RESEARCH ARTICLE

A Drosophila model of dominant inclusion body myopathy type 3shows diminished myosin kinetics that reduce muscle power andyield myofibrillar defectsJennifer A Suggs1 Girish C Melkani1 Bernadette M Glasheen2 Mia M Detor2 Anju Melkani1Nathan P Marsan2 Douglas M Swank2 and Sanford I Bernstein1

ABSTRACTIndividuals with inclusion body myopathy type 3 (IBM3) displaycongenital joint contractures with early-onset muscle weakness thatbecomes more severe in adulthood The disease arises from anautosomal dominant point mutation causing an E706K substitution inmyosin heavy chain type IIa We have previously expressed thecorresponding myosin mutation (E701K) in homozygous Drosophilaindirect flight muscles and recapitulated the myofibrillar degenerationand inclusion bodies observed in the human disease We have alsofound that purified E701K myosin has dramatically reduced actin-sliding velocity andATPase levels Since IBM3 is a dominant conditionwe now examine the disease state in heterozygoteDrosophila in orderto gain a mechanistic understanding of E701K pathogenicity MyosinATPase activities in heterozygotes suggest that approximatelyequimolar levels of myosin accumulate from each allele In vitro actinsliding velocity rates for myosin isolated from the heterozygotes werelower than the control but higher than for the pure mutant isoformAlthough sarcomeric ultrastructure was nearly wild type in youngadults mechanical analysis of skinned indirect flight muscle fibersrevealed a 59 decrease in maximum oscillatory power generationand an approximately 20 reduction in the frequency at whichmaximum power was produced Rate constant analyses suggest adecrease in the rate of myosin attachment to actin with myosinspending decreased time in the strongly bound state Thesemechanical alterations result in a one-third decrease in wing beatfrequency and marginal flight ability With aging muscle ultrastructureand function progressively declined Aged myofibrils showed Z-linestreaming consistent with the human heterozygote phenotype Basedupon the mechanical studies we hypothesize that the mutationdecreases the probability of the power stroke occurring andor altersthe degreeofmovement of themyosin leverarm resulting indecreasedin vitro motility reduced muscle power output and focal myofibrillardisorganization similar to that seen in individuals with IBM3

KEYWORDS Inclusion body myopathy type 3 Myosin heavy chainDrosophila melanogaster Myofibril Muscle mechanics

INTRODUCTIONInclusion body myopathy type 3 (IBM3) is a rare autosomaldominant disease caused by a mutation in the MYH2 gene thatresults in an E706K substitution in fast muscle myosin heavy chainIIa (Darin et al 1998 Martinsson et al 2000) In general jointcontractures are observed at birth resolving to mild myopathyduring childhood and progressing to muscle weakness duringmiddle age Muscle biopsies reveal progressive histopathologicalabnormalities including focal myofilament disruption Z-linestreaming and rimmed vacuoles with 15-20 nm diameter inclusionbodies (Tajsharghi et al 2002)

The charge change in IBM3 myosin is within the evolutionarilyconserved SH1-SH2 alpha helix of the motor domain (Martinssonet al 2000 Wang et al 2012) This short kinked and highlyflexible alpha helix contains the SH1 (Cys-707) and SH2 (Cys-697)cysteines which are sim19 nm apart at opposite ends of the helix(Bobkova et al 1999 Rayment et al 1993) The SH1-SH2 helixplays a key role in myosin conformational changes during forcegeneration (Huston et al 1988 Preller et al 2011) Crosslinkingthe cysteine residues reduces myosin ATPase rates and weakensactin affinity of the myosin molecules (Thompson et al 2008)Mutations in the SH1-SH2 helix region can depress themechanochemical activity of the myosin motor (Hu et al 2002Kad et al 2007 Preller et al 2011 Zeng et al 2004)Crystallographic analysis indicates that the SH1-SH2 helix meltsfollowing the power stroke to yield an internally uncoupled state(Himmel et al 2002) Our previous molecular modeling suggestedthat this state might be stabilized by the E706K mutation resultingin a reduced ability to proceed through the mechanochemical cycle(Wang et al 2012)

Investigation into the mechanism of E706K myosinpathogenicity has been hampered by a paucity of biopsied muscleand is further complicated by the presence of other myosin isoformsin mutant skeletal muscle fibers To clarify the means by which theIBM3 mutation causes muscle dysfunction we developed an IBM3disease model in Drosophila melanogaster (Wang et al 2012)Drosophila is an advantageous system for the study of myosinmyopathies (Swank et al 2000) because a single gene coupledwith alternative RNA splicing yields all muscle myosin heavy chainisoforms (Bernstein et al 1983 Rozek and Davidson 1983)Introduction of myosin transgenes and subsequent geneticmanipulation permits expression of mutant myosin heavy chainsin the absence of wild-type isoforms in either all or in subgroups offly muscles

Exclusive expression of the corresponding E701K IBM3 myosinmutation in Drosophila fast-twitch indirect flight muscles (IFMs)enabled us to define defects associated with the genetic lesionHomozygous expression recapitulated hallmarks of the diseaseReceived 29 September 2016 Accepted 27 February 2017

1Department of Biology andMolecular Biology Institute San Diego State UniversitySan Diego CA 92182-4614 USA 2Department of Biological Sciences and Centerfor Biotechnology and Interdisciplinary Studies Rensselaer Polytechnic InstituteTroy NY 12180-3590 USA

Authors for correspondence (swankdrpiedu sbernsteinmailsdsuedu)

SIB 0000-0001-7094-5390

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (httpcreativecommonsorglicensesby30) which permits unrestricted usedistribution and reproduction in any medium provided that the original work is properly attributed

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muscle fibers contained myofibrils that progressively degeneratedand rimmed vacuoles containing inclusions (Wang et al 2012)Biochemical analysis of purified E701K myosin revealed an 80reduction in actin-activated ATPase activity and in vitro actinfilament sliding velocity (Wang et al 2012) Furthermore motordomains of isolated full-length mutant myosin molecules werethermally unstable suggesting that aggregated myosin may beimplicated in inclusion body formation (Wang et al 2012)Here we take advantage of the Drosophila system to explore the

genetically dominant basis of IBM3 myosin pathogenicity inhumans and to gain insights into the disease mechanism throughbiochemical ultrastructural and fiber mechanical studies OurATPase assays on myosin isolated from E701K+ IFMs suggest theaccumulation of equimolar levels of wild-type and mutant myosinsDespite relatively normal ultrastructure flight ability is dramaticallyimpaired in young E701K+ organisms Mechanical analysis ofskinned heterozygous IFM fibers reveals severely decreased musclepower generation Evaluation of our data indicates that slowedE701K+ muscle fiber kinetics result from a reduced overall rate ofcross-bridge cycling that arises from slower steps associated withactin binding and the power stroke We interpret our results in lightof the observed effects on active muscle stiffness and indicate howthese changes may be implicated in the morphological andfunctional deterioration of muscles in individuals with IBM3

RESULTSHeterozygotes were generated by crossing E701K-3 or E701K-5homozygous flies that exclusively express IBM3 (E701K) mutantmyosin heavy chain in their IFMs and jump muscles to yw lsquowild-typersquo organisms The transgenes are expressed in theMhc10 geneticbackground which lacks muscle myosin in these muscle types(Collier et al 1990) Homozygotes were crossed to yw flies suchthat the progeny had one endogenous wild-typeMhc allele and onetransgene expressing E701K myosin heavy chain Similarly wecrossed females from a control line (Swank et al 2000) expressing atransgenic source of wild-type myosin in the Mhc10 background(w PwMhc2 Mhc10) with yw male flies to generate controlheterozygotes (w PwMhc2y Mhc10+ hereafter referred to asPwMhc2+) The heterozygous mutant fly lines lack a statisticallysignificant relative difference in levels of myosin expression[104plusmn57 (plusmnsem) for E701K-3+ 929plusmn90 for E701K-5+]compared with age-matched PwMhc2+ control heterozygotes(100plusmn58) and served as the basis of the current study(Studentrsquos t-test Pgt05)

ATPase activity assays indicate that E701K and wild-typemyosin accumulate at equimolar levels in heterozygotesWe previously reported that the IBM3 mutation induced poor basaland actin-activated ATPase activity as well as a significantreduction in catalytic efficiency in the fast IFM myosin isoform(Wang et al 2012) As myosin isolated from E701K+ IFMsconsists of a population of E701K mutant and wild-type moleculeswe predicted that biochemical properties would be the average forthose of wild-type IFM myosin and pure E701K myosinAlternatively it is possible that the instability of the mutantprotein in the heterozygote could lead to activity levels nearer towild type To examine this we compared the ATPase parameters ofmyosin isolated from E701K+ IFMs and from PwMhc2+ controlIFMs (plusmnsd) (Fig 1) with values defined for myosin from E701Khomozygotes (Wang et al 2012) CaATPase activity (Fig 1A) ofpure E701Kmyosin was roughly 11 of thewild-type control (084plusmn036 versus 786plusmn138 sminus1 Plt0001) whereas E701K+ myosin

activity increased to 60 of the control (474plusmn051 sminus1 Plt005 andPlt0001 compared with both wild type and homozygotesrespectively) Basal MgATPase activity (Fig 1B) of pure E701Kmyosin was sim13 of the control (003plusmn001 versus 023plusmn004 sminus1Plt0001) whereas E701K+ myosin increased the basal activity tonearly 70 of the control value (016plusmn003 sminus1 Plt005 and Plt0001compared with both wild type and homozygotes respectively)Actin-activated Vmax of myosin (Fig 1C) from the E701Khomozygotes was sim14 of the control (023plusmn006 versus 168plusmn026 sminus1 Plt0001) whereas E701K+ myosin was sim57 of thecontrol (096plusmn013 sminus1 Plt0001 compared with both wild type andhomozygotes) Km values (Fig 1D) the actin levels required for halfmaximal ATPase stimulation were not significantly changed in pureE701K myosin compared with control (039plusmn011 versus029plusmn005 μM) with the E701K+ myosin yielding a value that didnot differ significantly from either wild type or homozygotes(032plusmn008 μM) Catalytic efficiency (VmaxKm ratio Fig 1E) wasreduced in pure E701Kmyosin tosim11 of control (061plusmn016 versus588plusmn153 sminus1 microMminus1 Plt0001) In E701K+ myosin it improved tonearly 49 of the control value (278plusmn052 sminus1 microMminus1 Plt005compared with wild type Plt0001 compared with homozygote)Overall myosin isolated from E701K+ heterozygotes yieldedenzymatic activity values that are roughly the calculated mean ofthe wild-type control and the E701K homozygote levels (CaATPase474 versus 435 sminus1 mean basal MgATPase 016 versus 013 sminus1

mean Vmax 096 versus 096 sminus1 mean catalytic efficiency 278 vs317 sminus1 microMminus1 mean) This demonstrates that IBM3 myosin andwild-type myosin accumulate at approximately equimolar levels inIFMs from E701K+ flies

