Rad regulation of CaV1.2 channels controls cardiac fight-or-flight response

Abstract

Fight-or-flight responses involve β-adrenergic-induced increases in heart rate and contractile force. In the present study, we uncover the primary mechanism underlying the heart's innate contractile reserve. We show that four protein kinase A (PKA)-phosphorylated residues in Rad, a calcium channel inhibitor, are crucial for controlling basal calcium current and essential for β-adrenergic augmentation of calcium influx in cardiomyocytes. Even with intact PKA signaling to other proteins modulating calcium handling, preventing adrenergic activation of calcium channels in Rad-phosphosite-mutant mice (4SA-Rad) has profound physiological effects: reduced heart rate with increased pauses, reduced basal contractility, near-complete attenuation of β-adrenergic contractile response and diminished exercise capacity. Conversely, expression of mutant calcium-channel β-subunits that cannot bind 4SA-Rad is sufficient to enhance basal calcium influx and contractility to adrenergically augmented levels of wild-type mice, rescuing the failing heart phenotype of 4SA-Rad mice. Hence, disruption of interactions between Rad and calcium channels constitutes the foundation toward next-generation therapeutics specifically enhancing cardiac contractility.

Extended Data Fig. 1 |. Rad phosphorylation is required for augmentation of Ca²⁺ channel current.

a, Schematic depicting creation of 4SA-Rad mice by homologous recombination. Alanine-substitutions were introduced for four serine (Ser) residues, Ser and Ser in exon2 (Ex2), and Ser²⁷² and Ser³⁰⁰ in exon5 (Ex5). The neomycin cassette was removed by Cre recombinase. b, Anti-Rad and anti-β-actin antibody immunoblots. Lower: Graph of quantification of intensity of Rad band normalized to β-actin loading control. Data are mean ± s.e.m.; P = 0.61 by unpaired two-tailed t-test. N = 3 mice in each group. c, Anti-α_1C and anti-β subunit antibody immunoblots of WT and 4SA-Rad ventricular cardiomyocyte lysates. Lower: Graph of quantification of intensity of α_1C and β subunit bands normalized to β-actin. Data are mean ± s.e.m.; P = 0.99 and P = 0.41, from left to right, by unpaired two-tailed t-test. N = 3 mice in each group. d, Exemplar traces of normalized Ca²⁺ current in response to a step depolarization to +10 mV. e, Graph of τ₁ and τ₂ inactivation. Fits were obtained using two exponentials by Clampfit. Data are mean ± s.e.m.; P = 0.86 for τ₁ and P = 0.77 for τ₂ by unpaired two-tailed t-test. n = 61 WT cells and 31 4SA-Rad cells from 22 and 8 mice respectively. f-g, Exemplar current-voltage relationships of Ca²⁺ channels from WT (f) and 4SA-Rad cardiomyocytes (g) acquired in the absence (black trace) and presence of 10 μM forskolin (blue trace). Insets: Exemplar whole-cell Ca_V1.2 currents. Pulses from −60 mV to +50 mV before (black traces) and 3 minutes after (blue traces) forskolin. h, Ratio of Ba²⁺ current after forskolin to Ba²⁺ current before forskolin for WT, heterozygous 4SA-Rad, and homozygous 4SA-Rad cardiomyocytes. Data are mean ± s.e.m.; n = 25 WT cells from 6 mice; 26 heterozygous 4SA-Rad cells from 3 mice; 28 homozygous 4SA-Rad cells from 5 mice. i, Ba²⁺ current elicited by voltage ramp every 6 seconds, with black traces (control), green traces after PKA inhibitor Rp-8Br-cAMPS, and blue traces after calyculin A in presence of Rp-8Br-cAMPS in WT ventricular myocytes. Representative of 4 similar experiments.

