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

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.

the tissue chunks. Dissociated SAN cells were used for experiments at room temperature (RT, 22-25°C) or 36 ± 0.5 °C.

Single-molecule fluorescence in situ hybridization (smFISH)
SmFISH was performed as previously described (9) in WT and ACI KO SAN sections using probes for ACI, ACV, and ACVI. Isolated samples were incubated with 4% diethylpyrocarbonate (DEPC)-paraformaldehyde (PFA) for 24hr, followed by incubation with 30% sucrose for 2-3 days. Samples were then embedded in the optimal cutting temperature (OCT) compound and cryo-sectioned (10 µm) onto superfrost slides. RNAscope multiple fluorescent detection reagents v2 (Advanced Cell Diagnostics, ACD, #323111, Newark, CA) were used for the assay. Probe hybridisation was performed according to the manufacturer's instructions (ACD).
Sections were immersed in 4% PFA for 15 minutes at 4°C and serially dehydrated in 50%, 70%, and 100% ethanol for 5 minutes at RT. Sections were then treated with hydrogen peroxidase for 10 minutes at RT, followed by proteinase digestion using protease 4 for 30 minutes at RT. The following steps were performed at 40 °C. Probes were reacted for two hours. AMP1, AMP2, AMP3 reagents were used to amplify the signal. The appropriate horseradish peroxidase (HRP) reagent and Opal dye were used. HRP blocker was then added to complete the reaction. Slides were washed twice after every step at RT. Probes for Adcy1 (#451241), Adcy6 (#539861), GAPDH (#4471841), and controls were obtained from ACD. For subsequent immunofluorescent staining, slides were treated with 10% goat serum for 30 mins at RT, incubated with primary antibody specific for HCN4 (1:500 dilution, Alomone Labs, Jerusalem, Israel) overnight at 4°C, washed with TBS-0.005% Tween20 three times for 5 mins each, incubated with secondary antibody (fluorescein isothiocyanate (FITC), ThermoFisher Scientific, 1:500 dilution) for 2 hours at RT, and again washed with TBS-0.005% Tween20 three times for 5 mins each. Slides were incubated in 4′,6-diamidino-2-phenylindole (DAPI) solution for 30 secs at RT to label cell nuclei. They were then mounted on Fluoromount-G and sealed under a coverslip.

Whole-mount Immunohistochemistry
Whole-mount Immunohistochemistry was performed as described previously (10,11). SAN tissue was dissected and fixed with 4% PFA for 30 min and was then dehydrated through a graded ethanol series (25, 50, 75, 95, 100%), cleared and bleached for 2 hours with 20% dimethyl sulfoxide (DMSO) in ethanol and 12 hours with hydrogen peroxide (6%) in ethanol. Tissue was then rehydrated through a graded ethanol series, washed in PBS (3 × 10 min). SAN tissue was permeabilized with 0.5% Triton X-100 in PBS for 30 mins, blocked for 2 h with 5% normal donkey serum at RT and then incubated with primary antibodies in PBS at 4°C. The following primary antibodies were used: (1) anti-HCN4 (Abcam, Cambridge, MA, 1:200 dilution), a polyclonal antibody raised against rat HCN4, (2) anti-ACI (Santa Cruz Biotechnology, Inc., Dallas, TX, 1:100 dilution), a monoclonal antibody raised against mouse ACI. SAN tissue was washed with PBS (3 x 10 minutes) and then incubated with anti-rat and anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, 1:1000 dilution) for 4 hours at RT in the dark. It was then washed in PBS (3 × 10 min) and incubated for 2 h with 20% DMSO diluted in PBS. Coverslips were mounted on the slides with ProLong Diamond Antifade Mountant (ThermoFisher Scientific Inc.). The slides were sequentially imaged using a Zeiss 900 confocal laser-scanning microscope equipped with an Airyscan detector module, a Plan-Apo 63× 1.4 NA oil-immersion objective and 488/561 lasers. Imaris software (Bitplane, Zürich, Switzerland) was used to perform 3D reconstructions.

