Angiotensin II activates CaV 1.2 Ca2+ channels through β-arrestin2 and casein kinase 2 in mouse immature cardiomyocytes

Key points: Angiotensin II (AngII) is crucial in cardiovascular regulation in perinatal mammalians. Here we show that AngII increases twitch Ca²⁺ transients of mouse immature but not mature cardiomyocytes by robustly activating Ca_V 1.2 L-type Ca²⁺ channels through a novel signalling pathway involving angiotensin type 1 (AT₁ ) receptors, β-arrestin2 and Casein Kinase 2. A β-arrestin-biased AT₁ receptor agonist, TRV027, was as effective as AngII in activating L-type Ca²⁺ channels. Our results help understand the molecular mechanism by which AngII regulates the perinatal circulation and also suggest that β-arrestin-biased AT₁ receptor agonists may be valuable therapeutics for paediatric heart failure.

Abstract: Angiotensin II (AngII), the main effector peptide of the renin-angiotensin system, plays important roles in cardiovascular regulation in the perinatal period. Despite the well-known stimulatory effect of AngII on vascular contraction, little is known about regulation of contraction of the immature heart by AngII. Here we found that AngII significantly increased the peak amplitude of twitch Ca²⁺ transients by robustly activating L-type Ca_V 1.2 Ca²⁺ (Ca_V 1.2) channels in mouse immature but not mature cardiomyocytes. This response to AngII was mediated by AT₁ receptors and β-arrestin2. A β-arrestin-biased AT₁ receptor agonist was as effective as AngII in activating Ca_V 1.2 channels. Src-family tyrosine kinases (SFKs) and Casein Kinase 2α'β (CK2α'β) were sequentially activated when AngII activated Ca_V 1.2 channels. A cyclin-dependent kinase inhibitor, p27^Kip1 (p27), inhibited CK2α'β, and AngII removed this inhibitory effect through phosphorylating tyrosine 88 of p27 via SFKs in cardiomyocytes. In a human embryonic kidney cell line, tsA201 cells, overexpression of CK2α'β but not c-Src directly activated recombinant Ca_V 1.2 channels composed of C-terminally truncated α_1C , the distal C-terminus of α_1C , β_2C and α₂ δ₁ subunits, by phosphorylating threonine 1704 located at the interface between the proximal and the distal C-terminus of Ca_V 1.2α_1C subunits. Co-immunoprecipitation revealed that Ca_V 1.2 channels, CK2α'β and p27 formed a macromolecular complex. Therefore, stimulation of AT₁ receptors by AngII activates Ca_V 1.2 channels through β-arrestin2 and CK2α'β, thereby probably exerting a positive inotropic effect in the immature heart. Our results also indicated that β-arrestin-biased AT₁ receptor agonists may be used as valuable therapeutics for paediatric heart failure in the future.