Extended Data Fig. 2 |. Characterization of 4SA-Rad knock-in mice.

a, Graph of number of 292 pups from 43 litters of heterozygous 4SA-Rad mice crosses. Dashed lines are the expected number of pups representing 25% (WT and 4SA-Rad) and 50% (heterozygous 4SA-Rad). P = 0.2885 observed vs. expected by Chi-square. The discrepancy between observed and expected is not significant. b, Trichrome stain of cross-sections of WT and 4SA-Rad heart from 5-month-old mice. Representative of 15 WT mice and 24 4SA-Rad mice. c, Graph of heart weight normalized to body weight. Data are mean ± s.e.m.; P = 0.45 by unpaired two-tailed t-test. N = 20 WT mice and 26 4SA-Rad mice. d, Graph of lung weight normalized to body weight. Data are mean ± s.e.m.; P = 0.79 by unpaired two-tailed t-test. N = 20 WT mice and 27 4SA-Rad mice. e, Graph of body weight normalized to tibial length of mice. Data are mean ± s.e.m.; * P < 0.05 by unpaired two-tailed t-test. N = 20 WT mice and 27 4SA-Rad mice.

Extended Data Fig. 3 |. Myocardial transcriptional profiling of 4SA-Rad knock-in mouse model.

a, Hierarchical clustering of 3 homozygous 4SA-Rad and 3 WT mice based on significantly dysregulated transcripts. b, Gene ontology and KEGG pathway analysis of significantly upregulated and downregulated transcripts. c, Graphs of normalized counts for selected cardiac genes. P_adjusted shown in figure for those transcripts that are significantly changed between 4SA-Rad and WT mice (see Methods and Supplementary Table 2). d, Fold-change of transcripts in 4SA-Rad:WT mice.

Extended Data Fig. 4 |. Echocardiographic characteristics of WT and 4SA-Rad mice.

a, Graph of basal fractional area of change (FAC) of WT and 4SA-Rad mutant mice. Data are mean ± s.e.m.; ****P < 0.0001 by unpaired two-tailed t-test. N = 23 WT mice and 22 4SA-Rad mice. b, Graph of basal left ventricular (LV) end-diastolic (LVED) area. Data are mean ± s.e.m.; **P < 0.01 by unpaired two-tailed t-test. N = 22 WT mice and 23 4SA-Rad mice. c, Graph of LV end-systolic (LVES) area. Data are mean ± s.e.m.; ****P < 0.0001 by unpaired two-tailed t-test. N = 22 WT mice and 23 4SA-Rad mice. d, Graph of end-diastolic volume (EDV). Data are mean ± s.e.m.; **P < 0.01 by unpaired two-tailed t-test. N = 21 WT mice and 21 4SA-Rad mice. e, Graph of end-systolic volume (ESV). Data are mean ± s.e.m.; ****P < 0.0001 by unpaired two-tailed t-test. N = 21 WT mice and 21 4SA-Rad mice.

Extended Data Fig. 5 |. Disrupting β and Rad interaction by mutation of β_2B.

a, FRET two-hybrid binding isotherms were determined for N-terminal Cer-tagged WT β_2B, 2DA-β_2B and 3DA-β_2B, and N-terminal Ven-tagged WT Rad. Graph summarizing Kd,EFF for WT, 2DA-β_2B and 3DA-β_2B. Data are mean ± s.e.m.; **** P < 0.0001 by unpaired two-tailed t-test. n = 9, 4 and 4 independent replicates/experiments, from left to right. b, FRET two-hybrid binding isotherms were determined for N-terminal Cer-tagged WT β_2B, 2DA-β_2B and 3DA-β_2B, and N-terminal Ven-tagged I-II loop of α_1C. Graph summarizing Kd,EFF for WT, 2DA-β_2B and 3DA-β_2B. Data are mean ± s.e.m.; P = 0.36 and P = 0.66 by unpaired two-tailed t-test. n = 3 independent replicates/experiments for all groups. c, Schematic depicting approach for creation of 2DA-β_2B knock-in mouse. Guides and single-strand oligodeoxynucleotide as the donor template with Ala-substitutions for the two Asp residues in exon 11 were tested by Genome Engineering and iPSC Center (GEiC) at the Washington University in St. Louis. HA-L = homology arm-left, HA-R = homology arm-right. d, Ba²⁺ current elicited by voltage ramp every 6 s, with black traces obtained before and blue traces obtained after ISO in 2DA-β_2B and 3DA-β_2B ventricular myocytes.

