Sigma (Sigma-Aldrich, USA) supplied Rob, isoproterenol, dimethyl sulfoxide (DMSO), Metoprolol succinate, Triphenyl tetrazolium chloride (TTC), Hematoxylin, Eosin and dichlorodihydrofluorescein diacetate (DCFH-DA). Himedia (Maharashtra, India) supplied the antibiotic penicillin-streptomycin, trypsin (0.25%), and MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide]. Gibco (Gibco, Grand Island, NY, USA) supplied DMEM, PBS, and foetal bovine serum. H9c2 cell line was received from ATCC (Manassas, Virginia), and Trizol reagent was obtained from SRL (Mumbai, India). Assay kits were bought from Origin Diagnostics and Research in Kerala, India. Thermo Fisher Scientific Inc. (United States) supplied the fluorescent dyes, Pierce BCA Protein Assay Kit, GeneJET RNA purification kit, Power SYBR Green Master Mix, and Verso cDNA Synthesis Kit. APJ Scientific Solutions (Trivandrum, India) synthesised the forward and reverse real-time PCR primers. Antibodies were bought from Cell Signalling Technology (Massachusetts, USA). All of the compounds used were analytic grade.
24 male 6 week old Sprague Dawley rats weighing between 150 and 180 g were acquired from the Mannuthy College of Veterinary and Animal Sciences. They were housed in a specific pathogen-free, temperature and humidity-controlled environment (25 ± 2 °C, 50 ± 5% humidity) with a standard 12 h Light/12 h Dark cycle at University of Kerala's Department of Biochemistry. During the course of the trial, rats were given an unlimited supply of standard diet and water ad libitum.
Ethical statement The study was approved by University of Kerala Institutional Animal Ethical Committee (IAEC sanction no. IAEC 2-KU-01/2023-BCH-JA (08)), in accordance with the guidelines of the Committee for the Control and Supervision of Experiments on Animals (CCSEA), Government of India. All animal experiments were conducted following ARRIVE guidelines, ensuring compliance with current animal welfare regulations. Every effort was made to minimize the number of animals used and their suffering. The number of rats used in each experiment is specified in the corresponding figure legends.
H9c2 cells derived from rat embryonic cardiomyocytes were acquired from ATCC, Manassas and maintained in high glucose DMEM supplemented with 10% (v/v) heat-inactivated FBS, 100 µg/mL penicillin-streptomycin. H9c2 cardiomyoblasts were differentiated using the methods previously reported. In summary, H9c2 cells were treated with retinoic acid (10 nM) every day for 6-7 days in DMEM with 1% FBS.
H9C2 differentiation was investigated by monitoring morphological changes, performing gene expression analysis with qRT-PCR, protein expression with western blotting, and stimulating differentiated cells with ISO.
Cardiomyocytes were cultured in 96-well plates at a density of 2 × 10 cells/well and differentiated as previously described. After reaching 80% confluence, the cells were divided into five groups
The dose of ISO was determined by reviewing previous publications. The following studies were carried out on the cells after they had been grown for 24 h.
H9c2 cardiomyocytes were cultured in a 96-well plate and treated with 50 µg/ml Rob and Met for 24 h. The cultures were then treated with 100 µM ISO for 24 h. The MTT test was used to determine cell viability. The absorbance was measured at 570 nm with a microplate reader (BioTek Instruments Inc., Winooski, USA). The morphological changes in the cells were examined and documented using an inverted microscope (EVOS XL Core).
DCFH-DA, a cell-permeable fluorescent probe, was utilised to quantify intracellular ROS. H9c2 cells were pre-treated with Rob and Met, and then exposed to ISO for 24 h. The samples were incubated with 10 µM DCFH-DA for 20 min at 37 °C in the dark. After incubation, cells were gently rinsed with PBS thrice and subsequently fixed with 4% paraformaldehyde for 10 min at room temperature. Following fixation, nuclei were counterstained with DAPI for 5 min to visualize nuclear morphology. Images were captured using a microscope (ZOE Fluorescent Cell Imager, BioRad) at 100 x magnifications with a filter of emission wavelengths 510-550 nm and excitation wavelengths 470-490 nm and DAPI.
Intracellular ROS levels were measured by Fluorescence-activated cell sorting. After treatment, the cells were then trypsinised, collected, and washed three times with PBS before centrifugation at 1000 g for two minutes. The cell pellets were resuspended in 1 ml of cold PBS and analysed using a BD FACS Aria III (BD FACSDiva version 7.0). The results were reported as the percentage of fluorescence intensity relative to the control.
Rob's protective effect against ISO-induced necrosis and apoptosis was assessed using a fluorescent double-staining technique with AO/EB. 5 × 10 cells were seeded in a 12-well plate and differentiated as previously described. The groups were subsequently treated according to the procedures mentioned above. After treatment, adherent cells were washed twice with PBS (pH 7.4). Each group was re-suspended with a 1 ml working solution containing a 1:1 solution of AO (100 µg/ml)/EB (100 µg/ml) and incubated for 30 min at 37 °C in the dark. The unbounded stain was then rinsed thoroughly with PBS. A fluorescent microscope (ZOE Fluorescent Cell Imager, BioRad) was used to differentiate live and dead cells in each well.
