Experimental animal care and use complied with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 8th edition, 2011). All experiment procedures were approved by the Nanjing Medical University. Fifty male Sprague-Dawley rats weighting 200-250 g were purchased from Nanjing Medical University Laboratory Animal Center. All rats were caged in a room with controlled temperature and humidity with a 12-h light/dark cycle and provided a standard chow and drinking water ad libitum. Rats were randomly assigned to Control group (n = 10) and isoproterenol induced cardiomyopathy group (ISO, n = 40). Rats in ISO group were intraperitoneally injected 2.5 mg/kg/d isoproterenol hydrochloride (Sigma, Switzerland) dissolved in normal saline, once a day for 2 weeks [13, 14]. Echocardiography was performed at the end of the 2nd week and the 6th week. After echocardiography measurement at the end of the 2nd week, 28 survival rats in ISO group were randomized into three groups including ARNI (angiotensin receptor neprilysin inhibitor, n = 9), Dapa (Dapagliflozin, n = 9) and ISO (saline, n = 10) groups. ARNI (Novartis Pharma Schweiz AG, Chinese national medicine permission number J20190001) was administered intragastrically at a dose of 68 mg/kg, and Dapa (AstraZeneca Pharmaceuticals LP, Chinese national medicine permission number J20170040) was administered intragastrically at a dose of 3 mg/kg for 4 weeks, respectively. The medication method for ARNI and Dapa was adopted according to previously published literature [15, 16, 17].
Assay of Cardiac Function-related Parameters
Echocardiography was performed at the end of the 2nd week and the 6th week. The rats were anesthetized with ketamine, and then the cardiac function was evaluated using a Vevo 2100 (VisualSonics, Canada) system equipped with a MS-250, 16.0-21.0 MHz imaging transducer.
Electrical Programmed Stimulation
At the end of the 6th week, all rats underwent ventricular electrical programmed stimulation (EPS) for the evaluation of susceptibility of ventricular arrhythmias (VAs) before being sacrificed. They were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (50 mg/kg). Three needle electrodes were placed on the right upper limb and legs to perform electrocardiography. Then EPS was used to stimulate the left ventricular apex of the heart through a bipolar electrode and the incidence of ventricular arrhythmias (VA) was investigated. By a cycle length of 140 ms, the threshold potential for stable pacing was achieved. Pacing was started with twice as much as the threshold and a cycle length of 140 ms, which was the interval of eight stimuli (S1). An extra stimulus (S2) was applied until it failed to induce ventricular depolarization, while the interval between S1 and S2 was progressively shortened by 10 ms.
Samples and Histopathology
Animals were sacrificed after EPS immediately. Blood was collected from the descending aorta. After being weighed and washed with ice-cold PBS, one part of the heart was cut and fast frozen by liquid nitrogen, then moved to − 80 °C for further detection. The other part fixed in 4% paraformaldehyde was used for staining. Masson’s trichrome staining was performed to detect cardiac fibrosis. Five fields of each sample were randomly selected and collagen volume fraction (CVF) was assessed by Image-Pro Plus 6.0.
Measurement of Plasma Angiotensin II (Ang II)
Plasma level of Ang II was measured from the blood collected from the abdominal descending aorta. Blood was collected into tubes containing EDTA, and then centrifuged at 3000 rpm at 4 °C for 15 mins to separate the plasma. Plasma Ang II level was determined using enzyme linked immunosorbent assay (ELISA) kit. All steps were carried out in accordance with the manufacture’s specifications (Abcam Inc, UK). The final solution was read by a microplate reader (ELX800, BioTek, Vermont, USA).
Measurement of Plasma Glucose and Cardiac MDA Levels
Plasma level of glucose was detected by the glucose-oxidase method using a commercially available glucose assay kit from Jiancheng Bioengineering (Nanjing, Jiangsu, China). The level of MDA, in the heart tissue, was detected by using a lipid peroxidation (malondialdehyde; MDA) assay kit from Jiancheng Bioengineering (Nanjing, Jiangsu, China). Lipid peroxidation was determined by the reaction of MDA with thiobarbituric acid (TBA) to form a colorimetric product, proportional to the MDA present. The intensity of the color was measured spectrophotometrically at 505 nm for glucose and 532 nm for MDA.
Measurement of Superoxide Anions
The lucigenin-derived chemiluminescence method was used to examine superoxide anions level in the cardiac tissue. Superoxide anions can react with dark-adapted lucigenin (5 μM) resulting in photon emission which can be captured once every minute for 10 mins by a luminometer (20/20n, Turner, Sunnyvale, CA, USA). The superoxide anions levels were expressed as the mean light units (MLU) per minute per milligram of protein .
Measurement of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidase Activity
The enhanced lucigenin chemiluminescence method was used to detect NADPH oxidase activity. NADPH oxidase can react with NADPH substrate (100 μM) in the medium to generate superoxide anions which can react with lucigenin (5 μM) to produce light emission. A luminometer (20/20n, Turner, CA, USA) can capture the light emission once every minute for 10 mins. The NADPH oxidase activity could be expressed as the (MLU) per minute per milligram of protein .
Protein expressions of angiotensin II type-1 receptor (AT1R, antibody from Endo Life Science Inc, USA), the superoxide (O2-)-generating NADPH oxidase isoforms (NOX2 and NOX4, antibodies from Abcam, Burlingame, CA, USA), and inflammatory markers including TNFα, IL-1β, IL-6 and IL-10 ( antibodies from Proteintech, Chicago, IL, USA) in myocardial tissue were detected by Western blotting . Simply, total cardiac proteins in the homogenate were extracted and measured. Antibodies AT1, NOX2, NOX4, TNFα, IL-1β, IL-6 and IL-10 were applied according to the manufacturer’s instructions. Horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG were used as secondary antibody. Protein expression level was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, antibody from Proteintech, Chicago, IL, USA). The signals were quantified by using Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE).
Survival over the 6-week experiment was analyzed according to the daily recording of deaths by the standard Kaplan-Meier analysis with the log rank test.
Data are expressed as mean ± SEM and analyzed by GraphPad Prism v8.0.2 (GraphPad Software, CA). Comparisons between the two groups performed with two-tailed unpaired t test. For multiple-group comparisons, data were performed using one-way ANOVA followed by Bonferroni’s post-hoc test. A value of P<0.05 was considered statistically significant.