Atypical expression of circadian clock genes in denervated mouse skeletal muscle

The central circadian clock in the suprachiasmatic nucleus of the hypothalamus synchronizes peripheral clocks through neural and humoral signals in most mammalian tissues. Here, we analyzed the effects of unilateral sciatic denervation on the expression of circadian clock- and clock-controlled genes in the gastrocnemius muscles of mice twice per day on days 0, 3, 7, 9, 11 and 14 after denervation and six times on each of days 7 and 28 after denervation to assess the regulation mechanism of the circadian clock in skeletal muscle. Sciatic denervation did not affect systemic circadian rhythms since core body temperature (Day 7), corticosterone secretion (Days 7 and 28), and hepatic clock gene expression remained intact (Days 7 and 28). Expression levels of most circadian clock-related genes such as <i>Arntl, Per1, Rora, Nr1d1</i> and <i>Dbp</i> were reduced in accordance with the extent of muscle atrophy, although circadian <i>Per2</i> expression was significantly augmented (Day 28). Cosinor analysis revealed that the circadian expression of <i>Arntl</i> (Days 7 and 28) and <i>Dbp</i> (Day 28) was phase advanced in denervated muscle. The mRNA expression of <i>Clock</i> was significantly increased in denervated muscle on Day 3 when the severe atrophy was absent, and it was not affected by atrophic progression for 28 days. Sciatic denervation did not affect the expression of these genes in the contralateral muscle (Days 7 and 28), suggesting that humoral changes were not involved in denervation-induced muscle clock disruption. We then analyzed genome-wide gene expression using microarrays to determine the effects of disrupting the molecular clock in muscle on circadian rhythms at Day 7. Among 478 circadian genes, 313 lost rhythmicity in the denervated muscles. These denervation-sensitive genes included the lipid metabolism-related genes, <i>Nrip1, Bbs1, Ptgis, Acot1, Scd2, Hpgd, Insig1, Dhcr24, Ldlr</i> and <i>Mboat1</i>. Our findings revealed that sciatic denervation disrupts the circadian expression of clock and clock-controlled genes either directly or indirectly via muscle atrophy in the gastrocnemius muscles of mice in a gene-specific manner.

下丘脑视交叉上核（suprachiasmatic nucleus, SCN）中的中枢生物钟，可通过神经与体液信号在多数哺乳动物组织中同步外周生物钟。本研究针对小鼠腓肠肌（gastrocnemius muscles），分析单侧坐骨神经去神经支配（unilateral sciatic denervation）对生物钟（circadian clock）及钟控基因（clock-controlled genes）表达的影响：于坐骨神经去神经支配后第0、3、7、9、11、14天每日取样2次，于第7、28天每日取样6次，以解析骨骼肌生物钟的调控机制。坐骨神经去神经支配并未影响全身生物钟节律：第7天时的核心体温、第7与28天时的皮质酮分泌，以及第7、28天时的肝脏生物钟基因表达均保持完整。多数与生物钟相关的基因（如*Arntl*、*Per1*、*Rora*、*Nr1d1*及*Dbp*）的表达水平随肌肉萎缩程度降低，而第28天时的节律性*Per2*表达则显著上调。余弦分析法（Cosinor analysis）显示，去神经支配肌肉中，*Arntl*（第7、28天）与*Dbp*（第28天）的节律表达相位提前。在未出现严重肌肉萎缩的第3天，去神经支配肌肉中*Clock*的mRNA表达显著上调，且该表达不受后续28天肌肉萎缩进展的影响。坐骨神经去神经支配并未影响对侧腓肠肌中上述基因的表达（第7、28天），提示体液变化并未参与去神经支配诱导的肌肉生物钟紊乱。随后，本研究于第7天利用基因芯片（microarrays）开展全基因组基因表达分析，以明确肌肉分子生物钟紊乱对节律表达的影响。在478个节律相关基因中，有313个在去神经支配肌肉中丧失了节律性。这些对去神经支配敏感的基因涵盖脂质代谢相关基因，包括*Nrip1*、*Bbs1*、*Ptgis*、*Acot1*、*Scd2*、*Hpgd*、*Insig1*、*Dhcr24*、*Ldlr*及*Mboat1*。本研究结果表明，小鼠腓肠肌经坐骨神经去神经支配后，可通过直接或间接的肌肉萎缩介导途径，以基因特异性方式扰乱生物钟及钟控基因的节律性表达。