Circadian clock regulation of muscle stem cells and its potential therapeutic targeting

Abstract

Background: The circadian clock drives daily oscillations of stem cell behaviors. Molecular clock components orchestrate key myogenic properties of muscle stem cells (MuSCs) in muscle regeneration. Recent studies indicate circadian clock involvement in the pathogenesis of Duchene Muscular Dystrophy (DMD) and disease progression. We generated a novel mouse model with Bmal1-mediated clock gain-of-function in satellite cells to explore the effect of genetic clock activation in promoting MuSC regenerative response. Furthermore, our recent discovery of Chlorhexidine (CHX) as a clock-activating molecule with pro-myogenic properties provides a pharmacological tool to probe clock modulation for disease applications. Hypothesis: Genetic and pharmacological activation of the circadian clock may promote regenerative capacity via clock-controlled pro-myogenic mechanisms to mitigate dystrophic disease. Methods: Mice with satellite cell-selective ectopic expression of Bmal1 (BMKI) were subjected to cardiotoxin injury to elicit regenerative response. Muscle regeneration at 3, 7, 14, and 30 days post-injury was interrogated via Pax7 immunostaining to determine satellite cell expansion, embryonic myosin heavy chain for nascent myofiber formation and myogenic gene induction. In parallel, we carried out medicinal chemistry optimization of CHX scaffold that yielded structural derivatives with improved clock-enhancing activities. Their clock-modulatory and pro-myogenic effcacies were determined in primary myoblasts and a pre-clinical DMD model, the mdx mice. Results: Upon injury, Bmal1 knock in (BMKI) mice displayed a prolonged phase of myogenic activation coupled with enhanced neo-myofiber formation during regeneration. Ex vivo culture of primary myoblasts isolated from BMKI mice demonstrated augmented proliferative and myogenic properties. Based on the findings of clock gain-of-function in promoting regenerative myogenesis, we screened CHX analogs for improved clock-enhancing activities and identified a new compound, CM2, with increased effcacy on promoting myogenic differentiation and proliferative growth of C2C12 myoblasts, primary mouse myoblast and dystrophin-deficient myoblasts. Both CHX and CM2 were suffcient to promote nascent myofiber formation in vivo induced by injury. Lastly, direct administration of CHX or CM2 into dystrophic muscle markedly induced regenerative response with up-regulations of myogenic factors and clock components. Studies of these clock-activating compounds in bone marrow-derived microphages revealed significant inhibition of NLRP3 inflammasome activation, suggesting both anti-inflammatory and pro-myogenic mechanisms of pharmacological clock activation may contribute to improved dystrophic pathophysiology in the mdx mice. Conclusions: Through genetic and pharmacological approaches to augment clock function, our study establishes the mechanistic basis for targeting circadian clock to promote muscle regenerative repair. The pro-myogenic and anti-inflammatory actions of clock-enhancing molecules suggest their drug development potentials as novel intervention strategy for dystrophic diseases. Arthur Riggs Diabetes and Metabolism Research Institute Innovative Award, R56AG080294. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.