Titin-based adaptations of cardiac function Titin's I-band region functions as a molecular spring that together with the extracellular matrix (ECM) regulates the passive myocardial stiffness that is critically important for diastolic filling. Prevous work has shown that extensive differential splicing during postnatal development converts the giant fetal cardiac titin isoform into smaller and stiffer adult titin isoforms. The mechanical significance of postnatal isoform switching and the mechanical importance of changes in the ECM require in-depth study.
Aim 1 focuses on this topic in both the left ventricle (LV) and right ventricle (RV). For this work we made a mouse model in which a splice factor (RBM20) that is responsible for splicing stiff adult titins has been deleted and that expresses in adult myocardium titins similar in size to the giant fetal cardiac isoforms. This model mimics dilated cardiomyopathy (DCM) patients with RBM20 mutations in which fetal cardiac sized titins are expressed in the adult. Differential splicing and posttranslational modifications of titin will be studied, as well as changes in the ECM, and passive myocardial stiffness will be studied using multi-disciplinary experiments at a wide range of levels. Upregulating compliant titin isoforms might also affect cardiac hypertrophy in both concentric and eccentric remodeling (Aim 2). Recent work indicated that a stretch-sensing signalosome binds to titin that triggers hypertrophy in response to an increase in strain. This important concept requires critical testing and in-depth study. The expression of compliant titins in our new KO mouse model predict that hypertrophy is attenuated (because titin's strain is reduced) and that this contributes to the DCM phenotype.
Aim 2 will test this by studying hypertrophy in response to increased afterload (transverse aortic constriction (TAC) model) or increased preload (aortocaval fistula (ACF) surgery model), two contrasting models that result in the distinct concentric or eccentric hypertrophy phenotypes, respectively. Titin isoform switching is also likely to affect systole as titin- based passive tenson has been proposed to play an important role in the Frank-Starling mechanism (FSM) of the heart, i.e., the increase in contractility with length. Although the FSM is critical for adjusting stroke volume to match venous return, the mechanism(s) underlying the FSM have remained elusive.
Aim 3 will test the role that titin plays using the RBM20 mouse model that expresses giant titin isoforms in the adult heart and that provides an exciting opportunity to study the effet of a reduction in titin-based passive tension on the FSM under physiological conditions. Experiments will be performed at the cell, tissue, and whole heart levels. A novel mouse model was made for this application, novel reagents were developed, a strong team is in place, and results from pilot studies support the proposal's guiding hypotheses. The proposed studies will provide insights in the importance of titin and the ECM in diastolic filling, systolic performance, and hypertrophy signaling, topics of great interest from a basic science viewpoint and that have high clinical significance for pediatric heart disease, heart failure in adults, and hypertrophic ad dilated cardiomyopathy.

Public Health Relevance

An important focus of this proposal is on the role of titin, the largest protein known, in diastolic filling during neonatal development, and the proposed studies will examine isoform switching and post-translational modification of titin, relative to the change in ECM, in both the left and right ventricles of the heart. The role of titin in the heart's pump function and in hypertrophy of the muscular wall of the heart's pumping chambers will also be studied. This work will contribute to the understanding of mechanisms giving rise to diastolic dysfunction and reduced pump function in heart failure patients, and it will contribute to understanding of the disease mechanisms of heart failure patients with mutations in RBM20.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Myocardial Ischemia and Metabolism Study Section (MIM)
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Adhikari, Bishow B
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University of Arizona
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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
Bull, Mathew; Methawasin, Mei; Strom, Joshua et al. (2016) Alternative Splicing of Titin Restores Diastolic Function in an HFpEF-Like Genetic Murine Model (TtnΔIAjxn). Circ Res 119:764-72
Perkin, John; Slater, Rebecca; Del Favero, Giorgia et al. (2015) Phosphorylating Titin's Cardiac N2B Element by ERK2 or CaMKIIδ Lowers the Single Molecule and Cardiac Muscle Force. Biophys J 109:2592-601
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
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