The organization and composition of the extracellular matrix of the heart has a dramatic influence on its function, including diastolic stiffness. The extracellular matrix is synthesized and maintained by cardiac fibroblasts; at the single cell level, individual fibers are moved by these cells to create tension in the matrix. However, very little is known about the mechanisms by which the matrix fibers are manipulated by cells. Understanding these mechanisms is an important first step toward designing strategies for effective tissue engineering of cardiac tissue in vitro. To enable us to study the interactions between single fibers and individual cardiac fibroblasts, we have developed a novel technique for measuring the cellular forces generated on single collagen fibers. In preliminary studies, we have found that the lamellipodium can move a single fiber over many cell lengths and develop an isometric tension of 180-250 pN on a single fiber. We propose to extend these preliminary studies to characterize a number of important phenomena, including the long-term effects of tension on collagen-cell contacts; the recovery of force upon relaxation; and the influence of cyclic applied loads of 10-50% of the isometric tension value. Preliminary studies show that the tension is dependent upon myosin II and we will extend those studies to examine the effects of myosin light-chain kinase inhibitors or other modifications of cell motility. The roles of specific integrin-matrix interactions are not well known and we will use specific integrin blocking antibodies to determine which integrins are most involved in force generation or contact assembly. To further explore the roles of specific integrins, collagen fibers will be coated with different matrix molecules. Cell-cell interactions may play an important role in fiber motility and we will compare the forces generated on single cells versus cell pairs or larger aggregates. Important questions that we plan to answer are: 1) Do oscillations in force increase the average force that a cell generates on a fiber (is there an optimum frequency or magnitude of oscillation)? 2) Are contacts less dynamic after application of static or oscillatory forces? 3) Can two cells in contact generate a much greater tension than a single cell? 4) Do cardiac fibroblasts isolated from volume overloaded tissue generate greater matrix forces? From the answers to these and other questions we expect to better understand the process of extracellular matrix remodeling in the heart.
