Activation of aldehyde dehydrogenase 2 (ALDH2) confers cardioprotection in protein kinase C epsilon (PKCvarepsilon) knockout mice

Abstract

Acute administration of ethanol can reduce cardiac ischemia/reperfusion injury. Previous studies demonstrated that the acute cytoprotective effect of ethanol on the myocardium is mediated by protein kinase C epsilon (PKCvarepsilon). We recently identified aldehyde dehydrogenase 2 (ALDH2) as a PKCvarepsilon substrate, whose activation is necessary and sufficient to confer cardioprotection in vivo. ALDH2 metabolizes cytotoxic reactive aldehydes, such as 4-hydroxy-2-nonenal (4-HNE), which accumulate during cardiac ischemia/reperfusion. Here, we used a combination of PKCvarepsilon knockout mice and a direct activator of ALDH2, Alda-44, to further investigate the interplay between PKCvarepsilon and ALDH2 in cardioprotection. We report that ethanol preconditioning requires PKCvarepsilon, whereas direct activation of ALDH2 reduces infarct size in both wild type and PKCvarepsilon knockout hearts. Our data suggest that ALDH2 is downstream of PKCvarepsilon in ethanol preconditioning and that direct activation of ALDH2 can circumvent the requirement of PKCvarepsilon to induce cytoprotection. We also report that in addition to ALDH2 activation, Alda-44 prevents 4-HNE induced inactivation of ALDH2 by reducing the formation of 4-HNE-ALDH2 protein adducts. Thus, Alda-44 promotes metabolism of cytotoxic reactive aldehydes that accumulate in ischemic myocardium. Taken together, our findings suggest that direct activation of ALDH2 may represent a method of harnessing the cardioprotective effect of ethanol without the side effects associated with alcohol consumption.

Figure 1. Alda-44 selectively activates ALDH2 and protects against ALDH2 inactivation by 4-HNE

A) Recombinant ALDH2 activity (rate of NADH production at 340nm) was recorded in the absence and presence of 40µM Alda-44 B) Alda-44 (40µM) increased recombinant ALDH2 activity by 65% but was without effect on the related enzymes, ALDH3 and ALDH5. C) Inactivation of recombinant ALDH2 by 200µM 4-HNE was prevented by 40µM Alda-44. D) Western blot analysis of 4-HNE protein adducts on human recombinant ALDH2. Treatment with 40µM Alda-44 prevented 4-HNE adduct formation on ALDH2 induced by incubation with 200µM 4-HNE. Data are expressed as mean S.E.M, n=4, *p<0.05 vs. control.

Figure 2. Alda-44 activates ALDH2 in cardiac mitochondria isolated from WT and PKCε KO hearts

ALDH2 activity in purified mitochondria isolated from PKCε WT hearts (A) and PKCε KO hearts (B). C) Treatment with 40µM Alda-44 increased mitochondrial ALDH2 activity to a similar extent in WT and PKCε cardiac mitochondria, suggesting that PKCε is not required for Alda-44 activation of ALDH2. Data are expressed as mean±S.E.M, n=4, *p<0.05 vs. control.

Figure 3. Experimental protocols used for ischemia and reperfusion in isolated hearts

Hearts were exposed to 30 min global ischemia followed by 60 min of reperfusion or to normoxic perfusion for an equivalent duration. Ethanol (50mM) was applied for a period of 15 min, followed by 5 min washout, prior to ischemia. Alda-44 (40µM) was applied for 10 min prior to ischemia and for the first 10 min of reperfusion.

Figure 4. Effect of ethanol and Alda-44 on ischemia/reperfusion in WT and PKCε KO hearts

A) Representative sections of PKCε WT and PKCε KO hearts showing viable tissue (red) and necrotic tissue (white) after ischemia reperfusion, with and without treatment with 50mM ethanol or 40µM Alda-44. Ethanol reduced infarct size (B) and CK release (C) in WT but not PKCε KO hearts suggesting that ethanol preconditioning requires PKCε. Alda-44 reduced necrotic cell death in both WT and in PKCε KO hearts suggesting that Alda-44 confers protection in the absence of PKCε. Data expressed as mean±S.E.M, n=5, *p<0.05 vs. IR control.

Figure 5. Alda-44 preserves ALDH2 activity and reduces 4-HNE protein adducts induced by IR in WT and PKCε KO hearts

A) 4-HNE protein adducts in hearts exposed to normoxia, or IR injury, in the absence or presence of Alda-44. Treatment with Alda-44 reduced IR-induced increase in 4-HNE protein adducts in both WT and PKCε KO hearts B) ALDH2 activity in hearts exposed to normoxia or IR in the absence and presence of Alda-44. Treatment with Alda-44 prevented the IR-induced inhibition of ALDH2 in both WT and PKCε KO hearts. Data expressed as mean S.E.M., n=4, *p<0.05 vs. IR control.

Figure 6. Effect of Alda-44 on SAPK/JNK phosphorylation in PKCε WT and KO hearts

A) Western blot demonstrating absence of PKCε protein in PKCε KO hearts. B) Western blots of total and phosphorylated p46 and p54 SAPK/JNK and enolase in hearts exposed to normoxia or IR in the absence and presence of Alda-44. C) Quantification of p46 and p54 SAPK/JNK phosphorylation in hearts exposed to IR in the absence and presence of Alda-44. Data are expressed as mean±S.E.M, n=3, *p<0.05 vs. IR.

Figure 7. Schematic diagram of cardiprotection conferred by ALDH2

Ischemia/reperfusion results in ROS generation, leading to the accumulation of reactive aldehydes, such as 4-HNE, formed due to ROS-induced lipid peroxidation.4-HNE is oxidized by mitochondrial ALDH2 to the non-electrophilic metabolite, 4-HNA. However, at high concentrations, 4-HNE can also impair ALDH2 activity (red line). ALDH2 activity can be increased by ethanol preconditioning via PKCε-mediated phosphorylation. Alternatively, ALDH2 can be activated directly by Alda-44, which also prevents 4-HNE-induced inhibition of ALDH2 (green arrow). Thus, the mechanism of protection mediated by Alda-44 is likely to be due to a combination of increased ALDH2 activity and prevention of 4-HNE-mediated ALDH2 inactivation, resulting in reduced 4-HNE accumulation during ischemia/reperfusion.