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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL062881-23
Application #
8891471
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Adhikari, Bishow B
Project Start
1995-08-01
Project End
2017-07-31
Budget Start
2015-08-01
Budget End
2017-07-31
Support Year
23
Fiscal Year
2015
Total Cost
Indirect Cost
Name
University of Arizona
Department
Physiology
Type
Schools of Medicine
DUNS #
806345617
City
Tucson
State
AZ
Country
United States
Zip Code
85721
Methawasin, Mei; Granzier, Henk (2018) Softening the Stressed Giant Titin in Diabetes Mellitus. Circ Res 123:315-317
Tonino, Paola; Kiss, Balazs; Strom, Josh et al. (2017) The giant protein titin regulates the length of the striated muscle thick filament. Nat Commun 8:1041
Methawasin, Mei; Granzier, Henk (2017) Response by Methawasin and Granzier to Letter Regarding Article, ""Experimentally Increasing the Compliance of Titin Through RNA Binding Motif-20 (RBM20) Inhibition Improves Diastolic Function in a Mouse Model of Heart Failure With Preserved Ejection Frac Circulation 135:e681-e682
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Bull, Mathew; Methawasin, Mei; Strom, Joshua et al. (2016) Alternative Splicing of Titin Restores Diastolic Function in an HFpEF-Like Genetic Murine Model (Ttn?IAjxn). Circ Res 119:764-72
Methawasin, Mei; Strom, Joshua G; Slater, Rebecca E et al. (2016) Experimentally Increasing the Compliance of Titin Through RNA Binding Motif-20 (RBM20) Inhibition Improves Diastolic Function In a Mouse Model of Heart Failure With Preserved Ejection Fraction. Circulation 134:1085-1099
Pappas, Christopher T; Mayfield, Rachel M; Henderson, Christine et al. (2015) Knockout of Lmod2 results in shorter thin filaments followed by dilated cardiomyopathy and juvenile lethality. Proc Natl Acad Sci U S A 112:13573-8
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

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