Implication of Orai1 in the diastolic calcium dysregulation during Ischemia and Reperfusion in cardiomyocytes

Abstract

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agencia Estatal de Investigación Background It is well established that abnormalities in the regulation of the intracellular concentration of Ca2+ ([Ca2+]i) occurrs in diseased heart. Ischemia/Reperfusion (I/R) syndrome is characterized by [Ca2+]i overload that might be associated with the increase of left ventricular pressure, which might trigger diastolic dysfunction. Several studies have demonstrated that Orai1/2/3 isoforms of the Store Operated Calcium Channels (SOCC) and TRPC channels play a role in cardiac pathologies. Previously, we demonstrated that the expression of Orai1 increased after I/R although their role in the diastolic [Ca2+]i regulation is unknown. Objective The aim of this study was to examine the role of SOCC in the diastolic [Ca2+] after I/R in cardiomyocytes and the activation of transcription factors by I/R. Materials and Methods The experiments were performed in adult (ARVM) and neonatal rat ventricular cardiomyocytes (NRVM) and in a rat model of myocardial I/R. Ca2+ studies were realized using microfluorimetric technique in FURA-2AM loaded cardiomyocytes. To evoke [Ca2+]i transients, ARVM were field stimulated at 0.5Hz and NRVMs at 1Hz. Immunofluorescence was used to investigated CREB activation using a specific phospho-CREB antibody and protein expression was analysed by Western Blot. Orai1 expression was assessed in rat samples and in ventricular biopsies of patients with ischemic heart failure. Results We found that field stimulation of ARVM from I/R animal showed significant increase in the diastolic [Ca2+]i as compare to AVRM isolated from sham rats. Also, NRVM subjected to simulated protocol of I/R presented similar increase in the diastolic [Ca2+]i. These responses correlated with significant increase in the expression of Orai1 in both cardiomyocytes. Interestingly, NRVM and ARVM preincubation with SYNTA-66 and GSK-7975A to block SOCC successfully reduced the increase in the diastolic [Ca2+]I induced by IR. Moreover, we found that I/R promoted CREB activation through PKA activation, but not though EPAC2 or ERK1/2. Finally, we found that Orai1 is overexpressed in ventricle biopsies of patient with heart failure from ischemic origin. Conclusions Our results confirms that I/R induced an upregulation of Orai1 which was associated with diastolic dysfunction, suggesting that Orai1 plays a role in the I/R induced diastolic [Ca2+]i increase. Moreover, our data demonstrated the activation of the transcription factor CREB in cardiomyocytes after I/R, known to activate a plethora of genes involved in the adverse cardiac remodelling.