The 24-Hour Time Course of Integrated Molecular Responses to Resistance Exercise in Human Skeletal Muscle Implicates MYC as a Hypertrophic Regulator

Molecular control of recovery after exercise in muscle is temporally dynamic. A time course of biopsies around resistance exercise (RE) combined with -omics is necessary to better comprehend the molecular choreography of skeletal muscle adaptation in humans. We collected vastus lateralis biopsies before and 30 minutes, 3-, 8-, and 24 hours after acute RE. RNA-sequencing defined the transcriptome and a time-point matched biopsy only group were controls. DNA methylomics and computational approaches complimented the transcriptome data. Moreover, we tested whether cyclic transient overexpression of transcription factor MYC for 4 weeks were sufficient to induce skeletal muscle hypertrophy in female mice. Overall design: Thirteen recreationally active volunteers volunteered to participate in the study. Eight in the RE group (age of 32±5 years, height of 181±9 cm, weight of 83±8 kg, and body mass index (BMI) of 25.3±2.0), and five in the CON group (age of 30±4 years, height of 177±5 cm, weight of 85±12 kg, and finally a BMI of 27.3±3.6). Following an overnight fast, volunteers consumed a standardized amount of liquid formula for breakfast. One hour after the breakfast, skeletal muscle biopsies were collected from the vastus lateralis using a Bergström needle. Ninety minutes after breakfast, volunteers started a 45 minute standardized RE session (7 reps x 7 set on leg press and leg extension machines; RE group) or rested for 45 minutes (CON group). Biopsies were again collected at 30 minutes, 3-, 8-, and 24 hours after exercise cessation. Biopsies were time point matched in the CON group. A standardized lunch was administered following the biopsy at 3 hours post RE. Following the 8-hour biopsy, volunteers were sent home overnight and instructed to eat a healthy dinner. The morning after, volunteers again received a standardized breakfast prior to donating their final biopsy, 24 hours after RE. Approximately 25 mg of muscle tissue from each biopsy was homogenized in TRI Reagent (Sigma-Aldrich, St Louis, MO, USA). RNA isolated using bromochloropropane and centrifugation. Next, the RNA was processed using Direct-zol filter columns (Zymo Research, Irvine, CA, USA). Finally, the RNA was treated with DNAse and eluted in DEPC-treated water prior to storage at -80°C. The concentration and purity of the RNA were determined using a BioTek PowerWave XS microplate reader (BioTek Instruments Inc., Winooski, VT, USA). mRNA library preparation was done using Poly A enrichment, followed by RNA sequencing by an Illumina NovaSeq 6000 (Novogene Corp. Inc., Sacramento, CA, USA) using 150 bp paired-end sequencing.

肌肉运动后恢复的分子调控具有时间动态性。为更深入解析人类骨骼肌适应过程中的分子调控程序，需围绕抗阻运动（Resistance Exercise, RE）采集时序肌肉活检样本并结合多组学技术开展研究。本研究采集了受试者在急性抗阻运动前，以及运动后30分钟、3小时、8小时和24小时的股外侧肌活检样本；通过RNA测序（RNA-sequencing）解析转录组，并以仅在匹配时间点采集活检样本的组别作为对照组，同时采用DNA甲基化组学（DNA methylomics）与计算生物学方法辅助补充转录组数据。此外，本研究还探究了为期4周的转录因子MYC（Transcription Factor MYC）周期性瞬时过表达是否足以诱导雌性小鼠发生骨骼肌肥大。

实验设计：本研究共招募13名规律运动的健康志愿者，其中抗阻运动组8人（年龄32±5岁，身高181±9cm，体重83±8kg，体质量指数（Body Mass Index, BMI）25.3±2.0），对照组5人（年龄30±4岁，身高177±5cm，体重85±12kg，体质量指数（BMI）27.3±3.6）。所有受试者于夜间空腹后，统一摄入标准化剂量的液态配方餐作为早餐。早餐后1小时，采用Bergström穿刺针采集股外侧肌骨骼肌活检样本。早餐后90分钟，抗阻运动组受试者开始为期45分钟的标准化抗阻运动训练（腿推机与腿屈伸器械各7组，每组7次重复），对照组受试者则静息45分钟。运动结束后30分钟、3小时、8小时及24小时，再次采集活检样本；对照组则按照与抗阻运动组匹配的时间点采集活检样本。抗阻运动后3小时采集活检样本后，为受试者提供标准化午餐。完成8小时时间点的活检采样后，受试者可返家过夜，并被要求食用健康晚餐。次日清晨，受试者再次接受标准化早餐，随后在抗阻运动后24小时采集最后一批活检样本。

样本处理流程：每份活检样本取约25mg肌肉组织，在TRI Reagent（Sigma-Aldrich, St Louis, MO, USA）中进行匀浆。采用溴氯丙烷与离心法分离RNA，随后通过Direct-zol过滤柱（Zymo Research, Irvine, CA, USA）对RNA进行纯化处理。最后，用DNA酶对RNA进行消化，并用经焦碳酸二乙酯（Diethyl Pyrocarbonate, DEPC）处理的水洗脱，随后于-80℃条件下保存。采用BioTek PowerWave XS酶标仪（BioTek Instruments Inc., Winooski, VT, USA）测定RNA的浓度与纯度。

测序流程：采用Poly A富集法构建mRNA文库，随后使用Illumina NovaSeq 6000测序平台（Novogene Corp. Inc., Sacramento, CA, USA）进行150bp双端测序。