Variable N-terminal regions of muscle myosin heavy chain modulate ATPase rate and actin sliding velocity

We integratively assessed the function of alternative versions of a region near the N terminus of Drosophila muscle myosin heavy chain (encoded by exon 3a or 3b). We exchanged the alternative exon 3 regions between an embryonic isoform and the indirect flight muscle isoform. Each chimeric myosin was expressed in Drosophila indirect flight muscle, in the absence of other myosin isoforms, allowing for purified protein analysis and whole organism locomotory studies. The flight muscle isoform generates higher in vitro actin sliding velocity and solution ATPase rates than the embryonic isoform. Exchanging the embryonic exon 3 region into the flight muscle isoform decreased ATPase rates to embryonic levels but did not affect actin sliding velocity or flight muscle ultrastructure. Interestingly, this swap only slightly impaired flight ability. Exchanging the flight muscle-specific exon 3 region into the embryonic isoform increased actin sliding velocity 3-fold and improved indirect flight muscle ultrastructure integrity but failed to rescue the flightless phenotype of flies expressing embryonic myosin. These results suggest that the two structural versions of the exon 3 domain independently influence the kinetics of at least two steps of the actomyosin cross-bridge cycle.     

Alternative N-terminal regions of Drosophila myosin heavy chain tune muscle kinetics for optimal power output

We assessed the influence of alternative versions of a region near the N-terminus of Drosophila myosin heavy chain on muscle mechanical properties. Previously, we exchanged N-terminal regions (encoded by alternative exon 3s) between an embryonic (EMB) isoform and the indirect flight muscle isoform (IFI) of myosin, and demonstrated that it influences solution ATPase rates and in vitro actin sliding velocity. Because each myosin is expressed in Drosophila indirect flight muscle, in the absence of other myosin isoforms, this allows for muscle mechanical and whole organism locomotion assays. We found that exchanging the flight muscle specific exon 3 region into the embryonic isoform (EMB-3b) increased maximum power generation (P(max)) and optimal frequency of power generation (f(max)) threefold and twofold compared to fibers expressing EMB, whereas exchanging the embryonic exon 3 region into the flight muscle isoform (IFI-3a) decreased P(max) and f(max) to approximately 80% of IFI fiber values. Drosophila expressing IFI-3a exhibited a reduced wing beat frequency compared to flies expressing IFI, which optimized power generation from their kinetically slowed flight muscle. However, the slower wing beat frequency resulted in a substantial loss of aerodynamic power as manifest in decreased flight performance of IFI-3a compared to IFI. Thus the N-terminal region is important in tuning myosin kinetics to match muscle speed for optimal locomotory performance.     

The myosin converter domain modulates muscle performance

Myosin is the molecular motor that powers muscle contraction as a result of conformational changes during its mechanochemical cycle. We demonstrate that the converter, a compact structural domain that differs in sequence between Drosophila melanogaster myosin isoforms, dramatically influences the kinetic properties of myosin and muscle fibres. Transgenic replacement of the converter in the fast indirect flight muscle with the converter from an embryonic muscle slowed muscle kinetics, forcing a compensatory reduction in wing beat frequency to sustain flight. Conversely, replacing the embryonic converter with the flight muscle converter sped up muscle kinetics and increased maximum power twofold, compared to flight muscles expressing the embryonic myosin isoform. The substitutions also dramatically influenced in vitro actin sliding velocity, suggesting that the converter modulates a rate-limiting step preceding cross-bridge detachment. Our integrative analysis demonstrates that isoform-specific differences in the myosin converter allow different muscle types to meet their specific locomotion demands.     

