Isolation of primary fibroblasts
Tail-tip fibroblasts (TTFs)
Adult mouse tails were skinned and cut into small pieces which were plated on tissue culture dishes and cultured in DMEM supplemented with 15% FBS and antibiotics. The medium was changed every 2 to 3 days. TTFs migrated out from the explants after 2 or 3 days. One week later, TTFs were frozen or replated for viral transduction.
Cardiac fibroblasts (CFs)
Hearts from adult mice (older than 4 weeks of age) were minced into small pieces and plated on tissue culture dishes. Three minutes later, culture medium (DMEM:199 (4:1), 15% FBS and antibiotics) was gently added to the dishes. CFs started to migrate out of the minced heart tissue after two days. The medium was replaced every two days. Ten days later, CFs were frozen or replated for viral transduction.
Generation of retroviruses
Retroviral plasmid DNA was generated by subcloning EGFP, Myc-tagged Nkx2-5, GATA4, Tbx5, Hand2, MEF2C, and FLAG-tagged MesP1 cDNAs into the retroviral vector pBabe-X38
Ten micrograms of retroviral plasmid DNA was transfected using Fugene 6 (Roche) into Platinum E cells (Cell Biolabs) which were plated on a 10-cm tissue culture dish at a density of 3×106 cells per dish, 24 hours prior to transfection. Twelve hours after transfection, medium was changed to 12 ml of fresh medium (DMEM supplemented with 10% FBS and antibiotics). After 36 hours of transfection, viral medium was harvested and filtered through a 0.45 μm cellulose filter. The viral supernatant was mixed with polybrene (Sigma) to a final concentration of 6 μg/ml.
TTFs and CFs were plated on tissue culture dishes pre-coated with SureCoat (Cellutron) at a density of 0.8×104/cm2. After 24 hours, the fibroblast growth medium was replaced with freshly made viral mixture containing polybrene. Twenty four hours later, viral medium was replaced with induction medium, composed of 10% conditioned medium obtained from neonatal rat/mouse cardiomyocyte culture, DMEM/199 (4:1), 10% FBS, 5% horse serum, antibiotics, non-essential amino acids, essential amino acids, B-27, insulin-selinum-transferin, vitamin mixture, and sodium pyruvate (Invitrogen). Conditioned medium was filtered through a 0.22 μm pore size cellulose filter. Medium was changed every two days until cells were harvested.
qPCR, Western blot analysis and immunocytochemistry
Total RNA was extracted from cultured cells and cDNA was synthesized by reverse transcription. All qPCR probes were obtained from Applied Biosystems. Western blots were performed with anti-Myc (Santa Cruz, clone A-14 1:1000) and anti-FLAG (Sigma, 1:2000) antibody. For immunocytochemistry, cells were fixed in 4% paraformaldehyde and incubated with primary antibodies: anti-GFP (Torrey Pines Biolabs 1:400), anti-cTnT (Thermo Scientific 1:400), anti-Myc (Santa Cruze, clone A-14 1:200), and anti-α-actinin (Sigma, 1:400). After washing with PBS, Alexa fluorogenic secondary antibodies (Invitrogen) were used to detect the signal.
Total RNA was isolated from uninfected CFs, CFs transduced with either empty vector or GHMT retroviruses and adult heart. Microarray analysis was performed on the platform of Illumina Mouse-6 Beadchip by the DNA Microarray Core Facility at the University of Texas Southwestern Medical Center. Data were analyzed using GeneSpring GX software (Agilent).
For the initial assay to detect αMHC-GFP expression, adherent fibroblasts were washed with PBS and detached from culture dish by treatment with accutase (Millipore) for 10 min at 37°C. Cells were then washed with 2% FBS in PBS and filtered through a cell strainer. Cells were incubated with propidium iodide (1:1000 dilution in 1% FBS in PBS) for 15 min at room temperature. Dead cells were excluded by propidium iodide staining and live cells were analyzed for GFP expression using FACS Caliber (BD Sciences) and FlowJo software.
For intracellular staining of cardiac-specific markers, cells were fixed with 4% paraformaldehyde for 15 min after being harvested, as described above. Fixed cells were washed with PBS and permeabilized with saponin for 10 min at room temperature. After being washed with PBS, cells were incubated with 5% goat and donkey serum in PBS at room temperature for 30 min, followed by incubation with primary antibodies (rabbit polyclonal anti-GFP antibody (Invitrogen) at a 1:100 dilution and mouse monoclonal anti-cTnT antibody (Thermo Scientific) at a 1:400 dilution in 0.2% goat and donkey serum in PBS for 30 min at room temperature. After washing twice with PBS, cells were incubated with secondary antibodies for 30 min at room temperature. Secondary antibodies were goat anti-rabbit Alexa fluor 488 (Invitrogen) at a 1:200 dilution and donkey anti-mouse Cy5 (Jackson Laboratory) at a 1:400 dilution in PBS containing 0.2% goat and donkey serum. Cells were washed with PBS three times, and then analyzed for GFP and cTnT expression using FACS Caliber (BD Sciences) and FlowJo software.
