Animal details
Wistar albino rats of either sex was used in present study. Rats were kept under 12-h light dark cycle at room temperature (20-22°C) and humidity (50-60%). All experimental protocols and animal handling procedures were performed according to guidelines of CPSCEA, Ministry of Fisheries, Animal Husbandry and Dairying, Government of India and with permission of Institutional Animal Ethics Committee (181/IAEC-1/2019).
Induction of diabetic cardiomyopathy
Rats were orally fed with high fat diet (HFD) prepared by mixing powdered normal pellet diet (365 g), yeast powder (1 g), casein (250 g), lard (310 g), DL-methionine (3 g), cholesterol (10 g), vitamin and mineral mix powder (60 g) and sodium chloride (1 g) for two weeks followed by administration of single low dose of streptozotocin at a dose of 35 mg/kg, (intraperitoneal, i.p.), which was further followed by feeding of HFD for 9 weeks [8]. Rats with blood sugar >200 mg/dL after 1 week of administration of streptozotocin were included in the study. The dosing details for animals are given in Table 1.
Anthropometric measurements
Body weight of all rats were measured at the time of initiation of experiments and at the end of experiment before the surgery. Post-surgery, the hearts were dissected out and weighed, the ratio of heart weight to body weight of rat was calculated.
Glycemic status
Fasting blood glucose (FBS) and postprandial blood glucose (PPBS) levels were measured at the start of the study. Before the blood sample, the rats were fasted for overnight. Rats were restrained with rodent restrainer under aseptic conditions, with the tail exposed. The tail's distal end was then cleaned in 70% alcohol. After carefully drying the tail, it was penetrated with the lancet. A glucometer strip was used to assess glucose levels after the first drop of blood was wiped away. PPBS was measured after 2-h of meal consumption in rats. The glucose level was also recorded at the end of the experiments.
Echocardiography
Ketamine (80 mg/kg i.p.) and xylazine (8 mg/kg i.p.) were used to anaesthetize rats. Using a hand-held 12 MHz neonate cardiac probe transducer and a totally digitized machine (Philips HD11 XE), two-dimensional M-mode echocardiography pictures were generated from a short axis view at the level of the papillary muscles of the left ventricle. Left ventricular internal diastolic dimension (LVIDd) and internal systolic dimension (LVIDs) were measured from the trailing edge of the septum to the leading edge of the posterior wall of ventricular septum. The dimensions provided above were used to compute cardiac output and ejection fraction [9].
Quantification of TNF-α and IL-6 levels
The levels of TNF-α and IL-6 in cardiac tissue was determined by using enzyme-linked immunosorbent assay (ELISA) kits as per instructions of manufacturer. Results were expressed as pg/mg protein.
Western blot analysis
Western Blot was performed for estimating PDE-1 and TGF-β in cardiac tissue. Protein was extracted from heart tissue via homogenisation in total protein extraction kit (Real Gene; Cat no. 150001) as per manufacturer’s instructions. Using bicinchoninic acid protein assay kit, total protein concentrations in tissue extracts were measured (G-Biosciences). For immunoblotting, we employed an SDS-Polyacrylamide gel (10% Resolving gel) and a PVDF membrane. Washing buffer used was 1X TBST and blocking buffer was 5% skim milk prepared in washing buffer.
Hemodynamic parameters
Tracheostomy was performed on sedated rats, and the rats were cannulated using a 14G polythene cannula. A small animal ventilator (RWD, Shenzhen) was used to provide artificial breathing. The heart was exposed, and the left ventricular systolic pressure was measured using a 24-gauge stainless steel cannula, connected to a pressure recording system (Biopac, USA). All the pressure tracings were digitally captured.
Myocardial enzyme assay
At the end of the study, blood sample was taken by retro-orbital sinus. Serum was partitioned from blood by centrifugation at 3000 g for 15 min and kept at -20 ºC until analysis. The estimation of activity of the lactate dehydrogenase (LDH), creatine kinase‑MB (CK‑MB) and aspartate aminotransferase (AST) using commercially available test kits was done.
