Adenylyl cyclase isoform 1 contributes to sinoatrial node automaticity via functional microdomains
Lu Ren, … , Manuel F. Navedo, Nipavan Chiamvimonvat
Lu Ren, … , Manuel F. Navedo, Nipavan Chiamvimonvat
Published November 22, 2022
Citation Information: JCI Insight. 2022;7(22):e162602. https://doi.org/10.1172/jci.insight.162602.
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Research Article Cardiology

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

Abstract

Sinoatrial node (SAN) cells are the heart’s primary pacemaker. Their activity is tightly regulated by β-adrenergic receptor (β-AR) signaling. Adenylyl cyclase (AC) is a key enzyme in the β-AR pathway that catalyzes the production of cAMP. There are current gaps in our knowledge regarding the dominant AC isoforms and the specific roles of Ca2+-activated ACs in the SAN. The current study tests the hypothesis that distinct AC isoforms are preferentially expressed in the SAN and compartmentalize within microdomains to orchestrate heart rate regulation during β-AR signaling. In contrast to atrial and ventricular myocytes, SAN cells express a diverse repertoire of ACs, with ACI as the predominant Ca2+-activated isoform. Although ACI-KO (ACI–/–) mice exhibit normal cardiac systolic or diastolic function, they experience SAN dysfunction. Similarly, SAN-specific CRISPR/Cas9-mediated gene silencing of ACI results in sinus node dysfunction. Mechanistically, hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) channels form functional microdomains almost exclusively with ACI, while ryanodine receptor and L-type Ca2+ channels likely compartmentalize with ACI and other AC isoforms. In contrast, there were no significant differences in T-type Ca2+ and Na+ currents at baseline or after β-AR stimulation between WT and ACI–/– SAN cells. Due to its central characteristic feature as a Ca2+-activated isoform, ACI plays a unique role in sustaining the rise of local cAMP and heart rates during β-AR stimulation. The findings provide insights into the critical roles of the Ca2+-activated isoform of AC in sustaining SAN automaticity that is distinct from contractile cardiomyocytes.

Authors

Lu Ren, Phung N. Thai, Raghavender Reddy Gopireddy, Valeriy Timofeyev, Hannah A. Ledford, Ryan L. Woltz, Seojin Park, Jose L. Puglisi, Claudia M. Moreno, Luis Fernando Santana, Alana C. Conti, Michael I. Kotlikoff, Yang Kevin Xiang, Vladimir Yarov-Yarovoy, Manuela Zaccolo, Xiao-Dong Zhang, Ebenezer N. Yamoah, Manuel F. Navedo, Nipavan Chiamvimonvat

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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.

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ACI is the predominant Ca2+-activated isoform in the SAN, forming microd...
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, white, and red scale bars: 10, 0.4, and 0.2 μm, respectively. (E) Scatterplot analyses for the colocalization of ACI with Cav-3 and ACI with HCN4 from Airyscan confocal microscopic images, where the fluorescence intensity values of the 2 fluorochromes for each pixel are plotted against each other. (F) Additional analyses for the colocalization of ACI and Cav-3 and of ACI and HCN4 were performed using Pearson’s correlation coefficients (the ratio between the covariance of 2 variables and the product of their SDs) and overlap coefficients (the proportion of overlap between 2 probability distributions, as a measure of the similarity between distributions) from Airyscan images (n = 10–11 cells from 4 mice). (G) Representative super-resolution STED images of isolated SAN cells, triple-labeled for HCN4 (red), ACI (green), and Cav-3 (purple). Scale bar: 0.2 μm. (H) Upper panel: Scatterplot analyses for the colocalization of ACI with Cav-3 and ACI with HCN4 from STED images. Lower panel: Additional analyses for the colocalization of ACI and Cav-3 and of ACI and HCN4 were performed using Pearson’s correlation and overlap coefficients from STED images (n = 8 cells from 3 mice). (I) Representative 3D rendering of PLA in SAN cells for ACI with HCN4, Cav-3, Cav1.2, RyR2, β1-AR, and β2-AR. Supplemental Video 1 shows a 3D rendering of ACI and Cav-3. Scale bar: 5 μm. (J) Summary of PLA data, n = 12–24 cells from 4–6 mice per group. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001 by 1-way ANOVA for multiple comparisons, followed by Kruskal-Wallis post hoc analyses.