Because of the fact that this study involves data collected under Institutional Review Board approval, the data that support the findings of this study are available from the corresponding author on reasonable request at jtmaxwel@wakehealth.edu. This work has been reported in line with the ARRIVE guidelines 2.0.

Human sample acquisition and isolation of human CPCs

This study was approved by the Institutional Review Board at Children’s Healthcare of Atlanta and Emory University. Human c-kit positive cells (CPCs) used in this study were isolated from right atrial appendage tissue routinely removed during surgical repair of congenital heart defects as previously described (25). Atrial tissue was transported to the laboratory in Krebs‐Ringer solution containing 35 mM NaCl, 4.75 mM KCl, 1.2 mM KH2PO4, 16 mM Na2HPO4, 134 mM sucrose, 25 mM NaHCO3, 10 mM glucose, 10 mM HEPES, and 30 mM 2,3‐butanedione monoxime, pH = 7.4 with NaOH. Child CPCs (cCPCs) are categorized as being isolated from patients aged 12 months to 5 years. Neonatal CPCs (nCPCs) are categorized as being isolated from patients aged 1 day to 1 month (25). Both populations of CPCs were created by pooling isolated cells from 3 distinct donors to prevent patient-specific effects. Information on CPC marker expression and phenotypic characterization of human CPCs and how these markers change with electrical stimulation can be found in our previous reports (22, 26, 27). Human bone marrow-derived mesenchymal stromal cell (MSCs) were purchased from Lonza (PT-2501, Morrisville, NC). These cells have been verified by the supplier to differentiate down the adipogenic, chondrogenic, and osteogenic lineages and to be CD29+, CD44+, CD73+, CD90+, CD105+, CD166+, CD14−, CD19−, CD34−, CD45−, and HLA-DR−. Two distinct batches of cells (19TL155677, 18TL113327) were pooled together to prevent batch or patient-specific effects.

Cell culture

MSCs were maintained in culture in Mesenchymal Stem Cell Basal Medium (PT-3238, Lonza, Morrisville, NC) supplemented with Mesenchymal Stem Cell Growth Medium SingleQuots (PT-4105, Lonza, Morrisville, NC) containing Mesenchymal Stem Cell Growth Supplement, L-glutamine, and GA-1000. All populations of CPCs were maintained in culture in Ham's F12 medium (11,765,054, ThermoFisher Scientific, Waltham, MA) supplemented with 10% of fetal bovine serum (FBS), 100 U/ml of penicillin/streptomycin, 2 mmol/l of l‐glutamine, and 0.01 μg/ml of basic fibroblast growth factor (bFGF). cCPCs also underwent a protocol of electrical stimulation (ES-CPCs) as previously reported (26). For electrical stimulation of cCPCs, cells were seeded onto 6‐well dishes at ∼1,000,000 cells per well in calcium‐supplemented media (CPC culture media plus 2 mmol/l CaCl2). A C‐dish electrode array in conjunction with C‐Pace Electrical Stimulation System (Ion Optix, Westwood, MA) was used to apply chronic electrical pulses to the cells at 1 Hz frequency, 10 ms duration, and 10 V amplitude for 7 days. Media was replaced every 24–48 h.

Rat pulmonary artery banding (PAB) model

All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of Emory University and conform to the guidelines from the NIH Guide for the Care and Use of Laboratory Animals. Male adolescent (3–4 weeks old) athymic rats (Crl:NIH‐Foxn1 rnu) (∼150 g) were obtained from Charles River Laboratories (Wilmington, MA). All rats exhibited normal RV function on echocardiography at the time of pulmonary artery banding (PAB) surgery (3–4 weeks old). On the day of surgery, rats were intubated and mechanically ventilated (VentStar Small Animal Ventilator, Braintree Scientific). Rats were anesthetized with 2% isoflurane until no response from toe pinch reflex and a limited left thoracotomy was performed to expose the pulmonary artery (PA). The PA was dissected from the aorta and partially ligated over an 18‐gauge angiocatheter. The sizer was then promptly removed to allow for antegrade flow through the banded area, and thoracotomy performed was closed under positive pressure ventilation to evacuate pleural air (n = 20). Sham operated control (CTL) animals underwent the same procedure without banding the pulmonary artery (n = 4). Animals were double housed.

