Mesp1 Marked Cardiac Progenitor Cells Repair Infarcted Mouse Hearts

Mesp1 directs multipotential cardiovascular cell fates, even though it’s transiently induced prior to the appearance of the cardiac progenitor program. Tracing Mesp1-expressing cells and their progeny allows isolation and characterization of the earliest cardiovascular progenitor cells. Studying the biology of Mesp1-CPCs in cell culture and ischemic disease models is an important initial step toward using them for heart disease treatment. Because of Mesp1’s transitory nature, Mesp1-CPC lineages were traced by following EYFP expression in murine Mesp1Cre/+; Rosa26EYFP/+ ES cells. We captured EYFP+ cells that strongly expressed cardiac mesoderm markers and cardiac transcription factors, but not pluripotent or nascent mesoderm markers. BMP2/4 treatment led to the expansion of EYFP+ cells, while Wnt3a and Activin were marginally effective. BMP2/4 exposure readily led EYFP+ cells to endothelial and smooth muscle cells, but inhibition of the canonical Wnt signaling was required to enter the cardiomyocyte fate. Injected mouse pre-contractile Mesp1-EYFP+ CPCs improved the survivability of injured mice and restored the functional performance of infarcted hearts for at least 3 months. Mesp1-EYFP+ cells are bona fide CPCs and they integrated well in infarcted hearts and emerged de novo into terminally differentiated cardiac myocytes, smooth muscle and vascular endothelial cells.


Myocardial Infarction (MI) Surgery.
An animal model of ischemic myocardial infarction (MI) in 8-10 week old adult male C.B-17 SCID mice was induced by chronic (permanent) ligation of the left anterior descending (LAD) artery (McConnell et al., 2009) and as we have similarly performed in rats (Wu et al., 2011).
Prior to surgery, all instruments were sterilized in a dry bead sterilizer and the animal was prepared for sterile surgery. Anesthetized mice (2% isoflurane in 100% oxygen) were placed in a supine position and an endotracheal polyethylene tube (BD Intramedic™ Polyethylene Sterile Tubing, PE 90) was inserted into the trachea, visually guided by use of a dissecting microscope. Once proper position was confirmed, the endotracheal cannula was then connected to a volume-cycled rodent ventilator (Harvard Apparatus Inspira Advanced Safety Ventilator), which was supplemented with 100% oxygen and with a tidal volume approximately 0.15 -0.25 ml and a respiratory rate of 100 -125 breaths per minute. Once steady breathing was established, and deep anesthesia was confirmed by the limb withdrawal response and then placed onto a heated surgical table and draped to prevent contamination from the area. Thoracotomy was performed by a small incision at the third intercostal space and the intercostal muscles were separated and dissected to the side in order to expose the ribs. Self-retaining micro-retractors were placed to separate the third and fourth ribs in order to expose the heart and visualize the LAD artery. An 8.0-prolene suture (Ethicon, Johnson & Johnson) was used to ligate the LAD artery (double ligation) at 1.5 mm distal to the left atrial appendage, which was identified directly following bifurcation of the left main coronary artery. Occlusion of the LAD artery was confirmed by observing blanching (pale coloring) of myocardial tissue distal to the suture and dysfunction of the anterior wall motion. Sham-operated control animals underwent the identical procedure, but without the ligation. Un-operated control animals did not undergo surgery. Immediately after LAD ligation, CPCs were injected within the myocardium. Control animals, that underwent LAD ligation, were injected with Phosphate Buffered Saline (PBS) within the myocardium. The rib cage was then closed with 5.0 prolene RB-1 suture (Suture Express) and the retracted muscles were placed in position and the skin was closed using 5.0 sutures. Animals were extubated and allowed to recover from anesthesia in a warm space under observation. The animals were closely monitored for any abnormal signs of pain or labored breathing before being returned to clean housing and the animal room.

Intramyocardial Injection of Mesp1-CPCs.
A mouse model of ischemic MI in 8-10 week old adult male C.B-17 SCID mice was induced by permanent ligation of the LAD artery, as described above. Ligation was verified by immediate myocardial blanching and anterior wall dysfunction. All animals with anterior wall motion dysfunction consistent with the infarct were randomized into the various groups: (1) sham-operated, (2) MI + PBS, (3) MI + Mesp1-CPCs, and (4) un-operated-controls.
CPCs were injected using 5 (2 µl) injections (total volume = 10 µl) each with 20,000 CPCs (total number of cells = 0.1x106 cells) into the infarct and border zone areas, immediately following LAD artery ligation. Control groups included sham-operated mice, MI-operated mice injected with PBS and un-operated control mice. Following CPC injection, the chest was closed and the animals were monitored closely. An echocardiogram was performed 24-hr post MI to determine the extent of the induced MI. In selected mice, CPCs in mouse hearts were imaged live using bioluminescence at 24-hr post-cell injection.

