Animals

This study was approved by the Institutional Animal Care and Use Committee (IACUC) of the American University of Beirut (AUB). Five-month old WT C57BL/J6 fertile female and male mice (25–30 g) were purchased from Charles River Laboratories (Wilmington, MA, USA) and housed under pathogen-free conditions with constant temperature and humidity control at the AUB animal care facility. Mice were provided sterile bedding and ad libitum access to water and rodent chow.

Study timeline and experimental design

The experimental design of this study is shown in Fig. 1. Baseline echocardiography (echo) was performed. Mice were then enrolled into 8 weeks of smoking exposure in parallel with subcutaneous (SQ) injections of EE or vehicle (V), with echo recorded every other week. Mice were sacrificed at week 8 and the organs were collected. Ovariectomy was performed as previously described [15], 3 weeks before being used in experiments.

Fig. 1

Study timeline. EE, ethinylestradiol; V, vehicle; CS, chronic cigarette smoking; WT, wild type; SQ, subcutaneous; TPM, total particulate matter; Cig, cigarette; Echo, echocardiography

Echocardiography

Echocardiography was performed using the VEVO 2100 (FUJIFILM VisualSonics, Inc., Toronto, ON, Canada) with a frequency of 15.0 MHz. For imaging, animals were anaesthetized with isoflurane 2 to 3% in an oxygen mix chamber and placed on an electrically heated platform in a supine position. Body temperatures (maintained at 37 °C), heart rates and respiratory rates were continuously monitored throughout the procedure via an Indus Mouse-Monitor Heated Surgical Platform and the depth of anaesthesia was adjusted to maintain heart rate at 400–500 beats per minute. M-mode and B-mode echocardiography images of the left ventricle (LV) were obtained in the parasternal long-axis and short-axis views. Measurements at baseline and at 8 weeks right before sacrifice were acquired. For all animals three to four beats were measured using the same transducer position and mean calculations were obtained from three consecutive cardiac cycles. Fractional shortening (FS), ejection fraction (EF), left ventricular end‐systolic diameter (LVESD), left ventricular end‐diastolic diameter (LVEDD), left ventricular end‐systolic volume (LVESV) and left ventricular end‐diastolic volume (LVEDV) were recorded.

Chronic smoking exposure

Upon arrival to the smoking exposure room and following a short acclimatization period, female mice were exposed to cigarette smoke (CS) using an exposure apparatus (ONARES, CH Technologies, USA). This apparatus includes a smoke generator with a mixing/conditioning chamber and a “nose only” rodent exposure carousel. It allows for exposure to mainstream smoke from a cigarette in conscious, restrained rodents. This system has been extensively used to study smoking-related diseases. Mice in the smoking groups (V + CS and EE + CS) received CS twice daily (7 days/week) for 8 weeks. Cigarettes were placed into the cigarette puffer, and a peristaltic pump was used to generate puffs at a frequency of 1 puff/minute, duration of 2.5 s, and a volume of 5 ml per puff generated from 3R4F cigarettes (University of Kentucky, Lexington, KY, USA). 3R4F are scientifically prepared cigarettes concentrated with toxins and chemicals, making the study timeline suitable to observe the effects of smoking on mice. Animals received two 60 min CS sessions per day allowing a total particular matter (TPM) concentration of about 100g/cm3/mouse/session (100 mg TPM, 9.4 mg tar, and 0.726 mg nicotine per cigarette).

Drug delivery

Stock solutions of EE (Sigma-Aldrich, St. Louis, MO) for injections were dissolved in a vehicle (V) of 0.1% solution of polyoxyethylated 12-hydroxystearic acid (Sigma-Aldrich) and additional dilutions were made using normal saline. Mice were subcutaneously injected with 25 μg/kg/day EE, daily for 21 days (3 weeks) per cycle, for a total of 2 cycles in 8 weeks. Vehicle was given to control groups (100 μL/day). EE contraceptives are widely adapted in rodent research and are used in this study at a human-equivalent dose. The EE dosage was selected based on previous reports and also to be comparable to clinically relevant levels during oral contraceptive therapy daily regimen that an average 60–70 kg woman would be prescribed in an oral contraceptive [15].

Necropsy

At the end of the study, all mice were sacrificed. Mice were anesthetized with 3% isoflurane vapor (Forane®) diluted with O2. They were then injected with heparin for 5 min prior to sacrifice to facilitate blood collection. After that, plasma was separated and mixed with protease inhibitors and snap-frozen in liquid nitrogen for further analysis. Hearts were isolated from all mice, the right ventricle was removed, and the rest was divided into 2 sections: one section was immediately suspended in formalin-containing storage vials for histology analysis and the other was snap-frozen in liquid nitrogen and stored at − 80 °C for molecular analysis.

