Li Meishan, Ogilvie Hannah, Ochala Julien, Artemenko Konstantin, Iwamoto Hiroyuki, Yagi Naoto, Bergquist Jonas, Larsson Lars
Department of Physiology and Pharmacology, Karolinska Institutet, SE-171 77, Stockholm, Sweden; Department of Clinical Neuroscience, Clinical Neurophysiology, Karolinska Institutet, SE-171 77, Stockholm, Sweden.
Aging Cell. 2015 Apr;14(2):228-35. doi: 10.1111/acel.12307. Epub 2015 Feb 2.
Novel experimental methods, including a modified single fiber in vitro motility assay, X-ray diffraction experiments, and mass spectrometry analyses, have been performed to unravel the molecular events underlying the aging-related impairment in human skeletal muscle function at the motor protein level. The effects of old age on the function of specific myosin isoforms extracted from single human muscle fiber segments, demonstrated a significant slowing of motility speed (P < 0.001) in old age in both type I and IIa myosin heavy chain (MyHC) isoforms. The force-generating capacity of the type I and IIa MyHC isoforms was, on the other hand, not affected by old age. Similar effects were also observed when the myosin molecules extracted from muscle fibers were exposed to oxidative stress. X-ray diffraction experiments did not show any myofilament lattice spacing changes, but unraveled a more disordered filament organization in old age as shown by the greater widths of the 1, 0 equatorial reflections. Mass spectrometry (MS) analyses revealed eight age-specific myosin post-translational modifications (PTMs), in which two were located in the motor domain (carbonylation of Pro79 and Asn81) and six in the tail region (carbonylation of Asp900, Asp904, and Arg908; methylation of Glu1166; deamidation of Gln1164 and Asn1168). However, PTMs in the motor domain were only observed in the IIx MyHC isoform, suggesting PTMs in the rod region contributed to the observed disordering of myosin filaments and the slowing of motility speed. Hence, interventions that would specifically target these PTMs are warranted to reverse myosin dysfunction in old age.
为了在运动蛋白水平上揭示与衰老相关的人类骨骼肌功能损伤背后的分子事件，已经开展了包括改良的单纤维体外运动分析、X射线衍射实验和质谱分析在内的新型实验方法。老年对从单个人类肌肉纤维段提取的特定肌球蛋白亚型功能的影响表明，I型和IIa型肌球蛋白重链（MyHC）亚型在老年时运动速度显著减慢（P < 0.001）。另一方面，I型和IIa型MyHC亚型的力产生能力不受老年影响。当从肌肉纤维中提取的肌球蛋白分子暴露于氧化应激时，也观察到了类似的效果。X射线衍射实验未显示任何肌丝晶格间距变化，但揭示了老年时肌丝组织更加紊乱，如赤道1,0反射宽度更大所示。质谱（MS）分析揭示了八种与年龄相关的肌球蛋白翻译后修饰（PTM），其中两种位于运动结构域（Pro79和Asn81的羰基化），六种位于尾部区域（Asp900、Asp904和Arg908的羰基化；Glu1166的甲基化；Gln1164和Asn1168的脱酰胺化）。然而，仅在IIx MyHC亚型中观察到运动结构域中的PTM，这表明杆状区域中的PTM导致了观察到的肌球蛋白丝无序和运动速度减慢。因此，如果要逆转老年时的肌球蛋白功能障碍，有必要针对这些PTM进行特异性干预。
