85. epigenetic silencing of abcb1 (p-glycoprotein) expression in cardiomyocytes as a potential mechanism of clozapine-induced myocarditis

Clozapine is the most effective antipsychotic medication for the management of treatment-resistant schizophrenia. However, the use of clozapine is limited due to severe and sometimes fatal adverse events, including inflammation in the heart (myocarditis). At present, there are no clinically useful markers for identifying those patients at greatest risk for myocarditis. Studies of clozapine dosing and genetic studies have not identified robust risk markers. This suggests that understudied epigenetic factors may be involved. The aim of our study is to investigate the potential epigenetic mechanism by which clozapine induces myocarditis using genome-wide profiling of DNA methylation, histone modifications, and RNA sequencing in a novel patient-derived in vitro model of clozapine-induced myocarditis. Peripheral mononuclear blood cells derived from patients with (cases) and without (controls) a history of clozapine-induced myocarditis were reprogrammed into induced pluripotent stem cells (iPSCs) and then differentiated into beating cardiomyocytes (iPSC-CMs). These cells were exposed to clozapine at a physiologically relevant concentration (2.8 µM) for 24 hours. Before and after clozapine treatment, RNA from the iPSC-CMs were sequenced (RNA-seq), DNA was assessed for methylation (EPIC array) and histone changes at H3K4me3 and H3K27ac (ChIP-seq), and cell media was assessed for proinflammatory cytokine levels. The RNA analysis showed gene expression of ABCB1, which encodes for an efflux drug transporter known as p-glycoprotein, was significantly increased in controls (fold change = 3.97, adjusted p-value = 0.00005) but not in cases (fold change = 0.002, adjusted p-value = 1) following treatment with clozapine, despite equivalently low expression levels prior to treatment in controls and cases. Triangulation of these results with the methylation and histone modification data showed that the H3K4me3 (known as an activation mark) modification was significantly associated with the ABCB1 increased gene expression in the controls compared to cases (p-value = 0.04), after clozapine exposure. Methylation patterns also indicated that the gene promoter was targeted by clozapine, resulting in DNA methylation loss, (cg02364454, p-value =0.007), corroborating with upregulation of ABCB1 expression found in RNA-seq data, and activation mark peaks found in ChIP-seq experiments. Furthermore, pro-inflammatory cytokines (IL-6, IL-8, MCP-1, and IL-23) were increased exclusively among the cases following clozapine treatment, in a clinically relevant concentration. Our findings suggest an epigenetic-mediated pharmacokinetic mechanism by which clozapine induces myocarditis. ABCB1 (p-glycoprotein) is a crucial transporter involved in the efflux of substrate drugs (e.g., clozapine) out of the cell. Importantly, evidence suggests that p-glycoprotein is the only efflux transporter expressed in cardiomyocytes and as such, perturbations to this transporter will impair the cell's ability to clear clozapine thereby increasing exposure and the probability of cardiotoxicity. Additional studies to validate our findings and further elucidate this potential mechanism are warranted.