Alternative versions of the myosin relay domain differentially respond to load to influence Drosophila muscle kinetics

We measured the influence of alternative versions of the Drosophila melanogaster myosin heavy chain relay domain on muscle mechanical properties. We exchanged relay domain regions (encoded by alternative versions of exon 9) between an embryonic (EMB) isoform and the indirect flight muscle isoform (IFI) of myosin. Previously, we observed no effect of exchanging the EMB relay domain region into the flight muscle isoform (IFI-9b) on in vitro actin motility velocity or solution ATPase measurements compared to IFI. However, in indirect flight muscle fibers, IFI-9b exhibited decreased maximum power generation (P_max) and optimal frequency of power generation (f_max) to 70% and 83% of IFI fiber values. The decrease in muscle performance reduced the flight ability and wing-beat frequency of IFI-9b Drosophila compared to IFI Drosophila. Previously, we found that exchanging the flight muscle specific relay domain into the EMB isoform (EMB-9a) prevented actin movement in the in vitro motility assay compared to EMB, which does support actin movement. However, in indirect flight muscle fibers EMB-9a was a highly effective motor, increasing P_max and f_max 2.5-fold and 1.4-fold, respectively, compared to fibers expressing EMB. We propose that the oscillatory load EMB-9a experiences in the muscle fiber reduces a high activation energy barrier between two strongly bound states of the cross-bridge cycle, thereby promoting cross-bridge cycling. The IFI relay domain's enhanced sensitivity to load increases cross-bridge kinetics, whereas the EMB version is less load-sensitive.     

Kinetic analysis of Drosophila muscle myosin isoforms suggests a novel mode of mechanochemical coupling

The molecular mechanism of myosin function was addressed by measuring transient kinetic parameters of naturally occurring and chimeric Drosophila muscle myosin isoforms. We assessed the native embryonic isoform, the native indirect flight muscle isoform, and two chimeric isoforms containing converter domains exchanged between the indirect flight muscle and embryonic isoforms. Myosin was purified from the indirect flight muscles of transgenic flies, and S1 was produced by alpha-chymotryptic digestion. Previous studies in vertebrate and scallop myosins have shown a correlation between actin filament velocity in motility assays and cross-bridge detachment rate, specifically the rate of ADP release. In contrast, our study showed no correlation between the detachment rate and actin filament velocity in Drosophila myosin isoforms and further that the converter domain does not significantly influence the biochemical kinetics governing the detachment of myosin from actin. We suggest that evolutionary pressure on a single muscle myosin gene may maintain a fast detachment rate in all isoforms. As a result, the attachment rate and completion of the power stroke or the equilibrium between actin.myosin.ADP states may define actin filament velocity for these myosin isoforms.     

Alternative relay domains of Drosophila melanogaster myosin differentially affect ATPase activity, in vitro motility, myofibril structure and muscle function

The relay domain of myosin is hypothesized to function as a communication pathway between the nucleotide-binding site, actin-binding site and the converter domain. In Drosophila melanogaster, a single myosin heavy chain gene encodes three alternative relay domains. Exon 9a encodes the indirect flight muscle isoform (IFI) relay domain, whereas exon 9b encodes one of the embryonic body wall isoform (EMB) relay domains. To gain a better understanding of the function of the relay domain and the differences imparted by the IFI and the EMB versions, we constructed two transgenic Drosophila lines expressing chimeric myosin heavy chains in indirect flight muscles lacking endogenous myosin. One expresses the IFI relay domain in the EMB backbone (EMB-9a), while the second expresses the EMB relay domain in the IFI backbone (IFI-9b). Our studies reveal that the EMB relay domain is functionally equivalent to the IFI relay domain when it is substituted into IFI. Essentially no differences in ATPase activity, actin-sliding velocity, flight ability at room temperature or muscle structure are observed in IFI-9b compared to native IFI. However, when the EMB relay domain is replaced with the IFI relay domain, we find a 50% reduction in actin-activated ATPase activity, a significant increase in actin affinity, abolition of actin sliding, defects in myofibril assembly and rapid degeneration of muscle structure compared to EMB. We hypothesize that altered relay domain conformational changes in EMB-9a impair intramolecular communication with the EMB-specific converter domain. This decreases transition rates involving strongly bound actomyosin states, leading to a reduced ATPase rate and loss of actin motility.     

