Date of Award

Fall 8-22-2025

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Physiology

First Advisor

David Barefield

Abstract

Cardiomyopathies, such as hypertrophic and dilated subtypes, are leading causes of heart failure and are often driven by genetic mutations that disrupt the structure and function of the cardiac sarcomere. While much of the research has focused on ventricular involvement, the role of atrial sarcomere dysfunction remains unexplored. The regulation of contractile function by sarcomeric proteins in the atria remains poorly understood, especially the impact of protein stoichiometry among myosin binding proteins. Cardiac myosin binding protein-C (cMyBP-C) and its atrial paralog, myosin binding protein H-like (MyBP-HL), have been shown to have a stoichiometric relationship within the atrial sarcomere. These two proteins co-localize within the C-zone of the thick filament and compete for the same binding sites. Unlike in the ventricle, where cMyBP-C is dominant, the atria exhibit near-equal expression of cMyBP-C and MyBP-HL. Given that cMyBP-C is a well-established regulator of contractility through its modulation of myosin activity, any disruption in the balance between cMyBP-C and MyBP-HL could have significant consequences for atrial biophysics. Thus, understanding how shifts in this stoichiometry due to mutations, deletions, or overexpression impact atrial myofilament function is critical to uncovering mechanisms of atrial dysfunction in cardiomyopathy. To investigate this, we used human missense variants in MYBPC3 and MYBPHL and screened their effects on sarcomere localization via immunofluorescence and immunoblotting. Certain missense variants in both MyBP-C and MyBP-HL impaired sarcomere localization. To study if these missense variants could alter myosin binding protein stoichiometry, we designed and validated a novel T2A/P2A bicistronic system to express our Mini-C and MyBP-HL constructs in a consistent and reproducible manner. Expression of these missense variants within the T2A/P2A construct caused no shifts in expression of the myosin binding proteins. To determine the effects of myosin binding protein stoichiometric changes on myosin relaxation states within the atria, Mant-ATP assays were performed on wild-type and MYBPHL-null atria, with and without alkaline phosphatase to control for cMyBP-C phosphorylation. Phosphatase treatment increased myosin heads in the super-relaxed state in wild-type but not MYBPHL-null atria. Together, these results reveal a novel role of MyBP-HL in regulating sarcomere composition and function, underscoring its importance in atrial contractile function.

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