Pi YeQing, Zhang Dahua, Kemnitz Kara R, Wang Hao, Walker Jeffery W
Department of Physiology, University of Wisconsin, Madison, WI 53706 USA.
J Physiol. 2003 Nov 1;552(Pt 3):845-57. doi: 10.1113/jphysiol.2003.045260. Epub 2003 Aug 15.
Cardiac troponin I (cTnI) is a phosphoprotein subunit of the troponin-tropomyosin complex that is thought to inhibit cardiac muscle contraction during diastole. To investigate the contributions of cTnI phosphorylation to cardiac regulation, transgenic mice were created with the phosphorylation sites of cTnI mutated to alanine. Activation of protein kinase C (PKC) by perfusion of hearts with phorbol-12-myristate-13-acetate (PMA) or endothelin-1 (ET-1) inhibited the maximum ATPase rate by up to 25 % and increased the Ca2+ sensitivity of ATPase activity and of isometric tension by up to 0.15 pCa units. PKC activation no longer altered cTnI phosphorylation, depressed ATPase rates or enhanced myofilament Ca2+ sensitivity in transgenic mice expressing cTnI that could not be phosphorylated on serines43/45 and threonine144 (PKC sites). Modest changes in myosin regulatory light chain phosphorylation occurred in all mouse lines, but increases in myofilament Ca2+ sensitivity required the presence of phosphorylatable cTnI. For comparison, the beta-adrenergic agonist isoproterenol caused a 38 % increase in maximum ATPase rate and a 0.12 pCa unit decrease in myofilament Ca2+ sensitivity. These beta-adrenergic effects were absent in transgenic mice expressing cTnI that could not be phosphorylated on serines23/24 (protein kinase A, PKA, sites). Overall, the results indicate that PKC and PKA exert opposing effects on actomyosin function by phosphorylating cTnI on distinct sites. A primary role of PKC phosphorylation of cTnI may be to reduce the requirements of the contractile apparatus for both Ca2+ and ATP, thereby promoting efficient ATP utilisation during contraction.
心肌肌钙蛋白I（cTnI）是肌钙蛋白 - 原肌球蛋白复合物的一种磷蛋白亚基，被认为在舒张期抑制心肌收缩。为了研究cTnI磷酸化对心脏调节的作用，构建了将cTnI磷酸化位点突变为丙氨酸的转基因小鼠。用佛波醇 - 12 - 肉豆蔻酸酯 - 13 - 乙酸酯（PMA）或内皮素 - 1（ET - 1）灌注心脏激活蛋白激酶C（PKC），可使最大ATP酶速率降低多达25%，并使ATP酶活性和等长张力的Ca2 +敏感性增加多达0.15个pCa单位。在表达丝氨酸43/45和苏氨酸144（PKC位点）不能被磷酸化的cTnI的转基因小鼠中，PKC激活不再改变cTnI磷酸化、降低ATP酶速率或增强肌丝Ca2 +敏感性。所有小鼠品系中肌球蛋白调节轻链磷酸化都有适度变化，但肌丝Ca2 +敏感性增加需要存在可磷酸化的cTnI。作为比较，β - 肾上腺素能激动剂异丙肾上腺素使最大ATP酶速率增加38%，肌丝Ca2 +敏感性降低0.12个pCa单位。在表达丝氨酸23/24（蛋白激酶A，PKA，位点）不能被磷酸化的cTnI的转基因小鼠中，这些β - 肾上腺素能效应不存在。总体而言，结果表明PKC和PKA通过在不同位点磷酸化cTnI对肌动球蛋白功能发挥相反作用。cTnI的PKC磷酸化的主要作用可能是降低收缩装置对Ca2 +和ATP的需求，从而在收缩过程中促进有效的ATP利用。