Fig. 1 |. Adrenergic agonist-induced stimulation of Ca²⁺ current requires phosphorylation of Rad in ventricular and atrial cardiomyocytes.

a,b, Exemplar current–voltage relationships of Ca²⁺ channels from WT and 4SA-Rad cardiomyocytes in the absence (black trace) and presence (blue trace) of isoproterenol (ISO). Inset: exemplar whole-cell Ca_V1.2 currents. Pulses from −60 mV to +50 mV before (black traces) and 3 min after (blue traces) 200 nM isoproterenol. c, Ba²⁺ current elicited by voltage ramp from −60 mV to +30 mV, with black traces obtained before and blue traces after isoproterenol in WT and 4SA-Rad ventricular myocytes. d, Graph of maximal conductance density (at +10 mV) for WT (172 cells, 22 mice) and 4SA-Rad cardiomyocytes (56 cells, 11 mice), acquired via the step protocol. Data are the mean ± s.e.m. ***P < 0.001 by unpaired, two-tailed Student’s t-test. Specific P values can be found in the associated Source data (see Supplementary Information). e, Fold-change in peak current at −20 mV caused by isoproterenol or forskolin (FSK). Data are mean ± s.e.m. ****P < 0.0001 by unpaired, two-tailed Student’s t-test. WT: isoproterenol, 30 cells, 8 mice and forskolin, 71 cells, 15 mice; 4SA-Rad: isoproterenol, 21 cells, 4 mice and forskolin, 35 cells, 7 mice. f, V₅₀ before and after isoproterenol or forskolin. Data are the mean ± s.e.m. ****P < 0.0001 for WT, P = 0.67 (isoproterenol), P = 0.41 (forskolin) for 4SA-Rad by unpaired, two-tailed Student’s t-test. NS, not significant. WT: isoproterenol, 30 cells, 8 mice and forskolin, 76 cells, 15 mice; 4SA-Rad: isoproterenol, 20 cells, 4 mice and forskolin, 36 cells, 7 mice. g, Same as c except with atrial cardiomyocytes. h, Diary plot of normalized fold-change of conductance at −20 mV for WT and 4SA-Rad atrial and ventricular myocytes. Data are mean ± s.e.m. Ventricle WT: 22 cells, 3 mice; 4SA-Rad: 31 cells, 3 mice; atrial WT: 29 cells, 3 mice; 4SA-Rad: 20 cells, 3 mice. i, Graph of ratio of Ba²⁺ conductance after to Ba²⁺ conductance before isoproterenol treatment versus voltage. Data are mean ± s.e.m. Ventricle WT: 29 cells, 3 mice; 4SA-Rad: 35 cells, 3 mice; atrial WT: 34 cells, 3 mice; 4SA-Rad: 29 cells, 3 mice.

Fig. 2 |. Augmentation of Ca²⁺ influx by phosphatase inhibitors is Rad dependent.

