Development of chronic hypobaric hypoxia-induced mouse model. All animal procedures were approved by the Experimental Animal Ethics Committee of the Army Medical University and conformed to the regulations of the Guide for the Care and Use of Laboratory Animals. Male C57BL/6J mice (6–8 weeks old) were housed in a temperature-controlled environment with a 12-hour light/dark cycle. The mice were subjected to HH condition (10.0% oxygen content and 46.3 kPa air pressure) in a chamber (AIPUINS XBS-03, Hangzhou, China) or were housed in normobaric normoxia (NN, 20.9% oxygen content and 101.3 kPa air pressure) as controls for 6 weeks (Fig. 1a).
Blood preparation and enzyme-linked immunosorbent assay. Detection kits for mouse plasma B-type natriuretic peptide (BNP), troponin I (TnI), and creatine kinase-MB (CK-MB) were purchased from Jiangsu Jingmei Biological Technology Co., Ltd. (Jiangsu, China). Approximately 1.5 mL of blood was drawn from each mouse and stored in procoagulant tubes. Plasma was separated by centrifugation (3000×g, 20 min) after coagulation at room temperature for 10 min. The plasma levels of BNP, TnI, and CK-MB were measured using a commercially available BNP enzyme-linked immunosorbent assay (ELISA) kit (JM-02343M2, 210727B8), TnI ELISA kit (JM-02662M2, 210727I4), and CK-MB ELISA kit (JM-03084M2, 210727C6), respectively, following the manufacturer’s instructions.
Hemodynamic monitoring. Right heart catheterization (RHC) was performed using a pressure detecting device (ADInstruments Mikro-Tip®, MPVS Ultra RSBMIL002/M) after a 6-week HH or NN exposure. The mice were placed on a heated pad and anesthetized with 2% isoflurane. The right jugular vein was exposed, and a 1F needle (ADInstruments Mikro-Tip®, SPR-1000) was slightly bent inwards to conduct the cannula containing the catheter into the jugular vein. The cannula was maneuvered to the right ventricle, with its tip pointing towards the heart until an RV pressure curve could be identified using LabChart 7 software. Next, the cannula tip was manipulated to the left and upward. The catheter was advanced into the main pulmonary artery, passing through the pulmonary valve. When the catheter enters the main pulmonary artery, the diastolic pressure rises on the monitor, and a pulmonary artery pressure curve appears. When the curve was constant, the related indices, such as the mean pulmonary artery pressure (mPAP), maximum positive time derivative of left ventricular pressure (max dP/dt), and RV velocity time integral (VTI) and electrocardiograms were measured.
Evaluation of right ventricular hypertrophy. After the hemodynamic measurement, the mice were sacrificed by cervical dislocation, and their hearts were removed quickly and weighed. The free wall of the right ventricle was dissected from the left ventricle and interstitial septum. Whole heart weight (normalized by body weight) and Fulton’s index (right ventricle / [left ventricle + interstitial septum]) were used as indices of cardiac hypertrophy.
Histological analysis. The hearts from the mice exposed to NN and HH were excised, placed in 4% paraformaldehyde, dehydrated in graded concentrations of ethanol, immersed in xylene, and embedded in paraffin. Sections of 5-µm thickness were cut on a microtome with a disposable blade, stained with hematoxylin-eosin and Masson’s trichrome stain, and examined by light microscopy. The cardiomyocyte cross-sectional area (CSA) was analyzed by staining the heart sections with a wheat germ agglutinin–Alexa Fluor® 647 conjugate (W32466, Invitrogen). Six mice from each group were included in the histological analysis. A minimum of five cross-sections of each heart were examined, and the measurements were averaged for statistical analysis. ImageJ software (RRID:SCR_003070) was used to quantify all the histological endpoints.