Echocardiography

Transthoracic echocardiography was performed prior to surgery and at 2 weeks, 4 weeks, and 6 weeks post-banding using a Vevo 2100 digital high‐frequency ultrasound system (FujiFilm Visualsonics, Toronto, ON, Canada) equipped with a probe (MS250) suited for rat imaging. Tricuspid annular plane systolic excursion (TAPSE) was measured in the apical four‐chamber view in M‐mode. As previously shown, this surgery produces severe right ventricular dysfunction within two weeks post-banding (26, 27). Right ventricular dysfunction was confirmed as at least a 35% reduction in TAPSE. Ultrasound acquisition and analysis was performed blinded with only PI of study knowing group allocation.

Cell therapy treatments

Two weeks post-banding, animals were randomized into cohorts without (Sham and PAB) or with cell therapy (MSC, cCPC, nCPC, ES-CPC). All cells for therapy were expanded to passage 4 in culture to normalize passage number across treatment groups. Cells were harvested in sterile saline and labeled with DiR (1,1’-dioctadecyl-3,3,3,’,3’-tetramethylindotricarbocyanine iodide; Thermo Fisher, Waltham, MA) per manufacturer’s protocol. Cells were injected under echocardiographic guidance into the RV free wall using a 27‐gauge BD Insulin Syringe with 12.7 mm BD Micro‐Fine short bevel needle mounted on a stereotactic frame (BD Medical Technology) into 3 spots on the right ventricular free wall, totaling 500,000 cells per heart and ~ 50 µL per injection. Successful delivery of CPCs into the rat myocardium was confirmed by echocardiography. The PAB cohort received saline injections equal to the volume of cell injections for the cell therapy groups. Cell retention was tracked using an IVIS Spectrum in vivo imaging system (Perkin Elmer, Waltham, MA). DiR fluorescence in the rat heart was measured as radiant efficiency and compared between rats as percentage retention (100% on day 0) over time (26, 27). Animal cohorts are identified as CTL (Sham), PAB (banded, saline injected), MSC (PAB + MSC injection), nCPC (PAB + nCPC injection), cCPC (PAB + cCPC injection), or ES-CPC (PAB + ES-CPC injection). N = 4 animals/hearts used for each group. No inclusion or exclusion criteria were used for animals or resulting data points. All animals were processed simultaneously for treatments and analysis. The method of euthanasia was exsanguination, as rats were anesthetized with ketamine (0.1 mg/g) and xylazine (0.01 mg/g) via intraperitoneal injection, and hearts were excised for analysis at 6 weeks post-banding or sham surgery.

Mass spectrometry

Hearts were rinsed and flushed with cold phosphate buffered saline, and the right ventricle was excised and snap frozen in liquid nitrogen. For the protein isolation, 300 µL of urea lysis buffer (8 M urea, 10 mM Tris, 100 mM NaH2PO4, pH 8.5), including 3 µL (100 × stock) HALT(-EDTA) protease and phosphatase inhibitor cocktail (Pierce) was added to the tissue. Samples were sonicated (Sonic Dismembrator, Fisher Scientific) 3 times for 5 s each with 5 s intervals of rest at 30% amplitude to disrupt nucleic acids and were subsequently centrifuged at 4° C. Protein concentration was determined by the bicinchoninic acid (BCA) method, and samples were frozen in aliquots at -80 °C. Protein homogenates (100 µg) were treated with 1 mM dithiothreitol (DTT) at room temperature for 30 min, followed by 5 mM iodoacetimide at room temperature for 30 min in the dark. Protein samples were digested with 1:100 (w/w) lysyl endopeptidase (Wako) at room temperature for overnight. Next day, samples were diluted with 50 mM NH4HCO3 to a final concentration of less than 2 M urea and were further digested overnight with 1:50 (w/w) trypsin (Promega) at room temperature. Resulting peptides were desalted with HLB column (Waters) and were dried under vacuum.