In vitro motility of actin filaments is reduced for myosin fromE701K+ heterozygotesWe used in vitro actin sliding assays to determine the unloadedvelocity of fluorescently labeled actin filaments generated byE701K E701K+ and PwMhc2 transgenic control myosin (plusmnsd)(Fig 1F) We have previously shown that E701K myosintranslocates fluorescently labeled actin filaments at 20 of thevelocity of wild-type fast muscle myosin (134plusmn005 versus 667plusmn018 microm sminus1 Plt0001) (Wang et al 2012) Myosin isolated fromE701K+ heterozygote IFMs moves actin filaments at a velocity of341plusmn036 microm sminus1 amounting to 51 of wild-type velocity(Plt0001 compared with both wild type and homozygotes) Thisis sim15 below the average velocity of the wild-type and E701Khomozygote myosins (400 microm sminus1) The disparity in actin filamentvelocity variance is graphically depicted in Fig S1

Ultrastructural analysis shows generally normal myofibrilassembly followed by moderate structural degeneration inE701K+ IFMsTo determine the effect of heterozygous expression of IBM3myosinon IFM myofibril assembly and stability we studied age-matchedcontrol and E701K+ mutant IFMs using transmission electronmicroscopy (Fig 2) Longitudinal sections of both heterozygotes(E701K-3 and E701K-5) revealed occasional gaps in themyofibrillar lattice at 2 days of age (Fig 2BC) These aberrationsoccurred with increasing frequency as flies aged from 2 daysthrough 6 weeks as did defects in Z-lines including non-linearityand abnormal sarcomeric localization (Fig 2EFHIKL) Suchdefects were not observed in the PwMhc2+ wild-type control(Fig 2ADGJ) Transverse views of myofibrils from adults at2 days of age revealed that some E701K+ myofibrils hadminor disruptions with occasional gaps in the myofilament lattice

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(Fig 2BprimeCprime) These defects increased during the aging process(Fig 2EprimeFprimeHprimeIprimeKprimeLprime) as did the propensity for mislocalization ofZ-line material to the filament-containing region of the myofibril(Fig 2KprimeLprime) Notably we saw neither the rapid ultrastructuraldegradation nor the vacuoles and autophagic vesicles that we hadfound in IBM3 homozygotes (Wang et al 2012) Althoughhomozygote IFMs were too structurally damaged to allow flight orpermit mechanical analysis the nearly wild-type structure ofheterozygote IFMs at 2 days of age allowed us to assess flightperformance and determine fiber mechanical parameters

Locomotion assays reveal reduced wing beat frequency anddecreased flight ability in E701K+ heterozygotesWe examined the wing beat frequency of female IBM3heterozygotes (E701K-3+ and E701K-5+) at 2-3 days of ageValues for both E701K+ heterozygous lines significantlydecreased by sim25 compared with control fly values at both15degC and 25degC (Table 1) In contrast to homozygous IBM3 mutantflies which demonstrate no flight ability (Wang et al 2012)E701K+ heterozygotes exhibited weak flight Flight indices weresignificantly decreased by 75 and 57 compared with controlvalues at 15degC and 22degC respectively (Table 1) We also examinedthe flight ability of E701K+ flies during aging at 22degC and foundprogressive disability (Table 1) with statistically significantreductions of 20 at 1 week (Plt001 Studentrsquos t-test) and afurther reduction of 36 at 3 weeks (Plt0001) In contrast the

control line showed no decline at 1 week and no statisticallysignificant decrease over the entire period

Mechanical analysis of E701K+ fibers reveals reduced fiberstiffness lower power output and altered myosin kineticsTo determine the effect of the E701K mutation on muscleviscoelastic properties we used small-amplitude sinusoidalanalysis Using these results we calculated elastic modulus (in-phase stiffness) and viscous modulus (out-of-phase stiffness) for themutant heterozygote and control lines The elastic modulus wassignificantly reduced in the mutant lines over the frequency range of5-220 Hz with the largest reduction occurring around 140 Hz(Fig 3A) Comparing dip frequency values (lowest value from allfrequencies tested) between mutant heterozygote and control linesthe mutantrsquos elastic modulus Ee was about 40 lower than thecontrol value (Table 2) These in-phase stiffness results suggest thatfewer heads are bound to actin (lower duty ratio) or that there is lessforce generation per head The viscous modulus was significantlyless negative at the lowest frequencies (05-15 Hz) and also at 70 to170 Hz (Fig 3B) At the lowest viscous modulus values Ev theviscous modulus values of the mutant lines were about 50 less thanthe control (Table 2) As viscousmodulus is inversely proportional towork performed by the muscle less work was generated by themutant heterozygote fibers at these frequencies which includes thewing beat frequency range ofDrosophila at 15degC (Table 1) The fibermechanical assays were also performed at this temperature

Fig 1 ATPase activities and in vitro motility values for myosinfrom E701K+ heterozygotes are intermediate between those ofhomozygotes and wild-type controls (A) CaATPase activitiesThe value for the E701K+ heterozygote is significantly differentfrom both PwMhc2+ control and E701KE701K homozygotes(B) MgATPase activities The value for the E701K+ heterozygote issignificantly different from both control and E701KE701Khomozygotes (C) Vmax for actin-activated MgATPase activities Thevalue for the E701K+ heterozygote is significantly different fromboth control and E701KE701K homozygotes (D) Km values areactin concentrations at which half-maximal actin-activatedMgATPase activities (Vmax) are exhibited No significant differencesare exhibited among the samples (E) Catalytic efficiency (ratio ofVmax to Km) The value for the E701K+ heterozygote issignificantly different from both control and E701KE701Khomozygotes (F) In vitro velocity of actin filaments propelled bymyosins of each genotype The value for the E701K+ heterozygoteis significantly different from both control and E701KE701Khomozygotes In all E701KE701K homozygote assays n=4 n=3for all other samples except control in vitro motility (n=5) E701KE701K homozygote data median values and wild-type motilitymedian values are fromWang et al (2012) Each ATPase data pointis a biological replicate that is the mean of duplicate technicalreplicates In vitromotility biological replicates represent the mean ofover 20 actin filaments per sample Statistical significance wasmeasured using Studentrsquos t-test (Plt005 Plt001 Plt0001ns=not significant) All values are meanplusmnsd

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Sinusoidal analysis also showed that the mutation decreasedmuscle power generation and shifted optimal power production tolower frequencies (Fig 4A) Power was reduced by 59 and fmaxwas reduced by about 20 due to the mutation (Table 2) Powergenerated by the mutant heterozygote was almost zero at the wingbeat frequency at which control flies beat their wings (sim145 Hz at15degC) The decrease in fmax suggests that the mutation slowedoverall muscle and myosin kinetics This slowing of kinetics wassupported by the elastic and viscous modulus results Both mutantmodulus graphs show a leftward shift in their dip frequencies fEe

and fEv (lowest values) compared with the control (Fig 3 andTable 2)To gain insight into how the cross-bridge cycle might be

altered we determined the influence of the E701K mutation onapparent muscle mechanical rate constants and amplitudes Thiswas carried out by fitting the complex modulus equation that wepreviously refined for Drosophila IFMs to Nyquist plots of thecomplex modulus (Swank et al 2006) The mutationsignificantly changed these rate constants and their associatedamplitudes (Table 3) Based on interpretations from previousstudies using this analysis (Kawai and Brandt 1980 Miller et al

2010 Palmer et al 2007) we interpreted the changes as follows(Fig S2) (1) the decrease in the A process suggests a decrease instiffness of the passive viscoelastic components of the musclepotentially including myosin (2) the decrease in rate constant2πb suggests that cross-bridge transition rates involving myosinattachment to actin work production are slowed whereas theincrease in 2πc suggests rate constants associated with workabsorption and myosin detachment from actin have increased (3)the amplitude of work-producing steps (B process) and absorbingsteps (C process) of the cycle are both reduced suggesting adecrease in the number of myosins producing force at any giventime andor cross-bridge stiffness during strongly bound steps ofthe cycle

We gained additional insight into cross-bridge kinetics byvarying ATP and Pi concentrations in conjunction with sinusoidalanalysis Varying Pi concentration did not alter 2πb values of thecontrol or mutants fibers (Fig 4B) According to our previousmodeling (Swank et al 2006) this suggests the rate-limiting cross-bridge step for work production was not changed by the mutationPlotting [ATP] versus fmax and fitting the graph with a Michaelis-Menton curve showed that in addition to decreasing fmax the

Fig 2 Ultrastructure of myofibrils from control and E701K+ heterozygote IFMs during aging shows progressive defects in filament arrangement andZ-line organization Transmission electron micrographs of longitudinal and transverse sections through IFM myofibrils from adult flies aged 2 days 2 weeks4 weeks or 6 weeks after eclosion PwMhc2wild-type transgenic controls (AAprimeDDprimeGGprimeJJprime) assemble into well-organized sarcomeres (A) and this structure isretained as the flies age (DGJ) PwMhc2 myofilaments are packed in a rigid double hexagonal array (Aprime) that is consistent during aging (DprimeGprimeJprime) Two-day-oldmyofibrils from E701K-3+ and E701K-5+ IFMs also assemble well-ordered sarcomeres (BC) with double-hexagonal filament packing (BprimeCprime) althoughoccasional minor gaps in the microfilament arrays are observed (asterisks) These gaps are exacerbated during aging in longitudinal sections (EFHIKL) andare particularly evident in transverse sections (EprimeFprimeHprimeIprimeKprimeLprime) As the heterozygotes age Z-lines become non-linear (arrowheads) and Z-line streaming whereZ-line material is mislocalized is observed (KprimeLLprime arrows) Scale bars 1 microm (longitudinal sections) 05 microm (transverse sections)

