Animals
Our study chose rabbit as experimental subject because the rabbit heart shares some physiological commonalities with the human heart. Thirty healthy New Zealand white rabbits (weight 2.5-3.5Kg), were provided by the Experimental Animal Center of the Hebei Medical University. Those animals were randomly divided into three groups; sham operation group (sham group), heart failure group (HF group), and CCM group (CCM group), with n=10 for each group. Rabbits only receive thoracotomy in the sham operation group. Thoracotomy and ascending aortic cerclage were performed in the heart failure group. In the CCM group, rabbits underwent thoracotomy, ascending aortic cerclage and receive 4 weeks CCM after the formation of chronic heart failure.
Induction of heart failure
The procedure of trans aortic constriction for the creation of the HF model has been established and described in our previous study. Briefly, all animals were anesthetized with 3% sodium pentobarbital (30 mg/kg) via the ear venous. The thoracic cavity was opened. After the ascending aorta was dissected, it was occluded to make a constriction to 60% of the original circumference. A pediatric temporary pacing lead (Medtronic 6491) was used to deliver CCM was sutured to left ventricular anterior wall and the other end of the electrode was fixed to the neck for later use. After 12 weeks, left ventricular ejection fraction £ 40% meant that the heart failure model had been successfully established.
CCM protocols
EPS320 Cardiac Stimulator (BARD Micro Pace, Inc, USA) was used to deliver CCM stimulation to the absolute refractory period of heart by sensed R-wave. As detailed previously, the signals consisted of a biphasic square-wave pulses with phase duration=2 ms, stimulus amplitude=7 V, and 30 ms delay after R-wave sensing[13, 14]. The CCM signals was conducted 6 hours per day for consecutive 4 weeks (Fig. 1A).
Electrocardiography
Needle electrodes were placed subcutaneously in the four limbs for surface ECG recordings as described previously[15]. The lead II of ECG was selected of further analysis. QT interval mainly focus on the repolarization of the action potential of the heart. Currently, the beat-to-beat instability of the QT interval is viewed as ECG biomarker that detect cardiac arrhythmias, mainly focus on the repolarization of the action potential of the heart. The QT interval was defined as the duration from the beginning of the Q wave to the end of T wave. In addition, heart rate-corrected QTc calculation was obtained by using the Carlson’s formula (QTc=QT-0.175(RR-300))[16].
Electrophysiology study
Programmed electrical stimulation protocols was performed by EPS320 Cardiac Stimulator (BARD Micro Pace, Inc, USA) to assess the electrophysiological characteristic. The effective refractory prior (ERP) was measured (2 times the diastolic threshold, 2-milliseconds pulse width) at basic cycle lengths of 250 milliseconds with a train of 8 basic (S1) stimuli followed by a premature (S2) stimulus with 2-milliseconds decrements(Fig. 1B). The longest S1S2 interval that failed to produce a propagated ventricular response was taken as the ERP[17]. ventricular tachycardia (VT) inducibility was assessed with 8 beat drive trains(S1) at 200 milliseconds, followed by 1-3 ventricular extra-stimuli. Single (S2), or double (S2-S3) premature stimuli were applied with a coupling interval of 160 milliseconds, progressively shortened in 2-milliseconds steps until VT induction or until ventricular effective refractory period was reached (Fig. 1C). VT was defined as 8 consecutive ventricular beats at a cycle length≦150 ms[18].
Histological observation
At the end of the study, the rabbits were sacrificed by exsanguination under anesthesia. The heart from anesthetized rabbits was rapidly excised, and fixed in 4% paraformaldehyde solution. The heart was sectioned transversely across myocardial papillary muscle, and then embedded with paraffin. Myocardial tissue sections (5μm) were cut from the paraffin blocks and stained with picrosirius red liquid dye for 60 min. Hematoxylin was used as a nuclear counterstain for 10 min. The stained sections were observed under the polarized light which could distinguish type I from type III collagen by different colors. Yellow and red color indicated type I collagen, and green color indicated collagen III. Digital photographs were taken at 400 ´ magnification for 10 random fields from each section, and percentages of CVF (collagen volume fraction = collagen area/total area ×100%) was detected by Image-ProPlus5.1 (Media Cybernetics). Similarly, type I and III collagen was also observed under a polarization microscope.
Hydroxyproline Assay
Myocardial hydroxyproline content was analyzed using a commercially available kit (Jiancheng Bioengineering Institute, Nanjing, China) according to manufacturer’s instructions. Briefly, the myocardial tissue (30–100 mg) was digested at 95 °C for 20 min in an acidic buffer solution. After the hydrolysis, the samples were centrifuged at 3500 rpm for 10 min. Absorbance of the final solution was determined using a microplate spectrophotometer at 550 nm, and hydroxyproline content was calculated as μg per mg of tissue according to the protocol.
Western blot analysis
The total protein samples were extracted from myocardia tissues and quantified using the bicinchoninic acid (BCA) protein assay (Beyotime, Shanghai, China). Samples were separated with 10% SDS–PAGE electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. After blocked with 5% fat-free milk for 2 h, the membrane was incubated with primary antibodies overnight at 4°C.
The following primary antibodies were independently used to detect specific proteins: Gal-3 (1:1000 dilution, Abcam, USA), CTGF (1:1000 dilution, Santa Cruz, USA), Kv4.3 (1:400 dilution, Santa Cruz, USA), KCNH2 (1:500 dilution, Santa Cruz, USA), KCNQ1 (1:500 dilution, Santa Cruz, USA), CX43 (1:500 dilution, Abcam, USA). An antibody against β-actin (1:1000 dilution, Santa Cruz, USA) was used as an internal control. Following washing with PBS, membranes were treated with horseradish peroxidase-conjugated secondary antibodies (dilution, 1:1000 dilution, Santa Cruz, USA). The immunoreactive bands were visualized using the enhanced chemiluminescence kit (ECL Millipore Corp., Bedford, MA, USA). Developed films were scanned and Image-ProPlus 5.1 was used for quantitative analysis.
Statistical analysis
Continuous data were reported as mean value ± SE. Categorical data were presented as absolute values and percentages. Differences among multiple groups were compared with single factor analysis of variance (ANOVA) while the comparison between two groups was detected with LSD method. The Fisher exact test was used to check for the significance of frequency data. Analyses were performed using the Software Package for Statistical Science (SPSS for Windows; Version 22, SPSS Inc.; Chicago, IL). P < 0.05 is considered statistically significant.