Donnelly Centre for Cellular & Biomolecular Research, Department of Molecular Genetics, University of Toronto, Ontario M5S 3E1, Canada.
Donnelly Centre for Cellular & Biomolecular Research, Department of Molecular Genetics, University of Toronto, Ontario M5S 3E1, Canada.
J Mol Cell Cardiol. 2017 Oct;111:61-68. doi: 10.1016/j.yjmcc.2017.08.001. Epub 2017 Aug 4.
Endurance exercise improves cardiac performance and affords protection against cardiovascular diseases but the signalling events that mediate these benefits are largely unexplored. Phosphorylation is a widely studied post-translational modification involved in intracellular signalling, and to discover novel phosphorylation events associated with exercise we have profiled the cardiac phosphoproteome response to a standardised exercise test to peak oxygen uptake (VO2peak). Male Wistar rats (346±18g) were assigned to 3 independent groups (n=6, in each) that were familiarised with running on a motorised treadmill within a metabolic chamber. Animals performed a graded exercise test and were killed either immediately (0h) after or 3h after terminating the test at a standardised physiological end point (i.e. peak oxygen uptake; VO2peak). Control rats were killed at a similar time of day to the exercised animals, to minimise possible circadian effects. Cardiac proteins were digested with trypsin and phosphopeptides were enriched by selective binding to titanium dioxide (TiO2). Phosphopeptides were analysed by liquid chromatography and high-resolution tandem mass spectrometry, and phosphopeptides were quantified by MS1 intensities and identified against the UniProt knowledgebase using MaxQuant (data are available via ProteomeXchange, ID PXD006646). The VO2peak of rats in the 0h and 3h groups was 66±5mlkgmin and 69.8±5mlkgmin, respectively. Proteome profiling detected 1169 phosphopeptides and one-way ANOVA found 141 significant (P<0.05 with a false discovery rate of 10%) differences. Almost all (97%) of the phosphosites that were responsive to exercise are annotated in the PhosphoSitePlus database but, importantly, the majority of these have not previously been associated with the cardiac response to exercise. More than two-thirds of the exercise-responsive phosphosites were different from those identified in previous phosphoproteome profiling of the cardiac response to β-adrenergic receptor stimulation. Moreover, we report entirely new phosphorylation sites on 4 cardiac proteins, including S81 of muscle LIM protein, and identified 7 exercise-responsive kinases, including myofibrillar protein kinases such as obscurin, titin and the striated-muscle-specific serine/threonine kinase (SPEG) that may be worthwhile targets for future investigation.
耐力运动可改善心脏功能，并提供心血管疾病的保护，但介导这些益处的信号事件在很大程度上仍未被探索。磷酸化是一种广泛研究的翻译后修饰，参与细胞内信号转导，为了发现与运动相关的新磷酸化事件，我们对标准运动测试至峰值摄氧量（VO2peak）的心脏磷酸化组蛋白反应进行了分析。雄性 Wistar 大鼠（346±18g）被分配到 3 个独立的组（每组 6 只），这些组在代谢室内的电动跑步机上进行了跑步适应。动物进行了分级运动测试，在达到标准生理终点（即峰值摄氧量；VO2peak）后立即（0h）或 3h 后处死。对照组动物在与运动组相似的时间点处死，以最大程度地减少可能的昼夜节律影响。用胰蛋白酶消化心脏蛋白，并通过选择性结合二氧化钛（TiO2）富集磷酸肽。通过液相色谱和高分辨率串联质谱分析磷酸肽，并通过 MS1 强度进行定量，并使用 MaxQuant 对 UniProt 知识库进行鉴定（数据可通过 ProteomeXchange 获得，ID PXD006646）。0h 和 3h 组大鼠的 VO2peak 分别为 66±5mlkgmin 和 69.8±5mlkgmin。蛋白质组学分析检测到 1169 个磷酸肽，单因素方差分析发现 141 个有显著差异（P<0.05，假发现率为 10%）。对运动有反应的几乎所有（97%）磷酸化位点都在 PhosphoSitePlus 数据库中注释，但重要的是，其中大多数以前与心脏对运动的反应无关。超过三分之二的运动反应性磷酸化位点与先前心脏对β-肾上腺素能受体刺激反应的磷酸化组蛋白分析中鉴定的不同。此外，我们报告了 4 种心脏蛋白上的全新磷酸化位点，包括肌 LIM 蛋白的 S81，以及 7 种运动反应性激酶，包括肌原纤维蛋白激酶，如 obscurin、titin 和横纹肌特异性丝氨酸/苏氨酸激酶（SPEG），这些激酶可能是未来研究的有价值的靶点。
