Stimulation of AT1 Angiotensin Receptors Activates CaV1.2 L-Type Ca2+ Channels through Beta-Arrestin2 and Casein Kinase 2 in Immature Cardiomyocytes

Abstract

Angiotensin II (AngII) plays important roles in cardiovascular regulation in the perinatal period. Here, we examined the effect of AngII on cardiac CaV1.2 L-type Ca2+ channels. As assessed in the whole-cell configuration of the patch-clamp method, 2-hour treatment of AngII (3 μM) doubled the activity of CaV1.2 channels in mouse neonatal ventricular cardiomyocytes (NVCM) and an atrial myocyte line HL-1, but not in adult ventricular cardiomyocytes (AVCM). An AT1 receptor antagonist, candesartan (10 μM) and knockdown of β-arrestin2, but not an AT2 receptor antagonist, PD123319 (3 μM) or knockdown of β-arrestin1 or Gq/11 significantly suppressed the AngII-mediated activation of CaV1.2 channels in HL-1 cardiomyocytes. To elucidate possible involvement of protein kinases in this system, we examined the effects of array of kinase inhibitors in NVCM. Then, we found that a casein kinase 2 (CK2) inhibitor, quinalizarin (3 μM) almost completely inhibited the AngII-mediated activation of CaV1.2 channels. Knockdown of CK2α, α’ or β subunit in HL-1 cardiomyocytes indicated requirement of CK2 α’ and β but not α in this response. Immunoblotting revealed that the expression levels of CK2α’ was 7 and 4.5 times higher in NVCM and HL-1 cardiomyocytes than in AVCM, respectively. In tsA201 cells, overexpression of CK2α’β significantly increased the activity of recombinant CaV1.2 channels composed of C-terminally truncated α1C, the distal C-terminus of α1C, β2C and α2δ1 subunits; however, it failed to activate CaV1.2 channels containing α1C whose threonine 1704 was substituted with alanine. These results indicate that stimulation of AT1 receptors activates CaV1.2 L-type Ca2+ channels through β-arrestin2 and casein kinase 2 in immature but not adult cardiomyocytes. This response may play an important role in cardiovascular regulation in the perinatal period.