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Location and function of transient receptor potential canonical channel 1 in ventricular myocytes.

ABSTRACT: Transient receptor potential canonical 1 (TRPC1) protein is abundantly expressed in cardiomyocytes. While TRPC1 is supposed to be critically involved in cardiac hypertrophy, its physiological role in cardiomyocytes is poorly understood. We investigated the subcellular location of TRPC1 and its contribution to Ca²⁺ signaling in mammalian ventricular myocytes. Immunolabeling, three-dimensional scanning confocal microscopy and quantitative colocalization analysis revealed an abundant intracellular location of TRPC1 in neonatal rat ventricular myocytes (NRVMs) and adult rabbit ventricular myocytes. TRPC1 was colocalized with intracellular proteins including sarco/endoplasmic reticulum Ca²⁺ ATPase 2 in the sarcoplasmic reticulum (SR). Colocalization with wheat germ agglutinin, which labels the glycocalyx and thus marks the sarcolemma including the transverse tubular system, was low. Super-resolution and immunoelectron microscopy supported the intracellular location of TRPC1. We investigated Ca²⁺ signaling in NRVMs after adenoviral TRPC1 overexpression or silencing. In NRVMs bathed in Na⁺ and Ca²⁺ free solution, TRPC1 overexpression and silencing was associated with a decreased and increased SR Ca²⁺ content, respectively. In isolated rabbit cardiomyocytes bathed in Na⁺ and Ca²⁺ free solution, we found an increased decay of the cytosolic Ca²⁺ concentration [Ca²⁺]_i and increased SR Ca²⁺ content in the presence of the TRPC channel blocker SKF-96365. In a computational model of rabbit ventricular myocytes at physiological pacing rates, Ca²⁺ leak through SR TRPC channels increased the systolic and diastolic [Ca²⁺]_i with only minor effects on the action potential and SR Ca²⁺ content. Our studies suggest that TRPC1 channels are localized in the SR, and not present in the sarcolemma of ventricular myocytes. The studies provide evidence for a role of TRPC1 as a contributor to SR Ca²⁺ leak in cardiomyocytes, which was previously explained by ryanodine receptors only. We propose that the findings will guide us to an understanding of TRPC1 channels as modulators of [Ca²⁺]_i and contractility in cardiomyocytes.

SUBMITTER: Hu Q

PROVIDER: S-EPMC7085981 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Location and function of transient receptor potential canonical channel 1 in ventricular myocytes.

Hu Qinghua Q Ahmad Azmi A AA Seidel Thomas T Hunter Chris C Streiff Molly M Nikolova Linda L Spitzer Kenneth W KW Sachse Frank B FB

Journal of molecular and cellular cardiology 20200123

Transient receptor potential canonical 1 (TRPC1) protein is abundantly expressed in cardiomyocytes. While TRPC1 is supposed to be critically involved in cardiac hypertrophy, its physiological role in cardiomyocytes is poorly understood. We investigated the subcellular location of TRPC1 and its contribution to Ca<sup>2+</sup> signaling in mammalian ventricular myocytes. Immunolabeling, three-dimensional scanning confocal microscopy and quantitative colocalization analysis revealed an abundant int ...[more]

PMID: 31982426

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Project description:BackgroundFibroblast proliferation and differentiation are central in atrial fibrillation (AF)-promoting remodeling. Here, we investigated fibroblast regulation by Ca(2+)-permeable transient receptor potential canonical-3 (TRPC3) channels.Methods and resultsFreshly isolated rat cardiac fibroblasts abundantly expressed TRPC3 and had appreciable nonselective cation currents (I(NSC)) sensitive to a selective TPRC3 channel blocker, pyrazole-3 (3 μmol/L). Pyrazole-3 suppressed angiotensin II-induced Ca(2+) influx, proliferation, and α-smooth muscle actin protein expression in fibroblasts. Ca(2+) removal and TRPC3 blockade suppressed extracellular signal-regulated kinase phosphorylation, and extracellular signal-regulated kinase phosphorylation inhibition reduced fibroblast proliferation. TRPC3 expression was upregulated in atria from AF patients, goats with electrically maintained AF, and dogs with tachypacing-induced heart failure. TRPC3 knockdown (based on short hairpin RNA [shRNA]) decreased canine atrial fibroblast proliferation. In left atrial fibroblasts freshly isolated from dogs kept in AF for 1 week by atrial tachypacing, TRPC3 protein expression, currents, extracellular signal-regulated kinase phosphorylation, and extracellular matrix gene expression were all significantly increased. In cultured left atrial fibroblasts from AF dogs, proliferation rates, α-smooth muscle actin expression, and extracellular signal-regulated kinase phosphorylation were increased and were suppressed by pyrazole-3. MicroRNA-26 was downregulated in canine AF atria; experimental microRNA-26 knockdown reproduced AF-induced TRPC3 upregulation and fibroblast activation. MicroRNA-26 has NFAT (nuclear factor of activated T cells) binding sites in the 5' promoter region. NFAT activation increased in AF fibroblasts, and NFAT negatively regulated microRNA-26 transcription. In vivo pyrazole-3 administration suppressed AF while decreasing fibroblast proliferation and extracellular matrix gene expression.ConclusionsTRPC3 channels regulate cardiac fibroblast proliferation and differentiation, likely by controlling the Ca(2+) influx that activates extracellular signal-regulated kinase signaling. AF increases TRPC3 channel expression by causing NFAT-mediated downregulation of microRNA-26 and causes TRPC3-dependent enhancement of fibroblast proliferation and differentiation. In vivo, TRPC3 blockade prevents AF substrate development in a dog model of electrically maintained AF. TRPC3 likely plays an important role in AF by promoting fibroblast pathophysiology and is a novel potential therapeutic target.

Dataset Information

Location and function of transient receptor potential canonical channel 1 in ventricular myocytes.

Publications

Location and function of transient receptor potential canonical channel 1 in ventricular myocytes.

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