Cardiotonic bipyridine amrinone slows myosin-induced actin filament sliding

at saturating [MgATP]

J. KLINTH1, A. ARNER2 and A. MANSSON1,*1Department of Chemistry and Biomedical Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden;2Department of Physiological Sciences, University of Lund, BMC F11, Tornavagen 10, SE-221 84 Lund, Sweden

Received 5 March 2002; accepted in revised form 19 July 2002

Abstract

Previously reported effects of amrinone on skeletal muscle function suggest that the drug reduces the rate constantof myosin cross-bridge dissociation. We have used the in vitro motility assay to further elucidate the mechanismunderlying this effect and to aid these studies a new, improved, filament tracking software was developed in theMatlab� environment. The experiments were carried out at 30�C using heavy meromyosin from fast rabbit muscleand rhodamine–phalloidin labeled actin filaments. A slowing effect of amrinone on filament sliding velocity at1 mM MgATP was observed at drug concentrations >0.3 mM. This effect showed signs of saturation at the highestdrug concentrations (1–2 mM) that could be readily tested. The sliding velocity exhibited hyperbolic dependence on[MgATP] with a Vmax of 7.2 ± 0.9 lm/s and a KM of 0.18 ± 0.02 mM. Amrinone (1 mM) reduced Vmax by32 ± 5% (P < 0.01) and KM by 42 ± 8% (P < 0.05; n¼ 4). These results are accounted for in the moststraightforward way by a model where amrinone acts directly on the actomyosin system and reduces the rateconstant of MgADP release. Such a well-defined effect on the myosin cross-bridge cycle makes the drug apotentially useful pharmacological tool for further studies of myosin function both in vitro and in the orderedfilament array of a living muscle fiber.

Introduction

Amrinone is a bipyridine compound that was developedas a cardiotonic agent (Alousi et al., 1979) but the drughas also been shown to affect several aspects of skeletalmuscle function (Mansson and Edman, 1984, 1985;Mansson et al., 1989; Morner and Mansson, 1990;Bottinelli et al., 1993). In both intact frog muscle fibersand in intact lumbrical muscles of the mouse themaximum tetanic force was increased by the drug andthe maximum velocity of shortening was reduced(Mansson and Edman, 1985; Mansson et al., 1989).These effects were associated with a reduced curvatureof the force–velocity relationship and with greaterconformity of this relationship to a hyperbola at highforce.Furthermore the rate of redevelopment of force after

a release during the tetanus plateau was reduced(Mansson and Edman, 1985; Mansson et al., 1989).All these effects strongly suggest that amrinone affectsmyosin cross-bridge function in both the frog andmouse skeletal muscle. In the frog muscle preparationthe twitch force was also potentiated (Mansson andEdman, 1985), an effect that was not observed in themouse muscle (Mansson et al., 1989). This suggests thatin frog muscle there may be an additional effect of

amrinone on the excitation–contraction coupling in-creasing the amount of available activator calcium inresponse to an action potential (see Morner andMansson, 1990).An analysis (Mansson and Edman, 1985) using the

Huxley (1957) model suggested that the effects ofamrinone on the force–velocity relationship and on therate of force redevelopment are compatible with areduced rate constant of both cross-bridge dissociationand association. However, even though the model ofHuxley (1957) provides a very nice approximation ofcross-bridge function, it does not account for all aspectsof muscle contraction (cf. Eisenberg et al., 1980; Piazzesiand Lombardi, 1995; Edman et al., 1997). Additionalstudies were therefore needed to identify the mechanismof action of amrinone in greater detail.To this end Bottinelli et al. (1993) further investigated

the effects of amrinone on skeletal muscle function infast and slow skinned skeletal muscle fibers of the rat.These studies confirmed the effects of amrinone on themaximum isometric force and on the maximum velocityof shortening observed in the intact muscle prepara-tions. Furthermore Bottinelli et al. (1993) presentedevidence suggesting that amrinone shifts the relationshipbetween the maximum velocity of shortening and[MgATP] towards higher MgATP concentrations. Onthe other hand, the velocity of shortening extrapolatedto infinite [MgATP] was not appreciably affected. Theseresults may be interpreted to mean that amrinone acts as

*To whom correspondence should be addressed: Tel.: +46-480-

446243; Fax: +46-480-446262; E-mail: alf.mansson@hik.se

Journal of Muscle Research and Cell Motility 24: 15–32, 2003. 15� 2003 Kluwer Academic Publishers. Printed in the Netherlands.

a competitive inhibitor of the MgATP binding tomyosin. However, Bottinelli et al. (1993) also foundthat amrinone activated the myosin ATPase activity.The latter effect is not immediately explainable on basisof an inhibition of the MgATP binding.We have, in the present study, used the in vitro

motility assay to further investigate the mechanism ofaction of amrinone on actomyosin function. Our results,interpreted in terms of a kinetic model of the cross-bridge cycle, suggest that amrinone reduces the rateconstant of MgADP release rather than competitivelyinhibiting the binding of MgATP to myosin as proposedpreviously. The possibility is discussed that the effectarises from a stabilizing effect of the drug on myosin–ADP and actomyosin–ADP states. Interestingly such astabilizing effect may also be consistent with an in-creased rate of phosphate release and thus an increasedmyosin–ATPase rate in the presence of amrinone.Our findings, using rabbit myosin, are partly contra-

dictory to those of Bottinelli et al. (1993) and arediscussed in relation to their results obtained on skinnedmuscle fibers of the rat. We also discuss the possibility touse amrinone, like hitherto 2,3-butanedionemonoxime(BDM) as a pharmacological tool in the study of musclefunction (Bagni et al., 1992; Herrmann et al., 1992;McKillop et al., 1994). Like BDM, but unlike severalother substances affecting cross-bridge function (e.g.other nucleotides than ATP), amrinone freely diffusesinto the cytoplasm of living cells and its effects arereversible (Mansson and Edman, 1985; Mansson et al.,1989). These properties make the drug useful for inves-tigating aspects of myosin II function that require thehighly ordered filament lattice of a living muscle fiber.Preliminary accounts of the present work have beengiven recently (Widen et al., 2000; Klinth et al., 2001).

Model

A mathematical model was developed for the interpre-tation of in vitro motility data obtained at differentconcentrations of substrate and MgADP. The model,although focused on the in vitro motility assay, is alsouseful for interpretation of the MgATP-, and MgADP-dependence of the velocity of unloaded shortening inskinned fibers and myofibrils. In some aspects thepresent model is similar to earlier kinetic models usedfor this purpose (Ferenczi et al., 1984; Pate et al., 1992)and to some models used in studies of isometric steady-state properties and transient mechanical responses toperturbation of substrate and product concentration(e.g. Dantzig et al., 1991). The present model was,however, derived in a slightly different way and featuresaspects more specific to the present study. Explicitanalytical equations are derived to relate the velocity offilament sliding to the concentrations of MgATP andMgADP.It is assumed in the modeling that a large number of

myosin molecules act simultaneously to propel an actin

filament. We have focused on this situation since thecondition is likely to be met in our experiments (seeDiscussion). Furthermore, the possibility is consideredin the model that rigor-like heads are present and brakefilament sliding.When actin filaments slide at constant velocity (vf) the

forces acting to propel and brake the filaments exactlybalance. A simple mathematical formulation of thiscondition is given by

WUðx; yÞhavk ¼ WUðx; yÞ vfg2

k þ ð1� Uðx; yÞÞkhdrag

0OUðx; yÞO1 ð1Þ

where F(x, y) is the fraction of properly functioning (notrigor-like) myosin heads at a position (x, y) on the flow-cell surface and Y is the fraction of these heads that arein a strongly bound state at a given time. The quantityhav is the average length of the cross-bridge workingstroke, k denotes cross-bridge stiffness and g2 is the rateconstant for cross-bridge detachment at the end of thepower-stroke. Finally hdrag is the average strain of anon-functional rigor-like cross-bridge when it detachesfrom actin.The quantity Y is thought to be rather low (<0.05;

e.g. Howard, 2001) for fast rabbit myosin during slidingat maximum velocity. The numerical value of hav wastaken as 5 nm on basis of single molecule experimentsusing optical tweezers (Veigel et al., 1998) and thepossibility was considered that this value is reduced byamrinone. Higher values than 5 nm (up to 10 nm ormore) have been observed both in single proteinexperiments (Tanaka et al., 1998; Tyska et al., 1999)and in intact muscle fibers (Huxley and Simmons, 1971).However, the assumed size of the power-stroke distanceis not expected to significantly affect the simulatedpercentage effects of amrinone on vf (see Eqs. 2–3below). On the other hand, the absolute values obtainedfor some rate constants (k4 and K1k2 below) would be50% lower if the model is fitted using hav ¼ 10 nm.Cross-bridge stiffness is tentatively assumed to beconstant and similar for cross-bridges in different states.The value of the rate constant g2 depends on [MgATP],[MgADP] and, possibly, the amrinone concentration(see below).Using data in the literature hdrag may be estimated to

be about 20 nm. This may be inferred from thefollowing reasoning. At zero strain the detachment rateconstant of rigor cross-bridges has been shown to bevery low. For actomyosin in solution a value of 0.01 s)1

was obtained (Marston, 1982) and in fibers the rate ofdetachment of cross-bridges of low strain is even lower(Schoenberg and Eisenberg, 1985). The rate constant ofdetachment was increased to about 1 s)1 at a load ofabout 10 pN (Nishizaka et al., 1995). This load corre-sponds to a sliding distance of 17 nm if cross-bridgestiffness is 0.6 pN/nm (Nishizaka et al., 1995; Veigelet al., 1998). At a sliding rate > 1000 nm s)1 the dis-tance of 17 nm is traversed in less than approximately

