Heart failure with preserved ejection fraction (HFpEF) is a major health care problem for which there are no known treatments that improve long-term outcomes. Most patients have a history of hypertension (HTN) and concentric left ventricular (LV) remodeling, a combination we term hypertensive heart disease (HHD). The vast majority of patients also have LV diastolic dysfunction (DD) resulting in increased chamber stiffness. Worsening DD parallels progression from concentric remodeling to symptomatic HFpEF. This multi-PI application is designed to elucidate the changes at the myofilament level that contribute to DD using myocardium obtained by intra-operative biopsy from controls and patients with HHD who are either non-failing (HHD-NF) or failing (HFpEF). We focus on titin (Granzier) and actomyosin dynamics (LeWinter), two major determinants of diastolic stiffness in HFpEF. Titin is a giant elastic myofilament that together with the extracellular matrix (ECM) determines passive myocardial stiffness. Recent studies reveal alterations in titin in HFpEF patients that might contribute to DD.
Aims 1 and 2 focus on measuring titin-based stiffness in skinned myocardial strips. (An additional outcome will be the first evaluation of ECM-based stiffness in HFpEF.) To address titin-based mechanisms we focus on isoform expression, PKA/PKG phosphorylation of titin's N2B element that decreases passive stiffness and the newly discovered PKCa phosphorylation of the PEVK element that increases passive stiffness. Parallel experiments will be carried out on wild-type (WT) mice and mice with genetically altered titin compliances, without and with experimental HFpEF. Actomyosin cross-bridge dynamics will be studied using sinusoidal length perturbation in skinned myocardial strips and measurement of force relaxation kinetics in single myofibrils. Preliminary data reveal prolonged cross-bridge attachment time (ton) at submaximal [Ca2+] and reduced phosphorylation of cardiac troponin I (cTnI) and myosin binding protein C (cMyBPC) in HHD patients. Ton is a key determinant of relaxation rate. The guiding hypothesis is that hypo- phosphorylation of PKA sites on cTnI and/or cMyBP-C causes prolonged ton and contributes to DD in HHD and is most severe in HFpEF patients. We will determine if prolonged ton is associated with slowed myofibrillar relaxation kinetics, severity of concentric remodeling, worsening DD and progression to HFpEF. Mechanistic studies include replacing native cTnI with phospho-mimetic cTnI mutants. Transgenic mice with cTnI and cMyBPC PKA phosphorylation site substitutions with and without experimentally induced HFpEF will be studied to determine if phosphorylation state alters ton in the way our hypothesis predicts and whether substitutions simulating complete phosphorylation rescue the ton phenotype. The proposed work is integrative, combining studies in human tissue with genetic mouse models. These approaches were identified by recent NHLBI working groups as priorities for HF prevention. The long-term goal of this work is to provide novel therapeutic targets through a mechanistic understanding of the myofilament basis of HFpEF.

Public Health Relevance

Heart failure with preserved ejection fraction (HFpEF) is a clinical problem of major importance. For the >50% of HF patients with HFpEF, no treatments exist that influence long-term outcomes. Little is known about the underlying basis of this form of heart failure. The research proposed in this application is the first systematic study to define how myofilaments contribute to increased passive stiffness and slowed relaxation in HFpEF patients. This research will fill a major knowledge gap in our understanding of HFpEF.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
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Adhikari, Bishow B
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University of Arizona
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Methawasin, Mei; Granzier, Henk (2018) Softening the Stressed Giant Titin in Diabetes Mellitus. Circ Res 123:315-317
Meyer, Markus; Rambod, Mehdi; LeWinter, Martin (2018) Pharmacological heart rate lowering in patients with a preserved ejection fraction-review of a failing concept. Heart Fail Rev 23:499-506
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
LeWinter, Martin M (2017) Pericardiectomy to Treat Heart Failure With Preserved Ejection Fraction: Unrestrained Enthusiasm? Circ Heart Fail 10:e003971
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LeWinter, Martin M; Zile, Michael R (2016) Could Modification of Titin Contribute to an Answer for Heart Failure With Preserved Ejection Fraction? Circulation 134:1100-1104
Pulcastro, Hannah C; Awinda, Peter O; Methawasin, Mei et al. (2016) Increased Titin Compliance Reduced Length-Dependent Contraction and Slowed Cross-Bridge Kinetics in Skinned Myocardial Strips from Rbm (20?RRM) Mice. Front Physiol 7:322
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

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