Bowling N, Huang X, Sandusky G E, Fouts R L, Mintze K, Esterman M, Allen P D, Maddi R, McCall E, Vlahos C J
Cardiovascular Research, Discovery Research, Eli Lilly and Company, Indianapolis, IN 46285, USA.
J Mol Cell Cardiol. 2001 Apr;33(4):789-98. doi: 10.1006/jmcc.2000.1349.
We have previously demonstrated that protein kinase C (PKC)- alpha expression is significantly elevated in failing human left ventricle, with immunostaining showing increased PKC- alpha localization at the intercalated disks of cardiomyocytes. In the present study we sought to determine, in the failing heart, if PKC- alpha interacted with connexin-43 (Cx-43) both spatially and functionally, and to compare the association of PKC- alpha/Cx-43 with that of PKC- epsilon, a PKC isozyme that does not significantly increase in failing hearts. The possibility of a PKC- alpha or PKC- epsilon/Cx-43 association in non-failing hearts was also investigated. Co-immunoprecipitation of PKC- alpha or PKC- epsilon and Cx-43 in non-failing and failing left ventricle was achieved using antibodies to PKC- alpha or Cx-43. Confocal microscopy confirmed that PKC- alpha distribution within the cardiomyocyte included co-localization with connexin-43 in both failing and non-failing myocardium. In a similar manner, confocal imaging of PKC- epsilon showed cardiomyocyte distribution in both cytosol and membrane, and colocalization of PKC- epsilon with Cx-43. Recombinant PKC- alpha or - epsilon increased PKC activity significantly above endogenous levels in the co-immunoprecipitated Cx-43 complexes (P<0.05). However, phosphorylation of purified human Cx-43 (isolated from failing human left ventricle) by recombinant PKC- alpha or PKC- epsilon resulted in only PKC- epsilon mediated Cx-43 phosphorylation. Thus, in the human heart PKC- alpha, PKC- epsilon, and Cx-43 appear to form a closely associated complex. Whereas only PKC- epsilon directly phosphorylates Cx-43, both PKC isoforms result in increased phosphorylation within the Cx-43 co-immunoprecipitated complex.
我们之前已经证明，蛋白激酶C（PKC）-α在衰竭的人类左心室中表达显著升高，免疫染色显示PKC-α在心肌细胞闰盘处的定位增加。在本研究中，我们试图确定在衰竭心脏中PKC-α是否在空间和功能上与连接蛋白-43（Cx-43）相互作用，并比较PKC-α/Cx-43与PKC-ε（一种在衰竭心脏中无显著增加的PKC同工酶）之间的关联。我们还研究了在非衰竭心脏中PKC-α或PKC-ε/Cx-43关联的可能性。使用针对PKC-α或Cx-43的抗体，在非衰竭和衰竭的左心室中实现了PKC-α或PKC-ε与Cx-43的共免疫沉淀。共聚焦显微镜证实，在衰竭和非衰竭心肌中，心肌细胞内PKC-α的分布包括与连接蛋白-43的共定位。以类似的方式，PKC-ε的共聚焦成像显示其在细胞质和细胞膜中的心肌细胞分布，以及PKC-ε与Cx-43的共定位。重组PKC-α或-ε使共免疫沉淀的Cx-43复合物中的PKC活性显著高于内源性水平（P<0.05）。然而，重组PKC-α或PKC-ε对纯化的人Cx-43（从衰竭的人类左心室分离）的磷酸化仅导致PKC-ε介导的Cx-43磷酸化。因此，在人类心脏中，PKC-α、PKC-ε和Cx-43似乎形成了一个紧密相关的复合物。虽然只有PKC-ε直接磷酸化Cx-43，但两种PKC同工型都导致Cx-43共免疫沉淀复合物内的磷酸化增加。
