Human IMD (IMD1-53) with the sequence His-Ser-Gly-Pro-Arg-Arg-Thr-Gln-Ala-Gln-Leu-Leu-Arg-Val-Gly-Cys-Val-Leu-Gly-Thr-Cys-Gln-Val-Gln-Asn-Leu-Ser-His-Arg-Leu-Trp-Gln-Leu-Met-Gly-Pro-Ala-Gly-Arg-Gln-Asp-Ser-Ala-Pro-Val-Asp-Pro-Ser-Ser-Pro-His-Ser-Tyr-NH2 with an intramolecular disulfide bond between Cys16-Cys21 (17) was synthesized by ShineGene Bio-Technologies (Shanghai, China).

Adult male Sprague Dawley rats, (weight, 280–320 g; n=60), were supplied by Sino-British Sippr/BK Lab Animal Ltd. (Shanghai, China) The animal experiment was in compliance with the National Research Council's protocol for the Care and Use of Laboratory Animals, and was approved by the Animal Care Committee of Shanghai General Hospital (Shanghai, China). The HF model was induced in Sprague Dawley rats by ligation of the left anterior descending (LAD) coronary artery. Briefly, all rats were anesthetized with intraperitoneal injection of 1% sodium pentobarbital (40 mg/kg; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), endotracheal intubated and mechanically ventilated with a small animal ventilator. Then all rats underwent thoracotomy and pericardiotomy, and the LAD coronary artery was ligated by a 6–0 prolene suture at the origin. Successful myocardium ischemia was verified by ST-segment elevation on an electrocardiogram. The sham group rats underwent same procedure without LAD coronary artery ligation.

The rats that survived 24 h after surgery were randomly assigned to the following 3 groups: Sham (n=10), where rats were administrated subcutaneously with saline (0.6 µg/kg/h) by a mini-osmotic pump (Alzet model 2004; DURECT Corporation, Cupertino, CA, USA) for 4 weeks; HF (n=18), where rats were administrated subcutaneously with saline (0.6 µg/kg/h) by a mini-osmotic pump for 4 weeks; and HF rats with IMD treatment (HF+IMD group; n=20), where rats were daily administrated subcutaneously with IMD (0.6 µg/kg/h) (13) by a mini-osmotic pump for 4 weeks. After 4 weeks, rats underwent echocardiographic examination, haemodynamic measurement and ventricular fibrillation threshold (VFT) determination. After that, animals were sacrificed and hearts were excised for further study.

Rats were lightly anesthetized with 1% sodium pentobarbital, (40 mg/kg; Sinopharm Chemical Reagent Co., Ltd.) and transthoracic echocardiography was performed with a 30 MHz high frequency transducer (VisualSonics Vevo770; VisualSonics, Inc., Toronto, ON, Canada) as previously described (18). End-diastolic and end-systolic left ventricle diameters were measured by M-mode tracing. Left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) were calculated. Following echocardiography study, haemodynamic parameters were measured using the BL-420E Biological system (Chengdu Tai-Meng Science and Technology Co., Ltd., Chengdu, Sichuan, China). Briefly, a catheter was inserted into the right common carotid artery to monitor the arterial blood pressure. Subsequently the catheter was inserted into the left ventricle to record the left ventricular end-diastolic pressure (LVEDP) and the maximal rate of left ventricular pressure increase and decrease (±LVdp/dt_max).

A total of 5 rats in the HF+IMD group and 7 rats in the HF group succumbed during the haemodynamic measurement and anesthesia prior to the second thoracotomy. Therefore, 10 rats in each group were selected for the determination of VFT. VFT measurement was performed as previously described (19). A bipolar needle pacing electrode was penetrated into the peri-infarct zone, which was defined as the zone with a <3-mm width located between the infarct zone and the non-infarct area. A train of rectangular 4 ms pacing pulses was performed for 5 sec at a frequency of 50 HZ using the BL-420E Biological system. The interval between pulse trains was 1 min. The pacing voltage of the first pulse train started at 4 V and progressively augmented by a step of 1 V. The VFT was the minimum voltage which induced ventricular fibrillation (sustained >2 sec).

