β-adrenergic Receptor-stimulated Cardiac Myocyte Apoptosis: Role of Cytochrome P450 ω-hydroxylase

Prolonged or excessive beta-adrenergic activation leads to cardiac myocyte loss and heart dysfunction; however, the underlying cellular mechanisms are still unclear. Therefore, we first confirmed the effect of isoproterenol (ISO), a beta-adrenergic receptor agonist, on cardiac toxicity using TUNEL and caspase activity assays in cultured rat cardiomyocytes. ISO treatment significantly increased cardiomyocyte apoptosis. Persistent ISO stimulation of cardiomyocytes also increased the expression of CYP4A3, a major CYP450 v-hydroxylase that produces 20-hydroxyeicosatetraenoic acid (20HETE) in a time-dependent manner. Next, we examined the effect of ISO and 20-HETE on cardiomyocyte apoptosis using annexin V and propidium iodide staining. Treatment with either 20-HETE or ISO significantly increased cardiomyocyte apoptosis, and inhibition of 20-HETE production using 17-ODYA, a CYP450 omega-hydroxylase inhibitor, dramatically attenuated ISO-induced cardiomyocyte apoptosis. To determine the apoptotic pathway involved, the mitochondrial membrane potential (Delta Psi m) was measured by detecting the ratio of JC-1 green/red emission intensity. The results demonstrated that 17-ODYA significantly abolished ISO-induced disruption of Delta Psi m and that 20-HETE alone induced a marked disruptive effect on Delta Psi m in cardiomyocytes. In addition, 20-HETE-induced disruption of Delta Psi m and apoptosis was significantly attenuated by KN93, a CaMKII inhibitor. Taken together, these results demonstrate that 20-HETE treatment induces significant apoptosis via mitochondrial-dependent pathways, and that inhibition of 20-HETE production using 17-ODYA attenuates ISO-induced cardiomyocyte apoptosis.