Animals
All animal experiments were conducted according to the institutional animal ethical committee guidelines, which conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Eighth edition 2011) (Ethics 47–07-2019). We used 350–400 gr. (11–12 weeks old) male Sprague–Dawley rats (Envigo Ltd, Jerusalem, Israel), which were maintained at a constant temperature and relative humidity under a regular light–dark schedule (12 h:12 h), fed with normal rodent diet and with tap water ad libitum.
Study design
All animals underwent baseline cardiac function evaluation by echocardiography, blood pressure (BP) and kidney function. Subsequently, the animals underwent MI procedure. Then, we divided the animals into 2 experimental groups. The first group of untreated rats served as control; the second group received empagliflozin 30 mg/kg/day. Therapy was administered for four consecutive weeks.
We monitored BP once a week and cardiac function was re-evaluated every 2 weeks (week 2 and 4), and kidney function was re-evaluated at the end of the experiment (week 4). Right before sacrifice, we measured cardiac hemodynamics directly by means of the Millar pressure–volume (P–V) system. Finally, after euthanasia, the heart was excised and preserved for additional analyses.
Echocardiography
We performed echocardiographic scans as previously described [18]. Briefly, under light sedation with 29 mg/kg ketamine and 4.3 mg/kg xylazine, the rats were placed in a left decubitus position and scanned via a commercial echo-scanner (Vivid i). Echocardiography at frequency of 9 MHz, depth 2.5 cm and frame rate 315 frames/sec, was used to scan two parasternal short axis sections at the apical (AP) and papillary muscle (PM) levels. Two-dimensional (2D) echocardiography scans took place at baseline before MI, 2 weeks after surgery and at the end of the experiment (4 weeks post-MI). The echocardiographic analysis included diastolic and systolic structural parameters. For ventricular function assessment, we calculated three parameters: (1) Fractional shortening (FS) which takes into consideration a 2D cross-section of the heart. Specifically, in the M-mode cines, the left ventricle (LV) intra-ventricular diameters were measured at systole and diastole (LVIDs and LVIDd, respectively), and FS was calculated as follows: FS = (LVIDd-LVIDs)/LVIDd; (2) Fractional area change (FAC) was calculated as the change in LV area during cardiac cycle. Particularly, LV end diastolic area (LVEDA) and LV end systolic area (LVESA) are measured in 2D cines and FAC were calculated as follows: FAC = (LVEDA-LVESA)/ LVEDA; (3) EF was determined by the Vivid i LV function software. The echocardiographic analysis was performed for each level (AP and PM) separately, since the extent of cardiac damage is level specific.
BP measurements
We monitored BP in conscious rats by a validated tail-cuff plethysmography method using CODA non-invasive BP system (Kent Scientific Corporation, Torrington CT, USA).
Urine collection
Each rat was placed for 24 h in a metabolic chamber (Techniplast S.p.A., Buguggiate, Italy) in which urine was collected for the determination of its volume, and glucose concentration. Subsequentially, blood sample was withdrawn from the tail vein to asses blood glucose and creatinine levels.
MI induction
Rats underwent MI procedure, as previously described by Bhindi [19]. Briefly, we intubated the rats under deep anesthesia with a mixture of 87 mg/kg ketamine and 13 mg/kg xylazine and ventilated them at a rate of 80–90 breaths per minute, and 1 to 2 ml/100 gr tidal volume. Using intercostal space left thoracotomy, the chest was opened, and the pericardial sac was dissected. A stich was placed through myocardium at a slightly greater depth than the perceived level of the left anterior descending (LAD) artery. Next, we tightened the suture to ensure complete LAD occlusion, the chest was closed, the skin stitched, and the rat was placed in its cage for recovery.
Empagliflozin treatment
Empagliflozin powder was generously provided by Boehringer-Ingelheim GmbH, Germany. Drug dosage of 30 mg/kg/day was adopted from Zhou and Wu, as they showed that this rather high dosage affected cardiac physiology as well as cellular biochemistry [20]. Accordingly, rats of group #2 were weighted weekly to adjust their dosage. First drug bolus was given immediately (~ 10–15 min) after MI by subcutaneous injection of the drug dissolved in 0.5 ml DDW. Then, for consecutive 4 weeks, drug was given by 0.5 ml gavage of empagliflozin dissolved in drinking water.
