CYP2J2 Regulation in Adult Ventricular Myocytes: Cell-wide Consequences and Effects on Stress Responses

Abstract

Cytochrome P450 2J2 (CYP2J2) is drug metabolizing member of the cytochrome P450 (CYP) superfamily. While CYPs involved in drug metabolism are generally highly expressed in “drug clearance” organs such as the small intestine, liver and kidneys, CYP2J2 is predominantly expressed in the heart. In ventricular myocytes, CYP2J2 is the primary arachidonic acid (AA) CYP epoxygenase and thus the primary source of the AA metabolites, the epoxyeicosatrienoic acids (EETs). EETs have been extensively investigated in disease settings and a large body of evidence suggests that EETs are cardioprotective against a wide array of cardiovascular conditions. While the exact mechanisms of this protection are largely unknown, EETs have been shown to act as signaling molecules, activating many pathways that prevent cellular damage. Given the critical functions of EETs in cardiac cells, CYP2J2 is an important enzyme to study, particularly in how various cellular and disease stressors affect the expression of this enzyme and ultimately, how these changes alter cardiomyocyte homeostasis. The overall aim of this dissertation was to investigate the regulation of the CYP2J2 gene in adult human ventricular myocytes under various stressors caused by pathophysiological conditions. Quantifying CYP2J2 mRNA and protein in cardiomyopathic ventricular tissues suggests that CYP2J2 levels are decreased as a result of cardiac disease, specifically in ischemia and diabetes. In vitro studies with ventricular myocytes confirm that hypoxia and type 2 diabetes downregulate CYP2J2 expression. Interestingly, increasing oxidative stress has the opposite effects, instead up-regulating CYP2J2. Focusing on the consequences of down-regulation, we inhibited CYP2J2 enzyme activity or silenced gene expression of CYP2J2 and then subjected ventricular myocytes to oxidative stress, hypoxia and human type 2 diabetic serum. Findings indicate that when this gene is silenced, cardiomyocytes become more susceptible to toxicity and death associated with these stresses. This increased sensitivity could be mitigated and reversed with the addition of external EETs. The in vitro cell work provide support for CYP2J2 as a cardioprotective enzyme via its metabolism of AA and EETs formation. Separately, the effects of CYP2J2 silencing on the transcriptome of ventricular myocytes was investigated using RNA-sequencing. Viability assays suggest that silencing CYP2J2 gene expression is not in of itself toxic to the cells. RNASeq results, however, indicate that there are cell-wide consequences to gene expression when downregulating CYP2J2. Many pathways are affected, including genes involved in maintaining and controlling ion channels and cell electrophysiology. These findings were further investigated and confirmed by patch clamp experiments. Pharmacological inhibition of CYP2J2 in ventricular myocytes have direct and immediate consequences on the action potential duration (APD). Further investigation suggests that the resulting aberrant APD is likely due to potassium channel current blockage or inhibition. In summary, CYP2J2 expression is adversely affected by cell stressors associated with disease states. The resulting downregulation limits the cardioprotection conferred by CYP2J2 to ventricular myocytes, likely through its bioactivation of AA to EETs, and makes cells more susceptible to toxicity by disease stressors. While the downregulation of CYP2J2 in ventricular myocytes does not seem to outwardly affect cardiomyocyte health by itself, without a secondary external stressor, there are many alterations in the cells’ internal signaling, with detrimental effects on cardiomyocyte health and function.