Publication Date

2019-12-12

Availability

Open access

Embargo Period

2019-12-12

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2019-12-09

First Committee Member

Danuta Szczesna-Cordary

Second Committee Member

Claudia De Oliveira Rodrigues

Third Committee Member

Peter Buchwald

Fourth Committee Member

Arun Malhotra

Fifth Committee Member

Fangliang Zhang

Sixth Committee Member

Audrey Chang

Abstract

Background: Hypertrophic Cardiomyopathy (HCM) is one of the most prevalent heart diseases characterized by ventricular and septal hypertrophy, myocardial fibrosis and myofibrillar disarray. Genetic forms of HCM are mainly caused by mutations in major sarcomeric proteins such as β-myosin heavy chain (β-MHC), myosin protein binding C (MyBP-C), regulatory and essential light chains (RLC/ELC), troponin, tropomyosin and actin. My Ph.D. proposal focuses on myosin RLC and two RLC mutations: Arginine-to-Glutamine, R58Q, and Aspartic acid-to-Valine, D166V, that cause HCM and strives to provide insight into the importance of human cardiac myosin RLC phosphorylation site at Ser-15 for normal heart function and its potential rescue role in HCM. A plethora of research studies recognize the role of Ser-15 phosphorylation in cardiac muscle contraction, playing a crucial role in the function of the heart under normal and disease conditions, and studies from heart failure (HF) patients and RLC animal models of HCM show severely reduced levels of cardiac myosin phosphorylation that coincide with depressed heart function. Methods: First, I used a phosphomimic recombinant RLC variant where the Ser-15 at the phosphorylation site is substituted with aspartic acid (S15D) and placed in the background of R58Q to assess whether I could rescue/mitigate R58Q-induced structural/functional abnormalities in porcine reconstituted system in vitro. Secondly, I used a highly effective Adeno-Associated Virus, serotype-9 (AAV9), to deliver phosphomimic S15D-RLC construct into the hearts of D166V and R58Q mice models and examine the recovery of heart function by echocardiography, strain analysis and muscle fiber mechanics. Finally, I investigated the underlying molecular mechanisms by assessing the effects of R58Q mutation on super relaxed (SRX) state of myosin cross-bridge population which represents a reserve of slowly cycling myosin heads with very low ATP-hydrolysis rates that is thought to be cardioprotective during times of stress, e.g. cardiomyopathy. Findings: I have shown rescue of several R58Q-exerted adverse phenotypes in RLC-depleted and mutant S15D-R58Q-reconstituted porcine cardiac muscle preparations. A low level of maximal isometric force observed for R58Q- versus WT-reconstituted fibers was restored by S15D-R58Q. Significant beneficial effects were also observed on the Vmax of actin-activated myosin ATPase activity in S15D-R58Q versus R58Q-reconstituted myosin, along with its binding to fluorescently-labeled actin, and in the ability of myosin motors to develop maximal tension. In another set of experiments, a significant improvement of heart function was observed in AAV-treated versus PBS-injected mice, including an increase in cardiac output and stroke work and a decrease in relaxation constant, Tau, shown to be prolonged in HCM-D166V mice. Strain analysis showed enhanced myocardial longitudinal shortening in AAV-treated versus control mice. Increased maximal contractile force was observed in skinned papillary muscles from AAV-injected HCM-D166V hearts. A rescue in fractional shortening and intact maximal force was also observed in AAV-injected R58Q mice. In mechanistic studies, I show that R58Q mutation of RLC promotes the OFF state of myosin, in both reconstituted porcine fibers and transgenic mouse papillary muscles. Rescue experiments performed in S15D-R58Q-reconstituted porcine muscle fibers showed a mild destabilization of the SRX state, suggesting an S15D-mediated shift in disordered-relaxed (DRX)↔SRX equilibrium towards the DRX state of myosin. I also show that AAV-S15D delivery in R58Q lead to disruption of SRX compared to PBS-treated R58Q animals. Interpretations: Our data suggest that myosin RLC phosphomimic may be used as a potential rescue strategy to abrogate/alleviate the RLC mutation-induced adverse cardiac phenotypes and is a likely candidate for therapeutic intervention in vivo.

Keywords

Hypertrophic cardiomyopathy; Super-relaxed state; Regulatory light chain; S15D phosphomimic; AAV gene therapy; Muscle contraction

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