The goals of this grant are twofold: The first is the development of the principal investigator as an independent researcher. The second goal is to determine the role of the myocardial toll-like receptors (TLR) in lipopolysaccharide (LPS, endotoxin) signal transduction in cardiomyocytes in vitro and in vivo. The myocardium is known to be an endotoxin responsive organ yet the membrane protein responsible for transmitting the LPS signal to the cardiomyocyte interior remains unknown. Recently published work implicated the TLR4 as the cell membrane LPS receptor responsible for initiating cell signaling in the macrophage. This proposal will evaluate what role TLRs play in transmitting the LPS binding signal to the cardiomyocyte interior. First, the expression of TLRs in the myocardium will be characterized. We will determine which TLR isoforms, if any, are expressed under basal conditions in mouse myocardium, human myocardium, a cardiomyocyte cell line (HL-1 cells), and mouse macrophages. Once the specific myocardial isoforms expressed are known, the regulation of those TLR isoforms will be investigated. The systemic LPS response is modulated by LPS, repeated LPS administration, lipoteichoic acid (LTA) and burn serum. TLR mRNA and protein levels will be determined by northern and western blots after exposed to the above conditions. We will then determine the importance of LPS signaling via TLRs in the pathogenesis of myocardial dysfunction in septic shock, using in vitro and in vivo systems. The HL-1 cardiomyocyte cell line will be transfected with a non-functional TLR resulting in over-expression of this TLR. Response to LPS will be determined by evaluating the LPS signal transduction pathway via MEK1, ERK2 and NF-kappaB. Finally, a transgenic mouse over-expressing a cardiac specific non-functional TLR will be produced. Cardiac function will be evaluated. Langedorff preparation, histology and animal health/survival following LPS, and LTA administration.