Activation of ɛ 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 ɛPKC, but not δ or αPKC. 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 ɛPKC 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 ɛPKC activation may lead to new therapies to protect against ischemia–reperfusion injury in the heart and other organ systems.

Epidemiological studies show that drinking alcohol decreases mortality due to ischemic heart disease, primarily by reducing the incidence of myocardial infarction (MI) (1–4). This decreased incidence of MI is likely due to ethanol’s pleotropic effects on lipids, platelets, and fibrinolytic activity (5–9). Recent epidemiological data suggest that drinking alcohol may also improve survival after MI (10–12), but the mechanisms underlying this cardioprotective effect of ethanol remain uncertain.

Survival after MI is directly related to myocardial recovery (13). Reducing ischemia–reperfusion injury improves myocardial recovery after MI. The increasing use of emergent reperfusion therapies during acute MI, including thrombolysis and coronary angioplasty, provides impetus for finding therapies to reduce ischemia–reperfusion injury. We recently found that regular ethanol consumption induces long-term protection against ischemia–reperfusion injury in guinea pig hearts (14). We showed that this cardioprotective effect of ethanol requires adenosine A₁ receptor activation at the time of ischemia (14), like experimental ischemic preconditioning (15–19). Ischemic preconditioning occurs when brief periods of ischemia and reperfusion protect hearts against injury from subsequent prolonged ischemia–reperfusion. A recent study demonstrated that hearts from rats fed ethanol for 8 wk could be preconditioned with a single 5-min episode of ischemia prior to prolonged ischemia–reperfusion, whereas hearts from control animals were not protected (20). However, the short-term protective effect of ischemic preconditioning is not clinically applicable, necessitating the search for long-term therapies which maintain protection against ischemia–reperfusion injury in patients at risk for MI.

The protective effect of ischemic preconditioning has been associated with activation of protein kinase C (PKC) in several animal models and human myocardium (21–27). Recent reports correlate activation of specific PKC isozymes, including ɛ, δ, and αPKC, with ischemic preconditioning (26–29). Gray et al. (26), directly implicated ɛPKC in ischemic preconditioning’s protection of neonatal cardiomyocytes using PKC isozyme-specific inhibitors. We now report evidence that PKC activity is necessary for mediating ethanol’s protective effect against ischemia–reperfusion injury at the time of ischemia. We also observed that regular ethanol consumption causes sustained translocation of a distinct PKC isozyme in myocytes, potentially contributing to the long-term cardioprotection against ischemia–reperfusion injury induced by ethanol.

METHODOLOGY

Perfused Heart Study.

Male Hartley guinea pigs (275–300 g) were fed a nutritionally supplemented liquid diet (Dyets, Bethlehem, PA) containing 15% or 0% ethanol-derived calories for 8 wk. This approximates the upper limits of moderate ethanol consumption in humans (<45 g of ethanol/day, at 7 kcal/g ethanol, results in ≈15% ethanol-derived calories in a 2000 kcal diet; see ref. 4). Control animals received the same number of calories as the paired ethanol-fed animals had consumed over the previous 24 hr. Serum ethanol levels were drawn after 8 wk in five animals.

Hearts were isolated and perfused via the Langendorff method as described previously (14) using a Krebs-Henseleit perfusate (pH 7.4, 37°C) bubbled with 95%O₂/5%CO₂. Left ventricular (LV) pressures were measured using a 2-French, high-fidelity micromanometer (Millar Instruments, Houston, TX) at a LV end diastolic pressure of 10 mmHg and pacing rate of 240 beats per minute. Coronary flow was measured by an in-line flow meter (Gilmont Instruments, Barrington, IL). Creatine kinase (CK) release during reperfusion was measured with a commercially available kit (Sigma). Values were corrected for dry heart weight and coronary flow rates and expressed in units per ml per g dry weight (units/ml × gdw).

Ischemia–Reperfusion Protocol.

Ethanol was removed from the liquid diet 12–16 hr prior to sacrifice to avoid a direct effect of ethanol on hearts. After a 20-min equilibration period, hearts were subjected to 45 min of no-flow ischemia and 48 min of reperfusion as previously described (14) (n = 9 ethanol; n = 9 control). After reperfusion, hemodynamic measurements were repeated every 6 min for 48 min. Hearts were then dried for 24 hr at 80°C and weighed. To exclude the possibility that ethanol withdrawal might contribute to findings, additional experiments were performed using animals allowed to consume ethanol until sacrifice (n = 5).

