Pirarubicin, purity ≥98%, was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd.. Canagliflozin was obtained from Janssen-Cilag International NV. Brain natriuretic peptide (BNP; cat. no. MB-1608A), creatine kinase MB (CK-MB; cat. no. MB-6930A) and cardiac troponin T (cTnT; cat. no. MB-7278A) test kits were purchased from Shanghai Meixuan Biological Science and Technology Ltd. Malondialdehyde (MDA; cat. no. A003-1-2), superoxide dismutase (SOD; cat. no. A001-3-2) and lactate dehydrogenase (LDH; cat. no. A020-2-2) test kits were obtained from Nanjing Jiancheng Bioengineering Institute. SGLT2 inhibitor (SGLT2i) was purchased from MedChemExpress. The antibodies for SOD2 (1:3,000; cat. no. 13141T), pro/cleaved-caspase- (1:1,000; cat. no. 14220T/9664T), Bcl-2/Bax (1:1,000; cat. no. 4223T/2772T) were obtained from Cell Signaling Technology, Inc. The antibody for NOX2 (1:1,000; cat. no. 19013-1-AP) was obtained from ProteinTech Group, Inc.. All chemicals and reagents were analytical grade.

The present study was performed according to the Guide for the Care and Use of Laboratory Animals (14) and was approved by the Animal Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (CMU; approval no. 20195101). A total of 40 Male Sprague Dawley (SD) rats (180–200 g; age, 6 weeks) were obtained from the CMU experimental animal center. SD rats were housed at 23±2°C with humidity of 40–60% and a 12/12-h light/dark cycle. Rats were randomly divided equally into 4 groups (n=10 in each group): normal group (CON; normal-diet-fed rats), canagliflozin group (canagliflozin-diet-fed rats, 60 mg•kg⁻¹), THP group (normal-diet-fed rats; 3 mg•kg⁻¹ THP was injected via caudal vein once a week) and canagliflozin + THP group (canagliflozin-diet-fed rats, 60 mg•kg⁻¹; 3 mg•kg⁻¹ THP was injected via caudal vein once a week). The food consumption and body weight was measured twice a week.

The experiment ended at week 8. The rats were anesthetized with inhaled isoflurane (2%, maintenance dose was also 2%). Needle electrodes were inserted subcutaneously into the right upper limb, right lower limb and left lower limb respectively. The lead IV electrocardiography (ECG) was recorded by BL-420F biological function measurement system (Chengdu Taimeng Technology Company). The hair of the precordial region was removed and the Doppler echocardiography was measured by Vivid E95 ultrasonic diagnostic apparatus (General Electric Company).

At the end of the 8th week, the rats were weighed after fasting overnight and sacrificed by cervical dislocation under anesthesia (inhalation of 2% isoflurane). Blood samples (1–2 ml per rat) were collected from abdominal aorta immediately after sacrifice and centrifuged at 314 × g, 4°C for 30 min within 6 h. The supernatant was frozen in a −80°C refrigerator and serum LDH, BNP, CK-MB, cTn-T, SOD and MDA contents were determined as soon as possible according to the operation procedure of the kit. Heart samples were excised and weighed. The left ventricular part of the heart was immersed in 10X its volume of 4% paraformaldehyde solution and stored for 4 h in a refrigerator. The rest of the left ventricular portion of the heart was stored in −80°C refrigerator for follow-up experiments. The next day, the heart tissue was dehydrated, dewaxed, embedded in paraffin and cut into 5 µm sections. Hematoxylin and eosin staining was performed according to the instructions of the kit (30°C, 30 min). TUNEL apoptosis detection kit (green fluorescence) was purchased from Beyotime Institute of Biotechnology. The paraffin section was dewaxed in xylene, dehydrated with absolute alcohol, washed with distilled water and then 20 µg/ml proteinase K without DNase added (37°C for 30 min), before washing with PBS for three times. TUNEL solution (50 µl) was added to the target area and incubated at 37°C for 60 min. DAPI staining solution (100%; Beyotime Institute of Biotechnology) was used to stain the nuclei (37°C, 3–5 min). After washing with PBS 3 times, an anti-fluorescence quenching sealing solution was used to seal the plates, which were observed under a fluorescence microscope (magnification, ×200). A total of three fields of view were observed. Apoptosis level=apoptotic cells/total cells ×100%.

