The mechanism of the force enhancement by mgADP under simulated isChaemic conditions in rat cardiac myocytes

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Previous investigations on the mechanical effects of

ATP, its breakdown products and other metabolites have

contributed greatly to our understanding of the actomyosin

interaction in skeletal (e.g. Bremel & Weber, 1972; Cooke

& Pate, 1985; Hibberd et al. 1985) and cardiac muscle

preparations (e.g. Fabiato & Fabiato, 1975, 1978). However,

most of our knowledge concerning the effects of MgADP on

muscle mechanics originates from experiments performed

on skeletal muscle preparations at physiological pH in the

absence of inorganic phosphate (Pi; Siemankowski et al.1985; Cooke & Pate, 1985; Hoar et al. 1987; Westerblad et al.1998; Lu et al. 2001).

The effects of MgADP on the cross-bridge cycle in cardiac

tissue are particularly of interest during advanced stages

of cardiac ischaemia when intracellular Pi and H+

accumulation diminish force generation (Elliott et al.1992). It has been suggested that after the depletion of the

phosphocreatine (CP) store, MgATP hydrolysis increases

MgADP significantly in the myofibrillar compartment at the

expense of MgATP. Thus MgADP may potentially contribute

to the development of ischaemic contracture (Ventura-

Clapier & Veksler, 1994; Veksler et al. 1997; Stapleton &

Allshire, 1998). However, in the presence of Pi and low pH,

quantitative data on the effects of [MgADP] on cardiac force

generation and on the cross-bridge cycle are sparse.

We therefore decided to investigate the interactions

between [MgATP], [MgADP] and both Ca2+-dependent

and Ca2+-independent force production under simulated

ischaemic conditions (pH 6.2 and 30 mM Pi). Progression

of the metabolic disturbance during ischaemia was

mimicked by substituting MgADP for MgATP ([MgATP] +

[MgADP] = 5 mM) in a concentration-dependent manner.

In additional experiments the effects of a decrease in MgATP

concentration without an increase in MgADP concentration

were tested to isolate the MgADP-dependent component of

ischaemic force restoration.

The mechanism of the force enhancement by MgADP undersimulated ischaemic conditions in rat cardiac myocytesZoltán Papp, Ágnes Szabó, Jan Paul Barends * and G. J. M. Stienen *

Department of Physiology, University of Debrecen, Medical and Health Science Center, Medical School, H-4012 Debrecen, Hungary and *Laboratoryfor Physiology, Institute for Cardiovascular Research, Vrije Universiteit, 1081 BT Amsterdam, The Netherlands

In this study, the effects of MgADP and/or MgATP on the Ca2+-dependent and Ca2+-independent

contractile force restoration were determined in order to identify the origin of the tonic force

increase (i.e. ischaemic contracture) which develops during advanced stages of ischaemia.

Experiments were performed at 15 °C during simulated ischaemic conditions in Triton-skinned

right ventricular myocytes from rats. In the presence of 5 mM MgATP the maximal Ca2+-

dependent force (Po) of 39 ± 2 kN m_2 (mean ± S.E.M.) under control conditions (pH 7.0, 15 mM

phosphocreatine (CP)) decreased to 8 ± 1 % during simulated ischaemia (pH 6.2, 30 mM inorganic

phosphate (Pi), without CP). This change was accompanied by a major reduction in Ca2+ sensitivity

(pCa50 4.10 vs. 5.62). Substitution of MgADP for MgATP restored isometric force production and

its Ca2+ sensitivity (pCa50 4.74 at 4 mM MgADP and 1 mM MgATP). In addition, it shifted the

MgATP threshold concentration of Ca2+-independent force development to higher levels in a

concentration-dependent manner. However, Ca2+-independent force was facilitated less by MgADP

than Ca2+-dependent force. The MgADP-induced increase in force was accompanied by marked

reductions in the velocity of unloaded shortening and the rate of tension redevelopment. These data

and simulations using a model of cross-bridge kinetics suggest that the ischaemic force is not a

consequence of a reduction in intracellular MgATP concentration, but identify MgADP as a key

modulator of the cross-bridge cycle under simulated ischaemic conditions in cardiac muscle, with a

much lower inhibition constant (0.012 ± 0.003 mM) than in skeletal muscle. Therefore, MgADP has

a high potential to stabilize the force-generating cross-bridge state and to facilitate the development

of ischaemic contracture, possibly involving a Ca2+ activation process in the ischaemic

myocardium.