Generation of cardiomyocyte-specificPacs2knockout mice. Cardiomyocyte-specific Pacs2 knockout (Pacs2−/−) mice were generated on a C57BL/6J background by the CRISPR/Cas9 system at cyagen. The gRNA to mouse Pacs2 gene, the donor vector containing loxP sites, and Cas9 mRNA were co-injected into fertilized mouse eggs to generate targeted conditional knockout offspring. Pacs2flox/flox mice in which the Pacs2 gene was flanked by loxP sites within introns 1 and 3 (KO region: ~1842 bp) were crossed with α-myosin heavy chain (α-MHC) promoter-Cre transgenic mice (Cyagen Biosciences) to obtain Pacs2flox/+/CreαMHC+/− mice. F0 founder animals were identified by PCR followed by sequence analysis, which were bred to wildtype mice to test germline transmission and F1 animal generation. F1 founders, including Cardiomyocyte-specific Pacs2 knockout (Pacs2flox/flox/CreαMHC+/−) mice, were genotyped by tail genomic PCR.
Generation of cardiomyocyte-specificPacs2knock-in mice. The cardiomyocyte-specific Pacs2 knock-in in C57BL/6J mice was created using CRISPR/Cas-mediated genome engineering (Cyagen Biosciences). The Hipp11 locus is located within an intergenic region between the Eif4enif1 and Drg1 genes on mouse chromosome 11. The mouse Pacs2 gene (NCBI Reference Sequence: NM_001291444.1) is located on mouse chromosome 12. For the KI model, the “alphaMHC_long promoter-Kozak-Mouse Pacs2 CDS-rBG pA” cassette was inserted into Hipp11 locus (~0.7 kb 5' of Eif4enif1 gene and ~4.5 kb 3' of the Drg1 gene). To engineer the targeting vector, homology arms were generated by PCR using BAC clone as the template. Cas9 and gRNA were co-injected into fertilized eggs with a targeting vector for mice production. The pups were genotyped by PCR followed by sequencing analysis.
Echocardiography. Cardiac geometry and function were examined using ultrasonography (GE Vivid 7 Dimension, L15/6-MHz transducer). The mice were anesthetized with 2% isoflurane while maintaining proper body temperature (36–37°C) and heart rate (450–550 beats/ minute). The temporal frame rate in the echo mode was set to 60 Hz. A 1.0-mm sampling gate was used to obtain the inflow and outflow velocities, and the maximal sweep speed was 200 mm/s. RV end-diastolic (ED) and end-systolic (ES) areas were measured using ImageJ from the apical or basal 4-chamber views at end-diastole or end-systole. The RV fractional area change (FAC) was calculated as follows: FAC = ([ED RV area – ES RV area] / ED RV area) × 100%. For Tei index calculation, the tricuspid closure opening time (TCO) and ejection time (ET) were measured from tissue Doppler myocardial velocity images, as follows: Tei index = (TCO – ET) / ET. Data were collected from six mice per group and represented the average of minimum of five separate scans in a random blind fashion. To avoid bias, the researcher performed all echocardiography procedures blinded to the experimental treatments.
Cell culture and RNA transfection. Rat H9C2 cardiomyocytes (BFN60804388) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cardiomyocytes were cultivated in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, Louis, MO, USA) and 10% fetal bovine serum (Hy Clone Laboratories, PA, USA), and supplemented with 1% antibiotic-antimycotic (1000 U/mL penicillin and 100 µg/mL streptomycin). H9C2 cardiomyocytes in the NN group were incubated at 37°C with 5% CO2. HH conditions were achieved by using a HH chamber (Billups-Rothenberg) flushed with a pre-analyzed gas mixture of 1% O2, 5% CO2, and 94% N2. To maintain cardiomyocyte cultures, the medium was changed every 2 days. LVVs carrying Pacs2 RNA system were constructed by Gene Pharma Technology (Shanghai, China). The LVVs were added to the cells at a multiplicity of infection of 100. The transfection medium was changed 2 days later, and the cells were continuously cultured in fresh medium. Real-time quantitative reverse transcription-PCR and western blotting were used to detect the efficiency of Pacs2 overexpression in cardiomyocytes.