Additional experiments were performed with chelerythrine chloride (10 μM; Sigma) added to the perfusate 10 min prior to ischemia (n = 9 ethanol, n = 9 control). This concentration of chelerythrine, a potent and specific inhibitor of PKC (30), was used as described previously (28, 31, 32). Chelerythrine decreased baseline LV developed pressure (LVDP) by 14% and increased coronary flow by 7% in both ethanol and control hearts (Table 1). Similar changes in hemodynamics have been reported in previous studies with PKC inhibitors (28, 31, 32).

Table 1.

PKC activity is important in mediating ethanol’s cardioprotective effect against ischemia–reperfusion injury

	Preischemia		Reperfusion
	Control	Ethanol	Control	Ethanol
Developed pressure (mmHg)	117  ±  4	117  ±  2	25  ±  4	49  ±  4*
Diastolic pressure (mmHg)	10  ±  0	10  ±  0	58  ±  5	35  ±  6*
Perfusion pressure (mmHg)	70  ±  0	70  ±  0	76  ±  1	76  ±  1
Coronary flow (ml/min)	35  ±  2	36  ±  1	23  ±  4	23  ±  2
(+) CHE
Developed pressure (mmHg)	104  ±  4†	102  ±  4†	24  ±  3	30  ±  3†
Diastolic pressure (mmHg)	10  ±  0	10  ±  0	53  ±  4	49  ±  4†
Perfusion pressure (mmHg)	70  ±  0	70  ±  0	77  ±  1	76  ±  1
Coronary flow (ml/min)	39  ±  1†	39  ±  1†	19  ±  2	20  ±  2

Hemodynamic data from experiments in perfused guinea pig hearts subjected to 45 min of global ischemia and 48 min of reperfusion. Hearts from guinea pigs consuming 15% ethanol-derived calories for 8 wk were compared to hearts from pair-fed controls (ethanol, n = 9; control, n = 9). Experiments were repeated in the presence of the PKC antagonist, chelerythrine [(+)CHE; ethanol, n = 9; control, n = 9]. Data are presented as mean ± SEM. P < 0.05 for all reperfusion data in controls and ethanol-treated animals versus preischemia values. 

*P < 0.05 ethanol versus control. 

 

^†P < 0.05 (+)CHE versus (−)CHE. 

An additional group of hearts were studied from guinea pigs consuming 1.25% ethanol in their drinking water for 8 wk to document that milder levels of regular ethanol consumption also produce protection against ischemia–reperfusion injury (n = 10 ethanol; n = 8 control). These experiments were performed in an identical manner to those described above.

Cardiomyocyte Studies.

Male Hartley guinea pigs (275–300 g; n = 9 ethanol; n = 9 control) were given 10% (a dose more consistent with moderate consumption) or 0% ethanol and solid food ad libitum (Lab Diet: PMI Feeds, St. Louis) for 8 wk as described previously (14). Isolated hearts were perfused for 10 min with Krebs-Henseleit perfusate (pH 7.4, 37°C) bubbled with 95%O₂/5%CO₂. Perfusate was changed to a nominally Ca²⁺-free Krebs-Henseleit perfusate (pH 7.4) with 0.5 mg/ml bovine albumin for 10 min and then Krebs-Henseleit perfusate containing 0.7 mg/ml collagenase B (Boehringer Mannheim), 25 mmol/liter CaCl₂, and 0.5 mg/ml BSA at a constant flow of 5 ml/min for 20 min. Two hearts (one ethanol and one control) were deemed unusable due to insufficient collagenase digestion and were eliminated from analysis with the heart from their paired animal. Ventricles were minced in Kraftbruhe buffer (pH 7.2), triturated with a Pasteur pipette, filtered through a stainless mesh, and centrifuged for 5 min at 1000 × g. Myocytes were isolated from nonmyocytes by resuspending the pellet in 20 ml of 4% Ficoll-400 (Sigma)/Kraftbruhe buffer and centrifuging for 5 min at 40 rpm. The myocyte pellet was resuspended in 5 ml of Kraftbruhe buffer.

