Animals
Neonatal CM isolation and culture in vitro
Neonatal hearts were dissected from P0–1 C57bl/6 pups, washed with ice-cold PBS and dissociated using standard procedures. Neonatal CMs were seeded in culture plates precoated with fibronectin (0.8–1 million per 3.5-cm dish or 0.25 to 0.4 million per well of 2-well chamber slides) and cultured in primary neonatal CM culture medium (80% DMEM with 4.5 g l−1 glucose, 20% Medium 199, 5% FCS and 100 U ml−1 penicillin and streptomycin). After overnight culture, chemicals were added to the medium and cells were further cultured for 72–96 h before collection. Concentrations of chemical were as follows: etomoxir 100 μM (Cayman, 11969); octyl-αKG 500 μM (Cayman, 11970); CPI-HCl 25 μM (Selleckchem, S8287); R2HG 500 μM (Cayman, 16366). Kdm5b knockdown was achieved through transfection of pooled Kdm5b short interfering RNA (Dharmacon) with DharmaFECT 1 Transfection Reagent. Non-targeting pooled short interfering RNAs were used for the control group.
Immunofluorescence and histological analysis
Hearts were immediately fixed in 4% PFA after dissection. For trichrome staining after I–R injury, hearts were embedded in paraffin and continuously sectioned from the apex to the ligation site. Every second section from hearts of each group was used for staining and quantification. In vitro-cultured neonatal CMs were fixed with 4% PFA for 10 min at room temperature and permeabilized (0.3% Triton X-100 and 5% BSA) for 1 h at room temperature. To determine the surface area of CMs, approximately 120 CMs randomly selected from 5–6 paraffin sections of each heart sample were measured using the ImageJ software tool. The sarcomere density of isolated adult CMs was analysed using the ImageJ software tool. For other quantification procedures, using either isolated CMs or tissue sections, hundreds of CMs were analysed for each individual heart. Values from each group of CMs (or sections) were averaged and are presented as one sample (n = 1). For experiments with neonatal CMs, values for each sample represent the results obtained from one isolation of CMs from pooled neonatal hearts. Antibodies for immunofluorescence staining are listed in the Supplementary Information. Microscopic images were acquired with a fluorescence stereomicroscope (Leica M205 FA). Regular immunofluorescence images were acquired with a fluorescence microscope (Zeiss Imager Z1) and processed with ZEN 2 imaging software. Confocal immunofluorescence images were acquired with a Leica SP8 confocal microscope and processed with LAS X software 3.5.7.23225. Acquisition of histological images was carried out with a light microscope (Zeiss Axioplan2).
EdU incorporation assay
EdU and other reagents were prepared according to the manufacturer’s instructions (ThermoFisher C10339). In vivo EdU incorporation assays were carried out according to previous publications45. To analyse EdU incorporation in cultured neonatal CMs, cells in 2-well chamber slides were labelled with 10 µM EdU for 12 h. After two washes with pre-warmed PBS, cells were fixed with 4% PFA for 10 min at room temperature and EdU incorporation was visualized using the Click-iT EdU kit (Invitrogen), following the manufacturer’s protocol.
Western blot assays
Freshly isolated or cultured cells were washed with ice-cold PBS and lysed in cell lysis buffer (20 mM Tris (pH 7.5), 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1× Complete Protease Inhibitor Cocktail (Roche Diagnostics)) for 10 min on ice, followed by sonication using the Bioruptor (Dianagene) at 4 °C for 5 min. Proteins were separated by SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Millipore). Proteins detected by antibodies were visualized using an enhanced chemiluminescence detection system (GE Healthcare) and quantified using the ChemiDoc gel documentation system (Bio-Rad). Antibodies and dilutions used in this study are listed in the Supplementary Information.
Adult CM isolation and in vitro culture
Isolation of adult CMs was carried out as described previously46. In brief, dissected hearts were cannulated through the aorta and retrogradely perfused with calcium-free buffer. Cannulated hearts were enzymatically digested by perfusion with enzyme buffer solution and cut off from the cannula. Atria were separated, and ventricles were minced in enzyme buffer. After gentle pipetting, myocytes were centrifuged at 500 r.p.m. for 1 min and cell pellets containing CM fractions were resuspended in stop buffer. The calcium content of the cell suspension was then stepwise adjusted to 1 mM and CM-containing cell pellets were resuspended in M199 cell culture medium, supplemented with creatinine, l-carnitine, HEPES, penicillin–streptavidin, 5% FCS and insulin–transferrin–sodium selenite medium supplement. Cells were seeded in dishes precoated with laminin and maintained in a humidified incubator at 37 °C and 5% CO2. To determine CM numbers in adult hearts, the dissected heart was washed with ice-cold PBS and fixed with 1% PFA overnight. After washing with ice-cold PBS, hearts were cut into 1–2-mm3 pieces and incubated with digestion buffer (PBS containing 0.5 U ml−1 of collagenase B (Roche no. 11088807001) and 0.2% NaN3) with constant shaking at 1,000 r.p.m. at 37 °C. Every 12–24 h, digested CMs were collected, and new digestion buffer was added until the heart was fully digested. CMs were pooled, plated into a Sedgewick rafter chamber and counted therein.
Measurement of oxygen consumption rate with Agilent Seahorse XF
Adult CMs were isolated and seeded at a density of 6,000 cells per well in a 96-well plate for Seahorse measurements (Agilent Seahorse XFe96 Analyzer). Cells were washed with PBS and Seahorse base medium after attachment to the plate. The following substrates were added as energy substrates to the medium 1 h before measurements of the oxygen consumption rate: glucose 5 mM (Sigma), pyruvate 0.2 mM (Sigma), glutamine 4 mM (Sigma), palmitate–BSA 0.2 mM (Agilent), BSA control (Agilent), carnitine 0.2 mM (Sigma), valine 1 mM (Sigma), isoleucine 1 mM (Sigma), leucine 1 mM (Sigma), sodium propionate 0.05 mM (Sigma), sodium acetate 0.05 mM (Sigma), sodium octanoate 0.1 mM (Sigma), sodium decanoate 0.1 mM (Sigma). The oxygen consumption rate was measured using the Mito Stress Test kit (Agilent). The following inhibitors were injected: oligomycin (2 µM), FCCP (2 µM), rotenone and antimycin A (1 µM).
Magnetic resonance imaging and data processing
Cardiac magnetic resonance imaging (MRI) measurements were carried out using a 7.0T Bruker Pharmascan (Bruker) equipped with a 760 mT m−1 gradient system, using a cryogenically cooled four-channel phased array element 1H receiver coil (CryoProbe), a 72-mm room-temperature volume resonator for transmission, and the IntraGate self-gating tool47. Electrocardiogram parameters were adapted for one heart slice and transferred afterwards to the navigator signals of the remaining slices. Thus, in-phase reconstruction of all pictures was guaranteed. Measurements are based on the gradient echo method (repetition time = 6.2 ms; echo time = 1.3 ms; field of view = 2.20 × 2.20 cm; slice thickness = 1.0 mm; matrix = 128 × 128; oversampling = 100). The imaging plane was localized using scout images showing the two- and four-chamber view of the heart, followed by acquisition of images in short-axis view, orthogonal on the septum in both scouts. Multiple contiguous short-axis slices consisting of 7 to 10 slices were acquired for complete coverage of the left and right ventricle. Mice were measured under isoflurane (1.5–2.0% in oxygen and air with a flow rate of 1.0 l min−1) anaesthesia. Body temperature was maintained at 37 °C by a thermostatically regulated water flow system during the entire imaging protocol. MRI data were analysed using Qmass digital imaging software (Medis Imaging Systems, Leiden, the Netherlands).
FACS-based isolation of cardiac nuclei
Ventricles were washed with ice-cold PBS after dissection and snap frozen in liquid N2. For isolation of cardiac nuclei, the frozen ventricle was thawed in 3 ml lysis buffer (5 mM CaCl2, 3 mM MgAc, 2 mM EDTA, 0.5 mM EGTA and 10 mM Tris-HCl, pH 8) in M-tubes (Miltenyi Biotec) and homogenized using the gentleMACS Dissociator (Miltenyi Biotec), following the manufacturer’s protocol (protein_01). The resultant homogenate was mixed with lysis buffer containing 0.4% Triton X-100, incubated on ice for 10 min, and subsequently filtered through 40-μm cell strainers (BD Bioscience). The flow-through was centrifuged at 1,000g for 5 min at 4 °C to collect nuclei. Nuclei were further purified by centrifugation at 1,000g for 5 min at 4 °C through a 1 M sucrose cushion (3 mM MgAc, 10 mM Tris-HCl, pH 8) and then stained with a PCM1 antibody in nuclei stain buffer (DPBS, 1% BSA, 0.2% Igepal CA-630, 1 mM EDTA). DNA was stained by DAPI before FACS. FACS was carried out using a FACSAria III (BD Biosciences). Quantification of PCM+ cardiac nuclei and DNA content was carried out with the LSR Fortessa (BD Biosciences) analyser. Data acquisition and analysis were accomplished using the BD FACS Diva v8 software. The gating strategy is shown in the Supplementary Information.
RNA-seq and data analysis
RNA was extracted from isolated adult CMs using the Direct-zol Total Kit (Zymo Research) combined with on-column DNase digestion (DNase-Free DNase Set, Qiagen) to avoid contamination by genomic DNA. RNA and library preparation integrity were verified using the LabChip Gx Touch 24 (Perkin Elmer). A 200 ng quantity of total RNA was used as input for the SMARTer Stranded Total RNA Sample Prep Kit – HI Mammalian (Clontech) following the manufacturer’s instructions. Sequencing was carried out on a NextSeq500 instrument (Illumina) using v2 chemistry, resulting in an average of 22 million reads per library with a 1 × 75 bp single-end setup. Raw reads were assessed for quality, adapter content and duplication rates with FastQC 0.11.8 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Trimmomatic version ≥ 0.36 was used to trim reads after a quality drop below a mean of Q15 in a window of five nucleotides48. Only reads of at least 15 nucleotides were cleared for subsequent analyses. Trimmed and filtered reads were aligned versus mouse genome version mm10 (GRCm38.p5) using STAR ≥ 2.5.4b with the parameters –outFilterMismatchNoverLmax 0.1 –alignIntronMax 200000 (ref. 49). The number of reads aligning to genes was counted with featureCounts ≥ 1.6.0 from the Subread package50. Only reads mapping at least partially inside exons were admitted and aggregated per gene. Reads overlapping multiple genes or aligning to multiple regions were excluded. Differentially expressed genes were identified using DESeq2 version ≥ 1.14.0 (ref. 51). Genes were classified as significantly differentially expressed with a P value < 0.05. Annotations were enriched using UniProt data (release 24.03.2017) based on Ensembl gene identifiers (Activities at the Universal Protein Resource (UniProt)).
ChIP, ChIP–seq and data analysis
Chromatin was prepared using the truChIP Chromatin Shearing Kit (COVARIS) and sheared to an average size of 200–500 bp by sonication (Diagenode Bioruptor). Protein–DNA complexes were immunoprecipitated with IgG or KDM5B antibodies, followed by incubation with Protein A/G magnetic beads (Dynabeads, Invitrogen). For ChIP–qPCR, beads were washed and protein–DNA complexes were eluted and purified using 10% Chelex-100 (w:v, Bio-Rad Laboratories) in Tris–EDTA. Immunoprecipitated chromatin was analysed by qPCR using SYBR Green quantitative real-time analysis with primers that are listed in the Supplementary Information. A detailed description of ChIP–seq analysis is provided in the Supplementary Information.
I–R injury and measurement of AAR and infarct area out of AAR
Animals were anaesthetized using 4.5% isoflurane and endotracheally intubated with a 22-gauge intravenous catheter. Mice were placed on a 37 °C heating plate in the supine position and ventilated at a rate of 225 strokes min−1 and a stroke volume of 250 µl with a mixture of oxygen and 1.5% isoflurane using a MiniVent rodent ventilator. Chest hair was removed, and skin was disinfected and opened with a small incision of several millimetres in length from the left armpit to the sternal border. Pectoralis major and minor muscles were separated, the chest was opened in the third intercostal space, and retractors were inserted. Next, the pericardium was opened to access the heart. The left coronary artery was ligated for 30 min and reopened for reperfusion in a proximal position using a prolene suture (7-0). The retractors were removed, and the chest wall was closed by bringing together the second and third rib using a vicryl suture (5-0). The muscles were placed into their original position, and the skin incision was closed with vicryl (5-0). Mice were ventilated with oxygen until awakening, followed by extubation, and placement into their cages. At 24 h after I–R surgery, the animals were euthanized for AAR and infarct area out of AAR measurement, for which hearts were removed and the aorta quickly cannulated for an injection of 500 µl 1% Evan’s blue solution into the ventricle. Hearts were kept on ice-cold saline for further investigations. Afterwards, the heart was frozen and sliced at 0.5 mm. Heart sections were subsequently stained with TTC solution (1% in PBS) at 37 °C for 30 min and then fixed with formalin solution.
Viability assay of adult CMs under hypoxic conditions
Freshly isolated adult CMs seeded in chamber slides were cultured either under normoxia or in a hypoxia chamber with 1% O2, 5% CO2 at 37 °C for 18 h. Cells were washed at room temperature with PBS and incubated with PBS containing ethidium homodimer 1 (4 µM) and calcineurin (2 µM) for 45 min at room temperature. Immunofluorescence images were acquired with a Zeiss Imager Z1 microscope.
