Developing a Protocol to Fractionate and Identify Cardiac Extracellular Matrix Proteins Crucial to Cardiomyocyte Proliferation Using Size-Exclusion Chromatography.
Madungwe, Ngonidzashe B.
- Engineered heart tissue is one of the solutions currently being investigated as an option for regenerating and replacing cardiac tissue post-myocardial infarction (MI). The constructs that are needed for this treatment option vary depending on the source of the cells used, the culture conditions and the scaffold material used. Although a number of polymers, both natural and synthetic are being use... read mored, decellularized extracellular matrix (ECM) is becoming more important in these studies as it is a blueprint of how nature creates scaffolds. Its usefulness has been found to include promoting cardiomyocyte proliferation, among other advantages as a scaffold biomaterial. The important factors utilized by ECM in its function are not well understood. Therefore this study sought to examine whether the molecular weight of ECM peptides was an important factor in cardiomyocyte proliferation. Using size-exclusion chromatography to fractionate digested ECM, a protocol was designed to separate the peptides by their molecular sizes. The fractions were characterized by concentration and molecular weight, before being coated onto tissue culture plastic (TCP) and studied for how they influence proliferation of cardiomyocytes cultured on them. The results showed statistical significance in the proliferation of cardiomyocytes on the fractions as compared to standards of TCP and collagen I. Overall, there was no significant difference in the number of proliferating cells between the fractions; while a larger percentage of proliferating cardiomyocytes tended to be on the fractions than the standards. Based on these findings, it was concluded that despite there not being one specific peptide size responsible for proliferation, the peptides obtained in fractions 5 to 7 (with molecular weight sizes of 1 kDa to 6.2 kDa) showed a little more potential to promote proliferation than the other fractions.read less