ROLE OF PHOSPHORYLATION OF CTNL IN THE REGULATION OF MYOCARDIUM

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 心脏收缩力的肾上腺素能调节部分通过蛋白激酶A（PKA）肌原纤维蛋白磷酸化发生。 PKA靶向心肌肌钙蛋白I（cTnI）、心肌肌球蛋白结合蛋白C（cMyBP-C）和肌联蛋白的N-末端。 为了分离cTnI磷酸化与cMyBP-C/肌联蛋白磷酸化对力-[Ca 2 +]关系的影响，将大鼠皮肤小梁中的内源性心肌肌钙蛋白（cTnI）被动交换为WT cTnI、含有不可磷酸化cTnI（S23/24 A）突变体的cTnI或磷酸化模拟cTnI（S23/24 D）突变体。 PKA不能磷酸化cTnI突变体，因此MyBP-C和肌联蛋白成为这些制剂中唯一的PKA靶点。 在2.3和2.0 μ m SL下测量力-[Ca 2 +]关系和Ca 2+敏感性（pCa 50）。 在长和短SL下，WT Tn和含cTnI（S23/24 A）Tn小梁之间的pCa 50相似，在短SL下pCa 50降低。 WT和cTnI（S23，24 A）交换小梁的PKA治疗在两种SL下均降低了pCa 50，但在2.3 μ m SL下的程度更大，消除了两种条件下pCa 50的SL依赖性。与含cTnI的cTnI（S23, 24 D）交换可降低SL下的pCa 50（与WT和cTnI（S23，24 A）相比），并消除SL对pCa 50的影响。总之，cTnI和心脏C蛋白/肌联蛋白的磷酸化都独立地降低pCa 50，但在更长的SL时程度更大，从而降低了力的Ca 2+敏感性的长度依赖性。为了深入了解为什么会这样，进行了小角度X射线衍射，以确定pCa 50的这些变化是否与肌丝间距或相互作用的变化相关。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. ¿-adrenergic modulation of cardiac contractility occurs, in part, through protein kinase A (PKA) myofibrillar protein phosphorylation. PKA targets the N-terminus of cardiac troponin I (cTnI), cardiac myosin-binding protein C (cMyBP-C) and titin. To isolate the effects of cTnI phosphorylation from cMyBP-C/titin phosphorylation on force-[Ca2+] relations, endogenous cardiac troponin (cTn) in rat skinned trabeculae was passively exchanged with either WT cTn, cTn containing a non-phosphorylatable cTnI(S23/24A) mutant, or a phosphomimetic cTnI(S23/24D) mutant. PKA cannot phosphorylate either cTnI mutant, thus leaving MyBP-C and titin as the sole PKA targets in those preparations. Force-[Ca2+] relationships and Ca2+-sensitivity (pCa50) were measured at 2.3 and 2.0 ¿m SL. pCa50 was similar between WT Tn and Tn containing cTnI(S23/24A) trabeculae at both long and short SL, with decreased pCa50 at short SL. PKA treatment of WT and cTnI(S23,24A) exchanged trabeculae reduced pCa50 at both SL, but to a greater extent 2.3 um SL, eliminating the SL-dependence of pCa50 for both conditions. Exchange with cTn containing cTnI(S23/24D) reduced pCa50 at both SL (compared to WT and cTnI(S23,24A)) and also eliminated the influence of SL on pCa50. In summary, phosphorylation of cTnI and cardiac C-protein/titin both independently reduced pCa50, but to greater extents at longer SLs, thus reducing length dependence of Ca2+ sensitivity of force. In order to get insight into why this is so, small-angle x-ray diffraction was performed to determine whether these shifts in pCa50 were associated with changes in myofilament spacing or interaction.