Table 1 Locomotion analysis of control and IBM3 myosin heterozygotes

Wing beat frequency (2-3 days) Flight index young (2-3 days) Flight index aged

15degC 25degC 15degC 22degC 1 week 22degC 3 weeks 22degC

PwMhc2+ 1456plusmn14 (30) 1768plusmn26 (20) 36plusmn013 (118) 47plusmn02 (46) 47plusmn02 (46) 42plusmn03 (46)E701K-3+ 1093plusmn14 (28) 1276plusmn20 (20) 09plusmn008 (162) 20plusmn01 (150) 15plusmn01 (150) 10plusmn01 (50)E701K-5+ 1060plusmn11 (30) 1204plusmn37 (20) 09plusmn009 (146) 20plusmn01 (150) 18plusmn01 (150) 11plusmn01 (50)

All values are meanplusmnsem Numbers of flies tested are indicated in parenthesesStatistically different from the age-matched PwMhc2+ control flies (Studentrsquos t-test Plt005)

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Sinusoidal analysis also showed that the mutation decreasedmuscle power generation and shifted optimal power production tolower frequencies (Fig 4A) Power was reduced by 59 and fmaxwas reduced by about 20 due to the mutation (Table 2) Powergenerated by the mutant heterozygote was almost zero at the wingbeat frequency at which control flies beat their wings (sim145 Hz at15degC) The decrease in fmax suggests that the mutation slowedoverall muscle and myosin kinetics This slowing of kinetics wassupported by the elastic and viscous modulus results Both mutantmodulus graphs show a leftward shift in their dip frequencies fEe

and fEv (lowest values) compared with the control (Fig 3 andTable 2)To gain insight into how the cross-bridge cycle might be

altered we determined the influence of the E701K mutation onapparent muscle mechanical rate constants and amplitudes Thiswas carried out by fitting the complex modulus equation that wepreviously refined for Drosophila IFMs to Nyquist plots of thecomplex modulus (Swank et al 2006) The mutationsignificantly changed these rate constants and their associatedamplitudes (Table 3) Based on interpretations from previousstudies using this analysis (Kawai and Brandt 1980 Miller et al

2010 Palmer et al 2007) we interpreted the changes as follows(Fig S2) (1) the decrease in the A process suggests a decrease instiffness of the passive viscoelastic components of the musclepotentially including myosin (2) the decrease in rate constant2πb suggests that cross-bridge transition rates involving myosinattachment to actin work production are slowed whereas theincrease in 2πc suggests rate constants associated with workabsorption and myosin detachment from actin have increased (3)the amplitude of work-producing steps (B process) and absorbingsteps (C process) of the cycle are both reduced suggesting adecrease in the number of myosins producing force at any giventime andor cross-bridge stiffness during strongly bound steps ofthe cycle

We gained additional insight into cross-bridge kinetics byvarying ATP and Pi concentrations in conjunction with sinusoidalanalysis Varying Pi concentration did not alter 2πb values of thecontrol or mutants fibers (Fig 4B) According to our previousmodeling (Swank et al 2006) this suggests the rate-limiting cross-bridge step for work production was not changed by the mutationPlotting [ATP] versus fmax and fitting the graph with a Michaelis-Menton curve showed that in addition to decreasing fmax the

Fig 2 Ultrastructure of myofibrils from control and E701K+ heterozygote IFMs during aging shows progressive defects in filament arrangement andZ-line organization Transmission electron micrographs of longitudinal and transverse sections through IFM myofibrils from adult flies aged 2 days 2 weeks4 weeks or 6 weeks after eclosion PwMhc2wild-type transgenic controls (AAprimeDDprimeGGprimeJJprime) assemble into well-organized sarcomeres (A) and this structure isretained as the flies age (DGJ) PwMhc2 myofilaments are packed in a rigid double hexagonal array (Aprime) that is consistent during aging (DprimeGprimeJprime) Two-day-oldmyofibrils from E701K-3+ and E701K-5+ IFMs also assemble well-ordered sarcomeres (BC) with double-hexagonal filament packing (BprimeCprime) althoughoccasional minor gaps in the microfilament arrays are observed (asterisks) These gaps are exacerbated during aging in longitudinal sections (EFHIKL) andare particularly evident in transverse sections (EprimeFprimeHprimeIprimeKprimeLprime) As the heterozygotes age Z-lines become non-linear (arrowheads) and Z-line streaming whereZ-line material is mislocalized is observed (KprimeLLprime arrows) Scale bars 1 microm (longitudinal sections) 05 microm (transverse sections)

Table 1 Locomotion analysis of control and IBM3 myosin heterozygotes

Wing beat frequency (2-3 days) Flight index young (2-3 days) Flight index aged

15degC 25degC 15degC 22degC 1 week 22degC 3 weeks 22degC

PwMhc2+ 1456plusmn14 (30) 1768plusmn26 (20) 36plusmn013 (118) 47plusmn02 (46) 47plusmn02 (46) 42plusmn03 (46)E701K-3+ 1093plusmn14 (28) 1276plusmn20 (20) 09plusmn008 (162) 20plusmn01 (150) 15plusmn01 (150) 10plusmn01 (50)E701K-5+ 1060plusmn11 (30) 1204plusmn37 (20) 09plusmn009 (146) 20plusmn01 (150) 18plusmn01 (150) 11plusmn01 (50)

All values are meanplusmnsem Numbers of flies tested are indicated in parenthesesStatistically different from the age-matched PwMhc2+ control flies (Studentrsquos t-test Plt005)

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mutation reduced ATP Km values by 50 (Fig 4C and Table 3)The reduced Km values suggest an increase in ATP affinityThe changes in 2πb 2πc and ATP Km suggest a decrease in

myosin duty ratio A decrease in duty ratio the time myosin spendsstrongly bound to actin would be predicted to cause a decrease inisometric tension generation We observed that active isometrictension was decreased by about 35 (Table 2) It is alternativelypossible that the decrease in tension resulting from the mutationcould arise from changed cross-bridge stiffness Measuring theelastic modulus of the fibers in the absence of ATP (rigorconditions where all myosin heads are bound to actin) shouldindicate a change in cross-bridge stiffness We did not observe achange in rigor elastic modulus in the mutant heterozygote fibersrelative to control (Fig 4D)

Effects of increased or decreased E701Kwild-type generatioWe genetically increased the ratio of mutant (E701K) to wild-type(+) myosin alleles to 21 in an attempt to discern the lsquotipping pointrsquofor production of high levels of myofibrillar disarray and vesicleformation found in both the IBM3 human heterozygotes and in theDrosophila homozygotes Increased relative gene copy number ofE701K myosin resulted in poor flight ability compared with acontrol line with three copies of wild-type myosin The averageflight index of E701KE701K+ was reduced by sim87 comparedwith PwMhc2PwMhc2+ at all ages tested (Table S1) Thiscompares with an average of a 65 reduction in flight ability inE701K heterozygotes relative to control heterozygotes (Table 1)The ultrastructure of 4-week-old flight muscle revealed a dramaticincrease in the frequency of Z-line disruption and focaldiscontinuities in the myofilamentous lattice (Fig 5) comparedwith heterozygous E701K+ muscle at the same age (Fig 2)However in contrast to flies expressing only E701K myosin (Wanget al 2012) the aged IFMs did not display obvious vacuole orautophagic vesicle formation When the ratio of mutant (E701K) towild-type (+) myosin alleles was altered to 12 flight ability wasessentially unchanged compared with the three-copy control(Table S1) further indicating that the dose of the mutant allele isdirectly correlated to the severity of the mutant phenotype Thissuggests that increased expression of endogenous or transgenicwild-type myosin genes could prove therapeutic in IBM3 patients

DISCUSSIONWe modeled the dominant effects of the human IBM3 myosinmutation in Drosophila muscle and obtained insights into the basisof both the mechanical and structural defects caused by thismutation Our data indicate that equimolar levels of mutant myosinaccumulate in young heterozygotes in agreement with thetranscriptional level results in human patients (Tajsharghi et al2002) We observed a 51 decrease for in vitro motility of actinfilaments driven by myosin isolated from Drosophilaheterozygotes which mimics the 56 reduction observed inmyosin isolated from biopsies of humans expressing the mutation(Li et al 2006) Despite essentially normal myofibrillar structure inyoung Drosophila IFMs fiber power output and muscle functionare dramatically reduced During aging we observed myofibrillardegeneration including Z-disc streaming accompanied by a furtherreduction in muscle function The progressive nature of theDrosophila IBM3 heterozygote phenotype mimics the decreasedmuscle function and increased myofibril disarray observed in aginghuman heterozygotes (Tajsharghi et al 2002)

Human IBM3 disease presentation is complicated compared withDrosophila in that multiple fiber types are present andco-expression of myosin isoforms occurs in human mixed fibermuscles (Schiaffino and Reggiani 1994) In individuals with IBM3

Fig 3 The effect of muscle length oscillation frequency on elasticmodulus and viscous modulus of IBM3 myosin heterozygote IFM fibersdiffers from control fibers (A) Elastic modulus and (B) viscous modulus ofcontrol (PwMhc2+) and IBM3 mutant (E701K-3+ and E701K-5+) IFM fibersfrom 05 to 650 Hz Muscle length change was 025 peak to peakExperiments were performed at 15degC Meanplusmnsem n=7 for each genotypeAsterisks and horizontal lines indicate the frequencies at which the valuesshow statistically significant differences between the mutant heterozygote andcontrol fibers one-way ANOVA Plt005

Table 2 Dynamic properties from sinusoidal analysis and isometric tension

Power (Wm3) fmax (Hz)Complex stiffness(kNm2) Ee (kNm2) fEe (Hz) minusEv (kNm2) fEv (Hz)

Active tension(mNm2)

PwMhc2+ 1236plusmn95 1321plusmn86 6049plusmn523 5711plusmn486 2136plusmn96 2231plusmn309 964plusmn124 508plusmn069 (5)E701K-3+ 519plusmn46 1093plusmn93 3587plusmn264 3449plusmn265 1729plusmn121 1089plusmn54 843plusmn111 365plusmn018E701K-5+ 519plusmn50 1011plusmn69 4074plusmn376 3933plusmn363 1629plusmn104 1206plusmn128 750plusmn75 294plusmn049 (4)

fmax is the frequency at which power generation was highest Complex stiffness was measured at fmax Elastic modulus (Ee) and viscous modulus (minusEv) minimumvalues were measured at the oscillation frequency (dip frequency) that produced the lowest value for each individual fiber fEe and fEv are the dip frequencies forthe elastic and viscous moduli respectively Active isometric tension wasmeasured at pCa 50 All values are meanplusmnsem n=7 fibers for each line except wherenumbers in parentheses indicate the number of fibers testedStatistically different from PwMhc2+ control fibers (Studentrsquos t-test Plt005)

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increased fiber size variability as well as increases in mass andfunction of non-type IIa fibers have been documented (Li et al2006 Tajsharghi et al 2002) likely as being compensatory for theabnormalities found in type IIa fibers Cross-sectional areameasurements of dorsolongitudinal IFM fibers in IBM3heterozygotes did not show a statistically significant differencecompared with control organisms at 2-3 days of age (Table S2)indicating a lack of hypertrophic compensation AsDrosophila doesnot have the ability to undergo fiber growth through use of satellitestem cells (Demontis et al 2013) or through activation of othermyosin heavy chain genes (Bernstein et al 1983 Rozek andDavidson 1983) this result is not surprising Although theDrosophila system does not mimic all properties of human

skeletal muscle it has the ability to directly define thebiochemical and mechanical origins of the disease phenotypeDrosophila has the added advantage of genetic homogeneity that islacking in human patients who often display different diseaseseverities likely arising from disparate modifying alleles (Li et al2006)

Previous work that modeled the IBM3 mutation in Dictyosteliumnon-muscle myosin II (Zeng et al 2004) suggested that productrelease rate particularly that of ADP is reduced as a result of theSH1-SH2 helix mutation This would result in myosin spendingincreased time bound to actin yielding a higher duty ratio (thefraction of the chemomechanical cycle in which actin is bound tomyosin) This could explain the slower actin motility we observed

Fig 4 E701K+ heterozygote IFM fibers and control fibers display differences in power output parameters but not in rigor stiffness (A) The powergenerated by maximally activated control (PwMhc2+) and IBM3 mutant heterozygote (E701K-3+ and E701K-5+) IFM fibers when oscillated at 025 peak-to-peak strain over a frequency range of 05-200 Hz at 15degC Vertical dashed lines indicate the frequency at which maximum power was generated ( fmax) Dataaremeanplusmnsem n=7 for each genotype (B) The response of 2πb to phosphate concentration Data aremeanplusmnsem n=7 (C) Effect of MgATP concentration onthe frequency at which maximum power ( fmax) is produced in control (PwMhc2+) and IBM3 mutant (E701K-3+ and E701K-5+) IFM fibers Data are meanplusmnsem n=7 (D) Rigor stiffness determined by measuring elastic modulus at pCa 45 and ATP=0 mM from 05 Hz to 650 Hz No significant differences in rigorelastic modulus were observed between the mutant and control fibers one-way ANOVA Plt005 Data are meanplusmnsem n=6 for each mutant heterozygote andn=5 for control

Table 3 Apparent muscle rate constants and amplitudes and the response of fiber kinetics to varying ATP concentrations

A (kNm2) k B (kNm2) 2πb (sminus1) C (kNm2) 2πc (sminus1) ATP Vmax ATP Km (mNm2)

PwMhc2+ 598plusmn76 0094plusmn0006 4070plusmn930 1156plusmn131 3915plusmn912 1947plusmn153 234plusmn8 170plusmn020E701K-3+ 420plusmn26 0058plusmn0005 847plusmn127 788plusmn129 1033plusmn107 2726plusmn192 171plusmn4 101plusmn010E701K-5+ 497plusmn49 0053plusmn0005 672plusmn71 567plusmn52 919plusmn102 3188plusmn237 165plusmn7 078plusmn014

See Materials and Methods for a full description of the significance of rate constant and amplitude values ATP Vmax and Km were determined by plotting fmax

versus [ATP] fitting with the Michaelis-Menten curve and averaging the resultant Vmax and Km values All values are meanplusmnsem n=7Statistically different from PwMhc2+ control fibers (Studentrsquos t-test Plt005)

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with both homozygous and heterozygous mutant myosin andwould be in keeping with the reduced overall muscle kinetics thatyielded a reduced fmaxA number of our findings however do not support a model for

IBM3 muscle myosin spending increased time in strongly actin-bound states In contrast to the increases in active stiffness and activeforce generation expected for augmented actin binding we foundthat both elastic and viscous modulus decreased (Fig 3) and thatisometric force generation was either unchanged or reduced(Table 2) Additionally our sinusoidal analysis uncovered anincrease in the apparent rate constant 2πc (Table 3) a parameter thatis inversely proportional to time strongly bound to actin (Kawai andBrandt 1980 Palmer et al 2007) This suggests that the mutantmyosin spends less time in strongly bound states such as the ADPstate Furthermore varying ATP concentration while performingsinusoidal mechanical analysis revealed a slightly higher affinity ofthe heterozygous myosin for ATP again suggesting fastertransitions through the final steps prior to myosin dissociationfrom actin Finally we did not observe a dramatic reduction ofin vitro actin filament velocity in heterozygote myosin below the

average of levels for the control andmutant homozygote despite thefact that our ATPase activity assays indicate the presence ofequimolar levels of IBM3 and wild-type myosin (Fig 1) Asequimolar mixtures of skeletal muscle myosins (which are regulatedby ADP off-rate) translocate actin at the rate of the slower isoform(Cuda et al 1997 Sata et al 1993) our observations suggest thatthe mechanical results do not arise from an increase in the timemyosin spends strongly bound to actin

Our data instead support a mechanism in which the power strokeitself or the rates of steps associated with the power stroke have beenaltered in the IBM3 heterozygote so that fewer or shorter powerstrokes occur The IBM3 mutation severely decreased rate constant2πb (Table 3) which is influenced primarily by work-generatingsteps associated with actin attachment and the power stroke Inconcordance with this we observed no change in the response of2πb to Pi concentration for the heterozygote (Fig 2B) If ADPrelease rate had been substantially decreased as suggested abovethen it would have become rate limiting and 2πb should haveincreased with Pi concentration (Kawai and Brandt 1980) Inaddition the decreased rate of actin translocation in the in vitro

Fig 5 Ultrastructure of myofibrils expressing two IBM3 E701K Mhc genes and one wild-typeMhc gene show enhanced myofibrillar degeneration andZ-line streaming Transmission electron micrographs of longitudinal and transverse sections through IFM myofibrils from 4-week-old adult flies PwMhc2PwMhc2+ transgenic control myofibrils (top row) contain well-organized sarcomeres (A) with a double-hexagonal lattice of thick and thin filaments (Aprime) Myofibrilsfrom (B)E701K-3E701K-3+ and (C)E701K-5E701K-5+ IFMs show severely disordered sarcomeres with non-linear Z-lines (arrowheads) and Z-line streamingAlthough some normally structured sarcomeres are present the overall phenotype is dramatically worsened compared with mutant heterozygotes (Fig 2) Intransverse sections (BprimeCprime) myofibrillar disorder and mislocalization of Z-line material is evident (arrows) Scale bars 2 microm (longitudinal sections) 1 microm(transverse sections)

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motility assay would be expected to yield a more linear relationshipbetween motility and isoform level if fewer or shorter power strokesresult from the mutation compared with when the slower IBM3myosin remains bound for a longer period due to a reduced ADPrelease rate Finally and in agreement with these conclusions theobserved lower B C and stiffness values for the IBM3heterozygotes (Tables 2 and 3) suggest that less myosin is boundat any given time The observed lower stiffness and force in theheterozygote do not arise from reduced cross-bridge stiffness asrigor stiffness was not changed in our assays (Fig 4D)The slower kinetics and less-stiff IFM fibers explain the decrease

in flight ability observed in IBM3 heterozygotes (Table 1) Becausea decreased elastic modulus correlates with decreased wing beatfrequency (WBF) (Ramanath et al 2011) the lower active stiffnesscaused a lower WBF in the mutants (Table 1) reducingaerodynamic power for flight Attempts by IBM3 heterozygotes atbeating their wings at a higher frequency would provide inadequatepower for flight as muscle power output for the mutantheterozygote is negligible at the wild-type WBF (Fig 4A)The modest ultrastructural degradation observed in the IBM3

heterozygote (Fig 2) is in consonance with a mechanism for IBM3that involves a reduced power stroke size andor number Theresulting reduced duty ratio should not be as detrimental toultrastructure as an increase in the time spent in strongly bound actinstates as is the case when an embryonic isoform of myosin isexpressed in IFMs (Wells et al 1996) or when unregulated thinfilaments are present (Beall and Fyrberg 1991) A single copy ofthe wild-type myosin gene is even sufficient to permit relativelynormal ultrastructure to be maintained in the presence of two IBM3myosin alleles (Fig 5) Furthermore well-aligned thick filamentswere seen in an IBM3 C elegans model that co-expresses a wild-type isoform of myosin heavy chain (Tajsharghi et al 2005) Thelsquoprotectiversquo effects of wild-type myosin are seen in human patientswhere focal disruptions in ultrastructure occur rather than overallsarcomere degradation (Martinsson et al 2000 Oldfors 2007) Bycontrast the more severe ultrastructural abnormalities observed inIBM3 homozygotes (Wang et al 2012) which are not capable ofproducing mechanical power output (data not shown) are likely toarise from serious defects in assembly due to the lack of wild-typemyosin moleculesAs in the human condition where functional defects precede

