Animal model
Mongrel dogs (male = 7, female = 11) weighing between 15 and 18 kg were purchased through the Experimental of Animal Care Center of Tianjin Medical University, there was no gender difference observed in our study. All the experiments were conducted in accordance with the guide for the Care and the Use of Laboratory Animials and were approved by the ethical committee of Tianjin Medical University.
Experimental groups
All experimental dogs were randomly assigned into three groups: (1) control group (n = 6) received only a coronary angiogram; (2) model group (n = 6) that underwent an established procedure to induce MI and four weeks later underwent a renal arteriogram; (3) treatment group (n = 6) first underwent MI-inducing procedure and then underwent RDN four weeks later.
Establishment of MI model
Dogs in model and treatment groups were anesthetized with sodium pentobarbital (30 mg/kg IV), intubated, and ventilated using a respirator with room air supplemented with oxygen. Continuous ECG monitoring was carried out. After femoral artery access was established and 1000 IU of heparin was injected, 0.2 ml of anhydrous alcohol was injected distally to the first diagonal of the left anterior descending coronary (LAD) as described in a previous study, resulting in left ventricular (LV) damage.
Renal sympathetic denervation
In the treatment group dogs, the ablation electrode (6F saline irrigated catheter tip) was inserted into each artery via the fermoral artery under fluroscopy, and radiofrequency (RF) energy was applied to the endothelial lining with an irrigation flow rate of 12 ml/min. The temperature and power of the radiofrequency ablation instrument was set at 43 °C and 10 W.The target sites were in different directions,lasted for at least 90 s.The catheter was then retreated1–2 cm to generate another ablation location. This procedure was repeated four times in each renal artery, and then, the similar RF energy was applied to the contralateral renal artery.
Transthoracic echocardiography
All of the dogs underwent transthoracic ecchocardiography at baseline, 4 weeks after MI and 4 weeks after RDN (CX50, Philips, Netherlands). The left ventricular ejection fraction (LVEF), left ventricular end-systolic dimension (LVESD), left ventricular end-diastolic dimension (LVEDD), left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure(LVEDP) were recorded. Three consecutive cardiac cycles were observed, and the average values were recorded as the final cardiac parameters. The LVEF was calculated by the formula: (LVVmax- LVVmin) / LVVmax.
Evaluation of plasma NT-BNP and creatinine level
Venous blood were collected in vacutainers for the NT-BNP and Cr assays. Samples for the NT-BNP and Cr assays were centrifuged at 3000 g for 10 min at 4 °C, and the plasma was separated kept in microtubes and stored at − 70 °C until assay. NT-BNP and Cr levels were examined by using enzyme-linked immunosorbent assay (ELISA). All assays were performed twice.
Histological evaluation
After perfusion with ice-cold PBS, the hearts were cut and fixed in 4% phosphate buffered formalin for 48–72 h at 4 °C, subsequently, the tissues were then dehydrated and embedded in paraffin. Infarcted myocardial tissue was flash-frozen by liquid nitrogen then stored at − 80 °C. Masson’s trichrome staining were performed to detect cardiac fibrosis.Connective tissue was differentiated on the basis of its color. Immunohistochemical staining was performed using the Power VisionTM two-step method. The sections were stained with MMP-2(polyclonal rabbit anti-MMP-2 antibody,Abcam, USA) and MMP-9(polyclonal rabbit anti-MMP-9 antibody Santa Cruz, USA). Immunohistochemical score (IHS) was utilized. This method has been shown to approximate data generated from image analysis-based scoring systems as described in a previous study [5]. HE staining was used to detect the renal artery.
TUNEL staining
TUNEL staining was performed using a commercial kit (In Situ Cell Death Detection FITC Kit or TMR red, Roche). Myocardial tissues were fixed in 4% paraformaldehyde and dehydrated with ethyl alcohol. Paraffin sections were incubated with anti-α-actin in antibodies (1:200 dilution; Sigma-Aldrich) in a humidified chamber. The cells that exhibited condensed nuclei with an irregular form or nuclei split into green particles were considered to be TUNEL-positive cells. The apoptotic index (AI) was calculated according to the following formula: AI = (number of apoptotic cells/total number of nuclei) × 100%.
Western blot
Protein expression levels of Bcl-2 (MDL, China), Bax (MDL, China), caspase 3 (MDL, China), and GRP78 (MDL, China) were assessed by western blotting. All antibodies were applied according to the manufacturer’s instructions. The integrated optical densities of the protein bands were obtained by an imaging system.
RT-PCR
mRNA levels were assessed by using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted from myocardial tissue. Purified RNA was quantified, and cDNA was synthesized using an I-script cDNA synthesis kit (Bio-Rad).
TaqMan primers from Life Technologies were used in the quantitative real-time RT-PCR. The sequences for the primers are summarized in supplemental Table 1. The values were corrected based on ß-actin levels, and the final mRNA level was calculated using the formula x=2-ΔΔCT, where x is the fold change relative to the control.
Statistical analysis
The data were presented as the mean ± SD. Group comparisons were subjected to analysis of variance (ANOVA), followed by the least significant difference (LSD) test to identify differences among various groups,and a probability value < 0.05 was required for statistical significance(version 20.0 SPSS).