Animals and drugs
Male Sprague-Dawley rats (330–355 g) were obtained from Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). Animals were kept under standard laboratory conditions in a specific pathogen-free room at a constant temperature (20°C–22°C) with 12 hours of light/12 hours of dark exposure. All animal experiments were carried out in accordance with the ARRIVE guideline [22]. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University.
Empagliflozin was purchased from MedChemExpress lnc. (Monmouth Junction, USA). Empagliflozin was dissolved in 10% dimethyl sulfoxide, 20% kolliphor (Sigma Aldrich, USA) and 80% phosphate buffer saline as a stock solution (10 mg/mL).
CA model and experimental protocol
The animals were surgically prepared as previously described [23]. CA was induced by ventricular fibrillation (VF) through a transoesophageal electrode. The electrode was positioned to ensure constant ventricular capture and was given a 30 V voltage and 30 Hz current. The current flow continued for 3 minutes to prevent a spontaneous reversal of VF. After 6 minutes of untreated VF, CPR was started with precordial compression (PC) at a rate of 250/minutes and mechanical ventilation with 100% O2. After 2 minutes of PC, epinephrine (0.01 mg/kg) was administrated and 2 J biphasic waveform shocks was performed after 4 minutes of CPR. ROSC was defined as the return of supraventricular rhythm with a MAP ≥ 60 mmHg for a minimum of 5 minutes. If VF persisted, another 2 J shock was given after another 1 minutes of PC. Resuscitation was declared a failure when there was no ROSC after 6 minutes of CPR. After ROSC, mechanical ventilation was continued with 100% O2 at 30 minutes, 50% O2 at 20 minutes, and 30% O2 at 10 minutes.
After achieved ROSC, the animals were randomized into three groups: CA+ empagliflozin (EMP) group (n = 18), CA + vehicle group (n = 22) and Sham group. The animals in Sham group underwent the same operation with the other groups but without inducing CA. The rats in the CA + EMP group received 10 mg/kg of empagliflozin at 10 minutes after ROSC by intraperitoneal injection. Meanwhile, the rats in Vehicle group received placebo (10% dimethyl sulfoxide, 20% kolliphor and 80% phosphate buffer saline) for control. An illustration of the protocol used is provided (Fig. 1).
Histopathologic analysis
The heart tissues were embedded in paraffin and sectioned at 5 μm thickness. Tissue slices of the hearts were stained with hematoxylin-eosin (HE) and Masson’s trichrome (Servicebio, Guangzhou, China) for histological evaluation according to the manufacturer’s instructions. Ten fields of vision (magnification of ×400) were randomly sampled from each heart tissue sample under an optical microscope. The fibrotic tissue area stained blue (%) by Masson staining was analyzed using counting software (Image J, National Institute of Health, USA).
Serum cardiac troponin I (cTnI) levels
Blood samples were collected from the abdominal aorta at 24 hours after ROSC in rats. The blood was allowed to clot for 30 minutes at room temperature. The clot was removed by centrifugation at 1500 g for 15 minutes and the serum was stored at -80°C until use. Serum cTnI levels were detected using a rat cTnI enzyme-linked immunosorbent assay kit (Cusabio, China) according to the manufacturer’s instructions.
Echocardiography
Rats were anesthetized with 2% to 3% isoflurane, and in vivo cardiac function was assessed by transthoracic echocardiography using a high-resolution imaging system equipped with a 30-MHz transducer (Vevo 3100, VisualSonics, Toronto, Canada), as previously described [24]. The left ventricular (LV) function was assessed by the ejection fraction (EF) and cardiac output (CO). All measurements were conducted by a single investigator who was blinded to the experimental groups.
Oxidative stress analysis
The oxidative stress in the heart tissue at 24 hours after CA was evaluated by immunohistochemical staining of 4-hydroxynonenal (4-HNE) to assess lipid peroxidation and 8-hydroxy-2′-deoxyguanosine (8-OHdG) to detect the extent of nucleic acid oxidation. Heart tissues were sectioned and embedded in paraffin. For immunohistochemistry, the fixed sections were immunostained overnight at 4°C using a primary monoclonal antibody against 4-HNE and 8-OHdG (Abcam, Cambridge, United Kingdom), followed by corresponding secondary antibody for 2.5 h at room temperature. Ten fields of vision (magnification of ×400) were randomly sampled from each heart tissue sample under an optical microscope. 4-HNE and 8-OHdG relative intensity area (%) were detected by automated counting software (Image J, National Institute of Health, USA). The ROS levels in the heart at 6 hours after CA were measured using a tissue ROS assay kit (Genmed Scientifics, Wilmington, DE, USA) that utilized 2′,7′-dichlorofluorescein diacetate as the oxidative fluorescent probe. The ROS signals were detected by a microplate reader (Molecular Devices, Sunnyvale, USA).
Western blot analysis
For immunoblotting, 𝛽-hydroxy butyrate dehydrogenase 1 (BDH1) (diluted 1:1000; Abcam, Cambridge, United Kingdom) and GAPDH (diluted 1:2000; Cell Signaling Technologies, USA) antibodies were used. Protein bands were detected using an enhanced chemiluminescence kit (Cell Signaling Technologies, USA). GAPDH was used to normalize protein loading. The densities of protein blots were quantified by using Image J software (National Institutes of Health, Bethesda, USA) and normalized to control.
Transmission electron microscopy and analysis
Heart samples were collected and fixed in 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4) overnight at 4°C. The heart tissues were cut into 500-μm thick transverse slices and were post-fixed in 1% osmium tetroxide for 1 hour. Tissue slices were then dehydrated in ascending series of ethanol and embedded in epon. After dehydration, the specimens were conventionally processed and examined under a transmission electron microscope (Tecnai G2, FEI, Hillsboro, USA) through 13,500× objectives. Area of individual mitochondria was quantified by using Image J software (National Institutes of Health, Bethesda, USA).
Mitochondrial complex Ⅰ activity
The activity of mitochondrial complex Ⅰ was measured in heart homogenates (1 mg) using the complex I enzyme activity assay kit (Colorimetric) (Abcam, Cambridge, United Kingdom) according to the manufacturer’s instructions.
Measurement of metabolites levels
ATP levels were measured in heart homogenates with an ATP bioluminescent assay kit (Beyotime, China) according to the manufacturer’s instructions. β-hydroxybutyrate (BHB) concentrations in heart homogenates and serum were measured using a BHB colorimetric assay kit (Biovision, Milpitas, CA, USA) according to the manufacturer’s instructions. Nicotinamide adenine dinucleotide (NAD+)/reduced form of NAD+ (NADH) levels in heart homogenates were measured using a NAD+/NADH quantification colorimetric kit (Biovision, Milpitas, CA, USA) according to the manufacturer’s instructions. Serum glucose and lactate concentrations were measured by a biochemistry analyzer (YSI, Yellow Springs, OH, USA).
Statistical analysis
All data were presented as mean ± standard deviation (SD), and analyzed using GraphPad Prism 8 (GraphPad, San Diego, CA, USA). The unpaired t test was used to compare the same parameters between two groups. Normally distributed data were analyzed by one- or two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Lipid droplets number was analyzed by the Kruskal-Wallis test with Dunn’s post hoc analyses between multiple groups. The survival curve among the groups was obtained using Kaplan-Meier survival analysis and compared with a log-rank test. A two-tailed P < 0.05 was considered significant.