Wolska B M, Keller R S, Evans C C, Palmiter K A, Phillips R M, Muthuchamy M, Oehlenschlager J, Wieczorek D F, de Tombe P P, Solaro R J
Departments of Physiology and Biophysics and Medicine, Section of Cardiology, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA.
Circ Res. 1999 Apr 16;84(7):745-51. doi: 10.1161/01.res.84.7.745.
We compared the dynamics of the contraction and relaxation of single myocytes isolated from nontransgenic (NTG) mouse hearts and from transgenic (TG-beta-Tm) mouse hearts that overexpress the skeletal isoform of tropomyosin (Tm). Compared with NTG controls, TG-beta-Tm myocytes showed significantly reduced maximal rates of contraction and relaxation with no change in the extent of shortening. This result indicated that the depression in contraction dynamics determined in TG-beta-Tm isolated hearts is intrinsic to the cells. To further investigate the effect of Tm isoform switching on myofilament activity and regulation, we measured myofilament force and ATPase rate as functions of pCa (-log of [Ca2+]). Compared with controls, force generated by myofilaments from TG-beta-Tm hearts and myofibrillar ATPase activity were both more sensitive to Ca2+. However, the shift in pCa50 (half-maximally activating pCa) caused by changing sarcomere length from 1.8 to 2.4 microm was not significantly different between NTG and TG-beta-Tm fiber preparations. To test directly whether isoform switching affected the economy of contraction, force versus ATPase rate relationships were measured in detergent-extracted fiber bundles. In both NTG and TG-beta-Tm preparations, force and ATPase rate were linear and identically correlated, which indicated that crossbridge turnover was unaffected by Tm isoform switching. However, detergent extracted fibers from TG-beta-Tm demonstrated significantly less maximum tension and ATPase activity than NTG controls. Our results provide the first evidence that the Tm isoform population modulates the dynamics of contraction and relaxation of single myocytes by a mechanism that does not alter the rate-limiting step of crossbridge detachment. Our results also indicate that differences in sarcomere-length dependence of activation between cardiac and skeletal muscle are not likely due to differences in the isoform population of Tm.
我们比较了从非转基因（NTG）小鼠心脏和过表达原肌球蛋白（Tm）骨骼肌异构体的转基因（TG-β-Tm）小鼠心脏分离出的单个心肌细胞的收缩和舒张动力学。与NTG对照组相比，TG-β-Tm心肌细胞的最大收缩和舒张速率显著降低，而缩短程度没有变化。这一结果表明，在TG-β-Tm分离心脏中确定的收缩动力学抑制是细胞固有的。为了进一步研究Tm异构体转换对肌丝活性和调节的影响，我们测量了肌丝力和ATP酶速率作为pCa（[Ca2+]的负对数）的函数。与对照组相比，TG-β-Tm心脏的肌丝产生的力和肌原纤维ATP酶活性对Ca2+都更敏感。然而，在NTG和TG-β-Tm纤维制剂中，肌节长度从1.8微米变为2.4微米引起的pCa50（半最大激活pCa）的变化没有显著差异。为了直接测试异构体转换是否影响收缩经济性，在去污剂提取的纤维束中测量了力与ATP酶速率的关系。在NTG和TG-β-Tm制剂中，力和ATP酶速率都是线性的且相关性相同，这表明横桥周转率不受Tm异构体转换的影响。然而，TG-β-Tm的去污剂提取纤维显示出比NTG对照组显著更低的最大张力和ATP酶活性。我们的结果提供了第一个证据，即Tm异构体群体通过一种不改变横桥 detachment限速步骤的机制调节单个心肌细胞的收缩和舒张动力学。我们的结果还表明，心肌和骨骼肌之间激活的肌节长度依赖性差异不太可能是由于Tm异构体群体的差异。