16

20 ms and a detachment rate constant of 1 s)1 wouldnot lead to any significant detachment in this time.However, by considering the dimensions of the myosinhead it seems unlikely that the drag stroke of rigor cross-bridges could be longer than about 20 nm, here taken asthe numerical value of hdrag.Effects of viscous drag between the actin filament and

the surface are neglected in Eq. 1 since such effects arelikely to be small compared to the forces due to myosincross-bridges (e.g. Howard, 2001). Also resistive dragforces due to weakly bound cross-bridges are neglected(see further Discussion).By rearranging Eq. 1 and defining F ” F(x,y) (as-

suming homogeneous myosin function on the surface):

vf ¼ g2 hav �1� UUW

hdrag

� �ð2Þ

which transforms into

vf ¼ havg2 if U ! 1: ð3Þ

Inspection of Eq. 2 suggests that percentage effects onvf of changes in g2 would be independent of the fractionof functional ‘non-rigor’ cross-bridges in the prepara-tion. Furthermore, it is immediately clear from Eq. 2that sliding stops when (1)F)/(F) ‡ Yhav/hdrag.For fitting of the model to experimental data Eq. 3

was used rather than the more complex Eq. 2. This wasjustified since there was evidence that the fraction ofrigor cross-bridges was low in the present experiments(see Discussion).It is now of interest to find an expression for g2 as a

function of [MgATP], [MgADP] and amrinone concen-tration. In the derivation of this expression it is assumedthat the rate constant of cross-bridge dissociation duringfilament sliding is determined by transitions betweencross-bridge states at the end of the power-stroke (cf.Ferenczi et al., 1984; Siemankowski et al., 1985; Pateet al., 1992; Weiss et al., 2001). The relevant transitionsand states are shown in the following kinetic scheme:

A-M-ADP

ADP

A-M-ATPA-M M-ATP

ATP

k4([amr]) k2([amr])

Kamr

A-M-amr

amr

k -4([amr])

K1([amr])

Scheme 1.

Here k4([amr]) and k2([amr]) are dissociation rateconstants whereas k)4([amr]) is an association rateconstant for ADP binding. The quantities K1([amr])and Kamr, on the other hand, are binding constants forthe binding of ATP and amrinone, respectively to theactive site of myosin. Actin, myosin and amrinone aredenoted by A, M and amr respectively. It is implicit thatthe nucleotide in Scheme 1 exists as a complex withMg2+.

The possibility that amrinone might compete withATP binding to myosin is represented by the stateA–M–amr and the binding constant Kamr. The constantsk4([amr]), k)4([amr]), k2([amr]) and K1([amr]) have‘[amr]’ as an argument to indicate that they might varywith the amrinone concentration. Such effects of amri-none could occur if the drug binds to a site involved inthe structural changes that accompany transitions be-tween actomyosin states (see further Discussion). Thisdrug binding is, in the modeling, assumed to occurwithout competition with nucleotide binding. A moredetailed treatment of this case would be to introduce athird row in Scheme 1 with actomyosin–nucleotide–amrinone states. Furthermore, a binding constant foramrinone binding, which might vary with the nucleotidespecies should be introduced. However, in the presentwork we applied modeling to the situations eitherwithout amrinone or with saturating concentrations ofthe drug. Under these conditions the binding constantgoverning the transition from an actomyosin–nucleotidecomplex to an actomyosin–nucleotide–amrinone com-plex would not appear in the modeling of the data.Instead the actomyosin states would be completelyshifted towards the amrinone-free and amrinone boundspecies, respectively in the two conditions justifying theuse of amrinone dependent rate constants k4([amr]),k)4([amr]), k2([amr]) and K1([amr]). Furthermore, weonly have to consider two sets of values for theconstants, one set in the absence of amrinone and oneset in the presence of drug. With this in mind wesimplify: k4 ” k4([amr]), k)4 ” k)4([amr]), k2 ” k2([amr])and K1 ” K1([amr]).The cross-bridge states in Scheme 1 (A–M–ADP,

A–M, A–M–ATP and, A–M–amrinone) may be lumpedtogether into one state As.Thus

½As� ¼½AMADP� þ ½AM� þ ½AM amrinone�þ ½AMATP� ð4Þ

where [As], [AMADP], [AM], [AM amrinone] and[AMATP] represent the fraction of the myosin cross-bridges that are in the states As, A–M–ADP, A–M,A–M–amrinone and A–M–ATP, respectively. A disso-ciation constant g2 during filament sliding may then bedefined by

d½As�=dt ¼ �g2½As�: ð5Þ

At high [MgATP] ðk2K1½MgATP�Þ � k4. Hence (aftersetting [AM]tot¼[AM] + [AM amrinone]) the followingrelationship would be valid for most of the range ofcross-bridge strains for which detachment occurs duringshortening:

d½AM�tot=dt ¼k4½AMADP� � ðk�4½AM�½MgADP�þ K1k2½AM�½MgATP�Þ � 0: ð6Þ

Now, by applying Eqs. 4–6 and rapid equilibrium con-ditions for the transitions at the end of the power-stroke

17

and, furthermore, assuming that Kamr> 0 it can beshown that g2 may be approximated by:

g2 �k4½MgATP�

k4K1k2

þ ½MgATP� þ k�4

K1k2½MgADP� þ Kamrk4

K1k2½amrinone�

ð7Þ

Using similar reasoning as for high [MgATP] it is clearthat Eq. 7 is approximately valid also at low [MgATP](where k2K1 ½MgATP� � k4).Eq. 7 may thus be used to approximate g2 as a

function of [MgATP], [MgADP] and amrinone concen-tration at all levels of [MgATP]. In our experiments[MgADP] is close to zero and the corresponding term inthe denominator of Eq. 7 is neglected in the analysis. Byinserting Eq. 7 into Eq. 3 it is also clear that vf as afunction of the MgATP concentration is well approxi-mated by a hyperbolic relationship with Michaelianconstants KM¼k4/(K1k2) and Vmax¼k4hav. The inhibi-tion constant Ki for the inhibitory effect of MgADP isapproximately given by (see also Pate et al., 1992)

Ki ¼k4k�4

ð8Þ

Materials and methods

Protein preparations and SDS–PAGE

Myosin was obtained from white leg muscle (�20 g)from New Zeeland white rabbits (female, �2 kg). Theanimals were anaesthetized with an intramuscular injec-tion of Hypnorm� (fentanyl 0.1 mg/kg, fluanisone3 mg/kg; Janssen Pharmaceutical Ltd, Oxford, UK)followed by intraperitoneal injection of Dormicum�(midazolam, 2 mg/kg; Roche, Basel, Switzerland) andkilled with an intravenous injection of pentobarbital(6 mg/kg; Apoteksbolaget AB, Umea, Sweden). Themuscle was immediately washed in ice cold 0.15 M NaCland myosin was then extracted and purified as describedby Sata et al. (1993). The isolated myosin was eitherused directly for preparation of heavy meromyosin(HMM) or stored for up to 1 week at )20�C before use.The storage solution contained 50% (v/v) glycerol andhigh KCl concentration (0.6 M). HMM was preparedessentially as described by Kron et al. (1991) usingTLCK-treated a-chymotrypsin (Sigma, St Louis, MO)and was used in motility assay within 48 h.Actin was purified from bovine heart muscle following

the protocol of (Pardee and Spudich, 1982) and wasstored as F-actin either on ice for at most 1 week orfrozen in liquid nitrogen and stored at )80�C for up to3 months.Protein concentration was determined spectrophoto-

metrically at 280 nm (myosin and HMM) or 290 nm(actin) using extinction coefficients (dm3 g)1 cm)1) equalto those given by Kron et al. (1991): 0.53 (myosin), 0.60(HMM) and 0.62 (actin). The purity of the final protein

preparations was confirmed by sodium dodecyl sul-phate–polyacrylamide gel electrophoresis (SDS–PAGE).In order to assess the purity of actin and myosinpreparations and the light chain composition of themyosin preparations we used Bio Rad 12% Tris-HClprecast minigels (BioRad Laboratories, Hercules, CA).The electrophoresis and Coomassie brilliant blue R-250staining was carried out according to manufacturersrecommendations and 5 lg of protein was applied toeach lane. The separation and identification of differentmyosin heavy chain isoforms was also performed usingSDS–PAGE and comassie blue staining essentially asdescribed by Talmadge and Roy (1993). The separatinggels were thus prepared to contain 8% acrylamide-bis(50:1) and the running conditions were 70 V for 22 h ina minigel system. The amount of protein was 2 lg perlane in this case.