Generation of α-MHC-GFP transgenic mice
A plasmid containing a 5.5 kb genomic fragment upstream of the mouse α-MHC gene plus exons 1–3 and intronic sequences39
, inserted upstream of a neomycin-resistance cassette followed by an Internal Ribosomal Entry Sequence (IRES) and a GFP reporter was linearized and micro-injected into the pronucleus of zygotes. The founders were crossed to C57BL/6 mice to get stable lines.
Induction of Cre by tamoxifen administration
Tamoxifen (Sigma) was dissolved in sesame oil (90%) and ethanol (10%) at a concentration of 50 mg/ml. To induce Cre activity, tamoxifen (0.2 mg/g body weight) was administered by gavage with a 22-gauge feeding needle into mice bearing Tcf21iCre/+
/Rosa26-LacZ for three to five or seven consecutive days, respectively. Mice were analyzed or subjected to MI surgery at day 8 post-oral gavage of the last dosage.
Sorting of Tcf21-expressing cells and gene expression
Three-month old Tcf21iCre/+
mice were induced with 0.2mg/g tamoxifen for 3 consecutive days by gavage, and a week later hearts were isolated and processed (atria and aorto-pulmonary trunk were removed) to generate single cell suspensions for FACS sorting, as described previously41
. The suspension was filtered through tissue strainers, centrifuged at 400 × g for 5 minutes and resuspended in 10% CM media (10% Hyclone FBS, 3:1 DMEM/M-199, 10 mM HEPES, 1.2% antibiotic/antimycotic) before sorting with a MoFlo flow cytometer (Cytomation Inc) using Summit software. For transcript analysis, sorted cells were collected into lysis buffer for RNA extraction (RNAqueous Micro kit from Ambion). A fraction of each sample was also collected into PBS for post-sort assessment of purity. Complimentary DNA was synthesized using Superscript III reverse transcriptase (Invitrogen) and random hexamers (Roche). Gene expression profiles were generated using standard qPCR methods with iTAQ SYBR Green master mix (Bio-Rad) on a CFX96 instrument (Bio-Rad). Samples were run in triplicate and normalized to cyclophilin expression. Fold enrichment was determined with respect to the unsorted population.
MI surgery and intramyocardial injection of retroviruses
Mice were anesthetized with isoflurane, intubated with a polyethylene tube (size 60), and then ventilated with a volume-cycled rodent respirator with a 2–3 ml/cycle at a respiratory rate of 120 cycles/min. Thoracotomy was performed at the third intercostal space and self-retaining microretractors were placed to separate the third and fourth rib to visualize the LAD. A 7.0 prolene suture (Ethicon, Johnson & Johnson, Brussels, Belgium) was then passed under the LAD at 1.5 mm distal to the left atrial appendage, immediately after the bifurcation of the left main coronary artery. The LAD was doubly ligated. The occlusion was confirmed by the change of color (becoming paler) of the anterior wall of the left ventricle. Sham-operated mice underwent the same procedure without ligation. Immediately after ligation of the LAD, 50 μl of concentrated retrovirus was injected into the border zone of the infarct at 5 different areas using a gastight 1710 syringe (Hamilton). The chest wall was then closed with a 5.0 Dexon absorbable suture (Tyco Healthcare, United States Surgical, USA), and the skin was closed with Topical Tissue Adhesive (Abbott Laboratory, IL, USA). Mice were extubated and allowed to recover from surgery under a heating lamp. The mouse surgeon was blinded to the study. Mice with FS>30% at day 1 post-MI were removed from the study.
Cardiac function was evaluated by two-dimensional transthoracic echocardiography on conscious mice using a VisualSonics Vevo2100 imaging system. Fractional shortening (FS) and ejection fraction (EF) were used as indices of cardiac contractile function. M-mode tracings were used to measure LV internal diameter at end diastole (LVIDd) and end systole (LVIDs). FS was calculated according to the following formula: FS (%) = [(LVIDd − LVIDs)/LVIDd] × 100. EF is estimated from (LVEDV-LVESV)/LVEDV 100%. Left ventriclular end systolic volume, LVESV; end diastolic volume, LVEDV. All measurements were performed by an experienced operator blinded to the study.
Six and twelve weeks after MI, the cardiac function of mice was re-evaluated by cardiac MRI using a 7T small animal MR scanner (Varian, Inc, Palo Alto, CA) with a 38 mm birdcage RF coil. Under anesthesia by inhalation of 1.5 – 2% isoflurane mixed with medical-grade air via nose-cone, the animals were placed prone on a mouse sled, (Dazai Research Instruments) equipped with a pneumatic respiratory sensor and ECG electrodes for cardiac sensing, head first with the heart centered with respect to the center of the RF coil. The mouse chests were shaved and a conducting gel was applied to optimize ECG contact between electrodes and mouse. All MRI acquisitions were gated using both cardiac and respiratory triggering. The bore temperature was kept at 35°C to assure adequate and constant heart rate.
