Basic mechanisms of infarct repair

The recent development of a murine model of myocardial infarction by coronary artery ligation is a useful tool to characterize the normal wound healing process. As genetically modified mice become increasingly more available to query the role of molecules, it is important to establish the baseline features of murine infarct repair in terms of the migration and proliferation of myofibroblasts and endothelial cells, ventricular remodeling, and cardiac function.

The methods and results we have to date are as follows:

Mouse surgery:

Briefly, in an anesthetized mouse, we visualize the coronary artery under a microscope and passed an 8-0 monofilament PE suture under it using a 6mm tapered needle. Successful ligation of the coronary artery resulted in blanching of the ischemic vascular bed and visible dyskinesis. The animals are then closed and recover sternal recumbency in a heated chamber. At the time of sacrifice, hearts are placed into a zinc-based fixative for at least 6 hours prior to routine processing and embedding. We routinely perform this procedure, infarcting 30% of the left ventricle with 85-90% survival. We have chosen to analyze the following time points: 4days, 1 week, 2 weeks, and 4 weeks.

Cell Kinetics:

Fibroblasts: Activated fibroblasts transiently express smooth muscle alpha-actin (also found in smooth muscle cells of vascular wall). In conjunction with BrdU labeling, proliferating fibroblasts can be identified and counted. Our results indicate that the peak of fibroblast proliferation in mice is 15% and occurs at 4 days post-MI.

Endothelial cells: The surface marker CD31 labels endothelial cells. As with the fibroblasts, double-labeling with BrdU permits detection of proliferating endothelial cells. Our results show that the peak of endothelial cell proliferation is 3% and also occurs at 4 days post-MI. More information about our staining methods can be viewed in our paper (Journal of Cardiovascular Pathology).

Macrophages: CD45-positive leukocytes are present throughout the time course of our studies (4 weeks) and do not proliferate to any significant extent at any time.

Morphometry:

Using the Scion imaging program, we measure infarct size, necrosis, granulation tissue, and scar and express these as a percentage of the left ventricle.

The detailed results of our characterization of this model can be viewed in our recently published paper in AJPath (American Journal of Pathology)

Cardiac Function:

To measure ventricular pressure 4 weeks post-MI, we advance a 1.4 French Millar catheter into the left ventricle via the carotid artery. From the ventricular pressure tracing, we calculate dP/dt and use this to compare our experimental groups.

We are currently using these methods to investigate the role of bFGF in murine myocardial infarct repair. Our preliminary results indicate that bFGF is required for fibroblast proliferation and so deletion of this gene leads to impaired wound contraction, which, in turn, exacerbates cardiac dysfunction.