Solutions and assay conditions

Amrinone (obtained as a gift from Sanofi-SynthelaboInc, Paris, France) was dissolved in 0.5 M lactic acidgiving a stock solution with concentration 214 mm. Thewash solution, used in several preincubation and rinsingsteps, contained (mM): KCl 25, MgCl2 4.0, EGTA 1.0,DTT 1.0, imidazole-HCl 25, pH 7.4.The composition of the assay buffer was as follows

(mM): KCl 67–84, MgCl2 3.9–6.9, Na2ATP 0.01–3.0,EGTA 1.0, dithiotreitol (DTT) 11, lactic acid 2.5,imidazole-HCl 25, pH 7.2. The adjustment of pH withNaOH increased the sodium concentration by 12.8 mMin both the wash solution and the assay buffer. Theassay buffer also contained 0.6% methylcellulose and ananti-bleach mixture composed of glucose (3 mg/ml),glucose oxidase (0.1 mg/ml) and catalase (0.02–0.2mg/ml depending on activity). In three out of the fourexperiments where the MgATP concentration wasvaried creatine phosphate (CP; 10 mM; disodium salt)and creatine kinase (CK; 0.1 mg/ml; Sigma) were alsopresent in the assay buffer and the concentration of KClwas varied to keep the ionic strength at approximately160 mM. In other experiments the ionic strength wasapproximately 130 mM and no CP or CK was present.A computer program (http://www.stanford.edu/

�cpatton/maxc.html) was used to calculate ionicstrength and free concentrations of Mg2+ and MgATP.The stability constants used in the calculations werethose given by Godt and Lindley (1982) and Andrewset al. (1991). The motility assay was performed, inprinciple, as described by Kron et al. (1991). A flow cell(volume �30 ll), with one surface coated with nitrocel-lulose (Colloidon; Electron Microscopy Sciences, FortWashington, PA) was used. This cell was incubated withHMM (60 lg/ml) in wash buffer for 2 min. Next, twoblocking solutions were added. First, free proteinbinding surfaces on the nitrocellulose film were blockedby incubation with bovine serum albumin (BSA; Sigma;0.5–1 mg/ml) in wash buffer and second, non-functionalrigor-like heads were blocked by incubation with unla-

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beled (non-fluorescent) actin filaments (5 lM) in thepresence of 1 mM MgATP. The unlabeled actin fila-ments were sheared through a hypodermic needle (G27)prior to use. Before infusion of actin filaments (20 nM)labeled with rhodamine–phalloidin (RhPh; MolecularProbes, Eugene, OR) the flow cell was rinsed in twovolumes of wash solution to remove unlabeled actinfilaments and MgATP. Before addition of assay buffer,with or without amrinone, the flow cell was preincubat-ed for 30 s with a wash solution containing either thedrug or vehicle alone (2.5 mM lactic acid). The temper-ature varied between 28 and 32�C between differentexperiments but was kept constant to ±0.5�C during agiven experiment.The velocity of filament sliding (vf) was studied at 3–5

different positions in a given flow cell. These positionswere, in all cases (test and control), chosen close to theflow-cell opening at which solutions were infused and allmeasurements were taken within 15 min after infusionof assay buffer.

Observation system

In all experiments actin filaments were imaged using epi-fluorescence illumination and a Nikon 100·, 1.4 N.A.temperature regulated oil immersion objective. Initialexperiments were performed using a Nikon TMDinverted microscope (Nikon Inc., Tokyo, Japan) asdescribed in Svensson et al. (1997). In a majority of theexperiments, however, a Nikon Eclipse TE 300 invertedmicroscope was employed. In these experiments actinfilaments were recorded using an ORCA-ER (C-4742-95-12ER; Hamamatsu, Hamamatsu-City, Japan) cooledCCD-camera without image intensifier (8-bit images).The frame rate was 2–4.2 s)1 depending on the velocityof filament sliding. Each frame consisted of 252 · 252pixels and the size of each pixel corresponded to a0.26 · 0.26 lm2 square piece of the imaged object.

Analysis of assay data – tracking and analysis software

In a few early experiments the video was replayedthrough the argus-10 system and 10–12 filaments wereselected from each flow-cell for analysis. Analysis andcalculation of filament sliding velocities were thenperformed essentially as described by Svensson et al.(1997). In the following this method of analysis will bedenoted the ‘manual method’.For the majority of the experiments program routines,

created in the Matlab� environment, were used foreither ‘semiautomatic’ or fully automated analysis ofimage sequences recorded by the ORCA-ER camera.Matlab v. 6.0 (The MathWorks, Natick, MA) was usedfor the program development.In the case of ‘semiautomatic’ analysis neither filtering

nor other processing was applied to the image sequence.Furthermore the filaments to be analyzed were selectedby the experimenter and their positions were defined byselecting the coordinates of their leading or trailing end

using the computer mouse. When using this method ofanalysis the velocity of at least 10 filaments wasmeasured for each flow-cell studied.In the fully automated analysis each frame in a

sequence was first filtered (Wiener2 filter) to removepixel noise, then treated to equalize the backgroundillumination and filtered again (unsharp contrast en-hancement filter). Thereafter automatic thresholdingwas performed in order to convert the sequence of grayscale images into a black and white (binary) imagesequence. The threshold level was selected to fulfill twocriteria. First the number of white pixels should be lessthan a pre-selected value (1000–10,000) and secondly,the number of white objects should be less than anotherpre-selected value, normally 100. The appropriatethreshold level was obtained by first iteratively, in stepsof five, increasing it from zero up to 75 and counting thenumber of resulting white objects and white pixels,respectively. The threshold level was then selected as thelowest level for which the selection criteria above werefulfilled. Finally the binary image was treated bymorphological operations to bridge white pixels sepa-rated by single black pixels. The final output in form ofthe binary image sequence could be viewed and if it wasnot satisfactory the image processing was repeated.Next this binary image sequence was analyzed with

the aim of tracking individual actin filaments fromframe-to-frame and calculating their velocity. Filamentsin the first frame of the sequence were labeled using anobject detection routine of the Matlab Image AnalysisToolbox. A given filament in the first frame was foundin the second frame by first twice dilating the image ofthe filament (adding pixels along the filament perimeter)and then searching for a filament in the second framethat overlapped this dilated image. In the third framethe same filament was found as an object overlappingthe filament image of the second frame once dilated andshifted along a vector defining the velocity between thefirst and second frame. The latter procedure was repeatedover 13 additional frames to track the filament for morethan 3 s. An event where two filaments were crossing wasdetected through a sudden jump in the centroid positionof the filament. In this case the standard deviation (SD)of the frame-to-frame velocity was greatly increased andthe filament was normally excluded from the analysis onthis basis (see below). Overlapping filaments that sepa-rated during the analysis procedure were also identified.One of these separating filaments was tracked and itsvelocity was measured. The program was constructed toavoid duplicating the tracking of this filament during alater phase of the analysis.The distance that a filament moved between conse-

cutive frames, and the frame-to-frame velocity, were, inthe automatic analysis, calculated either on basis ofchanges in the position of the filament centroid (‘cent-roid method’; Homsher et al., 1992) or using the ‘pathreconstruction/perimeter method’ (Hamelink et al.,1999). The calculations were performed essentially asdescribed earlier (Homsher et al., 1992; Hamelink et al.,

19

1999) but the increase in perimeter was, in the pathreconstruction method, calculated using built-in func-tions of the Matlab Image Analysis Toolbox.In both the automatic and semiautomatic methods the

position or path length traced out by a filament wasobtained in at least 15 consecutive frames and a runningaverage of the frame-to-frame velocity was calculatedover eight frames. The velocity finally considered to bestrepresent smooth filament movement was that obtainedin the eight-frame window where the ratio between theSD and the mean (vmean) of the frame-to-frame velocityattained its minimum. No measured velocities wereincluded in the analysis if the ratio SD/vmean was >0.20(vf > 1 lm/s) or 0.30 (vf £ 1 lm/s).Errors in velocity estimation or spurious filament

paths may be introduced in automatic analysis due tovarious sources of noise (cf. Homsher, 1992). Sucherrors or false filament paths were largely eliminated inthe present analysis by the SD/vmean exclusion criterion(see above). However, in order to further reduce the riskof errors in the measurements filaments were alsoexcluded from the analysis on basis of the followingtwo criteria indicating negligible movement: 1. the rootmean square distance (Davg) of the filament from thestarting point (in all frames studied) was less than orequal to 1 lm or 2. a ratio DR=DS � 0:5. Here

DR ¼Xni¼2

xi

���������� ð9Þ

where n is the number of frames and xi is the vectorrepresenting the filament centroid position in the framei relative to the position in the first frame (i¼1). Thequantity DS, on the other hand, is the value of DS if thefilament had been moving with the constant velocityvmean along a straight path. The two exclusion criteriatogether with the criterion based on SD/vmean werefound to eliminate all stationary filaments (94 out of 94)from the analysis in one test experiment. In thisexperiment the filaments were either locked to thesurface in the absence of ATP or non-specificallyadsorbed to a BSA coated surface in the absence ofmyosin. In the latter case the filaments exhibitedsignificant Brownian movement. The above three crite-ria also lead to exclusion of several filaments thatfollowed strongly curved paths.The results of the automatic and semiautomatic

analysis were compared for three different myosinpreparations in the presence and absence of amrinoneand at different concentrations of MgATP. The result ofthe comparison is shown in Figure 1 where the velocitiesobtained using the centroid method or the path recon-struction method (see above) are plotted against thevelocities obtained using the semiautomatic method. Itcan be seen that the correlation between the semiauto-matic measurements and the two sets of automaticmeasurements is good (r � 0:97 and 0.98) and the slopeof the regression lines are not significantly different fromone as expected for the case of complete correspondence.

In the present work manual or semiautomatic meth-ods of analysis were used in the majority of theexperiments whereas automatic analysis was used in afew of the late experiments.

Analysis of the relationship between MgATPconcentration and velocity of filament sliding

If not otherwise stated hyperbolae (Michaelis–Mentenequations) were fitted to all data points at MgATPconcentrations of 1 mM and below.In three of the experiments, where [MgATP] was

varied, CK and CP were present in the assay buffer andthe ionic strength was 160 mM. These experimentalconditions differed from those of the remaining experi-ments of the study (ionic strength 130 mM and no CKand CP present). However these differences do notappear to affect the results to any significant degree.This is suggested by two findings: 1. In one experimentwhere CK and CP were absent both the absolute valuesof Vmax and KM and the effects of amrinone on theseparameters fitted well in among the results of the otherthree experiments (see Figure 5b); 2. Neither vf in thecontrol solution nor the effect of amrinone on vf at1 mM MgATP seemed to be affected by the smalldifference in ionic strength and the presence of CK andPCr (see Table 1).