a,b, Volcano plots of fold-change for relative protein quantification by TMT MS of α_1C-APEX2 cardiomyocytes treated with vehicle, isoproterenol or calyculin A (CAL). The data shown are means for five pairs of biologically independent samples. a, Comparison between isoproterenol-treated and vehicle. b, Comparison between calyculin A and vehicle. A nonadjusted, unpaired, two-tailed Student’s t-test was used. PPP1R12A, protein phosphatase 1 regulatory subunit 12A. See Supplementary Table 1 for complete dataset. c,g, Schematics of the FRET pairs, Cer–β_2B with Ven–WT-Rad (c) or Ven–4SA-mutant Rad (g) (where Ven is the Venus tag and Cer is the Cerulean tag). d,e, FRET efficiency (E_D) is plotted against the free concentration Ven–WT Rad in the absence of (d) and 10 min after (e) exposure to calyculin A. a.u., arbitrary units. Solid line fits a 1:1 binding isotherm. f, Graph summarizing K_d,EFF for the binding of WT Rad and the β_2B-subunit. Data are the mean ± s.e.m. (n = 3 for pre- and post-calyculin). **P < 0.01 by unpaired, two-tailed Student’s t-test. h,i, Same as d and e except that Ven–4SA-Rad was used. j, Graph summarizing K_d,EFF for the binding of 4SA-Rad and the β_2B-subunit. Data are the mean ± s.e.m. (n = 3 for pre- and post-calyculin; P = 0.96 (NS) by unpaired, two-tailed Student’s t-test). k,l, Ba²⁺ current elicited by voltage ramp every 6 s, with black traces obtained before and blue traces obtained after calyculin A in WT (k) and 4SA-Rad (l) ventricular myocytes. m, Graph of ratio of Ba²⁺ conductance after calyculin A treatment to Ba²⁺ conductance before treatment versus voltage of ventricular cardiomyocytes isolated from WT and 4SA-Rad mice. Data are mean ± s.e.m. (WT: 21 cells, 3 mice; 4SA-Rad: 19 cells, 3 mice). n, V₅₀ before and after calyculin A. Black dashed line in V₅₀ for WT + isoproterenol. Data are the mean ± s.e.m. **P < 0.01 for WT, P = 0.52 for 4SA-Rad by unpaired, two-tailed Student’s t-test. WT: 18 cells, 3 mice; 4SA-Rad: 18 cells, 3 mice.

Fig. 3 |. Rad phosphorylation is required for adrenergic agonist-induced augmentation of Ca²⁺ transient.

a, Model depicting the targets of β-adrenergic agonist stimulation in cardiomyocytes. TnI: troponin I, P: phosphorylated protein. Schematic created using BioRender.com. b, Schematic of experimental protocol. Cardiomyocytes from WT and 4SA-Rad cardiomyocytes were loaded with Fura2-AM and subsequently field stimulated at 1 Hz. Fluorescence was acquired for 10 s before and after superfusion with vehicle, 100 nM isoproterenol (ISO) or 5 μM forskolin (FSK). SR Ca²⁺ load was determined after infusion of caffeine. c, Graph of fold-change after vehicle infusion of Ca²⁺ transient amplitude versus basal amplitude in WT (black) and 4SA-Rad (red) ventricular myocytes. WT: 128 cells, 3 mice; 4SA-Rad: 175 cells, 3 mice. d, Graph of isoproterenol-induced fold-change of Ca²⁺ transient amplitude versus basal amplitude in WT (black) and 4SA-Rad (red) ventricular myocytes. WT: 225 cells, 4 mice; 4SA-Rad: 183 cells, 5 mice. e, Graph of forskolin-induced fold-change of Ca²⁺ transient amplitude versus basal amplitude in WT (black) and 4SA-Rad (red) ventricular myocytes. WT: 109 cells, 3 mice; 4SA-Rad: 149 cells, 5 mice. f, Graph of isoproterenol- and forskolin-induced fold-change in amplitude of Ca²⁺ transient. Data are mean ± s.e.m. P < 0.0001 by one-way ANOVA, ****P < 0.0001 by Sidak’s multiple-comparison test (n = 128, 225, 109, 175, 183 and 149 cardiomyocytes from 3, 4, 3, 3, 5, 5 mice, respectively, from left to right). g, Same as d except with atrial cardiomyocytes. Atrial myocytes: WT: 133 cells, 4 mice; 4SA-Rad: 75 cells, 4 mice. h, Isoproterenol-induced fold-change in Ca²⁺ transient amplitude. Data are the mean ± s.e.m. ****P < 0.0001 by unpaired, two-tailed Student’s t-test (n = 133, 75 atrial cardiomyocytes from 4 and 4 mice, from left to right). i, Amplitude of basal Ca²⁺ transient for ventricular and atrial cardiomyocytes. Data are mean ± s.e.m. Unpaired, two-tailed Student’s t-test. ****P < 0.0001; *P < 0.05 (n = 462, 507, 133 and 75 cardiomyocytes from 8, 10, 4 and 4 mice, respectively, from left to right).

