Costamere Structure, Membrane Stability and Integrin Trafficking in the Normal and Diseased Heart

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 DESCRIPTION (provided by applicant): Increased fragility of cardiac myocyte (CM) membranes during pathological processes such as pressure overload or ischemia/reperfusion, can lead to CM dysfunction, membrane rupture and ultimately CM death. While this phenomenon has been studied extensively, the mechanisms underlying it remain incompletely defined. Preservation of CM membrane integrity requires strong and stable connections of CMs with the surrounding extracellular matrix (ECM). CM attachment to the ECM is mediated by integrin complexes, which are in part localized at the unique site within CMs termed costameres. Integrins bind directly to ECM proteins but require adaptor proteins to link with the actin cytoskeleton and sarcomeres within the cell. A critical adapter that is also crucial for functional activation of integrins in most cells, is Talin, the fous of this proposal. Preliminary data show that Talin is essential for the structural support of costameres and thus for CM membrane stability. It is hypothesized that talin plays a role in CMs as an integrin-actin linker, and also regulates integrin protein trafficking. As such it contribute to maintenance of costamere structure and preserves the integrity of cells. Further, when talin protein is cleaved by calpain proteases during insults such as myocardial infarction, cellular fragility, cellular rupture and even cell death, can occur. This in turn can lead to elaboration of ECM production and deleterious remodeling responses which includes fibrosis. Much of this remodeling is propagated by cardiac fibroblasts (CF), where talin is also highly expressed and where it may play an important role in cell growth. This proposal will pursue a series of studies to assess the function of talin in the heart. First the mechanism(s) that lead to heart failure in he talin deficient heart will be evaluated with a focus on how integrin trafficking is involved in thi process. Study of the mechanisms that lead to increased integrin endocytosis and degradation will be specifically evaluated. Understanding this process is important since it can affect cell adhesion of CMs to the ECM within the heart. None of these details has been studied previously in CMs. Studies will also define how cell tension that is altered in talin deficient myocytes, can lead to costameric disruption. Preliminary studies using atomic force microscopy show that talin deletion from CMs reduces membrane tension. Studies will test how decreased tension in talin deficient CMs increases integrin turnover at the costameres, potentially producing weaker cell-ECM connections; leading to cellular and whole heart dysfunction. Cleavage of proteins by calpain proteases has been linked to CM dysfunction. Talin protein is cleaved by calpain. This in part lead to the hypothesis that calpain-mediated cleavage of talin can cause sarcolemmal rupture and cell death, and that abolition of this process can protect the heart from damage. To test this, a calpain-resistant talin transgene will be used in the context of in vivo ischemia-reperfusion, and ex vivo hypoxia/re-oxygenation models. Calpain activation and talin cleavage usually occurs in these models. Studies will be performed using wild-type mice prone to talin cleavage, and transgenic ones expressing the talin variant which is resistant to calpain cleavage. These unique models, and cells derived from them, will allow direct testing if prevention of talin cleavage will protect CMs and the whole heart from stress-related damage. Finally, studies will be pursued detailing how talin expression in CFs influences proliferation, growth, elaboration of ECM and ultimately, the fibrotic response of the heart. This is important since cardiac fibrosis occurs as part of multiple cardiac pathologies, and can lead to deleterious cardiac function and arrhythmias, even if myocardial function is preserved. It is suggested that talin may play important roles to modulate myocardial fibrosis. The clinical significance of this project is that heart failure of varied causes is found in a large number of VA patients, necessitating frequent hospitalizations and attention to outpatient care. Identification of root causes of cardiac dysfunction and importantly, studies which could lead to novel therapeutics for heart failure are essential, and will be the focus of this proposal.