Fig. 1. Velocity of filament sliding in automatic analysis (vaf ) plottedvs. velocity in semiautomatic analysis (vsaf ) in the same image

sequences. Either the centroid method (open circles) or the path

reconstruction method (filled circles) was used in the automatic

analysis. Data obtained in three different myosin preparations at

different concentrations of MgATP and amrinone. Each data point

represents average velocity (±SEM) obtained in measurements on

10–33 filaments using the semiautomatic method and 10–127 filaments

using either of the automatic methods. Error bars shown for

semiautomatic measurement only if SEM was greater than in the

automatic analysis. When no error bars are shown SEM was smaller

than the size of the symbol. The straight lines (partly overlapping) were

derived by linear regression of vaf on vsaf for the centroid method and

for the path reconstruction method. Dotted and dashed lines represent

95% confidence intervals of the regression lines.

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Statistical analysis and curve fitting

The statistical analysis of the data in Figure 3 wasperformed in the software Statistica v 5 (StatSoft, Tulsa,OK) using repeated measures analysis of variance. Thisanalysis procedure was followed by Tukey’s post hoc testfor difference between individual drug concentrations.The analysis was performed on logarithmically trans-formed velocity data. The reason for this approach wasthat the percentage effects of amrinone (but not theabsolute effects) on filament velocity were independent ofthe velocity in the control solution (cf. Figure 4, below).The logarithmic transformation also had the effect to

make the variance of the filament velocity more homo-geneous at different concentrations of amrinone. In theother experiments of this study statistical significance ofdifferences between means was tested using Student’st-test for paired observations. Linear and non-linearregression was performed using the Graph Pad Prismsoftware, (GraphPad Software Inc., San Diego, CA). Innon-linear regression curves were fitted to the experi-mental data using a Marquardt algorithm to minimizethe sum of squared differences between the fitted lineand the experimental data. Statistical significance of anydeviation of experimental data from a fitted curve wastested using a run’s test. Here the number of ‘runs’ ofresiduals is compared to the number expected if thedeviations of the experimental data points from thefitted curve is due to random variations. A ‘run’, in thisconnection, is a sequence of data points that are eitherall above or all below the fitted curve.All data are given as mean ± standard error of the

mean. The number (n) of experiments correspondsdirectly to the number of separate myosin preparationstested.

Results

Characterization of proteins

HMM was obtained from rabbit leg muscle thatcontained mainly fast myosin. The dominance of fastmyosin is clear from the typical gels in Figure 2 showingmyosin and HMM preparations from a given muscle.The myosin light chain composition (Figure 2a) ischaracteristic for fast muscle as suggested by compari-son with psoas and soleus muscles (not shown; see alsoPate et al., 1992). Furthermore the 8% SDS–PAGE ofFigure 2b suggests that the myosin heavy chains from

the leg muscle (‘vastus’) were of the fast types IIx andIIb (cf. Tikunov et al., 2001). The concentration ratiobetween the two isoforms varied only slightly betweendifferent preparations as suggested by the integratedintensity of the corresponding bands (IIx/IIb: �0.6–1)studied in five randomly selected preparations. It canalso be seen, in the somewhat overloaded gels inFigure 2a, that the concentration of most proteins otherthan actin and proteolytic fragments of myosin was lowin the HMM preparation. Also, the actin preparationsexhibited a high degree of purity (Figure 2a, lane 3).

Table 1. Effects of 1 mM amrinone on the velocity of filament sliding (vf) at 1 mM MgATP in the presence and absence of CK and CP

No CK/CP CK/CP All experiments

No amrinone (vf, lm/s) 4.82 ± 0.54 (n = 7) 5.98 ± 0.57 (n = 4) 5.24 ± 0.42 (n = 11)

Amrinone effect (%) )26.3 ± 3.4* (n = 7) )26.3 ± 5.2*** (n = 3) )26.3 ± 2.7* (n = 10)

* statistical significance, P < 0.001 in t-test for paired observations.

*** statistical significance, P < 0.05 in t-test for paired observations.

Fig. 2. SDS–PAGE of myosin, HMM and actin preparations. (A)

acrylamide 12% (wt/vol) gel showing preparations of myosin, HMM

and actin. The HMM preparation was obtained from the myosin

preparation analyzed in the left lane. Molecular weight markers are

shown to the right. From top: 206, 119, 91, 51, 35, 28, 20 kD. (B)

acrylamide 8% (wt/vol) gel illustrating typical myosin heavy chain

composition of the myosin preparations (‘vastus’) used in the present

work (middle lane). Myosin preparations from rabbit soleus and rabbit

psoas also shown for comparison. Myosin isoforms indicated to the

right of gel: IIx, myosin heavy chain IIx; IIb, myosin heavy chain IIb

and I, myosin heavy chain I.

21

Effects of amrinone on filament sliding velocity at 1mMMgATP

Table 1 summarizes the effects of 1 mM amrinone onthe velocity of filament sliding (vf) at 1 mM MgATP(tested in all experiments of this study). It can be seenthat the drug reduced vf by 26.3 ± 2.7% (P < 0.001)from a value of 5.2 lm/s in the control solution. Theeffect was of similar magnitude whether or not CP andCK were present in the assay solution. The observedeffect is in good agreement (also quantitatively) with theeffect of 1 mM amrinone on the unloaded shorteningvelocity of intact skeletal muscle preparations (Manssonand Edman, 1985; Mansson et al., 1989).The effects of amrinone on vf were investigated for

different concentrations of the drug. In these experi-ments no CP or CK was present and [MgATP] was1 mM. It is clear from Figure 3 that there was nosignificant effect of amrinone at drug concentrations of0.3 mM and below. On the other hand, when theconcentration increased from 0.3 to 1 mM there was asharp reduction in velocity. This reduction appeared tosaturate at a value of about 25% at drug concentrationsof 1 mM and higher. The statistical analysis of the datain Figure 3 was limited to data at 0, 0.1, 1 and 2 mMamrinone since these concentrations were tested in allexperiments. The mean velocity differed significantlyfrom the control value (P < 0.05) both at 1 and 2 mMamrinone but not at 0.1 mM. Furthermore the velocitiesat both 1 and 2 mM amrinone differed significantlyfrom the velocity measured at 0.1 mM amrinone(P < 0.01). Due to limited solubility of the drug, atthe pH used, higher amrinone concentrations than2 mM were not studied. We can therefore not fully

exclude the possibility that the concentrations in ourexperiments were below the level of saturation. How-ever, it seems unlikely in view of the very similar effectsrecorded at 1 and 2 mM amrinone (not significantdifference, P � 0:9).There was some variability, between different myosin

preparations, in the velocity of filament sliding. Thisvariability is presumably due to the presence of a smallbut variable amount of non-functional, rigor-like, myo-sin heads (see further Discussion). If this is the correctinterpretation and if amrinone reduces the rate constantfor cross-bridge dissociation, as proposed previously(Mansson and Edman, 1985), Eq. 2 predicts that thepercentage effects of amrinone would be similar inmagnitude independent on the velocity. This predictionis confirmed by the experimental data in Figure 4 whereit can be seen that the slope of the regression line of Dvf(%) upon vf is not significantly different from zero(P � 0:2).

Effects of amrinone on velocity of filament slidingat varied [MgATP]

Effects of amrinone on the velocity of filament slidingwere studied in four myosin preparations at concentra-tions of MgATP ranging from 0.03 to 3 mM. The resultsare shown in Figure 5a. Here the average velocity in thepresence and absence of amrinone is plotted as afunction of [MgATP]. Fitting of rectangular hyperbolato the experimental data suggest that amrinone reducedboth the sliding velocity (Vmax) at infinite concentrationof MgATP and the concentration of MgATP (KM) atwhich velocity is equal to Vmax/2. In this connection it isof interest to note that a runs test suggested nosignificant deviation of the data from the hyperbolicmodel neither in the absence (P¼ 1.00) nor in thepresence of amrinone (P¼ 0.30). The effects on Vmax

and KM are also seen if the data in Figure 5a (except the

Fig. 3. The effect of different concentrations of amrinone on filament

sliding velocity (vf). Bars represent standard error of the mean and

figures in parentheses are the number of myosin preparations.

Filament velocity normalized to velocity in the absence of the drug

before calculation of averages. The concentration of the vehicle (lactic

acid) was 2.5 mM for all concentrations of amrinone. Line represents

equation vf¼A+(B)A)/(1+10([Amrinone]-log EC50)) fitted to the data

with A¼ 0.724, B¼ 1.178, EC50¼ 0.5. Statistical significance of

difference from control (*P < 0.05) or from velocity at 0.1 mM

amrinone (*P < 0.01). Only data at 0, 0.1, 1 and 2 mM amrinone

were compared in the statistical analysis.

Fig. 4. The percentage reduction (Dvf) caused by 1 mM amrinone in

the velocity of filament sliding (vf) plotted as a function of vf in the

control solution. Full line (regression line) and dashed lines (95%

confidence interval) obtained by regression analysis (r � 0.45). Same

data as in Table 1.

