Adenylyl cyclase isoform 1 contributes to sinoatrial node automaticity via functional microdomains

Figures

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  Figure 1. ACI is the predominant Ca2+-activated isoform in the SAN, forming microdomains with key Ca2+ handling proteins and HCN4 channels. Summary data of relative abundance of ACI-VIII in single cells isolated from 3 regions of the heart. (A) Single-cell qPCR from SAN, atria, and ventricles (n = 23, 11, 7 cells, respectively, from n = 3–5 mice for each group). (B) Representative smFISH images of ACI mRNA expressions in the SAN tissue. HCN4 (green) was used as a counterstain for SAN tissue, and DAPI (blue) was used to stain the nuclei. Images were obtained from SAN tissues that were cryo-sectioned (10 μm) onto super-frost slides. Approximately 20–30 sections were obtained for each sample. Scale bar: 20 μm. (C) Representative high-resolution Airyscan immunofluorescence images of whole-mount SAN tissues, stained with anti-HCN4 (red) and ACI (green) antibodies. Images at higher magnifications are shown in the second rows for each group. Scale bars: 5 μm. (D) Representative high-resolution Airyscan images of isolated SAN cells, stained with anti-HCN4 (red), ACI (green), and Cav-3 (blue) antibodies. Images at higher magnifications are shown in the second and third rows for each group. Yellow,…

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  Figure 2. ACI–/– mice exhibit sinus node dysfunction. (A–D) Representative M-mode echocardiographic images (A) for WT and ACI–/– mice. Summary data from echocardiography for heart rate in beats per minute (bpm) (B), fractional shortening (FS) (C), and mitral valve (MV) E/A ratio (D). (E) Representative ECG tracings of WT and ACI–/– mice at baseline and after β-AR stimulation. (F) Heart rates (bpm) over 24-hour period are plotted for WT and ACI–/– mice (data are expressed as mean ± SEM; n = 23 and 19 mice for WT and ACI–/–, respectively). Heart rates were averaged after every hour for a 24-hour recording. (G) Heart rate variability, plotted as RR intervals (RR-I) for n beat and n + 1 beat in ms. (H) Heart rate histograms with numbers of values for each RR-I. (I) Representative heart rate traces of WT and ACI–/– mice after injection of atropine, followed by propranolol to block autonomic nervous system. (J) Summary data of the maximum, minimum, median, and intrinsic heart rates from I (n = 10 and 14 mice for WT and ACI–/–, respectively). Summary data in B–D and J are shown as violin plots with symbols within the plots representing individual data points with median as well as quartiles…

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  Figure 3. SAN-specific CRISPR/Cas9-mediated ACI gene silencing (CRISPR ACI–/–) exhibit sinus node dysfunction. (A) Representative surface ECG recordings for SAN-specific CRISPR/Cas9-mediated ACI gene silencing mice at baseline and after ISO stimulation. Delivery of scrambled sgRNA was used as control. (B) Summary data for heart rate (bpm) at baseline and after ISO injection (n = 6 mice for each group). (C) SAN isolated from liposome-treated mice with the following landmarks. LA, left atrium; RAA, right atrial appendage; IVC, inferior vena cava; SVC, superior vena cava. (D) Representative images of SAN tissue with mCherry signals from the reporter gene and green fluorescence signals from the genetically encoded HCN4-GCaMP8 transgenic mice. (E) Ca2+ signals are shown for WT and SAN-specific CRISPR/Cas9-mediated ACI gene silencing SAN tissues. Supplemental Video 2 shows Ca2+ signals from a SAN tissue from SAN-specific CRISPR/Cas9-mediated ACI gene silencing. (F) Representative CaT traces from SAN-specific CRISPR/Cas9-mediated ACI gene silencing SAN tissue compared with control mice (treated with scrambled sgRNA) at baseline and after ISO stimulation. (G) Summary data for CaT frequency (bpm).…

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  Figure 4. ACI–/– SAN cells exhibit reduced AP firing frequency. (A and B) Representative traces of spontaneous APs at baseline and after ISO from (A) WT and (B) ACI–/– SAN cells. Dotted lines in the zoomed-in traces indicate 0 mV. (C) Scattered plots of interspike intervals for n beat and n + 1 beat in ms. (D) The number of values for each interspike interval was plotted as histograms. (E) Summary data of beating rate (bpm). (F) AP duration at 90% repolarization (APD90). (G) Peak potentials. (H) Maximum diastolic potentials. (I) Percentages of nonfiring duration. (J) Percentages of cells that exhibited nonfiring activity. n = 12–15 cells from n = 6–7 mice per group. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 using 2-way ANOVA with Holm-Sidak multiple-comparison post hoc analyses.

