Study design

Twenty-eight male Wistar rats, aged 2 months and weighing ~ 200 g, were randomly assigned to two experimental groups: Control (n = 14) and PAH (n = 14). All animals were housed in a temperature-controlled room (approximately 22 °C) with 60% relative humidity, maintained on a 12/12-h light/dark cycle, and provided with free access to water and standard rodent chow. The experimental protocol was approved by the Ethics Committee on Animal Use of the Federal University of Viçosa (CEUA/UFV; approval number 11/2022), in accordance with the Guide for the Care and Use of Laboratory Animals.

Induction of experimental pulmonary arterial hypertension

For the induction of experimental PAH, animals in the PAH group received a single intraperitoneal injection of 60 mg/kg body weight of MCT (Sigma-Aldrich, St. Louis, MO, USA) dissolved in saline (140 mM NaCl; pH 7.4). Control animals received an equivalent volume of saline (140 mM NaCl; pH 7.4) (Natali et al. 2015).

Echocardiography

The echocardiographic evaluation was performed on the 24th day after the administration of MCT. The animals were anesthetized with 1.5% isoflurane dissolved in 100% oxygen at a constant flow of 1 L/min (Isoflurane, BioChimico, RJ, Brazil). The images were acquired with the animals in the lateral decubitus position. Two-dimensional studies with a fast-sampling rate of 120 fps in M mode were performed using the MyLabTM30 ultrasound system (Esaote, Genoa, Italia) and 11 MHz nominal frequency transducers. The two-dimensional transthoracic echocardiography and M-mode was obtained at a scanning speed of 200 mm adjusted according to heart rate. Tricuspid annular plane systolic excursion (TAPSE) was evaluated and used as an indicator of RV function.

Sample collection

The animals from two experimental groups (Control and PAH) were euthanized by decapitation on the 25th day after the injection of MCT. After euthanasia, the heart and RV were dissected and processed for the analyzes of interest, as described below.

Analysis of chemical elements

A scanning electron microscope (Leo 1430 V P, Carl Zeiss, Jena, Thuringia, Germany) with an attached x-ray detector system (Tracor TN5502, Middleton, WI, USA) was used, as previously described (Bastos et al. 2020), to obtain images of the distribution of chemical elements and to determine the proportion of carbon (C), sodium (Na), potassium (k), calcium (Ca), iron (Fe), magnesium (Mg), copper (Cu), zinc (Zn), and selenium (Se) in cardiac tissue (RV) in a model of MCT- induced PAH. For this end, fragments (4X3X2.5 mm) from the RV were fixed in 2.5% glutaraldehyde, dehydrated in ethanol, submitted to critical point drying (CPD 030, Bal-tec, Witten, North Rhine-Westphalia, Germany) and coated with evaporated carbon (Quorum Q150 T, East Grinstead, West Sussex, England, UK). The EDS microanalysis was performed at × 1000 magnification with an accelerating voltage of 20 kV.

Protein expression

The expression of type 2 ryanodine receptors (RyR2), Phospholamban (PLB), Phospho-Phospholamban, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA2) and sodium/calcium exchanger (NCX1) proteins was determined by Western blotting, as previously described (Pelozin et al. 2022). Frozen fragments of the RV (100 mg) were homogenized in RIPA buffer, composed of 150 mM NaCl, 50 mM Tris–HCl (pH 8.0), 0.1% sodium dodecyl sulfate, 1% NP-40, in addition to a protease and phosphatase inhibitor cocktail (1:100; Sigma-Aldrich, St. Louis, MO; PhosSTOP 1:10; Roche, Basel, Switzerland). Insoluble cardiac tissues were removed by centrifugation at 3,000 g, 4 °C, for 10 min. Samples were loaded and subjected to SDS-PAGE on 8–10% polyacrylamide gels. After electrophoresis, proteins were electrotransferred to a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ). Equal loading of samples (40 μg) and even transfer efficiency were monitored with the use of 0.5% Ponceau S staining of the blot membrane. The blot membrane was then incubated in a blocking buffer (5% nonfat dry milk, 10 mM Tris·HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20) for 2 h at room temperature and incubated with specific antibodies overnight at 4 °C. The Odyssey® Fc Imaging System (LI-COR; Lincoln, NE, USA) was used to detect labeled proteins.