22

data points at 0 and 30 lM MgATP) are replotted in adouble reciprocal diagram (Figure 5b). Here the inter-cept on the horizontal axis is shifted to the left by 1 mMamrinone consistent with a reduction of KM. This

change is associated with a shift upwards of the intercepton the vertical axis in accordance with the decrease ofVmax. The quantities Vmax and KM (estimated using non-linear regression with respect to the untransformed data)were found to be reduced by 32.1 ± 5.2% (P < 0.01)and 42.3 ± 7.6% (P < 0.05), respectively (n¼ 4; pairedobservations). The average values of Vmax and KM in thecontrol solution were 7.2 ± 0.9 lm/s and 0.18 ± 0.02mM, respectively. It can be seen in Table 2 that similarresults were obtained whether the data points at 3 mMMgATP were included in the curve fitting or not.In consistence with results in the preceding section it is

also shown in Table 2 that vf at 1 mM MgATP wassignificantly reduced by 1 mM amrinone. Furthermore,1 mM amrinone also reduced the filament velocity at3 mM MgATP (n¼ 2) to about the same extent al-though the change was not statistically significant. At0.1 mM MgATP (below KM), on the other hand, vf wasnot altered by the drug. These findings constituteevidence, independent of curve fitting, that amrinonereduced both Vmax and KM.The effects of the drug on KM may be more conve-

niently judged from plots of normalized velocity against[MgATP]. This is illustrated in Figure 5c where theaverage values of vf from the four myosin preparationshave been normalized to vf at 1 mM MgATP in bothtest and control. It is clear from the plots of normalizedvelocity that there was a tendency for all normalizedvalues of vf at [MgATP] £ 0.3 mM to be higher in thepresence of amrinone than in its absence. If amrinoneproduced no change in KM one would, at least to a firstapproximation, expect that 50% of the normalizedvelocity data points would be reduced and 50% wouldbe increased by the drug. In contrast, the ratio betweenthe numbers of data points that showed reduced andincreased normalized velocity was 0/3, 0/4, 0/4 and 1/3,respectively for the four myosin preparations.In all experiments of Figure 5 ‘manual’ or ‘semiauto-

matic methods’ (see Materials and methods) were usedto track filaments in the in vitro motility experiments.Since tracking methods where filaments are selected bythe observer may be subject to bias it was important toconfirm the results using an alternative method ofanalysis. A computer program was therefore developedto automatically analyze velocities of smoothly movingfilaments.Evidence that no appreciable bias was introduced in

the semiautomatic analysis is presented in Figure 1 thatshows excellent correlation between results obtainedusing automatic and semiautomatic analysis. However,a comparison of the semiautomatic tracking and the twodifferent methods of automatic tracking was alsoperformed to directly confirm the effects of amrinoneon sliding velocities at different [MgATP]. The resultsare presented in Figure 6. It is clear that the agreementbetween the results obtained using the different methodsis, in general, very good. This applies both to theindividual data points and to the amrinone inducedchanges of KM and Vmax. One exception is the control

Fig. 5. Effects of amrinone on vf at different concentrations of

MgATP. (A) The average filament velocity vf plotted vs. [MgATP].

Data normalized to velocity at 1 mM MgATP in the control solution

(vf1c) for each myosin preparation before calculation of averages. Open

circles: control solution. Filled circles: 1 mM amrinone. Rectangular

hyperbolae fitted to the averaged data shown by full line (control

solution: Vmax � 1.19 and KM � 0.18 mM) and dashed line (amri-

none: Vmax � 0.82 and KM � 0.098 mM). Bars represent standard

error of the mean. The data points refer to averages from four myosin

preparations except for the data points at 0.06 and 3 mM MgATP that

refer to three and two myosin preparations, respectively. (B) Double

reciprocal plot of the data in A (except data points at 0 and 0.03 mM

MgATP). Control solution: open circles and full line. Amrinone

(1 mM): filled circles and dashed line. Lines fitted using linear

regression. (C) Data in A at [MgATP] £ 0.3 mM replotted after

normalization, in both test and control, to the velocity at 1 mM

MgATP (vf,1) in respective solution.

23

data point at 3 mM MgATP obtained using the ‘cent-roid method’ (Figure 6a). However, since this data point

is based on only 10 useful filament paths the result maybe attributed to random variation. This explanation wasconfirmed by semiautomatically measuring the velocityof all filaments (to avoid selection bias). The result (opensquare in Figure 6a) was in good agreement with thesemiautomatic measurements on 10 selected filamentsand with the measurements obtained using the otherautomatic method (Figure 6b). The experimental dataof Figures 1 and 6 thus support the view that semi-automatically measured filament velocities are reliableeven if they are obtained from just 10 filaments selectedby the observer. The data in Figure 6 also support theabove conclusion that both KM and Vmax are reduced byamrinone.

The sliding velocity does not depend on fılament length

The automatic tracking program was used to study therelationship between filament velocity and the apparentarea of the filament image, which is likely to be roughlyproportional to filament length. Study of altogether 270filament images in two experiments showed no correla-tion (r � �0:02) between vf and filament area. Similarresults were obtained in the presence of 1 mM amri-none. These findings suggest that the sliding velocity inour experiments did not vary with the length of thefilaments and the use of the model (Eqs. 3, 7) is appro-priate from this point of view (see further Discussion).

Modeling of results

The relationship between [MgATP] and vf in thepresence and absence of amrinone was modeled onbasis of Scheme 1 and Eq. 1. In the fitting of the model(Eqs. 3,7) to the data the constant hav was taken as5 nm. The best fit of Eqs. 7 and 3 to the experimentaldata in the absence of amrinone was then obtained byvarying the rate constants K1k2 and k4 in the non-linearregression. The result is illustrated in Figure 7a (fullline) where k4 � 1690 s�1 and K1k2 � 9850 mM)1 s)1

(r2 � 0.994). As mentioned in the Model section thevalues of these rate constants would have been 50%lower if hav had been assumed to be 10 nm instead of5 nm. However, the interpretation of the amrinoneeffects in terms of proportional changes in different

Table 2. Effects of 1 mM amrinone on the relationship between MgATP concentration and the velocity of filament sliding, vf

0–1 mM MgATPa (n = 4) 0–3 mMa (n = 2) and 0–1 mMa

(n = 2) MgATP, pooled

Dvf at different [MgATP]

DVmax, % DKM, % DVmax, % DKM, % 0.1 mM 1 mM 3 mM

)32 ± 5%* )42 ± 8%** )31 ± 6%** )40 ± 10%** +5.2 ± 5.0% )29 ± 5%* )29 ± 4%

(n ¼ 4) (n¼ 4) (n¼ 2)

a Michaelis–Menten equation fitted to data in this range of MgATP concentrations. Amrinone induced changes in Vmax (DVmax) and KM (DKM)

given as percentage of control value.

* statistical significance, P < 0.01 in t-test for paired observations.

** statistical significance, P < 0.05 in t-test for paired observations.

Fig. 6. Different automatic and semiautomatic methods used to track

and measure velocity of actin filaments (vf) at different concentrationsof MgATP in the absence (open symbols and crosses) and presence of

1 mM amrinone (filled symbols). Data from one myosin preparation.

Bars represent standard error of the mean. Lines represent hyperbola

(Michaelis–Menten equation) fitted to the data. (A) Automatic

‘centroid’ method (crosses or filled squares; 8–41 filaments per point)

compared to semiautomatic method with analysis of 10–33 selected

filaments per point (circles and full lines) or analysis of all smoothly

moving filaments (open square; 55 filaments). Dashed lines represent

Michaelis–Menten equations fitted to data obtained using automatic

method, except for the control curve in A. Here the data point at

3 mM MgATP was that based on semiautomatic measurements on all

filaments (open square). (B) Automatic ‘path reconstruction’ method

(diamonds and dashed lines; 20–100 filaments per point) compared to

semiautomatic method as in A (circles and full lines; 10–33 filaments

per point). Amrinone reduced the Michaelian constant Vmax by

25–30% according to the different methods and produced a reduction

of KM by 22% (semiautomatic), 12% (dashed lines in A) or by 22%

(path reconstruction method).

24

constants are not expected to be appreciably affected bythe precise value of hav (see Eqs. 2–3).Different hypotheses for the effects of amrinone were

considered. First, as suggested by Mansson and Edman(1985) and Bottinelli et al. (1993) amrinone might act asa competitive inhibitor of MgATP binding. This wouldcorrespond to Kamr > 0 in Eq. 7 and would lead to arightward shift of the relationship between the MgATPconcentration and vf. On the other hand, the velocity(Vmax) at infinite [MgATP] would be unaffected. Sucheffects are clearly inconsistent with the experimental

data in Figure 5 and Table 2. Consequently, a ratherpoor fit (r2 � 0.71) of the model to the data wasobtained on the assumption of competition of amrinonewith MgATP binding. Another possibility to considerwould be that amrinone reduces the second order rateconstant K1k2 for actomyosin dissociation. However,inspection of Eq. 7 shows that the effects would besimilar to those of competitive inhibition of MgATPbinding. Thus, KM would be increased whereas Vmax

would not be affected.The two simplest remaining possibilities for the

mechanism of action of amrinone are that the drugeither reduces the rate constant k4 for MgADP releaseor the average power-stroke distance, hav. A reductionof the rate constant for MgADP release, k4, would beexpected to produce quantitatively similar reductions ofKM and Vmax. This is illustrated in Figure 7a where themodel (Eqs. 3 and 7) is fitted to the experimental data onthe assumption that k4 is the only model parameter thatis altered by the drug. The best fit (r2 � 0.976; dashedline in Figure 7a) was obtained if k4 was reduced by29% from the control value.In contrast to a reduction of k4 an isolated reduction

of hav by amrinone would not affect KM. Such a lack ofchange in KM is in conflict with what we observed and,consequently, the fitting of the model to the data gave afit (r2 � 0.938) that was less good than that obtained ifk4 was the only parameter varied.The modeling above thus suggests that the effect of

amrinone on the actin filament sliding velocity in vitro ismediated by a reduced rate constant, k4, of MgADPrelease. If amrinone affects sliding velocity only throughthis mechanism one would expect a proportional reduc-tion in Vmax and KM in each given experiment (Eq. 7).This was also observed. It can thus be seen in Figure 7bthat the experimental data for the amrinone inducedchange in KM vs. the change in Vmax are close to the linerepresenting a reduction of k4 in the model. It is also ofinterest to note that even if the 95% confidence interval(not shown) for the regression line is large the dashedline in Figure 7b, representing a decrease in hav, isoutside this confidence interval.

