MuSC function. The objective of this study was to investigate if supplementation with NR and PT (NRPT) promotes skeletal muscle regeneration after muscle injury in elderly individuals by improved recruitment of MuSCs. METHODS: Thirty-two elderly individuals (55–80 years of age) were randomized to daily supplementation with either NRPT (1,000 mg NR and 200 mg PT) or matched placebo. Two weeks after initiation of supplementation, skeletal muscle injury was induced by electrically induced eccentric muscle work. Skeletal muscle biopsies were obtained before, 2 hours after, and 2, 8, and 30 days after injury. RESULTS: A substantial skeletal muscle injury was induced by the protocol and associated with release of myoglobin and creatine kinase, muscle soreness, tissue edema, and a decrease in muscle strength. MuSC content, proliferation, and cell size revealed a large demand for recruitment after injury, but this was not affected by NRPT. Furthermore, histological analyses of muscle fiber area, central nuclei, and embryonic myosin heavy chain showed no NRPT supplementation effect. CONCLUSION: Daily supplementation with 1,000 mg NR and 200 mg PT is safe but does not improve recruitment of the MuSC pool or other measures of muscle recovery in response to injury or subsequent regeneration in elderly individuals. TRIAL REGISTRATION: ClinicalTrials.gov NCT03754842. FUNDING: Novo Nordisk Foundation (NNF17OC0027242) and Novo Nordisk Foundation CBMR.

Significance: The replication stress response protein, ATR, is active in quiescent muscle stem cells in response to DNA:RNA hybrids, and this activity maintains quiescence by ensuring degradation of key cell-cycle transition factors by the E3 ubiquitin ligase cyclin F–SCF complex. This is critical for understanding how stem cells regulate a state of prolonged and reversible cell-cycle arrest and how genome integrity is maintained over time. Together, these studies offer a unique picture of a molecular mechanism controlling stem cell quiescence.

Aging and type 2 diabetes mellitus (T2DM) are associated with impaired skeletal muscle function and degeneration of the skeletal muscle microenvironment. However, the origin and mechanisms underlying the degeneration are not well described in human skeletal muscle. Here we show that skeletal muscles of T2DM patients exhibit pathological degenerative remodeling of the extracellular matrix that was associated with a selective increase of a subpopulation of fibro-adipogenic progenitors (FAPs) marked by expression of THY1 (CD90) - the FAP ^CD90+ . We identified Platelet-derived growth factor (PDGF) signaling as key regulator of human FAP biology, as it promotes proliferation and collagen production at the expense of adipogenesis, an effect accompanied with a metabolic shift towards glycolytic lactate fermentation. FAPs ^CD90+ showed a PDGF-mimetic phenotype, with high proliferative activity and clonogenicity, increased production of extracellular matrix production and enhanced glycolysis. Importantly, the pathogenic phenotype of T2DM FAP ^CD90+ was reduced by treatment with the anti-diabetic drug Metformin. These data identify PDGF-driven conversion of a sub-population of FAPs as a key event in the pathogenic accumulation of extracellular matrix in T2DM muscles.

Mitochondrial dysfunction has been implicated as a central mechanism in the metabolic myopathy accompanying critical limb ischemia (CLI). However, whether mitochondrial dysfunction is directly related to lower extremity ischemia and the structural and molecular mechanisms underpinning mitochondrial dysfunction in CLI patients is not understood. Here, we aimed to study whether mitochondrial dysfunction is a distinctive characteristic of CLI myopathy by assessing mitochondrial respiration in gastrocnemius muscle from 14 CLI patients (65.3 ± 7.8 y) and 15 matched control patients (CON) with a similar comorbidity risk profile and medication regimen but without peripheral ischemia (67.4 ± 7.4 y). Furthermore, we studied potential structural and molecular mechanisms of mitochondrial dysfunction by measuring total, sub-population, and fiber-type-specific mitochondrial volumetric content and cristae density with transmission electron microscopy and by assessing mitophagy and fission/fusion-related protein expression. Finally, we asked whether commonly used biomarkers of mitochondrial content are valid in patients with cardiovascular disease. CLI patients exhibited inferior mitochondrial respiration compared to CON. This respiratory deficit was not related to lower whole-muscle mitochondrial content or cristae density. However, stratification for fiber types revealed ultrastructural mitochondrial alterations in CLI patients compared to CON. CLI patients exhibited an altered expression of mitophagy-related proteins but not fission/fusion-related proteins compared to CON. Citrate synthase, cytochrome c oxidase subunit IV (COXIV), and 3-hydroxyacyl-CoA dehydrogenase (β-HAD) could not predict mitochondrial content. Mitochondrial dysfunction is a distinctive characteristic of CLI myopathy and is not related to altered organelle content or cristae density. Our results link this intrinsic mitochondrial deficit to dysregulation of the mitochondrial quality control system, which has implications for the development of therapeutic strategies.