Abstract
Store-operated Ca(2+) entry (SOCE) has emerged as an important mechanism in cardiac pathology. However, the signals that up-regulate SOCE in the heart remain unexplored. Clinical trials have emphasized the beneficial role of mineralocorticoid receptor (MR) signaling blockade in heart failure and associated arrhythmias. Accumulated evidence suggests that the mineralocorticoid hormone aldosterone, through activation of its receptor, MR, might be a key regulator of Ca(2+) influx in cardiomyocytes. We thus assessed whether and how SOCE involving transient receptor potential canonical (TRPC) and Orai1 channels are regulated by aldosterone/MR in neonatal rat ventricular cardiomyocytes. Molecular screening using qRT-PCR and Western blotting demonstrated that aldosterone treatment for 24 h specifically increased the mRNA and/or protein levels of Orai1, TRPC1, -C4, -C5, and stromal interaction molecule 1 through MR activation. These effects were correlated with a specific enhancement of SOCE activities sensitive to store-operated channel inhibitors (SKF-96365 and BTP2) and to a potent Orai1 blocker (S66) and were prevented by TRPC1, -C4, and Orai1 dominant negative mutants or TRPC5 siRNA. A mechanistic approach showed that up-regulation of serum- and glucocorticoid-regulated kinase 1 mRNA expression by aldosterone is involved in enhanced SOCE. Functionally, 24-h aldosterone-enhanced SOCE is associated with increased diastolic [Ca(2+)]i, which is blunted by store-operated channel inhibitors. Our study provides the first evidence that aldosterone promotes TRPC1-, -C4-, -C5-, and Orai1-mediated SOCE in cardiomyocytes through an MR and serum- and glucocorticoid-regulated kinase 1 pathway.
Highlights
Store-operated Ca2؉ entry (SOCE) has emerged as an important mechanism in cardiac pathology
Quantitative determination of the different mRNA expression levels was performed with a CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad) with either gene-specific primers or primers for YWHAZ, RPL32, and TBP as endogenous controls (Table 1) from Eurofins MWG Operon (France). mRNA levels were normalized to housekeeping genes and were expressed as -fold change of that determined in untreated neonatal rat ventricular cardiomyocytes (NRVMs) for each culture
NRVMs were incubated for 24 h in the absence or presence of 1 nM, 10 nM, 100 nM, or 1 M Aldo alone or in combination with 10 M selective mineralocorticoid receptor (MR) antagonist RU. mRNA levels were normalized to housekeeping genes and expressed as -fold change of that determined in untreated NRVMs for each culture
Summary
Aldosterone-mediated cardiac action may cause various responses associated with elevation of [Ca2ϩ]i, including the direct or indirect increase of the Ca2ϩ-dependent phosphatase 2B calcineurin and its downstream transcription factor NFATc3 in the development of cardiac hypertrophy [40], the negative feedback on cAMP-response element-binding protein via protein phosphatase 2A in promoting apoptosis [41], and more recently the activation of the multifunctional Ca2ϩ/ calmodulin-dependent protein kinase II associated with poor outcomes after myocardial infarction [42]. We showed that aldosterone promotes an MR-specific enhancement of STIM1-dependent SOCE activities, which correlated with the increased expression and activity of TRPC1, -C4, -C5, and Orai channels This effect was dependent on the up-regulation of serum- and glucocorticoid-inducible kinase 1 (SGK1). This is the first demonstration of the cardiac SOCE activation by mineralocorticoid signaling, an effect that might contribute to the benefit of MR blockade in cardiac diseases
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