Activation of epsilon protein kinase C correlates with a cardioprotective effect of regular ethanol consumption

Abstract

In addition to decreasing the incidence of myocardial infarction, recent epidemiological data suggest that regular alcohol consumption improves survival after myocardial infarction. We recently found that chronic ethanol exposure induces long-term protection against cardiac ischemia-reperfusion injury, which improves myocardial recovery after infarction. Furthermore, this cardioprotection by ethanol is mediated through myocyte adenosine A1 receptors. We now determine the role of protein kinase C (PKC) in ethanol's protective effect against ischemia-reperfusion injury. Using perfused hearts of ethanol-fed guinea pigs, we find that improved contractile recovery and creatine kinase release after ischemia-reperfusion are abolished by PKC inhibition with chelerythrine. Western blot analysis and immunofluorescence localization demonstrate that regular ethanol consumption causes sustained translocation (activation) of epsilonPKC, but not delta or alphaPKC. This same isozyme is directly implicated in ischemic preconditioning's protection against ischemia-reperfusion injury. Our findings suggest (i) that regular ethanol consumption induces long-term cardioprotection through sustained translocation of epsilonPKC and (ii) that PKC activity is necessary at the time of ischemia to mediate ethanol's protective effect against ischemia-reperfusion injury. Studying this selective effect of ethanol on epsilonPKC activation may lead to new therapies to protect against ischemia-reperfusion injury in the heart and other organ systems.

Figure 1

LVDP prior to 45 min of global ischemia and during reperfusion in four groups of perfused guinea pig hearts (n = 9 for each group): 1, following 8 wk 15% ethanol-derived calories (○); 2, pair-fed controls (▵); 3, following 8 wk of ethanol, before and after 10 mM chelerythrine (•); and 4, pair-fed controls, before and after chelerythrine (▪). LVDP recovery is significantly greater in hearts from ethanol-treated animals (P < 0.05 at each 6-min interval). Chelerythrine abolished ethanol’s protective effect on LVDP recovery. Data are presented as mean ± SEM (SEM not included for group 2 but lie well within SEM of groups 3 and 4).

Figure 2

CK release during the first 18 min of postischemic reperfusion from hearts of ethanol-treated (shaded bars) and control (black bars) animals (units/ml × gdw; n = 9 for each group; mean ± SEM). CK release was significantly less from hearts of ethanol-treated animals (∗, P < 0.05). Chelerythrine abolished ethanol’s reduction of CK release during reperfusion.

Figure 3

Representative Western blots depicting ɛPKC translocation in vehicle- (group 1), 100 nM 4α-PMA- (group 2), or 100 nM 4β-PMA-treated (group 3) myocytes following isolation from one pair of control and ethanol-fed animals. Myocytes were subjected to fractionation by centrifugation to buffer-soluble (cytosol) and Triton X-100-soluble (particulate) fractions.

Figure 4

PKC isozyme translocation in vehicle-treated myocytes. (Left) Depicted are Western blots for ɛ, α, and δPKC from the particulate fraction of each of the seven control (C) and ethanol-fed (E) animal pairings used in this study (numbered 1 through 7). (Right) Depicted is each average corresponding PKC isozyme level in both the cytosolic and particulate fractions of these pairings (±SEM, n = 7). ɛ, α, and δPKC levels for all treatment groups are normalized to the vehicle-treated paired control for each group and to the average cell viability of each treatment group following PMA (or vehicle) treatment. ∗, P < 0.05.

Figure 5

Confocal indirect immunofluorescence images of vehicle-treated myocytes of a control animal (A), 4β-PMA-treated myocytes of a control animal (B), and vehicle-treated myocytes of an ethanol-fed animal (C). (B and C) Depict activated ɛPKC translocated to myofibrillar structures; such cells were scored as having a translocated ɛPKC pattern of immunofluorescence. A, however, depicts inactive ɛPKC in a diffuse cytosolic staining pattern; such cells were scored as inactive. Images were acquired at ×60, 0.42-mm resolution in the z axis. Insets, ×3.5.

Figure 6

Quantitative ɛPKC translocation assessed by immunofluorescence localization. One hundred myocytes from each treatment group were scored using the criteria described in the legend to Fig. 5 (also see Results) as either having an activated ɛPKC translocation pattern or an inactive pattern. Data are mean ± SEM from the same seven animal pairs used in the Western blot analysis.