Discussion

Summary of the results and outline of the discussion

A major result of the present study is that amrinoneslows myosin-induced actin filament sliding in vitro athigh (>0.1 mM) but not at low [MgATP]. Modelingsuggests that amrinone exerts these effects by reducingthe rate constant of MgADP release. In the following wewill discuss the experiments and the validity of themodel. We will then consider the postulated effect ofamrinone on cross-bridge function in relation to resultsin the literature. Finally the potential usefulness ofamrinone as a pharmacological tool in the study ofcross-bridge function will be considered.

Fig. 7. Modeling of results. (A) Experimental data in Figure 5

replotted using the same symbols. Model parameters varied in Eqs. 7

and 3 to obtain the best fit to the data in the control solution. In the

fitting procedure the parameter hav was assumed to be fixed at 5 nm.

Furthermore, [MgADP]¼ 0 mM and Kamr¼ 0. The best fit to the

control data (full line) was then obtained for k4 � 1690 s)1 and

K1k2 � 9850 mM)1 s)1 (r2 � 0.994). The absolute values of the rate

constants were obtained by scaling the simulated results to the absolute

value of Vmax (7.2 lm/s). The effects of 1 mM amrinone were

simulated on the assumption that 1 mM amrinone reduces the rate

constant k4 without additional effects. The best fit is illustrated by

dashed line (k4 down by 28.8%; r2 � 0.976). Other constants as for

control curve (full line). (B) Percentage effect of 1 mM amrinone (filled

squares) on KM (DKM) plotted vs. percentage effect of the drug on Vmax

(DVmax). The DVmax and DKM data shown by filled squares were

obtained from four different myosin preparations. Full line derived by

linear regression of DKM on DVmax (slope:1.03 ± 0.73; r2 � 0.50).

Dashed horizontal line (DKM¼ 0) represents expected form of rela-

tionship between DKM and DVmax if the only effect of amrinone is a

reduction of hav. Dashed line with slope 1 represents expected

relationship between DKM and DVmax if amrinone reduces k4 without

other effects.

25

Validity of model

The model derived in this paper assumes that a largenumber of myosin molecules act simultaneously topropel an actin filament. This condition must be met toallow the use of average values for working stroke andtime (1/g2) spent by the cross-bridges in an attachedstate that resists shortening. The assumption of a largenumber of simultaneously working myosin heads islikely to be correct under our experimental conditions.Thus a majority of the actin filaments studied had alength clearly exceeding 1 lm and the flow cells wereincubated for 2 min with a HMM concentration of60 lg/ml. Uyeda et al. (1991) showed that the velocityof filament sliding saturated at filament lengths >0.5 lmif the concentration of HMM in the incubation solutionwas 40 lg/ml or greater. It might be argued that thehigher ionic strength in our experiments, than in thoseof Uyeda et al. (>100 mM compared to 25 mM) wouldrequire longer filaments and higher HMM-concentra-tion for saturation. However, if saturation were notreached one would expect considerably higher velocitiesfor long than for short actin filaments. This was notobserved in our experiments.In the absence of ‘rigor-like’ heads the sliding velocity

as a function of MgATP may be approximated by Eq. 7inserted into Eq. 3. This equation, like similar equationsused previously for interpretation of skinned fiberexperiments, predicts that: (1) the velocity of filamentsliding at high MgATP concentration is limited by therate constant of MgADP release, (2) Km and Vmax forthe hyperbolic relationship between sliding velocity andthe MgATP concentration should vary roughly inproportion if the average power-stroke distance, havand the product K1k2 are approximately constant and(3) the apparent inhibition constant, Ki, for the inhibi-tion of the sliding velocity by MgADP is given by theratio k4/k)4.The first prediction above has been corroborated by a

wealth of experimental data (e.g. Ferenczi et al., 1984;Siemankowski et al., 1985; Pate et al., 1992; Ma andTaylor, 1994; Weiss et al., 2001). The second predictionis also in general agreement with results in the literature.Thus, Stienen et al. (1988) found a direct proportional-ity between Vmax and KM in skinned fast and slowskeletal muscle fibers from the frog Xenopus laevis. Alsoin other studies using in vitro motility assays (Yamashitaet al., 1994) or skinned fibers (Pate et al., 1992; Lofgrenet al., 2001) a co-variation of Vmax and KM wasdemonstrated. However, in these studies there was nodirect proportionality between the two parameters. Pateet al. (1992), in their experiments on rabbit skeletalmuscle, found that, compared to fast muscle fibers, KM

for the unloaded velocity of shortening is reduced byabout 10 times in slow muscle whereas Vmax decreasesonly by a factor of about three. Qualitatively similarresults were obtained by Yamashita et al. (1994) in theircomparison of skeletal and cardiac myosin using thein vitro motility assay. The co-variation is generally

consistent also with results obtained in fast and slowsmooth muscle preparations. Thus, Lofgren et al.(2001), in experiments on skinned smooth muscles fromthe guinea pig, found that Vmax for the unloadedshortening velocity was reduced proportionally morethan KM (about 7 vs. 4·) in a slow muscle (aorta)compared to a fast one (Taenia coli). It should be noted,however, that the results of Pate et al. (1992); Yamash-ita et al. (1994); Lofgren et al. (2001) are not inconsis-tent with the present model even if there is no directproportionality between Vmax and KM. These effectsmay be interpreted within the framework of the model ifthere, in addition to differences in k4, also are differ-ences, between fast and slow muscle fibers, in the secondorder dissociation rate constant K1k2 or in the power-stroke distance, hav.The third prediction of the model, i.e. that

Ki, ADP¼k4/k)4 is also well consistent with experimentaldata. If k4¼500 s)1 and k)4¼5000 mM)1 s)1 (at 22�C;cf. Sellers, 1999) then Ki, ADP would be 0.1 mM which isin reasonable agreement with experimental results ob-tained using the in vitro motility assay (Homsher et al.1992: 0.12 mM; Yamashita et al., 1994: 0.48 mM) orskinned fibers (Pate et al., 1992: 0.2 mM; Seow andFord, 1997: 0.33 mM).The effect of contamination of the myosin preparation

with rigor-like heads was also modeled. The modelingsuggests that the relationship between the ratio (non-functional HMM-molecules/functional HMM-mole-cules) and the velocity of sliding is rectilinear, reachingzero velocity when the ratio exceeds Whav/hdrag � 0.05 �5/20 � 0.01 (see Eq. 2 and text in the Model section). Ifthe properties of N-ethylmaleimide (NEM) treatedmyosin heads are considered similar to those of rigorheads (cf. King and Greene, 1987) this prediction is ingood agreement with the experimental data of Warshawet al. (1990). These authors found that the actin filamentmovement along myosin filaments was completelyblocked if 1% of NEM-myosin was co-polymerized inthe filaments with unmodified skeletal muscle myosin.Since the average sliding velocity in our experiments

was higher than 5 lm/s there is a reason to believe thatthe fraction of rigor-like heads was considerably lowerthan 1%. This is also consistent with our observationthat the fraction of moving filaments normally exceeded75%. In line with these observations we neglected anyeffects of rigor-like bridges and used Eq. 3 rather thanEq. 2 in our modeling of the experimental data.In two recent papers (Stehle and Brenner, 2000;

Amitani et al., 2001) it was proposed that the maximumactin filament sliding velocity to a large extent is limitedby resistive drag forces caused by weakly bound cross-bridges. Such effects have been neglected in the presentmodeling for several reasons. First the proposed mech-anism, based on weakly bound bridges, does not seem tobe consistent with several results from the literature (seeabove). These results include the correlation of the rateconstant of ADP release with the sliding velocity (e.g.Ferenczi et al., 1984; Siemankowski et al., 1985; Pate

26

et al., 1992; Ma and Taylor, 1994; Weiss et al., 2001)and the inhibiting effect of increased ADP concentrationon velocity (Homsher et al., 1992; Pate et al., 1992;Yamashita et al., 1994). The latter effect, on the otherhand, is accounted for very well (also quantitatively) bythe model used here. Also other results seem to be atvariance with the idea that resistive effects of weaklybound bridges limit sliding velocity, at least under thepresent experimental conditions of relatively high ionicstrength (130–160 mM). Thus, Homsher et al. (1992)found that the sliding velocity was virtually independentof ionic strength within the range 75–150 mM. A similarresult was obtained by Gulati and Podolsky (1981) for arange of ionic strengths between 50 and 190 mM inskinned fibers. If weakly bound cross-bridges had asignificant limiting effect on the sliding velocity in theseranges of ionic strengths the velocity would be expectedto increase with increasing ionic strength since thenumber of weakly bound cross-bridges would be ex-pected to decrease under these conditions (cf. Schoen-berg, 1988). Other findings by Homsher et al. (1992)also support the idea that there are only minor effects ofweakly bound cross-bridges on sliding velocity. Thus,neither increased concentration of inorganic phosphatenor the addition of ATP-cS had any appreciable effecton sliding velocity in the in vitro motility assay. Bothinterventions would be expected to increase the fractionof weakly bound cross-bridges on the expense ofstrongly bound force-producing ones. The nucleotideATP-cS is presumed to bind to actomyosin and ‘lock’the myosin cross-bridges in a state corresponding to theweakly bound actomyosin–ATP state. Inorganic phos-phate, on the other hand, would be expected to increasethe fraction of weakly bound cross-bridges by reversingthe cross-bridge power-stroke.

