IN VITRO PURIFICATION OF CARDIOMYOPATHY-ASSOCIATED MUTATIONS IN HUMAN ? -MYOSIN HEAVY CHAIN

Abstract

Hypertrophic cardiomyopathy (HCM) and Left Ventricular Non-compaction (LVNC) are two distinct forms of genetic cardiomyopathies that can arise from point mutations within the cardiac sarcomere. HCM is typically characterized by thick ventricular walls and stiffening of the ventricles, while LVNC is characterized by irregular trabeculation and sponge-like appearance of the ventricular wall; both lead to impaired heart function. Interestingly, similar point mutations in the enzymatic thick filament protein ?-Myosin Heavy Chain (?-MyHC), have been implicated in both forms of cardiomyopathies. One such example is the HCM-associated I457T and LVNC-associated I467T, both of which are found in close proximity to the ATP-binding pocket of the motor domain. We hypothesize the replacement of a hydrophobic isoleucine with a non-polar threonine in a known hydrophobic ATP-binding pocket induces a structural alteration that subsequently affects ATPase activity of the motor domain. To date, I457T and I467T mutants were made following site-directed mutagenesis via recombinant PCR of the MYH7 gene. Mutants were cloned into a plasmid containing a viral genome for amplification of cDNA in vitro. Subsequent infection of C2C12 myoblasts with virus containing MYH7 gene allowed for chaperone-mediated production of functional myosin protein. Column chromatography was used to purify the myosin protein.