Circadian Clock in Muscle Disease Etiology and Therapeutic Potential for Duchenne Muscular Dystrophy.

Circadian clock and clock-controlled output pathways exert temporal control in diverse aspects of skeletal muscle physiology, including the maintenance of muscle mass, structure, function, and metabolism. They have emerged as significant players in understanding muscle disease etiology and potential therapeutic avenues, particularly in Duchenne muscular dystrophy (DMD). This review examines the intricate interplay between circadian rhythms and muscle physiology, highlighting how disruptions of circadian regulation may contribute to muscle pathophysiology and the specific mechanisms linking circadian clock dysregulation with DMD. Moreover, we discuss recent advancements in chronobiological research that have shed light on the circadian control of muscle function and its relevance to DMD. Understanding clock output pathways involved in muscle mass and function offers novel insights into the pathogenesis of DMD and unveils promising avenues for therapeutic interventions. We further explore potential chronotherapeutic strategies targeting the circadian clock to ameliorate muscle degeneration which may inform drug development efforts for muscular dystrophy.

Anti-adipogenic properties of clock activator chlorhexidine and a new derivative.

Background: The circadian clock exerts temporal control of metabolic pathways to maintain homeostasis, and its disruption leads to the development of obesity and insulin resistance. In adipose tissue, key regulators of clock machinery orchestrate adipogenic processes via the Wnt signaling pathway to impact mature adipocyte development. Methods: Based on the recent finding of chlorhexidine as a new clock activator, we determined its potential anti-adipogenic activities in distinct adipogenic progenitor models. Furthermore, we report the structural optimization of chlorhexidine leading to the discovery of analogs with improved efficacy in inhibiting adipogenesis. Results: In adipogenic progenitors with Per2::dLuc luciferase reporter, Chlorhexidine shortened clock period length with induction of core clock components. Consistent with its clock-activating function, Chlorhexidine robustly suppressed the lineage commitment and maturation of adipogenic mesenchymal precursors, with comparable effect on inhibiting preadipocyte terminal differentiation. Mechanistically, we show that Chlorhexidine induces signaling components of the Wnt pathway resulting in activation of Wnt activity. Via modification of its chemical scaffold, we generated analogs of chlorhexidine that led to the identification of CM002 as a new clock- activating molecule with improved anti-adipogenic activity. Conclusions: Collectively, our findings uncovered the anti-adipogenic functions of a new class of small molecule clock activators. These compounds provide novel chemical probes to dissect clock function in maintaining metabolic homeostasis and may have therapeutic implications in obesity and associated metabolic disorders.

Transcription Repression of CRY2 via PER2 Interaction Promotes Adipogenesis.

The circadian clock is driven by a transcriptional-translational feedback loop, and cryptochrome 2 (CRY2) represses CLOCK/BMAL1-induced transcription activation. Despite the established role of clock in adipogenic regulation, whether the CRY2 repressor activity functions in adipocyte biology remains unclear. Here we identify a critical cysteine residue of CRY2 that mediates interaction with Period 2 (PER2). We further demonstrate that this mechanism is required for repressing circadian clock-controlled Wnt signaling to promote adipogenesis. CRY2 protein is enriched in white adipose depots and robustly induced by adipogenic differentiation. Via site-directed mutagenesis, we identified that a conserved CRY2 cysteine at 432 within the loop interfacing with PER2 mediates heterodimer complex formation that confers transcription repression. C432 mutation disrupted PER2 association without affecting BMAL1 binding, leading to loss of repression of clock transcription activation. In preadipocytes, whereas CRY2 enhanced adipocyte differentiation, the repression-defective C432 mutant suppressed this process. Furthermore, silencing of CRY2 attenuated, while stabilization of CRY2 by KL001 markedly augmented adipocyte maturation. Mechanistically, we show that transcriptional repression of Wnt pathway components underlies CRY2 modulation of adipogenesis. Collectively, our findings elucidate a CRY2-mediated repression mechanism that promotes adipocyte development, and implicate its potential as a clock intervention target for obesity.

Targeted screening and identification of chlorhexidine as a pro-myogenic circadian clock activator.

