Klotho is a circulating protein, and Klotho deficiency disturbs endothelial integrity, but the molecular mechanism is not fully clarified. We report that vascular endothelium in Klotho-deficient mice showed hyperpermeability with increased apoptosis and down-regulation of vascular endothelial (VE)-cadherin because of an increase in VEGF-mediated internal calcium concentration ([Ca 2+ ]i) influx and hyperactivation of Ca 2+ -dependent proteases. Immunohistochemical analysis, the pull-down assay using Klotho-fixed agarose, and FRET confocal imaging confirmed that Klotho protein binds directly to VEGF receptor 2 (VEGFR-2) and endothelial, transient-receptor potential canonical Ca 2+ channel 1 (TRPC-1) and strengthens the association to promote their cointernalization. An in vitro mutagenesis study revealed that the second hydrolase domain of Klotho interacts with sixth and seventh Ig domains of VEGFR-2 and the third extracellular loop of TRPC-1. In Klotho-deficient endothelial cells, VEGF-mediated internalization of the VEGFR-2/TRPC-1 complex was impaired, and surface TRPC-1 expression increased 2.2-fold; these effects were reversed by supplementation of Klotho protein. VEGF-mediated elevation of [Ca 2+ ]i was sustained at higher levels in an extracellular Ca 2+ -dependent manner, and normalization of TRCP-1 expression restored the abnormal [Ca 2+ ]i handling. These findings provide evidence that Klotho protein is associated with VEGFR-2/TRPC-1 in causing cointernalization, thus regulating TRPC-1–mediated Ca 2+ entry to maintain endothelial integrity.

In the catheter ablation for AF, we found no significant reduction in the 1-year incidence of recurrent atrial tachyarrhythmias by ATP-guided PVI compared with conventional PVI.

Background-Aldosterone has recently attracted considerable attention for its involvement in the pathophysiology of heart failure, in which apoptotic cell loss plays a critical role. This study examined whether aldosterone directly induces myocyte apoptosis via its specific receptors. Methods and Results-Neonatal rat cardiac myocytes were exposed to aldosterone (10 Ϫ8 to 10 Ϫ5 mol/L). Nuclear staining with Hoechst 33258 showed that aldosterone induced myocyte apoptosis in a dose-and time-dependent fashion. Treatment of myocytes with 10 Ϫ5 mol/L aldosterone significantly increased the percentage of apoptosis (15.5Ϯ1.4%) compared with serum-deprived control (7.3Ϯ0.6%). Radio ligand binding assay revealed the existence of plasma membrane receptor with high affinity (K d , 0.2 nmol/L) for aldosterone in cardiac myocytes but not in fibroblasts. Aldosterone rapidly (Ϸ30 seconds) mobilized [Ca 2ϩ ] i that was blocked by neomycin. Aldosterone induced dephosphorylation of the proapoptotic protein Bad, enhancement of mitochondrial permeability transition, decrease in mitochondrial membrane potential, and release of cytochrome c from the mitochondria into the cytosol with concomitant activation of caspase-3. These effects of aldosterone were inhibited by concurrent treatment with either an L-type Ca

These data suggest that IL-1beta-induced NO production in cardiac myocytes lowers energy production and myocardial contractility through a direct attack on the mitochondria, rather than through cGMP-mediated pathways.