Microvascular rarefaction in the sinoatrial node

This study tested the hypothesis that sinoatrial node microvascular rarefaction occurs in a model of heart failure with preserved ejection fraction (HFpEF). While it has been established that the sinoatrial node exhibits vascular heterogeneity, it was not known whether microvessel density changes regionally or is altered overall during HFpEF. Data provided here demonstrates vascular and pacemaker cell densities under normal conditions with representations at low and high magnifications. 

Methods
Whole-Mount Immunohistochemistry

Following confirmation of isoflurane anesthesia, we rapidly excised hearts and placed them in prewarmed (37 °C) heparinized (0.2 mg/mL) Tyrode III solution (in mM: 140 NaCl, 5.4 KCl, 1.0 MgCl₂, 1.8 CaCl₂, 5.0 HEPES, and 10 d-glucose, pH adjusted to 7.4 with NaOH). We then dissected the SAN and surrounding regions, including portions of the right atrial appendage, superior vena cava, and inferior vena cava. We pinned and stretched the tissues in a 35 mm Sylgard-coated dish, rapidly rinsed them three times in phosphate-buffered saline (PBS), and fixed them in 2 mL of 4% paraformaldehyde (0.01 M PBS) for 1 hour at room temperature. After fixation, we washed the tissues three times with PBS and transferred them to a 50 mL tube containing PBS for 12 hours at 4°C. We performed tissue dehydration, bleaching, and clearing in dimethyl sulfoxide (DMSO) to reduce tissue autofluorescence, as previously described [PMID: 25262932, 16806289]. Briefly, we dehydrated tissues through an EtOH gradient (25, 50, 75, 95, 100%), cleared for 2 hours in 20% DMSO in EtOH, and bleached tissues for 12 hours in 6% hydrogen peroxide in EtOH. We achieved rehydration via a reverse ethanol gradient, followed by three 10-minute PBS washes. We permeabilized tissues with 0.5% Triton X-100 in PBS (3 x 10min). We blocked non-specific binding with 5% normal donkey serum in 0.5% Triton X-100 PBS for 2 hours. After PBS washes (3 x 10min), we incubated the tissues for 48 hours at 4 °C in a 200μL volume of primary antibody cocktail, which included: goat anti-mouse CD31 (1:50; AF3628, R&D Systems) and rabbit anti-mouse HCN4 (1:200; ab289962, Abcam) diluted in PBS. We washed primary antibodies three times in PBS followed by incubation in 1 mL of secondary antibodies cocktail containing donkey anti-goat Alexa Fluor 488 (1:1000, ab150129, Abcam), and donkey anti-rabbit Alexa Fluor 594, (1:1000, ab150076; Abcam) for 3 hours at room temperature with minimal light exposure. After labeling, we washed tissues in PBS, and incubated them for 2 hours in a 25% DMSO PBS solution. We then mounted tissues in Vectashield vibrance medium (Vector Laboratories) with a coverslip and allowed mounting media hardening for 12 hours before acquiring images.

Immunohistochemistry Image Acquisition

For confocal tissue imaging, we used an inverted Leica Stellaris 5 microscope (Leica Microsystems, Wetzlar, Germany) equipped with 10x objective (Leica HC PL APO 10x, NA 0.40 CS2) for imaging the entire dissected tissue, 20x objective (Leica HCX PL FL 20x, NA 0.40 CS2) for focusing on the entire SAN region and, 63x oil-immersion objective (Leica HC PL APO 63x, NA = 1.4 Oil CS2) for specific sSAN and iSAN regions. We captured sSAN and iSAN images at 0.18 x 0.18x 0.30 μm/pixel resolution (231 μm2 field of view) using Leica Application Suite X (LAS X) software. We acquired Z-stacks to span the entire SAN tissue depth (approximately 406 Z-planes). We used the LAS X Navigator to perform stitching with an overlap of approximately 15%.