Echocardiography
Echocardiography to assess systolic and diastolic function were performed using Vevo 2100 (VisualSonics, Fujifilm, Toronto, ON, Canada) imaging system and a MS 550D probe (22-55 MHz) (12,13). Systolic function was assessed using M-mode and two-dimensional measurements in conscious mice. Diastolic function was assessed in mice anesthetized with 1% isoflurane. The measurements represented the average of six selected cardiac cycles from at least two separate scans performed in a blinded fashion with papillary muscles used as a point of reference for consistency in level of scan. End diastole was defined as the maximal left ventricular (LV) diastolic dimension and end systole was defined as the peak of posterior wall motion.
Fractional shortening (FS), a surrogate of systolic function, was calculated from LV dimensions as follows: Fractional shortening (FS), a surrogate of systolic function, was calculated from LV dimensions as follows: FS = ((EDD-ESD)/EDD) x100%, where EDD and ESD are LV end diastolic and end systolic dimension, respectively. A pulsed Doppler velocity profile of inflow across the mitral valve (MV) was performed to assess the diastolic function. E/A ratio (E wave/A wave) was quantified to define diastolic function.

Hemodynamic monitoring
Mice were anesthetized by intraperitoneal injection of 80 mg/kg of ketamine and 5 mg/kg of xylazine and maintained at 37 ºC. Hemodynamic monitoring was performed as previously described (14). The arterial catheter was inserted retrogradely into the left ventricle via carotid artery. The recording of pressure and volume was performed by using Millar Pressure-Volume System MPVS-300 (Millar, Inc., Houston, TX), Power Lab, and Lab Chart 6.0 software (AD Instruments, Colorado Springs, CO). The pressure and volume were calibrated before recordings.
The volume calibration used fresh heparinized 37 °C mouse blood and a cuvette (P/N 910-1049, Millar, Inc.). To change the preload, a gentle and quick abdominal compression was applied to occlude inferior vena cava.

Electrocardiography (ECG) Telemetry
All telemetry placements were performed 1 week before the start of each experiment. Mice were anesthetized with ketamine/xylazine (80 mg/kg /5 mg/kg) before placement of a transmitter (Data Sciences International (DSI), New Brighton, MN) into the abdominal cavity with subcutaneous electrodes in the lead I configuration. Baseline measurements were recorded for 24 hours and followed by intraperitoneal injection of isoproterenol (ISO, 0.1 mg/kg, IP) in ACI -/-, ACVIII -/and WT animals. Atropine (2 mg/kg, ip) and propranolol (1 mg/kg, ip) were used to block the heart's autonomic control. The analog telemetric ECG signals were digitised at 1 kHz and recorded using PONEMAH software (DSI). R peaks of the ECG signal were detected, and the mean HR was calculated from the RR interval and averaged for 1 min. For baseline recordings, t=0 corresponds to noon, while t=24 corresponds to midnight. HR variability (HRV) was plotted as RR interval (RR-I) against the next RR interval.

SAN-specific CRISPR/Cas9-mediated gene silencing of ACI
A transgenic mouse model expressing a fluorescent Ca 2+ indicator (GCaMP8) under the control of the Hcn4 promoter was previously generated and used for the study (15). CRISPR/Cas9 system containing 3X sgRNA (GeneCopoeia, Rockville, MD) was used to specifically target the ACI isoform, followed by in vivo delivery using liposome and SAN painting technique (16,17). A vector containing a scrambled sequence was used as control. Both the targeting and control vectors contained mCherry and were encapsulated in liposomes. The liposomal emulsion was delivered onto the SAN region under direct visualization. ECG and echocardiograms were performed 5-7 days after surgery at baseline and after ISO injection. Light Sheet-Based Fluorescence Microscopy (LSFM) was performed in freshly dissected SAN to confirm that the in vivo gene delivery was successful. Green fluorescence protein (GFP) and mCherry signals were simultaneously detected during live SAN imaging.

Light Sheet-Based Fluorescence Microscopy (LSFM)
Freshly dissected tissues were placed in normal Tyrode's solution, immersed in 1.5% agarose in a capillary tube, and mounted inside the Lattice Lightsheet 7 microscope (Carl Zeiss, Jena, Germany). During experiments, tissue was maintained at 37 °C and constantly gassed with 95% O2/5% CO2. Baseline measurements were taken before the application of 1 µM of ISO. Imaris software (Bitplane, Zürich, Switzerland) was used to perform 3D reconstructions.

Immunofluorescence Confocal Microscopy
Immunofluorescence labeling was performed as previously described (18). Isolated SAN cells were allowed to adhere to coverslips for 10 minutes before fixing with 4% PFA. Cells were then washed with phosphate-buffered saline (PBS, 3 x 10 minutes). Cells were permeabilized for 10 minutes with 1% Triton X-100% and then blocked with 5% donkey serum for 1h at RT. The

