Abstract
ABSTRACTIndividuals with inclusion body myopathy type 3 (IBM3) display congenital joint contractures with early-onset muscle weakness that becomes more severe in adulthood. The disease arises from an autosomal dominant point mutation causing an E706K substitution in myosin heavy chain type IIa. We have previously expressed the corresponding myosin mutation (E701K) in homozygous Drosophila indirect flight muscles and recapitulated the myofibrillar degeneration and inclusion bodies observed in the human disease. We have also found that purified E701K myosin has dramatically reduced actin-sliding velocity and ATPase levels. Since IBM3 is a dominant condition, we now examine the disease state in heterozygote Drosophila in order to gain a mechanistic understanding of E701K pathogenicity. Myosin ATPase activities in heterozygotes suggest that approximately equimolar levels of myosin accumulate from each allele. In vitro actin sliding velocity rates for myosin isolated from the heterozygotes were lower than the control, but higher than for the pure mutant isoform. Although sarcomeric ultrastructure was nearly wild type in young adults, mechanical analysis of skinned indirect flight muscle fibers revealed a 59% decrease in maximum oscillatory power generation and an approximately 20% reduction in the frequency at which maximum power was produced. Rate constant analyses suggest a decrease in the rate of myosin attachment to actin, with myosin spending decreased time in the strongly bound state. These mechanical alterations result in a one-third decrease in wing beat frequency and marginal flight ability. With aging, muscle ultrastructure and function progressively declined. Aged myofibrils showed Z-line streaming, consistent with the human heterozygote phenotype. Based upon the mechanical studies, we hypothesize that the mutation decreases the probability of the power stroke occurring and/or alters the degree of movement of the myosin lever arm, resulting in decreased in vitro motility, reduced muscle power output and focal myofibrillar disorganization similar to that seen in individuals with IBM3.
Highlights
Inclusion body myopathy type 3 (IBM3) is a rare autosomal dominant disease caused by a mutation in the MYH2 gene that results in an E706K substitution in fast muscle myosin heavy chain IIa (Darin et al, 1998; Martinsson et al, 2000)
Heterozygotes were generated by crossing E701K-3 or E701K-5 homozygous flies that exclusively express IBM3 (E701K) mutant myosin heavy chain in their indirect flight muscles (IFMs) and jump muscles to yw ‘wildtype’ organisms
ATPase activity assays indicate that E701K and wild-type myosin accumulate at equimolar levels in heterozygotes We previously reported that the IBM3 mutation induced poor basal and actin-activated ATPase activity, as well as a significant reduction in catalytic efficiency in the fast IFM myosin isoform (Wang et al, 2012)
Summary
Inclusion body myopathy type 3 (IBM3) is a rare autosomal dominant disease caused by a mutation in the MYH2 gene that results in an E706K substitution in fast muscle myosin heavy chain IIa (Darin et al, 1998; Martinsson et al, 2000). The charge change in IBM3 myosin is within the evolutionarily conserved SH1-SH2 alpha helix of the motor domain (Martinsson et al, 2000; Wang et al, 2012). This short, kinked and highly flexible alpha helix contains the SH1 (Cys-707) and SH2 (Cys-697) cysteines, which are ∼1.9 nm apart, at opposite ends of the helix (Bobkova et al, 1999; Rayment et al, 1993). Our previous molecular modeling suggested that this state might be stabilized by the E706K mutation, resulting in a reduced ability to proceed through the mechanochemical cycle (Wang et al, 2012)
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