Animal model
All animal experiments were carried out in accordance with the recommendations of national and international animal care and ethical guidelines and were approved by the Ethics Committee for Animal Research of Xiangya Hospital of Central South University (permit code: 2103590). Monocrotaline (MCT, 55 mg/kg; C2401, Sigma-Aldrich) was intraperitoneally injected in Sprague-Dawley (SD) rats (weighing 220–250 g) to establish PAH as described previously[17]. Transthoracic echocardiography was performed every seven days to measure the maximum velocity of tricuspid regurgitation before day 21, and every two days after day 21. A maximum tricuspid regurgitation velocity greater than or equal to 2.5 m/s was the criterion for successful establishment of the PAH model. Right ventricular function was determined by measuring the fractional area change (FAC), the ratio between the difference in end-diastolic and end-systolic area and the end-diastolic area of the right ventricle. Right ventricular tissues were harvest at day 28.
Analysis of right ventricular systolic pressure and cardiac function
We used transthoracic echocardiography to collect and analyze right ventricular systolic pressure and function data in rats as previously described[17]. All rats were anaesthetized by inhalation of 6–8% (v/v) sevoflurane, and anaesthesia was maintained with 2% (v/v) sevoflurane. All rats were placed on a heating pad to maintain a body temperature of 37°C. Echocardiography was performed with the Vivid E7 system (General Electric Vingmed). Right ventricular systolic pressure (RVSP) was equal to cross-valve pressure plus an estimated right atrial pressure (RAP) of 10 mmHg. Cross-valve pressure was estimated by calculating the maximum velocity of the tricuspid regurgitant (TRmax) jet using the modified Bernoulli equation. Right ventricular function was determined by measuring the fractional area change (FAC), the ratio between the value of end-diastolic right ventricular area (RVA d) minus end-systolic right ventricular area (RVA s) and end-diastolic area. The rats in control group had no tricuspid regurgitation, we directly assessed the right ventricular end-systolic pressure using right ventricular catheterization technique. The monitoring data was collected and analyzed using LabChart 8 (ADInstruments).
Quantitative mass spectrometry
According to the data measured by echocardiography, right ventricular tissue (male, 6–8 weeks old, n = 3) were collected at day 28 from euthanized rats and shipped to Jingjie PTM-Biolab for 4D label-free quantitative proteomic analysis. Samples were subjected to standard protein extraction and trypsin digestion. The tryptic peptides were desalted by C18 SPE column and were subsequently dissolved in solvent A (0.1% formic acid, 2% acetonitrile), directly loaded onto a home-made reversed-phase analytical column (25-cm length, 75/100 µm i.d.). Peptides were separated with a gradient from 6–24% solvent B (0.1% formic acid in acetonitrile) over 70 min, 24–35% in 14 min and climbing to 80% in 3 min then holding at 80% for the last 3 min, all at a constant flow rate of 450 nL/min on a nanoElute UHPLC system (Bruker Daltonics). The peptides were subjected to capillary source followed by the timsTOF Pro (Bruker Daltonics) mass spectrometry.
TEPP-46 treatment
The PAH rats were randomly divided into two groups on the 21st day after MCT injection. The experimental group was given TEPP-46 (30 mg/kg; MedChem Express, HY-18657) gavage treatment every day, and the control group was given corresponding solvent gavage operation. Cardiac function data were obtained every 2 days, and right ventricular tissues were harvested on day 28.
Isolation, culture, and treatment of primary cardiac myocytes
Neonatal rat ventricular myocytes (NRVMs) were isolated from 1-day-old SD rats by using a neonatal heart dissociation kit (Miltenyi Biotec, 130-098-373) following the manufacturer’s instructions and cultured in dulbecco's modified eagle medium (DMEM) (Gibco, 11965092) containing 10% fetal bovine serum (FBS) (Hyclone, SV30087.03), and 1% penicillin & streptomycin (Abiowell, AWH0529a). The cells were grown at 37°C in a humidified atmosphere with 5% CO2, and the medium was changed every two days. primary cardiac myocytes were exposed to arginine vasopressin (AVP, 1 µM, HY-P0049; MedChem Express) for up to 48 hours to induce hypertrophy and TEPP-46 (100 µM) for 24 hours to promote PKM2 tetramerization.
Western blotting analysis
Total protein was extracted from right ventricular tissues or myocytes with RIPA buffer (NCM, WB2100) containing 1 mM PMSF (Byotime, ST507), and protein concentration was determined by BCA protein assay kit (NCM, WB6501). Total proteins were separated on BeyoGel Plus PAGE Tris-Gly system (Byotime, 0469S) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, ISEQ00010). The membrane was blocked with 5% skim milk in PBS with 0.1% Tween 20 (PBST) at room temperature for 1 hours and incubated with primary antibodies (Table S1) at 4°C overnight. The membrane was then incubated with peroxidase affiniPure goat anti-rabbit IgG (H + L) (1:10000, Jackson, 111-035-144) at room temperature for 1 hour. Finally, enhanced chemiluminescence (ECL) reagent (Advansta, K-12045-D50) was used to detect the bands.
Crosslinking to determine PKM2 tetramer
As descirbed in previous studies[18, 19], an equal amount of fresh heart right ventricle tissue or primary myocytes was used for crosslinking. Myocardial tissue was cut into 1 mm3 pieces with surgical scissors, then was crosslinked with 2.5 mM DSS in PBS for 30 min at room temperature, with constant rotating. For primary myocytes, we used 500 µM DSS in PBS to crosslink. Samples were separated by BeyoGel Plus PAGE 4–20% Tris-Gly Gel (Beyotime, P0469S).