A total of 20 neonatal SD rats (male, 1–3 days, CMU Experimental Animal Center) were anesthetized with ketamine (55 mg/kg) plus xylazine (15 mg/kg) and disinfected with 75% ethanol. After the neonatal rats were sacrificed by cervical dislocation, the ventricles were quickly separated under aseptic conditions. The blood clots, blood vessels, fat and other tissues were washed 3 times in PBS buffer and then cut into sections with diameter <1 mm, digested by trypsin and II collagenase and then filtered, centrifuged, resuspended and seeded. Finally, primary rat cardiomyocytes were obtained by differential adhesion method [following 1.5 h culture in DMEM (Gibco; Thermo Fisher Scientific, Inc.) with 10% FBS (PAN-Biotech GmbH) and penicillin/streptomycin at 37.5°C with 5% CO₂, the culture supernatant containing cardiomyocytes was collected and re-seeded to obtain primary cardiomyocytes]. The primary cardiomyocytes were divided into four groups: Normal group (CON), canagliflozin group (canagliflozin, 60 µm, 14 h), THP group (THP, 10 µm, 12 h), THP and canagliflozin co culture group (canagliflozin, 60 µm, 14 h + THP, 10 µm, 12 h). In canagliflozin +THP group, the cells were pre incubated with canagliflozin (60 µm) for 2 h and then co cultured with THP (10 µm) for 12 h.

Heart tissue and primary rat cardiomyocytes was lysed in radioimmunoprecipitation (RIPA) lysis buffer. BCA kit was used to determine the protein concentration in the supernatant. Then ~50 µg heart tissue lysate or 20 µg of cell lysate was used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% gel) and proteins were then transferred to an FL PVDF membrane (EMD Millipore) at 4°C for 1.5 h. After blocking with 5% blocking protein powder (room temperature), the first antibody was incubated overnight at 4°C and the second antibody was incubated at room temperature for 1.5 h. The western blotting results were analyzed by BeyoECL Plus (Beyotime Institute of Biotechnology) in Image Lab (version: 5.2.1; Bio-Rad Laboratories, Inc.). The specific protein expression levels were normalized to GAPDH.

Data were presented as mean ± SD. The significance of differences between groups were analyzed statistically using one or two-way analysis of variance (ANOVA), followed by a Tukey's multiple-comparison post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The body weight (Fig. 1A, P<0.05 vs. CON) and food intake (Fig. 1B, P<0.05 vs. CON) of THP rats began to decrease in the third and fourth weeks, especially in the fifth and sixth week (P<0.01 vs. CON). However, there was no significant improvement in the above changes after treatment with canagliflozin (Fig. 1, P>0.05 vs. THP).

At 8 weeks after THP injection, a series of ECG and echocardiographic (Fig. 2) changes occurred in SD rats, including: Ejection fraction (Fig. 2A) and fractional shortening Fig. 2B) decreased, left ventricular internal diameter end diastole (Fig. 2C) and left ventricular internal diameter end systole (Fig. 2D) increased; R wave (Fig. 2E) and T wave (Fig. 2F) increased; S wave (Fig. 2G) decreased; QT interval (Fig. 2H) was prolonged.

Following canagliflozin treatment, the above changes were not significantly improved (Fig. 2A-H; P>0.05 vs. THP).

As shown in Fig. 3, the arrangement of cardiomyocytes was disordered, the intercellular space was enlarged and the cardiomyocytes were focal vacuolization or steatosis in the rats injected with THP alone. Compared with THP group, the treatment of canagliflozin showed no significant improvement on cardiac tissue.

TUNEL staining (Fig. 3) showed that there was no cardiomyocyte apoptosis in CON and canagliflozin group, but there was regional cardiomyocyte apoptosis in THP injection group. The treatment of canagliflozin had no effect on THP-induced cardiomyocyte apoptosis. The quantitative results are shown in Fig. 3A.

The SD rats were sacrificed after 8 weeks. Blood and heart tissue samples were collected and tested.

In blood, THP caused the decrease of SOD level (Fig. 4A) and the increase of MDA (Fig. 4B), LDH (Fig. 4C), CK-MB (Fig. 4D), cTnT (Fig. 4E) and BNP (Fig. 4F). However, the treatment of canagliflozin did not effectively improve the above changes (Fig. 4A-F; P>0.05 vs. THP).

The same was true of heart tissue, THP-induced the decrease of SOD level (Fig. 4G) and the increase of MDA (Fig. 4H), LDH (Fig. 4I), CK-MB (Fig. 4J), cTnT (Fig. 4K) and BNP (Fig. 4L) in rat heart. However, the treatment of canagliflozin does not effectively improve the above changes (Fig. 4G-L, P>0.05 vs. THP).

As shown in Fig. 5, THP injection for 8 weeks led to the decrease of the protein expression of SOD2, pro-caspase- and Bcl-2/Bax and the increase of the protein expression of NOX2 and cleaved-caspase-, which suggested that THP caused oxidative stress and increased apoptosis in rat heart. However, treatment with canagliflozin does not effectively improve the above changes (Fig. 5; P>0.05 vs. THP). Further evidence was provided by quantitative analysis (Fig. 5).

The same applied in vitro. As shown in Fig. 6: THP treatment of cardiomyocytes led to the decrease of the protein expression of SOD2, pro-caspase- and Bcl-2/Bax and the increase of the protein expression of NOX2 and cleaved-caspase-, which suggested that THP caused oxidative stress and increased apoptosis in rat cardiomyocytes. However, the treatment of SGLT2i does not effectively improve the above changes (Fig. 6; P>0.05 vs. THP). Further evidence was provided by quantitative analysis (Fig. 6).