structural abnormalities (Li et al 2006) Drosophila IBM3heterozygotes show exacerbation of muscle structural andfunctional defects during aging (Fig 2 Table 1) Most notablyfocal defects in sarcomere structure with streaming Z-line materialwere observed in both the model and the disease state (Martinssonet al 2000 Oldfors 2007) It is possible that this arises whenfunctional defects cause abnormal stress on myofibrillarcomponents resulting in structural degradation However we didnot detect an increase in the stress response in the heterozygoteLevels of the protein aggregate marker Ref(2)P (p62 in mammals)and the ubiquitin protein degradation signal are not increased inheterozygotes compared with controls (JAS GCM AM E PRatliff D B Foster and SIB unpublished data) although levelsof these proteins increase in IBM3 homozygotes (Suggs et al2015) Previous studies have shown that Ref(2)P levels increaseduring both stress and aging inDrosophila and that ubiquitin levelsincrease during muscle aging (Demontis and Perrimon 2010Dialynas et al 2015 Landis et al 2012) As aging leads tostructural deterioration inDrosophila IFMs (Miller et al 2008) it ispossible that other aspects of the aging response (reviewed byDemontis et al 2013) are triggered by the E701Kmyosin mutation

leading to the observed phenotypic abnormalities Anotherpossibility is that the mutant myosin is more prone to proteolysispossibly as a result of its tendency to aggregate (Wang et al 2012)Tajsharghi et al speculated that proteolytic degradation productscould contribute to toxic effects on muscle structure as theyobserved a disproportionally high level of myosin IIA mRNAcompared with protein levels in patients suggesting increasedprotein turnover (Tajsharghi et al 2002) In contrast to themyofibrillar defects observed in the Drosophila heterozygote thatmirror the human condition we did not discern the rimmed vacuolesobserved in patients It is notable however that such structures arenot detected in all human type IIa muscles being most prevalent inolder patients who are more seriously disabled (Martinsson et al2000 Tajsharghi et al 2002)

The location of the IBM3 mutation within the SH1-SH2 helixplaces it in proximity to the nucleotide binding pocket to the centraltransducer region that communicates with the actin-binding site andto the relay domain that is important for inducing lever armmovement through interaction with the converter domainAlterations of the SH1-SH2 helix such as crosslinking of theactive sulfhydryl residues (Thompson et al 2008) or variousmutations (Hu et al 2002 Kad et al 2007 Preller et al 2011 Zenget al 2004) affect ATP hydrolysis andor transduction of its chemicalenergy into the power stroke In accordance with these observationswe detected decreased ATPase rates in mutant myosin (Fig 1) Thiscould arise from a decrease in rate-limiting Pi release This wouldcorrelate with the observed reduction in 2πb and lead to a decrease inlength or probability of occurrence of the power stroke Our previousmodeling results (Wang et al 2012) predicted that the melted state ofthe SH1-SH2 helix that occurs after the power stroke and uncouplesthe lever arm from the motor (Himmel et al 2002) is stabilized bythe IBM3mutation This abnormal interaction could indeed decreasethe probability of transitioning through the power stroke or reduce itssize as predicted from our mechanical data Based upon ourfindings pharmacological agents that enhance the kinetics of myosinbinding to actin and steps of the cycle associated with the powerstroke might be expected to improve the functioning of type IIa-containing fibers in young IBM3 patients and perhaps reduce thestructural and functional degradation that occurs during aging

MATERIALS AND METHODSTransgenic lines and genetic crossesTheDrosophila melanogaster E701Kmutation in theMhc gene was createdby site-directed mutagenesis of an E coli plasmid and introduced into the flygenome by P element-mediated germline transformation along with a w+

eye color marker (Wang et al 2012) The transgene was crossed into theMhc10 genetic background which contains a null allele that results in flieslacking myosin expression in the indirect flight and jump muscles (Collieret al 1990) Two mutant fly lines homozygous for Mhc10 and the E701Ktransgene on the third chromosome (w Mhc10 E701K-3 and w Mhc10E701K-5) were shown to express wild-type levels of E701K protein in theIFMs (Wang et al 2012) A line carrying a transgene encoding full-lengthwild-type myosin heavy chain on its first chromosome PwMhc2Mhc10 isused as a control (Swank et al 2000)

For studies of heterozygotes the lines outlined above were each crossedwith a lsquowild-typersquo yw fly line to create progeny with one wild-typeendogenous Mhc+ allele one Mhc10 allele and one copy of the respectivetransgene ie w PwMhc2ywMhc10+ for the control line wyw Mhc10+E701K-3+ and wyw Mhc10+ E701K-5+ for the two IBM3 heterozygouslines Female progeny were aged and used for subsequent experiments

In addition to analyzing heterozygotes with 11 E701KMhc+ genotypicratio we analyzed flies with 12 or 21 E701KMhc+ genotypic ratios Theformer were produced by crossing male yw Mhc+Mhc+ E701KE701Kflies to female yw flies to create yw Mhc+Mhc+ E701K+ progeny

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Similarly control male yw PwMhc2 Mhc+Mhc+ flies were crossed withfemale yw to create yw PwMhc2ywMhc+Mhc+ control progeny Flies witha 21 E701KMhc+ genotypic ratio were produced by crossing male ywMhc+Mhc+ E701KE701K flies to female ww Mhc10Mhc10 E701KE701K flies yielding wyw Mhc10Mhc+ E701KE701K These flies werecompared with control flies with two transgenic wild-type myosin genes andone endogenous gene PwMhc2PwMhc2 Mhc10 Mhc+

Protein expressionRelative levels of myosin expression were determined by measuring myosin-to-actin ratios in upper thorax homogenates subjected to one-dimensionalSDS polyacrylamide gel electrophoresis (OrsquoDonnell et al 1989) Sixdissected upper thoraces from 2-day-old adult female flies for each line werehomogenized in 360 μl of SDS gel 1times Laemmli Sample Buffer (Bio-RadHercules CA) containing 5 2-mercaptoethanol Samples were boiled for5 min prior to loading at 2-5 μl on a 15-well 10 Mini-PROTEAN TGXpolyacrylamide gel (Bio-Rad) Gels stained with Coomassie Brilliant BlueR-250 were digitally scanned using an Epson Expression 636 flatbedscanner Myosin heavy chain and actin levels were determined by UN-SCAN-IT gel software (Silk Scientific) for three separate lanes per sampleThe median myosin-to-actin ratio in mutant thoraces was compared with themedian ratio found in thoraces from aged-matched flies expressing thePwMhc2 control transgene in the corresponding genetic background

Protein purificationMyosin was purified from the dorso-longitudinal set of IFMs ofsim120 youngfemale flies as previously described (Kronert et al 2008 2014 Swanket al 2001) Briefly fibers were collected in York modified glycerol[20 mM potassium phosphate (pH 70) 2 mM MgCl2 1 mM EGTA20 mM DTT and a protease inhibitor mixture] pelleted and thendemembranated for 30 min on ice in York modified glycerol containing2 (vv) Triton-X 100 After centrifugation and washing the solution wasreplaced with a high-salt extraction buffer [1 M KCl 50 mM potassiumphosphate (pH 68) 5 mM MgCl2 05 mM EGTA 10 mM sodiumpyrophosphate 20 mM DTT plus a protease inhibitor mixture] Thesoluble material was subjected to a low-salt precipitation to pellet myosinvia centrifugation a high-salt precipitation to remove residual contaminantsand another low-salt precipitation to isolate purified myosin Protein wassuspended in myosin storage buffer [05 M KCl 20 mM MOPS (pH 70)2 mM MgCl2 and 20 mM DTT] Myosin concentration was determined bymeasuring the absorption at 280 nm (Margossian and Lowey 1982) Atypical yield was sim100 microg of myosin Samples were immediately used forATPase and in vitro motility assays

G-actin was isolated from acetone powder of chicken breast muscle (Pardeeand Spudich 1982) by cycles of polymerization and depolymerization aspreviously described (Kronert et al 2014) Following dialysis actinconcentration was determined by absorption at 280 nm F-actin wasprepared by adding one volume of 10times polymerization buffer [50 mMTris-Cl (pH 8) 05 M KCl 20 mMMgCl2 10 mMATP] to nine volumes ofG-actin Actin stored on ice at 4degC was used within 1 month of preparation

ATPase activityATPase activities were determined using 2 microg of freshly prepared myosinand 1 mM [γ-32P]ATP Basal ATPase activities were assessed for 15 min inMgATPase buffer (10 mM imidazole pH 60 20 mM KCl 2 mM MgCl201 mMCaCl2) or CaATPase buffer (10 mM imidazole pH 60 01 MKCl10 mM CaCl2) at 25degC Following sample extraction with 18 M HClO4incorporation of radiolabel was determined by scintillation counting Actin-activated MgATPase activity was assessed by the addition of chickenskeletal muscle actin (01ndash2 μM) to samples prior to incubation Followingsubtraction of basal MgATPase activity from each data point the ATPaseactivity versus actin concentration was graphed The Michaelis-Mentenequation was used in conjunction with SigmaPlot (Systat Software SanJose CA US) to define Vmax Km and catalytic efficiency (VmaxKm) valuesDetailed procedures for ATPase activity determination have been previouslydescribed (Kronert et al 2008 2014 Swank et al 2001)

In vitro actin sliding velocityIn vitro actin sliding velocity assays were implemented as previouslydescribed (Kronert et al 2008 2014 Swank et al 2001) Brieflynitrocellulose-coated coverslips were treated with myosin at 05 μgμl andmyosin heads that irreversibly bound to actin were blocked with unlabeledactin filaments F-actin labeled with fluorescent phalloidin was added to thecoverslip and filament movement was digitally captured under fluorescentoptics in the presence of ATP Computational analysis of actin filamentmovement permitted determination of actin sliding velocity