(Received 10 April 2002; accepted after revision 27 May 2002)

Corresponding author Z. Papp: Department of Cardiology, University of Debrecen, Medical and Health Science Center,Medical School, PO Box 1, H-4004 Debrecen, Hungary. Email: [email protected]

Journal of Physiology (2002), 543.1, pp. 177–189 DOI: 10.1113/jphysiol.2002.022145

© The Physiological Society 2002 www.jphysiol.org

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The interpretation of skinned fibre experiments in the

presence of MgADP and the absence of CP is complicated

by intracellular adenine nucleotide gradients caused by

ATP hydrolysis and diffusion of the reactants (e.g. Cooke

& Pate, 1985). In order to minimize the intracellular

concentration gradients, isolated Triton-permeabilized

myocytes were used in this study. In addition, model

calculations were employed to evaluate the influence of the

adenine nucleotide concentration profiles inside the

cardiomyocytes.

The MgADP-specific changes in the kinetics of cross-

bridge interaction were assessed by measuring the

unloaded shortening velocity (Vo) and rate constant of

tension redevelopment (ktr). These parameters allowed the

recognition of the dominant role of MgADP on cross-

bridge transition rates in the presence of Pi, low pH and

[MgATP], and the determination of a significantly lower

inhibitory constant (Ki) of MgADP for myofibrillar

ATPase in cardiac muscle than in skeletal muscle (Sleep &

Glyn, 1986).

In the Appendix, a cross-bridge model is presented which

accounts for the influence of the MgADP and MgATP

concentrations on the isometric force under ischaemic

conditions in cardiac muscle. This model is based on

the formalism described by Pate & Cooke (1989) and

incorporates more recent experimental (Kawai et al. 1993)

and theoretical developments (Slawnynch et al. 1994).

These modelling efforts suggest that the enhancement of

the force-promoting role of MgADP under ischaemic

conditions is a natural consequence of thermodynamic

changes in the free energy profiles of the different cross-

bridge states.

Thus, experimental results and model calculations both

indicate that MgADP accumulation may result in a more

pronounced tonic force increase under ischaemic

conditions than under physiological conditions in the

myocardium.

METHODS Myocyte isolation and experimental set-upExperiments were performed in accordance with local ethicalguidelines. Myocytes were isolated mechanically as describedpreviously (Van der Velden et al. 1998). Briefly, hearts of Na-pentobarbitone-anaesthetized (20–35 mg (kg body weight)_1, I.P.,Sanofi, Libourne, France) Wistar rats (300–600 g) of either sexwere rapidly excised. After excision, the hearts were perfusedaccording to Langendorff with Tyrode solution (equilibrated with95 % O2–5 % CO2) containing (mM): NaCl 128.3, KCl 4.7,CaCl2 1.36, MgCl2 1.05, NaHCO3 20.2, NaH2PO4 0.42, glucosemonohydrate 11.1 and 2,3-butanedione monoxime (BDM) 30(Sigma, St Louis, MO, USA) (20 °C). The free right ventricularwall was then immersed in relaxing solution for cell isolationand mechanically disrupted within 5–10 s, using a tissuehomogenizer. The resultant suspension of small clumps ofmyocytes, single myocyte-sized preparations and cell fragments

