The ß2-Subunit of Voltage-Gated Calcium Channels Regulates Cardiomyocyte Hypertrophy.

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 Cavß, Cavα2δ, and Cavγ. Cavß 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 Cavß silencing, we demonstrate that Cavß2 downregulation enhances α1-adrenergic receptor agonist-induced cardiomyocyte hypertrophy. We report that a pool of Cavß2 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 Cavß2 in cardiomyocytes inhibits in vitro-induced hypertrophy. Quantitative proteomic analyses showed that Cavß2 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, Cavß2-downregulated cardiomyocytes had a 2-fold increase in calpain activity as compared to control cells. Furthermore, inhibition of calpain activity in Cavß2-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 Cavß2 participates in cellular functions that are independent of LTCC activity. They also indicate that a downregulation of nuclear Cavß2 during cardiomyocyte hypertrophy promotes the activation of calpain-dependent hypertrophic pathways.

Nanoenviroments of the ß-Subunit of L-Type Voltage-Gated Calcium Channels in Adult Cardiomyocytes.

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 Cavα1, Cavß, Cavα2δ and Cavγ subunits. Here, using ascorbate peroxidase (APEX2)-mediated proximity labeling and quantitative proteomics, we identified 61 proteins in the nanoenvironments of Cavß2 in cardiomyocytes. These proteins are involved in diverse cellular functions such as cellular trafficking, cardiac contraction, sarcomere organization and excitation-contraction coupling. Moreover, pull-down assays and co-immunoprecipitation analyses revealed that Cavß2 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 Cavß2 and is necessary for an effective pacing frequency-dependent increase of the Ca2+-induced Ca2+ release mechanism in cardiomyocytes.

FKBP52 regulates TRPC3-dependent Ca2+ signals and the hypertrophic growth of cardiomyocyte cultures.

The transient receptor potential (TRP; C-classical, TRPC) channel TRPC3 allows a cation (Na+/Ca2+) influx that is favored 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, also known as FKBP4) 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 (NRCs). A similar effect was achieved by overexpressing peptidyl-prolyl isomerase (PPIase)-deficient FKBP52 mutants. Mechanistically, expression of the FKBP52 truncation mutants elevated TRPC3-mediated currents and Ca2+ fluxes, and 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.

Water channel pore size determines exclusion properties but not solute selectivity.

Aquaporins (AQPs) are a ubiquitous family of transmembrane water channel proteins. A subgroup of AQP water channels also facilitates transmembrane diffusion of small, polar solutes. A constriction within the pore, the aromatic/arginine (ar/R) selectivity filter, is thought to control solute permeability: previous studies on single representative water channel proteins suggest narrow channels conduct water, whilst wider channels permit passage of solutes. To assess this model of selectivity, we used mutagenesis, permeability measurements and in silico comparisons of water-specific as well as glycerol-permeable human AQPs. Our studies show that single amino acid substitutions in the selectivity filters of AQP1, AQP4 and AQP3 differentially affect glycerol and urea permeability in an AQP-specific manner. Comparison between in silico-calculated channel cross-sectional areas and in vitro permeability measurements suggests that selectivity filter cross-sectional area predicts urea but not glycerol permeability. Our data show that substrate discrimination in water channels depends on a complex interplay between the solute, pore size, and polarity, and that using single water channel proteins as representative models has led to an underestimation of this complexity.

Hypotonic activation of the myo-inositol transporter SLC5A3 in HEK293 cells probed by cell volumetry, confocal and super-resolution microscopy.

Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol P ino [m/s] and expression/localization of SLC5A3. P ino values were determined by cell volumetry over a wide tonicity range (100-275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200-275 mOsm), P ino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ∼ 3 nm/s at 100-125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in P ino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200-2000 localizations/µm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80-800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells.