Animals. The study protocol was approved by the Commission of Ethics in the Use of Animals of the Medical School of the University of Sao Paulo (protocol #1150/2018). All experiments were performed in accordance with the ethical principles of animal research of the Brazilian College of Animal Experimentation and all methods are reported in accordance with ARRIVE guidelines. Female and male Wistar rats (2 months old) weighing 200–250 g were obtained from the University of São Paulo Medical School, São Paulo, SP, Brazil. Rats at a female:male ratio of 2:1 were randomly mated in one cage. After 4 days, the female rats were individually placed in other cages until they gave birth at 22 days' gestation. The animals were maintained in a temperature- and humidity-controlled environment with a 12-h dark/light cycle at the Heart Institute (InCor) animal facility. Food and water were supplied ad libitum.
Rat neonatal cardiomyocytes isolation and culture. Ventricular cardiomyocytes were obtained from 1- to 2-day-old neonatal Wistar rat hearts (n = 137) as previously described (57) with slight modifications. Briefly, the animals were euthanized by decapitation, and the hearts were excised and washed in an ice-cold ADS solution (ddH2O supplemented with 6.8 g/l NaCl, 4.76 g/l HEPES, 0.12 g/l NaH2PO4, 1 g/l Glucose, 0.4 g/l KCl, and 0.1 g/l MgSO4). The ventricles were minced, and tissue pieces were dissociated in an ADS solution containing collagenase type II (0.4 mg/ml; Worthington Biochemical Corporation, New Jersey, USA) and pancreatin (0.2 mg/ml) at 37°C with shaking for 10 min. This digestion procedure was repeated 5–6 times. For each digestion cycle, the supernatant was removed, centrifuged at 230 x g for 5 min, and suspended in culture medium [low-glucose DMEM and 199/EBSS (4:1) supplemented with 10% horse serum (HRS), 5% newborn calf serum (NBCS), 50 U/ml penicillin-streptomycin, and 1% sodium pyruvate; all from Thermo Fisher Scientific, Waltham, MA, USA]. After that, the cells were preplated in 100-mm culture dishes for 45 min to reduce the fibroblast content. The supernatant, after preplating, was centrifuged at 230 x g for 5 min. Cells were resuspended in a culture medium with 1% bromodeoxyuridine (BrdU), counted, plated on laminin- or fibronectin-coated culture surfaces depending on the experiment, and incubated at 37°C in a humidified atmosphere containing 5% CO2.
After 2 days, the culture medium was changed. Four days after cell plating, flow cytometric analyses were performed to evaluate the efficiency of the cardiomyocyte isolation protocol (Supplementary Material). Hypoxia conditions were chemically induced using 200µM cobalt chloride (CoCl2) (Merck, Darmstadt, Germany) (added to new culture medium) for 4 h. Then, the cells were treated with vehicle (dimethyl sulfoxide; DMSO) or 1 µM EMPA (AdooQ Bioscience, Irvine, CA, USA) (added to new culture medium) for 24 h. The EMPA concentration (1 µM) was in the range of the typical plasma concentration during the 25 mg q.d. oral treatment of patients (58). After that, experiments were carried out to analyze electrical potentials, mRNA (Supplementary Material), protein expression, Ca2+ transients, and contractility. For Ca2+ transients and contractility experiments, cells were incubated with the NHE1 inhibitor cariporide (10 µM) for 20 min in combination or not with EMPA. This concentration was chosen based on previous studies which show that 10 µM cariporide effectively inhibits NHE1-mediated Na+/H+ exchange in ventricular myocytes isolated from different species (29, 53, 54, 59, 60). The study design is depicted in Supplementary Figure S1.
Cardiomyocytes derived from iPSC. All procedures were performed in accordance with relevant guidelines and regulations and approved by the National Institute of Cardiology ethics review board under number 27044614.3.0000.5272. The iPSCs used in this study were obtained from the reprogramming of erythroblasts derived from a healthy individual, as described previously (61). Briefly, mononuclear cells were cultivated in an enrichment medium for erythroblasts, and after 12 days, cells were infected with CytoTune-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific). Then, the cells were cultured on a murine embryonic feeder layer in DMEM/F12, GlutaMAX supplement, with 20% KSR, 1% penicillin-streptomycin, 100 µM NEEA, 0.1 mM β-mercaptoethanol, 10 ng/ml of bFGF. After confirming pluripotent state (61), the iPSCs were maintained as feeder-free culture by using 1% Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Thermo Fisher Scientific) in StemFlex medium (Thermo Fisher Scientific) with 1% penicillin-streptomycin.
The differentiation protocol of iPSCs into cardiomyocytes was based on the modulation of Wnt pathway signaling described by Lian and colleagues (62). iPSCs were dissociated with TrypLE™ Express Enzyme (Thermo Fisher Scientific), and 4 x 105 iPSCs were seeded in each well of a 48-well plate coated with 1% Geltrex™ and cultured for 2 days. The Wnt signaling was activated on day 0 of the protocol by treatment with 9 µM of CHIR99021 (Tocris) diluted in basal medium (RPMI 1640 supplemented with B-27 without insulin (Thermo Fisher Scientific)). On days 1 and 2, cells were maintained in basal medium. On days 3 and 4, cells were treated, respectively, with 10 µM and 5 µM of XAV939 (Tocris) for Wnt inhibition. On day 5, XAV939 was removed from the medium, and, finally, on day 7, the medium was changed for RPMI plus B-27 with insulin (Thermo Fisher Scientific). iPSC-derived cardiomyocytes were cultured until day 24 with medium changes every 3 days. Lactic acid 4 µM (Sigma-Aldrich) in RPMI 1640 with insulin without glucose/B-27 was added to the cell culture media at differentiation day 24 and maintained for 6 days with medium changes every 2 days. From day 30, cell cultures were subjected to normoxia or chemical hypoxia induced by 250 µM CoCl2 for 4 hours. Then, the cells were treated with vehicle (DMSO) or 1 µM EMPA for 24 h. After that, cells were used for action potential recordings.
Action potential recordings. The 35-mm laminin-coated plates containing 5×105 cardiomyocytes derived from neonatal rats (n = 3 for each of 3 independent experiments) or iPSCs (n = 3 for each of 2 cardiac differentiation) were transferred to the stage of an inverted microscope (Nikon Instruments Inc., Melville, NY, USA). The action potential recording method was adapted from a previously described protocol (61). Cells were superfused with Tyrode's solution containing 150.8 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 11 mM D-glucose and 10 mM HEPES (pH 7.4 adjusted with NaOH). The superfusion was carried out at 37.0 ± 0.5°C using a temperature controller (Harvard Apparatus) saturated with oxygen at a perfusion flow rate of 0.5 ml/min (Minipuls 3). The transmembrane potential was recorded using glass microelectrodes (40–80 MΩ DC resistance) filled with 2.7 M KCl connected to a microelectrode amplifier (MultiClamp 700B; Molecular Devices, USA). Amplified signals were digitized (1440 Digidata A/D interface) and stored on a computer for future analysis using LabChart 7.3 software (ADInstruments). Paced (1 Hz) and/or spontaneous action potentials records were acquired. The following parameters were recorded: resting membrane potential (RMP), action potential amplitude (APA), dV/dtmax, and APD at 30, 50, and 90% repolarization (APD30, APD50, and APD90). Measures were obtained automatically. In rat cardiomyocytes, APDs were corrected by the beat rate. In human iPSC-derived cardiomyocytes, action potential recordings were analyzed from ventricle-like cells, classified by the following parameters: dV/dtmax > 10 V/s and APD30/APD90 > 0.3 (63). The starting point of the action potential was determined by the software as a 10% variation in voltage from the maximum diastolic potential.
Cytosolic calcium transient analysis. Calcium transient recording experiments were performed as previously described (57) with some modifications. Briefly, 3 × 105 cardiomyocytes derived from neonatal rats were plated on laminin-coated glass coverslips (25 × 25 mm; Corning, New York, NY, USA) inside 60 mm culture plates (n = 3). Cells were moved to a fresh medium supplemented with 3 µM Fura-2 AM (F1201, Thermo Fisher Scientific) and incubated for 15 min at 37°C. The medium was refreshed once again with a medium free of Fura-2 AM, and the coverslip was transferred to a recording chamber. All the recordings were performed under electrical stimulation at 1 Hz for 4 ms and 10 V (IonOptix, Milton, MA, USA). The range in fluorescence emission wavelength was 340–380 nm, and the fluorescence signal was collected with a photomultiplier tube via the ×40 oil objective during continuous excitation at 510 nm with a 75-W Xenon lamp. We used the IonOptix Contractility System to record calcium transients. The recordings were assessed by an expert blinded to the experimental groups. Data were processed and analyzed using IonWizard (Core and Analysis - IonOptix). The raw traces were filtered to reduce noise, and representative peaks from each cell were selected. Measurements of [Ca2+]i transient amplitude, time to peak, and time to 50% relaxation (DT50) were performed. Mean curves of [Ca2+]i transients for each experimental group were created by aligning the fluorescence signals of each cell recorded and calculated by the average of the fluorescence per timepoint.
Contractility analysis. To measure contractile properties, cardiomyocytes derived from neonatal rats (1x104 cells/well) were seeded onto laminin-coated 96-well plates (n = 3). Cells were incubated in a thermostatic chamber (21% O2 and 5% CO2 at 37°C) to maintain physiologic conditions. The cardiomyocytes derived from neonatal rats were visualized using EVOS Cell Imaging System (ThermoFisher Scientific), equipped with a 20x objective. Cells were allowed to stabilize for at least 10 min before any recordings. All the recordings were performed under electrical stimulation at 1 Hz for 20 ms and 20 V (IonOptix) and obtained as previously described (64). Movie images of beating cardiomyocytes were acquired with a duration of 10 s for each position, at 30 frames per second, 2048 x 1536 pixel resolution, and a pixel size of 0.307 µm/pixel with a depth of 8 bits. Data were processed and analyzed using Contractionwave, an open-source software for large-scale analysis of cardiomyocyte contraction (65), allowing us to determine the contraction and relaxation times (CT and RT, respectively).
SDS-PAGE and immunoblotting. Cardiomyocytes were seeded onto 35-mm laminin-coated plates at 105 cells/cm2. The cells were washed 3 times in an ice-cold PBS buffer (150 mM sodium chloride, 2.8 mM monobasic sodium phosphate, 7.2 mM dibasic sodium phosphate, pH 7.4). Subsequently, the cells were lysed in RIPA lysis buffer (10×) containing protease inhibitors (1 mM phenylmethanesulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin) and phosphatase inhibitor cocktails 2 and 3 (1:100 dilution). The cell lysate was mixed by vortexing for 30 s and centrifuged at 15,800 × g for 15 min at 4°C. The supernatant was isolated and stored at -80°C. Protein concentration was determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific) following the manufacturer's instructions. Then, samples of the cardiomyocyte proteins were solubilized in the Laemmli sample buffer and separated by SDS-PAGE using 7.5% polyacrylamide gels. For immunoblotting, proteins were transferred to PVDF membranes (Millipore Immobilon-P, Millipore, Bedford, MA) and incubated with a blocking solution containing 5% skim milk or BSA and 0.1% Tween 20 in PBS (pH 7.4) for 1 h to block nonspecific antibody binding, followed by overnight (4°C) incubation with specific monoclonal antibodies against HIF-1α (D2U3T) (Cell Signaling Technology, 14179, 1:1000 dilution), phospholamban (PLN) [2D12] (Abcam, ab2865, 1:1,000 dilution), actin (Abcam, ab179467, 1:5,000 dilution) or a polyclonal antibody against PLN (phospho S16) (Abcam, ab15000, 1:1,000 dilution). The PVDF membranes were washed five times in a blocking solution and incubated for 1 h at room temperature with the horseradish peroxidase-conjugated immunoglobulin secondary antibody (Jackson ImmunoResearch Laboratories, Inc, 1:2,000 dilution). After washing five times with blocking solution and twice in PBS (pH 7.4), the PVDF membranes were incubated for 1 min with an enhanced chemiluminescence detection (ECL) system (Cytiva, Marlborough, MA, USA) for visualization of the bound antibodies. The visualized bands were digitized using ImageScanner III (Cytiva) and quantified using ImageJ software (National Institutes of Health, Bethesda, MD). The unedited gel images are included within the supplementary material of the manuscript.
Statistical analyses. The results are reported as the mean ± standard error of the mean (SEM). For the analysis of action potential, statistical significance was determined by using unpaired t-tests. For analyses of proportions, Fisher's exact test was used. For calcium transients, contractility, and immunoblot analyses, statistical significance was determined using one-way ANOVA followed by a post hoc Tukey analysis with multiple comparisons with 95% confidence. For real-time RT-PCR, statistical significance was determined using two-way ANOVA followed by a post hoc Tukey analysis with multiple comparisons with 95% confidence. A P value < 0.05 was used to indicate significance. All statistical analyses were performed using GraphPad Prism version 8.0 (GraphPad Software, La Jolla, CA).