Thursday, November 21, 2024
11/21/2024
Abstract. Cardiac fibrosis and inflammation are hallmarks of alcoholic cardiomyopathy. A mediator of cardiac fibrosis, Larp6 interacts with the 5’ side loop (5’SL) of collagen I mRNA as an adapter protein which functions to upregulate collagen expression. In our studies, we target the interaction of Larp6 and collagen I mRNA using 1) genetic manipulation of Larp6 expression in cardiac fibroblasts, 2) a genetically modified mouse model, and 3) a novel small molecular inhibitor. End points include cardiac functional measures and inflammatory and fibrotic biomarkers in the heart and in isolated cardiac cells. First, we reduce Larp6 in cardiac fibroblasts (using shRNA delivered by a lentiviral vector) and use mutated cells and novel mice lacking functional 5’SL collagen I mRNA (5’SL mice). These interventions prevent Larp6 binding to collagen mRNA. In vivo exposure to alcohol follows the NIAAA chronic+binge model, and we compare the 5’SL mice to wildtype controls via serial echocardiography every two weeks until terminal, invasive left heart catheterization. Following extraction of the heart, we stain for collagen, marking fibrotic changes, while comparing left ventricular size to tibial length, as an index of cardiac hypertrophy. In vitro exposure to alcohol is up to 48 hours at various doses corresponding to blood alcohol concentration typical of clinical alcohol abuse. Using qPCR, Western blotting, and immunohistochemistry, we assess the following series of biomarkers in cardiac fibroblasts isolated from our mice: TRAF3IP2, IL1b, IL6, NFkB, and TNFa (to mark inflammation) and collagens I and III protein and mRNA, a-SMA, TGFβ, LOX and its activity, and hydroxyproline (to mark fibrosis). In addition to the genetic manipulation, we administer the novel small molecular inhibitor C9 - C9 inhibits the interaction of Larp6 and the 5’SL units of collagen I mRNA. The same cardiac functional measures (echocardiography and invasive catherization generating PV curves) are used for wildtype mice and endpoint assays of inflammatory and fibrotic markers are performed on cardiac fibroblasts. These data will establish the role of Larp6 in alcoholic cardiomyopathy, characterizing the cardiac fibroblast response to ethanol, and illustrating how Larp6 affects inflammatory, fibrotic, functional, and structural cardiac developments in alcoholic cardiomyopathy. Ultimately, these studies may identify novel, therapeutic targets for the treatment of alcoholic cardiomyopathy.
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