High throughput sequencing of skeletal muscle-specific Site-1 Protease knockout mouse gastrocnemius and soleus

The processes by which mitochondria respond to cellular energy demands brought on by physiologic and pathophysiologic stimuli are essential to cellular adaptation and organismal survival. Disrupted mitochondrial function is implicated in several human disease states, underscoring the need to elucidate the molecular mechanisms that control mitochondrial function and metabolism. We previously described a patient with a gain-of-function mutation in Site-1 Protease (S1P) who exhibited idiopathic hyperCKemia, myoedema, and altered muscle mitochondrial morphology. To date, S1P has been heavily characterized in liver and bone, with very little known about its potential function in skeletal muscle. In our current study, we generated a skeletal muscle-specific S1P knockout mouse line (S1PsmKO) to examine the role of S1P in skeletal muscle. S1PsmKO mice were overtly normal in their appearance and health. However, examination of 12-week-old S1PsmKO mice showed increased gastrocnemius muscle mass and aged S1PsmKO mice exhibited increased muscle mass in both gastrocnemius and soleus relative to age-matched controls. Moreover, isolated muscle from S1PsmKO exhibited increased maximal mitochondrial respiration relative to floxed littermates (controls). RNA-Seq analysis showed increased transcript numbers of the mitochondrial-resident gene, Mss51 in S1PsmKO gastrocnemius relative to control mice. Mss51 is a member of the TGF-beta/MSTN pathway, which controls mitochondrial metabolism. Similar to S1PsmKO muscle, S1P-depleted C2C12 cells also had increased maximal respiration, which was reversed by exogenous MSS51 expression. These data identify S1P as a regulator of muscle mass and mitochondrial metabolism, and implicate S1P as a novel modulator of TGF-beta signaling. Method: Male mice were maintained on standard chow diet until 12 weeks of age. Examination of gastrocnemius and soleus from Site-1 Protease floxed (control) and Site-1 Protease floxed HSACre+ (skeletal muscle-specific Site-1 Protease knockout) mice on standard chow diet, fasted for 4 h prior to organ harvesting. n=4/group线粒体响应生理及病理生理刺激引发的细胞能量需求的过程，对于细胞适应与机体存活至关重要。线粒体功能异常与多种人类疾病状态相关，这凸显了阐明调控线粒体功能与代谢的分子机制的必要性。 我们此前曾报道过一位携带位点1蛋白酶（Site-1 Protease, S1P）功能获得性突变的患者，该患者表现为特发性高肌酸激酶血症、肌水肿以及骨骼肌线粒体形态异常。 迄今为止，S1P的研究多集中于肝脏与骨骼组织，其在骨骼肌中的潜在功能尚鲜有报道。 在本研究中，我们构建了骨骼肌特异性S1P敲除小鼠品系（S1PsmKO），以探究S1P在骨骼肌中的作用。 S1PsmKO小鼠的外观与健康状况均无明显异常。然而，对12周龄S1PsmKO小鼠的检测显示，其腓肠肌质量增加；而老年S1PsmKO小鼠的腓肠肌与比目鱼肌质量，均较同龄对照小鼠有所提升。 此外，相较于floxed同窝仔鼠（对照），分离自S1PsmKO小鼠的骨骼肌表现出更高的最大线粒体呼吸速率。 RNA测序（RNA-Seq）分析显示，相较于对照小鼠，S1PsmKO小鼠腓肠肌中线粒体驻留基因Mss51的转录本数量显著上调。 Mss51属于转化生长因子-β/肌生长抑制素（TGF-beta/MSTN）通路成员，该通路可调控线粒体代谢。 与S1PsmKO小鼠骨骼肌表型一致，经S1P敲降的C2C12细胞同样表现出最大呼吸速率升高，而外源性过表达MSS51可逆转这一表型。 上述数据表明，S1P是骨骼肌质量与线粒体代谢的调控因子，并提示S1P是转化生长因子-β信号通路的新型调节因子。 方法：将雄性小鼠饲喂标准维持饲料，饲养至12周龄。采集位点1蛋白酶floxed（对照）小鼠以及位点1蛋白酶floxed且HSACre+（骨骼肌特异性位点1蛋白酶敲除）小鼠的腓肠肌与比目鱼肌，所有小鼠均饲喂标准维持饲料，并在器官取材前禁食4小时。每组n=4。