Pluteanu Florentina, Boknik Peter, Heinick Alexander, König Christiane, Müller Frank U, Weidlich Adam, Kirchhefer Uwe
Department of Anatomy, Animal Physiology and Biophysics, University of Bucharest, Bucharest, Romania.
Institute of Pharmacology and Toxicology, University of Münster, Münster, Germany.
Am J Physiol Heart Circ Physiol. 2022 Mar 1;322(3):H427-H441. doi: 10.1152/ajpheart.00539.2021. Epub 2022 Feb 4.
Protein phosphatase 2A (PP2A) represents a heterotrimer that is responsible for the dephosphorylation of important regulatory myocardial proteins. This study was aimed to test whether the phosphorylation of PP2A-B56α at Ser by PKC is involved in the regulation of myocyte Ca cycling and contraction. For this purpose, heart preparations of wild-type (WT) and transgenic mice overexpressing the nonphosphorylatable S41A mutant form (TG) were stimulated by administration of the direct PKC activator phorbol 12-myristate 13-acetate (PMA), and functional effects were studied. PKC activation was accompanied by the inhibition of PP2A activity in WT cardiomyocytes, whereas this effect was absent in TG. Consistently, the increase in the sarcomere length shortening and the peak amplitude of Ca transients after PMA administration in WT cardiomyocytes was attenuated in TG. However, the costimulation with 1 µM isoprenaline was able to offset these functional deficits. Moreover, TG hearts did not show an increase in the phosphorylation of the myosin-binding protein C after administration of PMA but was detected in corresponding WT. PMA modulated voltage-dependent activation of the L-type Ca channel (LTCC) differently in the two genotypes, shifting by +1.5 mV in TG and by -2.4 mV in WT. In the presence of PMA, inactivation remained unchanged in TG, whereas it was slower in corresponding WT. Our data suggest that PKC-activated enhancement of myocyte contraction and intracellular Ca signaling is mediated by phosphorylation of B56α at Ser, leading to a decrease in PP2A activity. The importance of the serine-41 phosphorylation site on B56α in reducing PP2A activity was demonstrated for the first time using a transgenic mutation model. Direct activation of PKC inhibits PP2A, leading to increased phosphorylation of MyBP-C in cardiomyocytes. The increased phosphorylation of contractile proteins is influenced by the PKC-phosphoB56α-PP2A signaling cascade resulting in improved intracellular Ca handling and enhanced contractility and relaxation. PKC-mediated inhibition of PP2A also leads to modulation of the LTCC activation and inactivation kinetics.
蛋白磷酸酶2A（PP2A）是一种异源三聚体，负责重要的心肌调节蛋白的去磷酸化。本研究旨在测试蛋白激酶C（PKC）介导的PP2A-B56α丝氨酸位点磷酸化是否参与心肌细胞钙循环和收缩的调节。为此，给予野生型（WT）和过表达非磷酸化S41A突变体形式（TG）的转基因小鼠心脏制剂直接PKC激活剂佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA），并研究其功能效应。PKC激活伴随着WT心肌细胞中PP2A活性的抑制，而TG中不存在这种效应。一致地，WT心肌细胞中给予PMA后肌节长度缩短和钙瞬变峰值幅度的增加在TG中减弱。然而，1μM异丙肾上腺素的共刺激能够抵消这些功能缺陷。此外，TG心脏在给予PMA后未显示肌球蛋白结合蛋白C的磷酸化增加，但在相应的WT中检测到。PMA对两种基因型的L型钙通道（LTCC）电压依赖性激活的调节不同，在TG中正向移动+1.5 mV，在WT中负向移动-2.4 mV。在PMA存在下，TG中的失活保持不变，而在相应的WT中则较慢。我们的数据表明，PKC激活增强的心肌细胞收缩和细胞内钙信号传导是由B56α丝氨酸位点磷酸化介导的，导致PP2A活性降低。首次使用转基因突变模型证明了B56α上丝氨酸-41磷酸化位点在降低PP2A活性中的重要性。PKC的直接激活抑制PP2A，导致心肌细胞中MyBP-C磷酸化增加。收缩蛋白磷酸化的增加受PKC-磷酸化B56α-PP2A信号级联的影响，导致细胞内钙处理改善，收缩性和舒张性增强。PKC介导的PP2A抑制还导致LTCC激活和失活动力学的调节。
