Congratulations Garrett Elmore, PhD

On Friday, January 31, 2025 Garrett Elmore successfully defended his dissertation and earned his doctoral degree in physiology. Congratulations, Dr. Elmore!

RAD Reduction Reveals Therapeutic Potential of Targeting Cardiomyocyte L-type Calcium Channel Regulation for Heart Failure

Calcium is a second messenger integral to muscle contraction, neurotransmitter and hormone release, electrical autorhythmicity, cell growth and migration, and gene expression regulation. Voltage-gated calcium channels are integral to Ca²⁺ signaling; their (dys)function is key to neurological, hormonal, skeletal, and cardiovascular physiology and disease. While calcium channels blockers have been implemented in medicine, there are currently no positive modulators in use.

Heart failure (HF) is prevalent and is predicted to increase with the aging population. Dilated cardiomyopathy (DCM) is a common cause of heart failure with reduced ejection fraction. Current therapies for systolic HF fail to address principal issues: compromised systolic function and dysfunctional Ca²⁺ handling. Dysfunctional Ca²⁺ handling and compromised excitation-contraction drive hypocontractility and pathological remodeling in HF and DCM. Thus, attractive therapeutic targets are Ca²⁺ cycling proteins, such as the cardiac L-type calcium channel, Ca_v1.2. 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 has emerged as a key mediator of the b-adrenergic receptor (b-AR) signaling cascade that contributes to increased heart rate (chronotropy), contractile strength (inotropy), and rate of relaxation (lusitropy). RAD is a downstream phosphorylated target of the b-AR/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway, regulated by sympathetic tone. RAD overexpression results in LTCC inhibition whereas knockout positively modulates LTCC activity sympathomimetically. Cardiomyocyte-restricted, inducible deletion of RAD (cRAD^Δ/Δ) functions as a calcitrope—augmenting systolic function by modulating LTCC activity. 

This work sought to test RAD knockdown’s efficacy as a positive inotrope in the context of dilated cardiomyopathy and systolic heart failure, thus unveiling a new approach of positive modulation of the LTCC. Being downstream of b-AR/cAMP signaling, cRAD^Δ/Δ more directly targets the LTCC and bypasses other inotropes’ limitations of depending on b-AR/cAMP signaling. Using mouse and human models, the hypothesis that after the onset of dilated cardiomyopathy, inducible myocardial RAD deletion rescues heart dysfunction was tested. 

The muscle lim protein knockout mouse (MLPKO) is a model of dilated cardiomyopathy and HF, that by 2 months of age, shows eccentric hypertrophy with compromised systolic function. Cardiomyocytes display compromised Ca²⁺ cycling and sarcomere function, and transcriptomic analysis reveals pro-hypertrophic and pathological remodeling signaling typical of HF models as early as 3 weeks that degenerates overtime. The experimental timeline used in this study was to induce cRAD^Δ/Δ after onset of DCM (2.5 months of age) and follow subjects for up to 2 months. Cardiac imaging techniques were used to test the treatment effect of cRAD^Δ/Δ in a before and after manner. Pre-treatment, MLPKO mice recapitulated the DCM and cardiac dysfunction priorly reported. One- and two-months post-treatment, cRAD^Δ/Δ intervention rescued systolic function. To confirm the putative mechanism of action being positive modulation of the LTCC, electrophysiological recordings of isolated cardiomyocytes were performed and showed augmented LTCC activity yet enhanced calcium-dependent inactivation (as hastened current decay) in cRAD^Δ/Δ-MLPKO relative to MLPKO. Live cell imaging demonstrated rescue of cardiomyocyte dysfunctional Ca²⁺ handling and contractile function, displaying both positive inotropy and lusitropy. Bulk RNAseq of hearts demonstrated downregulated pathological signaling cascades and pro-hypertrophic gene expression which comported with the reduction in eccentric hypertrophy observed with cardiac imaging and histology. RRAD knockdown effects translate from mouse to human heart. Ventricle slices from systolic HF patients were treated with lentiviral shRNA targeting RRAD and recapitulated the inotropic effects observed in the mouse model of DCM. Induction of cardiomyocyte-specific RAD knockout in MLPKO mice after onset of DCM rescued cardiac dysfunction and attenuated pathological remodeling. cRAD^Δ/Δ intervention provided positive inotropy and reverted transcriptional signatures towards healthy myocardium. This study reveals a profound cRAD^Δ/Δ effect on excitation-contraction, however, beyond junctional dyadic LTCC, does RAD participate directly in excitation-transcription pathways? 

RAD’s localization and (diss)association to the LTCC is critical to its regulation of Ca²⁺ current. Studies suggest upon phosphorylation by protein kinase A, RAD dissociates from the LTCC local domain from its interaction with Ca_vß. Immortalized cell lines showed membrane and nuclear localizations while suggesting calmodulin and serine phosphorylation-status could potentially regulate translocation. To study localization and translocation, a Flag epitope was introduced to the N-terminus of the endogenous mouse Rrad gene. The polybasic amphipathic C-terminus tail has been shown to be important for membrane anchoring in immortalized cell lines. Full-length 3xFlag-RAD (Flag-RAD) mice were compared with a second transgenic mouse model, in which the C-terminus of 3xFlag-RAD was truncated at alanine 277 (Flag-RADΔCT). The primary hypotheses that in cardiomyocytes, the polybasic carboxyl-terminus of RAD confers t-tubular localization and membrane targeting for LTCC regulation were tested. Ventricular cardiomyocytes were isolated for anti-Flag-RAD immunocytochemistry and ex vivo electrophysiology. Full-length Flag-RAD demonstrated t-tubular expression whereas Flag-RADΔCT lacked apparent membrane localization. Computational modelling corroborated confocal and super resolution structured illumination microscopy, predicting a basic amphipathic helix interaction with the membrane. Recapitulating cRAD^Δ/Δ mouse models, Flag-RADΔCT LTCC current was sympathomimetically modulated, and mice displayed a positive inotropic effect in echocardiography study. To test b-AR/cAMP/PKA phosphorylation of RAD’s potential translocation, isoproterenol was applied to isolated ventricular cardiomyocytes. Of full-length Flag-RAD cells, no overt nuclear translocation was observed, although a reduction in t-tubular expression corroborated prior reported reductions in RAD-Ca_vß binding. In summary, the polybasic amphipathic motif in RAD’s predicted helix 8, affords membrane anchoring and is required for LTCC regulation. 

RAD knockdown offers a new means of directly targeting cardiomyocyte L-type calcium channels in a highly specific manner. This study reveals the potential benefit of cardiomyocyte LTCC modulation that is direct—bypassing current calcitropes’ limitation of depending on ß-AR/cAMP. Leveraging emerging in vivo gene manipulation technology, RAD knockdown could be used as a cell-specific positive LTCC modulator.