Isolated myocytes were divided into three groups and treated for 15 min with active phorbol ester to maximally translocate PKC [4β-phorbol 12-myristate 13-acetate (4β-PMA), 100 nM; LC Laboratories, Woburn, MA), inactive phorbol ester as a control (4α-PMA, 100 nM), or vehicle (dimethyl sulfoxide, 0.04%). Myocytes were then washed three times in 15 ml of PBS and pelleted after each wash by centrifugation at 800 × g for 30 sec. Cell viability was determined by trypan blue exclusion.

Western Blot Analysis.

Vehicle and PMA-treated myocytes were homogenized in 1.0 ml of PBS with protease inhibitors (20 mg/ml each of phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, leupeptin, and aprotinin) by trituration 15 times with a syringe and 22-gauge needle and then centrifuged for 30 min at 100,000 × g (4°C). Supernatants were collected as the soluble (cytosolic) fraction and the pellets were rehomogenized by identical trituration in 1.0 ml of PBS/protease inhibitor solution with 1% Triton X-100. Homogenates were centrifuged and the resultant Triton X-100-soluble fraction was collected. Equal amounts of cytosolic and Triton X-100-soluble fractions were loaded onto SDS/PAGE gels after addition of SDS–Laemmli sample buffer. Western blots were probed with either anti-ɛPKC (1:100)- or anti-δPKC (1:100)-specific polyclonal IgG (Santa Cruz Biotechnology) or anti-αPKC (1:300) monoclonal IgG (Seikagaku America, Rockville, MD). Quantitation of Western blots was performed by densitometric analysis of digitized enhanced chemiluminescence-exposed films using NIH Image v1.58. Accuracy of this method and linearity of detection within the measured sample range was confirmed by quantitation of the same Western blots by enhanced chemifluorescence detection using a Molecular Dynamics STORM 850 fluorescence scanner and Molecular Dynamics ImageQuant v1.1 software (Sunnyvale, CA).

Immunofluorescence Localization of ɛPKC.

Myocytes were fixed immediately after vehicle or PMA treatment with 5% gluteraldehyde for 15 min. Fixed myocytes were incubated for 1 hr with 1% normal goat serum in PBS containing 0.1% Triton X-100 and then incubated with anti-ɛPKC IgG (Research and Diagnostic Antibodies, Berkeley, CA) diluted 1:100 in PBS containing 0.1% Triton X-100 and 2 mg/ml BSA overnight at 4°C. Myocytes were washed three times with PBS and incubated for 2 hr with fluorescein-conjugated rabbit IgG secondary antibody (Organon Technika, West Chester, PA) diluted 1:1000. Myocytes were washed three times with PBS, mounted on glass slides using Vecta Shield (Vector Laboratories), and viewed with a Zeiss IM35 microscope (Zeiss) with a ×40 water immersion objective. Images were recorded on Kodak Tmax 400 film with an exposure time of 45 sec for photomicrographs. The percentage of myocytes showing a predominantly cytosolic/perinuclear fluorescence (nontranslocated ɛPKC) or predominantly cross-striated fluorescence (translocated ɛPKC) were determined by blind counting of 100 myocytes in each treatment group for each heart. The preabsorbed anti-ɛPKC IgG used in this study do not exhibit any specific staining in similarly treated and fixed myocytes (26, 33).

Statistical Analysis.

All data are expressed as mean ± SEM. Comparisons between groups were made using repeated measures analysis of variance with multiple grouping factors. A Student–Newman–Keuls post hoc test was used to confirm the significance of differences between groups. P < 0.05 was considered to be statistically significant.

RESULTS

Serum ethanol levels were 10 ± 2 mg/dl (≈2 mM; 9–11 a.m.) after 8 wk of feeding with a nutritionally supplemented liquid diet containing 15% ethanol-derived calories. Body weights (728 ± 9 g vs. 728 ± 11 g) and dry heart weight to body weight ratios (4.02 ± 0.09 × 10⁻⁴ vs. 4.11 ± 0.09 × 10⁻⁴) were the same after 8 wk in ethanol-fed and control animals, suggesting no development of LV hypertrophy as is seen with heavy ethanol consumption (34–36). Baseline LVDP, coronary flow, and perfusion pressure were the same in hearts from ethanol-fed and control animals (Table 1).

