Cloning and preparation of integrating and non-integrating lentivirus.
cDNA of CAG promoter, TNNT2 promoter, Cre recombinase, AurKB promoter, CCNB, CCND, CDK1, and CDK4 were cloned into the pLenti-MCS-SV-puro backbone (Addgene). To produce the Lentivirus particles, 5X106 HEK293 cells were transfected using FuGENE HD transfection reagent (Promega) along with 5µg pMD2.g, 5µg psPAX2 (Integrating lentivirus) or psPAX2-D64V (non-integrating Lentivirus) (Addgene) and 10µg of the expression pLenti vector encoding the gene of interest for 48 h. The media containing the virus were collected and filtered through a Nalgene syringe filter 0.45 µm. For in vitro experiments, the virus was mixed with polybrene transfecting reagent (1µg/ml) (Millipore Cat# 1003). The virus was then used to treat 60 days old hiPS-CMs. For in vivo injections, the virus solution was centrifuged at 20,000xg at 4oC for 2 h and the pellet was resuspended in PBS. Modified RNA was synthesized and purchased from Bio-Synthesis, Inc. RNA modifications, transfection reagents, and procedures were conducted following the protocol described in10.
Preparation and maintenance of hiPS-CMs
The human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) were purchased from Cellular Dynamics (Currently FujiFilm Inc.). These hiPS-CMs have been selected after differentiation using an a-MHC-Blastocidin selection cassette. This strategy yields nearly 100% pure hiPS-CMs. hiPS-CMs were re-plated on fibronectin-coated plates and cultured in R.P.M.I. medium with a B27 supplement. Then the cardiomyocytes were maintained in RPMI/B27 supplement with insulin for 2–3 months. The cells were transferred every 2–3 weeks on fresh fibronectin.
Bulk RNAseq analysis
RNA from hiPS-CMs were isolated using the Qiagen, miRNeasy Micro Kit, #210874, following homogenization of the tissue in QIAzol (Qiagen). Using the Ovation RNA-seq System v2 Kit (NuGEN), total RNA (20–50 ng) was reverse transcribed to synthesize the first-strand cDNA using a combination of random hexamers and a poly-T chimeric primer. The RNA template was then partially degraded by heating, and the second-strand cDNA was synthesized using DNA polymerase. Double-stranded DNA was then amplified using single primer isothermal amplification (S.P.I.A.). Random hexamers were then used to amplify the second-strand cDNA linearly. cDNA samples were fragmented to an average size of 200 bp using the Covaris S2 sonicator. Libraries were made from the fragmented cDNA using the Ovation Ultralow V2 kit (NuGen).
Following end-repair and ligation, the libraries were PCR amplified with 9 cycles. A Bioanalyzer assessed library quality on High-Sensitivity DNA chips (Agilent), and concentration was quantified by qPCR (K.A.P.A.). The libraries were sequenced on a Novaseq sequencer with a single-read, 50-cycle sequencing run (Illumina). We utilized the RNAseq-analysis pipeline reported previously15. For the readers’ convenience and completeness of the current manuscript, we review the pipeline's critical steps/features and statistics below. Available adapters and low-quality regions of reads were trimmed using Fastq-mcf (http://code.google.com/p/ea-utils). Sample QC was assessed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were aligned to the GRCh38 genome using Tophat 2.0.13. Gene expression was tallied by Subread feature Counts using Ensemble's gene annotation for Sscrofa11.1. Finally, we calculated differential expression P-values using edgeR 8. Here, we first filtered out any genes without at least two samples with a C.P.M. (counts per million) between 0.5 and 5000. C.P.M.s below 0.5 indicate nondetectable gene expression, and C.P.M.s above 5000 are typically only seen in mitochondrial genes. If these high-expression genes were not excluded, their counts would disproportionately affect the normalization. After excluding these genes, we renormalized the remaining ones using "calcNormFactor" in edgeR, then calculated P-values for each gene with differential expression between samples using edgeR's assumed negative–binomial distribution of gene expression. We calculated the false discovery rates (FDRs) for each P-value with the Benjamini-Hochberg method based on the built-in R function "p.adjust".
