Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.
J Mol Cell Cardiol. 2010 May;48(5):954-63. doi: 10.1016/j.yjmcc.2010.01.002. Epub 2010 Jan 15.
Protein kinase A (PKA)-mediated phosphorylation of Ser23/24 of cardiac troponin I (cTnI) causes a reduction in Ca(2+)-sensitivity of force development. This study aimed to determine whether the PKA-induced modulation of the Ca(2+)-sensitivity is solely due to cTnI phosphorylation or depends on the phosphorylation status of other sarcomeric proteins. Endogenous troponin (cTn) complex in donor cardiomyocytes was partially exchanged (up to 66+/-1%) with recombinant unphosphorylated human cTn and in failing cells similar exchange was achieved using PKA-(bis)phosphorylated cTn complex. Cardiomyocytes immersed in exchange solution without complex added served as controls. Partial exchange of unphosphorylated cTn complex in donor tissue significantly increased Ca(2+)-sensitivity (pCa(50)) to 5.50+/-0.02 relative to the donor control value (pCa(50)=5.43+/-0.04). Exchange in failing tissue with PKA-phosphorylated cTn complex did not change Ca(2+)-sensitivity relative to the failing control (pCa(50)=5.60+/-0.02). Subsequent treatment of the cardiomyocytes with the catalytic subunit of PKA significantly decreased Ca(2+)-sensitivity in donor and failing tissue. Analysis of phosphorylated cTnI species revealed the same distribution of un-, mono- and bis-phosphorylated cTnI in donor control and in failing tissue exchanged with PKA-phosphorylated cTn complex. Phosphorylation of myosin-binding protein-C in failing tissue was significantly lower compared to donor tissue. These differences in Ca(2+)-sensitivity in donor and failing cells, despite similar distribution of cTnI species, could be abolished by subsequent PKA-treatment and indicate that other targets of PKA are involved the reduction of Ca(2+)-sensitivity. Our findings suggest that the sarcomeric phosphorylation background, which is altered in cardiac disease, influences the impact of cTnI Ser23/24 phosphorylation by PKA on Ca(2+)-sensitivity.
蛋白激酶 A（PKA）介导的肌钙蛋白 I（cTnI）丝氨酸 23/24 磷酸化导致力发展的 Ca2+敏感性降低。本研究旨在确定 PKA 诱导的 Ca2+敏感性调节是否仅归因于 cTnI 磷酸化，还是取决于其他肌节蛋白的磷酸化状态。供体细胞中的内源性肌钙蛋白（cTn）复合物与重组未磷酸化的人 cTn 部分交换（高达 66+/-1%），在衰竭细胞中，使用 PKA-（双）磷酸化的 cTn 复合物实现类似的交换。未添加复合物的心肌细胞浸入交换溶液中作为对照。供体组织中未磷酸化 cTn 复合物的部分交换显著增加 Ca2+敏感性（pCa（50））至 5.50+/-0.02，相对于供体对照值（pCa（50）=5.43+/-0.04）。用 PKA 磷酸化 cTn 复合物交换衰竭组织不会改变 Ca2+敏感性相对于衰竭对照（pCa（50）=5.60+/-0.02）。随后用 PKA 的催化亚基处理心肌细胞显著降低供体和衰竭组织的 Ca2+敏感性。磷酸化 cTnI 种的分析显示，供体对照和用 PKA 磷酸化 cTn 复合物交换的衰竭组织中存在相同分布的未磷酸化、单磷酸化和双磷酸化 cTnI。衰竭组织中肌球蛋白结合蛋白-C 的磷酸化明显低于供体组织。尽管 cTnI 种的分布相似，但供体和衰竭细胞之间 Ca2+敏感性的这些差异可被随后的 PKA 处理消除，并表明 PKA 的其他靶标参与了 Ca2+敏感性的降低。我们的发现表明，心脏疾病中改变的肌节磷酸化背景会影响 PKA 对 Ca2+敏感性的 cTnI 丝氨酸 23/24 磷酸化的影响。
