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.

Public Health Relevance

We focus on the role of titin, the largest protein known, in heart failure with preserved ejection fraction (HFpEF), a frequently occurring disease that results in poor filling of the heart. The underlying disease mechanisms are not understood and as a result no effective treatments exist. Recent work suggests that the giant protein titin plays an important role in the disease and we therefore focus on this protein. We use several mouse models in which the titin gene has been altered, allowing us to study how alterations in titin affect the filling phase of the heart, and in addition we use a HFpEF mouse model. We compare results to those of large mammals with HFpEF, including human patients. Because patients with filling problems often have hypertrophy (thickened walls of the heart chambers) we also study whether titin initiates hypertrophy. We expect that this work will greatly improve our fundamental understanding of the filling phase of the heart and the mechanisms that cause hypertrophy and that this will provide insights in new treatment strategies for heart disease patients.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-CVRS-M (02))
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Adhikari, Bishow B
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University of Arizona
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Hutchinson, Kirk R; Saripalli, Chandra; Chung, Charles S et al. (2015) Increased myocardial stiffness due to cardiac titin isoform switching in a mouse model of volume overload limits eccentric remodeling. J Mol Cell Cardiol 79:104-14
Granzier, Henk L; Hutchinson, Kirk R; Tonino, Paola et al. (2014) Deleting titin's I-band/A-band junction reveals critical roles for titin in biomechanical sensing and cardiac function. Proc Natl Acad Sci U S A 111:14589-94
Rain, Silvia; Bos, Denielli da Silva Goncalves; Handoko, M Louis et al. (2014) Protein changes contributing to right ventricular cardiomyocyte diastolic dysfunction in pulmonary arterial hypertension. J Am Heart Assoc 3:e000716
Hidalgo, Carlos; Saripalli, Chandra; Granzier, Henk L (2014) Effect of exercise training on post-translational and post-transcriptional regulation of titin stiffness in striated muscle of wild type and IG KO mice. Arch Biochem Biophys 552-553:100-7
Elhamine, Fatiha; Radke, Michael H; Pfitzer, Gabriele et al. (2014) Deletion of the titin N2B region accelerates myofibrillar force development but does not alter relaxation kinetics. J Cell Sci 127:3666-74
Methawasin, Mei; Hutchinson, Kirk R; Lee, Eun-Jeong et al. (2014) Experimentally increasing titin compliance in a novel mouse model attenuates the Frank-Starling mechanism but has a beneficial effect on diastole. Circulation 129:1924-36
LeWinter, Martin M; Granzier, Henk L (2014) Cardiac titin and heart disease. J Cardiovasc Pharmacol 63:207-12
Hidalgo, Carlos G; Chung, Charles S; Saripalli, Chandra et al. (2013) The multifunctional Ca(2+)/calmodulin-dependent protein kinase II delta (CaMKIIýý) phosphorylates cardiac titin's spring elements. J Mol Cell Cardiol 54:90-7
Hidalgo, Carlos; Granzier, Henk (2013) Tuning the molecular giant titin through phosphorylation: Role in health and disease. Trends Cardiovasc Med 23:165-71
Lee, Eun-Jeong; Nedrud, Joshua; Schemmel, Peter et al. (2013) Calcium sensitivity and myofilament lattice structure in titin N2B KO mice. Arch Biochem Biophys 535:76-83

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