The mammalian heart has a very limited regenerative capacity and, hence, heals by scar formation. Recent reports suggest that haematopoietic stem cells can transdifferentiate into unexpected phenotypes such as skeletal muscle, hepatocytes, epithelial cells, neurons, endothelial cells and cardiomyocytes, in response to tissue injury or placement in a new environment. Furthermore, transplanted human hearts contain myocytes derived from extra-cardiac progenitor cells, which may have originated from bone marrow. Although most studies suggest that transdifferentiation is extremely rare under physiological conditions, extensive regeneration of myocardial infarcts was reported recently after direct stem cell injection, prompting several clinical trials. Here, we used both cardiomyocyte-restricted and ubiquitously expressed reporter transgenes to track the fate of haematopoietic stem cells after 145 transplants into normal and injured adult mouse hearts. No transdifferentiation into cardiomyocytes was detectable when using these genetic techniques to follow cell fate, and stem-cell-engrafted hearts showed no overt increase in cardiomyocytes compared to sham-engrafted hearts. These results indicate that haematopoietic stem cells do not readily acquire a cardiac phenotype, and raise a cautionary note for clinical studies of infarct repair.

Embryonic stem (ES) cells are promising for cardiac repair, but directing their differentiation toward cardiomyocytes remains challenging. We investigated whether the heart guides ES cells toward cardiomyocytes in vivo and whether allogeneic ES cells were immunologically tolerated. Undifferentiated mouse ES cells consistently formed cardiac teratomas in nude or immunocompetent syngeneic mice. Cardiac teratomas contained no more cardiomyocytes than hind-limb teratomas, suggesting lack of guided differentiation. ES cells also formed teratomas in infarcted hearts, indicating injury-related signals did not direct cardiac differentiation. Allogeneic ES cells also caused cardiac teratomas, but these were immunologically rejected after several weeks, in association with increased inflammation and up-regulation of class I and II histocompatibility antigens. Fusion between ES cells and cardiomyocytes occurred in vivo, but was rare. Infarct autofluorescence was identified as an artifact that might be mistaken for enhanced GFP expression and true regeneration. Hence, undifferentiated ES cells were not guided toward a cardiomyocyte fate in either normal or infarcted hearts, and there was no evidence for allogeneic immune tolerance of ES cell derivatives. Successful cardiac repair strategies involving ES cells will need to control cardiac differentiation, avoid introducing undifferentiated cells, and will likely require immune modulation to avoid rejection.

Granulation tissue formation is a critical step in infarct repair, however, the kinetics of cell replication and the molecules that regulate this process are poorly understood. In uninjured mouse hearts and at 2 days post-infarction, very little DNA synthesis (measured by incorporation of a BrdU pulse) was detected in any cell type. Four days after permanent coronary occlusion, the rates of myofibroblast (smooth muscle ␣-actin and BrdU double-positive) and endothelial cell (CD31 and BrdU double-positive) proliferation were 15.4 ؎ 1.1% and 2.9 ؎ 0.5%, respectively. Most proliferating cells were located at the interface of the infarct and viable tissue. By 1 week, fibroblast and endothelial cell proliferation declined to 4.1 ؎ 0.6% and 0.7 ؎ 0.1%, respectively. In the 2-week infarct, the remaining necrosis had been phagocytosed, and fibroblast and endothelial cell proliferation were <0.5%. Although leukocytes were abundant throughout infarct repair, no significant proliferation was detected at any time in cells expressing CD45 or mac-3. Infarct size at 4 days was 38 ؎ 5% of the left ventricle and contracted to 20 ؎ 4% by 4 weeks. After 4 days, the chamber dilated to four times that of the control hearts and remained so for the duration of the time course. The vascular density (per mm 2 ) declined from 3643 ؎ 82 in control hearts to 2716 ؎ 197 at 1 week and 1010 ؎ 47 at 4 weeks post-myocardial infarction (MI). The average percent area occupied by vessels did not change significantly between the groups but the area/vessel (m 2 ) increased from 14.1 ؎ 0.3 in control hearts to 16.9 ؎ 1.9 at 1 week and 38.7 ؎ 7.9 at 4 weeks post-MI. These data indicate that mitogens for fibroblasts and endothelial cells peak within 4 days of infarction in the mouse heart. This provides the basis for identifying the responsible molecules and developing strategies to alter wound repair and improve cardiac function. Following coronary artery occlusion, extensive cardiomyocyte necrosis and remodeling of the ventricular wall occurs. Macrophages phagocytose the necrotic myocardium, and, concomitantly, myofibroblasts and endothelial cells proliferate and migrate into the infarct zone.

Cardiovascular disease is a leading cause of death, and translational research is needed to understand better mechanisms whereby the left ventricle responds to injury. Mouse models of heart disease have provided valuable insights into mechanisms that occur during cardiac aging and in response to a variety of pathologies. The assessment of cardiovascular physiological responses to injury or insult is an important and necessary component of this research. With increasing consideration for rigor and reproducibility, the goal of this guidelines review is to provide best-practice information regarding how to measure accurately cardiac physiology in animal models. In this article, we define guidelines for the measurement of cardiac physiology in mice, as the most commonly used animal model in cardiovascular research.Listen to this article’s corresponding podcast at http://ajpheart.podbean.com/e/guidelines-for-measuring-cardiac-physiology-in-mice/.