Histology

After formalin (10%) fixation for 2 days in a biopsy cassette and at room temperature, left ventricular transactional cuts were dehydrated and embedded in paraffin. Tissue sections from paraffin blocks (4 μm thickness) were mounted on glass slides for further different staining.

Hematoxylin and Eosin (H & E) staining

Briefly, heart mid sections of 5 μm thickness were taken for each mouse, de-paraffinized, rehydrated, and stained with H & E stain for 10 min. The slides were then washed and examined under a light microscope. Cross-sectional area of 100 cells of each of the 4 groups: V, V + CS, EE, and EE + CS was measured using Image J software (https://imagej.nih.gov/ij/).

Masson’s trichrome staining

In order to detect collagen deposition, tissue slides were stained with Masson’s Trichrome. Briefly, after dewaxing and hydration according to manufacturer’s protocol (Connective Tissue Stain, Abcam, ab150686) tissues were soaked in Boudin solution for 1 h at 56 °C, washed in running tap water, and then rinsed in distilled water. A second washing step was necessary after 10 min of incubation in hematoxylin, and then slides were stained in Biebrich scarlet acid fuchsin for 10 min. Sections were rinsed in water and incubated in phosphomolybdic phosphotungstic acid solution for 10 min. After that, they were transferred to aniline blue solution and stained for 5 min and finally mounted and observed using light microscopy. Fibrosis were measured using Image J software. For each section, ten fields were randomly imaged at 10 × magnification. Perivascular fibrosis was excluded from the analysis. The average measurement for all mice within the same group was then calculated.

Western blotting

Snap-frozen left ventricular sections of the heart were ground in liquid nitrogen with a mortar and pestle and then homogenized in lysis buffer containing protease and phosphates inhibitors. This step was followed by centrifugation (12,000xg at 4 °C) for 10 min. Protein concentrations were quantified using a DCTM Protein Assay II kit (Bio-Rad, Hercules, CA, USA). Equal amounts of protein samples were loaded in wells of a 10 or 12% gel and run until the dye front reached the bottom of the gel. Separated proteins were transferred onto methanol-activated PVDF membranes at 4 °C for 1.5 h at 90 V. The membranes were blocked with 5% fat-free milk prepared in Tween PBS solution at room temperature for 1h and incubated with primary antibodies and glyceraldehyde 3-phosphate dehydrogenase (GAPDH); the latter was used to ensure equal loading of samples. Immunoblots were then probed with the appropriate secondary antibody. All antibodies are listed in Table 1. The immunoreactive bands were visualized with ECL chemiluminescence detection kit (Thermo Fisher Scientific). Image J software (https://imagej.nih.gov/ij/) was used to quantify the intensity of bands and to normalize GAPDH protein levels in each group to confirm any up or down-regulation of the targeted protein.

Table 1 List of antibodiesReverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Snap-frozen heart tissues were used for RNA extraction. Briefly, mid-sections from the left ventricle were grounded in liquid nitrogen by a mortar and pestle then total RNA was isolated using TRIzol according to manufacturer’s instructions (Thermo Fisher Scientific, Grand Island, NY, USA). NanoDrop® ND-1000 UV–Vis spectrophotometer was used for RNA quantification. The purity of extracted RNA was assessed using the absorbance ratio of 260 to 280 nm, where a value of 1.8–2.0 indicates good-quality of RNA. To remove contaminating DNA, RNAs were treated with deoxyribonuclease I (Thermo, USA). qPCR was used to quantify differences in mRNA expression. Gene expressions were monitored using SYBR® Green PCR Master Mix (Bio-Rad, Hercules, CA, USA). GAPDH expression was used to normalize gene expressions between different samples. cDNA was synthesized from RNA using Revert Aid 1st Strand cDNA synthesis kit (Thermo, USA), followed by real-time PCR analysis in a CFX96 real-time PCR instrument (Bio-Rad, Germany). For the qPCR reaction, cDNA was loaded in duplicates of each of the forward and reverse primers of the gene of interest and mixed with SYBR Green. Negative control (RNAs free water) was used to check for nonspecific amplification. Fold expressions were normalized relative to the control and were calculated and plotted using Bio-Rad CFX Manager to compare differential gene expressions. The sequences of primers are listed in Table 2.

Statistical analysis

Data are shown as mean ± SEM. Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, San Diego, CA; https://www.graphpad.com). A difference of P < 0.05 was considered significant. The data for female mice was analyzed using 2-way ANOVA, while the data for male mice was analyzed using unpaired t-test. Prior to analysis, normality tests including the D'Agostino-Pearson omnibus, Anderson–Darling, Shapiro–Wilk, and Kolmogorov–Smirnov test were applied to ensure data distribution met assumptions for parametric testing. With all datasets passing at least one normality test, validation for the application of a 2-way ANOVA was achieved. In case of significant interaction or if one or both main effects was significant, a Tukey post-test was performed. When representative images are shown, the selected images were those that most accurately represented the average data obtained in all the samples.