Regular Ethanol Consumption Reduces Ischemia–Reperfusion Injury.

LVDP recovered to 42% of preischemic levels in hearts from ethanol-fed animals compared with 22% in controls (P < 0.05; Fig. 1). Increased LV end diastolic pressure, an index of myocyte contracture and irreversible injury, was lower during postischemic reperfusion in hearts from ethanol-fed animals (350%) compared with controls (580%; P < 0.05; Table 1). CK release, a measure of myocyte necrosis and/or loss of membrane integrity, was significantly lower from hearts of ethanol-fed animals compared with controls (260 ± 40 vs. 469 ± 74 units/ml × gdw, P < 0.05; Fig. 2). There were no differences between groups in coronary flow or coronary perfusion pressure after ischemia–reperfusion (Table 1), suggesting that ethanol’s protection did not involve vasodilation but is mediated at the myocyte level.

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.

In the above experiments, ethanol feeding was discontinued 12–16 hr before sacrifice to avoid a direct effect of ethanol on hearts. To determine whether ethanol withdrawal contributed to the observed cardioprotection, additional experiments were also performed using hearts from five animals consuming ethanol until sacrifice. LVDP recovered to 45% of preischemic levels (LVDP = 118 ± 4 mmHg preischemia; 54 ± 8 mmHg at 48 min reperfusion), LV end diastolic pressure increased to 290% of preischemic levels, and CK release during reperfusion was 279 ± 59 units/ml × gdw. These data suggest that ethanol’s cardioprotective effect is the same whether ethanol is present in the serum or withdrawn 12–16 hr before the ischemic insult. Furthermore, the presence of ethanol is not deleterious to the heart during ischemia–reperfusion.

To demonstrate that ethanol was cardioprotective at milder levels of consumption, hearts from guinea pigs drinking 1.25% ethanol for 8 wk were also studied. Weights were the same in ethanol-treated and control animals (850 ± 20 vs. 870 ± 20 g). LVDP recovery was greater (50 ± 7 vs. 34 ± 5 mmHg at 48-min reperfusion, P < 0.05), LV end diastolic pressure rise was less (21 ± 4 vs. 42 ± 5 mmHg at 48-min reperfusion; P < 0.05), and CK release during reperfusion was reduced (178 ± 26 vs. 313 ± 29 units/ml × gdw, P < 0.05) in hearts from ethanol-treated animals compared with controls. These data suggest that ethanol’s cardioprotective effect is also present at milder levels of regular ethanol consumption.

Role of PKC in Ethanol’s Cardioprotective Effect: Perfused Heart Study.

We next determined whether PKC activity is important in mediating ethanol’s protective effect against ischemia–reperfusion injury at the time of ischemia. In the presence of the PKC inhibitor chelerythrine, LVDP and end diastolic pressure (Table 1) and CK release (415 ± 42 units/ml × gdw vs. 484 ± 44 units/ml × gdw) were similar during postischemic reperfusion in hearts from ethanol-fed and control animals. Furthermore, these parameters were similar to data from hearts of control animals not exposed to chelerythrine (Figs. 1 and 2), suggesting that chelerythrine abolished ethanol’s protective effect.

Role of PKC in Ethanol’s Cardioprotective Effect: Cardiomyocyte Studies.

We next determined whether ethanol’s long-term cardioprotective effect against ischemia–reperfusion injury is due to sustained activation of selective PKC isozymes implicated in ischemic preconditioning (26–28). Translocation of PKC isozymes from the soluble to the particulate myocyte fraction correlates with activation (37). We determined the subcellular distribution of ɛ, δ, and αPKC using Western blot analysis in myocytes isolated from hearts of ethanol-fed and control animals. Isolated myocytes were exposed to β-PMA (phorbol ester activator of PKC), α-PMA (inactive phorbol ester, control), or dimethyl sulfoxide (control). β-PMA induced translocation of PKC isozymes from the cytosolic (inactive PKC) to particulate fractions (active PKC), as shown previously (26, 38). Representative autoradiographs of Western blots probed for ɛPKC are shown in Fig. 3. Individual autoradiographs of particulate fractions for each animal pair, as well as the mean data, are shown in Fig. 4. Response to β-PMA was similar in myocytes from ethanol-fed and control animals. Subcellular distribution of δ and αPKC did not differ in myocytes from ethanol-fed and control animals in the presence of dimethyl sulfoxide vehicle or α-PMA (Fig. 4). In contrast, the ratio of particulate to cytosolic ɛPKC was greater in myocytes from ethanol-fed compared with control animals (2.7 ± 0.1:1 vs. 1.5 ± 0.4:1, P < 0.05; Fig. 4). Total ɛPKC was not significantly different from total ɛPKC in myocytes from control animals (82 ± 15% of control levels, P, not significant).