Contractile function monitoring of hiPS-CMs using CardioExcyte 96 system
hiPS-CMs were plated on fibronectin-coated NSP-96, CardioExcyte 96 Sensor Plates with extra stimulation electrode. The NSP-96 plate was maintained in the CardioExcyte 96 system at 37˚C and 5% CO2. The cells were electrically stimulated at 1Hz during the experiments. Impedance recordings were performed every 30 minutes for 96 h. The data were analyzed with CardioExcyte 96 analysis software.
Electrophysiological recordings of the ionic current of hiPS-CMs
Sodium current (INa) was recorded in the whole-cell configuration of the patch-clamp technique. All recordings were performed at room temperature (20° to 22° C). The pipette solution consisted of (in mmol/L): 120 KCl, 1 MgCl2, 3MgATP, 10 Hepes, 10 EGTA, pH 7.3. Bath solution contained (in mmol/L) 140 NaCl, 5.4 KCl, 1.2 KH2PO4, 5 HEPES, 5.55 glucose, 1 MgCl2, 1.8 CaCl2, pH 7.4. Rseries was compensated >80%; no corrections were made for liquid junction potentials. Data were analyzed with either Clampfit 11 (Molecular Devices) or Igor, dPatch/SutterPatch (Sutter Instruments).
Single-Cell RNA-Sequencing Library Generation
hiPS-CMs either infected with LacZ or 4F for the indicated time were filtered using a 70µM filter, resuspended in 1% B.S.A. in PBS, and counted immediately before library preparation. G.E.M. Generation and Barcoding were performed following the Chromium Single Cell" Reagents Kits v3 Rev A User Guide with Chromium Single Cell" G.E.M., Library and Gel Bead Kit v3 (10X Cat # 1000092). Briefly, the Chromium chip was loaded, and the Chromium Single Cell B program was selected. The chip was ejected immediately following completion of the program. 100 µL of recovered G.E.M.s was slowly pipetted and transferred to tubes precooled on ice. The G.E.M.s were then incubated for the R.T. reaction using a thermal cycler at 53˚C for 45 min, then 85˚C for 5 min, followed by a 4˚C hold. Samples were then stored at -20˚C overnight. All libraries were pooled and sequenced using the NovaSeq to a read depth of at least 50,000 reads per cell. The sequenced reads for each sample were mapped to the GRCh38 genome to generate the (gene-by-cell) count matrix using cell ranger count (Cell Ranger version 3.1.0 from 10x Genomics) with default parameters. The counts" matrices across the samples were aggregated using cell ranger aggr. The resulting files were processed in R using the package Seurat (version 3.1.3)36. All cells with at least three detected genes and less than 30% of reads from mitochondrial genes and all detected genes in at least 200 cells were used in the further analyses. The remaining data were normalized using the "LogNormaliz" method. Principal Component Analysis for the subset of the 2000 most variable genes (Seurat function FindVariableFeatures) was then performed on the scaled data. The cells were clustered using the Louvain Algorithm with the resolution parameter value of 0.5 (Seurat function FindClusters) after determining the shared nearest neighbor graph using the first ten principal components (Seurat function FindNeighbors). The data were visualized using the UMAP algorithm with the first ten principal components as input (Seurat function RunUMAP). The cells were grouped into ten clusters based on the distribution of expression of the cell cycle genes of interest. Differential analysis between all pairs of clusters was performed using the Wilcoxon rank-sum test to identify the differentially expressed genes (Seurat function FindMarkers). The dimensionality reduction results were reformatted for compatibility with the learn_graph function in the R package monocle3, used for trajectory analysis37. This analysis was done for five groups of cells – 24 h unique cluster, 48 h pre-mitotic cells, 48 h mitotic cells, 72 h unique cluster, and 48 h quiescent cells.