Direct measurements of cardiac hemodynamics
Direct cardiac hemodynamic parameters measurements were obtained by the Millar P–V system (MPVS-300, Millar Instruments, Houston, TX, USA). The Millar P–V System simultaneously and continuously measures LV pressure and volume from the intact beating heart, producing characteristic P–V loops readings. From the P–V loops the following cardiovascular parameters were derived, systolic and diastolic BPs, heart rate (HR), stroke volume (SV), cardiac output (CO), EF, stroke work (SW), dP/dtmax and dP/dtmin. Briefly, rats were anesthetized with a combination of 87 mg/kg ketamine and 13 mg/kg xylazine and placed on controlled heating pads. Next, the rats were tracheotomized and intubated (0.5 cm long 50 PE tube) to facilitate breathing. The right carotid was exposed and ligated distally, the artery was clamped and incised, a 2-Fr Mikro-Tip® catheter (SPR-838, Millar Instruments, Houston, TX, USA) was advanced through the artery into the LV under pressure control; a ligature was then tightened around the catheter to avoid blood leakage and loss [21]. After stabilization for 5 min, signals were continuously sampled at a sampling rate of 1000 samples/sec by the MPVS-300 system, recorded for 15–20 min, and displayed on a personal computer by the PowerLab System and Chart5 software (AD Instruments, Colorado Springs, CO, USA). At the end of each experiment, 4–6 boluses of 100 μL of hypertonic (30%) saline were injected intravenously, and from the averaged shift of P–V relations, parallel conductance volume (Vp) was calculated by the software and used for the correction of the cardiac mass volume. Thereafter, the catheter was withdrawn, and the animal was sacrificed by overdose anesthetic.
Organ harvesting
After sacrifice, the heart was excised, and dissected transversally at the PM level and its upper section, i.e., AP was fixed in 4% buffered formaldehyde and then embedded in paraffin for histology.
Histology
Five μm sections were stained with hematoxylin and eosin, photographed and analyzed for structural measurements: Cross-sectional area, LV cross-sectional area, LV cavity area, and septal and anterior wall widths using the ImageJ freeware (NIH, Bethesda, MD, USA).
Picro-Sirius red was used to distinct collagen from healthy muscle in the histological sections thus evaluate collagen deposition. Briefly, after de-paraffinization and re-hydration, we rinsed the slides in distilled water and stained nuclei with Weigert’s hematoxylin solution for 5 min. Then, we placed the slides in Picro-Sirius red stain for 60 min. Next, we rinsed in 2 changes of 0.5% acetic acid water followed by dehydration quickly through 3 changes of absolute alcohol and xylene. Finally, slides covered with coverslip using a permanent mounting medium. Collagen area was measured in scar and remote regions under microscope (Nikon Eclipse Ci-L, Nikon Corporation, Tokyo, Japan) using the NIS Elements software (NIS Elements 4.0, Nikon Corporation, Tokyo, Japan).
We assessed the mean collagen area of 6 fields from each slide, for each photograph, the red color (collagen), the total tissue area and total picture size were measured. Subsequently, we calculated the percentage of the collagen volume fraction (CVF) according to the formula:
$$\% {\text{CVF}} = \frac{{collagen~area}}{{\left( {total~picture~size - ~non~tissue~area} \right)}}{\text{*}}100$$
Immunohistochemical staining
Sections of myocardial tissue were dewaxed and rehydrated in a graded alcohol series. Antigen retrieval was achieved by heating the specimens for 10 min at 94 °C in citric acid buffer (0.01 mol/l; pH 6.0). Sections were then incubated with 3% hydrogen peroxide at room temperature for 20 min to inactivate endogenous peroxidase activity. After rinsing with PBS buffer (pH 7.2), primary antibodies against TGFβ1 (1:200; cat. no. Ab92486; abcam, Cambridge, MA, USA) or Smad3 (1:200; cat. no. Ab40854; abcam, Cambridge, MA, USA), were added and incubated at room temperature for 1 h. Following further rinsing, anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:1,000; cat. no. D13-6; GBI Labs, Bothell, WA, USA) was added and incubated at room temperature for 15 min. Samples were subsequently incubated with DAB solution at room temperature for 6 min until color development. A total of 5 different fields of view were randomly selected under a light microscope with magnification of × 400. The average absorbance of TGF-β1 was analyzed using the NIS-Elements Software BR analysis system version 4.10.00 (NIS Elements 4.0, Nikon Corporation, Tokyo, Japan).
Collagen concentration measurement
Collagen concentration was estimated in remote MI LV muscle samples by measuring the hydroxyproline (HOP) concentration [22]. Briefly, frozen cardiac samples were minced and then hydrolyzed at 120℃ for 20 min in 1N HCl, 450 μl of Chloramine-T was added to the hydrolysate, oxidation continued for 25 min in RT. We added 500 μl of Ehrlich’s aldehyde reagent to the samples and incubated at 65℃ for 20 min. Optical density was measured at 550 nm. All samples were measured in duplicates.
Statistical analysis
Data are presented as mean ± SD. Comparisons between groups were performed by 2-way analysis of variance (ANOVA) with repeated measures, in which the treatment and the time point were the independent variables. Whenever the ANOVA was significant, a multiple comparison was performed using the Holm-Sidak as post-hoc test. Single measured data i.e., final measurements, P–V loop derived parameters, and histological analysis were compared between groups using student's t-test. P value of < 0.05 was considered significant.