Annexin V detects early and late apoptosis in cells by binding to the phosphatidylserine (PS) on the cell's outer leaflet. 2 × 10 cells were seeded in a six-well plate and treated as mentioned above. The cells were rinsed with 1X binding buffer and stained with Annexin V (5 µL) and propidium iodide (10 µL) as instructed by the Apoptosis Detection Kit (Invitrogen, USA). The apoptotic cells of each group were quantified through BD FACS Aria III (BD FACSDiva version 7.0). Early apoptotic cells were identified as Annexin V⁺/PI⁻, while late apoptotic or necrotic cells were Annexin V⁺/PI⁺.
The animals were randomly divided into 4 groups of 6 animals each and the experimental period was 14 days.
Group I (Control group): Rats were injected with 100 µl normal saline for 14 days intravenously.
Group II (ISO group): Rats were injected with 100 µl normal saline for 14 days intravenously and ISO (85 mg/kg, s.c.) in normal saline on 13th and 14th day at an interval of 24 h.
Group III: (Rob group): Rats were injected with Rob in normal saline (20 mg/kg, i.v) for 14 days intravenously.
Group IV: (Rob + ISO group): Rats were injected with Rob in normal saline (20 mg/kg, i.v) for 14 days intravenously and ISO (85 mg/kg, s.c.) in normal saline on the 13th and 14th days at an interval of 24 h.
The rats were euthanized using 5% isoflurane, administered through an induction chamber, followed by cervical dislocation to ensure humane euthanasia.
Heart tissues and H9c2 cells were collected, sonicated, and their protein concentration was measured with a Pierce BCA Protein Assay Kit. The cells were then collected in accordance with the manufacturer's instructions supplied by Origin Diagnostics and Research (Kerala, India) to assess the level of MDA (2202-01), CAT (3303-01), and SOD (3302-01). Each test was conducted in quadruples.
MI size was determined using the direct triphenyl tetrazolium chloride (TTC) technique. The process entailed cutting the heart transversely across the left ventricle with a thickness of 2 to 3 mm, then incubating it in a 1% TTC solution in phosphate buffer (pH 7.4) for 30 min at 37 °C. The sections were then preserved using 10% formalin. The infarcted myocardium was pale grey or white, while the non-ischemic myocardium was dyed red.
Heart tissue was extracted and fixed in 4% paraformaldehyde for 24 h, then dried, embedded, and sliced to 5 μm. Routine H&E was done. After sealing, the alterations in myocardial histopathology were examined under an optical microscope (EVOS XL Core).
Blood samples were collected and centrifuged to obtain serum. Myocardial injury indexes of LDH (6603-01), SGOT (11408005), and creatine kinase (11404001) in serum and LDH in cultural supernatant in vitro were determined by a commercial kit purchased from Origin and Agappe Diagnostics Ltd, India.
Trizol reagent was used to extract total RNA from tissues and cells. Gene expression levels of α-MHC, β-MHC, α-actinin, CHOP, Bip, ATF 6, PDI, Cas 3, Cas 9, BAX, and Bcl-2 were analysed following treatments. The RNA content and purity were estimated using a nanoplate reader and the ratio of absorbance readings at 260 and 280 nm (A260/A280) was taken. The Verso cDNA Synthesis kit was used to reverse transcribe 1 µg of total RNA extracted from each sample into cDNA, as per the manufacturer's instructions. Power SYBR Green Master Mix was used to create PCRs for each group's real-time experiment. The manufacturer's instruction for the heat cycler was followed to conduct reactions in a 20 µL volume. DNA targets were amplified and analysed using a Quant Studio 5 Real-Time PCR system. Table 1 contains the primer sequences used to amplify target genes. ΔΔCt was computed for each sample, and mRNA expression levels were shown by 2-ΔΔCt. The amount of PCR product was assessed as fluorescence signal intensity after standardising to β-actin as an internal reference.
The expression of the target proteins was analyzed using western blotting. In summary, total protein was obtained by lysing cells/tissue following treatment with RIPA. After centrifugation, the supernatant was collected, and the protein content was determined using the Pierce BCA Protein Assay Kit. Equivalent protein-containing samples were electrophoresed for 90 min on 10% SDS-PAGE gels, and the separated proteins were wet-transferred to nitrocellulose (NC) membranes. Immunoblots were performed with appropriate antibodies: Calsequestrin (1:1000 CST), Troponin I (1:1000), Bip (1:1000 CST), CHOP (1:1000 CST), PDI (1:1000 CST), AKT (1:1000 CST), pAKT (1:1000 CST), pGSK 3β (1:1000 CST), GSK 3β (1:1000 CST), ERK1/2 (1:1000 CST), p-ERK1/2 (1:1000 CST), Caspase 9 (1:1000 CST), and β-actin (1:1000 CST). The membranes were incubated with the primary antibodies overnight at 4 °C after blocking with 5% skim milk in tris-buffered saline (TBST) for 1 h at 37 °C. The following day, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG secondary antibodies (1:2500, CST) for 1 h at room temperature after being rinsed with TBST three times. Protein bands were visualized using enhanced chemiluminescence (Biorad ChemiDoc™ MP Imaging System). The optical density (OD) of the reaction zones was calculated using ImageJ image analysis software (NIH, Bethesda, MD, USA).
GraphPad Prism, version 9 for PC (GraphPad Software, San Diego, CA, USA), was utilized for statistical analyses. Data are presented as mean ± SD. Unless otherwise specified, all experiments were performed at least four times. Statistical analyses included one-way ANOVA, t-test, and Tukey's test.