Metabolic flux assessment
The bioenergetics of hiPS-CMs were measured using a Seahorse Bioscience XF96e Flux Analyzer. For these experiments, the assay medium consisted of unbuffered phenol red-free DMEM pH 7.4, supplemented with 5 mM glucose, 1 mM glutamine, and 100 µM L-carnitine, 100 nM insulin, and 100 µM BSA-palmitate. Following microplate insertion, the XF96e automated protocol consisted of a 12 min delay followed by baseline oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements. All experiments were conducted at 37°C. Data were normalized to the protein content.
Stable isotope-resolved metabolomics (SIRM)
hiPS-CMs were incubated in growth medium containing 5.5 mM 13C6-glucose in 6-well plates for 8 h, after which cell reactions were quenched in cold acetonitrile, and extracted in acetonitrile: water: chloroform (v/v/v, 2:1.5:1), and processed as described previously for metabolite assessments using mass spectropmetry38-42. Stable isotope data analyses were performed by obtaining the mass spectrometer .raw files, which are first converted to .mzML format with msConvert tool, a part of an open-source ProteoWizard suite, described in detail by Adusumilli and Mallick43. Isotopologue peak deconvolution and assignments were performed using El-MAVEN. Peaks were assigned using a metabolite library first generated and verified using full-scan MS and MS/MS spectra of unlabeled samples, as described previously41, 42, 44. The library contained metabolite names and corresponding molecular formulae used to generate theoretical m/z values for all possible isotopologues and retention times. The El-MAVEN parameters for compound library matching were as follows: EIC Extraction Window ± 7 ppm; Match Retention Time ± 0.60 min. For 13C isotopologue peak detection, the software criteria were set as follows: Minimum Isotope-parent correlation 0.5; Isotope is within seven scans of the parent; Abundance threshold 1.0; Maximum Error To Natural Abundance 100%. All assignments were visually inspected and compared with unlabeled samples for reference. Any peak groups assigned in error, e.g., not present or having different retention times than in the unlabeled samples, were deleted, and correct peak assignments were added manually, as described in42. Finally, the peak list with corresponding abundances was exported to a comma-separated (CSV) file and uploaded to the Polly workflow to perform natural abundance correction using Polly Isocorrect. Finally, the data were analyzed and plotted with GraphPad Prism 8.0 (GraphPad Software, San Diego, Ca, U.S.A.).
Immunocytochemistry and immunohistochemistry
The hiPS-CMs were fixed in 4% formaldehyde for 20 min (Thermos Scientific Cat#28908). Fixed cells were washed three times with PBS. For animal experiments, the heart was isolated and perfused with 1M KCL to arrest the heart, then washed with PBS and perfused with 4% paraformaldehyde (Electron microscopy sciences Cat# 15713-S). The hearts were cut longitudinally into half and kept in 4% paraformaldehyde for 48 h. The hearts were then washed with PBS, then placed in 10% sucrose solution for 1 h followed by 20% sucrose solution for 1 h at room temperature, then placed in 30% sucrose solution overnight at 4oC. The hearts were then processed into frozen OCT blocks and kept at -80oC for 24 h. The heart was sectioned using a cryostat (Leica Inc.) in 8µm thick sections, placed on slides, and kept at -20oC until staining. To start staining, the OCT was removed from the section by heating at 95oC for 5 min then washing in PBS for 30 min.
