Table_5_The Comparative Methylome and Transcriptome After Change of Direction Compared to Straight Line Running Exercise in Human Skeletal Muscle.XLSX

The methylome and transcriptome signatures following exercise that are physiologically and metabolically relevant to sporting contexts such as team sports or health prescription scenarios (e.g., high intensity interval training/HIIT) has not been investigated. To explore this, we performed two different sport/exercise relevant high-intensity running protocols in five male sport team members using a repeated measures design of: (1) change of direction (COD) versus; (2) straight line (ST) running exercise with a wash-out period of at least 2 weeks between trials. Skeletal muscle biopsies collected from the vastus lateralis 30 min and 24 h post exercise, were assayed using 850K methylation arrays and a comparative analysis with recent (subject-unmatched) sprint and acute aerobic exercise meta-analysis transcriptomes was performed. Despite COD and ST exercise being matched for classically defined intensity measures (speed × distance and number of accelerations/decelerations), COD exercise elicited greater movement (GPS-Playerload), physiological (HR), metabolic (lactate) as well as central and peripheral (differential RPE) exertion measures compared with ST exercise, suggesting COD exercise evoked a higher exercise intensity. The exercise response alone across both conditions evoked extensive alterations in the methylome 30 min and 24 h post exercise, particularly in MAPK, AMPK and axon guidance pathways. COD evoked a considerably greater hypomethylated signature across the genome compared with ST exercise, particularly at 30 min post exercise, enriched in: Protein binding, MAPK, AMPK, insulin, and axon guidance pathways. Comparative methylome analysis with sprint running transcriptomes identified considerable overlap, with 49% of genes that were altered at the expression level also differentially methylated after COD exercise. After differential methylated region analysis, we observed that VEGFA and its downstream nuclear transcription factor, NR4A1 had enriched hypomethylation within their promoter regions. VEGFA and NR4A1 were also significantly upregulated in the sprint transcriptome and meta-analysis of exercise transcriptomes. We also confirmed increased gene expression of VEGFA, and considerably larger increases in the expression of canonical metabolic genes PPARGC1A (that encodes PGC1-α) and NR4A3 in COD vs. ST exercise. Overall, we demonstrate that increased physiological/metabolic load via COD exercise in human skeletal muscle evokes considerable epigenetic modifications that are associated with changes in expression of genes responsible for adaptation to exercise.

目前，针对与团队运动、健康处方场景（如高强度间歇训练/HIIT）等运动情境在生理与代谢层面相关的运动后甲基化组与转录组特征，尚未开展系统性研究。

为探索该研究空白，我们采用重复测量设计，对5名男性团队运动员实施了两种与运动/训练相关的高强度跑步方案：（1）变向跑（COD），以及（2）直线跑（ST），两次试验之间设置至少2周的洗脱期。

分别于运动后30分钟与24小时，从受试者股外侧肌采集骨骼肌活检样本，采用850K甲基化芯片进行检测，并与近期未匹配受试者的短跑与急性有氧运动转录组荟萃分析结果开展对比分析。

尽管经典运动强度指标（速度×距离、加减速次数）匹配了变向跑与直线跑的运动强度，但相较于直线跑，变向跑的GPS运动负荷、心率（HR）、乳酸水平等生理代谢指标，以及中枢与外周的差异自觉用力程度评分（RPE）均显著更高，提示变向跑的实际运动强度更高。

两种运动方案均在运动后30分钟和24小时引发了甲基化组的广泛改变，尤其富集于丝裂原活化蛋白激酶（MAPK）、腺苷酸活化蛋白激酶（AMPK）以及轴突导向通路。

相较于直线跑，变向跑在全基因组范围内诱导出更为显著的低甲基化特征，尤其在运动后30分钟最为明显，该特征富集于蛋白质结合、MAPK、AMPK、胰岛素以及轴突导向通路。

与短跑转录组的甲基化组对比分析显示二者存在大量重叠：在变向跑后出现表达改变的基因中，有49%同时存在差异甲基化修饰。

通过差异甲基化区域分析，我们发现血管内皮生长因子A（VEGFA）及其下游核转录因子NR4A1的启动子区域存在显著富集的低甲基化修饰。VEGFA与NR4A1在短跑转录组以及运动转录组荟萃分析中均显著上调。

我们还证实，相较于直线跑，变向跑中VEGFA的基因表达水平有所升高，且经典代谢基因过氧化物酶体增殖物激活受体γ辅激活因子1α（PPARGC1A，编码PGC1-α）与NR4A3的表达上调幅度更为显著。

综上，本研究证实，人类骨骼肌通过变向跑施加更高的生理/代谢负荷时，会引发大量表观遗传修饰，这些修饰与运动适应性相关基因的表达改变密切相关。