Western blotting. Cardiomyocyte MAM fractions in vitro and MAM fractions of the hearts were isolated following a previously described protocol49. Western blotting was used to evaluate protein expression in different fractions. Briefly, the protein concentrations of different fractions after isolation were detected using the BCA assay (Beyotime Biotechnology, P0012). The same mass of total protein was separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% nonfat milk in Tris-buffered saline (Boster Biological Technology, AR0031) containing 0.5% Tween-20 (Solarbio, T8220), and membrane-bound proteins were probed with primary antibodies purchased from Abcam against the following antigens: PACS2 (ab222316, Abcam), mitofusin 2 (MFN2; ab124773, Abcam), voltage-dependent anion-selective channel protein 1 (VDAC1; ab14734, Abcam), acyl-CoA synthetase 4 (FACL4; ab92501, Abcam), mitochondrial fission 1 (FIS1; ab156865, Abcam), calnexin (ab133615, Abcam), microtubule-associated protein 1 light chain 3 beta (MAP1LC3B; ab192890, Abcam), translocase of outer mitochondrial membrane 20 (TOMM20; ab186735, Abcam), and actin beta (ACTB; ab8226, Abcam). Protein bands were visualized by chemiluminescence detection and quantified using the Image QuantTL software (GE Healthcare, Sweden).
Measurement of mitochondrial calcium in intact cells. Cardiomyocytes were seeded on glass-bottomed cell culture dishes and incubated with 1 µM of the calcium indicator Rhod2AM (ab142780, Abcam) at 37°C in the dark for 30 min, as per the manufacturer’s guidelines. Next, the cells were washed twice with calcium-free HBSS and imaged under a laser scanning confocal microscope (LSCM, Leica TCS-SP5). The fluorescence intensity (F) was normalized to the baseline fluorescence value F0 (F/F0) and expressed as mitochondrial calcium concentration ([Ca2+]m). We measured Fmax and Fmin, as previously described50. Fmax was obtained by perfusion with 10 µM ionomycin and 5 mM CaCl2; Fmin was measured by perfusion with 10 mM EGTA and 20 µM BAPTA-AM (B1205, Molecular probes) in HBSS. 2-APB (ab120124, Abcam), TG (T9033, Sigma-Aldrich), and ATP (A1852, Sigma-Aldrich) were also added to the external solution at a proper final concentration. The fluorescence intensity of Rhod2-AM was measured using LSCM. The fluorescence intensity was converted to [Ca2+] using the following formula: [Ca2+]m = Kd × (F − Fmin) / (Fmax − F), where Kd is the equilibrium dissociation constant of Rhod2 for Ca2+, which was 570 nM.
Immunofluorescence. Cardiomyocytes were stained with MitoTracker Deep Red FM (500 nM; M22426, Invitrogen) and fixed in 4% paraformaldehyde (P0099, Beyotime Institute of Biotechnology) at room temperature for 10 min. They were then permeabilized with 0.1% Triton 100-X (P0096, Beyotime Institute of Biotechnology) at room temperature for 30 min. Cells were washed with phosphate-buffered saline (PBS) three times and blocked in blocking buffer (P0102, Beyotime Institute of Biotechnology) for immunostaining at 37°C for 30 min. Samples were incubated with anti-MAP1LC3B antibody (1:100) or anti-ERP72 antibody (ab155800, Abcam; 1:100) at 4°C overnight and then washed in PBS twice, before staining with the secondary antibody (31561, Invitrogen; 1:500) at 37°C for 2 h. Co-localization of fluorescence was measured at 100–400 Hz under the LSCM. Samples without primary antibodies were used as negative controls. Images were analyzed using LAS X software (Leica) and Image-Pro Plus 5.0 (Media Cybernetics). Co-localization represented in Pearson’s correlation coefficient was measured using automatic thresholding, as previously described51.
Measurement of mitochondrial bioenergetics and FAO metabolism. The cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse XF96 extracellular flux analyzer (Seahorse Bioscience, North Billerica, USA). Briefly, cells with/without HH exposure or transfected with LVVs-Pacs2 were plated in XF96-well microplates (6000 cells/well, Seahorse Bioscience). After reaching the proper cell density, the cells were incubated with XF assay medium without CO2 at 37ºC for 1 h. For OCR measurement, cells were then serially exposed to 1 µM oligomycin (mitochondrial/ATP synthase inhibitor), 2 µM of trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP, a mitochondrial uncoupler), and 0.5 µM of rotenone/antimycin A (respiratory chain inhibitor), provided in the XF Cell Mito Stress Test Kit (Seahorse Bioscience). Three measurements were performed for each cycle (4 min mixing, followed by 3 min detection). Basal respiration, maximal respiration, proton respiration, and coupled respiration were collected using Seahorse XF Extracellular Flux analyzer software following the manufacturer’s protocol. To measure the FAO, cells were cultured replaced by FAO assay medium containing palmitate-BSA according to the manufacturer’s instructions. Other conditions were consistent with the normal OCR measurement. OCR or ECAR experiments were conducted at 37 ℃ and adjusted pH to 7.4. Following an XF assay, the number of cells were determined and used to normalize OCR and ECAR.