The fixed cells or cleaned sections were permeabilized with 0.1% Triton X-100 for 15 min (Millipore Cat# 55163804) and then blocked with 3% bovine serum albumin (B.S.A.) in PBS for 60 min at room temperature (V.W.R. Cat# 0332). The cells or tissue sections were then probed with primary antibody (1:200 in 1% B.S.A.) for 1.5 h. Then washed three times with PBS. They were then labeled with secondary fluorescent antibody (1:200 in 1% B.S.A.). Table 1 showed a list of primary and secondary antibodies used in this study. Cells/ tissue sections were then washed three times with PBS and stained with DAPI 1µg/ml (Biotium Cat# 40043) to stain the nucleolus blue. For EDU detection, the cells were also treated with 5µM 5-ethyl-2-deoxyuridine (EDU) for the course of the experiment, which will incorporate into the newly synthesized DNA. After fixation, permeabilization, and blocking of the cells/ tissue sections, the EDU incorporation was visualized using Click it EDU-Alexa-Flour647 imaging kit (Thermo Fisher Cat# C10340). For live-cell imaging, the cells were treated with NucBlue live cells stain (Thermo Fisher ) for 20 minutes.
Imaging was conducted for the whole well using the high content imaging instrument, Cytation 1. The percentage of co-localization of PHH3, EDU, GFP, or gene expression and Troponin-T was quantified using Gen 5.05 software.
Specific protein
|
Primary antibody
|
Cat #
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Cardiac Troponin
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Mouse Monoclonal Cardiac Troponin T Antibody
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Thermos Fisher (MA5-12960)
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Cardiac troponin
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Rabbit monoclonal Anti-Cardiac Troponin T antibody
|
Abcam ab209813
|
Phospho Histon H3 (PHH3)
|
Rabbit monoclonal Anti-Histone H3 (phospho S10) antibody - ChIP Grade
|
Abcam ab5176
|
CDK4
|
Rabbit monoclonal Anti-CDK4
|
Abcam ab199728
|
CCNB
|
Rabbit monoclonal Anti-CCNB
|
Abcam ab32053
|
CCND
|
Rabbit monoclonal Anti-CCND
|
Abcam ab134175
|
CDK1
|
Mouse monoclonal Anti-CDK1
|
Abcam ab18
|
Arura Kinase B
|
Rabbit polyclonal anti-Aurora B
|
Abcam ab2254
|
Ds Red
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Mouse monoclonal anti R.F.P.
|
Abcam ab150115
|
Secondary antibody
|
Cat #
|
Goat anti-Mouse IgG (H+L), F.I.T.C.
|
Thermos fisher A16079
|
Texas Red Goat anti-Rabbit IgG (H+L)
|
Thermos fisher T-6391
|
Alexa FlourTM 647 donkey anti-rabbit IgG
|
Thermos fisher A31573
|
Texas Red -X goat anti-mouse IgG
|
Thermos fisher T862
|
Anti-GFP, rabbit polyclonal antibody Alexa FluorTM 448 conjugate
|
Thermos fisher A21311
|
qRT-PCR
A piece of the left ventricle at the injection area was collected for RNA extraction. The heart was homogenized with QlAzol lysis reagent ( Qiagen Cat# 79306), then RNA was extracted following the miRNeasy micro kit protocol (Qiagen Cat# 217084). The concentration of the RNA was calculated using the Cytation 1 reader. 1ug of each RNA sample was used for reverse transcription using a mixture of oligo(dT) and random hexamer primers (SuperScript IV VILO Master Mix, ThermoFisher Scientific Cat # 11756050). Real-time PCR analysis was conducted with Taqman fast advanced master mix (Thermo Fisher 4444557), and primers specific to human CDK1, CDK4, CCNB, CCND genes (Hs00938777, Hs00364847, Hs01030099, Hs00765553 respectively, Applied Biosystem ), and the expression was normalized to rat GAPDH expression (Rn01775763 Applied Biosystem) using the Quant studio 5 real-time PCR detection system (Applied Biosystems).
Animal experiments
Animal studies were performed following the University of Louisville animal use guidelines, and the protocols were approved by the Institutional Animal Care and Use Committee (IACUC) and were accredited by the Association for Assessment and Accreditation of Laboratory Animal Care.
