Myocardial-restricted ablation of the GTPase RAD results in a pro-adaptive heart response in mice

Abstract

Existing therapies to improve heart function target β-adrenergic receptor (β-AR) signaling and Ca2+ handling and often lead to adverse outcomes. This underscores an unmet need for positive inotropes that improve heart function without any adverse effects. The GTPase Ras associated with diabetes (RAD) regulates L-type Ca2+ channel (LTCC) current (ICa,L). Global RAD-knockout mice (gRAD−/−) have elevated Ca2+ handling and increased cardiac hypertrophy, but RAD is expressed also in noncardiac tissues, suggesting the possibility that pathological remodeling is due also to noncardiac effects. Here, we engineered a myocardial-restricted inducible RAD-knockout mouse (RADΔ/Δ). Using an array of methods and techniques, including single-cell electrophysiological and calcium transient recordings, echocardiography, and radiotelemetry monitoring, we found that RAD deficiency results in a sustained increase of inotropy without structural or functional remodeling of the heart. ICa,L was significantly increased, with RAD loss conferring a β-AR–modulated phenotype on basal ICa,L. Cardiomyocytes from RADΔ/Δ hearts exhibited enhanced cytosolic Ca2+ handling, increased contractile function, elevated sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2a) expression, and faster lusitropy. These results argue that myocardial RAD ablation promotes a beneficial elevation in Ca2+ dynamics, which would obviate a need for increased β-AR signaling to improve cardiac function.

Reconstitution of β-adrenergic regulation of Ca V 1.2: Rad-dependent and Rad-independent protein kinase A mechanisms

“…Rad tonically inhibits Ca V 1.2, largely via an interaction with Ca V β (31,32). Ablation of Rad in murine heart was shown to increase basal Ca V 1.2 activity and rendered the channel insensitive to β-AR regulation, probably through a "ceiling" effect (33,34). Liu et al (20) reconstituted a major part of the Ca V 1.2 regulation cascade, initiated by forskolinactivated adenylyl cyclase in mammalian cells, ultimately attaining an approximately twofold increase in Ca 2+ current.…”

Adult spiny mice (Acomys) exhibit endogenous cardiac recovery in response to myocardial infarction

“…Ventricular cardiomyocytes were isolated using the Langendorff method as previously described 23 and are further detailed in the supplementary methods. Images used to analyze cardiomyocyte surface area and nucleation were acquired using an Olympus IX-71 and Olympus BX53 microscopes (Olympus, Tokyo, Japan) at 10x magnification.…”

“…I Ca,L was recorded from freshly isolated cardiomyocytes using the whole-cell configuration of the patch clamp technique, and cytosolic Ca 2+ transients were collected using fura2-AM as previously described. 23…”

“…Ventricular cardiomyocytes were isolated as previously described. 19 Briefly, mice received an i.p. injection of heparin (0.3ml of 1000 units/ml) prior to sacrifice.…”

“…ICa,L was recorded in the whole-cell configuration of the patch clamp technique as previously described. 19 All recordings were performed at room temperature (…”