BACKGROUND: The circadian clock is an evolutionarily conserved mechanism that exerts pervasive temporal control in stem cell behavior. This time-keeping machinery is required for orchestrating myogenic progenitor properties in regenerative myogenesis that ameliorates muscular dystrophy. Here we report a screening platform to discover circadian clock modulators that promote myogenesis and identify chlorhexidine (CHX) as a clock-activating molecule with pro-myogenic activities. METHODS: A high-throughput molecular docking pipeline was applied to identify compounds with a structural fit for a hydrophobic pocket within the key circadian transcription factor protein, Circadian Locomotor Output Cycles Kaput (CLOCK). These identified molecules were further screened for clock-modulatory activities and functional validations for pro-myogenic properties. RESULTS: CHX was identified as a clock activator that promotes distinct aspects of myogenesis. CHX activated circadian clock that reduced cycling period length and augmented amplitude. This action was mediated by the targeted CLOCK structure via augmented interaction with heterodimer partner Bmal1, leading to enhanced CLOCK/Bmal1-controlled transcription with upregulation of core clock genes. Consistent with its clock-activating function, CHX displayed robust effects on stimulating myogenic differentiation in a clock-dependent manner. In addition, CHX augmented the proliferative and migratory activities of myoblasts. CONCLUSION: Our findings demonstrate the feasibility of a screening platform to discover clock modulators with myogenic regulatory activities. Discovery of CHX as a pro-myogenic molecule could be applicable to promote regenerative capacities in ameliorating dystrophic or degenerative muscle diseases.

The Clock-modulatory Activity of Nobiletin Suppresses Adipogenesis Via Wnt Signaling.

The circadian clock machinery exerts transcriptional control to modulate adipogenesis and its disruption leads to the development of obesity. Here, we report that Nobiletin, a circadian clock amplitude-enhancing molecule, displays antiadipogenic properties via activation of Wnt signaling pathway that is dependent on its clock modulation. Nobiletin augmented clock oscillatory amplitude with period lengthening in the adipogenic mesenchymal precursor cells and preadipocytes, accompanied by an induction of Bmal1 and clock components within the negative feedback arm. Consistent with its clock-modulatory activity, Nobiletin strongly inhibited the lineage commitment and terminal differentiation of adipogenic progenitors. Mechanistically, we show that Nobiletin induced the reactivation of Wnt signaling during adipogenesis via transcriptional up-regulation of key components within this pathway. Furthermore, Nobiletin administration in mice markedly reduced adipocyte hypertrophy, leading to a significant loss of fat mass and reduction of body weight. Last, Nobiletin inhibited the differentiation of primary preadipocytes, and this effect was dependent on a functional clock regulation. Collectively, our findings uncover a novel activity of Nobiletin in suppressing adipocyte development in a clock-dependent manner, implicating its potential application in countering obesity and associated metabolic consequences.

Transcription repression of Cry2 via Per2 interaction promotes adipogenesis.

The circadian clock is driven by a transcriptional-translational feedback loop, and Cryptochrome 2 (Cry2) represses CLOCK/Bmal1-induced transcription activation. Despite the established role of clock in adipogenic regulation, whether the Cry2 repressor activity functions in adipocyte biology remains unclear. Here we identify a critical cysteine residue of Cry2 that mediates interaction with Per2, and demonstrate that this mechanism is required for clock transcriptional repression that inhibits Wnt signaling to promote adipogenesis. Cry2 protein is enriched in white adipose depots and was robustly induced by adipocyte differentiation. Via site-directed mutagenesis, we identified that a conserved Cry2 Cysteine at 432 within the loop interfacing with Per2 mediates heterodimer complex formation that confers transcription repression. C432 mutation disrupted Per2 association without affecting Bmal1 binding, leading to loss of repression of clock transcription activation. In preadipocytes, whereas Cry2 enhanced adipogenic differentiation, the repression-defective C432 mutant suppressed this process. Furthermore, silencing of Cry2 attenuated, while stabilization of Cry2 by KL001 markedly augmented adipocyte maturation. Mechanistically, we show that transcriptional repression of Wnt pathway components underlies Cry2 modulation of adipogenesis. Collectively, our findings elucidate a Cry2-mediated repression mechanism that promotes adipocyte development, and implicate its potential as a clock intervention target for obesity.

The clock-modulatory activity of Nobiletin suppresses adipogenesis via Wnt signaling.

The circadian clock machinery exerts transcriptional control to modulate adipogenesis and its disruption leads to the development of obesity. Here we report that Nobiletin, a clock amplitude-enhancing molecule, displays anti-adipogenic properties via activating a clock-controlled Wnt signaling pathway that suppresses adipocyte differentiation. Nobiletin augmented clock oscillation with period length shortening in the adipogenic mesenchymal precursor cells and preadipocytes, accompanied by an induction of Bmal1 and core clock components. Consistent with its circadian clock-modulatory activity, Nobiletin inhibited the lineage commitment and terminal differentiation of adipogenic progenitors. Mechanistically, we show that Nobiletin induced the re-activation of Wnt signaling during adipogenic differentiation via transcriptional up-regulation of key components of this pathway. Furthermore, Nobiletin administration in mice markedly reduced adipocyte hypertrophy, leading to a significant loss of fat mass and body weight reduction. Lastly, Nobiletin inhibited the maturation of primary preadipocytes and this effect was dependent on a functional clock regulation. Collectively, our findings uncover a novel activity of Nobiletin in suppressing adipocyte development, implicating its potential therapeutic application in countering obesity and its associated metabolic consequences.