The long-term goals of this work are to understand the roles of titin in both diastolic and systolic dysfunction, and to provide important new insights applicable to human cardiac function and disease. Titin is the third myofilament of the sarcomere, with critical roles in myofibrillar assembly and generation of passive and restoring forces. In normal hearts, titin-based forces are the main determinants of passive myocardial stiffness at physiological sarcomere lengths. Our work has shown that mammals may tune passive myocardial stiffness by co-expressing varying mixes of stiff and compliant titin isoforms, obtained via post-transcriptional switching of splice patterns. To understand the role of titin in diastolic dysfunction, we propose to study various heart disease models and test the hypothesis that adjusting the titin isoform expression ratio is a widely used mechanism for passive stiffness modulation in heart disease. In addition, titin's elastic properties may be adjusted via post-translational processes (e.g., PKA-based phosphorylation), mechanisms that are faster than those that require isoform switching. Hence, we will also study how post-translational modification affects stiffness of the different cardiac titin isoforms. Our hypothesis is that as the expression of stiff titins increases, passive stiffness becomes more sensitive to post-translational regulation. Because titin may play a role in the Frank-Starling (FS) mechanism of the heart, by sensing sarcomere stretch, changes in passive tension due to post-transcriptional and post-translational processes may not only modulate passive stiffness, but also the FS mechanism. Thus, titin may also impact systolic function in heart failure and disease. Therefore, we will study the length dependence of calcium sensitivity in myocardium with different isoform expression ratios, to test the hypothesis that changes in titin isoform expression in disease may affect contractile performance. Finally, in addition to acquired alterations in titin expression in heart disease, genetic defects of the titin filament, including a recently reported frame shift mutation that eliminates titin's M-line region with its kinase domain, cause familial dilated cardiomyopathy (DCM). To understand the role of mutant titins in this type of DCM, mouse models will be studied in which gene targeting is used to remove segments of titin's M-line region. We will test the hypothesis that titin's M-line region is critically important for the structural integrity of contracting sarcomere. ? ?
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