The main objective of this work is to investigate the relationship between Purkinje-myocardial conduction and characteristics of membrane action potentials in the endocardial layer. It is postulated that the pattern of connections between the peripheral conduction system and the myocardium electrotonically influences 1) uniformity of propagation, 2) local membrane excitability, 3) local repolarization properties and 4) response to rapid heart rate, in the endocardial layer. The proposed work will explain whether the system of Purkinje-myocardial transmission sites contributes to the initiation of ventricular arrhythmias through electrotonic mechanisms.
Four specific aims will be addressed:
(Aim 1) Develop a combined experimental-computer modeling strategy to study interactions between the peripheral conduction system and the myocardium.
(Aim 2) Examine relationships between Purkinje-myocardial transmission site density and excitation in the endocardial layer of the myocardium.
(Aim 3) Characterize relationships between Purkinje-myocardial transmission site density and the spatial distribution of repolarization properties in the endocardial layer of the myocardium.
(Aim 4) Identify characteristics of Purkinje-myocardial conduction that promote initiation of ventricular arrhythmias in response to premature stimuli. The ultimate goal of the work is to predict the response of the myocardium to endocardial packing as a function of the density of Purkinje-myocardial connections. A firm understanding of how the myocardium responds to endocardial stimuli is clinically important to predict the efficiency of pharmacological interventions and programmed premature stimulation in patients with myocardial ischemia or infarction. The results of the studies described here will have implications for implantable devices used to terminate reentry and ventricular fibrillation. Finally, because the studies will describe the pattern of connections between the peripheral conduction system and the myocardium, they will allow development of future computational and theoretical models to study action potential propagation in the heart. This will prove useful to validate the modeling approach presented in the proposal and to explain the mechanisms that establish ventricular arrhythmias. GANT=R01HL54462 The studies will focus on members of thrombospondin family and are based on the findings that thrombospondin 1 and 2 (TSP1 and TSP2) modulate growth and differentiation of endothelial cells. We will express full length, truncated, or in vitro mutagenized TSP1, TSP2, or TSP3 and also segments of TSP1. Production will be in Spodoptera frugiperda cells with recombinant baculoviruses. The recombinant proteins will be characterized, used to make antibodies, and tested in angiogenesis assays.
Specific aims are to: 1) Learn the mechanism(s) by which TSP1 and TSP2 inhibit growth of bovine aortic endothelial (BAE) cells. We will define both the active site(s) in the TSPs and the receptor(s) on BAE cells. 2) Learn whether TSPs also inhibit growth of endothelial cells cultured from normal or malignant mouse mammary tissue. 3) Extend the analysis of the effects of TSP1 and TSP2 to the entire process of angiogenesis, including in vitro correlates such as cell migration and tube formation, and especially angiogenesis in vivo. Special emphasis will be placed on mammary gland- and mammary tumor-associated angiogenesis and on identification of TSP derivatives that are anti-angiogenic in all assays. 4) Learn how the anti-angiogenic activity of TSP1 and TSP2 may synergize or antagonize other determinants of angiogenesis in mammary tumors. In particular, we will relate the anti-angiogenic activity to other relevant activities of TSPs, especially activation of latent TGFbeta, inhibition of proteases, and modulation of cell adhesion. Longer range goals will be to evaluate the hypothesis that expression of TSP1 and/or TSP2 will correlate inversely with density of microvessels in human breast carcinoma and then to plan experiments to evaluate the hypothesis that the TSP pathway is a suitable target for treatment of breast carcinoma based on inhibition of vascularization.