Ultrastructural analysisPreparation of samples for transmission electron microscopy was carried outas previously described (Suggs et al 2007 Wang et al 2012) Sampleswere viewed on a Tecnai 12microscope at 120 kV and images were capturedwith a XR-41S 2k digital camera and software from Advanced MicroscopyTechniques

For muscle fiber measurements transverse sections at sim200 nm werecollected from 2- to 3-day-old fly thoraces prepared for ultrastructuralanalysis and embedded in resin as indicated above Sections were transferredonto a standard glass microscope slide and briefly heated to insureadherence This was followed immediately by staining in 1 MethyleneBlue and 1 borax Samples were viewed and digitally photographed at 40timesunder a light microscope A stage micrometer with 001 mm divisions wassimilarly imaged for scale calibration Cross-sectional areas of each memberof collateral pairs of DLM fibers 45d 45e and 45f (numbering according toMiller 1950) from three flies per genotype were measured using ImageJVersion 151j (httpsimagejnihgovijdownloadhtml) Mean values wereassessed for statistically significant differences using a two-way ANOVAwith Dunnettrsquos multiple comparisons

Flight assaysFlight assays were conducted on female flies at 15degC 22degC and 25degC Theflight phenotype was determined by observing whether each fly tested flewupward (U) horizontal (H) down (D) or displayed no flight (N) whenreleased in a flight chamber (Drummond et al 1991) and quantified using aflight index equal to 6 UT+4HT+2DT+0NT where T is the total numberof flies tested (Tohtong et al 1995) Wing beat frequency (WBF) wasmeasured on 2- to 3-day-old female flies using an optical tachometer (Hyattand Maughan 1994)

Fiber mechanicsA pair of IFM bundles containing six fibers each were dissected out of 2- to3-day-old female fly thoraces and demembranated for 1 h at 4degC inskinning solution [5 mM ATP 1 mM free Mg2+ 025 mM phosphate5 mM EGTA 20 mM N N-Bis (2-hydroxyethyl)-2-aminoethanesulfonicacid (BES) at pH 70 1 mM DTT 50 glycerol and 05 Triton X-100pCa 80 and ionic strength 175 mM adjusted with Na methane sulfonate]Tungsten wire probes were used to separate and split the individual fibersAn aluminum clip was wrapped around each end of the fiber preparationThe preparations were attached to a mechanical measurement apparatusand bathed in relaxing solution (5 mM ATP 8 mM phosphate 15 mMcreatine phosphate 300 Uml creatine phosphokinase 1 mM free Mg2+5 mM EGTA 20 mM BES at pH 70 1 mM DTT pCa 80 ionic strength200 mM adjusted with Na methane sulfonate) Fibers were stretched by5 from resting length then subjected to sinusoidal analysis (see below) inrelaxing solution (pCa 80) before a partial exchange with activatingsolution (same as relaxing solution but pCa adjusted to 40) in the chamberto bring pCa to 50 The active fiber was further stretched (in 2increments of fiber length between clips) until maximum power generationwas obtained At this optimized length different relaxing activating orrigor solutions were exchanged into the chamber and further sinusoidalanalysis tension measurements or [ATP] and [Pi] variations wereperformed Sinusoidal analysis was run at the beginning and end of eachexperiment to ensure the performance of a fiber did not decrease by morethan 10 Detailed methods have been previously presented (Swank2012)

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motility assay would be expected to yield a more linear relationshipbetween motility and isoform level if fewer or shorter power strokesresult from the mutation compared with when the slower IBM3myosin remains bound for a longer period due to a reduced ADPrelease rate Finally and in agreement with these conclusions theobserved lower B C and stiffness values for the IBM3heterozygotes (Tables 2 and 3) suggest that less myosin is boundat any given time The observed lower stiffness and force in theheterozygote do not arise from reduced cross-bridge stiffness asrigor stiffness was not changed in our assays (Fig 4D)The slower kinetics and less-stiff IFM fibers explain the decrease

in flight ability observed in IBM3 heterozygotes (Table 1) Becausea decreased elastic modulus correlates with decreased wing beatfrequency (WBF) (Ramanath et al 2011) the lower active stiffnesscaused a lower WBF in the mutants (Table 1) reducingaerodynamic power for flight Attempts by IBM3 heterozygotes atbeating their wings at a higher frequency would provide inadequatepower for flight as muscle power output for the mutantheterozygote is negligible at the wild-type WBF (Fig 4A)The modest ultrastructural degradation observed in the IBM3

heterozygote (Fig 2) is in consonance with a mechanism for IBM3that involves a reduced power stroke size andor number Theresulting reduced duty ratio should not be as detrimental toultrastructure as an increase in the time spent in strongly bound actinstates as is the case when an embryonic isoform of myosin isexpressed in IFMs (Wells et al 1996) or when unregulated thinfilaments are present (Beall and Fyrberg 1991) A single copy ofthe wild-type myosin gene is even sufficient to permit relativelynormal ultrastructure to be maintained in the presence of two IBM3myosin alleles (Fig 5) Furthermore well-aligned thick filamentswere seen in an IBM3 C elegans model that co-expresses a wild-type isoform of myosin heavy chain (Tajsharghi et al 2005) Thelsquoprotectiversquo effects of wild-type myosin are seen in human patientswhere focal disruptions in ultrastructure occur rather than overallsarcomere degradation (Martinsson et al 2000 Oldfors 2007) Bycontrast the more severe ultrastructural abnormalities observed inIBM3 homozygotes (Wang et al 2012) which are not capable ofproducing mechanical power output (data not shown) are likely toarise from serious defects in assembly due to the lack of wild-typemyosin moleculesAs in the human condition where functional defects precede

structural abnormalities (Li et al 2006) Drosophila IBM3heterozygotes show exacerbation of muscle structural andfunctional defects during aging (Fig 2 Table 1) Most notablyfocal defects in sarcomere structure with streaming Z-line materialwere observed in both the model and the disease state (Martinssonet al 2000 Oldfors 2007) It is possible that this arises whenfunctional defects cause abnormal stress on myofibrillarcomponents resulting in structural degradation However we didnot detect an increase in the stress response in the heterozygoteLevels of the protein aggregate marker Ref(2)P (p62 in mammals)and the ubiquitin protein degradation signal are not increased inheterozygotes compared with controls (JAS GCM AM E PRatliff D B Foster and SIB unpublished data) although levelsof these proteins increase in IBM3 homozygotes (Suggs et al2015) Previous studies have shown that Ref(2)P levels increaseduring both stress and aging inDrosophila and that ubiquitin levelsincrease during muscle aging (Demontis and Perrimon 2010Dialynas et al 2015 Landis et al 2012) As aging leads tostructural deterioration inDrosophila IFMs (Miller et al 2008) it ispossible that other aspects of the aging response (reviewed byDemontis et al 2013) are triggered by the E701Kmyosin mutation

leading to the observed phenotypic abnormalities Anotherpossibility is that the mutant myosin is more prone to proteolysispossibly as a result of its tendency to aggregate (Wang et al 2012)Tajsharghi et al speculated that proteolytic degradation productscould contribute to toxic effects on muscle structure as theyobserved a disproportionally high level of myosin IIA mRNAcompared with protein levels in patients suggesting increasedprotein turnover (Tajsharghi et al 2002) In contrast to themyofibrillar defects observed in the Drosophila heterozygote thatmirror the human condition we did not discern the rimmed vacuolesobserved in patients It is notable however that such structures arenot detected in all human type IIa muscles being most prevalent inolder patients who are more seriously disabled (Martinsson et al2000 Tajsharghi et al 2002)

The location of the IBM3 mutation within the SH1-SH2 helixplaces it in proximity to the nucleotide binding pocket to the centraltransducer region that communicates with the actin-binding site andto the relay domain that is important for inducing lever armmovement through interaction with the converter domainAlterations of the SH1-SH2 helix such as crosslinking of theactive sulfhydryl residues (Thompson et al 2008) or variousmutations (Hu et al 2002 Kad et al 2007 Preller et al 2011 Zenget al 2004) affect ATP hydrolysis andor transduction of its chemicalenergy into the power stroke In accordance with these observationswe detected decreased ATPase rates in mutant myosin (Fig 1) Thiscould arise from a decrease in rate-limiting Pi release This wouldcorrelate with the observed reduction in 2πb and lead to a decrease inlength or probability of occurrence of the power stroke Our previousmodeling results (Wang et al 2012) predicted that the melted state ofthe SH1-SH2 helix that occurs after the power stroke and uncouplesthe lever arm from the motor (Himmel et al 2002) is stabilized bythe IBM3mutation This abnormal interaction could indeed decreasethe probability of transitioning through the power stroke or reduce itssize as predicted from our mechanical data Based upon ourfindings pharmacological agents that enhance the kinetics of myosinbinding to actin and steps of the cycle associated with the powerstroke might be expected to improve the functioning of type IIa-containing fibers in young IBM3 patients and perhaps reduce thestructural and functional degradation that occurs during aging

MATERIALS AND METHODSTransgenic lines and genetic crossesTheDrosophila melanogaster E701Kmutation in theMhc gene was createdby site-directed mutagenesis of an E coli plasmid and introduced into the flygenome by P element-mediated germline transformation along with a w+

eye color marker (Wang et al 2012) The transgene was crossed into theMhc10 genetic background which contains a null allele that results in flieslacking myosin expression in the indirect flight and jump muscles (Collieret al 1990) Two mutant fly lines homozygous for Mhc10 and the E701Ktransgene on the third chromosome (w Mhc10 E701K-3 and w Mhc10E701K-5) were shown to express wild-type levels of E701K protein in theIFMs (Wang et al 2012) A line carrying a transgene encoding full-lengthwild-type myosin heavy chain on its first chromosome PwMhc2Mhc10 isused as a control (Swank et al 2000)

