Angiotensin II Increases Expression of α1C Subunit of L-Type Calcium Channel Through a Reactive Oxygen Species and cAMP Response Element–Binding Protein–Dependent Pathway in HL-1 Myocytes

Abstract

Abstract-Angiotensin II (Ang II) is involved in the pathogenesis of atrial fibrillation (AF). L-type calcium channel (LCC)expression is altered in AF remodeling. We investigated whether Ang II modulates LCC current through transcriptional regulation, by using murine atrial HL-1 cells, which have a spontaneous calcium transient, and an in vivo rat model. Ang II increased LCC ␣1C subunit mRNA and protein levels and LCC current density, which resulted in an augmented calcium transient in atrial myocytes. An Ϸ2-kb promoter region of LCC ␣1C subunit gene was cloned to the pGL3 luciferase vector. Ang II significantly increased promoter activity in a concentration-and time-dependent manner.Truncation and mutational analysis of the LCC ␣1C subunit gene promoter showed that cAMP response element (CRE) (Ϫ1853 to Ϫ1845) was an important cis element in Ang II-induced LCC ␣1C subunit gene expression. Transfection of dominant-negative CRE binding protein (CREB) (pCMV-CREBS133A) abolished the Ang II effect. Ang II (1 mol/L, 2 hours) induced serine 133 phosphorylation of CREB and binding of CREB to CRE and increased LCC ␣1C subunit gene promoter activity through a protein kinase C/NADPH oxidase/reactive oxygen species pathway, which was blocked by the Ang II type 1 receptor blocker losartan and the antioxidant simvastatin. In the rat model, Ang II infusion increased LCC ␣1C subunit expression and serine 133 phosphorylation of CREB, which were attenuated by oral losartan and simvastatin. In summary, Ang II induced LCC ␣1C subunit expression via a protein kinase C-, reactive oxygen species-, and CREB-dependent pathway and was blocked by losartan and simvastatin. [2][3][4] Moreover, blockade of the renin-angiotensin system has been shown to be an effective treatment of AF. 5 Ang II increases spontaneous calcium sparks 6,7 and L-type calcium channel (LCC) current (I CaL ) in cardiomyocytes. 7,8 However, downregulation of I CaL 9 and channel expression 10,11 are observed in AF. Whether Ang II increases or decreases I CaL channel subunit expression in atrial myocytes and the detailed signaling mechanisms are unknown. Accordingly, in the present study, we explored the chronic effect of Ang II on the transcriptional regulation of LCC channel subunits in atrial myocytes. We used a murine atrial cell line HL-1, which is the only available atrial myocyte cell line that continuously divides and maintains a differentiated cardiac phenotype with spontaneous depolarization. 10 We also used an in vivo rat model of Ang II infusion to verify the results obtained in the cellular study. We first found that Ang II increased LCC channel ␣1C subunit expression and augmented I CaL and calcium transient amplitude in HL-1 atrial myocytes. The detailed signaling mechanisms by which Ang II regulates the expression of LCC ␣1C subunits were also studied. Materials and Methods Culture and Transfection of HL-1 CardiomyocytesHL-1 myocytes were cultured in the Claycomb medium. Transient transfection of the HL-1 myocytes was performed using Lipo-

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