Measurement of mitophagy levels using the mitochondria-targeted Keima reporter. We used the mtKeima reporter to measure the mitophagy levels. Cardiomyocytes were transfected with mitochondria-targeted monomeric Keima-Red-hyg (mtKeima; AM-V0251HM, Medical and Biological Laboratories Co., Ltd.), which contained hygromycin B-resistance gene. Hygromycin B infection was used to screen and obtain cardiomyocytes stably expressing mtKeima; cardiomyocytes were seeded on glass-bottom dishes and observed under an LSCM to evaluate mitophagy levels. The wavelengths of excitation and emission filters used were as follows: cytoplasmic Keima: 488 nm, 650–760 nm, lysosomal Keima: 561 nm, 570–630 nm52. Images were analyzed using ImageJ software. Briefly, the cardiomyocytes and mtKeima were segmented, and the areas of cytoplasmic and lysosomal mtKeima were determined. The mitophagy index was calculated as the ratio of the total area of lysosomal mitochondria to the total area of cytoplasmic mitochondria per well.
Transmission electron microscopy. The right myocardium or H9C2 cardiomyocytes were fixed in 2.5% glutaraldehyde for 2 h and immersed in 1% osmic acid for 2 h at 4°C. The fixed samples were then washed in PBS, dehydrated in a graded series of ethanol. Subsequently, the samples were embedded in Epon 812 (SPI Supplies, West Chester, PA, USA) and placed in a model for polymerization. After the semi thin section was used for positioning, the ultrathin section was made and collected for microstructure analysis, followed by counterstaining with 3% uranyl acetate and 2.7% lead citrate. Next, we observed the sections using a HT7800 TEM (HITACHI, Tokyo, Japan) operating at 100 kV.
LC-MS metabolomics analysis. We weighed 60 mg of sample and added 20 µL of 2-chloro-l-phenylalanine (0.3 mg / mL, dissolved in methanol) and 0.6 mL of mixed solution (methanol/water = 7/3 (v: v)) into the 1.5 mL EP tube. The samples were homogenized for 2 min and then extracted 30 min by sonication. They were then placed at -20°C for 20 min and centrifuged at 13000 g for 15 min (4℃). LC-HRMS was performed on a Waters UPLC I-class system equipped with a binary solvent delivery manager and a sample manager, coupled with a Waters VION IMS Q-TOF Mass Spectrometer equipped with an electrospray interface (Waters Corporation, Milford, USA). The injection volume was 3.00 µL, and the column temperature was set at 45℃. The mass spectrometric data was collected using a Waters VION IMS Q-TOF Mass Spectrometer equipped with an electrospray ionization source operating in either positive or negative ion mode. The source and desolvation temperatures were set at 120℃ and 500℃, respectively, with a desolvation gas flow of 900 L/h. Centroid data was collected from 50 to 1000 m/z with a scan time of 0.1 s and an interscan delay of 0.02 s over a 13-min analysis time.
