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Abstract P3137: The C-terminus Of Rad Is Required For Regulation Of The Cardiac Ca V 1.2 Channel

Abstract only Background: Heart failure with reduced ejection fraction (HFrEF) is a leading cause of hospitalization and death. Current therapies fail to address loss of contractile force. Targeting dysfunctional Ca 2+ handling proteins involved in excitation-contraction, such as Ca v 1.2 may offer...

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Published in:Circulation research 2023-08, Vol.133 (Suppl_1)
Main Authors: Elmore, Garrett, Ahern, Brooke M, Sebastian, Andrea, Lohano, Sarisha, McVay, Nicholas, Levitan, Bryana, Dupont, Alec, Andres, Doug A, Satin, Jonathan
Format: Article
Language:English
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Summary:Abstract only Background: Heart failure with reduced ejection fraction (HFrEF) is a leading cause of hospitalization and death. Current therapies fail to address loss of contractile force. Targeting dysfunctional Ca 2+ handling proteins involved in excitation-contraction, such as Ca v 1.2 may offer a means to improve quality of life and attenuate adverse cardiac remodeling. RAD is a key target bridging sympathetic modulation of Ca v 1.2 function. During stress, the beta-adrenergic (β-AR) effector, protein kinase A (PKA), phosphorylates RAD, modulating Ca v 1.2 current (I Ca,L ) and increasing cardiac contraction. RAD inhibits current, whereas Rad-knockout phenocopies a β-AR/PKA phosphorylated modulated current. Across RGK family members the highly conserved C-terminus is necessary for membrane association and block of I Ca,L in immortalized cell lines. The RAD C-terminus significance is unknown in adult mammalian cardiomyocytes. Hypothesis: RAD lacking a C-terminus will be unable to localize to surface membrane Ca v 1.2 channels; thus Ca v 1.2 activity will be modulated. Methods and Results: A 3xFLAG epitope was added to the N-terminus of the endogenous mouse Rrad gene. Full-length FLAG-RAD mice were compared to mice with truncated RAD (A277X). Ventricular cardiomyocytes were isolated for immunocytochemistry and ex vivo electrophysiology. Confocal microscopy was used to visualize localization of FLAG-tagged RAD. Full-length FLAG-RAD showed a repeating t-tubular pattern that co-localizes with Ca v 1.2 staining whereas FLAG-RAD A277X failed to show this pattern. A277X I Ca,L showed significantly hyperpolarized activation midpoint (-17 mV) and 2-fold increase in maximal conductance. Additionally, current decay was faster in A277X showing a significant 2-fold decrease in the current 30 ms after peak current. Conclusions: Myocardial Ca v 1.2 channels in a RAD C-terminal deletion model phenocopy Ca v 1.2 function modulated in response to β-AR stimulation. Mechanistically, the polybasic RAD C-terminus confers Ca v 1.2 regulation via membrane association of RAD. Interfering with RAD at the C-terminus residues constitute a specific target for boosting heart function as a treatment for HFrEF.
ISSN:0009-7330
1524-4571
DOI:10.1161/res.133.suppl_1.P3137