ROLE OF PHOSPHORYLATION OF CTNL IN THE REGULATION OF MYOCARDIUM

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 肾上腺素能调节心肌收缩能力，部分是通过蛋白激酶A(PKA)的肌原纤维蛋白磷酸化实现的。PKA靶向于心肌肌钙蛋白I(CTnI)、心肌肌球蛋白结合蛋白C(cMyBP-C)和肌动蛋白的N端。为了从cMyBP-C/titin磷酸化中分离cTnI磷酸化对力-[Ca+]关系的影响，将大鼠皮肤小梁内源性心肌肌钙蛋白(CTn)与WT cTn、含有非磷酸化cTnI(S23/24A)突变体的cTn或拟磷酸化cTnI(S23/24D)突变体被动交换。PKA不能磷酸化任何一个cTnI突变体，因此使MyBP-C和Titin成为这些制剂中唯一的PKA靶标。在2.3和2.0？m SL时，测量了力-[Ca~(2+)]关系和Ca~(2+)敏感性(PCa50)。在长SL和短SL时，WT TN和含有cTnI(S23/24A)的TN的pCa50相似，而在短SL时pCa50降低。PKA处理WT和cTnI(S23，24A)交换的小梁都降低了两个SL处的pCa50，但在更大程度上降低了2.3um SL，消除了两种情况下pCa50对SL的依赖性。与含有cTnI的cTn(S23/24D)交换后，SL的pCa50降低(与WT和cTnI(S23，24A)相比)，也消除了SL对pCa50的影响。总之，cTnI和心肌C蛋白/titin的磷酸化都独立地降低了pCa50，但随着SLS的延长而降低的程度更大，从而降低了钙离子对力的敏感性的长度依赖性。为了深入了解这一现象的原因，进行了小角X射线衍射，以确定pCa50的这些移位是否与肌丝间距或相互作用的变化有关。

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.