Stimulation of β₁-AR activates CaMKII in a β-arrestin–dependent and PKA-independent manner. (A) Tg mice overexpressing mouse WT–β₁-AR, GRK⁻–β₁-AR, or PKA⁻–β₁-AR were administered with saline (NS) or 5 mg/kg ISO by i.v. infusion for 5 min. LV homogenates were immunoblotted with anti-pCaMKII and anti-CaMKII antibodies. The CaMKII activation was quantified, expressed as fold increase over NS-treated WT mice, and shown as mean ± SEM (n = 5). Increased ISO-mediated CaMKII was observed in WT–β₁-AR and PKA⁻–β₁-AR Tg mice. *, P < 0.01 versus nonstimulation; ^#, P < 0.01 versus ISO-treated WT–β₁-AR Tg mice. (B) HEK-293 cells stably expressing GRK⁻–β₁-AR were transfected with CaMKII-δC and Flag-Epac1. Serum-starved cells were stimulated with 10 µM ISO at 37°C. The CaMKII activation was quantified, expressed as fold increase over nonstimulated cells, and shown as mean ± SEM (n = 5). ISO stimulation of WT–β₁-AR is a positive control. *, P < 0.01 versus ISO stimulation of GRK⁻–β₁-AR. IP, immunoprecipitation. (C and D) HEK-293 cells stably expressing WT–β₁-AR were transfected with CaMKII-δC and Flag-Epac1. Serum-starved cells were pretreated without or with PKI or KN-92 or -93 before stimulation with 10 µM ISO (C) or 5 µM 8-CPT (D) at 37°C. The CaMKII activation was quantified, expressed as fold increase over nonstimulated cells, and shown as mean ± SEM (n = 5). Inhibited ISO- and 8-CPT–mediated CaMKII activation were observed in cells pretreated with KN-93, a specific CaMKII inhibitor, but not in cells pretreated with PKI. *, P < 0.01 versus ISO stimulation; ^#, P < 0.01 versus 8-CPT stimulation. Forsk., forskolin; T-CaMKII, total CaMKII.