Visualization of cardiac ventricular myosin heavy chain homodimers and heterodimers by monoclonal antibody epitope mapping

Abstract

Two mAbs, one specific for cardiac α-myosin heavy chains (MHC) and the other specific for cardiac β-MHC, were used to investigate the heavy-chain dimeric organization of rat cardiac ventricular myosin. Epitopes of the two mAbs were mapped on the myosin molecule by electron microscopy of rotary shadowed mAb-myosin complexes. mAbs were clearly identifiable by the different locations of their binding sites on the myosin rod. Thus, myosin molecules could be directly discriminated according to their α- or β-MHC content. αα-MHC and ββ-MHC homodimers were visualized in complexes consisting of two molecules of the same mAb bound to one myosin molecule. By simultaneously using the α-MHC-specific mAb and the β-MHC-specific mAb, αβ-MHC heterodimers were visualized in complexes formed by one molecule of each of the two mAbs bound to one myosin molecule. Proportions of αα- and ββ-MHC homodimers and αβ-MHC heterodimers were estimated from quantifications of mAb-myosin complexes and compared with the proportions given by electrophoreses under nondenaturing conditions. This visualization of cardiac myosin molecules clearly demonstrates the arrangement of α- and β-MHC in αα-MHC homodimers, ββ-MHC homodimers, and αβ-MHC heterodimers, as initially proposed by Hoh, J.F.Y., G.P.S. Yeoh, M.A.W. Thomas, and L. Higginbottom (1979).