Cardiovascular disease can lead to myocardial infarction (Ml) and subsequent heart failure. There are currently a number of therapies aimed at preventing or treating heart failure post-MI. Only heart transplantation replaces infarcted myocardium to restore heart function, but there is a paucity of donor hearts and the incidence of cardiovascular disease continues to rise. A new and innovative option is the use of """"""""heart patches"""""""" (a.k.a. myocardial equivalents) created in vitro for implantation in vivo. The research proposed here aims to address a critical issue pertaining to the function and eventual use of such heart patches - the lower force generation capacity of myocardial equivalents when compared to native tissue. Our hypothesis is that cell-induced contraction of fibrin gel-based myocardial equivalents results not only in tissue alignment but also improved excitation-contraction coupling and greater tissue contraction force than what would be expected merely from aligning contracting cardiomyocytes. We propose that these enhancements are the result of two mechanisms: 1) increased gap junction formation associated with myocyte alignment, and 2) increased myocyte-fibroblast-myocyte conduction pathways.
The specific aims of the proposed research are thus as follows: 1) Evaluate how cell-mediated contraction and alignment of a fibrin gel induces myocyte alignment, which promotes formation of gap junctions and, as a result, increases contraction force, and 2) Determine the role that cardiac fibroblasts play in transducing the excitation signal between myocytes in these tissue constructs. To complete Aim 1 we will create aligned and isotropic constructs by entrapping neonatal rat cardiomyocytes and cardiac fibroblasts (in their native proportions) in fibrin gels in two different geometries: rectangular slab and tubular. We will measure the formation of gap junctions via immunohistochemistry and their function both optically (dye transfer, conduction delay via intracellular Calcium) and in terms of contraction of the constructs (i.e. excitation threshold, contraction force interval). To complete Aim 2 we will create constructs similar to the methods in Aim 1, however, we will alter the proportion of cardiac fibroblasts by pre-plating to remove fibroblasts. We will then pre-label the fibroblasts to distinguish them in optical measurements and create constructs with varying ratios of fibroblasts to myocytes, recording the formation and function of gap junctions between the two cell types. In addition, we will record the contractile properties of the entire construct and correlate them with fibroblast density. We expect that cell-induced contraction, alignment, and remodeling of fibrin gel will result in a more contractile construct and that the cellular mechanism for this enhancement will be elucidated. ? ? ?
