Our long-term objective is to understand the roles of titin in cardiac function and disease. Titin is the third myofilament of striated muscle and contains an extensible region, located in the I-band of the sarcomere, which functions as a molecular spring that, when extended, develops force. This force underlies a large fraction of the force developed by myocardium during the filling phase of the heart. Human myocardium expresses both stiff and compliant titin isoforms. Altered isoform expression profiles have been found in patients with dilated cardiomyopathy (DCM) and coronary artery disease. These are accompanied by altered passive stiffness. Furthermore, recent studies have discovered several titin mutations that lead to DCM. We propose to study the molecular basis of titin's extensibility, including the effects of posttranslational modifications on extensibility. We will use recombinant proteins representing titin's various spring elements and study their mechanical and structural properties, using atomic force microscopy and spectroscopic techniques. The effects of calcium and phosphorylation on mechanics and structure will be examined. Because titin isoform expression may regulate calcium sensitivity and beta-adrenergic control of titin-based elasticity, we will develop a titin exon microarray to characterize exon-splicing patterns. We will also develop and characterize several mouse knockout models in which spring elements have been excised in order to study the following hypothesized functions: 1) glutamate rich PEVK elements are calcium binding sites that can alter titin-based passive force in a calcium-dependent manner; 2) the cardiac unique N2B sequence can alter titin-based passive force via phosphorylation. This research will provide important and novel insights into the molecular basis of titin's elasticity and its adjustments via calcium and phosphorylation. Considering the multitude of 'titinopathies' that are rapidly being discovered, our findings will be important for understanding human cardiac function and disease.
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