The following primary antibodies were used: α- Tubulin (#2144) and GAPDH (#97,166), both from Cell Signalling Technology (Danvers, MA, USA). RyR2 ((#TA5-87,416), PLB (#PA5-85,268), p-PLB (Ser16, Thr17) (#711,401), SERCA2 (#MA3-919) were from Invitrogen Life Technologies (Strathclyde, UK). NCX1 (#151,608) were from Abcam (Cambridge, UK). For all primary antibodies were used at 1:1000 dilution, the secondary antibodies used were IRDye® 680RD or 800CW (1:15,000 LICOR, Lincoln, NE, USA). Bands were analyzed with Image J software (ImageJ based on NIH Image).

Isolation of right ventricular myocytes

Myocytes from the RV were enzymatically isolated as previously described (Natali et al. 2001). Briefly, after euthanasia, the heart was rapidly dissected and attached to a Langendorff-retrograde perfusion system via aorta and perfused with Tyrode solution containing (in mM): 130 NaCl, 1.43 MgCl2, 5.4 KCl, 0.75 CaCl2, 5.0 Hepes, 10.0 glucose, 20.0 taurine and 10.0 creatine, pH 7.4 until for about 5 min. The Tyrode solution was thus exchanged to Tyrode solution containing EGTA (0.1 mM) for 5 min. Subsequently, the heart was perfused with Tyrode solution containing 1 mg/ml collagenase type II (Worthington, USA) and 0.1 mg/ml protease (Sigma-Aldrich, USA) for about 12 min. Following this process, the RV of the digested heart was separated and cut into small fragments. These fragments were placed into a conical flask containing the enzymatic solution (collagenase and protease) and mechanically separated by shaking the flask for 5 min. Thereafter, the dispersed cells were separated from the non-dispersed tissue by filtration. After centrifugation, the resulting cells were suspended in Tyrode solution. The non-dispersed tissue was subjected to the mechanical dispersion process again. All solutions used in the isolation procedure were oxygenated (100% O2—White Martins, Brazil) and maintained at 37 °C. The isolated cells were stored at 5 °C until use and were utilized within 2–3 h after isolation.

Measurement of cardiomyocyte contractility

The contractile function of RV myocytes was measured by using an edge detection system (Ionoptix, Milton, USA) mounted on an inverted microscope (Nikon Eclipse—TS100, Japan) as previously described [14]. In summary, myocytes were placed in a bath on the stage of the inverted microscope and superfused with Tyrode solution containing (in mM): 137 NaCl, 5.4 KCl, 0.33 NaH2PO4, 0.5 MgCl2, 5 HEPES, 5.6 glucose, and 1.8 CaCl2, (pH 7.4) at 37 ± 1 °C. Only cardiomyocytes that presented a sarcomere with a clear and regular striated pattern, that did not contract spontaneously in the absence of external stimulation and that responded to 1 Hz stimulation with a single contraction were analyzed. The RV myocytes were externally stimulated (40 V, 5 ms duration) to contract at a frequency of 5 Hz using an electric field stimulator (Myopacer, Ionoptix, Milton, USA). The cardiomyocytes were then visualized on a computer monitor using a CCD camera (Myocam, Ionoptix, Milton, USA) and the images of cell contractions were recorded. From the recordings, the amplitude of cell shortening was evaluated as a percentage of the resting cell length. Cell departure velocity and return velocity were measured as the maximal rate of sarcomere shortening and relaxation, respectively as a percentage of the control group (Hobai et al. 2016). All these parameters were analyzed using the IonWizard 6.3 software (Ionoptix, Milton, USA).

Statistical analysis

The normality of the data was tested using the Shapiro–Wilk test. Differences between groups were tested using Student’s t test. Data are presented as mean ± SD. P < 0.05 was considered for statistically significant differences. All analyzes were performed using GraphPad Prism, version 6.01 (San Diego, CA, USA).