Department of Zoology, La Trobe University, Melbourne, Victoria, 3086, Australia.
J Physiol. 2012 Mar 15;590(6):1443-63. doi: 10.1113/jphysiol.2011.224535. Epub 2012 Jan 16.
Oxidation can decrease or increase the Ca2+ sensitivity of the contractile apparatus in rodent fast-twitch (type II) skeletal muscle fibres, but the reactions and molecular targets involved are unknown. This study examined whether increased Ca2+ sensitivity is due to S-glutathionylation of particular cysteine residues. Skinned muscle fibres were directly activated in heavily buffered Ca2+ solutions to assess contractile apparatus Ca2+ sensitivity. Rat type II fibres were subjected to S-glutathionylation by successive treatments with 2,2′-dithiodipyridine (DTDP) and glutathione (GSH), and displayed a maximal increase in pCa50 (−log10 [Ca2+] at half-maximal force) of ∼0.24 pCa units, with little or no effect on maximum force or Hill coefficient. Partial similar effect was produced by exposure to oxidized gluthathione (GSSG, 10 mM) for 10 min at pH 7.1, and near-maximal effect by GSSG treatment at pH 8.5. None of these treatments significantly altered Ca2+ sensitivity in rat type I fibres. Western blotting showed that both the DTDP–GSH and GSSG–pH 8.5 treatments caused marked S-glutathionylation of the fast troponin I isoform (TnI(f)) present in type II fibres, but not of troponin C (TnC) or myosin light chain 2. Both the increased Ca2+ sensitivity and glutathionylation of TnI(f) were blocked by N-ethylmaleimide (NEM). S-nitrosoglutathione (GSNO) also increased Ca2+ sensitivity, but only in conditions where it caused S-glutathionylation of TnI(f). In human type II fibres from vastus lateralis muscle, DTDP–GSH treatment also caused similar increased Ca2+ sensitivity and S-glutathionylation of TnI(f). When the slow isoform of TnI in type I fibres of rat was partially substituted (∼30%) with TnI(f), DTDP–GSH treatment caused a significant increase in Ca2+ sensitivity (∼0.08 pCa units). TnIf in type II fibres from toad and chicken muscle lack Cys133 present in mammalian TnIf, and such fibres showed no change in Ca2+ sensitivity with DTDP–GSH nor any S-glutathionylation of TnI(f) (latter examined only in toad). Following 40 min of cycling exercise in human subjects (at ∼60% peak oxygen consumption), TnI(f) in vastus lateralis muscle displayed a marked increase in S-glutathionylation (∼4-fold). These findings show that S-glutathionylation of TnI(f), most probably at Cys133, increases the Ca2+ sensitivity of the contractile apparatus, and that this occurs in exercising humans, with likely beneficial effects on performance.
氧化可以降低或增加啮齿动物快肌（II 型）骨骼肌纤维收缩装置的 Ca2+敏感性，但涉及的反应和分子靶点尚不清楚。本研究探讨了 Ca2+敏感性的增加是否归因于特定半胱氨酸残基的 S-谷胱甘肽化。在重缓冲 Ca2+溶液中直接激活去皮肌纤维，以评估收缩装置的 Ca2+敏感性。用 2,2′-二硫代二吡啶（DTDP）和谷胱甘肽（GSH）连续处理大鼠 II 型纤维，使 pCa50（最大力的一半时 Ca2+的负对数 [Ca2+]）最大增加约 0.24 pCa 单位，对最大力或 Hill 系数几乎没有影响或没有影响。暴露于氧化谷胱甘肽（GSSG，10 mM）10 分钟在 pH 7.1 下产生类似的部分效果，在 pH 8.5 下用 GSSG 处理产生近最大效果。这些处理均未显著改变大鼠 I 型纤维的 Ca2+敏感性。Western blot 显示，DTDP-GSH 和 GSSG-pH 8.5 处理均导致 II 型纤维中存在的快肌钙蛋白 I 同工型（TnI(f))的明显 S-谷胱甘肽化，但不影响肌钙蛋白 C（TnC）或肌球蛋白轻链 2。增加的 Ca2+敏感性和 TnI(f)的谷胱甘肽化均被 N-乙基马来酰亚胺（NEM）阻断。S-亚硝基谷胱甘肽（GSNO）也增加了 Ca2+敏感性，但仅在其导致 TnI(f)的 S-谷胱甘肽化的情况下。在人类股外侧肌 II 型纤维中，DTDP-GSH 处理也导致 TnI(f)的类似增加的 Ca2+敏感性和 S-谷胱甘肽化。当大鼠 I 型纤维中的慢同工型 TnI 被部分替代（约 30%）为 TnI(f)时，DTDP-GSH 处理导致 Ca2+敏感性显著增加（约 0.08 pCa 单位）。II 型纤维中的 TnIf 来自蟾蜍和鸡肌肉，缺乏哺乳动物 TnIf 中的 Cys133，此类纤维的 Ca2+敏感性无变化与 DTDP-GSH 也没有 TnI(f)的任何 S-谷胱甘肽化（后者仅在蟾蜍中检查）。在人类受试者进行 40 分钟的循环运动（约为最大耗氧量的 60%）后，股外侧肌中的 TnI(f)显示出明显的 S-谷胱甘肽化增加（约 4 倍）。这些发现表明 TnI(f)的 S-谷胱甘肽化（很可能在 Cys133 处）增加了收缩装置的 Ca2+敏感性，并且这种情况发生在运动中的人身上，可能对性能有有益的影响。
