Estrogen deprivation aggravates intracellular calcium dyshomeostasis in the heart of obese-insulin resistant rats

2.1 Animal preparation and ethical approval

This study was approved by the Institutional Animal Care and Use Committee of the Faculty of Medicine, Chiang Mai University (approval no. 5/2559 on May 5, 2016). All rat experiments conformed to the ARRIVE and the United State NIH guidelines. Thirty-two female Wistar rats (6 weeks of age, weighing 200–220 g) were obtained from the National animal center (Salaya campus, Mahidol University, Bangkok, Thailand). The rats were housed in a temperature-controlled room (25°C) with a 12-hr dark/light cycle setting and were given 1 week to acclimatize for.

2.2 Experimental protocol

Rats were randomly divided into two dietary groups: a normal diet group (ND, a diet containing 19.77% energy from fat), and a high-fat diet group (HFD, a diet containing 59.28% energy from fat; Pratchayasakul, Chattipakorn, & Chattipakorn, 2011). Thirteen weeks after the specific feeding, rats in each dietary group were divided into two subgroups to undergo either a sham operation (NDS or HFS) or a bilateral ovariectomy (NDO or HFO). Body weight and food intake of all rats were recorded throughout the experimental period. At the end of the sixth week after surgery, blood samples were collected from the tail vein for determination of metabolic parameters and estrogen levels. An oral glucose tolerance test (OGTT) and echocardiography were carried out. At the end of the study protocol, rats were deeply anesthetized with isoflurane, and the hearts were rapidly removed allowing single ventricular myocyte isolation (Palee, Apaijai, Shinlapawittayatorn, Chattipakorn, & Chattipakorn, 2016). The isolated cardiomyocytes were used for the measurement of intracellular Ca²⁺ transients. Cardiac Ca²⁺ regulatory proteins were investigated and biochemical studies were carried out.

2.3 Ovariectomy

In the ovariectomized group, rats were anesthetized using a rodent anesthesia machine (2% isoflurane vaporizer and 2 L/min oxygen flow) and then placed lying on the left and right lateral decubitus for left and right ovariectomy, respectively. After hair shaving and skin cleaning, a bilateral ovariectomy was carried out by initially making a midline dorsal skin incision. The incision was centered between the inferior crest of the rib cage and superior base of the thigh. The abdominal-pelvic cavity was accessed then the uterine tubes and ovaries were identified. Both ovaries were removed, and uterine horns were returned into the cavity. In the sham group, all rats underwent the same anesthesia and surgical preparation procedures as the OVX rats. Bilateral ovaries of sham rats were identified and exposed; the ovaries were not excised in these cases (Minta et al., 2018; Sivasinprasasn et al., 2017).

2.4 Cardiac function assessment

Echocardiography was used to determine LV function as a non-invasive method. Animals were given light anesthesia using 2% isoflurane with oxygen (2 L/min). An echocardiography probe (S12, GE Healthcare, CT) was placed on the chest at the parasternal short axis and connected to an echocardiography machine (GE vivid-i, GE healthcare). An M-mode echocardiogram at LV papillary muscle level was recorded, and %fractional shortening (%FS) was determined. A pulsed-wave Doppler spectrum of mitral flow was recorded from the apical four-chamber view using a color Doppler for guidance. The mitral E/A ratio was measured in each rat.

2.5 Determination of oxidative stress

Malondialdehyde (MDA) concentrations in cardiac tissues were measured using a high-performance liquid chromatography (HPLC) system (Thermo Scientific, Bangkok, Thailand) as described previously (Apaijai et al., 2014). Proteins from cardiac tissues were mixed with 10% trichloroacetic acid (TCA) containing BHT then heated at 90°C for 30 min and cooled to room temperature. The mixture was centrifuged, and the supernatant was mixed with 0.44 M H₃PO₄ and 0.6% thiobarbituric acid solution to generate thiobarbituric acid reactive substances (TBARS). The solution was filtered through a syringe filter (polysulfone type membrane, pore size 0.45 μm, Whatman International, Maidstone, UK) and analyzed using the HPLC system. Data were analyzed using BDS software (BarSpec Ltd., Rehovot, Israel), and plasma TBARS concentration was determined directly from a standard curve generated from a standard reagent for MDA at different concentrations and reported as MDA equivalent concentration.