Software for automatic analysis of in vitro motility data

A computer program for automatic analysis of thein vitro motility data was developed in the Matlabenvironment utilizing several built-in functions of theMatlab Image Analysis Toolbox. The program per-formed well as suggested by comparison with trackingand analysis routines where the filament positions wereselected by the experimenter using the computer mouse.The performance was good both for the version of theprogram that was based on the centroid method (e.g.Homsher et al., 1992) and for the version that was basedon the path reconstruction method of Hamelink et al.(1999). The former method, however, had the advantageof faster analysis.The results in Figure 1 suggest that the present

program may be used with high accuracy and precisioneven in situations where only few filaments (<20) can beanalyzed. This fact may be attributed to the use ofseveral exclusion criteria to exclude spurious filamentpaths. If only an exclusion criterion SD/vmean > 0.30 (cf.Homsher et al., 1992) or SD/vmean > 0.5 and vmean <0.8 lm/s (Homsher et al., 1996) was employed several

stationary filaments may be erroneously included in theanalysis (unpublished observations). Moreover thesestationary filaments often exhibit an apparent velocitythat is considerably higher or lower than the meanderived from the smoothly moving filaments. Theaddition of the two exclusion criteria based on DS/DS

and Davg also add new constraints for the characteriza-tion of filament sliding as stationary, erratic or smooththus extending the usefulness of the automatic analysis.

Possible sources of experimental errors

A. Possible bias in the selection of filaments for analysisWe observed that in some experiments the quality of themotility varied between different parts of a flow cell. Ithas also been shown that there may be a 10% differencein the average velocity between different cells in spite ofother experimental conditions being similar (Homsheret al., 1996). For practical reasons only about 10filaments from each flow cell were usually selected for‘manual’ and ‘semiautomatic’ analysis. In this procedurethe risk of errors due to bias must be considered,particularly since the effects of amrinone on velocity arenot considerably larger than the normal variationbetween flow cells. However, it is highly unlikely thatany of the effects of amrinone described in this paper isthe result of bias in filament selection. First, the effectsof amrinone were detected by casual observers. Second,in a few experiments both semiautomatic and differentmethods of automatic analysis of experiments wereperformed and the agreement between the sets of datawas excellent (see Figs. 1, 6). Finally, the filamentsanalyzed were located in predetermined and correspond-ing positions in the flow cells in test and control.

B. Effects related to sampling rateIn early experiments filament position was sampled at arate of 0.5–3 s)1 the lowest sampling rates being usedonly at low sliding velocities. If the filaments had notbeen selected for straight paths a sampling rate of3 s)1at the highest sliding velocities might have lead toan underestimation of the velocity (Homsher et al.,1992). However, filaments were selected for relativelystraight paths in the manual measurements when thesampling rate of 0.5–3 s)1 was employed. This wouldreduce the error in the velocity estimation. Moreimportantly the lower sampling rate in the earlyexperiments would not be expected to contribute tothe effects of amrinone described in this paper. First, atlow velocity of sliding (i.e. [MgATP] �0.1 mM; velocity� 3 lm/s) the effects of a low sampling rate on velocitywould be very small. Second, at high velocity of sliding alow sampling rate would be expected to cause greaterunderestimation of the velocity the higher the truevelocity of sliding. Thus the velocity would be expectedto be reduced more in the absence of amrinone than inits presence and the effect of the drug on Vmax would beunderestimated rather than overestimated. It is, in thisconnection, important to note that we observed no

27

significant difference in the effects of amrinone in theearly and late experiments where the sampling ratediffered up to twofold. This suggests that any compli-cations related to different sampling rate in someexperiments would be of minor significance.The sampling rate of 4.2 s)1 used in later experiments

is sufficient to obtain an accurate estimate of even thehighest velocities observed in the present study. This issuggested by studies of Homsher et al. (1992) whoshowed that the velocity (at about 5 lm/s) was under-estimated by 7%, using the centroid method, when thesampling rate was 2 s)1 and by, at most, 2% when thesampling rate was 5 s)1. This underestimation is likelyto be even less in the present study since the most curvedfilament paths are excluded from the present analysis bythe condition that DS/Pfinal £ 0.5 and Davg < 1 lm.

C. Complicating effects caused by the presenceof non-functional ‘rigor-heads’Modeling (Eq. 2) shows, as expected, that the presenceof ‘rigor heads’ would reduce the velocity of filamentsliding considerably and above a critical fraction of suchheads sliding would cease altogether. More importantly,the modeling suggests that the percentage effects ofamrinone would be virtually independent of any pres-ence of rigor heads in the preparation if amrinonereduces the rate constant of cross-bridge dissociation assuggested previously (Mansson and Edman, 1985;Mansson et al., 1989; Bottinelli et al., 1993). This fitswith the observation that the slope of the regression linebetween velocity of sliding and the percentage ofamrinone induced reduction in velocity was not signi-ficantly different from zero. The variability in velocity,on the other hand, may very well be due to a variablepresence of rigor heads in different preparations sincethe myosin isoform composition seemed to be fairlyconstant between preparations. This idea is consistentwith our observation (not shown) that a lower averagevelocity in general correlated with a larger fraction ofnon-moving filaments.

Mechanism of action of amrinone and relation to earlierwork

Strong evidence was presented in previous work thatamrinone exerts its effects on muscle mechanics throughactions independent of changes in the activation mech-anism. Mansson and Edman (1985) thus presentedevidence that the effects of amrinone on maximumtetanic tension, maximum velocity of shortening and theshape of the force–velocity relationship occurred also atfull calcium activation of the contractile machinery.These findings were later confirmed in skinned fastskeletal muscle fibers from the rat. Furthermore, aspointed out by Bottinelli et al. (1993) the fact that theeffects are present in the skinned fiber suggests that theyare not mediated by binding of the drug to cytosolicproteins since these are likely to be absent in themembrane free preparation. One such protein that

would otherwise have been a potential target foramrinone is phosphodiesterase. The inhibition of thisenzyme by the drug is underlying its positive inotropiceffects on heart muscle (e.g. Endoh et al., 1982).The conclusion in the earlier investigations (Mansson

and Edman, 1985; Bottinelli et al., 1993) that amrinoneproduces its effects through an action directly on themyosin cross-bridges is strongly supported by thepresent work. First, considering the concentration ofHMM and actin in the flow cells, the procedures forisolation of these proteins and for performing themotility assay it seems highly unlikely that even traceamounts of cytosolic proteins should remain in afunctional form in the motility assay. In agreement withthese ideas the SDS–PAGE suggested very little co-purification of other proteins in the actin, myosin andHMM preparations. Second, by knowing the proteinconcentration of actin and HMM solutions added to theflow cell and by further examining the gels in Figure 2 italso seems unlikely that the effects of the drug aremediated indirectly by binding to a myofibrillar protein(e.g. tropomyosin, troponin etc.) other than actin ormyosin. The concentration of these proteins relative toHMM and actin were reduced significantly compared tothe situation in a muscle fiber. The maximum velocity offilament sliding, on the other hand, was reduced byamrinone to about the same extent in our experiments asin the rat skinned fiber (Bottinelli et al., 1993) and as inintact muscle fibers of the frog (Mansson and Edman,1985).The modeling in the present work suggests that a

reduced rate constant of MgADP release in the myosincross-bridge cycle (reduction of k4; Scheme 1) providesthe most straightforward explanation for the effects ofamrinone on the velocity of filament sliding. Thus, thesimultaneous and roughly proportional reduction ofVmax and KM is accounted for in the simplest way if themain effect of amrinone is a reduction of k4 with onlyminor, if any, effects on other parameters such as K1k2and hav (see Eq. 7).The possibility that amrinone exerts its actions on the

filament sliding velocity mainly by competitive inhibi-tion of MgATP binding, as proposed earlier (Manssonand Edman, 1985; Bottinelli et al., 1993), is clearlyinconsistent with our results that suggest a decrease ofboth KM and Vmax in response to the drug. A compet-itive inhibition of MgATP-binding would instead beexpected to increase KM and produce no change in Vmax.Effects of amrinone of the latter type were observed byBottinelli et al. (1993) in their experiments on skinnedrat muscle fibers. The reason for the discrepancybetween our results and those of Bottinelli et al. isunclear. For instance it seems inconceivable that thepresence of the ordered filament lattice of the musclefiber and the presence of accessory myofibrillar proteinssuch as troponin, tropomyosin etc. would transform aninhibition of MgADP release in the in vitro motilityassay into inhibition of MgATP binding in the musclefiber. Thus, although this possibility cannot be excluded,

