C fos

All rats were sacrificed 30 days after MI, and tissues including the heart, SCG, and brain were dissected quickly and then fixed with 4% paraformaldehyde in preparation for histological staining. To determine the ischemic injury, Masson staining (Servicebio, Wuhan, China) was performed in heart sections. Evaluating the expression of α-SMA (Servicebio, Wuhan, China) can reveal the degree of cardiac fibrosis. Tyrosine hydroxylase (TH, Abcam, Cambridge, Massachusetts) and synaptophysin (SYN, Life Technologies, Grand Island, New York) were used to assess the sympathetic activity (20 (link)). To identify the virus-EGFP positive cells in the SCG, TH expression was detected by immunohistochemical staining. The immunofluorescence of c-Fos (Servicebio, Wuhan, China) was used to evaluate the neuronal activity (21 (link)). The fluorescent dye DAPI (4′,6-diamidino-2-phenylindole) was used to locate the position of the nucleus. All analyses were quantitatively carried out with commercially available software (Image Pro Plus, Media Cybernetics, Inc., Rockville, MD).

According to a rat brain atlas (Watson, 2004 ), brains were divided into three blocks. Blocks containing the SSN and NTS were also embedded in paraffin. PPG samples were embedded in paraffin. Each of the paraffin blocks was sectioned in 5 μm-slices. The SSN and NTS were mounted on poly-lysine coated slides, deparaffinized and rehydrated sequentially. All tissues were washed with PBS, then were permeabilized in PBS containing 0.1% Triton X-100 and BSA for 1 h. Subsequently, tissues were incubated with primary antibody overnight at 4°C in c-fos (Servicebio, Wuhan, China, GB11069, 1:200) and VIP (Beyotime Institute of Biotechnology, Haimen, China, bs-0077R, 1:500). Tissue sections were mounted on glass slides and studied with a light microscope.

Animals were transcardially perfused with 0.9% saline under deep anesthesia, and then with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brain was separated immediately and postfixed in 4% paraformaldehyde for 24 h. Soon afterwards, the brain tissue was cut into sections (5 μm) after paraffin-embedded. Paraffin-embedded brain sections or cell climbing slices were washed with 0.1 M PBS and incubated with 0.3% triton X-100 (Servicebio) for 10 min. Then after washed with PBS again, the sections were blocked with 5% bovine serum albumin (BSA) and incubated with the primary antibody overnight at 4 °C, including c-fos (1:200, Servicebio), NeuN (1:200, Proteintech), voltage-dependent anion channel 1 (VDAC1, 1:200, Abcam), Calcitonin gene-related peptide (CGRP, 1:200, Santa-Cruz), Sirt3 (1:200, Novus Biologicals) and Pgc-1α (1:200, Novus Biologicals). The next day, the sections were incubated with the Cy3-/FITC-labeled anti-mouse/rabbit antibody (1:200, Servicebio) for 1 h at RT. Then the nuclei were stained by DAPI. Afterwards, images in the TNC (as shown in Supplementary Fig. 1E) were captured with an ECLIPSE Ti-U microscope/ FV1200 confocal microscopy (Olympus, Japan) using the NLS-Elements BR.3.0 software (Nikon, Melville, NY).