An alternative domain near the ATP binding pocket of Drosophila myosin affects muscle fiber kinetics

We examined the importance of alternative versions of a region near the ATP binding site of Drosophila myosin heavy chain for muscle mechanical properties. Previously, we exchanged two versions of this region (encoded by alternative exon 7s) between the indirect flight muscle myosin isoform (IFI) and an embryonic myosin isoform (EMB) and found, surprisingly, that in vitro solution actin-activated ATPase rates were increased (higher Vmax) by both exon exchanges. Here we examined the effect of increased ATPase rate on indirect flight muscle (IFM) fiber mechanics and Drosophila locomotion. IFM expressing EMB with the exon 7a domain replaced by the IFM specific exon 7d domain (EMB-7d) exhibited 3.2-fold greater maximum oscillatory power (Pmax) and 1.5-fold greater optimal frequency of power generation (fmax) versus fibers expressing EMB. In contrast, IFM expressing IFI with the exon 7d region replaced by the EMB exon 7a region (IFI-7a), showed no change in Pmax, fmax, step response, or isometric muscle properties compared to native IFI fibers. A slight decrement in IFI-7a flight ability was observed, suggesting a negative influence of the increased ATPase rate on Drosophila locomotion, perhaps due to energy supply constraints. Our results show that exon 7 plays a substantial role in establishing fiber speed and flight performance, and that the limiting step that sets ATPase rate in Drosophila myosin has little to no direct influence in setting fmax for fast muscle fiber types.     

Actomyosin kinetics and in vitro motility of wild-type Drosophila actin and the effects of two mutations in the Act88F gene.

Two missense mutations of the flight muscle-specific actin gene of Drosophila melanogaster, Act88F, assemble into normally structured myofibrils but affect the flight ability of flies and the mechanical kinetics of isolated muscle fibers. We describe the isolation of actin from different homozygous Act88F strains, including wild-type, an Act88F null mutant (KM88), and two Act88F single point mutations (E316K and G368E), their biochemical interactions with rabbit myosin subfragment 1 (S1), and behavior with rabbit myosin and heavy meromyosin in in vitro motility assays. The rabbit and wild-type Drosophila actins have different association rate constants with S1 (2.64 and 1.77 microM-1 s-1, respectively) and in vitro motilities (2.51, 1.60 microns s-1) clearly demonstrating an isoform-specific difference. The G368E mutation shows a reduced affinity for rabbit S1 compared with the wild type (increasing from 0.11 to 0.17 microM) and a reduced velocity in vitro (reduced by 19%). The E316K mutant actin has no change in affinity for myosin S1 or in vitro motility with heavy meromyosin but does have a reduced in vitro motility (15%) with myosin. These results are discussed with respect to the recently published atomic models for the actomyosin structure and our findings that G368E fibers show a reduced rate constant for delayed tension development and increased fiber stiffness. We interpret these results as possibly caused either by effects on A1 myosin light chain binding or conformational changes within the subdomain 1 of actin, which contains the myosin binding site. E316K is discussed with respect to its likely position within the tropomyosin binding site of actin.     

Five Alternative Myosin Converter Domains Influence Muscle Power,Stretch Activation,and Kinetics

Muscles have evolved to power a wide variety of movements. A protein component critical to varying power generation is the myosin isoform present in the muscle. However, how functional variation in muscle arises from myosin structure is not well understood. We studied the influence of the converter, a myosin structural region at the junction of the lever arm and catalytic domain, using Drosophila because its single myosin heavy chain gene expresses five alternative converter versions (11a–e). We created five transgenic fly lines, each forced to express one of the converter versions in their indirect flight muscle (IFM) fibers. Electron microscopy showed that the converter exchanges did not alter muscle ultrastructure. The four lines expressing converter versions (11b–e) other than the native IFM 11a converter displayed decreased flight ability. IFM fibers expressing converters normally found in the adult stage muscles generated up to 2.8-fold more power and displayed up to 2.2-fold faster muscle kinetics than fibers with converters found in the embryonic and larval stage muscles. Small changes to stretch-activated force generation only played a minor role in altering power output of IFM. Muscle apparent rate constants, derived from sinusoidal analysis of the chimeric converter fibers, showed a strong positive correlation between optimal muscle oscillation frequency and myosin attachment kinetics to actin, and an inverse correlation with detachment related cross-bridge kinetics. This suggests the myosin converter alters at least two rate constants of the cross-bridge cycle with changes to attachment and power stroke related kinetics having the most influence on setting muscle oscillatory power kinetics.     

Inhibition of the relative movement of actin and myosin by caldesmon and calponin.