Electrophysiology
Whole-cell L-type and T-type Ca 2+ currents (ICa,L and ICa,T), HCN currents (If) and Na + current (INa) were recorded at 36 ± 0.5 °C using conventional whole-cell patch-clamp techniques (19). Current-voltage relations were assessed before and after the application of ISO (1 µM). Cell Cells incubated with only one primary antibody were used as negative controls. PLA probes (antimouse MINUS and anti-rabbit PLUS) were used as secondary antibodies to bind to primary antibodies. Ligase was added to cells to allow hybridization with the probes, and polymerase was added for a rolling circle amplification reaction. Coverslips were mounted on a microscope slide with Duolink mounting medium. The fluorescence signal was detected using a Zeiss confocal LSM 700 microscope. Images were collected at different optical planes (z-axis step = 0.5 mm). The stack of images for each sample was combined into a single-intensity projection image used to analyze the number of puncta/µm 2 per cell. All the data were analyzed in a blinded fashion with the NIH ImageJ software v1.53c.

Whole-cell Ca 2+ Transient Measurements
IonOptix contraction system (IonOptix LLC, Westwood, MA) was used to detect spontaneous Ca 2+ transients from single isolated SAN cells. Freshly isolated SAN cells were loaded with 5 µM Fluo-4 AM (F14201, ThermoFisher Scientific) for 15 minutes at RT. Cells were then perfused with Tyrode's solution (36 ± 0.5 °C) continuously. Baseline measurements were taken before ISO was applied in both WT and ACI -/mice. The maximal Fluo-4 fluorescence (F) was measured at peak amplitude and was normalized to the average of baseline fluorescence (F0).
Background fluorescence was subtracted for each recording.

Local Ca 2+ Release and Ca 2+ Transient Detection via Confocal Line Scanning
Local Ca 2+ release and Ca 2+ transients were quantified as previously described (23).
Freshly isolated SAN cells were loaded with 5 µM Fluo-4 AM for 15 mins at RT. Cells were then perfused with Tyrode's solution (36 ± 0.5 °C) continuously. Line-scan images across the whole cell were obtained to quantify local Ca 2+ signals with 488 nm excitation and 510 nm emission from the SAN cells. Pixel time was 0.76 µs; line time was 0.91 bms. Pinhole was set at 1.00 AU (Airy unit). Baseline recordings were performed before ISO was applied in both WT and ACI -/mice.

Culture of SAN Cells
SAN cells were first isolated as described above and maintained in culture as we have previously described (7) (Supplementary Fig. 12A-C). We demonstrate that SAN cells maintained in our culture condition retain their elongated morphology and action potential (AP) waveform for up to 40 hours. The culturing condition does not change β-adrenergic-mediated cAMP signal as determined in freshly dissociated and cultured SAN cells from a cardiac-specific cAMP reporter mouse (7). supplemented with 1x penicillin-streptomycin-glutamate (PSG), 4 mM NaHCO3, 10 mM HEPES, 10% fetal bovine serum (FBS), 6.25 µM blebbistatin and plated on the pre-coated laminin coverslips and incubated for 4 hours at 37°C in 5% CO2, before the media was replaced with serum-free M1018 (24).

Adenoviral Transfection of cAMP Biosensors in SAN Cells and Confocal Imaging
For adenoviral transfection, the media was replaced with 500 µL of serum-free medium containing adenoviral vectors carrying different versions of the FRET-based cAMP Universal Tag for imaging experiments (CUTie) sensor (25). Accordingly, we employed the cytosolic CUTie, the membrane-targeted AKAP79-CUTie, and sarcoplasmic reticulum-targeted AKAP18δ-CUTie.
Cells infected with the desired adenoviral vectors were incubated at 37°C with 5% CO2 for 36 to 40 hours. Adenoviral vectors were produced using the AdEasy system (Qbiogene Inc., Carlsbad, CA).(26) A Zeiss LSM 700 laser scanning confocal microscope paired with a Zeiss 63x oil immersion lens (numerical aperture = 1.4) was used to collect images at different optical planes

Statistical Analysis
Data were analyzed using GraphPad Prism (San Diego, CA) software and presented as mean ± SEM. Data were assessed for potential outliers using the GraphPad Prism Outlier Test and for normality of distribution. Statistical significance was then determined using appropriate unpaired two-tailed Student's t-test, nonparametric tests, one-way analysis of variance (ANOVA) or two-way ANOVA for multiple comparisons with appropriate post hoc test. Two-way ANOVA was followed by Holm-Sidak multiple comparison test. General linear model was used for twoway repeated measures and mixed-effect model was used when there were missing values. p<0.05 was considered statistically significant.

Data availability
All data generated or analyzed in this study are included in the main manuscript and/or Supplementary Table S1. Test for normal distribution for Figure 6. Anderson-Darling test was used.
Test for Normal Distribution for Figure 6 Panel