Unit of Biology and Genetics, Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", Piazza Lauro de Bosis 15, 00135, Rome, Italy.
Department of Biomedicine, Neuroscience and Advanced Diagnostics (BiND), Section of Human Anatomy, University of Palermo, Palermo, Italy; Euro-Mediterranean Institutes of Science and Technology (IEMEST), Palermo, Italy.
Redox Biol. 2019 Jun;24:101183. doi: 10.1016/j.redox.2019.101183. Epub 2019 Apr 3.
Besides its substantial role in eye lens, αB-crystallin (HSPB5) retains fundamental function in striated muscle during physiological or pathological modifications. In this study, we aimed to analyse the cellular and molecular factors driving the functional response of HSPB5 protein in different muscles from mice subjected to an acute bout of non-damaging endurance exercise or in C2C12 myocytes upon exposure to pro-oxidant environment, chosen as "in vivo" and "in vitro" models of a physiological stressing conditions, respectively. To this end, red (GR) and white gastrocnemius (GW), as sources of slow-oxidative and fast-glycolytic/oxidative fibers, as well as the soleus (SOL), mainly composed of slow-oxidative type fibers, were obtained from BALB/c mice, before (CTRL) and at different times (0', 15', 30' 120') following 1-h of running. Although the total level of HSPB5 protein was not affected by exercise, we found a significantly increase of phosphorylated HSPB5 (p-HSPB5) only in GR and SOL skeletal muscle with a higher amount of type I and IIA/X myofibers. The fiber-specific activation of HSPB5 was correlated to its interaction with the actin filaments, as well as to an increased level of lipid peroxidation and carbonylated proteins. The role of the pro-oxidant environment in HSPB5 response was investigated in terminally differentiated C2C12 myotubes, where most of HSPB5/pHSPB5 pool was present in the cytosolic compartment in standard culture conditions. As a result of exposure to pro-oxidizing, but not cytotoxic, HO concentration, the p-38MAPK-mediated phosphorylation of HSPB5 resulted functional to promote its interaction with the myofibrillar components, such as β-actin, desmin and filamin 1. This study provides novel information on the molecular pathway underlying the HSPB5 physiological function in skeletal muscle, confirming the contribution of the pro-oxidant environment in HSPB5 activation and interaction with substrate/client myofibrillar proteins, offering new insights for the study of myofibrillar myopathies and cardiomyopathies.
除了在眼睛晶状体中发挥重要作用外，αB-晶体蛋白（HSPB5）在生理或病理改变过程中在横纹肌中保留基本功能。在这项研究中，我们旨在分析驱动 HSPB5 蛋白在经历非破坏性耐力运动急性发作的小鼠不同肌肉中的功能反应的细胞和分子因素，或在暴露于促氧化剂环境的 C2C12 成肌细胞中，分别作为“体内”和“体外”的生理应激模型。为此，从小鼠中获得了红色（GR）和白色比目鱼肌（GW）（作为慢氧化和快糖酵解/氧化纤维的来源），以及主要由慢氧化型纤维组成的比目鱼肌（SOL），在跑步前（CTRL）和跑步后 1 小时的不同时间（0'、15'、30'、120'）。尽管 HSPB5 蛋白的总水平不受运动影响，但我们发现仅在 GR 和 SOL 骨骼肌中发现 HSPB5 的磷酸化（p-HSPB5）显著增加，其中 I 型和 IIA/X 型肌纤维数量更多。HSPB5 的纤维特异性激活与肌动蛋白丝的相互作用以及脂质过氧化和羰基化蛋白水平的增加有关。在终末分化的 C2C12 成肌细胞中研究了促氧化剂环境对 HSPB5 反应的作用，在标准培养条件下，大多数 HSPB5/pHSPB5 池存在于细胞质部分。由于暴露于促氧化剂但非细胞毒性的 HO 浓度，HSPB5 的 p-38MAPK 介导的磷酸化导致其与肌原纤维成分（如β-肌动蛋白、结蛋白和细丝蛋白 1）的功能相互作用。这项研究提供了 HSPB5 在骨骼肌中生理功能的分子途径的新信息，证实了促氧化剂环境在 HSPB5 激活及其与底物/肌纤维蛋白的相互作用中的贡献，为肌原纤维肌病和心肌病的研究提供了新的见解。
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