PKC-ε mediates multiple endothelin-1 actions on systolic Ca2+ and contractility in ventricular myocytes.
PKC-ε mediates multiple endothelin-1 actions on systolic Ca2+ and contractility in ventricular myocytes.
复制标题
PKC-β 介导内皮素 1 对心室肌细胞收缩 Ca2 和收缩力的多种作用。
DOI：
10.1016/j.bbrc.2012.06.024
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发表时间：
2012
影响因子：
3.1
通讯作者：
Chung,KaYoung
中科院分区：
文献类型：
--
作者：
Kang,Misuk;Chung,KaYoung
内皮素-1 (ET-1) 可诱导心肌正性肌力（增强收缩力），但建立潜在的细胞机制一直存在争议，部分原因是 ET-1 靶向的信号通路和末端效应器数量不断增加。在这里，我们提供的证据表明，ET-1 通过增加收缩期 Ca2+ 和肌丝 Ca2+ 敏感性，在心室组织中诱导正性肌力作用。为了检查 PKC-δ 和 PKC-ε 在 ET-1 急性反应中的作用，在成年大鼠心室肌细胞中表达激酶失活显性失活 PKC (dn-PKC) 构建体。将黄色荧光蛋白 (YFP) 与 dn-PKC 构建体融合，以可视化活体肌细胞中 dn-PKC 的表达和定位。由于假底物位点中丙氨酸突变为谷氨酸，dn-PKC 组成型易位至锚定位点，并且不受激动剂或佛波酯处理的影响。 Dn-PKC-δ-YFP 主要分布在成体肌细胞的 Z 线和闰盘上，而 dn-PKC-ε-YFP 染色表面肌膜、T 管/Z 线和核周区域。表达 dn-PKC-δ-YFP 的肌细胞表现出正常的收缩期 Ca2+ 和对 ET-1 的收缩反应。相反，在表达 dn-PKC-ε-YFP 的肌细胞中，整个 ET-1 反应被阻断，包括增加的 Ca2+ 瞬态、增强的肌丝 Ca2+ 敏感性和正性肌力。该报告提供了直接证据，表明 PKC-ε 在 ET-1 刺激后早期并强烈激活，从而介导 ET-1 对心肌急性作用的多种细胞内变化。
Endothelin-1 (ET-1) induces positive inotropy (enhanced contractility) in cardiac muscle, but establishing underlying cellular mechanisms has been controversial in part because of a growing number of signaling pathways and end effectors targeted by ET-1. Here we present evidence that ET-1 induces positive inotropism in ventricular tissue by increasing both systolic Ca2+and myofilament Ca2+sensitivity. To examine the roles of PKC-δ and PKC-ε in these acute responses to ET-1, kinase inactive dominant negative PKC (dn-PKC) constructs were expressed in adult rat ventricular myocytes. Yellow fluorescent protein (YFP) was fused to dn-PKC constructs to visualize expression and localization of dn-PKC in living myocytes. Due to an alanine to glutamate mutation in the pseudosubstrate site, dn-PKCs constitutively translocated to anchoring sites and were unaffected by agonist or phorbol ester treatment. Dn-PKC-δ-YFP mainly distributed at Z-lines and at intercalated disks in adult myocytes, whereas dn-PKC-ε-YFP stained the surface sarcolemma, T-tubules/Z-lines and perinuclear region. Myocytes expressing dn-PKC-δ-YFP showed normal systolic Ca2+and contractile responses to ET-1. In contrast, the entire ensemble of ET-1 responses was blocked in myocytes expressing dn-PKC-ε-YFP including increased Ca2+transients, enhanced myofilament Ca2+sensitivity, and positive inotropy. This report provides direct evidence that PKC-ε is activated early and robustly following ET-1 stimulation and thus mediates multiple intracellular changes underlying the acute actions of ET-1 on myocardium.