was permeabilized with 0.3 % Triton X-100 (Sigma) for 5 min,washed and kept in relaxing solution for cell isolation at 0 °C for6–24 h. A single myocyte was attached with silicone adhesive(100 % silicone, Dow Corning) to two thin stainless-steel needleswhile viewed by means of an inverted microscope. One needle wasattached to a force transducer (SensoNor, Horten, Norway)and the other to a piezoelectric motor (Physike Instrumente,Waldbrunn, Germany), both connected to joystick-controlledmicromanipulators. After curing for 50 min, the preparation wastransferred from the mounting area to a small temperature-controlled well (volume 68 ml) containing control relaxingsolution (Table 1), from which the myocyte could be transferredto a similar temperature-controlled well containing activatingsolution. During cell attachment and subsequent forcemeasurements, myocytes were viewed at a magnification of w 320.Images were captured by means of a charged coupling device(CCD) video camera and stored on a personal computer. Theaverage sarcomere length was determined by means of a spatialFourier transform as described previously (Fan et al. 1997) andadjusted to 2.2 mm. The diameters of the preparation weremeasured microscopically, in two perpendicular directions.Cross-sectional area was calculated by assuming an ellipticalcross-section. All chemicals were obtained from Merck(Darmstadt, Germany), unless indicated otherwise.

SolutionsThe composition of the solution used for cell isolation was (mM):MgCl2 1, KCl 100, EGTA 2, Na2ATP 4 and imidazole 10 (pH 7.0,adjusted with KOH). All solutions for force measurementscontained 1,2-bis(2-aminophenoxy)ethane-N,N,N‚N‚-tetraaceticacid (BAPTA; Acros Organics, Fairlawn, NJ, USA) as a Ca2+ buffer,because its Ca2+-chelating properties are less affected by low pHthan those of EGTA. The purity of the BAPTA was determined asdescribed by Harrison & Bers (1987) and was found to be 97.3 %.The compositions of the solutions for force measurements(shown in Table 1) were calculated as described previously(Stienen et al. 1999), using the stability constants listed byBrooks & Storey (1992) with appropriate temperature and ionicequivalent corrections (Harrison & Bers, 1989). The calculatedfree Mg2+ and total MgATP and MgADP concentrations ofsolutions were 1 and 5 mM, respectively, unless indicatedotherwise. Physiological conditions were mimicked by applyingsolutions with a pH of 7.0 in the presence of CP (15 mM) and inthe absence of Pi. Ischaemic conditions were simulated bydecreasing the pH from 7.0 to 6.2 and by including 30 mM

KH2PO4. Phosphocreatine was also omitted from the ischaemicsolutions used to study the effects of MgADP (in combinationwith MgATP). The ionic strength of the solutions varied slightly(between 207 and 182 mM, depending on the solution type; seeTable 1). Solutions with intermediate free Ca2+ concentrationswere obtained by mixing the activating and relaxing solutions. Toachieve maximal Ca2+ activation in the ischaemic activatingsolutions, appropriate amounts of CaCl2 were added to cover theCa2+ concentration range up to pCa 3.0. Total [ATP] and [ADP]were increased to account for the simultaneous Ca2+ binding toadenine nucleotides.

Force measurementsIsometric force was measured after the preparation had beentransferred from the relaxing to the activating solution, by movingthe stage of the inverted microscope. When a steady force wasreached, the length of the myocyte was reduced by 20 % within2 ms using the piezoelectric motor (slack test). As a result of thisintervention, the force first dropped to zero and then started to