2.6 Cardiomyocyte isolation and calcium transient measurement

Cardiomyocytes were isolated from the hearts of rats using a method described previously (Palee et al., 2016). In brief, under deep anesthesia, the heart was removed immediately and placed into a Langendroff apparatus. The hearts were perfused with modified Krebs solution (130 mM NaCl, 4.5 mM KCl, 1.4 mM MgCl₂, 0.4 mM NaH₂PO₄, 0.75 mM CaCl₂, 4.2 mM HEPES, 20 mM taurine, 10 mM creatine and 10 mM glucose, pH 7.3 at 37°C) for 5 min, followed by a Ca²⁺-free solution (100 μM EGTA) for 4 min, and a modified Krebs solution containing 100 μM CaCl₂ and 1 mg/ml Type II collagenase for another 8 min. The ventricles were removed from the cannula, cut into small pieces and incubated in 10 ml of collagenase solution gassed with 100% oxygen for 7 min at 37°C, with regular triturating. The cardiomyocytes were separated from undigested ventricular tissues by filtering through a cell strainer and were allowed to settle into a loose pellet. Then, the supernatant was removed and replaced with modified Krebs solution containing 1% BSA and 500 μM CaCl₂. This process was repeated with modified Krebs solution containing 1 mM CaCl₂. After this procedure, the cardiomyocytes were ready for the recording process.

The isolated cardiomyocytes were used for Ca²⁺ transient measurement using the CELL^R imaging software (Olympus Soft Imaging Solutions GmbH, Germany). Isolated cardiomyocytes were loaded with Fura-2/AM, and fluorescent intensity was recorded during electrical pacing (1 Hz, 10-ms duration, 15 V). The ratio of the emissions wavelengths is directly related to the amount of intracellular Ca²⁺. When Ca²⁺ binds to a ratiometric indicator, it changes the optimum excitation or emission wavelength of the indicator. An elevation of Ca²⁺ concentration induces an increase in Fura-2 emission fluorescence when the indicator is excited at 340 nm, with a corresponding decrease in fluorescence at 380 nm excitation (Bootman, Rietdorf, Collins, Walker, & Sanderson, 2013; Waldeck-Weiermair et al., 2015). The experiments were performed in a temperature-controlled chamber system at 37°C. Intracellular Ca²⁺ transient decay rate which approximates the rate of Ca²⁺ uptake into the SR by SERCA, intracellular Ca²⁺ transient amplitude which approximates the intracellular Ca²⁺ level during the systolic period, and diastolic Ca²⁺ level which approximates the intracellular Ca²⁺ level during the diastolic period were all calculated.

2.7 Determination of the expression of cardiac calcium regulatory proteins

To determine cardiac Ca²⁺ regulatory proteins, western blot analysis was used to examine the expression of the SERCA, RYR and NCX proteins. Briefly, rat myocardial tissues were homogenized in a lysis buffer (containing 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate [SDS] in 1× PBS) to extract the proteins. Then, the homogenate was centrifuged at 13,000 rpm for 10 min. Total protein (50–80 mg) was mixed with the loading buffer consisting of 5% mercaptoethanol, 0.05% bromophenol blue, 75 nM Tris, 2% SDS, and 10% glycerol with pH 6.8, and the mixture was boiled for 5 min and loaded into 10% gradient SDS-polyacrylamide gel. Proteins were then transferred to a nitrocellulose membrane with the presence of a glycine/methanol transfer buffer (containing 20 mM Tris, 0.15 M glycine, and 20% methanol) in a transfer system (Bio-Rad). The membranes were incubated in 5% skim milk in 1× TBS-T buffer (containing 20 mM Tris [pH 7.6], 137 nM NaCl, and 0.05% Tween-20) for 1 hr at room temperature and then exposed to anti-SERCA, RYR and NCX (Cell Signaling Technology, Danvers, MA) and anti-actin (Sigma-Aldrich, St. Louis, MO) for 12 hr. Bound antibody was detected by the conjugation of horseradish peroxidase with anti-rabbit IgG. Enhanced chemiluminescence detection reagents were administered to visualize peroxidase reaction products.

2.8 Determination of metabolic parameters and hormone levels

Plasma was prepared from fasted blood samples and was kept frozen at −80°C until analysis of glucose, cholesterol, triglyceride, insulin, and estradiol levels could be completed. Plasma estrogen concentration was measured by using a competitive enzyme immunoassay kit (Cayman Chemical Company, MI). Plasma insulin level was detected using a sandwich ELISA kit (Millipore, MI). Plasma glucose and triglyceride levels were determined by colorimetric assay from a commercially available kit (Biotech, Bangkok, Thailand). Fasting plasma HDL and LDL were determined using commercially available kits (ERBA Diagnostic, Mannheim, Germany) (Sivasinprasasn et al., 2015).

