Cardiac Radiotherapy-Induced Epigenetic Memory Underlies Electrophysiologic and Metabolic Reprogramming [RNA-seq]

Stereotactic arrhythmia radiotherapy (STAR) is emerging as a highly effective treatment for ventricular tachycardia (VT). Growing evidence indicates that STAR favorably reprograms the electrical substrate via modulating ion channel expression, though the mechanisms whereby single-fraction radiation mediates durable changes in gene expression are incompletely understood. The DNA damage response (DDR) recruits epigenetic regulators during the repair process after irradiation (IR). Here, we demonstrate acute changes in the cardiomyocyte epigenome and transcriptome after IR in vivo and in vitro. A subset of changes persist as late as 6 weeks post-IR in mice, including increased Scn5a expression and chromatin accessibility (encoding the alpha subunit of the sodium channel, NaV1.5), demonstrating a role for epigenetic memory in conduction velocity increases observed after STAR. Transcriptomic and epigenetic sequencing further identify dynamic changes to gene expression and regulatory regions involved in cellular repolarization, calcium handling, and metabolism after IR. Gene expression changes are mirrored by dose-dependent and cell-autonomous changes in repolarization, calcium flux, and mitochondrial respiration after IR, highlighting important cellular processes which may mediate both therapeutic and toxic effects of STAR. Cardiomyocyte-specific knockout of p53 failed to blut conduction velocity changes post-IR, and improved ROS scavenging via SOD2 overexpression had minimal impact on the post-IR transcriptome. Overall, we find that cardiomyocytes exposed to a single fraction of high-dose IR exhibit epigenetic reprogramming that mediates broad and dynamic physiologic responses. Overall design: RNAseq profiliing was performed in control and 25 Gy irradiated hiPSC-cardiomyocytes from two independent sources : (1) commercially available iCell Cardiomyocytes^2 (female) and (2) WTC11-AAV-CAG-GCaMP6 (GCaMP6) differentiated via small-molecule Wnt modulation (male). RNA was prepared at 1 day, 7 days, 14 days, or 28 days (GCaMP6 only) after treatment. RNAseq profiling was also performed in mice after whole-heart irradiation with 25 Gy or sham treatment. Left ventricle tissue was dissociated, nuclei were isolated, and flow cytometry was used to select PCM1+ cardiomyocyte nuclei prior to sequencing. Samples were collected from sham mice and 6 weeks post-IR. Myosin Heavy Chain6-Superoxide dismutase (Myh6-SOD) mice overexpresses SOD2 in cardiomyocytes, resulting in enhanced ROS scavenging. After exposuring Myh6-SOD and wild-type FVB mice to 25 Gy whole-heart IR or sham conditions, we performed bulk RNA sequencing at 2 days and 6 weeks post-IR.