Cardiac CaV1.2 channels require β subunits for β-adrenergic–mediated modulation but not trafficking
Lin Yang, … , Henry M. Colecraft, Steven O. Marx
Lin Yang, … , Henry M. Colecraft, Steven O. Marx
Published November 13, 2018
Citation Information: J Clin Invest. 2019;129(2):647-658. https://doi.org/10.1172/JCI123878.
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Cardiac CaV1.2 channels require β subunits for β-adrenergic–mediated modulation but not trafficking

Abstract

Ca2+ channel β-subunit interactions with pore-forming α-subunits are long-thought to be obligatory for channel trafficking to the cell surface and for tuning of basal biophysical properties in many tissues. Unexpectedly, we demonstrate that transgenic expression of mutant α1C subunits lacking capacity to bind CaVβ can traffic to the sarcolemma in adult cardiomyocytes in vivo and sustain normal excitation-contraction coupling. However, these β-less Ca2+ channels cannot be stimulated by β-adrenergic pathway agonists, and thus adrenergic augmentation of contractility is markedly impaired in isolated cardiomyocytes and in hearts. Similarly, viral-mediated expression of a β-subunit–sequestering peptide sharply curtailed β-adrenergic stimulation of WT Ca2+ channels, identifying an approach to specifically modulate β-adrenergic regulation of cardiac contractility. Our data demonstrate that β subunits are required for β-adrenergic regulation of CaV1.2 channels and positive inotropy in the heart, but are dispensable for CaV1.2 trafficking to the adult cardiomyocyte cell surface, and for basal function and excitation-contraction coupling.

Authors

Lin Yang, Alexander Katchman, Jared Kushner, Alexander Kushnir, Sergey I. Zakharov, Bi-xing Chen, Zunaira Shuja, Prakash Subramanyam, Guoxia Liu, Arianne Papa, Daniel Roybal, Geoffrey S. Pitt, Henry M. Colecraft, Steven O. Marx

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Figure 1

AID-mutant α1C channels trafficking and function in cardiomyocytes.

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AID-mutant α1C channels trafficking and function in cardiomyocytes. (A) ...
(A) Schematic of rabbit cardiac α1C subunit topology showing β-subunit binding to α-interacting domain (AID) motif in I-II loop. WT and mutant-AID motif in the I-II loop of α1C. (B) Schematic representation of the binary transgene system. The αMHCMOD construct is a modified αMHC promoter containing the tet-operon for regulated expression of FLAG-tagged DHP-resistant (DHP*) α1C. (C) Anti-FLAG (upper) and anti-β immunoblots (lower) of anti–FLAG antibody immunoprecipitation of cardiac homogenates of nontransgenic (NTG), pWT α1C, and AID-mutant α1C mice. Representative of 3 experiments. (D) Immunostaining of pWT and AID-mutant α1C cardiomyocytes. Anti-FLAG and FITC-conjugated secondary antibodies, and nuclear labeling with Hoechst stain. Negative control omitted anti–FLAG antibody. Images obtained with confocal microscopy at ×40. Scale bars: 20 μm. (E) Exemplar whole-cell CaV1.2 currents recorded from freshly dissociated cardiomyocytes of NTG, pWT, and AID-mutant α1C transgenic mice. Pulses from –60 mV to 0 mV before (black traces) and 3 minutes after (red traces) administration of 300 nM nisoldipine. (F) Scatter plot showing current densities before and after administration of 300 nM nisoldipine. Mean ± SEM. *P < 0.05 NTG versus transgenic pWT α1C, ****P < 0.0001 NTG versus transgenic AID-mutant α1C and also NTG pre- versus post-nisoldipine, ***P < 0.001 pWT or AID-mutant α1C pre- versus post-nisoldipine. One-way ANOVA and Dunnett’s multiple comparison test. NTG, n = 8 cardiomyocytes from 5 mice; pWT, n = 21 cardiomyocytes from 7 mice; AID-mutant, n = 45 cardiomyocytes from 9 mice. (G–I) Representative time courses of changes in sarcomere length after superfusion of 300 nM nisoldipine-containing solution for cardiomyocytes isolated from NTG mice (G) and pWT (H) and AID-mutant transgenic α1C mice. Cardiomyocytes were field-stimulated at 1 Hz. (J) Scatter plot showing percentage of contraction of sarcomere length in the absence and presence of nisoldipine for cardiomyocytes isolated from NTG mice and pWT and AID-mutant α1C transgenic mice. NTG, n = 12 cells from 3 mice; pWT, n = 16 cells from 3 mice; AID-mutant, n = 18 cells from 3 mice.