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.

To corroborate ɛPKC Western blot data, we used immunofluorescence localization to assess the degree of ɛPKC translocation in myocytes from ethanol-fed animals compared with controls. Representative microphotographs of ɛPKC antibody fluorescence are shown in Fig. 5. As shown previously (26, 33, 37–39), immunolocalization of ɛPKC antibody fluorescence from the perinuclear and cytosolic regions of the myocyte to the cross-striations (possibly myofilaments) is indicative of ɛPKC translocation to activation sites. Translocation by immunofluorescence was not discernable in cells stained for either α or δPKC. As shown in Fig. 6, blinded scoring revealed that the percentage of myocytes demonstrating a predominant cross-striated pattern of fluorescence was greater in hearts from ethanol-fed animals compared with controls (61 ± 3% vs. 22 ± 5%, P < 0.05). α-PMA, an inactive phorbol ester, did not cause redistribution of ɛPKC in either group of myocytes. In contrast, β-PMA increased the percentage of control myocytes with cross-striated fluorescence (62 ± 2% vs. 22 ± 5%, P < 0.05), but had no significant effect on the percentage of myocytes from ethanol-fed animals with cross-striated fluorescence (68 ± 3% vs. 61 ± 3%, P, not significant). These data suggest that ɛPKC is already significantly translocated or redistributed with regular ethanol consumption and that ethanol and β-PMA may induce ɛPKC translocation by similar mechanisms. Note also that the translocation to cellular structures occurs from discrete sites in the cell where inactive ɛPKC resides (Fig. 5).

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.

DISCUSSION

Our data support the hypothesis that regular ethanol consumption improves cardiac recovery after ischemia–reperfusion. In this study, ischemia–reperfusion injury is reduced in hearts from guinea pigs drinking ethanol compared with isocalorically matched controls, suggesting that a starvation effect is not responsible for ethanol’s cardioprotective effect. A major new finding of this study is that the PKC antagonist chelerythrine abolishes ethanol’s cardioprotection, suggesting that PKC activity is important for mediating protection at the time of ischemia. A second major finding is that regular ethanol consumption selectively translocates ɛPKC, but not δ or αPKC, as shown by Western blot analysis and immunofluorescence localization. These data suggest an isozyme-specific role for ɛPKC in producing ethanol’s long-term cardioprotective effect against ischemia–reperfusion injury. This observation is consistent with recent studies in other cell types demonstrating ethanol-induced activation of ɛPKC (40–44).

In addition to decreasing the incidence of MI (1–4), recent epidemiological data suggest that regular ethanol consumption improves survival after MI (10–12). One potential mechanism by which regular drinking may improve survival after MI is to reduce ischemia–reperfusion injury, analogous to experimental ischemic preconditioning (15–19). The studies in guinea pigs described here show that regular ethanol consumption mimics ischemic preconditioning and reduces ischemia–reperfusion injury. Yet ethanol’s protection was present after 8 wk of ethanol feeding, whether animals were allowed to drink until sacrifice or were taken off ethanol 12–16 hr before. This suggests that it may be possible to use pharmacological agents to produce long-term cardioprotection against ischemia–reperfusion injury in patients at risk for MI.

Studies in animal and human myocardium demonstrate that cardioprotection by ischemic preconditioning is abolished by PKC inhibitors given prior to ischemia–reperfusion (21–23, 45), mimicked by PKC activators (22, 23), and that membrane PKC activity is enhanced during ischemia, suggesting PKC translocation (24, 46). Recent reports correlate activation of specific PKC isozymes, including ɛ, δ and αPKC, with ischemic preconditioning (26–29). Gray et al. (26), directly implicated ɛPKC in preconditioning’s protection of neonatal cardiomyocytes using PKC isozyme-specific inhibitors.