Quantification of oxidative stress in cardiac tissues
The excised rat hearts were homogenized for 5 min at 3000 rpm in 10% w/v ice-cold 0.1 M phosphate buffer (pH 7.4), centrifuged for 10 min at 6000 rpm, and the resulting 2 mL supernatant was collected for lipid peroxides assay. The proteins were extracted from the tissue using an equal volume of 5% trichloroacetic acid (TCA) and centrifuged for 10 min at 4000 rpm at 4°C. The supernatant obtained was used to calculate glutathione (GSH) level and superoxide dismutase (SOD) activity.
Thiobarbituric acid reactive substances (TBARS) assay
The method of Ohkawa et al., (1979) [10] was used to assay lipid peroxides using thiobarbituric acid. Briefly, acetic acid, thiobarbituric acid, and sodium dodecyl sulfate were added to a test tube containing 0.2 mL tissue homogenate. The contents were heated at 95°C for 60 min. After the contents were cooled, 5 mL of butanol and pyridine (15:1) were added. Contents were vortexed and centrifuged at 4000 rpm for 10 min. Absorbance was noted at 532 nm using a spectrophotometer. Results were presented as nanomoles per gram of tissue.
SOD assay
A 96-well plate layout was prepared for the blank, standard, and tissue samples. In all specified samples wells except blank, 10 µL tissue homogenate was added, followed by 280 µL of tris buffer, and finally, 10 µL of pyrogallol was added by multichannel pipette. In a multimode plate reader, the absorbance was noted at 420 nm for 3 min at 30 sec interval immediately after adding pyrogallol [11].
Catalase assay
The CAT activity was estimated according to the established protocol [12]. The decrease in absorbance at 240 nm is precisely proportional to the breakdown of H2O2. Catalase activity was measured by the difference in absorbance per unit time. After adding 1 mL of 50 mM phosphate buffer (pH 7.0) and 0.1 mL of 30 mM hydrogen peroxide to 50 µL tissue supernatants, a reduction in absorbance at 240 nm was monitored every 5 sec for 30 sec. Catalase activity was reported as units per mg of protein.
GSH assay
GSH assay was done in the cardiac supernatant using Ellman’s protocol (1959) [13]. Briefly, in the phosphate buffer, 0.5 mL of DTNB was added to 0.2 mL of supernatant and absorbance was noted at 412 nm. The results were presented as micrograms of GSH per gram of tissue.
Real-time polymerase chain reaction studies
Briefly, Trizol C was used to isolate, purify, and treat total RNA from heart tissues (441066; SRL, India). Using a Nano-Drop 1000TM, the quantitative and qualitative analysis of total RNA were measured spectroscopically (ThermoScientific). Absorbance ratio, A260/A280 was used to check for contamination. For later usage, purified RNA was kept at -80°C. Primers for animal and housekeeping genes, TGF-β, PDE-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively, were designed using Primer3Plus software. The specificity of primers was checked by running the BLAST tool against the rat genome database. Four primers were shortlisted for TGF-β, PDE-1 and one for GAPDH based on their specificity. For the gene, the shortlisted primers were optimised and standardised (Table 2). The effectiveness of primers was measured using a temperature gradient approach with Tm 50°C as the starting point.
The cDNA was synthesized from 1µg of total RNA, the protocol was done according to the manufacturer's iScript cDNA synthesis kit (1706891; Bio-Rad, USA). RT-PCR was done using iTaq Universal SYBR Green Supermix (1725124; Bio-Rad). The procedure was done on a Bio-Rad CFX 96 according to the following parameters: (i) 95°C for 30 sec, (ii) 39 cycles of 95°C for 5 sec, (iii) Tm for 15 sec, and (iv) 72°C for 15 sec. The 2Ct approach was used to compute the relative fold change in individual target mRNA transcript. To ensure that the results were accurate, each experiment was done in duplicates.
Histological studies
Formalin fixed cardiac sections were cut (5 µm thick) were stained with hematoxylin followed by counterstaining with eosin to observe the gross morphological changes in the cardiac sections of various groups employed in the present study. The slides were observed under microscope. For evaluation of cardiac hypertrophy, the short axis diameter of cardiac myocytes was determined using light microscopy for 10 myocytes per field at a magnification of 400 ×. The data of each set of ten myocytes yielded mean values [14].
Statistical analysis
All data was expressed as mean± standard error of mean (SEM). The significance data was analysed by one-way analysis of variance followed by Tukey-Kramer multiple comparison post-hoc test. The value of probability P <0.05 was considered as significant.