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A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors
Published on: August 17, 2019
Mechanisms of a novel regulatory light chain-dependent cardiac myosin inhibitor
Kristina Kooiker1,2,3,4, Qing-Fen Gan5, Ming Yu5
1Division of Cardiology, Medicine, University of Washington, Seattle, WA, USA.
Insights
A novel molecule, RLC-1, was found to improve hypertrophic cardiomyopathy (HCM) by reducing cardiac muscle force and speeding up relaxation. This small molecule targets the underlying contractile changes in the heart, offering a potential new treatment for HCM.
Area of Science:
- Cardiovascular Research
- Molecular Cardiology
- Pharmacology
Background:
- Hypertrophic cardiomyopathy (HCM) is a genetic heart disease characterized by left ventricle thickening, hypercontractility, and impaired relaxation.
- Current HCM treatments do not directly address the sarcomeric contractile dysfunction.
- Mutations in sarcomeric proteins, like beta myosin heavy chain, are primary causes of HCM.
Purpose of the Study:
- To investigate the effects of a novel small molecule, RLC-1, on cardiac muscle contractility.
- To determine if RLC-1 can modulate myosin ATPase activity and improve HCM-related contractile changes.
Main Methods:
- High-throughput screening identified RLC-1 from bovine cardiac myofibrils.
- Experiments utilized demembranated rat left ventricle (LV) trabeculae and isolated rat LV myofibrils.
- X-ray diffraction studies analyzed structural changes induced by RLC-1.
- Intact trabeculae and isolated cardiomyocytes were used to assess RLC-1's effects on twitch mechanics.
Main Results:
- RLC-1 decreased maximal Ca2+-activated force and Ca2+ sensitivity in demembranated rat LV trabeculae.
- RLC-1 significantly reduced maximal and submaximal Ca2+-activated force in isolated rat LV myofibrils.
- RLC-1 accelerated both fast and slow phases of relaxation in demembranated tissues.
- X-ray diffraction revealed RLC-1 moves myosin heads away from the thick filament backbone.
- RLC-1 treatment in intact tissues and cells decreased peak twitch magnitude and enhanced activation/relaxation kinetics.
Conclusions:
- RLC-1 accelerates cardiac muscle kinetics and reduces force production across various experimental models.
- The molecule's unique structural effects on myosin heads differentiate it from other inhibitors.
- RLC-1's ability to decrease cardiac twitch magnitude and improve relaxation kinetics suggests potential therapeutic benefits for HCM.
Abstract:
Hypertrophic cardiomyopathy (HCM) is a genetic disease of the heart characterized by thickening of the left ventricle (LV), hypercontractility, and impaired relaxation. HCM is caused primarily by heritable mutations in sarcomeric proteins, such as β myosin heavy chain. Until recently, medications in clinical use for HCM did not directly target the underlying contractile changes in the sarcomere. Here, we investigate a novel small molecule, RLC-1, identified in a bovine cardiac myofibril high-throughput screen. RLC-1 is highly dependent on the presence of a regulatory light chain to bind to cardiac myosin and modulate its ATPase activity. In demembranated rat LV trabeculae, RLC-1 decreased maximal Ca2+-activated force and Ca2+ sensitivity of force, while it increased the submaximal rate constant for tension redevelopment. In myofibrils isolated from rat LV, both maximal and submaximal Ca2+-activated force are reduced by nearly 50%. Additionally, the fast and slow phases of relaxation were approximately twice as fast as DMSO controls, and the duration of the slow phase was shorter. Structurally, x-ray diffraction studies showed that RLC-1 moved myosin heads away from the thick filament backbone and decreased the order of myosin heads, which is different from other myosin inhibitors. In intact trabeculae and isolated cardiomyocytes, RLC-1 treatment resulted in decreased peak twitch magnitude and faster activation and relaxation kinetics. In conclusion, RLC-1 accelerated kinetics and decreased force production in the demembranated tissue, intact tissue, and intact whole cells, resulting in a smaller cardiac twitch, which could improve the underlying contractile changes associated with HCM.
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