Abstract P3050: Cpne5 Is Necessary For Normal Murine Sinoatrial Node Function In Vivo

Abstract

Cpne5 ( Copine 5 ) is a Ca 2+ -dependent phospholipid binding protein implicated in membrane trafficking but has no known role within the heart to date. In single-cell RNA-sequencing of the developing mouse heart, we previously found Cpne5 gene expression to be highly enriched in the cardiac conduction system (CCS). Further, variants in CPNE5 were identified in 6 independent GWAS related to heart rate variability in humans. We therefore hypothesized that Cpne5 is necessary for normal CCS function through Ca 2+ -dependent membrane trafficking of ion channels. Whole mount immunostaining with 3D volumetric imaging (iDISCO+) of intact WT hearts demonstrated robust and specific protein expression of Cpne5 throughout all CCS components, including the sinoatrial node (SAN) and SAN-transitional cells. Next, to assess the necessity of Cpne5 within the CCS, systemic knockout mice (Cpne5KO) were generated using CRISPR/Cas9. Successful deletion of Cpne5 in CRISPR-generated KO mice was confirmed by PCR and, at the protein level, immunofluorescence. Homozygous Cpne5KO mice were viable, showed normal growth and allele frequencies segregated in a normal Mendelian fashion. No gross anatomic defects of the CCS were observed in Cpne5KO mice and transthoracic echocardiogram studies showed a slight reduction in left ventricular fractional shortening but otherwise a structurally normal heart. Notably, while surface electrocardiogram (ECG) demonstrated normal intervals (PR, QRS, QTc), Cpne5KO mice exhibited pronounced sinus arrhythmia, with notable sinus pauses, sinus bradycardia, and increased heart rate variability as compared to WT littermate controls on long term telemetry. Systemic loss-of-function of Cpne5 in mice resulted in marked sinus node dysfunction, consistent with human GWAS data, implicating a novel role for Cpne5 in CCS function and/or development. Additional phenotyping and molecular studies, including CPNE5 deletion in human induced-pluripotent cell-derived SAN-like cells, are ongoing to further elucidate its underlying mechanism.