Korzick Donna H, Kostyak John C, Hunter J Craig, Saupe Kurt W
Intercollege Program in Physiology, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Am J Physiol Heart Circ Physiol. 2007 Oct;293(4):H2056-63. doi: 10.1152/ajpheart.00403.2007. Epub 2007 Aug 3.
In adult heart, selective PKCepsilon activation limits ischemia (I)-reperfusion (R) damage and mimics the protection associated with ischemic preconditioning. We sought to determine whether local delivery of PKCepsilon activator peptide psiepsilon-receptor for activated C-kinase (psiepsilon-RACK) is sufficient to produce a similarly protected phenotype in aged hearts. Langendorff-perfused hearts isolated from adult (5 mo; n = 9) and aged (24 mo; n = 9) male Fisher 344 rats were perfused with psiepsilon-RACK conjugated to Tat (500 nM) or Tat only (500 nM) for 10 min before global 31-min ischemia. Western blotting was used to measure mitochondrial targeting of PKCepsilon, PKCdelta, phospho (p)-GSK-3beta (Ser(9)) and GSK-3beta in hearts snap-frozen during I. Recovery of left ventricular developed pressure was significantly improved by psiepsilon-RACK (P < 0.01) and infarct size reduced in 24-mo rats vs. age-matched controls (60% vs. 34%; P < 0.01). Mitochondrial PKCepsilon levels were 30% greater during I with psiepsilon-RACK in aged vs. control rats (P < 0.01). Interestingly, mitochondrial GSK-3beta levels were threefold greater in aged vs. adult rats during I, and psiepsilon-RACK prevented this increase (P < 0.01). Mitochondrial p-GSK-3beta levels were also greater in aged rats after psiepsilon-RACK (P < 0.01), and subsequent inhibition of GSK-3beta with SB-216763 (3 muM) before I/R elicited protection similar to that of psiepsilon-RACK (n = 3/group). Mitochondrial proteomic analysis further identified group differences in the F(1)-ATPase beta-subunit, and coimmunoprecipitation studies revealed a novel interaction with PKCepsilon. F(1)-ATPase-PKCepsilon association was affected by psiepsilon-RACK in adult but not aged rats. Our results provide evidence, for the first time, for PKCepsilon-mediated protection in aged rat heart after I/R and suggest a central role for mitochondrial GSK-3beta but not F(1)-ATPase as a potential target of PKCepsilon to limit I/R damage with aging.
在成年心脏中，选择性激活蛋白激酶Cε（PKCε）可限制缺血（I）-再灌注（R）损伤，并模拟与缺血预处理相关的保护作用。我们试图确定局部递送PKCε激活肽——活化C激酶的ψε受体（ψε-RACK）是否足以在老年心脏中产生类似的保护表型。从成年（5月龄；n = 9）和老年（24月龄；n = 9）雄性Fisher 344大鼠分离的Langendorff灌注心脏，在进行31分钟全心缺血前，用与Tat偶联的ψε-RACK（500 nM）或仅用Tat（500 nM）灌注10分钟。在缺血期间快速冷冻的心脏中，采用蛋白质免疫印迹法检测PKCε、PKCδ、磷酸化（p）-糖原合成酶激酶-3β（Ser(9)）和糖原合成酶激酶-3β的线粒体靶向性。ψε-RACK显著改善了左心室舒张末压的恢复（P < 0.01），与年龄匹配的对照组相比，24月龄大鼠的梗死面积减小（60%对34%；P < 0.01）。与对照组大鼠相比，老年大鼠在缺血期间用ψε-RACK处理后线粒体PKCε水平增加30%（P < 0.01）。有趣的是，在缺血期间，老年大鼠的线粒体糖原合成酶激酶-3β水平是成年大鼠的三倍，而ψε-RACK可阻止这种增加（P < 0.01）。ψε-RACK处理后，老年大鼠线粒体p-糖原合成酶激酶-3β水平也升高（P < 0.01），并且在缺血/再灌注前用SB-216763（3 μM）抑制糖原合成酶激酶-3β可引发与ψε-RACK类似的保护作用（每组n = 3）。线粒体蛋白质组学分析进一步确定了F1-ATP酶β亚基的组间差异，共免疫沉淀研究揭示了其与PKCε的新相互作用。在成年大鼠而非老年大鼠中，ψε-RACK影响F1-ATP酶与PKCε的结合。我们的结果首次为PKCε介导的老年大鼠心脏缺血/再灌注损伤保护作用提供了证据，并表明线粒体糖原合成酶激酶-3β而非F1-ATP酶作为PKCε限制缺血/再灌注损伤的潜在靶点发挥核心作用。