2.9 Statistical analysis

Data were expressed as mean ± standard error of the mean (SEM). Comparisons of variables were performed using the one-way analysis of variance followed by an LSD post-hoc test. The comparison of the % fractional shortening was performed using the Kruskal-Wallis test followed by Dunn's test. p < 0.05 was considered statistically significant.

3.1 Estrogen deprivation aggravated adverse changes of metabolic profiles and oxidative stress in obese-insulin resistant rats

After consumption of an HFD for 13 weeks, HFD rats developed insulin resistance as indicated by markedly increased body weight (318.20 ± 5.48 vs. 272.08 ± 2.69 g) and an increased HOMA index (14.28 ± 2.69 vs. 7.56 ± 1.34), when compared with ND rats. However, the fasting blood glucose level (143.54 ± 3.47 vs. 144.92 ± 4.27 mg/dl) and the amount of daily energy intake (58.85 ± 1.63 vs. 62.13 ± 2.21 kcal/day) was not significantly different between the HFD and ND rats.

Six weeks after ovariectomy (i.e., 19 weeks after HFD or ND consumption), both estradiol level and uterus weight showed a significant decrease in NDO and HFO groups, compared with NDS rats, confirming the endogenous estrogen-deprived condition resulting from the removal of ovaries. Moreover, the body weight and visceral fat deposition were increased in NDO, HFS, and HFO when compared with NDS rats (Table 1). However, there was an observable marked increase in both body weight and visceral fat deposition in HFO rats when compared with NDS, NDO and HFS rats (Table 1). Furthermore, plasma cholesterol levels in NDO, HFS, and HFO rats were significantly increased when compared with NDS rats (Table 1). Insulin sensitivity was decreased in NDO, HFS, and HFO groups when compared with NDS rats (Table 1) indicated by an increased area under the curve following an OGTT. However, HFO rats had the most severe impairment of insulin sensitivity (Table 1). Moreover, the cardiac oxidative stress indicated by cardiac tissue MDA and plasma MDA was markedly increased in NDO, HFS, and HFO rats when compared with NDS (Table 1). However, HFO rats had the most severe cardiac oxidative stress (Table 1).

3.2 Estrogen deprivation aggravated cardiac dysfunction in obese-insulin resistant rats

Six weeks after ovariectomy (i.e., 19 weeks after HFD or ND consumption); NDO, HFS, and HFO rats had increased SBP, DBP, MAP and heart rate, compared with NDS rats (Figure 1a–d). In all groups, HFO rats had the most severe impairment of BP and heart rate regulation (Figure 1a–d). Echocardiograms demonstrated that HFS, HFO and NDO also developed LV systolic dysfunction as indicated by decreased %FS and also LV diastolic dysfunction as indicated by a decreased mitral E/A ratio when compared with NDS rats (Figure 2a,b). Similar to observed BP impairment, HFO rats had the most severe LV dysfunction (Figure 2a,b). The representative images of cardiac M-Mode and cardiac doppler are shown in Figure 2c,d respectively.

3.3 Estrogen deprivation aggravated intracellular calcium dyshomeostasis in obese-insulin resistant rats

In this study, the intracellular Ca²⁺ transients were determined to assess intracellular Ca²⁺ homeostasis. Six weeks after ovariectomy (i.e., 19 weeks after HFD or ND consumption), intracellular Ca²⁺ transient amplitude, intracellular Ca²⁺ transient rising rate, and intracellular Ca²⁺ transient decay rate were markedly decreased in NDO, HFS, and HFO rats, compared with NDS rats (Figure 3a–c). In addition, the diastolic Ca²⁺ level was increased in NDO, HFS, and HFO rats, compared with NDS rats (Figure 3d). In all groups, HFO rats had the most severe impairment of intracellular Ca²⁺ transient amplitude, intracellular Ca²⁺ transient rising rate, intracellular Ca²⁺ transient decay and diastolic Ca²⁺ level (Figure 3a–d). The representative intracellular Ca²⁺ transient tracings are shown in Figure 3e.

3.4 Estrogen deprivation aggravated cardiac calcium handling protein dysregulation in obese-insulin resistant rats

In this study, the Ca²⁺ handling proteins were investigated to assess intracellular Ca²⁺ regulation. Six weeks after ovariectomy (i.e., 19 weeks after HFD or ND consumption), SERCA, NCX and RYR protein expression were markedly decreased in NDO, HFS, and HFO rats, compared with NDS rats (Figure 4a–c). In all groups, HFO rats had the most severe impairment of Ca²⁺ handling protein expression (Figure 4a–c) and representative Ca²⁺ handling protein expression is shown in Figure 4d.