Department of Bioengineering, University of Washington, Box 355061, 3720 15th Avenue NE, Seattle, WA 98195, USA.
J Mol Cell Cardiol. 2011 Jan;50(1):165-74. doi: 10.1016/j.yjmcc.2010.10.025. Epub 2010 Oct 28.
Tension development and relaxation in cardiac muscle are regulated at the thin filament via Ca(2+) binding to cardiac troponin C (cTnC) and strong cross-bridge binding. However, the influence of cTnC Ca(2+)-binding properties on these processes in the organized structure of cardiac sarcomeres is not well-understood and likely differs from skeletal muscle. To study this we generated single amino acid variants of cTnC with altered Ca(2+) dissociation rates (k(off)), as measured in whole troponin (cTn) complex by stopped-flow spectroscopy (I61Q cTn>WT cTn>L48Q cTn), and exchanged them into cardiac myofibrils and demembranated trabeculae. In myofibrils at saturating Ca(2+), L48Q cTnC did not affect maximum tension (T(max)), thin filament activation (k(ACT)) and tension development (k(TR)) rates, or the rates of relaxation, but increased duration of slow phase relaxation. In contrast, I61Q cTnC reduced T(max), k(ACT) and k(TR) by 40-65% with little change in relaxation. Interestingly, k(ACT) was less than k(TR) with I61Q cTnC, and this difference increased with addition of inorganic phosphate, suggesting that reduced cTnC Ca(2+)-affinity can limit thin filament activation kinetics. Trabeculae exchanged with I61Q cTn had reduced T(max), Ca(2+) sensitivity of tension (pCa(50)), and slope (n(H)) of tension-pCa, while L48Q cTn increased pCa(50) and reduced n(H). Increased cross-bridge cycling with 2-deoxy-ATP increased pCa(50) with WT or L48Q cTn, but not I61Q cTn. We discuss the implications of these results for understanding the role of cTn Ca(2+)-binding properties on the magnitude and rate of tension development and relaxation in cardiac muscle.
心肌中肌钙蛋白 C (cTnC) 与 Ca2+的结合调节着肌动蛋白丝的紧张和松弛，同时，肌球蛋白横桥也能与之紧密结合。然而，cTnC 与 Ca2+结合的动力学特性在心肌肌节的有序结构中对这些过程的影响尚不清楚，且可能与骨骼肌不同。为了研究这一点，我们生成了具有不同 Ca2+离解速率 (k(off)) 的 cTnC 单氨基酸变异体，该速率通过停流光谱法（I61Q cTn>WT cTn>L48Q cTn）在完整肌钙蛋白 (cTn) 复合物中进行测量，并将其交换到心肌纤维和去膜横管中。在肌纤维中，在饱和 Ca2+浓度下，L48Q cTnC 不会影响最大张力 (T(max))、细肌丝激活 (k(ACT)) 和张力发展 (k(TR)) 速率，或松弛速率，但会增加慢相松弛的持续时间。相反，I61Q cTnC 使 T(max)、k(ACT) 和 k(TR)降低了 40-65%，而松弛几乎没有变化。有趣的是，I61Q cTnC 时 k(ACT) 小于 k(TR)，且这种差异随着无机磷酸盐的加入而增加，表明 cTnC Ca2+亲和力的降低可能限制了细肌丝激活动力学。与 I61Q cTn 交换的横管张力降低、张力对 Ca2+的敏感性（pCa(50))、张力-pCa 的斜率 (n(H))，而 L48Q cTn 增加了 pCa(50)，降低了 n(H)。用 2-脱氧-ATP 增加横桥循环增加了 WT 或 L48Q cTn 的 pCa(50)，但不增加 I61Q cTn 的 pCa(50)。我们讨论了这些结果对理解 cTn Ca2+结合特性在心肌张力发展和松弛的幅度和速率中的作用的影响。