Cardiac Magnetic Resonance Imaging (MRI).
Cardiac function was determined by magnetic resonance imaging (MRI) using the noninvasive Bruker PharmaScan® 7T small animal Scanner at 1-week and 6-weeks following induction of MI and / or MI plus the intramyocardial injection of the CPCs. Mice were initially anesthetized with 4-5% isoflurane (mixed with oxygen) and maintained with 1-2% isoflurane during imaging. An animal-monitoring system (SA Instruments, Stony Brook, NY) was used to monitor the mouse's ECG, respiratory rate, and body temperature. The temperature of the MRI's bore was maintained at 35 o C in order to ensure sufficient and constant heart rate. Respiratory-and cardiac-gated images were acquired at end-diastole and end-systole (Costandi et al., 2007). The imaging parameters to acquire cardiac-and respiratory-gated spin echo images were as follows: repetition time (TR), 5.7 ms; echo time (TE), 2.80 ms; field of view, 4.0 cm; number of slices, 10; slice thickness, 1.0 mm; matrix, 128 × 128; and number of averages, 1. The multi-slice scan was performed in the axial orientation to visualize the left and right ventricles, and data were analyzed by using Amira (2.4 + LV-ID(s))) * LV-ID(s)^3. Ligation of the LAD artery, used to induce the MI, was confirmed by echocardiography. A decreased %LV-FS of less than 25%, at 24-hours post-MI versus baseline, was excluded from the study. Data acquisition was performed by an experienced sonographer and data analysis was measured by individuals that were blinded to the study's assignment groups.

Histology and Immunohistochemistry.
Hearts were collected from euthanized animals for histological and immunohistochemical studies. Animals were first heparinized, to prevent blood clotting prior to being anesthetized with 3% isoflurane, for 10 mins. The chest cavity was opened and the heart was excised, connective tissue removed, rinsed in PBS, blotted and weighted. The heart weights (HW) and body weights (BW) were measured and the HW to BW ratio was calculated, as a measure of cardiac hypertrophy.
For infarct size measurements, the hearts were harvested, sectioned, and stained with Masson trichrome to visualize the infarct scar. All histological sections were examined with a Nikon Eclipse microscope using a 4x objective lens. Images were captured and measured the lengths and areas of infarct and LV of each treatment group (n=3/group). The scar was measured in each section by an investigator who was blinded to the identity of the sections using the area measurement approach. Specifically, infarct scar area and the total area of LV myocardium were traced manually in the digital images and measured by the computer. Infarct size, expressed as a percentage, was calculated by dividing the sum of infarct areas from all sections by the sum of LV areas from all sections (including those without infarct scar) and multiplying by 100.
For single immunofluorescence staining, we fixed embryos in 4% paraformaldehyde/PBS, dehydrated and embedded them in paraffin for histological sections. For double immunofluorescence staining, we retrograde perfused the harvested adult mouse hearts through the aorta using a Langendorff perfusion apparatus with 10% formalin for 15 mins.
Then heart samples were dehydrated and embedded in paraffin for histological sections. For antigen retrieval, we boiled sections in sodium citrate buffer (pH 6.0) for 10 mins. Sections For immunohistochemistry staining, we boiled sections in sodium citrate buffer (pH 6.0) for 10 mins for antigen retrieval. After peroxidase blocking (3% H2O2 in PBS), sections were blocked in 2% goat serum/PBS and incubated with primary antibodies anti-GFP (Santa Cruz) overnight at 4ºC. Then sections were treated with biotinylated anti-rabbit secondary antibodies (Vector Laboratories) at RT for 1 hr, followed by treatment with Vectastain Elite ABC reagent (avidin horseradish peroxidase; Vector Laboratories). Horseradish Peroxidase (HRP) activity was revealed using the DAB kit (Vector laboratories). Sections were dehydrated, counter-stained with hematoxylin as needed, mounted with Permount and examined under a Nikon Eclipse Ti invert microscope.

Ethics Statement -Study Approvals and Consent.
All animal studies have been approved by the Institutional Animal Care and Use Cells cultured as EBs were exposed to a serial of BMP4 (0.5 ng/ml, 1 ng/ml, 4 ng/ml and 16 ng/ml) and Activin A dosage (2 ng/ml, 4 ng/ml and 8 ng/ml).
FACS was performed on day 4 cells. Increasing amount of BMP4 is correlated with more EYFP+ cells, while increasing amount of Activin A is not.