AKAP-mediated targeting of protein kinase A regulates contractility in cardiac myocytes
AKAP-mediated targeting of protein kinase A regulates contractility in cardiac myocytes
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DOI：
10.1161/01.res.88.3.291
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发表时间：
2001-02-16
影响因子：
20.1
通讯作者：
Bond, M
中科院分区：
文献类型：
--
作者：
Fink, MA;Zakhary, DR;Bond, M
cAMP依赖性蛋白激酶A（PKA）通过A-激酶锚定蛋白（AKAP）的区室化将PKA靶向许多细胞类型中的不同亚细胞位置。然而，AKAP介导的PKA锚定在心脏中是否调节心脏收缩功能的问题尚未得到解决。我们通过腺病毒介导的基因转移引入Ht 31，一种以高亲和力结合PKA调节亚基II型（RII）的肽，破坏了AKAP介导的PKA在心肌细胞中的锚定。该肽与内源性AKAP竞争RII结合。不结合RII的Ht 31 P（脯氨酸取代的衍生物）用作阴性对照。然后，我们研究了Ht 31表达对RII分布，Ca 2+循环，细胞缩短和PKA依赖性底物磷酸化的影响。通过共聚焦显微镜，我们发现RII的再分布从核周区域和周期性横纹在Ht 31 P表达细胞的扩散性胞质定位在Ht 31表达细胞。在10 nmol/L异丙肾上腺素存在下，与对照细胞相比，Ht 31表达的心肌细胞显示出细胞缩短和舒张的速率和幅度增加（未感染和表达Ht 31 P的肌细胞）;在异丙肾上腺素刺激下，我们观察到Ca 2+下降90%的时间缩短，但在Ca 2+循环速率或Ca 2+振幅方面，Ht 31表达细胞与对照细胞之间没有显著差异。短暂的与对照组相比，在Ht 31表达细胞中，肌钙蛋白I和肌球蛋白结合蛋白C对异丙肾上腺素刺激的PKA依赖性磷酸化的增加显著降低。我们的研究结果表明，在响应β-肾上腺素能刺激，心肌细胞功能和底物磷酸化PKA调节PKA的靶向。的AKAP。
Compartmentalization of cAMP-dependent protein kinase A (PKA) by A-kinase anchoring proteins (AKAPs) targets PKA to distinct subcellular locations in many cell types. However, the question of whether AKAP-mediated PKA anchoring in the heart regulates cardiac contractile function has not been addressed. We disrupted AKAP-mediated PKA anchoring in cardiac myocytes by introducing, via adenovirus-mediated gene transfer, Ht31, a peptide that binds the PKA regulatory subunit type II (RII) with high affinity. This peptide competes with endogenous AKAPs for RII binding. Ht31P (a proline-substituted derivative), which does not bind RII, was used as a negative control. We then investigated the effects of Ht31 expression on RII distribution, Ca2+ cycling, cell shortening, and PKA-dependent substrate phosphorylation. By confocal microscopy, we showed redistribution of RII from the perinuclear region and from periodic transverse striations in Ht31P-expressing cells to a diffuse cytosolic localization in Ht31-expressing cells. In the presence of 10 nmol/L isoproterenol, Ht31-expressing myocytes displayed an increased rate and amplitude of cell shortening and relaxation compared with control cells (uninfected and Ht31P-expressing myocytes); with isoproterenol stimulation we observed decreased time to 90% decline in Ca2+ but no Significant difference between Ht31-expressing and control cells in the rate of Ca2+ cycling or amplitude of the Ca2+ transient. The increase in PKA-dependent phosphorylation of troponin I and myosin binding protein C on isoproterenol stimulation was significantly reduced in Ht31-expressing cells compared with controls. Our results demonstrate that, in response to beta -adrenergic stimulation, cardiomyocyte function and substrate phosphorylation by PKA is regulated by targeting of PKA. by AKAPs.