PKC isozyme activation is associated with translocation to sites in the myocyte where isozymes bind to anchoring molecules termed receptors for activated C kinases (RACKs; ref. 37). Isozyme-selective RACKs are located on a variety of subcellular structures, including membranes and cytoskeletal elements (37). Since we have shown that the selective PKC inhibitor chelerythrine inhibits ethanol-induced protection, and because our previous studies showed that translocation is required and sufficient for activation (26, 47–49), these data are most consistent with translocation correlated with activation of ɛPKC. After binding to their selective RACKs, activated PKC isozymes phosphorylate protein substrates which may ultimately mediate the cardioprotection against ischemia–reperfusion injury (50). Potential mediators of ischemic preconditioning’s first window of protection (up to 3 hr) include PKC activation of ATP-sensitive potassium channels causing reduced calcium influx (31, 51–53) and activation of vacuolar proton ATPase reducing intracellular acidification (54). Potential mediators of ischemic preconditioning’s second window of protection (24–72 hr) include transcription factors activated via a PKC-mediated pathway which regulate expression of heat shock/stress proteins (55, 56).

Because regular ethanol consumption reduces ischemia–reperfusion injury in a manner analogous to ischemic preconditioning, we searched for evidence that PKC activation at the time of ischemia is important in ethanol’s cardioprotective effect. Perfusion with chelerythrine prior to ischemia abolished protection, suggesting a role for PKC in ethanol-induced cardioprotection. We are aware that chelerythrine may have other effects on myocyte function. For example, treatment with chelerythrine did produce a small decrease of LVDP prior to ischemia. Similar changes in hemodynamics have been reported in prior ischemia–reperfusion studies with several PKC inhibitors (28, 31, 32) and are thought to be due to blocking PKC potentiation of adrenergic α1-associated slow Ca²⁺ channels (57). A recent report in aortic rings suggests that chelerythrine can also affect cyclic nucleotide phosphodiesterases with an IC₅₀ ranging from 18 to 206 μM (58) (we used 10 μM). Importantly, in our study, contractile recovery and CK release after ischemia–reperfusion were the same in control hearts in the presence or absence of chelerythrine. This suggests that chelerythrine did not alter cardiac recovery in ischemia–reperfusion in control hearts. These data are also consistent with chelerythrine inhibiting PKC-induced cardioprotection against ischemia–reperfusion injury in hearts from ethanol-fed animals.

We next examined the possibility that regular ethanol consumption causes sustained activation of PKC isozymes implicated in ischemic preconditioning. We find that regular ethanol consumption translocates ɛPKC, but not δ or αPKC. Thus, in guinea pig hearts, regular ethanol consumption may induce long-term cardioprotection by causing sustained translocation of ɛPKC to its RACK (37). Such sustained translocation was proposed to occur by Downey and coworkers (59). Ethanol-induced activation of ɛPKC has been observed in other cell types (40–44). For example, in neural crest-derived PC12 cells, ethanol-induced activation of ɛPKC enhances nerve growth factor-induced signaling and neurite outgrowth (40).

We propose that regular ethanol consumption causes sustained translocation of ɛPKC to its RACK, where it is poised at the time of ischemia to phosphorylate an effector protein(s) which protects against ischemia–reperfusion injury. This hypothesis is supported by recent findings of Gray et al. (26) in a myocyte model of ischemia–reperfusion injury. This study showed that a translocation antagonist specific for ɛPKC (a peptide derived from ɛPKC or its RACK) causes inhibition of translocation of ɛPKC and prevents protection induced by PMA or preconditioning against myocyte death after hypoxia–reoxygenation. Future studies will further elucidate the role of ɛPKC in this long-term cardioprotective effect of regular ethanol consumption and its relationship to adenosine A₁ receptor activation (14).

ABBREVIATIONS

MI

myocardial infarction

PKC

protein kinase C

LV

left ventricular

CK

creatine kinase

LVDP

LV-developed pressure

PMA

phorbol 12-myristate 13-acetate