Contractile activity of myosin II in smooth muscle and non-muscle cells requires phosphorylation of myosin by myosin light chain kinase. In addition, these cells have the potential for regulation at the thin filament level by caldesmon and calponin, both of which bind calmodulin. We have investigated this regulation using in vitro motility assays. Caldesmon completely inhibited the movement of actin filaments by either phosphorylated smooth muscle myosin or rabbit skeletal muscle heavy meromyosin. The amount of caldesmon required for inhibition was decreased when tropomyosin is present. Similarly, calponin binding to actin resulted in inhibition of actin filament movement by both smooth muscle myosin and skeletal muscle heavy meromyosin. Tropomyosin had no effect on the amount of calponin needed for inhibition. High concentrations of calmodulin (10 microM) in the presence of calcium completely reversed the inhibition. The nature of the inhibition by the two proteins was markedly different. Increasing caldesmon concentrations resulted in graded inhibition of the movement of actin filaments until complete inhibition of movement was obtained. Calponin inhibited actin sliding in a more "all or none" fashion. As the calponin concentration was increased the number of actin filaments moving was markedly decreased, but the velocity of movement remained near control values.     

Myosin heavy chain isoforms regulate muscle function but not myofibril assembly.

Myosin heavy chain (MHC) is the motor protein of muscle thick filaments. Most organisms produce many muscle MHC isoforms with temporally and spatially regulated expression patterns. This suggests that isoforms of MHC have different characteristics necessary for defining specific muscle properties. The single Drosophila muscle Mhc gene yields various isoforms as a result of alternative RNA splicing. To determine whether this multiplicity of MHC isoforms is critical to myofibril assembly and function, we introduced a gene encoding only an embryonic MHC into Drosophila melanogaster. The embryonic transgene acts in a dominant antimorphic manner to disrupt flight muscle function. The transgene was genetically crossed into an MHC null background. Unexpectedly, transformed flies expressing only the embryonic isoform are viable. Adult muscles containing embryonic MHC assemble normally, indicating that the isoform of MHC does not determine the dramatic ultrastructural variation among different muscle types. However, transformed flies are flightless and show reduced jumping and mating ability. Their indirect flight muscle myofibrils progressively deteriorate. Our data show that the proper MHC isoform is critical for specialized muscle function and myofibril stability.     

Expression of the inclusion body myopathy 3 mutation in Drosophila depresses myosin function and stability and recapitulates muscle inclusions and weakness

Hereditary myosin myopathies are characterized by variable clinical features. Inclusion body myopathy 3 (IBM-3) is an autosomal dominant disease associated with a missense mutation (E706K) in the myosin heavy chain IIa gene. Adult patients experience progressive muscle weakness. Biopsies reveal dystrophic changes, rimmed vacuoles with cytoplasmic inclusions, and focal disorganization of myofilaments. We constructed a transgene encoding E706K myosin and expressed it in Drosophila (E701K) indirect flight and jump muscles to establish a novel homozygous organism with homogeneous populations of fast IBM-3 myosin and muscle fibers. Flight and jump abilities were severely reduced in homozygotes. ATPase and actin sliding velocity of the mutant myosin were depressed >80% compared with wild-type myosin. Light scattering experiments and electron microscopy revealed that mutant myosin heads bear a dramatic propensity to collapse and aggregate. Thus E706K (E701K) myosin appears far more labile than wild-type myosin. Furthermore, mutant fly fibers exhibit ultrastructural hallmarks seen in patients, including cytoplasmic inclusions containing aberrant proteinaceous structures and disorganized muscle filaments. Our Drosophila model reveals the unambiguous consequences of the IBM-3 lesion on fast muscle myosin and fibers. The abnormalities observed in myosin function and muscle ultrastructure likely contribute to muscle weakness observed in our flies and patients.     

