In vivo titin cleavage by AAV9-mediated gene transfer in a genetic mouse model to determine the link between titin-based mechanical function and cardiac performance

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The giant sarcomere protein titin encoded by TTN is important for cardiomyocyte structure and function and is pathologically altered in heart disease, including heart failure and cardiomyopathies. Titin contains an elastic segment in the sarcomeric I-band, which is thought to determine most of the viscoelasticity of a cardiomyocyte and be responsible, in part, for myocardial passive stiffness, as well as regulate active contraction. However, the role of the titin springs in healthy and diseased hearts is difficult to assess directly. We have developed a unique genetic mouse model, in which a tobacco etch virus (TEV) protease-recognition site and a HaloTag are cloned into elastic titin. This titin cleavage (TC) model allows the specific and acute severing of the titin springs in situ by application of TEV protease (TEVp), while the HaloTag can be used for titin visualization. Here, I hypothesize that the titin-based spring forces critically determine both the diastolic and the systolic function of the heart, which will be revealed by in vivo titin cleavage using the TC mouse model. To this end, titin will be cleaved in a graded manner in heterozygous and homozygous mutant TC mice via adeno-associated virus (AAV)9 mediated overexpression of TEVp in the cardiomyocytes. Changes to cardiac mechanical properties caused by in vivo titin cleavage will be measured in the living heart by echocardiography and cardiac magnetic resonance imaging and in isolated cardiomyocytes or cardiac fibers by sarcomere length-tension recordings and atomic force microscopy-based nanoindentation. Specific aims include the quantification of: (1) changes to the running activity, heart function of the living animal, and cardiac proteomic composition, upon graded in vivo titin cleavage; (2) associated changes to the mechanical and structural properties of the cardiac fibers and cardiomyocytes, including the stretch-dependent passive tension in both the longitudinal and the transverse direction, the Ca2+-dependent active tension, and the (sub)cellular organization as detected by confocal and electron microscopy; and (3) the extent of titin degradation, aggregation, and turnover in native cardiomyocytes following titin cleavage. Findings expected from this project should firmly establish the link between titin-based cellular functions and in vivo performance of the heart. Crucial insight into the interplay between passive and active tension generation as well as diastolic and systolic heart function can be gained and much learned about sarcomeric protein homeostasis. Results will also aid in our mechanistic understanding of myocardial remodeling in various types of heart disease.