Following determination of VFT, all rats were sacrificed by injecting a fatal dose of pentobarbital (200 mg/kg; Sinopharm Chemical Reagent Co., Ltd.). The heart was excised and rinsed in cold saline. The atria and right ventricle were trimmed off, and the left ventricle was weighed. A transverse section obtained from the papillary muscle level of the left ventricle was fixed in 10% formalin and embedded in paraffin. The section was then sectioned into 4-µm slices and stained with haematoxylin-eosin for infarct size measurement. The infarct size was determined as previously described (9). Rats with infarct size <30% were excluded from analysis (n=2 from the HF+IMD group; n=1 from the HF group) (20). Left ventricular tissues from the peri-infarct zone were fixed in 10% formalin for 24 h and embedded for immunohistochemical studies and frozen in liquid nitrogen for western blot analysis.

Paraffin-embedded tissues were sectioned into 4-µm slices using a microtome (Leica Microsystems, Wetzlar, Germany). Following deparaffinization and rehydration, slides were treated with 3% H₂O₂ and subjected to heat mediated antigen retrieval with citrate buffer. The slides were incubated with primary antibodies against tyrosine hydroxylase (TH; 1:200, cat no. ab75875) and growth associated protein 43 (GAP43; 1:500, cat no. ab75810) (both from Abcam, Cambridge, UK) overnight at 4°C. Following this, the slides were washed and incubated with a horseradish peroxidase (HRP)-conjugated immunoglobulin G secondary antibody (cat no. GK600705; Gene Tech Biological Technology, Inc., Shanghai, China) for 30 min at room temperature. The immunoreactivity was developed with 3, 3′-diaminobenzidine tetrahydrochloride (Biological Technology, Inc.). Slides were counterstained with hematoxylin and analyzed under a light microscope.

The densities of TH- and GAP43-positive nerve fibers were determined using Image-Pro Plus version 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) and expressed as the nerve area divided by the total area examined. For each heart, one slide was taken to study. Each slide was divided into four quadrants at a ×400 magnification and one microscopic field with the highest nerve density in each quadrant was selected for nerve counting. The mean nerve density of four selected fields was used to represent the nerve density of that slide.

Briefly, heart tissues were minced and then lysed for 20 min on ice with lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). The lysates were centrifuged at 10,000 × g for 15 min at 4°C and the supernatants were collected. Total protein was extracted, the concentrations were determined using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology, Haimen, China). Equal amounts of proteins (20 µg) were separated by SDS-PAGE (10% separating gel and 5% stacking gel; 80 V; 120 min) and subsequently transferred onto PVDF membranes (EMD Millipore, Billerica, MA, USA). After that, the membranes were blocked with 5% non-fat milk for 2 h at room temperature. Membranes were then incubated with rabbit monoclonal antibodies against TH (1:2,000) and GAP43 (1:10,000), a rabbit polyclonal antibody against NGF (1:2,000, cat no. ab6199; Abcam), and a mouse monoclonal antibody against GAPDH (1:5,000, cat no. 60004–1-Ig; ProteinTech Group, Inc., Chicago, IL, USA) overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated goat anti-rabbit (1:5,000, 111–035-003; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and anti-mouse IgG secondary antibodies (1:2,000, cat no. A0216; Beyotime Institute of Biotechnology) at room temperature for 1 h. The bands were visualized using an Enhanced Chemiluminescence kit (EMD Millipore). The protein expressions were normalized to GAPDH protein content.

On the day of sacrifice, 1 ml fresh blood was collected from all rats via the inferior vena cava and centrifuged at 1,500 × g for 15 min at 4°C. Then supernatants were collected and stored at −80°C. Plasma BNP levels were determined using an enzyme-linked immunosorbent assay (ELISA) kit (cat no. WEA541Ra; Wuhan USCN Business Co., Ltd., Wuhan, China).

