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Interactome of the -subunit of L-type voltage-gated calcium channels in adult cardiomyocytes

ABSTRACT: In cardiomyocytes, Ca2+ influx through L-type voltage-gated calcium channels (LTCCs) following membrane depolarization regulates crucial Ca2+-dependent processes including duration and amplitude of the action potentials and excitation-contraction coupling. LTCCs are heteromultimeric proteins composed of the Cav1, Cav, Cav2 and Cav subunits. Here, using ascorbate peroxidase (APEX)-mediated proximity labeling and quantitative proteomics, we identified 61 proteins in the nano-environments of Cav2 in cardiomyocytes. These proteins are involved in diverse cellular functions such as cellular trafficking, muscular contraction, sarcomere organization and excitation-contraction coupling. Moreover, pull-down assays, co-immunoprecipitation analyses and super-resolution imaging using direct stochastic optical reconstruction microscopy (dSTORM) revealed that Cav2 interacts with the ryanodine receptor 2 (RyR2) in adult cardiomyocytes, probably coupling LTCCs and the RyR2 into a supramolecular complex at the dyads. This interaction is mediated by the Src-homology 3 domain of Cav2 and is necessary for an effective pacing frequency‐dependent increase of the Ca2+-induced Ca2+ release mechanism in cardiomyocytes.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Rattus Rattus (black Rat)

TISSUE(S): Specialized Cardiac Myocyte, Cell Culture

DISEASE(S): Disease Free

SUBMITTER: Katalin Barkovits

LAB HEAD: Dr. Erick Miranda Laferte

PROVIDER: PXD021375 | Pride | 2021-12-14

REPOSITORIES: Pride

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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.

			Action	DRS
	OEI26016_C1.mgf	Mgf
	OEI26016_C1.msf	Msf
	OEI26016_C1.mzid.gz	Mzid
	OEI26016_C1.raw	Raw
	OEI26049_B1.mgf	Mgf

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Interactome of the -subunit of L-type voltage-gated calcium channels in adult cardiomyocytes

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