Two-dimensional (2D) gradient echo images on three orthogonal planes (transverse, coronal and sagittal) were acquired to determine the long-axis of the heart in each mouse. Axial images perpendicular to the long axis of the heart were chosen for cine-imaging. Cine images at 12 phases per cardiac cycle were obtained with an echo time of 2.75 ms, repetition time = EKG R-R interval/12, flip angle of 45°, and NEX= 4. Each scan consisted of seven to ten contiguous slices from apex to LV outflow with 1 mm thickness, a matrix size of 128 × 128, and a field of view of 30 × 30 mm.
Epicardial and endocardial borders were manually traced for calculation of LVESV, LVEDV using NIH ImageJ software. Total LV volumes were calculated as the sum of all slice volumes. Stroke volume was calculated by the equation, LVEDV-LVESV. EF was calculated by the equation, (LVEDV-LVESV)/LVEDV 100%. Investigators performing MRI acquisition and analysis were blinded to the assignment of mice group.
Isolation of adult mouse cardiomyocytes, measurement of cardiomyocyte sarcomere shortening and Ca2+ transients
Mouse cardiomyocytes were isolated using enzymatic digestion and mechanical dispersion methods previously described33
. In brief, after retrograde perfusion with Ca2+
-free Krebs-Ringer buffer (KR, 35 mM NaCl, 4.75 mM KCl, 1.19 mM KH2
, 16 mM Na2
, 134 mM sucrose, 25 mM NaCO3
, 10 mM glucose, 10 mM HEPES, pH 7.4, with NaOH) and digestion with collagenase solution (Collagenase II, 8 mg/mL), the LV myocytes were separated using a fine scalpel and scissors. After gentle trituration, cells were kept in KB solution (10 mM taurine, 70 mM glutamic acid, 25 mM KCl, 10 mM KH2
, 22 mM glucose, 0.5 mM EGTA,pH 7.2 with KOH) and studied within 6 hours at room temperature.
To examine myocyte contractile capacity, isolated cardiomyocytes were incubated with 33 μm C12
FDG for 30 min. The green C12
cardiomyocytes were identified by a fluorescence microscope. Adult cardiomyocytes from wild-type mice incubated with C12
FDG were used to determine autofluorescence of cardiomyocytes. C12
cardiomyocytes and C12
cardiomyocytes were field stimulated at 1 Hz while being superfused with extracellular buffer at room temperature. Images were acquired at 240 Hz through a 60xmicroscope objective using a variable field rate CCD camera (IonOptix, Milton, MA). Cell length was measured by a video edge-detection system, using an IonOptix interface system. Intracellular Ca2+
transients were measured as previously described33–36
. Analyses were carried out with the software (IonWizard, IonOptix Corp.). Onlyrod-shaped, clearly striated cardiomyocytes that were Ca2+
tolerant were used in the experiments.
Calcium transient measurements in spontaneous beating cardiomyocytes
Calcium imaging of beating iCLMs, cultured neonatal mouse ventricular cardiomyocytes was performed using the PTI (Photon Technology International) Ca2+
Imaging System (Birmingham, NJ) with an automated fluorescence microscope and a CCD camera, as described previously35
. Calcium transients in individual spontaneous beating cell were calculated by measurement of Ca2+
-induced fluorescence at both 340 and 380 nm.
Membrane potential recordings of iCLMs in vitro
Action potentials of beating iCLMs with di-4-ANEPPS (Invitrogen) were assessed, as described previously37
Action potential recordings of iCLMs isolated from adult mouse hearts
Tomato+ cardiomyocytes were identified under a Nikon inverted fluorescence microscope with a red filter. Whole-cell current clamp experiments (Axopatch 200B, Molecular Devices) were conducted to measure cardiomyocyte membrane action potentials (AP). Cells were plated in a chamber that was superfused at 1 to 2mL/min with standard solution A containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES (pH 7.4 with NaOH), and 10 mM glucose (310 mOsm). Recording pipettes were prepared with resistances of 2–3 mΩ when filled with internal solution containing 135 mM KCl, 10 mM EGTA, 10 mM HEPES, 5 mM glucose (pH7.2 with KOH; 300 mOsm). Action potentials were recorded in response to brief (1–2 ms) depolarizing current (1–2 nA) injections delivered at 1 Hz. All data were filtered at 5 kHz and analyzed by pCLAMP 9 software (Molecular Devices).
Histology and immunohistochemistry
Hematoxylin and eosin (H&E) and LacZ staining were performed as described37
. Frozen sections from ischemic hearts were stained with anti-GFP (Aves Labs 1:800) antibody according to manufacturer’s instructions. Paraffin-embedded sections were stained with anti-cTnT (1:400) and anti-Cx43 (Cell Signaling, 1:50). Frozen sections from tamoxifen-treated Tcf21iCre/+
mice (~5 week old) were stained with anti-P4HB (ProteinTech, 1:200), anti-cTnT (Thermo Scientific, 1:400), anti-isolectin B4 (Vector Labs, 1:100), and anti-SM22α (Abcam, 1:200). Signals were detected with Alexa fluorogenic secondary antibodies (Invitrogen).