For studies of heterozygotes the lines outlined above were each crossedwith a lsquowild-typersquo yw fly line to create progeny with one wild-typeendogenous Mhc+ allele one Mhc10 allele and one copy of the respectivetransgene ie w PwMhc2ywMhc10+ for the control line wyw Mhc10+E701K-3+ and wyw Mhc10+ E701K-5+ for the two IBM3 heterozygouslines Female progeny were aged and used for subsequent experiments

In addition to analyzing heterozygotes with 11 E701KMhc+ genotypicratio we analyzed flies with 12 or 21 E701KMhc+ genotypic ratios Theformer were produced by crossing male yw Mhc+Mhc+ E701KE701Kflies to female yw flies to create yw Mhc+Mhc+ E701K+ progeny

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isms

Similarly control male yw PwMhc2 Mhc+Mhc+ flies were crossed withfemale yw to create yw PwMhc2ywMhc+Mhc+ control progeny Flies witha 21 E701KMhc+ genotypic ratio were produced by crossing male ywMhc+Mhc+ E701KE701K flies to female ww Mhc10Mhc10 E701KE701K flies yielding wyw Mhc10Mhc+ E701KE701K These flies werecompared with control flies with two transgenic wild-type myosin genes andone endogenous gene PwMhc2PwMhc2 Mhc10 Mhc+

Protein expressionRelative levels of myosin expression were determined by measuring myosin-to-actin ratios in upper thorax homogenates subjected to one-dimensionalSDS polyacrylamide gel electrophoresis (OrsquoDonnell et al 1989) Sixdissected upper thoraces from 2-day-old adult female flies for each line werehomogenized in 360 μl of SDS gel 1times Laemmli Sample Buffer (Bio-RadHercules CA) containing 5 2-mercaptoethanol Samples were boiled for5 min prior to loading at 2-5 μl on a 15-well 10 Mini-PROTEAN TGXpolyacrylamide gel (Bio-Rad) Gels stained with Coomassie Brilliant BlueR-250 were digitally scanned using an Epson Expression 636 flatbedscanner Myosin heavy chain and actin levels were determined by UN-SCAN-IT gel software (Silk Scientific) for three separate lanes per sampleThe median myosin-to-actin ratio in mutant thoraces was compared with themedian ratio found in thoraces from aged-matched flies expressing thePwMhc2 control transgene in the corresponding genetic background

Protein purificationMyosin was purified from the dorso-longitudinal set of IFMs ofsim120 youngfemale flies as previously described (Kronert et al 2008 2014 Swanket al 2001) Briefly fibers were collected in York modified glycerol[20 mM potassium phosphate (pH 70) 2 mM MgCl2 1 mM EGTA20 mM DTT and a protease inhibitor mixture] pelleted and thendemembranated for 30 min on ice in York modified glycerol containing2 (vv) Triton-X 100 After centrifugation and washing the solution wasreplaced with a high-salt extraction buffer [1 M KCl 50 mM potassiumphosphate (pH 68) 5 mM MgCl2 05 mM EGTA 10 mM sodiumpyrophosphate 20 mM DTT plus a protease inhibitor mixture] Thesoluble material was subjected to a low-salt precipitation to pellet myosinvia centrifugation a high-salt precipitation to remove residual contaminantsand another low-salt precipitation to isolate purified myosin Protein wassuspended in myosin storage buffer [05 M KCl 20 mM MOPS (pH 70)2 mM MgCl2 and 20 mM DTT] Myosin concentration was determined bymeasuring the absorption at 280 nm (Margossian and Lowey 1982) Atypical yield was sim100 microg of myosin Samples were immediately used forATPase and in vitro motility assays

G-actin was isolated from acetone powder of chicken breast muscle (Pardeeand Spudich 1982) by cycles of polymerization and depolymerization aspreviously described (Kronert et al 2014) Following dialysis actinconcentration was determined by absorption at 280 nm F-actin wasprepared by adding one volume of 10times polymerization buffer [50 mMTris-Cl (pH 8) 05 M KCl 20 mMMgCl2 10 mMATP] to nine volumes ofG-actin Actin stored on ice at 4degC was used within 1 month of preparation

ATPase activityATPase activities were determined using 2 microg of freshly prepared myosinand 1 mM [γ-32P]ATP Basal ATPase activities were assessed for 15 min inMgATPase buffer (10 mM imidazole pH 60 20 mM KCl 2 mM MgCl201 mMCaCl2) or CaATPase buffer (10 mM imidazole pH 60 01 MKCl10 mM CaCl2) at 25degC Following sample extraction with 18 M HClO4incorporation of radiolabel was determined by scintillation counting Actin-activated MgATPase activity was assessed by the addition of chickenskeletal muscle actin (01ndash2 μM) to samples prior to incubation Followingsubtraction of basal MgATPase activity from each data point the ATPaseactivity versus actin concentration was graphed The Michaelis-Mentenequation was used in conjunction with SigmaPlot (Systat Software SanJose CA US) to define Vmax Km and catalytic efficiency (VmaxKm) valuesDetailed procedures for ATPase activity determination have been previouslydescribed (Kronert et al 2008 2014 Swank et al 2001)

In vitro actin sliding velocityIn vitro actin sliding velocity assays were implemented as previouslydescribed (Kronert et al 2008 2014 Swank et al 2001) Brieflynitrocellulose-coated coverslips were treated with myosin at 05 μgμl andmyosin heads that irreversibly bound to actin were blocked with unlabeledactin filaments F-actin labeled with fluorescent phalloidin was added to thecoverslip and filament movement was digitally captured under fluorescentoptics in the presence of ATP Computational analysis of actin filamentmovement permitted determination of actin sliding velocity

Ultrastructural analysisPreparation of samples for transmission electron microscopy was carried outas previously described (Suggs et al 2007 Wang et al 2012) Sampleswere viewed on a Tecnai 12microscope at 120 kV and images were capturedwith a XR-41S 2k digital camera and software from Advanced MicroscopyTechniques

For muscle fiber measurements transverse sections at sim200 nm werecollected from 2- to 3-day-old fly thoraces prepared for ultrastructuralanalysis and embedded in resin as indicated above Sections were transferredonto a standard glass microscope slide and briefly heated to insureadherence This was followed immediately by staining in 1 MethyleneBlue and 1 borax Samples were viewed and digitally photographed at 40timesunder a light microscope A stage micrometer with 001 mm divisions wassimilarly imaged for scale calibration Cross-sectional areas of each memberof collateral pairs of DLM fibers 45d 45e and 45f (numbering according toMiller 1950) from three flies per genotype were measured using ImageJVersion 151j (httpsimagejnihgovijdownloadhtml) Mean values wereassessed for statistically significant differences using a two-way ANOVAwith Dunnettrsquos multiple comparisons

Flight assaysFlight assays were conducted on female flies at 15degC 22degC and 25degC Theflight phenotype was determined by observing whether each fly tested flewupward (U) horizontal (H) down (D) or displayed no flight (N) whenreleased in a flight chamber (Drummond et al 1991) and quantified using aflight index equal to 6 UT+4HT+2DT+0NT where T is the total numberof flies tested (Tohtong et al 1995) Wing beat frequency (WBF) wasmeasured on 2- to 3-day-old female flies using an optical tachometer (Hyattand Maughan 1994)

Fiber mechanicsA pair of IFM bundles containing six fibers each were dissected out of 2- to3-day-old female fly thoraces and demembranated for 1 h at 4degC inskinning solution [5 mM ATP 1 mM free Mg2+ 025 mM phosphate5 mM EGTA 20 mM N N-Bis (2-hydroxyethyl)-2-aminoethanesulfonicacid (BES) at pH 70 1 mM DTT 50 glycerol and 05 Triton X-100pCa 80 and ionic strength 175 mM adjusted with Na methane sulfonate]Tungsten wire probes were used to separate and split the individual fibersAn aluminum clip was wrapped around each end of the fiber preparationThe preparations were attached to a mechanical measurement apparatusand bathed in relaxing solution (5 mM ATP 8 mM phosphate 15 mMcreatine phosphate 300 Uml creatine phosphokinase 1 mM free Mg2+5 mM EGTA 20 mM BES at pH 70 1 mM DTT pCa 80 ionic strength200 mM adjusted with Na methane sulfonate) Fibers were stretched by5 from resting length then subjected to sinusoidal analysis (see below) inrelaxing solution (pCa 80) before a partial exchange with activatingsolution (same as relaxing solution but pCa adjusted to 40) in the chamberto bring pCa to 50 The active fiber was further stretched (in 2increments of fiber length between clips) until maximum power generationwas obtained At this optimized length different relaxing activating orrigor solutions were exchanged into the chamber and further sinusoidalanalysis tension measurements or [ATP] and [Pi] variations wereperformed Sinusoidal analysis was run at the beginning and end of eachexperiment to ensure the performance of a fiber did not decrease by morethan 10 Detailed methods have been previously presented (Swank2012)

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Similarly control male yw PwMhc2 Mhc+Mhc+ flies were crossed withfemale yw to create yw PwMhc2ywMhc+Mhc+ control progeny Flies witha 21 E701KMhc+ genotypic ratio were produced by crossing male ywMhc+Mhc+ E701KE701K flies to female ww Mhc10Mhc10 E701KE701K flies yielding wyw Mhc10Mhc+ E701KE701K These flies werecompared with control flies with two transgenic wild-type myosin genes andone endogenous gene PwMhc2PwMhc2 Mhc10 Mhc+

Protein expressionRelative levels of myosin expression were determined by measuring myosin-to-actin ratios in upper thorax homogenates subjected to one-dimensionalSDS polyacrylamide gel electrophoresis (OrsquoDonnell et al 1989) Sixdissected upper thoraces from 2-day-old adult female flies for each line werehomogenized in 360 μl of SDS gel 1times Laemmli Sample Buffer (Bio-RadHercules CA) containing 5 2-mercaptoethanol Samples were boiled for5 min prior to loading at 2-5 μl on a 15-well 10 Mini-PROTEAN TGXpolyacrylamide gel (Bio-Rad) Gels stained with Coomassie Brilliant BlueR-250 were digitally scanned using an Epson Expression 636 flatbedscanner Myosin heavy chain and actin levels were determined by UN-SCAN-IT gel software (Silk Scientific) for three separate lanes per sampleThe median myosin-to-actin ratio in mutant thoraces was compared with themedian ratio found in thoraces from aged-matched flies expressing thePwMhc2 control transgene in the corresponding genetic background