Alternative S2 hinge regions of the myosin rod differentially affect muscle function, myofibril dimensions and myosin tail length

Muscle myosin heavy chain (MHC) rod domains intertwine to form alpha-helical coiled-coil dimers; these subsequently multimerize into thick filaments via electrostatic interactions. The subfragment 2/light meromyosin "hinge" region of the MHC rod, located in the C-terminal third of heavy meromyosin, may form a less stable coiled-coil than flanking regions. Partial "melting" of this region has been proposed to result in a helix to random-coil transition. A portion of the Drosophila melanogaster MHC hinge is encoded by mutually exclusive alternative exons 15a and 15b, the use of which correlates with fast (hinge A) or slow (hinge B) muscle physiological properties. To test the functional significance of alternative hinge regions, we constructed transgenic fly lines in which fast muscle isovariant hinge A was switched for slow muscle hinge B in the MHC isoforms of indirect flight and jump muscles. Substitution of the slow muscle hinge B impaired flight ability, increased sarcomere lengths by approximately 13% and resulted in minor disruption to indirect flight muscle sarcomeric structure compared with a transgenic control. With age, residual flight ability decreased rapidly and myofibrils developed peripheral defects. Computational analysis indicates that hinge B has a greater coiled-coil propensity and thus reduced flexibility compared to hinge A. Intriguingly, the MHC rod with hinge B was approximately 5 nm longer than myosin with hinge A, consistent with the more rigid coiled-coil conformation predicted for hinge B. Our study demonstrates that hinge B cannot functionally substitute for hinge A in fast muscle types, likely as a result of differences in the molecular structure of the rod, subtle changes in myofibril structure and decreased ability to maintain sarcomere structure in indirect flight muscle myofibrils. Thus, alternative hinges are important in dictating the distinct functional properties of myosin isoforms and the muscles in which they are expressed.     

In vitro movement of actin filaments on gizzard smooth muscle myosin: requirement of phosphorylation of myosin light chain and effects of tropomyosin and caldesmon

ATP-dependent movement of actin filaments on smooth muscle myosin was investigated by using the in vitro motility assay method in which myosin was fixed on the surface of a coverslip in a phosphorylated or an unphosphorylated state. Actin filaments slid on gizzard myosin phosphorylated with myosin light chain kinase (MLCK) at a rate of 0.35 micron/s, but did not slide at all on unphosphorylated myosin. The movement of actin filaments on phosphorylated myosin was stopped by perfusion of phosphatase. Subsequent perfusion with a solution containing MLCK, calmodulin, and Ca2+ enabled actin filaments to move again. The sliding velocities on monophosphorylated and diphosphorylated myosin by MLCK were not different. Actin filaments did not move on myosin phosphorylated with protein kinase C (PKC). The sliding velocity on myosin phosphorylated with both MLCK and PKC was identical to that on myosin phosphorylated only with MLCK. Gizzard tropomyosin enhanced the sliding velocity to 0.76 micron/s. Gizzard caldesmon decreased the sliding velocity with increase in its concentration. At a 5-fold molar ratio of caldesmon to actin, the movement stopped completely. This inhibitory effect of caldesmon was relieved upon addition of excess calmodulin and Ca2+.     

A myosin phosphatase targeting subunit isoform transition defines a smooth muscle developmental phenotypic switch

Smooth muscle myosin phosphatasedephosphorylates the regulatory myosin light chain and thus mediatessmooth muscle relaxation. The activity of this myosin phosphatase isdependent upon its myosin-targeting subunit (MYPT1). Isoforms of MYPT1have been identified, but how they are generated and their relationship to smooth muscle phenotypes is not clear. Cloning of the middle sectionof chicken and rat MYPT1 genes revealed that each gene gave rise toisoforms by cassette-type alternative splicing of exons. In chicken, a123-nucleotide exon was included or excluded from the mature mRNA,whereas in rat two exons immediately downstream were alternative. MYPT1isoforms lacking the alternative exon were only detected in maturechicken smooth muscle tissues that display phasic contractileproperties, but the isoform ratios were variable. The patterns ofexpression of rat MYPT1 mRNA isoforms were more complex, with threemajor and two minor isoforms present in all smooth muscle tissues atvarying stoichiometries. Isoform switching was identified in thedeveloping chicken gizzard, in which the exon-skipped isoform replacedthe exon-included isoform around the time of hatching. This isoformswitch occurred after transitions in myosin heavy chain and myosinlight chain (MLC₁₇) isoforms and correlated with aseveralfold increase in the rate of relaxation. The developmentalswitch of MYPT1 isoforms is a good model for determining the mechanismsand significance of alternative splicing in smooth muscle.