Metabolic flux assays and targeted metabolic analysis
Metabolic flux assays were carried out with isolated Langendorff-perfused hearts from CtrlCre and Cpt1bcKO mice. Hearts were quickly excised and cannulated through the aorta. The cannulated heart was connected to a perfusion column apparatus maintained at 37 °C using a temperature-controlled water bath. Hearts were perfused retrogradely for 60 min with Krebs–Henseleit buffer with the following substrates: glucose 8 mM; pyruvate 0.12 mM; palmitate–BSA 0.4 mM; isoleucine 0.176 mM. In each perfusion assay, only one metabolite was replaced by a 13C-labelled metabolite ([13C]glucose, [13C]isoleucine or [13C]palmitate–BSA). Subsequently, hearts were snap frozen, pulverized in liquid nitrogen and subjected to metabolite extraction (acyl-CoAs or metabolites of the Krebs cycle) and quantification using liquid chromatography with triple-quadrupole mass spectrometry. Metabolic flux assays and α-ketoacids measurements were carried out using tissue from isolated hearts after perfusion with different substrates as indicated. Measurements of Krebs cycle metabolites and standardized targeted metabolic analysis were carried out with isolated adult CMs. A detailed description of the quantification of acyl-CoAs, Krebs cycle metabolites, α-ketoacids and targeted metabolome analysis is provided in the Supplementary Information.
Lentiviral transduction of CMs
HEK293T cells were grown in DMEM (Sigma) supplemented with 10% FCS (Sigma), 2 mM l-glutamine, 100 U penicillin and 100 µg ml−1 streptomycin at 37 °C, 5% CO2. HEK293T cells (2 × 106 per 10-cm dish) were transfected with 5 µg pLJM1-Kdm5b, pLJM1-Idh3b or pLJM1-Idh3g, 4.5 µg psPAX2 (Addgene, no. 12260) and 0.5 µg pMD2.G (Addgene, no. 12259) using the Turbofect transfection reagent and Opti-MEM for 6–8 h. The supernatants containing lentiviral particles were collected at 48 and 72 h after transfection and pooled. Lentiviruses were filtered through a 0.45 µM cell strainer to remove HEK293T cells and concentrated with a Lenti-X concentrator according to the manufacturer’s instructions (TaKaRa, 631231). Primary neonatal CMs were infected in suspension with Polybrene (8 μg ml−1) for 6 to 8 h.
Analysis of gene expression using qPCR with reverse transcription and assessment of mitochondrial DNA copy numbers
Total RNA was extracted using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RNA was reverse transcribed with Superscript II (Invitrogen) following standard procedures. Real-time PCR was carried out with two technical replicates using the StepOne real-time PCR system and KAPA SYBR FAST qPCR Master Mix (KAPA Biosystems). Relative quantification of gene expression was carried out using the ∆∆CT method. The Ct values of the target genes were normalized to expression of the 36b4 gene using the equation ΔCt = Ctreference − Cttarget and expressed as ΔCt. Relative mRNA expression values were shown with the average from control samples set as 1. Mitochondrial DNA copy numbers were determined using DNA extracted from isolated adult CMs. The data were normalized to internal controls (H19 or Mx1) and cell numbers. Primers and PCR conditions are listed in the Supplementary Information.
Electron microscopy
Hearts were isolated and fixed in 1.5% glutaraldehyde (v/v), 1.5% PFA (v/w) in 0.15 M HEPES (v/w), pH 8.0 at 4 °C for at least 24 h, and subsequently incubated with 1% osmium tetroxide for 2 h. Samples were stained en bloc with 50%-saturated watery uranyl acetate, followed by sequential ethanol dehydration (30%, 50%, 75%, 95%), and embedded in Agar 100. Ultrathin sections were cut using an ultramicrotome and image acquisition was carried out with a Philips CM10 electron microscope. All images were captured with a slow-scan 2k CCD (charge-coupled device) camera.
Statistical analysis
For all quantitative analyses, a minimum of three biological replicates were analysed. Statistical tests were selected on the basis of the assumption that sample data are from a population following a probability distribution based on a fixed set of parameters. Student’s t-tests were used to determine the statistical significance of differences between two groups. One-way AVOVA was used for multiple comparison tests. The following values were considered to be statistically significant: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Calculations were carried out using the GraphPad Prism 9 software package. Data are always represented as mean ± the standard error of the mean. No statistical method was used to predetermine sample size.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
