PDF(Pubmed)
Abstract：
Stretch activation is defined as a delayed increase in force after rapid stretches. Although there is considerable evidence for stretch activation in isolated cardiac myofibrillar preparations, few studies have measured mechanisms of stretch activation in mammalian skeletal muscle fibers. We measured stretch activation following rapid step stretches [∼1%-4% sarcomere length (SL)] during submaximal Ca2+ activations of rat permeabilized slow-twitch skeletal muscle fibers before and after protein kinase A (PKA), which phosphorylates slow myosin binding protein-C. PKA significantly increased stretch activation during low (∼25%) Ca2+ activation and accelerated rates of delayed force development (kef) during both low and half-maximal Ca2+ activation. Following the step stretches and subsequent force development, fibers were rapidly shortened to original sarcomere length, which often elicited a shortening-induced transient force overshoot. After PKA, step shortening-induced transient force overshoot increased ∼10-fold following an ∼4% SL shortening during low Ca2+ activation levels. kdf following step shortening also increased after PKA during low and half-maximal Ca2+ activations. We next investigated thin filament regulation of stretch activation. We tested the interplay between cardiac troponin I (cTnI) phosphorylation at the canonical PKA and novel tyrosine kinase sites on stretch activation. Native slow-skeletal Tn complexes were exchanged with recombinant human cTn complex with different human cTnI N-terminal pseudo-phosphorylation molecules: 1) nonphosphorylated wild type (WT), 2) the canonical S22/23D PKA sites, 3) the tyrosine kinase Y26E site, and 4) the combinatorial S22/23D + Y26E cTnI. All three pseudo-phosphorylated cTnIs elicited greater stretch activation than WT. Following stretch activation, a new, elevated stretch-induced steady-state force was reached with pseudo-phosphorylated cTnI. Combinatorial S22/23D + Y26E pseudo-phosphorylated cTnI increased kdf. These results suggest that slow-skeletal myosin binding protein-C (sMyBP-C) phosphorylation modulates stretch activation by a combination of cross-bridge recruitment and faster cycling kinetics, whereas cTnI phosphorylation regulates stretch activation by both redundant and synergistic mechanisms; and, taken together, these sarcomere phosphoproteins offer precision targets for enhanced contractility.
摘要：
拉伸激活定义为快速拉伸后力的延迟增加。尽管有相当多的证据表明在分离的心脏肌原纤维制剂中拉伸激活，很少有研究测量哺乳动物骨骼肌纤维拉伸激活的机制。我们在蛋白激酶A（PKA）之前和之后，在大鼠透化慢抽搐骨骼肌纤维的次最大Ca2激活期间，测量了快速阶梯拉伸后的拉伸激活[〜1％-4％肌节长度（SL）]，磷酸化慢肌球蛋白结合蛋白C。PKA在低（〜25％）Ca2激活期间显着增加了拉伸激活，并在低和半最大Ca2激活期间加速了延迟力发展（kef）的速率。在步骤延伸和随后的力量发展之后，纤维被迅速缩短到原来的肌节长度，这通常会引起缩短引起的瞬态力超调。PKA之后,在低Ca2+激活水平下，阶跃缩短引起的瞬时力超调增加了约10倍，随后约4%SL缩短。在低和半最大Ca2激活期间，PKA后缩短步骤后的kdf也增加。接下来，我们研究了细丝对拉伸激活的调节。我们测试了典型PKA处的心肌肌钙蛋白I（cTnI）磷酸化与拉伸激活时的新型酪氨酸激酶位点之间的相互作用。将天然慢骨架Tn复合物与具有不同人cTnIN末端假磷酸化分子的重组人cTn复合物交换：1）非磷酸化野生型（WT），2)规范的S22/23DPKA站点，3)酪氨酸激酶Y26E位点，4)组合S22/23D+Y26EcTnI。所有三种假磷酸化cTnI均比WT引起更大的拉伸激活。拉伸激活后,一个新的,假磷酸化cTnI达到了拉伸诱导的稳态力。组合S22/23D+Y26E假磷酸化cTnI增加kdf。这些结果表明，慢速骨骼肌肌球蛋白结合蛋白-C（sMyBP-C）磷酸化通过交叉桥募集和更快的循环动力学的组合来调节拉伸激活，而cTnI磷酸化通过冗余和协同机制调节拉伸激活；并且，放在一起，这些肌节磷蛋白为增强收缩力提供了精确的目标.