Protein purificationMyosin was purified from the dorso-longitudinal set of IFMs ofsim120 youngfemale flies as previously described (Kronert et al 2008 2014 Swanket al 2001) Briefly fibers were collected in York modified glycerol[20 mM potassium phosphate (pH 70) 2 mM MgCl2 1 mM EGTA20 mM DTT and a protease inhibitor mixture] pelleted and thendemembranated for 30 min on ice in York modified glycerol containing2 (vv) Triton-X 100 After centrifugation and washing the solution wasreplaced with a high-salt extraction buffer [1 M KCl 50 mM potassiumphosphate (pH 68) 5 mM MgCl2 05 mM EGTA 10 mM sodiumpyrophosphate 20 mM DTT plus a protease inhibitor mixture] Thesoluble material was subjected to a low-salt precipitation to pellet myosinvia centrifugation a high-salt precipitation to remove residual contaminantsand another low-salt precipitation to isolate purified myosin Protein wassuspended in myosin storage buffer [05 M KCl 20 mM MOPS (pH 70)2 mM MgCl2 and 20 mM DTT] Myosin concentration was determined bymeasuring the absorption at 280 nm (Margossian and Lowey 1982) Atypical yield was sim100 microg of myosin Samples were immediately used forATPase and in vitro motility assays

G-actin was isolated from acetone powder of chicken breast muscle (Pardeeand Spudich 1982) by cycles of polymerization and depolymerization aspreviously described (Kronert et al 2014) Following dialysis actinconcentration was determined by absorption at 280 nm F-actin wasprepared by adding one volume of 10times polymerization buffer [50 mMTris-Cl (pH 8) 05 M KCl 20 mMMgCl2 10 mMATP] to nine volumes ofG-actin Actin stored on ice at 4degC was used within 1 month of preparation

ATPase activityATPase activities were determined using 2 microg of freshly prepared myosinand 1 mM [γ-32P]ATP Basal ATPase activities were assessed for 15 min inMgATPase buffer (10 mM imidazole pH 60 20 mM KCl 2 mM MgCl201 mMCaCl2) or CaATPase buffer (10 mM imidazole pH 60 01 MKCl10 mM CaCl2) at 25degC Following sample extraction with 18 M HClO4incorporation of radiolabel was determined by scintillation counting Actin-activated MgATPase activity was assessed by the addition of chickenskeletal muscle actin (01ndash2 μM) to samples prior to incubation Followingsubtraction of basal MgATPase activity from each data point the ATPaseactivity versus actin concentration was graphed The Michaelis-Mentenequation was used in conjunction with SigmaPlot (Systat Software SanJose CA US) to define Vmax Km and catalytic efficiency (VmaxKm) valuesDetailed procedures for ATPase activity determination have been previouslydescribed (Kronert et al 2008 2014 Swank et al 2001)

In vitro actin sliding velocityIn vitro actin sliding velocity assays were implemented as previouslydescribed (Kronert et al 2008 2014 Swank et al 2001) Brieflynitrocellulose-coated coverslips were treated with myosin at 05 μgμl andmyosin heads that irreversibly bound to actin were blocked with unlabeledactin filaments F-actin labeled with fluorescent phalloidin was added to thecoverslip and filament movement was digitally captured under fluorescentoptics in the presence of ATP Computational analysis of actin filamentmovement permitted determination of actin sliding velocity

Ultrastructural analysisPreparation of samples for transmission electron microscopy was carried outas previously described (Suggs et al 2007 Wang et al 2012) Sampleswere viewed on a Tecnai 12microscope at 120 kV and images were capturedwith a XR-41S 2k digital camera and software from Advanced MicroscopyTechniques

For muscle fiber measurements transverse sections at sim200 nm werecollected from 2- to 3-day-old fly thoraces prepared for ultrastructuralanalysis and embedded in resin as indicated above Sections were transferredonto a standard glass microscope slide and briefly heated to insureadherence This was followed immediately by staining in 1 MethyleneBlue and 1 borax Samples were viewed and digitally photographed at 40timesunder a light microscope A stage micrometer with 001 mm divisions wassimilarly imaged for scale calibration Cross-sectional areas of each memberof collateral pairs of DLM fibers 45d 45e and 45f (numbering according toMiller 1950) from three flies per genotype were measured using ImageJVersion 151j (httpsimagejnihgovijdownloadhtml) Mean values wereassessed for statistically significant differences using a two-way ANOVAwith Dunnettrsquos multiple comparisons

Flight assaysFlight assays were conducted on female flies at 15degC 22degC and 25degC Theflight phenotype was determined by observing whether each fly tested flewupward (U) horizontal (H) down (D) or displayed no flight (N) whenreleased in a flight chamber (Drummond et al 1991) and quantified using aflight index equal to 6 UT+4HT+2DT+0NT where T is the total numberof flies tested (Tohtong et al 1995) Wing beat frequency (WBF) wasmeasured on 2- to 3-day-old female flies using an optical tachometer (Hyattand Maughan 1994)

Fiber mechanicsA pair of IFM bundles containing six fibers each were dissected out of 2- to3-day-old female fly thoraces and demembranated for 1 h at 4degC inskinning solution [5 mM ATP 1 mM free Mg2+ 025 mM phosphate5 mM EGTA 20 mM N N-Bis (2-hydroxyethyl)-2-aminoethanesulfonicacid (BES) at pH 70 1 mM DTT 50 glycerol and 05 Triton X-100pCa 80 and ionic strength 175 mM adjusted with Na methane sulfonate]Tungsten wire probes were used to separate and split the individual fibersAn aluminum clip was wrapped around each end of the fiber preparationThe preparations were attached to a mechanical measurement apparatusand bathed in relaxing solution (5 mM ATP 8 mM phosphate 15 mMcreatine phosphate 300 Uml creatine phosphokinase 1 mM free Mg2+5 mM EGTA 20 mM BES at pH 70 1 mM DTT pCa 80 ionic strength200 mM adjusted with Na methane sulfonate) Fibers were stretched by5 from resting length then subjected to sinusoidal analysis (see below) inrelaxing solution (pCa 80) before a partial exchange with activatingsolution (same as relaxing solution but pCa adjusted to 40) in the chamberto bring pCa to 50 The active fiber was further stretched (in 2increments of fiber length between clips) until maximum power generationwas obtained At this optimized length different relaxing activating orrigor solutions were exchanged into the chamber and further sinusoidalanalysis tension measurements or [ATP] and [Pi] variations wereperformed Sinusoidal analysis was run at the beginning and end of eachexperiment to ensure the performance of a fiber did not decrease by morethan 10 Detailed methods have been previously presented (Swank2012)

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Sinusoidal analysis and muscle mechanical rate constantsTo measure muscle stiffness and mechanical rate constants of the fiberssinusoidal analysis was performed as described previously (Swank 2012Swank et al 2006) Briefly a 0125 muscle length peak-to-peakamplitude sine wave was applied to the fiber at 50 frequencies from 05 to650 Hz At each frequency the amplitude ratio and phase difference forforce and length were calculated The ratio was divided by the fiber cross-sectional area to obtain the complex elastic and viscous moduli

The complex modulus from each fiber was fitted to a 3-term equationThis equation is based on the equation originally developed by Kawai andBrandt (Kawai and Brandt 1980) for sinusoidal analysis and has beenslightly modified to be more suitable for IFMs (Swank et al 2006) Y(f )=A(2π ifα)kndashB if(b+if )+C if(c+if ) where f is the applied frequency ofoscillation (05-650 Hz) i is the square root ofminus1 α is defined as 1 Hz and kis a unit-less exponent (Swank et al 2006) The first term (A) reflects theviscoelastic properties of passive structures within the fiber while thesecond and third terms (B and C) reflect an outcome of transitions betweencross-bridge states (changes in complex moduli due to the strain sensitivityof cross-bridge rate constants) that are exponential in the time domainProcess B is primarily influenced by the work-producing steps of the cross-bridge cycle while process C is influenced by thework-absorbing steps priorto and including myosin detachment from actin (Fig S2) Processes B and Cappear as hemispheres in the Nyquist plot with characteristic frequencies band c (Swank et al 2006) In the time domain these frequencies correspondto rate constants 2πb and 2πc For a more-detailed explanation includinginformation on the relationship between these rate constants and thosederived from step analysis see Kawai and Brandt (Kawai and Brandt 1980)

Pi and ATP concentration experimentsTo obtain more information about the effect of the mutation on the cross-bridge cycle rate constants ATP and Pi concentrations were varied in thebathing solutions For the Pi experiments the following activating solutioncomponents were adjusted to 13 mM MgATP 37 mM creatine phosphate450 Uml creatine phosphokinase and an ionic strength of 260 mM For theATP experiments the following activating solution components wereadjusted to 0 mM Pi 37 mM creatine phosphate and 450 Uml creatinephosphokinase with an ionic strength of 260 mM The ATP response wasfit with an exponential rise to maximum curve

Competing interestsThe authors declare no competing or financial interests

Author contributionsConceptualization SIB DMS Methodology SIB JAS DMS ValidationGCM JAS DMS Formal analysis GCM JAS DMS InvestigationBMG MMD NPM GCM AM JAS Data curation BMG MMDNPM GCM AM JAS Writing SIB JAS DMS Supervision SIBGCM DMS Funding acquisition SIB

FundingResearch supported by grants from the Muscular Dystrophy Association(MDA217900) and the National Institutes of Health (NIGMS R01 32443) to SIB aswell as a National Science Foundation equipment grant (0308029) to the SDSUElectron Microscope Facility

Supplementary informationSupplementary information available online athttpdmmbiologistsorglookupdoi101242dmm028050supplemental

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