Quantitative real-time PCR
The total RNA of ventricular tissue was isolated with Total RNA Kit II (R6934-01, Omega Bio-tek) according to the manufacturer’s instructions. An RT Reagent Kit with gDNA Eraser (RR047A, Takara) was used for cDNA synthesis. Real-time PCR was performed using All-in-OneTM qPCR Mix (QP001, GeneCopoeia) according to the manufacturer’s protocol. Primer sequences are listed in Table S2.
Histology and immunochemistry
Paraffin-embedded sections were subjected to hematoxylin and eosin (HE) or Masson’s trichrome staining. Images were acquired using a Nikon Eclipse E100 and Nikon DS-U3 automated slide scanner.
TUNEL staining
The heart samples were separated and fixed in 10% phosphate-buffered formalin for 24 h, subsequently embedded in paraffin, sliced (4–5 µm). Terminal deoxynucleotidyl transferase-mediated dexoxyuridine triphosphate nick-end labeling (TUNEL) staining was performed using the TUNEL BrightGreen Apoptosis Detection Kit (A112-01, Roche Life Science) following manufacturer’s instructions. Apoptotic nuclei were labeled with green fluorescein staining and total cardiomyocyte nuclei were marked with 4′,6′-diamidino-2-phenylindole (DAPI) (SouthernBiotech, 0100 − 20). The slices of heart tissues were viewed by confocal microscopy (Eclipse C1, NIKON). Rate of apoptosis was displayed as percentage of TUNEL positive nuclei in DAPI-stained nuclei.
Flow cytometry analysis
Cells were digestion with trypsin without EDTA and washed twice with PBS. Apoptosis of myocytes was detected by PE Annexin V Apoptosis Detection Kit I (BD Biosciences, 559763) following the manufacturer’s instructions. Flow cytometry analysis was performed on a DxP AthenaTM instrument (Cytekbio), and the data were analysed using FlowJo software.
Transmission electron microscopy (TEM) and evaluation of damaged mitochondria
Fresh heart right ventricle samples were fixed in 2.5% glutaraldehyde (pH 7.4) for 2 h. Samples were then washed three times with 0.1 M phosphate buffer (pH 7.2) and fixed in 1% osmic acid at 4℃ for 2 h. Subsequently, the samples were gradient dehydrated in a graded series of ethanol, embedded in Epon-Araldite resin for penetration and placed in a mold for polymerization. After the semi thin section was used for positioning, the ultrathin section was made and collected for microstructure analysis, followed by the counterstaining of 3% uranyl acetate and 2.7% lead citrate. Data were acquired with a Hitach HT7800 transmission electron microscope.
Healthy mitochondria exhibit intact and well-defined membranes, along with abundant and regular cristae. Swollen mitochondria, on the other hand, display distorted membranes and disrupted or enlarged cristae. Vacuolar mitochondria are characterized by the separation of inner and outer mitochondrial membranes, accompanied by the absence of cristae. To quantify damaged mitochondria, the ratio of vacuolar and swollen mitochondria to the total number of mitochondria in the image was computed. Ruptured mitochondria exhibit a discontinuity in the outer mitochondrial membrane. To quantify ruptured mitochondria, the ratio of ruptured mitochondria to the total number of mitochondria in the image was calculated.
Advanced oxidation protein product (AOPP) measurement
AOPP test was used to assess oxidative stress within the right ventricular tissues according to the manufacturer’s instructions. Right ventricular myocardial tissue samples (20–30 mg) were harvest for homogenization. Then, we collected the supernatant for AOPP measurements using the AOPP assay kit (Abcam, ab242295). Results were normalized by protein concentration.
ROS measurement in primary cardiac myocytes
Cells were seeded in 35 mm glass-bottomed culture dish with a density of 5×104/ml. Cells were incubated with 5 µM MitoSOX Red (Invitrogen, M36008) for 10 minutes at 37℃. The cells were washed twice with PBS, and the fluorescence imaging of live primary cardiomyocyte was conducted at 510/580 nm on Axio Observer 7 microscope (Zeiss). The mean fluorescence intensity of cells was quantified using Fiji software.
Immunofluorescence
Cells were stained with 100 nM MitoTracker Red (Invitrogen, M7512) for 30 minutes, fixed for 10 min with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 10 min. For OPA1 staining, cells were incubated with specific primary antibody (CST, 67589S) overnight in 1% BSA at 4°C, followed by secondary antibody (Invitrogen, A11034) at room temperature for 1 hour. DNA was stained with DAPI. Images were acquired using Apotome microscope (Zeiss). As previously described, the means of area, perimeter, aspect ratio and form factor of mitochondria were quantified using Mitochondria Analyzer in Fiji software according to the published protocol[20–22].
Statistical Analysis
All replicate data are expressed as the means ± standard error of the mean (SEM). The distribution of the data was assessed with the Shapiro–Wilk normality test. The significance of difference between two independent experimental groups was assessed using unpaired Welch’s t-test, and the significance of difference among more than two groups was assessed using one-way ANOVA. The data between two groups with repeated measurements were compared using two-way ANOVA. For proteomic data analysis, the false discovery rate (FDR) adjusted p value was calculated based on the Benjamini and Hochberg (BH) method[23]. The value of statistical significance was set at p < 0.05. *, ** and *** represents p < 0.05, < 0.01, and < 0.001, respectively. Statistical analyses were performed using GraphPad Prism 9 software.