Abstract
The myosin isoform composition of the heart is dynamic in health and disease and has been shown to affect contractile velocity and force generation. While different mammalian species express different proportions of α and β myosin heavy chain, healthy human heart ventricles express these isoforms in a ratio of about 1:9 (α:β) while failing human ventricles express no detectable α-myosin. We report here fast-kinetic analysis of recombinant human α and β myosin heavy chain motor domains. This represents the first such analysis of any human muscle myosin motor and the first of α-myosin from any species. Our findings reveal substantial isoform differences in individual kinetic parameters, overall contractile character, and predicted cycle times. For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin. Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1. Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar. Sequence analysis points to regions that might underlie the basis for this finding.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-012-0927-3) contains supplementary material, which is available to authorized users.
Highlights
Conventional myosins are the best-known family of motors, consisting of two heavy chains (MyHC) and two pairs of light chains: regulatory light chains (RLC) and essential light chains (ELC)
A lever arm, stabilized by binding of the ELC and RLC, transfers the conformational changes occurring in the motor domain into directional movement along the actin filament [1]
Adenoviruses encoding human a- and b-subfragment 1 (S1) were used to infect C2C12 myotubes and the protein was purified using the His-tag on the C-terminus of the motor. a-S1 and b-S1 co-purify with endogenous mouse ELC and RLC from the C2C12 muscle cells which were previously identified by mass spectrometry of the purified proteins as MLC1F, MLC2F, MLC3F, and MLC1A [13]
Summary
Winkelmann et al [20] pioneered the use of the mammalian skeletal C2C12 muscle cell line to examine the role of chaperones in proper folding of the motor domain of sarcomeric myosins. This approach was first used by Wang et al [21] to express embryonic chicken myosin and to measure the in vitro motility of the isolated myosin. Given the high degree of identity between a and b human myosins, several events in the cross-bridge cycle differ by as much as ten-fold These include the ATP hydrolysis step, which controls the lifetime of the detached myosin and the ADP release from actinÁS1
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