Fig. 4 |. Adrenergic signaling pathways are fully functional in 4SA-Rad mice.

a, Graph of amplitude of Ca²⁺ transient after caffeine in control, isoproterenoland forskolin-treated cardiomyocytes. Data are the mean ± s.e.m. P < 0.0001 by one-way ANOVA; ****P < 0.0001, **P < 0.01 by Sidak’s multiple-comparison test (n = 121, 120, 66, 129, 165 and 129 cardiomyocytes from 3, 4, 5, 3, 4 and 5 mice, respectively). b, Anti-SR Ca²⁺-ATPase (SERCA) antibody western blot of protein lysates of cardiomyocytes isolated from WT and 4SA-Rad mice. Lower: graph of density normalized to β-actin. Data are the mean ± s.e.m. Unpaired, two-tailed Student’s t-test. P > 0.99 (n = 3 mice in each group). c, Same as b except with anti-NCX antibody western blot. Lower: graph of density normalized to β-actin. Data are the mean ± s.e.m. Unpaired, two-tailed Student’s t-test P = 0.38 (n = 3 mice in each group). d, Graph of time constant of Ca²⁺ transient relaxation under basal conditions and after either isoproterenol or forskolin. Data are the mean ± s.e.m. P < 0.0001 by one-way ANOVA, ****P < 0.0001 by Tukey’s multiple-comparison test (n = 462, 225, 109, 478, 183, 149 cardiomyocytes from 12, 4, 3, 13, 5 and 5 mice, respectively, from left to right). e, Anti-phospho-PLN (P-PLN) antibody (upper) and anti-PLN antibody (middle) western blots of protein lysates of cardiomyocytes. Lower: graph of P-PLN density normalized to PLN density. Data are the mean ± s.e.m. P = 0.43 and P = 0.89, from left to right, by unpaired, two-tailed Student’s t-test (n = 7 mice in each group). f, Same as in e except with anti-phospho-S2808 RyR2 antibody (upper) and anti-RyR2 antibody (middle) western blots. Lower: graph of P-RyR2 density normalized to RyR2 density. Data are the mean ± s.e.m. P = 0.66 and P = 0.97, from left to right, by unpaired, two-tailed Student’s t-test (n = 3 mice in each group). g, Same as in e except with anti-S23/S24 TnI antibody (upper) and anti-TnI antibody (middle) western blots. Lower: graph of P-TnI density normalized to TnI density. Data are mean ± s.e.m. P = 0.46 and P = 0.33, from left to right, by unpaired, two-tailed Student’s t-test (n = 4 mice in each group).

Fig. 5 |. Phosphorylation of Rad required for regulation of contractility.