28

the effects of some other perturbations on filamentvelocity, e.g. increase in the concentrations of MgADPor inorganic phosphate, Pi, seem to be similar in themuscle fiber and in the in vitro motility assay (cf.Homsher et al., 1992; Pate et al., 1992; Yamashita et al.,1994). It is also of interest to point out that the similaritybetween fibers and the in vitro motility assay seems to beparticularly well documented for high temperature andhigh ionic strength (Homsher, 1992), as used in thepresent work.Another possibility to account for the differences

between our results and those of Bottinelli et al. (1993)is that they are due to the species difference. We haveused fast skeletal muscle myosin from the rabbit whereasBottinelli et al. (1993) used muscle fibers from fastskeletal muscle of the rat. Such an explanation for thedifference would mean that amrinone affects myosinfrom quite closely related species through differentmechanisms. The idea may be worth considering furtherbut this is outside the scope of the present work. It mayalso be hypothesized that the difference between ourdata and those of Bottinelli et al. (1993) is a complica-tion caused by scatter in the experimental data. Suchscatter may lead to uncertainties in the fitting of ahyperbola to the data points. However, we havepresented evidence independent of curve fitting thatboth KM and Vmax were reduced by amrinone. Thus at0.1 mM MgATP (just below KM) 1 mM amrinoneproduced no change in the velocity of filament slidingcorresponding to a significant increase in the normalizedvelocity. At both 1 and 3 mM MgATP, on the otherhand, amrinone reduced the velocity to about the samedegree. These effects are clearly different from the effectsof a substance such as MgADP. This nucleotide isgenerally assumed to behave as a competitive inhibitorof MgATP binding (Pate et al., 1992; Yamashita et al.,1994; see Eq. 7) and, in consistence with this idea itsignificantly increases the apparent KM for the relation-ship between [MgATP] and vf (e.g. Homsher et al., 1992;Yamashita et al., 1994).An inhibition of MgADP release, and thereby slowing

of cross-bridge dissociation from a strongly bound state,would be consistent with the main contractile effects ofamrinone observed in an intact muscle fiber i.e. areduction of the maximum velocity of shortening and anincrease of the maximum tetanic tension (Mansson andEdman, 1985). However, one effect of amrinone, firstobserved by Bottinelli et al. (1993), at first sight, seemsdifficult to reconcile with the effects of the drug on cross-bridge detachment kinetics. Amrinone was thus foundto significantly increase the Ca2+dependent ATPaseactivity of myosin at high ionic strength.We have confirmed this effect of the drug on the

myosin ATPase by studying the Mg2+dependent ATP-ase (500 mM KCl) of rabbit myosin in preliminaryexperiments (Mansson et al., unpublished). The effectsof amrinone on the myosin ATPase suggest that the drugincreases the rate constant of the phosphate release sincethis step is known to be rate limiting for the myosin

ATPase (Ma and Taylor, 1994). This effect, at first sight,appears to be mechanistically unrelated to inhibition ofMgADP release. However, a simultaneous activation ofthe myosin ATPase and an inhibition of the MgADPrelease, actually point to the interesting possibility thatamrinone stabilizes the myosin–ADP and the actomyo-sin–ADP states. Such an effect may very well be achievedby binding of the drug to a single site on myosin. Thisinterpretation is consistent with the finding that onepoint mutation in Dictyostelium myosin II, which reduc-es both the rate of release of MgADP and the in vitrosliding velocity, also activates the myosin ATPase(Murphy et al., 2001). This mutation (S456L) affectedthe switch II loop near the nucleotide-binding site onmyosin and, in addition to the effects on myosin ATPaseand on the release of MgADP, it also produced a largereduction in the myosin step size (hav). However, the ideathat amrinone affects hav, in addition to the proposedeffects on k4, is not supported by the in vitromotility datadescribed here. Furthermore, a reduction of step size isnot readily consistent with the observations that amri-none increases the maximum isometric tension in rat(Bottinelli et al., 1993), mouse (Mansson et al., 1989)and frog muscle fibers (Mansson and Edman, 1985). Inaddition, preliminary experiments (Mansson, unpub-lished) suggest that amrinone does not reduce theamount of length change needed to bring the tension tozero in a frog single muscle fiber during maximumactivation. The latter effect would be expected if the stepsize were reduced (Huxley and Simmons, 1971).Irrespective of the effect on the step size, however, the

mutation result (Murphy et al., 2001), clearly shows thata change in a single amino acid is sufficient to simul-taneously activate the myosin ATPase and reduce boththe rate of MgADP release and the velocity of actinfilament sliding at high [MgATP]. The effect of severalother point mutations in Dictyostelium myosin II areconsistent with this result. Thus, mutations in both theswitch II loop (I455A: Sasaki and Sutoh, 1998) and in(or near) the SH1–SH2 helices (G691C: Patterson et al.,1997; C678G and C678A: Suzuki et al., 1997) have beenshown to activate the myosin ATPase while reducing thevelocity of actin filament sliding. Furthermore, just likeamrinone, the mutations of the SH2 cysteine (C678Gand C678A; Suzuki et al., 1997) also lead to increasedforce generation per actin filament length as measuredusing optical tweezers. It would be of interest to clarifywhether amrinone mimics the consequences of any ofthe mentioned mutations by binding to myosin close toone of the mutation sites.Dual effects of amrinone on myosin ATPase and on

the rate constant of MgADP release may, as mentionedabove, be consistent with a stabilizing effect of the drugon the M–ADP and on the A–M–ADP states (Scheme1). Such a mechanism exhibits resemblances to themechanism proposed by Herrmann et al. (1992) tounderlie the effect of 2,3-butanedionemonoxime (BDM)on myosin function. However, in the case of BDMthe intermediate that appears to be stabilized is the

29

M–ADP–Pi state. This effect was accompanied by anincreased equilibrium constant for the ATP hydrolysisstep (preceding the M–ADP–Pi state) and a reduced rateconstant for the transition out of the M–ADP–Pi state.This would be analogous to the effects of amrinone toincrease the rate of transition into the M–ADP state butto reduce the rate of ADP release.

Amrinone as a pharmacological probe of cross-bridgefunction

The properties of amrinone would make it a usefulpharmacological tool for further studies of the myosincross-bridge function in systems representing differentlevels of structural organization, from isolated proteinsto myofibrils and intact muscle cells. It may, for instance,be of interest to first study the effects of amrinone onmuscle fiber mechanics and then apply the in vitromotility assay to myosin from this fiber (cf. Canepariet al., 1999; Thedinga et al., 1999; Hook and Larsson,2000). The drug may prove to be a most useful probe inthe process of correlating the behavior of single mole-cules with the behavior of an ensemble of motor proteinsin the highly ordered filament lattice of the intact musclecell. Thus, like for instance BDM, amrinone diffuses intoliving muscle fibers and its contractile effects in thesepreparations are reversible (Mansson and Edman, 1985;Mansson et al., 1989). Furthermore, we have presentedevidence to suggest that amrinone specifically stabilizesthe actomyosin–ADP state without effects on ATPbinding and the power-stroke distance. A drug withsuch a well-defined mechanism of action would be usefulfor imposing additional constraints on cross-bridgemodels of muscle contraction (e.g. Eisenberg et al.,1980; Piazzesi and Lombardi, 1995; Edman et al., 1997).The possibility to perturb cross-bridge function in intactsingle muscle fibers is of particular value. Thus, the greatstability and sarcomere uniformity of such preparationsallow the study of contractile phenomena that cannot beobserved or are less pronounced in, for instance, skinnedmuscle fibers. One such phenomenon is the doublehyperbolic shape of the force–velocity relationship thathas been clearly established for intact frog muscle fibers(Edman, 1988; Edman et al., 1997) but has not beenobserved or is less pronounced in skinned fibers (cf. Louand Sun, 1993). In order to correctly simulate the non-hyperbolic force–velocity relationship Edman et al.(1997) found it necessary to assume that the rateconstant of cross-bridge attachment increases with thevelocity of shortening. However, an alternative interpre-tation would be that the two heads of a myosin moleculework independently at low velocities of shortening (nearisometric conditions) but act cooperatively in sequenceduring shortening at moderate velocities to increase thepower output. Such cooperativity has recently beenconsidered for myosin II by several authors (Conibearand Geeves, 1998; Ito et al., 1999; Tyska et al., 1999;Berger et al., 2001; Lauzon et al., 2001) and if it exists itmost likely depends on some fine tuning of the ADP

release from one of the heads with attachment of theother head (cf. Conibear and Geeves, 1998). Thissuggests that the cooperativity may be affected by adrug (e.g. amrinone) that affects the rate constant of theADP release step in the cross-bridge cycle. It is interest-ing, in this connection, that previous studies (e.g.Mansson and Edman, 1985; Mansson, 1989) haveprovided indications that amrinone affects the force–velocity relationship, to make the non-hyperbolic shapeless apparent. It would be of interest to make moredetailed studies of the effects of amrinone on muscle fibermechanics and then interpret the results by modifyingexisting mathematical models to include cooperativitybetween the myosin heads.

Acknowledgements

We are grateful to associate professor Bjorn Wohlfartfor initiating the work on a filament tracking software.We wish to thank Mrs Kerstin Frolander and MrsChristina Persson for excellent technical assistance andAssociate professor Sven Tagerud and Lecturer Kata-rina Gustafsson for ongoing discussions and valuableadvice during the course of this investigation. Discus-sions on statistical evaluation with Associate professorsStaffan Carius and Patrik Dinnetz are gratefully ac-knowledged as well as advice by professor CarloReggiani on the separation of myosin heavy chainisoforms on SDS–PAGE. We are also grateful to Dr ASonesson, Mr German Wannhoff-Guerra, Miss CeciliaWiden and Miss Emma Johansson–Karlsson for theirimportant contributions in the initiation phase of theproject. This study was supported by grants from theScience Faculty, University of Kalmar, The SwedishResearch Council (projects no. F 5107-20006076 [A.M.]and 04x-8268 [AA]) and the Crafoord Foundation.Finally we gratefully acknowledge the gift of amrinoneby the Sanofi-Synthelabo Inc.

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