Hypertrophic cardiomyopathy (HCM) is a disease involving the thickening of the ventricular walls of the heart. In infants, its presentation can be particularly severe, and it is the leading cause of sudden cardiac death in pediatric populations. Unfortunately, current therapy is limited to symptomatic relief for mild cases and heart transplant for severe cases. To develop targeted therapies, we must first understand how HCM-causing mutations in genes that encode the contractile proteins of the heart affect those proteins and result in HCM. It has been hypothesized that alteration of beta-cardiac myosin's power generation by HCM mutations represents the underlying pathophysiologic trigger that leads to the HCM disease phenotype.I will assess the effects of 5 pediatric-specific HCM mutations on the biomechanical function of human beta-cardiac myosin and compare them to HCM mutations that are not specific to pediatric populations. First I will assess the key parameters involved in myosin power production. I will measure the myosin intrinsic force at a single molecule level using optical tweezers, myosin velocity using the in vitro motility assay and myosin activity using an ATPase assay. Even though these measurements are insightful, they do not provide a rigorous measurement of myosin power output. For measuring power output, we need quantitative force-velocity measurements (power = force x velocity), a fundamental biophysical parameter for muscle contraction. Current loaded motility assays use actin binding proteins as a proxy for load, but do not provide a measure of the force. Moreover, using actin binding proteins leads to sticking and shredding of actin filaments, thereby complicating even the velocity measurements. To address this issue, I propose to develop a novel loaded in vitro motility assay, where myosin coated beads will move along aligned actin filaments. The movement of the beads can be tracked to measure velocity. Application of flow (against the direction of bead movement) will apply a drag force on the bead. I will measure the bead velocity at various applied forces to get the force-velocity relationship of the WT and mutant myosins. This will provide a robust, reproducible assay for quantitating force velocity measurements of purified populations of myosin, and such an assay, in future, may provide a high throughput platform for designing and testing therapeutics that can be used to treat HCM.
|Program type||Postdoctoral Fellowship|
|Effective start/end date||07/01/2016 → 06/30/2018|