a,b, Pacing-induced change in sarcomere length before and after superfusion of forskolin. c, Graph showing field-stimulation-induced percentage change in sarcomere length. Data are the mean ± s.e.m. P < 0.0001 by one-way ANOVA; ***P < 0.001, ****P < 0.0001 by Sidak’s multiple-comparison test (n = 29, 29, 38 and 38 cardiomyocytes from 9, 9, 8 and 8 mice, respectively, from left to right). d, Graph of single-exponential decay time constant of reduction in contraction. Data are the mean ± s.e.m. ****P < 0.0001 by unpaired, two-tailed Student’s t-test (n = 29, 29, 38 and 38 cardiomyocytes from 9, 9, 8 and 8 mice, respectively, from left to right). e,i, Representative M-mode echocardiographic recordings before (e) and after (i) intraperitoneal injection of isoproterenol (ISO). f,g, Graphs of ejection fraction and fractional shortening from echocardiograms. Data are the mean ± s.e.m. ****P < 0.0001 by unpaired, two-tailed Student’s t-test (n = 21 mice for each group). h, Graphs of GCS and GLS. Data are the mean ± s.e.m. **P < 0.01, ****P < 0.0001 by unpaired, two-tailed Student’s t-test (n = 20, 23, 20 and 21 mice, from left to right). j, Graph of ejection fraction before and after isoproterenol. Data are the mean ± s.e.m. ****P < 0.0001 and P = 0.29 by unpaired, two-tailed Student’s t-test (n = 7 and 8 mice, from left to right). k, Graph of fractional area of change before and after isoproterenol. Data are the mean ± s.e.m. ****P < 0.0001 and P = 0.27 by unpaired, two-tailed Student’s t-test (n = 7 and 8 mice, from left to right). l, Graph of GLS before and after isoproterenol. ***P < 0.001 and P = 0.69 by unpaired, two-tailed Student’s t-test (n = 7 and 8 mice, from left to right). m, Graph of exercise time for WT and 4SA-Rad mice using either no incline or 15° incline with progressively increasing speed. Data are mean ± s.e.m. *P < 0.05, ***P < 0.001 by Mann–Whitney U-test (n = 16 mice (7 male/9 female), 19 mice (10 male/9 female), 25 mice (12 male/13 female) and 29 mice (15 male/14 female), from left to right).

Fig. 6 |. Phosphorylation of Rad required for regulation of heart rate in vivo.

a, Graph of minimum, mean and maximum heart rate over a 24-h period. Data are the mean ± s.e.m. *P < 0.05 by unpaired, two-tailed Student’s t-test (n = 4 WT mice; 7 4SA-Rad mice). b, Graph of heart rate before and 10 min after intraperitoneal injection of isoproterenol. Data are the mean ± s.e.m. *P < 0.05, ***P < 0.001 by unpaired, two-tailed Student’s t-test. c, Representative diary plots of heart rate during 4-h period after intraperitoneal injection of isoproterenol. The black trace is a WT mouse and the red trace a 4SA-Rad mouse. Representative ECGs are shown for the indicated timepoint. The arrows point to atrial activations (P waves). d, Graph of fraction of time in which heart rates were <400 beats min⁻¹ (bpm), 380 bpm or 350 bpm during a 24-h period without isoproterenol and a 3-h period post-isoproterenol injection. Data are the mean ± s.e.m. *P < 0.05, **P < 0.01 by unpaired, two-tailed Student’s t-test. Specific P values can be found in the associated Source data (see Supplementary Information) (n = 4 WT and 7 4SA-Rad mice).

Fig. 7 |. Augmentation of contractility requires Rad-bound Ca²⁺ channels under basal conditions.

a, Schematic depicting mutation of the Ca_Vβ subunit (DA-β) that prevents Rad binding. b, Anti-β subunit antibody (upper) and anti-β-actin (middle) antibody western blots. Lower: graph of densitometry, normalized to β-actin. Data are the mean ± s.e.m. *P < 0.05 two-tailed, unpaired Student’s t-test (n = 3 mice in each group). c, Anti-α_1C, anti-β and anti-FLAG antibody western blots of anti-α_1C immunoprecipitation, representing three similar experiments. Ig, heavy chain of immunoglobulin. d, Graph of basal conductance density at −20 mV acquired using a ramp protocol. Data are the mean ± s.e.m. *P < 0.05; ****P < 0.0001 two-tailed, unpaired Student’s t-test (n = 28, 24 and 24 cardiomyocytes from 3, 4 and 3 mice, respectively, from left to right). e, Graph of V₅₀. P < 0.0001 by one-way ANOVA; ****P < 0.0001 by Sidak’s multiple-comparison test. WT: 29, 29, 23, 23, 27 and 27 cells from 3, 3, 4, 4, 3 and 3 mice, respectively, from top to bottom. f, Ratio of Ba²⁺ currents after isoproterenol to before isoproterenol versus voltage. Data are the mean ± s.e.m. Sample size same as e. g, Graph of basal Ca²⁺ transient amplitude. The dashed blue line is the mean for WT + isoproterenol. The WT data are the same as in Fig. 3i. Data are the mean ± s.e.m. ****P < 0.0001 by unpaired, two-tailed Student’s t-test (n = 462, 151, 245, 316 cardiomyocytes from 8, 3, 3 and 6 mice, respectively, from left to right). h, Scatter plot of isoproterenol-induced fold-change of Ca²⁺ transient amplitude versus basal transient amplitude in WT (gray—data same as Fig. 3d) and 2DA-β_2B (green) myocytes. The dashed black line is a single-exponential fit of WT data (n = 225 WT cells, 4 mice; 151 2DA-β_2B cells, 3 mice). i, Scatter plot of isoproterenol-induced fold-change of Ca²⁺ transient amplitude versus basal transient amplitude. The blue dashed line is a single-exponential fit of transgenic WT β_2B data (n = 106 WT-β_2B cells, 3 mice; 148 3DA-β_2B cells, 3 mice). j, Representative M-mode recordings. k, Graph of fractional area of change. The dashed lines are means for WT mice without and with isoproterenol. Data are the mean ± s.e.m. ****P < 0.0001 by unpaired, two-tailed Student’s t-test (n = 5 mice from each group).

