Abstract P3072: Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (iPSC-CMs) Respond To Inflammatory Stimuli Through Innate Immune Responses

Abstract

Introduction: Cells respond to inflammatory stimuli in settings of sterile and pathologic cardiac inflammation. Nuclear factor Kappa B (NFKB) is a transcriptional mediator of innate immune responses. Lipopolysaccharide (LPS) promotes NFKB activation through toll-like receptors (TLR) to initiate cell autonomous processes that resist infection. High-mobility box group 1 (HMGB1) is a chromatin associated protein released upon nuclear rupture that similarly promotes cytokine release in response to injury. Few studies have interrogated multiple inflammatory stimuli and their impacts on iPSC-CM function. We examined innate immune activation using distinct TLR agonists and assessed functional responses in both 2D iPSC-CM monoculture and 3D collagen-based engineered heart tissues. Methods and Results: We measured electric field potential duration (Nanion CardioExcyte 96) of iPSC-CM monolayers after exposure to LPS and HMGB1. We observed a four fold increase in downstroke velocity after exposure to HMGB1 (512.7±126.9 V/s; p=0.0002 n=12) and a 30% decrease in downstroke velocity after LPS treatment (-188.9±61.4 V/s; p=0.004 n=12). This response was associated with nuclear localization of phosphorylated NFKB. Collagen-based engineered heart tissues demonstrated contractile defects in response to 48h of exposure to TLR agonists. A 50% reduction in fractional shortening was observed following HMGB1 treatment (-0.0060±0.0009; p<0.0001 n=6) and an 80% reduction was associated with LPS exposure (-0.0088±0.0006; p<0.0001 n=6). To identify gene expression regulatory regions associated with this response, we examined transcriptional activity of three candidate enhancer regions implicated in cytokine production in iPSC-CMs. These regions demonstrated a 0.5- to 20-fold increase in enhancer activity in iPSC-CMs. One enhancer demonstrated a 22% increase in activity following 48h of LPS, consistent with a direct role for NFKB in innate immune activation in human iPSC-CMs; this enhancer regulates a gene with increased expression in dilated cardiomyopathy. Conclusion: These data indicate that cardiomyocytes respond directly to inflammatory stimuli and demonstrate the utility of 2D and 3D human tissue culture models for cardiac inflammation.