Sun C. 20-HETE increases NADPH oxidase-derived ROS production and stimulates the L-type Ca 2ϩ channel via a PKCdependent mechanism in cardiomyocytes. Am J Physiol Heart Circ Physiol 299: H1109 -H1117, 2010. First published July 30, 2010; doi:10.1152/ajpheart.00067.2010.-The production of 20-hydroxyeicosatetraenoic acid (20-HETE) is increased during ischemia-reperfusion, and inhibition of 20-HETE production has been shown to reduce infarct size caused by ischemia. This study was aimed to discover the molecular mechanism underlying the action of 20-HETE in cardiac myocytes. The effect of 20-HETE on L-type Ca 2ϩ currents (ICa,L) was examined in rat isolated cardiomyocytes by patch-clamp recording in the whole cell mode. Superfusion of cardiomyocytes with 20-HETE (10 -100 nM) resulted in a concentration-dependent increase in I Ca,L, and this action of 20-HETE was attenuated by a specific NADPH oxidase inhibitor, gp91ds-tat (5 M), or a superoxide scavenger, polyethylene glycol-superoxide dismutase (25 U/ml), suggesting that NADPH-oxidase-derived superoxide is involved in the stimulatory action of 20-HETE on ICa,L. Treatment of cardiomyocytes with 20-HETE (100 nM) increased both NADPH oxidase activity and superoxide production by approximately twofold. To study the molecular mechanism mediating the 20-HETE-induced increase in NADPH oxidase activity, PKC activity was measured in cardiomyocytes. Incubation of the cells with 20-HETE (100 nM) significantly increased PKC activity, and pretreatment of cardiomyocytes with a selective PKC inhibitor, GF-109203 (1 M), attenuated the 20-HETE-induced increases in I Ca,L and in NADPH oxidase activity. In summary, 20-HETE stimulates NADPH oxidase-derived superoxide production, which activates L-type Ca 2ϩ channels via a PKC-dependent mechanism in cardiomyocytes. 20-HETE and 20-HETE-producing enzymes could be novel targets for the treatment of cardiac ischemic diseases.20-hydroxyeicosatetraenoic acid; L-type calcium channel; protein kinase C; cardiac myocytes; reactive oxygen species 20-HYDROXYEICOSATETRAENOIC ACID (20-HETE) is a lipid metabolite of arachidonic acid that is produced by -hydroxylase enzymes of the cytochrome P-450 (CYP)4A and CYP4F families, which are relatively abundant and exert regulatory functions dependent on the tissue (18). For example, in the kidney, 20-HETE regulates renal functions, such as renal vascular tone, tubuloglomerular feedback, autoregulation of renal blood flow, tubular transport, and mitogenesis (20). In blood vessels, 20-HETE is a potent vasoconstrictor that activates L-type Ca 2ϩ channels and inhibits Ca 2ϩ -sensitive K ϩ channels in vascular smooth muscle cells (28,29). In pulmonary arteries, 20-HETE enhances NADPH oxidase-dependent production of ROS in endothelial cells (21). Thus, it was proposed that 20-HETE plays an important role in the control of apoptosis and angiogensis in vascular endothelial cells in the pulmonary microcirculation (13). Recently, 20-HETE and CYP -hydroxylase were also identified in hearts from the rat and dog (13,26...

