Abstract
Transient receptor potential canonical 6 (TRPC6) channels are non-selective cation channels that are thought to underlie mechano-modulation of calcium signaling in cardiomyocytes. TRPC6 channels are involved in development of cardiac hypertrophy and related calcineurin-nuclear factor of activated T cells (NFAT) signaling. However, the exact location and roles of TRPC6 channels remain ill-defined in cardiomyocytes. We used an expression system based on neonatal rat ventricular myocytes (NRVMs) to investigate the location of TRPC6 channels and their role in calcium signaling. NRVMs isolated from 1- to 2-day-old animals were cultured and infected with an adenoviral vector to express enhanced-green fluorescent protein (eGFP) or TRPC6-eGFP. After 3 days, NRVMs were fixed, immunolabeled, and imaged with confocal and super-resolution microscopy to determine TRPC6 localization. Cytosolic calcium transients at 0.5 and 1 Hz pacing rates were recorded in NRVMs using indo-1, a ratio-metric calcium dye. Confocal and super-resolution microscopy suggested that TRPC6-eGFP localized to the sarcolemma. NRVMs infected with TRPC6-eGFP exhibited higher diastolic and systolic cytosolic calcium concentration as well as increased sarcoplasmic reticulum (SR) calcium load compared to eGFP infected cells. We applied a computer model comprising sarcolemmal TRPC6 current to explain our experimental findings. Altogether, our studies indicate that TRPC6 channels play a role in sarcolemmal and intracellular calcium signaling in cardiomyocytes. Our findings support the hypothesis that upregulation or activation of TRPC6 channels, e.g., in disease, leads to sustained elevation of the cytosolic calcium concentration, which is thought to activate calcineurin-NFAT signaling and cardiac hypertrophic remodeling. Also, our findings support the hypothesis that mechanosensitivity of TRPC6 channels modulates cytosolic calcium transients and SR calcium load.
Highlights
Transient receptor potential canonical (TRPC) 6 channels are non-selective cation channels expressed in the mammalian heart (Bon and Beech, 2013)
neonatal rat ventricular myocytes (NRVMs) were infected 24 h after plating with an adenoviral vector containing human Transient receptor potential canonical 6 (TRPC6) attached to enhanced green fluorescent protein fused to the C-terminal and 6X HIS tag fused to the N-terminal (TRPC6eGFP) at 10 multiplication of infection (MOI)
Our study supports a role of TRPC6 in sarcolemmal Ca2+ signaling in cardiomyocytes
Summary
Transient receptor potential canonical (TRPC) 6 channels are non-selective cation channels expressed in the mammalian heart (Bon and Beech, 2013). TRPC6 channels are thought to be stretch-activated channels (SACs). Stretch activation of TRPC6 channels was linked to increases in cytosolic Ca2+ concentration ([Ca2+]i) (Dyachenko et al, 2008). TRPC6 was suggested to be involved in cardiac systolic mechanosignaling (Seo et al, 2014). Despite these findings, the precise role of TRPC6 channels in Ca2+ signaling and its mechanical modulation in cardiomyocytes remains unclear. The precise role of TRPC6 channels in Ca2+ signaling and its mechanical modulation in cardiomyocytes remains unclear It is unclear if in cardiomyocytes TRPC6 channels contribute to mechano-electrical feedback (MEF), i.e., alter cell electrophysiology in response to mechanical stimuli (Franz, 2000)
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