Abstract 807: Rad Ablation as a Treatment to Target Cardiac Inotropy via L-type Calcium Channel Function

Abstract

The L-type Ca 2+ channel current (I Ca,L ) provides trigger Ca 2+ to contribute to cardiac contraction. Rad GTPase associates with the L-type Ca 2+ channel (LTCC) and serves as an endogenous inhibitor of LTCC activity. Overexpression of Rad blocks I Ca,L ; absence of Rad increases I Ca,L . Rad attenuates β-adrenergic receptor (β-AR) signaling. Chronic β-AR stimulation associates with Ca 2+ mishandling and can promote signaling that progresses towards heart failure. Early studies of global, constitutive Rad-knockout mice (gRadKO) suggested that elevated Ca 2+ dynamics leads to pathological cardiac hypertrophy; however, Rad is also expressed in non-cardiac tissues. Our objective is to test the hypothesis that increased myocardial I Ca,L via Rad deletion safely enhances cardiac function without driving pathological remodeling. We created a cardiac-restricted inducible Rad knockout mouse (Rad Δ/Δ ). In vivo function was measured with echocardiography. We examined I Ca,L through whole cell configuration of the patch clamp technique, assessed Ca 2+ handling, and sarcomere dynamics. Unlike gRadKO, Rad Δ/Δ showed no elevation of fetal gene program, nor fibrosis, and no change to aortic pressure. Rad Δ/Δ had a sustained increase of inotropy without structural or functional remodeling (EF: Rad Δ/Δ =76 ± 2%, n=16; Rad fl/fl =59 ± 4%, n=7; p=0.001.) I Ca,L was significantly increased, with Rad loss mirroring a β-AR modulated phenotype on basal I Ca,L (max. conductance: Rad Δ/Δ =254 ± 19 pS/pF, n=15; Rad fl/fl =144 ± 12 pS/pF, n=18; p<10 -4 ). Contrary to models of chronic β-AR stimulation, Rad Δ/Δ retained β-AR signaling shown in vivo using isoproterenol, and by preserved phosphorylation of protein regulators of Ca 2+ reuptake and contractility. Rad Δ/Δ cardiomyocytes show enhanced cytosolic Ca 2+ handling (Decay of Ca 2+ transient: Rad Δ/Δ = 0.07 ± 0.003 (F 340 /F 380 )/s, n=67, Rad fl/fl = 0.10 ± 0.005, n=69; p<10 -4 ), increased contractile function, and elevated SERCA2a expression. These new findings challenge the canonical assumption that increased myocardial Ca 2+ necessarily promotes pathology. We conclude that cardiac hypertrophy in gRadKO was caused by non-cardiac tissue effects, and myocardial Rad deletion is a promising cardiac inotropic therapeutic direction.