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Hypertrophic Cardiomyopathy-Associated CRYABR123W Activates Calcineurin, Reduces Calcium Sequestration, Alters the CRYAB Interactome and the Proteomic Response to Pathological Hypertrophy

ABSTRACT: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular condition in the world affecting around 1:500 people. HCM is characterized by ventricular wall thickening, decreased ventricular chamber volume and diastolic dysfunction. Inherited HCM is most commonly caused by sarcomere gene mutations, however approximately 50% of patients do not present with a known mutation highlighting the need for further research into additional pathologic mutations. CRYABR123W was previously identified as a novel sarcomere-independent mutation causing HCM associated with pathologic NFAT signaling in the setting of pressure overload. We generated stable H9C2 cell lines expressing FLAG tagged wild type and mutant CRYAB, which demonstrated that CRYABR123W has increased calcineurin activity. Using Alphafold to predict structural and interaction changes we generated a model where CRYABR123W uniquely binds to the autoinhibitory domain of calcineurin. Co-immunoprecipitation using the CRYAB FLAG tag fol-lowed by mass spectrometry showed novel and distinct changes in the protein interaction patterns of CRYABR123W. Finally, mouse hearts extracted from our wild type and CRYABR123W model with and without pressure overload caused by transverse aortic constriction (TAC) were used in global proteomic and phosphoproteomic mass spectrometry analysis which showed dysregulation in cytoskeletal, metabolomic, cardiac and immune function. Our data illustrates how CRYABR123W drives calcineurin activation and exhibits distinct changes in protein interaction and cellular pathways during the development of HCM and pathological cardiac hypertrophy.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Michael T. Chin

PROVIDER: MSV000097115 | MassIVE | Fri Feb 14 06:49:00 GMT 2025

REPOSITORIES: MassIVE

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Project description:Aims: Cardiac hypertrophy is a compensatory response to pressure overload, leading to heart failure. Recent studies have demonstrated that Rho is immediately activated in left ventricles after pressure overload, and that Rho signaling plays crucial regulatory roles in actin cytoskeleton rearrangement during cardiac hypertrophic responses. However, the mechanisms by which Rho and its downstream proteins control actin dynamics during hypertrophic responses remain not fully understood. In this study, we identified the pivotal roles of mammalian homologue of Drosophila diaphanous (mDia) 1, a Rho-effector molecule, in pressure overload-induced ventricular hypertrophy. Methods and Results: Male wild-type (WT) and mDia1-knockout (mDia1KO) mice (10–12 weeks old) were subjected to a transverse aortic constriction (TAC) or sham operation. The heart weight/tibia length ratio, cardiomyocyte cross-sectional area, left ventricular wall thickness, and expression of hypertrophy-specific genes were significantly decreased in mDia1KO mice 3 weeks after TAC, and the mortality rate was higher at 12 weeks. Echocardiography indicated that mDia1 deletion increased the severity of heart failure 8 weeks after TAC. Importantly, we could not observe apparent defects in cardiac hypertrophic responses in mDia3-knockout mice. Microarray analysis revealed that mDia1 was involved in the induction of hypertrophy related genes, including immediate early genes (IEGs), in pressure overloaded hearts. Loss of mDia1 attenuated activation of the mechanotransduction pathway in TAC-operated mice hearts. We also found that mDia1 was involved in stretch-induced activation of the mechanotransduction pathway and gene expression of c-fos in neonatal rat ventricular cardiomyocytes (NRVMs). mDia1 regulated the F/G-actin ratio in response to pressure overload in mice. Additionally, increases in nuclear myocardin-related transcription factors (MRTFs) and serum response factor (SRF) were perturbed in response to pressure overload in mDia1KO mice and to mechanical stretch in mDia1 depleted NRVMs. Conclusions: mDia1, through actin dynamics, is involved in compensatory cardiac hypertrophy in response to pressure overload.

Project description:BACKGROUND: Cardiac hypertrophy compensates for increased biomechanical stress of the heart induced by prevalent cardiovascular pathologies, but can result in cardiac failure if left untreated. We hypothesized that the tail-anchored protein Dysferlin with multiple Ca2+-binding C2-domains is critical for the integrity of the transverse-axial tubule (TAT) network inside cardiomyocytes, and contributes to the proliferation of TAT endomembranes during pressure overload-induced cardiac hypertrophy. OBJECTIVE: To reveal the impact of the membrane fusion and repair protein Dysferlin on TAT network stabilization and proliferation necessary for the hypertrophic growth of cardiomyocytes. METHODS AND RESULTS: Super-resolution light and electron microscopy of mouse cardiomyocytes identified a specific localization of Dysferlin in a vesicular compartment in nanometric proximity to contact sites of the TAT network with the sarcoplasmic reticulum (SR), a.k.a. junctional complexes for Ca2+-induced Ca2+ release. Mass spectrometry was used to characterize the cardiac Dysferlin interactome, thereby identifying a novel protein interaction with the membrane-tethering SR protein Juncophilin-2, a known interactor of L-type Ca2+ channels and Ryanodine Receptor Ca2+ release channels in junctional complexes. While the Dysferlin knockout caused a mild progressive phenotype of dilated cardiomyopathy in the mouse heart, global proteome analysis revealed changes preceding systolic failure. Following transverse aortic constriction (TAC), Dysferlin protein expression was significantly increased in hypertrophied wild-type myocardium, while Dysferlin knockout animals presented markedly reduced left-ventricular hypertrophy. Live-cell membrane imaging demonstrated a profound reorganization of the TAT network in wild-type left-ventricular myocytes post-TAC with robust proliferation of axial tubules, which critically depended on the increased expression of Dysferlin within newly-emerging tubule components. CONCLUSIONS: Dysferlin represents a new molecular target in cardiac disease that protects the integrity of tubule-SR junctional complexes for regulated excitation-contraction coupling, and controls TAT network reorganization and tubular membrane proliferation in cardiomyocyte hypertrophy induced by pressure-overload.

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Hypertrophic Cardiomyopathy-Associated CRYABR123W Activates Calcineurin, Reduces Calcium Sequestration, Alters the CRYAB Interactome and the Proteomic Response to Pathological Hypertrophy

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