Titin is a giant elastic protein that is found in the cardiac sarcomere where it performs multiple important functions, including that of a molecular spring that develops passive stiffness during diastole. We have shown that titin-based passive stiffness is adjustable through differential splicing and post-translational modifications of titins spring elements. In this renewal we first focus on the role of these pathways in changing cellular stiffness in heart disease and how a change in titin-based cellular stiffness is manifested at the level of the left ventricular (LV) chamber. Aim 1 will study a novel mouse model in which 9 immunoglobulin-like (Ig-like) domains have been deleted from titin's tandem Ig segment (one of titin's three spring elements), the so-called IG KO. By performing integrative experiments, from molecular to in vivo levels, we will use this novel model to study how changes in cellular stiffness translate to changes in diastolic LV chamber stiffness. Aim 2 focuses on titin's role in heart failure with normal ejection fraction (HFpEF), a rapidly increasing public health problem of major and increasing scope that is characterized by diastolic stiffening. Using an integrative approach the role of titin in the increased diastolic stiffness in HFpEF will be investigated, including its gender and age dependencies. The experiments focus on differential splicing using a novel titin exon microarray and on posttranslational modifications in titin, including the distint stiffness modulation pathways consisting of PKA/PKG phosphorylation (reduced stiffness) and PKC phosphorylation (increased stiffness), using novel phospho-antibodies that we raised. HFpEF is accompanied by cardiac hypertrophy and Aim 3 addresses titin's role as a biomechanical sensor that triggers hypertrophy signaling. Hypertrophy will be investigated in response to increased pressure or increased volume overload, using the new IG KO model as well as our existing titin models that are deficient in either the N2B spring element (N2B KO) or the PEVK spring element (PEVK KO). Pilot studies support the novel hypothesis that titin's N2B element functions as a biomechanical sensor with increased N2B strain triggering hypertrophy and the N2B-binding protein FHL1 functioning as a critical link in the downstream signaling pathway. This hypothesis will be tested by breeding the titin spring element KO models with a FHL1 KO model and studying hypertrophy induced by pressure-overload and volume-overload. The proposal addresses innovative hypotheses and uses innovative animal models and experimental tools, including a novel exon microarray, novel phospho-antibodies and loaded intact cell mechanics. The research is expected to lead to an in-depth understanding of the functions of titin, relative to that of other players, in diastolic dysfunction and mechanosensing and hypertrophy signaling. We anticipate that this work will greatly increase the understanding of the functions of titin in the heart and that it might provide novel therapeutic targets for combating the clinically important HFpEF syndrome and pathological hypertrophy.