MADM mice experiment
For lineage tracing, we used mosaic analysis with double markers (MADM) transgenic mice were developed as prescribed in 4. All the surgeries were performed as described in 45, 46. Adult (about 12 weeks old) female MADM mice were anesthetized with sodium pentobarbital (60 mg/kg i.p.). After opening the chest through a left thoracotomy, a nontraumatic balloon occluder was implanted around the mid-left anterior descending coronary artery (L.A.D.) using an 8-0 nylon suture. Myocardial infarction was produced by 60-min coronary ischemia, followed by reperfusion (I/R). Rectal temperature was carefully monitored and maintained around 37°C throughout the experiment. Successful performance of coronary occlusion and reperfusion was verified by visual inspection and by observing S-T segment elevation and widening of the QRS on the electrocardiogram during ischemia and their resolution after reperfusion. Seven days after I/R, mice were re-anesthetized with sodium pentobarbital, 60 mg/kg I.P. and the chest reopened through a central thoracotomy. The mice were randomly selected to be injected with 20 ul of TNNT2-4Fpolycistronic-NIL, TNNT2-LacZ-NIL virus intramyocardially using a 30-gauge needle. The injections were made at the border between infarcted and non-infarcted myocardium as two injections 10 µL each (2x107 transducing units (T.U.) per mouse heart). Forty-eight hours after injection, mice received Tamoxifen (40mg/kg I.P.) for three days (Sigma Aldrich T5648). Mice were euthanized 14 days after MI, and the hearts were harvested for pathology study. The mice sergeant was blinded to all administered viruses.
MADM mice frozen hearts were sectioned Longitudinally into 180-210 sections (3 sections per slide collect one and throw away 2) 8µm thick. The frozen sections were fixed, permeabilized, and blocked as described in Immunocytochemistry and immunohistochemistry section, then stained with Dapi 1µg/ml (Biotium Cat# 40043) to stain the nucleolus blue. The Coverslips were mounted with Vectashield antifading medium (vector labs Cat# H-1000) onto slides and visualized using Keyence BZ9000 imaging system (10X magnification to the whole left ventricle). The percentage of single-colored cardiomyocytes from the total labeled cardiomyocytes was calculated using B.Z. analyzer software. Individuals analyzed the results were blinded to the treatment applied in each animal.
Rat experiments
All surgeries were performed as described in 47, 48. Briefly, Female Fischer 344 (F344) rats at the age ranging from 8-12 weeks were anesthetized with ketamine (37 mg/kg) and xylazine (5 mg/kg), intubated, and ventilated with a rodent respirator. Anesthesia was maintained with 1% isoflurane inhalation, and body temperature was kept at 37°C with a heating pad. All rats underwent a 2 h occlusion of the left anterior descending coronary artery, followed by reperfusion. Seven days after MI, echocardiography was performed to ensure the development of MI. All rats in this study had EF drop > 20 points from baseline. Rats were randomized into two groups (TNNT2-GFP-NIL, TNNT2-4Fpolycistronic-NIL). Rats were re-anesthetized with ketamine/xylazine, intubated, and ventilated. The chest was reopened to expose the heart. Viral vectors (1x108 T.U. per rat heart in 100 µl PBS) were injected into the left ventricle along the infarct border at five sites (20 µl/site) using a 30G needle. The rat surgeon was blinded to whether 4F or control non-integrated lentivirus was administered in each animal.
Cardiac function was assessed by serial echocardiography at baseline (before MI), one week after MI (before virus injection), and then four weeks after virus injection. Animals were anesthetized lightly with isoflurane, placed on the imaging table in the supine position, and prepared for imaging using the Vevo 2100 Imaging System (Visual Sonics) equipped with a 25-MHz transducer. Parasternal longitudinal axis images were acquired and analyzed by LV trace using the Vevo LAB 3.2.6 to obtain the LV functional parameters, including the end-diastolic and end-systolic area, volume, stroke volume, fractional shortening, and ejection fraction. Imaging and calculations were done by an individual who was blinded to the treatment, and the code was broken after all data acquired.
