Functional diversity among a family of human skeletal muscle myosin motors

Abstract

Human skeletal muscle fibers express five highly conserved type-II myosin heavy chain (MyHC) genes in distinct spatial and temporal patterns. In addition, the human genome contains an intact sixth gene, MyHC-IIb, which is thought under most circumstances not to be expressed. The physiological and biochemical properties of individual muscle fibers correlate with the predominantly expressed MyHC isoform, but a functional analysis of homogenous skeletal muscle myosin isoforms has not been possible. This is due to the difficulties of separating the multiple isoforms usually coexpressed in muscle fibers, as well as the lack of an expression system that produces active recombinant type II skeletal muscle myosin. In this study we describe a mammalian muscle cell expression system and the functional analysis of all six recombinant human type II skeletal muscle myosin isoforms. The diverse biochemical activities and actin-filament velocities of these myosins indicate that they likely have distinct functions in muscle. Our data also show that ATPase activity and motility are generally correlated for human skeletal muscle myosins. The exception, MyHC-IIb, encodes a protein that is high in ATPase activity but slow in motility; this is the first functional analysis of the protein from this gene. In addition, the developmental isoforms, hypothesized to have low ATPase activity, were indistinguishable from adult-fast MyHC-IIa and the specialized MyHC-Extraocular isoform, that was predicted to be the fastest of all six isoforms but was functionally similar to the slower isoforms.

Fig. 1.

Expression and purification of the human skeletal myosin II isoforms. (A) Met1-Pro840 of the myosin was expressed under the control of the CMV promoter, fused to the gene encoding an enhanced GFP (eGFP) with a C-terminal 6xHistidine tag. This construct included a linker (Ala-Ala-Ala) between S1 and eGFP. (B) C₂C₁₂ cells expressing the recombinant myosins (MyHC-IId-eGFP-6xHis in this image) display bright eGFP fluorescence 72 h postinfection. (C) Coomassie Brilliant Blue stained SDS-PAGE gel with fractions corresponding to the 488 nm chromatographic peak (excitation peak for eGFP) during a linear salt gradient elution for MyHC-IIa-eGFP-6xHis (Lane 1), MyHC-IIb-eGFP-6xHis (Lane 2), MyHC-IId-eGFP-6xHis (Lane 3), and MyHC-Perinatal-eGFP-6xHis (Lane 4).

Fig. 2.

F-actin-activated ATPase saturation curves for the human IIb (•), IId (▪), IIa (⧫), Extraocular (▴), Embryonic (▾), and Perinatal (○) skeletal myosin II isoforms. Averaged data from F-actin-activated ATPase assays with the recombinant S1-eGFP-6xHis proteins are plotted. Each data point is the average ATPase rate from assays performed in duplicate on at least three different preparations of protein (except MyHC-Perinatal where n = 2) and is a reaction rate (s^-1) calculated from the slope of a linear fit (r² > 0.95 in most cases) of product (nMol P_i) versus time (seconds) over the course of the assay, corrected for the basal Mg²⁺ ATPase activity at F-actin. The datasets for each preparation of an isoform across a range of 0– F-actin were fit to the Michaelis-Menten equation using KaleidaGraph.