iTRAQ proteomics analysis/nanoUHPLC-MS/MS analysis. Lysis buffer (1% SDS, 8 M urea, 1x Protease Inhibitor Cocktail [Roche Ltd. Basel, Switzerland]) was added into the samples and vibrated and milled for 400 s thrice. The samples were then lysed on ice for 30 min and centrifuged at 15000 rpm for 15 min at 4℃. The protein concentration of the supernatant was determined using the BCA protein assay; we then transferred 100 µg of protein/condition into a new Eppendorf tube and adjusted the final volume to 100 µL with 8 M urea. We added 2 µL of 0.5 M TCEP and incubated the sample at 37℃ for 1 h; next, 4 µL of 1 M iodoacetamide was added to the sample, and the incubation lasted 40 min protected from light at room temperature. Five volumes of -20℃ pre-chilled acetone were then added to precipitate the proteins overnight at -20℃. The precipitates were washed by 1-mL pre-chilled 90% acetone aqueous solution twice and then re-dissolved in 100 µL 100 mM TEAB. Sequence grade modified trypsin (Promega, Madison, WI) was added at the ratio of 1:50 (enzyme: protein, weight: weight) to digest the proteins at 37℃ overnight. The peptide mixture was desalted by C18 ZipTip, quantified by Pierce™ Quantitative Colorimetric Peptide Assay (23275), and lyophilized by SpeedVac.
The resultant peptide mixture was labeled with iTRAQ 8Plex labeling kit (Sciex) following the manufacturer’s instructions. The labeled peptide samples were then pooled and lyophilized in a vacuum concentrator. The peptide mixture was re-dissolved in the buffer A (20 mM ammonium formate in water, pH 10.0, adjusted with ammonium hydroxide), and then fractionated by high pH separation using Ultimate 3000 system (ThermoFisher Scientific, MA, USA) connected to a reverse-phase column (XBridge C18 column, Waters Corporation, MA, USA). High pH separation was performed using a linear gradient, starting from 5% B to 45% B in 40 min (B: 20 mM ammonium formate in 80% ACN, pH 10.0, adjusted with ammonium hydroxide). The peptides were re-dissolved in 5% ACN aqueous solution containing 0.5% formic acid and analyzed by on-line nanospray LC-MS/MS on Q Exactive™ HF-X coupled to EASY-nLC 1200 system (Thermo Fisher Scientific, MA, USA). The column flow rate was maintained at 250 nL/min. The electrospray voltage of 2 kV versus the inlet of the mass spectrometer was used.
Bioinformatics data analysis. The UPLC–Q-TOF/MS raw data were analyzed using progenesis QI (Waters CorporationMilford, USA) software. The parameters used were retention time (RT) range 0.5–14.0 min, mass range 50–1000 Da, and mass tolerance 0.01 Da. Isotopic peaks were excluded from the analysis, the noise elimination level was set at 10.00, the minimum intensity was set to 15% of base peak intensity, and RT tolerance was set at 0.01 min. The Excel file was obtained with three-dimension data sets including m/z, peak RT and peak intensities; RT–m/z pairs were used as the identifier for each ion. The resulting matrix was further reduced by removing any peaks with missing values (ion intensity = 0) in > 60% of samples. The internal standard was used for data QC (reproducibility). The positive and negative data were combined to yield a combined data set imported into SIMCA-P + 14.0 software package (Umetrics, Umeå, Sweden). Principle component analysis and (orthogonal) partial least-squares-discriminant analysis ([O] PLS-DA) were performed to visualize the metabolic alterations among the experimental groups, after mean centering and unit variance scaling. Tandem mass spectra were processed by PEAKS Studio version X (Bioinformatics Solutions Inc., Waterloo, Canada). Differentially expressed proteins were filtered if they contained ≥ 1 unique peptide with P ≤ 0.05 and fold change ≥ 1.2. The pathway analysis was performed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.
Statistical analysis and reproducibility. All statistical analyses were performed with SPSS 20.0 software. The measurement variables were presented as mean ± standard deviation (SD) in minimum triplicates. Statistical significance was determined using Student’s t-test between two groups and corrected for multiple comparisons (Least-Significant Difference) for more than two groups. Mann–Whiney test or nonparametric ANOVA (Kruskal–Wallis) followed by the Dunn multiple comparison post-hoc test was used when one or more datasets showed non-normal distribution. Imaging experiments and animal tests were assessed in a blinded fashion. Sample sizes were kept similar between experimental groups and replicates of experiments. The number of biological replicates and observations are described in the figure legends. Statistical significance was considered at P< 0.05, with *P < 0.05, **P < 0.01. For graphs, all data were analyzed using GraphPad Prism software (version 5.0 or 8.4.0).