At the end of the experiments (5 weeks after MI), animals were sacrificed, and their hearts were harvested for histological studies. The frozen hearts were sectioned longitudinally into 400-500 sections 8um thickness (take a section to throw one add two sections per slide), and one slide for every ten slides (20-25 slide per animal) were stained with Standard Masson's Trichrome staining to determine scar size. The stained sections were imaged using the Keyence BZ9000 imaging system (4X magnification). Image J software was used to measure the scar area (blue) and healthy area (red) on longitudinal sections. Individuals assessing scar area were blinded to the treatment applied in each animal.
Pig experiments
All surgeries were performed as described in49. Yorkshire pigs weighing 25-35 kg received 200 mg amiodarone orally daily for seven days pre-operatively. Pigs were premedicated with an intramuscular injection of a solution containing ketamine hydrochloride and xylazine.
Pigs were injected with a dose of buprenorphine S.R. before the procedure. To create myocardial infarction, the right neck's skin was cut to make a small opening, allowing access to the right carotid artery. A 7-8F fast-cath sheath was introduced into the carotid artery. The pig was injected with Heparin (300 units/kg I.V.) to prevent clotting of the sheaths and catheters during the procedure. After intubation, the pig was mechanically ventilated. Anesthesia was maintained with isoflurane. Body temperature was monitored continuously with a rectal probe attached to a thermocouple and maintained within physiology range using a veterinary blanket. The pigs were subjected to 5 minutes of stabilization, followed by baseline hemodynamics and echocardiography.
A 6-7F Hockey-stick catheter was guided to the left main coronary artery under fluoroscopy as following: the catheter engaged the left main coronary ostium, and an angioplasty-type balloon catheter and guidewire assembly were fluoroscopically guided into the L.A.D. Then, the wire was advanced into the distal L.A.D., and an appropriate balloon catheter was telescoped over the wire and positioned above the first diagonal branch (the entire L.A.D. territory was included for occlusion). The balloon's placement will be verified by intracoronary contrast dye injection (Contrast media) and documented by cine angiogram before inflation. The balloon was inflated to occlude the L.A.D. and the L.A.D. occlusion was maintained for 90 minutes to produce myocardial infarction, targeting an infarct size of >50% of the area at risk. Inflation and position of the balloon were verified by contrast angiogram again at the end of ischemia. If necessary, the balloon will be repositioned, and such "positional re-inflation" will be limited to less than 20 seconds to avoid any preconditioning. Once the balloon is inflated, external defibrillator pads were placed on the pig's chest for "hands-free" cardioversion if ventricular fibrillation occurs using a bipolar defibrillator (HP Codemaster XL+) at 300 Joules. After the 90 min ischemic period, the intracoronary balloon was deflated to initiate reperfusion. After the procedure of myocardial infarction, the balloon catheter was withdrawn, and a cine angiogram was taken to document the wide open of the L.A.D. artery. After withdrawing the Hockey-stick guide catheter, the arterial sheath catheter was removed, and the arterial was repaired by anastomosis. The skin incision was closed in 3 layers using 3-0 Vicryl for internal sutures and 3-0 P.D.S. for the final subcutaneous layer. The pig was weaned from anesthesia, and the animal was extubated when appropriate and allowed to recover. Animals received antimicrobial therapy cetiofur pre-operatively and every 24 hours for the first 48 hours postoperatively. Animals were prepped and draped in a routine sterile fashion. 5% dextrose and normal saline was continuously infused during the procedures
Seven days after the MI procedure, pigs were re-anesthetized as described above. Pigs were subjected to MRI scans and echocardiography. Anesthesia was maintained with isoflurane. Body temperature was monitored continuously with a rectal probe attached to a thermocouple and maintained within physiology range using a veterinary blanket. Animals will be prepped and draped in a routine sterile fashion. A dose of buprenorphine S.R. will be given before the procedure. The chest was opened through the initial skin incision and continued down to the sternum. To avoid a post-surgical pulmonary complication, a midline sternotomy to expose the heart in the intrapleural space without breaking the pleural membrane was performed with extra care to avoid rapture of the pleural membrane intact during the opening of the mediastinum. Then the heart suspended in a pericardial cradle as described in 50, 51.
