Stem cells in the dog heart are self-renewing, clonogenic, and multipotent and regenerate infarcted myocardium, improving cardiac function

Abstract

The purpose of this study was to determine whether the heart in large mammals contains cardiac progenitor cells that regulate organ homeostasis and regenerate dead myocardium after infarction. We report that the dog heart possesses a cardiac stem cell pool characterized by undifferentiated cells that are self-renewing, clonogenic, and multipotent. These clonogenic cells and early committed progeny possess a hepatocyte growth factor (HGF)-c-Met and an insulin-like growth factor 1 (IGF-1)-IGF-1 receptor system that can be activated to induce their migration, proliferation, and survival. Therefore, myocardial infarction was induced in chronically instrumented dogs implanted with sonomicrometric crystals in the region of the left ventricular wall supplied by the occluded left anterior descending coronary artery. After infarction, HGF and IGF-1 were injected intramyocardially to stimulate resident cardiac progenitor cells. This intervention led to the formation of myocytes and coronary vessels within the infarct. Newly generated myocytes expressed nuclear and cytoplasmic proteins specific of cardiomyocytes: MEF2C was detected in the nucleus, whereas alpha-sarcomeric actin, cardiac myosin heavy chain, troponin I, and alpha-actinin were identified in the cytoplasm. Connexin 43 and N-cadherin were also present. Myocardial reconstitution resulted in a marked recovery of contractile performance of the infarcted heart. In conclusion, the activation of resident primitive cells in the damaged dog heart can promote a significant restoration of dead tissue, which is paralleled by a progressive improvement in cardiac function. These results suggest that strategies capable of activating the growth reserve of the myocardium may be important in cardiac repair after ischemic injury.

Fig. 1.

Cardiac progenitor cells. (A and B) Individual progenitor cells positive for c-kit, MDR1, or Sca-1-like, when placed in single wells, generate multicellular clones (A, phase contrast microscopy). A clone generated by a single c-kit-positive cell (B, green) is also illustrated. (C) Clones expanded in differentiating medium give rise to myocytes, SMCs, and ECs. (Scale bars, 10 μm.) (D) Cardiac progenitor cells express HGF, c-Met, IGF-1, and IGF-1 receptors by Western blotting. Actin shows loading conditions.

Fig. 2.

Cardiac progenitor cells in the acutely infarcted heart. Sections of infarcted myocardium 8 h after coronary occlusion in a nontreated (A) and a GF-treated (B–F) heart. (A) c-kit-MDR1-positive cardiac progenitor cells (green, arrowheads) and myocytes are dead by apoptosis (hairpin 1, white). (B) The viable myocardium of the border zone (BZ) and the dead myocardium of the infarcted region (MI) are shown. c-kit-MDR1-positive cells (green, arrowheads) are viable as shown by the absence of hairpin 1 and the presence of propidium iodide staining of their nuclei. In contrast, myocytes are all apoptotic (hairpin 1, white). (C) Similarly, in the center of the infarcted segment, c-kit-Sca-1-like-positive cells (green, arrowheads) are viable and dispersed among dead myocytes. (D and E) Similarly, c-kit-MDR1-Sca-1-like-positive progenitor cells (green) in the border (D), infarct (E), and remote myocardium (RM) (F) of GF-treated hearts express Ki67 (yellow, arrowheads). In D and E, the replicating progenitor cells are located between dead myocytes (hairpin 1, white). (Scale bars, 10 μm.)

Fig. 3.

GFs improve regional cardiac performance after infarction. Myocardial contraction measured by sonomicrometer crystals is shown. (Left) Baseline conditions before coronary artery occlusion. (Center) Recordings at 2 days after infarction. (Right) Recordings at 28 days after infarction. (A) Data from an infarcted segment from a nontreated heart. (B and C) Data from infarcted segments from GF-treated hearts. All panels show data from infarcted segments with holosystolic bulging. In A, loss of function and paradoxical motion at 2 days (Bottom Center) is still present at 28 days (Bottom Right). Conversely, in B and C, the loss of function and paradoxical motion at 2 days (Bottom Center in each) is followed by significant recovery of contraction at 28 days (Bottom Right in each). LVP, left ventricular pressure; SL, segment length.

Fig. 4.

Myocardial regeneration after infarction in GF-treated hearts. Newly formed myocytes are clustered together (α-sarcomeric actin, red, arrowheads). Areas of scarring are visible in A (collagen I-III, blue). (C) Bright fluorescence in nuclei corresponds to BrdUrd labeling of accumulated newly formed myocytes. PI, propidium iodide (green). (Scale bar, 100 μm.)

Fig. 5.

Formation of coronary vessels after infarction in GF-treated hearts. (A) Higher magnification of regenerated myocytes (cardiac myosin heavy chain, red) and coronary arterioles in which SMCs are positive for α-smooth muscle actin (yellow) and ECs are positive for von Willebrand factor (green). (B and C) Arterioles (B) and capillaries (C) contain red blood cells (green). BrdUrd appears as white fluorescence dots in nuclei. (Scale bars, 10 μm.)

Fig. 6.

GFs improve cardiac performance after infarction. Shown are linear correlations between the magnitude of myocardial regeneration and indices of regional and global ventricular function.