Narine Sarvazyan Laboratory
ONGOING AND RECENTLY COMPLETED PROJECTS
ECTOPIC NEXUS. Our lab is exploring pathophysiological significance of a condition which we named an Ectopic Nexus. It refers to a functional state of an injured cardiac tissue in which multiple poorly-coupled ectopic sources form a transient "breeding" microenvironment in which ectopic activity develops from individual cells into slowly propagating local intercellular waves (LIWs) confined to the area of injury. We have shown that, at least in vitro, LIWs are not limited to a specific set of experimental conditions but instead is a generic phenomenon. We characterized the parameter space in which the generation of LIWs takes place. We described both numerically and experimentally how the LIWs are formed under dynamic conditions. Our current efforts are aimed at establishing LIWs existence on the level of the whole heart.
CARDIOTOXICITY OF DEHP. Di(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer found in a variety of polyvinyl chloride (PVC) medical products. Our laboratory is examining the potential adverse effects of DEHP by studying its effect on confluent, synchronously beating cultures of neonatal rat cardiomyocytes. Our studies have shown that exposure to DEHP leads to a decrease in conduction velocity and asynchronous cardiomyocyte cell beating. DEHP-treated samples have a lower expression level of connexin-43, a gap-junction protein that facilitates intercellular electrical communication between cardiomyocytes. Furthermore, the use of organelle-specific connexin-43 antibodies, IF1 and CT1, allowed us to analyze changes in intracellular distribution of connexin-43. In DEHP-treated samples the amount of gap-junctional connexin-43 (IF1-sensitive) was significantly decreased. In contrast, Golgi and perinuclear (CT1-sensitive) staining was more pronounced in DEHP-treated samples. Our future studies include studying the applicability of these findings to human patients, and investigating the mechanism by which DEHP exerts its effects.
N-CADHERIN-MEDIATED STEM CELL ADHESION. We have recently developed a mouse embryonic stem cell line that constitutively overexpresses N-cadherin, a major adhesion protein found in the intercalated disc. Our studies have shown that overexpression of N-cadherin leads to marked changes in the phenotype and adhesion of these cells, both during embryoid body formation and cell differentiation stages. We hypothesize that overexpressing N-cadherin would allow transplanted ESC to integrate more efficiently to a host myocardium, and that increased adhesion would facilitate gap junction connections between the two types of cells. These modified stem cells show elevated levels of p120, a protein involved in N-cadherin trafficking and stabilization, as well as elevated levels of connexin-43, a gap junction protein that facilitates intercellular communication. Next, we hope to use these cells in transplantation studies in order to assess their arrhythmogenicity and to compare it to unmodified stem cells.
IN VITRO MODEL OF CARDIAC FIBERS.. Using Matrigel, a commercially available basement membrane preparation (BD Biosciences), we developed a simple in vitro model that mimics the three-dimensional environment and mechanical load conditions of cardiac muscle. A semisolid pillow from concentrated Matrigel was overlaid with a suspension of neonatal rat cardiomyocytes in diluted Matrigel. This model provided an environment in which spontaneously formed multicellular fibers continuously contracted against mechanical load. Additionally, the Matrigel-cell mixture could be cut to form larger cardiomyocyte fibers. This approach allowed for functional monitoring of 20-300uM fibers by loading with a Ca2+ sensitive dye, as well as structural monitoring as the model provided easy access to epitopes for immunostaining.
Cor.At CELLS. We are working with Axiogenesis to examine the properties of Cor.At cells, cardiomyocytes that were genetically selected from differentiated mouse embryonic stem cells. These cells are morphologically and functionally similar to atrial myocytes. These cells are being used in our Matrigel model to generate three-dimensional fibers and to assess intercalated disc maturation.