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  Figure 5. ACI–/– SAN cells exhibit an impaired β-AR response of Ca2+ transients (CaTs) with Ca2+ alternans and a blunted response of local Ca2+ release (LCR) to β-AR stimulation. (A) Representative whole-cell CaT traces of SAN cells from WT and ACI–/– mice before and after ISO application. (B–G) Summary data of τrise (B), τdecay (C), percentages of nonfiring duration (D), normalized peak amplitude (E), beating rate (F), and percentage of cells exhibiting nonfiring activity (G). Number of symbols in the bar graphs represents number of cells. n = 19–33 cells from n = 6–7 mice per group. (H) Representative LCR recordings from WT and ACI–/– SAN cells before and after ISO application. (I) Representative 3D reconstructions of Ca2+ sparks in WT SAN cells. (J–M) Summary data of spark rate (spark numbers per AP cycle, normalized per 100 μm) (J), amplitude (K), full width at half maximum (FWHM) (L), and full duration at half maximum (FDHM) (M). Each symbol represents the average of sparks from 1 cell. n = 16–18 cells from n = 6–7 mice per group. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by 2-way ANOVA with repeated measures, followed by Holm-Sidak…

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  Figure 6. ACI–/– SAN cells demonstrate a significant decrease in β-AR responses of ICa,L but not ICa,T. (A) Representative traces of ICa,L at baseline (top panels) and after ISO perfusion (bottom panels) in WT (left panels) and ACI–/– SAN cells (right panels). Representative ICa traces were recorded using 400 ms test pulses from a holding potential of –55 mV to test potentials between –50 and +40 mV with 10 mV increments, using whole-cell patch-clamp mode. (B and C) Normalized I-V relationship of ICa,L before and after ISO stimulation in WT (B) and ACI–/– (C) SAN cells. The normalized conductance-voltage relationship from B and C are fitted with a Boltzmann function (V1/2 are –29.2 and –35.6 mV for WT SAN cells, while V1/2 are –30.8 and –33.6 mV for ACI–/– SAN cells, before and after ISO, respectively). (D) Superimposition of representative traces for each group at a test potential of –10 mV. (E) Summary data of current density at –10 mV. (F) Representative traces of ICa,T at baseline (top panels) and after ISO (bottom panels) in WT (left panels) and ACI–/– (right panels) SAN cells. (G and H) Normalized I-V relationship of ICa,T before and after ISO perfusion in WT (G) and ACI–/– (H) SAN…

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  Figure 7. ACI–/– SAN cells show a lack of response of If to β-AR stimulation. (A) Representative traces of If for WT and ACI–/– SAN cells before and after ISO administration. Zero-current levels are shown using dotted lines. The inset shows a diagram of the voltage-clamp protocol from –140 mV to –40 mV in 10 mV increments from a holding potential of –35 mV. (B) Superimposed individual If traces from each group at –70 mV at baseline and after ISO. (C) Summary data of current density at –70 mV. (D) Normalized conductance-voltage relationship before and after ISO application, fitted using a Boltzmann function. (E–I) Summary data for half-activation voltage (V1/2) (E), slope factor (F), τfast (G), τslow (H), and time constants of deactivation (I). n = 10–14 cells from n = 4–5 mice per group. Data are expressed as mean ± SEM in C and E–I. *P < 0.05 and **P < 0.01 by 2-way ANOVA, followed by Holm-Sidak multiple-comparison post hoc analyses. Results from normality tests are shown in Supplemental Table 2.

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  Figure 8. ACI differentially regulates local cAMP signaling at functional microdomains in SAN cells. (A) Representative confocal images of SAN cells expressing CUTie sensors localized to the cytosol, plasma membrane (AKAP79-targeted), and SR (AKAP18δ-targeted). (B and C) Representative time course of changes in FRET response (R/R0) in SAN cells expressing CUTie sensors localized to the cytosol (black), plasma membrane (AKAP79; pink) and SR (AKAP18δ; blue) upon application of the AC activator forskolin (10 μM) and the PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX, 100 μM) (B), and the β-AR agonist isoproterenol (ISO, 1 μM) (C). The FRET ratios were normalized to basal levels before treatment. (D–F) Time course of changes in the magnitude of normalized FRET responses (R/R0) in WT (black) and ACI–/– (red) SAN cells expressing the CUTie sensors after stimulation with ISO (1 μM). Bar graphs on the right show corresponding summary data for the maximal increase in the FRET ratio response to ISO. (G–J) Representative time course of changes in the magnitude of normalized FRET responses (R/R0) in WT (G and I) and ACI–/– (H and J) SAN cells expressing CUTie sensors localized to the cytosol (cyt),…