The Transient receptor potential (TRP; C-classical; TRPC) channel TRPC3 permeates a cation (Na+/Ca2+) influx that is favoured by the stimulation of Gq protein-coupled receptors (GPCRs). An enhanced TRPC3 activity is related to adverse effects including pathological hypertrophy in chronic cardiac disease states. In the present study we identified FK506 binding protein 52 (FKBP52) as a novel interaction partner of TRPC3 in the heart. FKBP52 was recovered from a cardiac cDNA library by a C-terminal TRPC3 fragment (amino acids 742-848) in a yeast two-hybrid screen. Downregulation of FKBP52 promoted a TRPC3-dependent hypertrophic response in neonatal rat cardiomyocytes (NRC). A similar effect was achieved by overexpressing PPIase-deficient FKBP52 mutants. Mechanistically, FKBP52 truncated mutants elevated TRPC3-mediated currents and Ca2+ fluxes, the activation of calcineurin and the nuclear factor of activated T-cells in NRCs. Our data demonstrate that FKBP52 associates with TRPC3 via an as yet undescribed binding site in the C-terminus of TRPC3 and modulates TRPC3-dependent Ca2+ signals in a PPIase-dependent manner. This functional interaction might be crucial for limiting TRPC3-dependent signaling during chronic hypertrophic stimulation.

Aim: The role of the Na+/K+-ATPase (NKA) in heart failure associated with myocardial infarction (MI) is poorly understood. The elucidation of its precise function is hampered by the existence of two catalytic NKA isoforms (NKA-α1 and NKA-α2). Our aim was to analyze the effects of an increased NKA-α2 expression on functional deterioration and remodeling during long term MI treatment in mice and its impact on Ca2+ handling and inotropy of the failing heart. Methods and Results: Wild-type (WT) and NKA-α2 transgenic (TG) mice (TG-α2) with a cardiac-specific overexpression of NKA-α2 were subjected to MI injury for 8 weeks. As examined by echocardiography, gravimetry and histology, TG-α2 mice were protected from functional deterioration and adverse cardiac remodeling. Contractility and Ca2+ transients (Fura 2-AM) in cardiomyocytes from MI-treated TG-α2 animals showed reduced Ca2+ amplitudes during pacing or after caffeine application. Ca2+ efflux in cardiomyocytes from TG-α2 mice was accelerated, and diastolic Ca2+ levels decreased. Based on these alterations, sarcomeres exhibited an enhanced sensitization and thus increased contractility. After the acute stimulation with the β-adrenergic agonist isoproterenol (ISO), cardiomyocytes from MI-treated TG-α2 mice responded with increased sarcomere shortenings and Ca2+ peak amplitudes. This positive inotropic response was absent in cardiomyocytes from WT-MI animals. Conclusions: Cardiomyocytes with NKA-α2 as predominant isoform minimize Ca2+ cycling but respond to β-adrenergic stimulation more efficiently during chronic cardiac stress. These mechanisms might improve the β-adrenergic reserve and contribute to functional preservation in heart failure.

L-type voltage-gated calcium channels (LTCCs) regulate crucial physiological processes in the heart. They are composed of the Cavα1 pore-forming subunit and the accessory subunits Cavβ, Cavα2δ, and Cavγ. Cavβ is a cytosolic protein that regulates channel trafficking and activity, but it also exerts other LTCC-independent functions. Cardiac hypertrophy, a relevant risk factor for the development of congestive heart failure, depends on the activation of calcium-dependent pro-hypertrophic signaling cascades. Here, by using shRNA-mediated Cavβ silencing, we demonstrate that Cavβ2 downregulation enhances α1-adrenergic receptor agonist-induced cardiomyocyte hypertrophy. We report that a pool of Cavβ2 is targeted to the nucleus in cardiomyocytes and that the expression of this nuclear fraction decreases during in vitro and in vivo induction of cardiac hypertrophy. Moreover, the overexpression of nucleus-targeted Cavβ2 in cardiomyocytes inhibits in vitro-induced hypertrophy. Quantitative proteomic analyses showed that Cavβ2 knockdown leads to changes in the expression of diverse myocyte proteins, including reduction of calpastatin, an endogenous inhibitor of the calcium-dependent protease calpain. Accordingly, Cavβ2-downregulated cardiomyocytes had a 2-fold increase in calpain activity as compared to control cells. Furthermore, inhibition of calpain activity in Cavβ2-downregulated cells abolished the enhanced α1-adrenergic receptor agonist-induced hypertrophy observed in these cells. Our findings indicate that in cardiomyocytes, a nuclear pool of Cavβ2 participates in cellular functions that are independent of LTCC activity. They also indicate that a downregulation of nuclear Cavβ2 during cardiomyocyte hypertrophy promotes the activation of calpain-dependent hypertrophic pathways.

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.