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A STICKY SITUATION: ESSENTIAL ROLES OF ADHESION G PROTEIN COUPLED RECEPTORS ADGRF5 AND ADGRG1 IN CARDIAC HOMEOSTASIS AND FAILURE
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Thesis/Dissertation
Date
2025-05
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Biomedical Sciences
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https://doi.org/10.34944/dfkz-jj45
Abstract
Rationale: Cardiovascular Disease (CVD) continues to be the leading cause of death both in the US and worldwide. This is due in part to the complex and diverse molecular causes of CVD, an aging population, and treatment strategies which address symptoms rather than the underlying molecular mechanisms that drive disease pathology. Therefore, there is a critical translational need to identify novel molecular targets which can be exploited for therapeutic potential. G-Protein Coupled Receptors (GPCRs) serve as pharmacological targets of more than 30% of all FDA-approved drugs, including key treatments for heart failure like β-blockers. GPCRs constitute the largest protein-coding superfamily in the human genome, with widespread tissue expression and vital roles in various functions, including sensory perception and immune response. However, a subgroup of GPCRs known as Adhesion GPCRs (AGPCRs) has yet to be explored for their pharmacological potential, representing a promising but underutilized area for drug development.
Objective: This thesis focuses on investigating roles of AGPCR family members ADGRF5 and ADGRG1 in cardiomyocytes, in both healthy and failing hearts, to identify novel targets which mediate cardiac pathophysiology.
Methods & Results: RNA sequencing was performed on healthy left ventricular tissue from adult C57BL/6 mice, allowing for the ranking of AGPCR family members by their expression levels. This analysis revealed ADGRF5 and ADGRG1 as novel targets of interest due to their high cardiac tissue expression. Further validation using previously published single-cell RNA sequencing data confirmed that both cardiac ADGRF5 and ADGRG1 expression is indeed specific to cardiomyocytes. To investigate their functional roles in the heart, cardiomyocyte-specific knockout mouse models were developed using the αMHC promoter to induce the genetic deletion of ADGRF5 or ADGRG1 through the Cre-LoxP recombination system.
First, cardiomyocyte-specific genetic deletion of ADGRG1 initially did not impact cardiac structure or function. However, these mice gradually developed increased systolic and diastolic left ventricular (LV) volumes and internal diameters over time. Importantly, when subjected to chronic pressure overload, the deletion of ADGRG1 in CMs accelerated the onset of cardiac dysfunction, accompanied by reduced CM hypertrophy, heightened cardiac inflammation, and increased mortality. These findings suggest that ADGRG1 plays a crucial role in the early adaptation to chronic cardiac stress. Overall, this study provided a compelling proof-of-concept that targeting CM-expressed AGPCRs may present a novel therapeutic strategy for managing the progression of heart failure.
In our next study, cardiomyocyte-specific genetic deletion of ADGRF5 did not initially alter cardiac structure or function. However, as these mice aged, we observed notable differences in cardiac remodeling, including increased fibrosis, myocyte hypertrophy, and higher mortality rates. Additionally, the knockout mice exhibited an increased incidence of premature ventricular contractions, which in some animals led to fatal ventricular arrhythmias. To investigate the underlying molecular mechanisms of these changes, bulk RNA sequencing was performed on left ventricular tissue from both knockout mice and their littermates, revealing 58 differentially expressed genes. Validation of these differentially expressed targets was confirmed in isolated adult mouse cardiomyocytes (AMCMs) versus controls and revealed upregulation of the various fetal cardiac genes, including ion channel subunits Scn1b, Cacnah1, and Hcn2 as well as hypertrophic genes, Anp and Bnp. Concurrent with these findings, in vitro studies using (NRVMs) established that indeed, upon increasing infection of adenovirus encoding ADGRF5 resulted in step wise decreases in expression of these genes. Additionally, using this in vitro system, we identified that ADGRF5 couples with Gαq/11-mediated signaling in healthy myocytes and with the use of a Gq/11 inhibitor, expression of these genes is restored, suggesting that ADGRF5 intracellular signaling contributes to their expression. Further mechanistic studies using Givinostat, a pan-HDAC inhibitor, revealed that these gene expression changes occurred in an HDAC sensitive matter. Additionally, when knockout mice and controls were interrogated via transverse aortic constriction, a model for pressure-overload induced HF in vivo, knockout mice possessed a slight acceleration toward functional cardiac failure and accelerated myocyte growth which coincided with enhanced expression of Nppa and Nppb gene expression. Conversely, by directing overexpression of ADGRF5-CTF to myocytes specifically via AAV9-cTnT-ADGRF5-CTF-Flag vs LacZ delivery, injected retro-orbitally 3-weeks post TAC-induction, revealed that ADGRF5 prevented the onset of cardiac failure in wild type mice. Taken together, this study reveals that ADGRF5 maintains cardiac homeostasis through repression of harmful developmental cardiac genes, thus repressing hypertrophic gene activation and electrophysiological imbalance.
Conclusion: In conclusion, this work displays the importance of uninvestigated AGPCRs and their critical (patho)physiological contributions. Additionally, AGPCRs may offer a new avenue for the development of CVD therapeutics.
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