More than the clock: distinct regulation of muscle function and metabolism by PER2 and RORalpha

Circadian rhythms, governed by the dominant central as well as various peripheral clocks, regulate almost all biological processes, including sleep-wake cycles, hormone secretion, and metabolism. In certain contexts, regulation and function of the peripheral oscillations can be decoupled from the central clock. However, the specific mechanisms underlying muscle-intrinsic clock-dependent modulation of muscle function and metabolism remain unclear. We investigated the outcome of perturbations of the primary and secondary feedback loops of the molecular clock in skeletal muscle by specific gene ablation of Period 2 (Per2) and RAR-related orphan receptor alpha (RORa), respectively. In both models, a dampening of core clock gene oscillation was observed, while the phase was preserved. Moreover, both loops seem involved in the homeostasis of amine groups. Very divergent outcomes were seen for overall muscle gene expression, primarily affecting circadian rhythmicity in the Per2, and non-oscillating genes in the RORα knockouts, leading to distinct outcomes in terms of metabolome and phenotype. These results highlight the entanglement of the molecular clock and muscle plasticity, and allude to specific functions of different clock components, i.e. the primary and secondary feedback loops, in this context. The reciprocal interaction between muscle contractility and circadian clocks might therefore be instrumental to determine a finely tuned adaptation of muscle tissue to perturbations in health and disease. Muscles from PER2 muscle-specific knockout mice (MKO), control (CTRL), and RORa MKO mice were collected at zeitgeber time (ZT) 0, 4, 8, 12, 16, and 20. 3-4 mice were used for seqeuencing replicated for each time-point and each genotype.昼夜节律（Circadian rhythms）由核心中枢钟（central clock）与多种外周钟（peripheral clock）共同调控，几乎覆盖机体所有生物学过程，包括睡眠-觉醒周期、激素分泌及新陈代谢等。在特定情境下，外周振荡（peripheral oscillations）的调控与功能可与中枢钟解耦。然而，依赖肌肉固有钟的肌肉功能与新陈代谢调控背后的具体机制仍未明确。本研究分别通过对周期蛋白2（Period 2, Per2）与视黄酸受体相关孤儿受体α（RAR-related orphan receptor alpha, RORa）实施特异性基因敲除（gene ablation），探究了骨骼肌中分子钟（molecular clock）一级与二级反馈环路扰动所产生的效应。在两种敲除模型中，均观察到核心钟基因的振荡幅度出现衰减，但相位得以保留。此外，两类反馈环路似乎均参与胺类基团的稳态维持。整体肌肉基因表达谱则呈现出显著差异：Per2敲除模型主要影响昼夜节律相关基因，而RORα敲除模型则主要影响非振荡基因，最终在代谢组（metabolome）与表型（phenotype）层面产生了截然不同的结果。上述结果凸显了分子钟与肌肉可塑性（muscle plasticity）之间的紧密关联，并暗示了不同钟组件（即一级与二级反馈环路）在此场景下的特异性功能。因此，肌肉收缩力与昼夜节律之间的双向互作，可能在决定肌肉组织针对健康与疾病状态下的扰动做出精准适应性调控中发挥关键作用。本研究在授时因子时间（zeitgeber time, ZT）0、4、8、12、16及20时，分别采集PER2肌肉特异性敲除小鼠（MKO）、野生型对照（CTRL）以及RORa MKO小鼠的肌肉组织。每个时间点与每种基因型均设置3-4只小鼠进行生物学重复测序。