TRPC4α and TRPC4β Similarly Affect Neonatal Cardiomyocyte Survival during Chronic GPCR Stimulation

Abstract

The Transient Receptor Potential Channel Subunit 4 (TRPC4) has been considered as a crucial Ca2+ component in cardiomyocytes promoting structural and functional remodeling in the course of pathological cardiac hypertrophy. TRPC4 assembles as homo or hetero-tetramer in the plasma membrane, allowing a non-selective Na+ and Ca2+ influx. Gαq protein-coupled receptor (GPCR) stimulation is known to increase TRPC4 channel activity and a TRPC4-mediated Ca2+ influx which has been regarded as ideal Ca2+ source for calcineurin and subsequent nuclear factor of activated T-cells (NFAT) activation. Functional properties of TRPC4 are also based on the expression of the TRPC4 splice variants TRPC4α and TRPC4β. Aim of the present study was to analyze cytosolic Ca2+ signals, signaling, hypertrophy and vitality of cardiomyocytes in dependence on the expression level of either TRPC4α or TRPC4β. The analysis of Ca2+ transients in neonatal rat cardiomyocytes (NRCs) showed that TRPC4α and TRPC4β affected Ca2+ cycling in beating cardiomyocytes with both splice variants inducing an elevation of the Ca2+ transient amplitude at baseline and TRPC4β increasing the Ca2+ peak during angiotensin II (Ang II) stimulation. NRCs infected with TRPC4β (Ad-C4β) also responded with a sustained Ca2+ influx when treated with Ang II under non-pacing conditions. Consistent with the Ca2+ data, NRCs infected with TRPC4α (Ad-C4α) showed an elevated calcineurin/NFAT activity and a baseline hypertrophic phenotype but did not further develop hypertrophy during chronic Ang II/phenylephrine stimulation. Down-regulation of endogenous TRPC4α reversed these effects, resulting in less hypertrophy of NRCs at baseline but a markedly increased hypertrophic enlargement after chronic agonist stimulation. Ad-C4β NRCs did not exhibit baseline calcineurin/NFAT activity or hypertrophy but responded with an increased calcineurin/NFAT activity after GPCR stimulation. However, this effect was not translated into an increased propensity towards hypertrophy but rather less hypertrophy during GPCR stimulation. Further analyses revealed that, although hypertrophy was preserved in Ad-C4α NRCs and even attenuated in Ad-C4β NRCs, cardiomyocytes had an increased apoptosis rate and thus were less viable after chronic GPCR stimulation. These findings suggest that TRPC4α and TRPC4β differentially affect Ca2+ signals, calcineurin/NFAT signaling and hypertrophy but similarly impair cardiomyocyte viability during GPCR stimulation.

TRPC Channels in Cardiac Plasticity

“…In addition, TRPC4b overexpression has nothing to do with resting or AngII-induced cardiac hypertrophy. In both TRPC4a-and TRPC4b-overexpressing cardiomyocytes, basal activity of the CaN/NFAT pathway is increased [86]. Consistent with these findings, the most important role of TRPC1/C4 seems to be the pathway for background Ca 2+ entry in cardiomyocytes to fine-tune systolic and diastolic intracellular Ca 2+ concentration [87].…”

TRPC Channels: Dysregulation and Ca2+ Mishandling in Ischemic Heart Disease

“…The resulting TRPC-mediated Ca 2+ influx is required for the activation of signaling pathways that play minor roles in the healthy heart. For instance, they are involved in the activation of transcription factors promoting cardiac hypertrophy, fibrosis, and/or arrythmia [5,13,28,55,[66][67][68][69]. Here, we discuss the role of TRPC channels in processes related to cardiac ischemic diseases.…”

The β2-Subunit of Voltage-Gated Calcium Channels Regulates Cardiomyocyte Hypertrophy

“…Neonatal rat cardiomyocytes were isolated as previously described by Kirschmer et al ( 16 ). Briefly, for each replicate of an experiment, 10–15 pups (1–3 day old) from 2 to 3 Wistar rats (Charles River Laboratory) were euthanized by decapitation and the hearts were cut into small pieces in a Petri dish containing calcium- and bicarbonate-free Hank's balanced salt solution with Hepes (CBFHBSS) (137 mM NaCl, 5.36 mM KCl, 0.81 mM MgSO 4 , 5.55 mM D-Glucose, 0.44 mM KH 2 PO 4 , 0.34 mM Na 2 HPO 4 , 20 mM Hepes, pH 7.4) supplemented with penicillin-streptomycin.…”

“…The measurement of the fluorometric Ca 2+ transients in NRCs was performed as described by Kirschmer et al ( 16 ). Briefly, cells were cultured on cover slides coated with laminin (Roche) and loaded for 20 min at room temperature with Fura-2 (2 mM) in a Ca 2+ -free normal Tyrode solution (140 mM NaCl, 4 mM KCl, 1 mM MgCl 2 , 5 mM Hepes, 10 mM glucose, pH 7.4).…”