In the case of the pleural membrane broken, efforts are made to close the tear by re-approximation of the pleural membrane with 6-0 Prolene and to reestablish a negative pressure in the pleural cavity using a withdrawal tube with a purse-string closure. After the chest was opened, the pericardium was cut vertically, and the heart was exposed and suspended in a pericardial cradle. Five intramyocardial injections (200ml each) (3x109 T.U. total/pig heart) of viral vectors were performed along the infarct border, and the site of injection was demarcated with a 6-0 Prolene suture. After completing these procedures, warm normal saline (approx. 500mL) was used for flushing the thoracic cavity. This flush was suctioned out before closing the chest. The pericardium was approximated as soon as possible. The sternum was closed with a 20G stainless steel suture and 5 Green Braided P.T.F.E. nonabsorbable surgical sutures. The chest was closed in layers (0 PDS II suture for the muscle and 2-0 PDS II suture subcutaneously), and a single mediastinal tube (18F catheter), 3-way valve, and 60cc syringe will be used to reestablish a negative intrapleural pressure and evacuate any remaining blood or irrigation solution. The chest tube (18F catheter) was removed before skin closing after no visible air leak or blood accumulation, and a purse-string suture (2-0 PDS II) was used to ensure an airtight seal. The skin incision was then glued with Vetbond adhesive. Then the chest was closed, and the inhaled anesthetic was turned off, the animal extubated when appropriate, and allowed to recover. Animals received antimicrobial therapy cetiofur pre-operatively and every 24 h for the first 48 h postoperatively.
Four weeks after virus injection animal was anesthetized with isoflurane as described before to perform the final cardiac MRI and echocardiography. Body temperature will be monitored and maintained within the physiological range. Arterial blood pressure and surface E.C.G. were monitored continuously. Then the animal was deeply anesthetized with 5% isoflurane. A bolus of 3-6 ml/kg of 3M KCl solution will be injected into the left atrium until the heart is arrested. After the cessation of vital signs, the heart will be harvested for postmortem procession.
T.T.C. stain of the pig heart
Directly after the heart was harvested, the aorta was perfused with normal saline (500-1000 ml) to flush out vascular blood. The heart was weighed and transversely sliced into 5-6 sections. Heart sections were incubated in 1% T.T.C. at 37oC for 5 min. Then right ventricular, atriums were removed, and LV sections were weighted. The pictures of LV slices were taken using a professional camera. Images were analyzed using Image-J software. Scar size percentages were calculated.
Statistical Analyses
For all assays, power analyses were performed to choose the group sizes, which will provide >80% power to detect a 10% absolute change in the parameter with a 5% Type I error rate. These power analyses indicated a minimum of 4 experimental replicates per group; therefore, we used a range of 4 – 15 experimental replicates per group for each assay. Special statistical consideration for RNAseq was detailed under the methods section. The Kolmogorov-Smirnov (K-S) test for normality was conducted; all data sets showed normal distribution. Then, differences between the two groups were examined for statistical significance with unpaired Student t-tests. However, to compare data consisting of more than two groups, we performed one- or Two-way ANOVA tests followed by Bonferroni post hoc multiple comparisons to get the corrected p-value. A value of P<0.05 was regarded as significant. Error bars indicate S.D. The person who performed the analysis was blinded to the experimental groups. Two blinded clinical cardiology fellows assessed all cardiac function Echocardiography and MRI analyses, and the data represented are the average of their analyses. Usually, there were no significant discrepancies between the two readings.