Fig. 8 |. Adrenergic augmentation of contractility is CaV1.2 dependent.

a, Maximal basal conductance (G_max) density. The black dashed line is the mean 3DA-β_2B G_max. Data are the mean ± s.e.m. ****P < 0.0001 unpaired, two-tailed Student’s t-test (n = 123 and 37 cells from 10 and 4 mice, from left to right). b, Graph of V₅₀ acquired via ramp protocol. Data are mean ± s.e.m. P < 0.0001 by one-way ANOVA, ****P < 0.0001 by Sidak’s multiple-comparison test (n = 29, 29, 35, 35, 26, 26 cells from 3, 3, 3, 3, 4 and 4 mice, respectively, from top to bottom). c, Isoproterenol-induced fold-change of Ca²⁺ channel conductance at −20 mV, acquired via ramp protocol. Data are mean ± s.e.m. P < 0.0001 by one-way ANOVA, ****P < 0.0001 by Sidak’s multiple-comparison test (n = 28, 35 and 26 cells from 3, 3 and 4 mice, respectively, from left to right). d, Basal Ca²⁺ transient amplitude. Data are the mean ± s.e.m. ****P < 0.0001 by unpaired, two-tailed Student’s t-test (n = 507 4SA-Rad cells, 10 mice (same data as Fig. 3i); 467 4SA-Rad/3DA-β_2B cells, 4 mice). e, Isoproterenol-induced fold-change of Ca²⁺ transient amplitude versus basal amplitude. The black dashed line is the exponential fit for the WT (n = 183 4SA-Rad cells, 5 mice (same as Fig. 3d); 160 4SA-Rad/3DA-β_2B cells, 4 mice). f, Isoproterenol-induced fold-change in Ca²⁺ transient amplitude. The WT and 4SA-Rad data same as Fig. 3f. The dashed blue line is mean for isoproterenol-treated 3DA-β_2B cardiomyocytes. Data are the mean ± s.e.m. ****P < 0.0001 by unpaired, two-tailed Student’s t-test (n = 225, 183 and 160 cells from 4, 5 and 4 mice, respectively, from left to right). g, Change in sarcomere length. Data are the mean ± s.e.m. P = 0.50 by unpaired, two-tailed Student’s t-test (n = 22 cells, 3 mice). h, Time constant of Ca²⁺ reuptake. Data are the mean ± s.e.m. ****P < 0.0001 by unpaired, two-tailed Student’s t-test (n = 148 and 160 cells, from 3 and 4 mice, from left to right). i, Fractional area of change. 4SA-Rad is same data as Fig. 5k. Dashed lines are means for WT without and with isoproterenol. Data are mean ± s.e.m. P = 0.27 and P = 0.12 by unpaired, two-tailed Student’s t-test, from left to right (n = 8 mice for each group).