Prior to the detection of the MDA levels and SOD activity the protein concentration was quantified using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology). Then the remaining ventricle samples were homogenized in saline and centrifuged at 1,000 × g for 15 min at 4°C. The supernatants were collected and the level of MDA and the activity of SOD were detected using commercial kits (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) according to the manufacturer's protocol. The level of MDA is expressed as nmol/mgprot and the activity of SOD is expressed as U/mgprot.

Data are presented as the mean ± standard deviation and data analysis was performed by SPSS 19.0 software (IBM SPSS, Armonk, NY, USA). Statistical differences between groups were analyzed using one-way analysis of variance followed by the Students Newman-Keuls post-hoc test after normality distribution was evaluated. P<0.05 was considered to indicate a statistically significant difference.

Representative echocardiograms are presented in Fig. 1A. Compared with the HF group, IMD treatment significantly increased LVEF (32.5±4.0 vs. 27.8±5.1%; P<0.05; Fig. 1B) and LVFS (15.8±2.2 vs. 13.6±2.6%; P<0.05; Fig. 1C). A significant increase in ±LVdp/dtmax and a decrease in LVEDP were observed in IMD treatment group in comparison with the HF group (Table I). In addition, IMD treatment significantly decreased arterial blood pressure compared with the sham group and the HF group (Table I).

A representative ventricle fibrillation induced by ventricle electric stimulation in an HF rat treated with saline is presented in Fig. 1A. The VFT of the HF group was significantly reduced compared with the sham group (7.8±1.3 vs. 14.1±2.1 V; P<0.05; Fig. 2B). IMD induced a rise of VFT compared with the HF group (10.2±1.8 vs. 7.8±1.3 V; P<0.05; Fig. 2B).

There were no significant differences in body weight between the three groups at the end of experiment (Table I). However, the left ventricle weight/body weight (LVW/BW) value was significantly increased in the HF group compared with the sham group (2.85±0.08 vs. 2.48±0.05; P<0.05; Fig. 3A and B). A significant decrease in LVW/BW was observed in the IMD treatment group compared with the HF group (2.76±0.09 vs. 2.85±0.08; P<0.05; Fig. 3B). However, the infarct size between the HF group and IMD treatment group was not significantly different (Fig. 3C).

Representative immunostaining for TH- and GAP43-positive nerve fibers in the peri-infarct zone are presented in Fig. 4A. The densities of TH- and GAP43-positive nerve fibers in the peri-infact zone of the HF group were significantly increased compared with the sham group (TH, 9534±1150 vs. 2509±281 µm²/mm²; GAP43, 11367±1375 vs. 5073±854 µm²/mm²; all P<0.05; Fig. 4B and C, respectively). However, a significant decrease in the densities of TH- and GAP43-positive nerve fibers was observed in the IMD treatment group compared with the HF group (TH, 7532±825 vs. 9534±1150 µm²/mm²; GAP43, 8554±615 vs. 11367±1375 µm²/mm²; all P<0.05; Fig. 4B and C).

Representative western blot images are presented in Fig. 5A. The protein expression levels of TH, GAP43 and NGF were significantly increased in the HF group compared with the sham group (TH, 0.710±0.053 vs. 0.419±0.034; GAP43, 0.422±0.018 vs. 0.197±0.010 and NGF: 0.439±0.013 vs. 0.213±0.013; all P<0.05; Fig. 5B-D, respectively). IMD treatment significantly decreased the protein expression levels of TH, GAP43 and NGF compared with the HF group (TH, 0.530±0.038 vs. 0.710±0.053; GAP43, 0.320±0.021 vs. 0.422±0.018 and NGF, 0.270±0.021 vs. 0.439±0.013; all P<0.05; Fig. 5B-D, respectively).

The plasma BNP level in the HF group was significantly increased compared with the sham group. IMD treatment significantly decreased the plasma BNP level compared with the HF group (Table II). The MDA level in the peri-infarct zone of the HF group was significantly increased, while the SOD activity was significantly decreased compared with the sham group. IMD significantly decreased the MDA level and increased SOD activity in comparison to the HF group (Table II).