Abstract-Although functional coupling between protein kinase C⑀ (PKC⑀) and mitochondria has been implicated in the genesis of cardioprotection, the signal transduction mechanisms that enable this link and the identities of the mitochondrial proteins modulated by PKC⑀ remain unknown. Based on recent evidence that the mitochondrial permeability transition pore may be involved in ischemia/reperfusion injury, we hypothesized that protein-protein interactions between PKC⑀ and mitochondrial pore components may serve as a signaling mechanism to modulate pore function and thus engender cardioprotection. Coimmunoprecipitation and GST-based affinity pull-down from mouse cardiac mitochondria revealed interaction of PKC⑀ with components of the pore, namely voltage-dependent anion channel (VDAC), adenine nucleotide translocase (ANT), and hexokinase II (HKII). VDAC1, ANT1, and HKII were present in the PKC⑀ complex at Ϸ2%, Ϸ0.2%, and Ϸ1% of their total expression, respectively. Moreover, in vitro studies demonstrated that PKC⑀ can directly bind and phosphorylate VDAC1. Incubation of isolated cardiac mitochondria with recombinant PKC⑀ resulted in a significant inhibition of Ca 2ϩ -induced mitochondrial swelling, an index of pore opening. Furthermore, cardiac-specific expression of active PKC⑀ in mice, which is cardioprotective, greatly increased interaction of PKC⑀ with the pore components and inhibited Ca 2ϩ -induced pore opening. In contrast, cardiac expression of kinase-inactive PKC⑀ did not affect pore opening. Finally, administration of the pore opener atractyloside significantly attenuated the infarct-sparing effect of PKC⑀ transgenesis. Collectively, these data demonstrate that PKC⑀ forms physical interactions with components of the cardiac mitochondrial pore. This in turn inhibits the pathological function of the pore and contributes to PKC⑀-induced cardioprotection.

Abstract-The importance of proteasomes in governing the intracellular protein degradation process has been increasingly recognized. Recent investigations indicate that proteasome complexes may exist in a species-and cell-type-specific fashion. To date, despite evidence linking impaired protein degradation to cardiac disease phenotypes, virtually nothing is known regarding the molecular composition, function, or regulation of cardiac proteasomes. We have taken a functional proteomic approach to characterize 26S proteasomes in the murine heart. Multidimensional chromatography was used to obtain highly purified and functionally viable cardiac 20S and 19S proteasome complexes, which were subjected to electrophoresis and tandem mass spectrometry analyses. Our data revealed complex molecular organization of cardiac 26S proteasomes, some of which are similar to what were reported in yeast, whereas others exhibit contrasting features that have not been previously identified in other species or cell types. At least 36 distinct subunits (17 of 20S and 19 of 19S) are coexpressed and assembled as 26S proteasomes in this vital cardiac organelle, whereas the expression of PA200 and 11S subunits were detected with limited participation in the 26S complexes. The 19S subunits included a new alternatively spliced isoform of Rpn10 (Rpn10b) along with its primary isoform (Rpn10a). Immunoblotting and immunocytochemistry verified the expression of key ␣ and ␤ subunits in cardiomyocytes. The expression of 14 constitutive ␣ and ␤ subunits in parallel with their three inducible subunits (␤1i, ␤2i, and ␤5i) in the normal heart was not expected; these findings represent a distinct level of structural complexity of cardiac proteasomes, significantly different from that of yeast and human erythrocytes. Furthermore, liquid chromatography/tandem mass spectroscopy characterized 3 distinct types of post-translational modifications including (1) N-terminal acetylation of 19S subunits (Rpn1, Rpn5, Rpn6, Rpt3, and Rpt6) and 20S subunits (␣2, ␣5, ␣7, ␤3, and ␤4); (2) N-terminal myristoylation of a 19S subunit (Rpt2); and (3) phosphorylation of 20S subunits (eg, ␣7)). Taken together, this report presents the first comprehensive characterization of cardiac 26S proteasomes, providing critical structural and proteomic information fundamental to our future understanding of this essential protein degradation system in the normal and diseased myocardium. T he proteasome is a key proteolytic enzymatic system governing the degradation of majority intracellular proteins. 1 Recent studies implicate proteasomes in cardiac diseases. [2][3][4][5][6][7][8][9][10][11] Proteasome inhibitors are widely used in cancer therapy, however, their impact on cardiac function remains unclear; investigations in the heart have reported conflicting results. 2-4,12 A major contributing factor to these controversies is the lack of information pertaining to the structural organization and protein composition of cardiac proteasomes, therefore prohibiting the identification of mol...

Abstract-Although activation of protein kinase C (PKC) ⑀ and mitogen-activated protein kinases (MAPKs) are known to play crucial roles in the manifestation of cardioprotection, the spatial organization of PKC⑀ signaling modules in naïve and protected myocardium remains unknown. Based on evidence that mitochondria are key mediators of the cardioprotective signal, we hypothesized that PKC⑀ and MAPKs interact, and that they form functional signaling modules in mitochondria during cardioprotection. Both immunoblotting and immunofluorescent staining demonstrated that PKC⑀, ERKs, JNKs, and p38 MAPK co-localized with cardiac mitochondria. Moreover, transgenic activation of PKC⑀ greatly increased mitochondrial PKC⑀ expression and activity, which was concomitant with increased mitochondrial interaction of PKC⑀ with ERKs, JNKs, and p38 as determined by co-immunoprecipitation. These complex formations appeared to be independent of PKC⑀ activity, as the interactions were also observed in mice expressing inactive PKC⑀. However, although both active and inactive PKC⑀ bound to all three MAPKs, increased phosphorylation of mitochondrial ERKs was only observed in mice expressing active PKC⑀ but not in mice expressing inactive PKC⑀. Examination of potential downstream targets of mitochondrial PKC⑀-ERK signaling modules revealed that phosphorylation of the pro-apoptotic protein Bad was elevated in mitochondria. Together, these data show that PKC⑀ forms subcellular-targeted signaling modules with ERKs, leading to the activation of mitochondrial ERKs.