Author ORCID Identifier

https://orcid.org/0000-0001-9355-1976

Date Available

10-1-2025

Year of Publication

2025

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Medicine

Department/School/Program

Physiology

Faculty

Dr. Jonathan Satin

Abstract

Heart failure (HF) is prevalent and is predicted to increase with the aging population. Dilated cardiomyopathy (DCM) is a common cause of systolic HF. Current therapies fail to address principal issues: compromised systolic function and dysfunctional Ca2+ handling. No therapeutic approach exists for direct, positive modulation of cardiomyocyte L-type calcium channels (LTCC). RAD (Ras associated with diabetes) is a key regulator of the cardiac LTCC. RAD is a downstream phosphorylated target of the ß-adrenergic receptor (ß-AR)/cAMP/protein kinase A pathway; phosphorylation augments LTCC activity, contributing to chronotropy, inotropy, and lusitropy. RAD overexpression results in LTCC inhibition, whereas knockout positively modulates LTCC activity sympathomimetically. Cardiomyocyte-restricted, inducible deletion of RAD (cRADΔ/Δ) functions as a calcitrope—stably augmenting systolic function by modulating LTCC activity.

This work sought to test RAD knockdown’s efficacy as a positive inotrope in the context of systolic HF, thus unveiling a new approach of positive modulation of the LTCC. Using mouse and human models, the hypothesis that, after the onset of DCM, cRADΔ/Δ rescues heart dysfunction was tested. The muscle lim protein knockout mouse (MLPKO) is a model of DCM and HF, developing eccentric hypertrophy with compromised systolic function. cRADΔ/Δ was induced after onset of DCM (2.5 mo. of age). Cardiac imaging showed rescued systolic function and attenuated hypertrophy in mice 1- and 2-months post cRADΔ/Δ-induction. Electrophysiological recordings showed positive modulation of the LTCC balanced by enhanced calcium-dependent inactivation in cRADΔ/Δ-MLPKO. Live cell imaging revealed rescued Ca2+ handling and contractile function. In human systolic HF ventricular slices, RRAD knockdown recapitulated the inotropy and lusitropy observed in murine cardiomyocytes, suggesting translational potential. To explore RAD’s localization, mice with FLAG-tagged RAD were generated. We also tested the hypothesis that the C-terminus tail is necessary for membrane anchoring and LTCC regulation by generating C-terminus truncated FLAG-RAD mice. Full-length FLAG-RAD localized to t-tubules, whereas Flag-RADΔCT lacked membrane anchoring. Computational modelling corroborated microscopy, predicting a basic amphipathic helix interaction with the membrane. Electrophysiology cellular and in vivo studies showed positively modulated ICa,L and sympathomimetic systolic function. FLAG-RAD cells incubated with a ß-AR agonist showed reductions in t-tubular expression. Phosphorylation of C-terminal serines may destabilize RAD’s polybasic helix 8’s interaction with the membrane, contributing to release from the LTCC.

RRAD knockdown offers a new means of directly targeting cardiomyocyte LTCC in a highly specific manner. This study reveals the potential benefit of cardiomyocyte LTCC modulation that is direct—bypassing current calcitropes’ limitations of dependence on ß-AR/cAMP and lack of target specificity. Leveraging emerging in vivo gene manipulation technology, RRAD knockdown could be used as a cardiomyocyte-specific, positive LTCC modulator.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2025.13

Funding Information

  • National Institutes of Health Training Grant (no.: 5T32GM118292-05), 2021-2022
  • Department of Defense (no.: PR191210), 2021-2023
  • Department of Defense (no.: PR22074), in 2023

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