The data acquisition by LC–MS/MS protocol was adapted from a published procedure (Seyfried, Dammer et al. 2017) and was performed by the Integrated Proteomics Core Facility at Emory University. Derived peptides were resuspended in 100 µL loading buffer (0.1% trifluoroacetic acid). Peptide mixtures (2 uL) were separated on a self-packed C18 (1.9 µm, Dr. Maisch, Germany) fused silica column (15 cm × 100 µm internal diameter (ID); New Objective, Woburn, MA) attached to an EASY-nLC™ 1200 system and were monitored on a Q-Exactive Plus Mass Spectrometer (ThermoFisher Scientific, San Jose, CA). Elution was performed over a 56 min gradient at a rate of 700 nL/min (buffer A: 0.1% formic acid in water, buffer B: 0.1% formic acid in acetonitrile): The gradient started with 1% buffer B and went to 40% in 56 min, then increased from 40 to 99% within 1 min and finally staying at 99% for 3 min. The mass spectrometer cycle was programmed to collect one full MS scan followed by 20 data dependent MS/MS scans. The MS scans (400–1600 m/z range, 1 × 106 AGC target, 100 ms maximum ion time) were collected at a resolution of 70,000 at m/z 200 in profile mode. The HCD MS/MS spectra (2 m/z isolation width, 28% collision energy, 1 × 105 AGC target, 50 ms maximum ion time) were acquired at a resolution of 17,500 at m/z 200. Dynamic exclusion was set to exclude previously sequenced precursor ions for 20 s within a 10 ppm window. Precursor ions with + 1, and + 7, or higher charge states were excluded from sequencing.

Label-free quantification analysis was adapted from a published procedure (28). Spectra were searched using the search engine Andromeda, integrated into MaxQuant, against rat Uniprot/Swiss-Prot database (8097 target sequences). Methionine oxidation (+ 15.9949 Da), asparagine and glutamine deamidation (+ 0.9840 Da) and protein N-terminal acetylation (+ 42.0106 Da) were variable modifications (up to five allowed per peptide); cysteine was assigned as fixed carbamidomethyl modification (+ 57.0215 Da). Only fully tryptic peptides with up to two miscleavages were considered in the database search. A precursor mass tolerance of ± 20 ppm was applied before mass accuracy calibration and ± 4.5 ppm after internal MaxQuant calibration. Other search settings included a maximum peptide mass of 6,000 Da, a minimum peptide length of six residues and 0.05-Da tolerance for high resolution MS/MS scans. The FDR for peptide spectral matches, proteins and site decoy fraction was set to 1%. Quantification settings were as follows: match full MS1 peaks between runs; use a 0.7-min retention time match window after an alignment function was found with a 20-min retention time search space. The LFQ algorithm in MaxQuant was used for protein quantitation. The quantitation method considered only razor and unique peptides for protein level quantitation. Data was prepared for presentation using Perseus software, including heat maps and principal component analysis. Proteins were consider significantly upregulated or downregulated at ± 1.3-fold, p < 0.05. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD051582.

Proteomic ingenuity pathway analysis and gene ontology

Differentially expressed proteins were submitted to Ingenuity Pathway Analysis (IPA, QIAGEN Bioinformatics, Hilden, Germany) for pathway and network analysis. Disease and cell therapy-associated networks (Networks tab), upstream regulators or proteome changes (Upstream Analysis tab, Upstream Regulators sub-heading), and enriched functional annotations (Diseases & Functions tab) associated with ‘Cardiovascular System Development and Function’ and ‘Cardiovascular Disease’ (Diseases and Bio Functions sub-heading) and cardiac adverse effects (‘Cardiotoxicity’ categories under the Tox Functions sub-heading) were derived, using Fisher’s exact test for p-value calculations. DAVID Functional Annotation Clustering Tool (http://david.abcc.ncifcrf.gov/) with Benjamini–Hochberg FDR correction for p-value calculations was used for gene ontology analysis.

Statistical analysis

Data are presented as mean ± SD unless otherwise noted in the legend. Statistical analysis was performed using unpaired t-test and ANOVA (GraphPad Prism, v9.2.0) as noted. Differences were considered statistically significant at P < 0.05 or as noted in the figure legend.