Background: It has been reported that 20-hydroxyeicosatetraenoic acid (20-HETE) aggravates myocardial ischemia/reperfusion (I/R) injury, but the exact mechanism of action is still unclear. Methods and Results:Experiments were performed in isolated rat hearts subjected to 35 min of ischemia followed by 40 min of reperfusion in Langendorff preparations. Perfusion with HET0016, an inhibitor of 20-HETE production, significantly improved I/R-induced reduction in cardiac contractility, myocardial infarction, and myocardial apoptosis. In contrast, administration of 20-HETE aggravated I/R-induced myocardial injury and enhanced apoptosis. I/R significantly increased production of reactive oxygen species (ROS) and oxidative stress, both of which were significantly inhibited by HET0016 and enhanced by 20-HETE administration. Apocynin, an inhibitor of NADPH oxidase, blocked 20-HETE-induced ROS production in the I/R hearts. 20-HETE increased the expression of gp91 phox and p22 phox , the subunits of NADPH oxidase; and stimulated NADPH oxidase activity. In addition, GF-109203 significantly attenuated the 20-HETE-induced increases in the NADPH oxidase expression and activity. Finally, in the Langendorff I/R preparation, both apocynin and tempol, ROS scavengers, significantly blocked 20-HETE-induced myocardial dysfunction. Conclusions:All of the results demonstrated that in isolated rat hearts 20-HETE stimulates NADPH oxidase-derived superoxide production, which aggravates I/R-induced myocardial injury via a PKC-dependent mechanism. (Circ J 2013; 77: 1807 -1816

Cardiomyocyte apoptosis is involved in a variety of cardiac stresses, including ischemia-reperfusion injury, heart failure, and cardiomyopathy. Both Angiotensin II (Ang II) and 20-hydroxyeicosatetraenoic acid (20-HETE) induce apoptosis in cardiomyocytes. Here, we examined the relationship between 20-HETE and Ang II in cardiomyocyte apoptosis. Apoptosis was examined using flow cytometry in primary cultured rat cardiomyocytes treated with control, Ang II, and Ang II plus HET0016 (a 20-HETE formation inhibitor). The results demonstrated that the treatment of cardiomyocytes with Ang II or 20-HETE significantly increased the percentage of apoptotic cells and that Ang II-induced apoptosis was markedly attenuated by HET0016 or losartan (an AT1 receptor antagonist). In apoptotic mechanism experiments, Ang II or 20-HETE treatment significantly reduced mitochondrial membrane potential, indicating that a mitochondria-dependent mechanism is involved. Ang II-induced alteration in mitochondrial membrane potential was significantly attenuated by HET0016. Treatment of cardiomyocytes with Ang II also increased superoxide production, and this effect of Ang II was attenuated by HET0016. Treatment of cardiomyocytes with Ang II significantly increased CYP4A1 expression and 20-HETE production, as measured by Western blot, real-time RT-PCR, and mass spectrometric analysis. All results suggest that 20-HETE may play a key role in Ang II-induced apoptosis in cardiomyocytes by a mitochondrial superoxide-dependent pathway.

Cardiomyocyte apoptosis has been documented involved in a variety of cardiac stresses, including ischemia-reperfusion injury, heart failure, and cardiomyopathy. Both angiotensin II (Ang II) and 20-Hydroxyeicosatetraenoic Acid (20-HETE), a hydroxylation product of arachidonic acid catalyzed by CYP450-ω hydroxylase, induce apoptosis in cardiomyocyte. However, the crosstalk between 20-HETE and Ang II in cardiomyocytes apoptosis process is unclear. In the current study, we examined apoptosis using flow cytometry in primary cultured neonatal rat ventricular myocytes treated with control, Ang II (100nM), Ang II plus HET0016 (10μM), HET0016, or 20-HETE (10nM) along. The results demonstrated that treatment with Ang II or 20-HETE significantly increased apoptosis and that Ang II-induced apoptosis were markedly attenuated by HET0016, a 20-HETE agonist. In addition, Ang II-induced increases of caspase-3 activity were significantly attenuated by 20.9±3.4% after co-treatment with HET0016. Our results also demonstrated that HET0016 significantly suppressed Ang II-induced increases of superoxide production by 27.52.3% and mitochondrial membrane potential by 64.5±6.3%. Finally, Ang II-induced nuclei crenation, chromatin condensation and fractionation were attenuated by 73.6±8.5% with HET0016 pre-treatment. In addition, treatment cardiomyocytes with Ang II increased CYP4A1 expression and 20-HETE production, measured by Western blot, real-time RT-PCR, and mass spectrometric analysis. All results suggest that 20-HETE may play a key role in Ang II-induced apoptosis in cardiomyocytes. 20-HETE and 20-HETE-producing enzymes could be novel targets for the treatment of Ang II related cardiac diseases.