Myocardial infarction (MI) is a clinical manifestation of ischemic heart disease. MI results in significant loss of viable cardiomyocytes that affects the healthy functioning of the myocardium. If left untreated, MI can lead to congestive heart failure. The current methods of treatment for end-stage heart failure, like heart transplant or mechanical left-ventricular assist devices do not aim at regenerating the lost cardiomyocytes and further still are accompanied by co-morbidity and limited effectiveness.
Cellular cardiomyoplasty (cell therapy) is currently being investigated as a potential long-term therapeutic option to treat MI. We have investigated skeletal myoblasts (SMs) and mesenchymal stem cells (MSCs) to treat MI. At first, we developed and validated a novel method based on electron paramagnetic resonance (EPR) oximetry, to determine oxygen concentration in the infarct tissue before and after stem-cell therapy. A thorough in vitro characterization of both SMs and MSCs was done using an oxygen-sensing paramagnetic probe (OxySpin). In vivo experiments using SMs and MSCs were done on mouse and rat models, respectively. The oxygen concentration (PO2) at the ischemic site was ~0.2% in the heart but it significantly increased following cell therapy.
Despite the improvement in oxygenation and reduction in scar tissue formation by SM cells, their inability to express gap-junction proteins results in the lack of electrical synchronization of the differentiated SM cells with the existing myocardium. We considered MSCs because of their ability to differentiate into cells of different lineages including cells of the cardiovascular lineage. On induction of MSCs to differentiate into cardiomyoctes in vitro, we observed the ability of MSCs to express the gap-junction protein connexin-43. This ensures that on successful differentiation and engraftment of MSCs they are able to integrate with the surrounding cardiomyocytes providing long-term stability.
To enhance the effects of therapy, in vitro preconditioning strategies were experimented on MSCs. One of the primary impediments that cell therapy has to overcome is that of excessive cell loss immediately after transplantion because of the severe hypoxic environment in the MI heart. The effect is further aggravated by the presence of reactive oxygen species and inflammatory factors. In order to equip the transplanted cell to survive in this hostile environment, we preconditioned MSCs in a sub-lethal hypoxic environment. The optimal time period for hypoxic preconditioning (HPC) and cell-seeding density were investigated. Also passages that responded positively to HPC were identified. We determined that culturing cells at 0.5% O2 for 24 h, MSCs of passage 3 to 5 showed an increase in the expression of pro-survival and pro-angiogenic factors. A high cell-seeding density was observed to upregulate the pro-survival proteins at a lower passage. We also studied pharmacological preconditioning of MSCs with a trimetazidine derivative drug having anti-oxidant properties. Although the drug induced a reduction in apoptosis, it failed to induce pro-survival and angiogenic factors.
Overall the research done for this dissertation focused on the monitoring of changes in oxygenation in infracted hearts before and after stem-cell therapy, and exploring preconditioning strategies for the enhancement of cell survival in the hostile ischemic environment