Functional analysis of novel MYH7 mutations with prevalence in early-onset patients

University Of Colorado, Boulder CO

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Abstract

? DESCRIPTION (provided by applicant): Myosin is the motor driving contraction in cardiac and skeletal muscle. For cardiac muscle, the primary isoform is ?-MyHC. Mutations have been identified for several decades in ?-MyHC and other sarcomeric myosins and there has been a lot of interest in studying their functionality and how they might lead diseases such as Familial Hypertrophic Cardiomyopathy or Familial Dilated Cardiomyopathy. Recently, there have been novel mutations identified in ?-MyHC and shown to be prevalent in early-onset patients. The clinical prognosis for these patients is very poor and their only alternative is heart transplant, hence the need for less invasive interventions. Understanding how these mutations lead to disease is crucial for developing treatments, but past studies of the sort are limited because they lack the human context, because almost all of these studies were performed in animals. Expressing human myosin in differentiated muscle cells enables purifying human ?-MyHC to homogeneity and was pioneered by the Leinwand Lab to overcome these obstacles. In this proposal I am hypothesizing that: (1) HCM causing mutations consistently lead to enhanced myosin activity and conversely DCM causing mutations lead to depressed activity; (2) Omecamtiv mecarbil, a small molecule myosin activator currently in clinical trials for treatment of heart failure, will induce stronger kinetic effects on ?-MyHC by increasing ATP-hydrolysis rate, ADP-release rates and actin affinity; (3) HCM-causing mutations increase duty ratio, therefore exhibiting longer cross-bridge kinetics and longer relaxation times, while DCM- causing mutations will show the opposite effect. I will complete these projects by expressing human myosin with known disease causing mutations and perform conventional kinetic assays such as ATPase and Stopped- Flow Kinetics to analyze how the different rates are affected. I will also perform contractility studies in isolated cardiomyocytes that express the mutations here proposed and relate the effect of the mutation to the biochemical rates. These aims should result in a comprehensive understanding of cardiomyopathy causing mutations and their functional and contractile characteristics.