The AKAP Cypher/Zasp contributes to β-adrenergic/PKA stimulation of cardiac CaV1.2 calcium channels

Abstract

Stimulation of the L-type Ca²⁺ current conducted by Ca_V1.2 channels in cardiac myocytes by the β-adrenergic/protein kinase A (PKA) signaling pathway requires anchoring of PKA to the Ca_V1.2 channel by an A-kinase anchoring protein (AKAP). However, the AKAP(s) responsible for regulation in vivo remain unknown. Here, we test the role of the AKAP Cypher/Zasp in β-adrenergic regulation of Ca_V1.2 channels using physiological studies of cardiac ventricular myocytes from young-adult mice lacking the long form of Cypher/Zasp (LCyphKO mice). These myocytes have increased protein levels of Ca_V1.2, PKA, and calcineurin. In contrast, the cell surface density of Ca_V1.2 channels and the basal Ca²⁺ current conducted by Ca_V1.2 channels are significantly reduced without substantial changes to kinetics or voltage dependence. β-adrenergic regulation of these L-type Ca²⁺ currents is also significantly reduced in myocytes from LCyphKO mice, whether calculated as a stimulation ratio or as net-stimulated Ca²⁺ current. At 100 nM isoproterenol, the net β-adrenergic-Ca²⁺ current conducted by Ca_V1.2 channels was reduced to 39 ± 12% of wild type. However, concentration-response curves for β-adrenergic stimulation of myocytes from LCyphKO mice have concentrations that give a half-maximal response similar to those for wild-type mice. These results identify Cypher/Zasp as an important AKAP for β-adrenergic regulation of cardiac Ca_V1.2 channels. Other AKAPs may work cooperatively with Cypher/Zasp to give the full magnitude of β-adrenergic regulation of Ca_V1.2 channels observed in vivo.

Figure 1.

Morphology and protein expression in WT and LCyphKO mice. (A) Representative WT (left) and LCyphKO (right) dissociated cardiac ventricular myocytes immunostained for α-actinin to mark the z-lines. Bar, 25 µm. (B) Representative WT (left) and LCyphKO (right) dissociated cardiac ventricular myocytes immunostained for Ca_V1.2. (C) Confocal sections at the cell surface of representative immunostained WT (left) and LCyphKP (right) myocytes. (D) Quantification of Ca_V1.2 channels on the cell surface of ventricular myocytes from WT and LCyphKO (Cypher-L KO) mice. (E) Immunoblots of the indicated proteins in ventricular tissue from WT and LCyphKO mice. (F) Quantification of the indicated proteins in ventricular tissue from WT (black bars) and LCyphKO (white bars) mice (n = 4–6 replicates; 8–10 mice). *, P < 0.05; ***, P < 0.005. Error bars indicate SEM.

Figure 2.

The amplitude of basal Ca_V1.2 current was reduced in myocytes from LCyphKO mice. (A) Representative families of Ca²⁺ currents evoked by depolarization from −40 mV to a range of potentials from −40 to 80 mV in 10-mV increments in cardiac myocytes from WT, LCyphHET (HET), and LCyphKO (KO) mice. (B) Mean peak current–voltage relationships recorded in cardiac myocytes derived from WT, LCyphHET, and LCyphKO mice (n = 8–14 mice). (C) Comparison of Ca²⁺ currents evoked by depolarization from −40 to 0 mV in cardiac myocytes from WT, LCyphHET, and LCyphKO mice (n = 8–12 mice). *, P < 0.05. Error bars indicate SEM.

Figure 3.

β-adrenergic regulation of Ca_V1.2 Ca²⁺ current was reduced in myocytes from LCyphKO mice. (A) Mean peak current–voltage relations in the absence or presence of 100 nM Iso recorded in cardiac myocytes derived from WT, LCyphHET (HET), and LCyphKO (KO) mice. (B) Stimulation ratio for Ca²⁺ currents after treatment with 100 nM Iso in WT, LCyphHET, and LCyphKO mice, calculated as total current/basal current at a test potential of 0 mV. (C) Net β-adrenergic–stimulated Ca²⁺ currents induced by 100 nM Iso in WT, LCyphHET, and LCyphKO mice calculated as Iso current/basal current. (D) Normalized net β-adrenergic–stimulated Ca²⁺ currents induced by 100 nM Iso in WT, LCyphHET, and LCyphKO mice calculated as Iso current/basal current. WT, −9.2 ± 1.0 pA/pF; LCyphHET, −6.9 ± 1.4 pA/pF; LCyphKO, −4.3 ± 1.3 pA/pF; P < 0.05. n = 6–7 cells for each I-V curve; n = 3–4 mice for each I-V curve. *, P < 0.05; **, P < 0.01. Error bars indicate SEM.

Figure 4.

Concentration–response curves for Iso-induced Ca²⁺ currents in myocytes from WT, LCyphHET, and LCyphKO mice. Representative Ca²⁺ currents evoked by depolarization from −40 to 0 mV after treatment of dissociated cardiac myocytes with the indicated concentrations of Iso. (A) WT mice. (B) LCyphHET (HET) mice. (C) LCyphKO (KO) mice. (D) Mean maximal β-adrenergic–stimulated Ca²⁺ currents (calculated as Iso current/basal current and normalized for reduced expression of Ca_V1.2 in LCyphKO mice) at a test potential of 0 mV from WT, LCyphHET, and LCyphKO mice. Concentration–response curves were fit by a Hill equation with n_H = 1. The resulting estimates of concentrations that give a half-maximal response for stimulation by Iso were 34.4 nM in WT, 53.5 nM in LCyphHET, and 25.7 nM in LCyphKO mice. n = 4–8 cells for each data point; n = 8–10 mice of each genotype. WT, −8.73 ± 0.81 pA/pF; LCyphHET, −7.5 ± 1.3 pA/pF; LCyphKO, −4.9 ± 1.1 pA/pF; P < 0.05. **, P < 0.01. Error bars indicate SEM.