Z. Papp, Á. Szabó, J. P. Barends and G. J. M. Stienen178 J. Physiol. 543.1

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redevelop. The rate of tension redevelopment was determinedfrom the force signals after the myocyte had been restretched to itsoriginal length (Lo; see below). About 3 s after the onset of forceredevelopment the myocyte was returned to the relaxing solution,where a slack test with a long slack duration (10 s) was performedto assess the passive force level. The active isometric force wascalculated by subtracting the passive force from the total peakisometric force. Force redevelopment after the restretch was fittedto a single exponential to estimate the rate constant of forceredevelopment (ktr) at saturating Ca2+ levels under both control(pCa 4.73) and ischaemic conditions (pCa 3.4; Fig. 4B). Theduration of the slack period was 21 ms under the controlconditions. Similarly short slack durations did not provide goodestimates of ktr during simulated ischaemia and in the presence ofMgADP. The slack duration was therefore increased to 900 ms inthese latter measurements. The unloaded shortening velocity (Vo)was determined under ischaemic conditions (at pCa 3.2) usingslack tests with a duration of 2.7 s, whereas the amplitude of thelength changes was varied between 19 and 23 % of Lo (in fivesteps). Vo was derived from the slope of the linear regression linebetween the relative length change and the duration of unloadedshortening (Fig. 4A).

Force and length signals were monitored by using an analogue penrecorder and were stored in a personal computer. The samplingrate during experiments was 20 Hz, while during slack tests it was1 kHz.

Experimental protocolThe temperature during the measurements was set to 15 °C inorder to maintain the mechanical stability of the permeabilizedmyocyte preparations. During the initial stage of the experiment,nonischaemic control parameters were determined. The firstcontracture was performed at saturating Ca2+ concentration(pCa 4.73). Thereafter, the sarcomere length was readjusted to2.2 mm, if necessary. The second measurement at pCa 4.73 wasused as a measure of the maximal force output (Po) of the

preparation. The next three to five test measurements were carriedout under various experimental conditions, followed by anothernonischaemic control activation at pCa 4.73. Measurements werecontinued unless maximal force output (Po) declined below 70 %.The force production at submaximal levels of activation wasnormalized to the nearest reference force value obtained atmaximal activation.

Mathematical modellingIn the absence of CP, the intracellular MgATP and MgADPconcentrations depend on the interplay between ATP hydrolysis(resulting in MgATP breakdown and MgADP production) anddiffusion. During simulated ischaemia the myokinase inhibitorP1,P5-di(adenosine-5‚) pentaphosphate (Boehringer Mannheim,Mannheim, Germany) did not modify the force recordings (seeResults), so the myokinase activity was neglected in ourcalculations. Hence, with the assumptions of cylindrical geometryfor the myocyte, with a radius (ro) of 10 mm (average radius ofmyocytes), and equal diffusion constants for MgATP and MgADP(Cooke & Pate, 1985), the MgATP concentration (y) at a distance rfrom the centre of the myocyte at a given time t is determined bythe following differential equation:

dy(r,t) d2y(r,t) dy(r,t)——— = D≤——— + ———dt dr2 r dr

ay(r,t)_ ———————————≥, (1)

Km(1 + [ADP]/Ki) + y(r,t)

where D denotes the diffusion coefficient (20 mm2 s_1 inside themyocyte (Cooke & Pate, 1985) and 270 mm2 s_1 in the unstirredmedium outside the myocyte (Yoshizaki et al. 1987)), a is the rateof MgATP consumption by myosin (0.2 mM s_1 at infinite MgATPconcentration; Ebus et al. 2001), Km (10 mM) is the Michaelis-Menten constant for MgATP binding (Ebus et al. 2001) and Ki isthe inhibition constant for MgADP, assuming competitiveinhibition for the same binding site. Calculations were performed

MgADP during ischaemiaJ. Physiol. 543.1 179

Table 1. Compositions of the solutions

pCa Na2ATP MgCl2 Ionic strength(mM) (mM) (mM)

Control solutions, MgATP 5 mM; CP 15 mM; pH 7.0; Pi 0 mM

Relaxing 10.00 6.75 9.46 207Activating 4.73 6.80 6.32 182

Ischaemic solutions, MgATP 5 mM; CP 0 mM; pH 6.2; Pi 30 mM






Ischaemic solutions, MgATP 0.01 mM; CP 15 mM; pH 6.2; Pi 30 mM


All solutions contained, in addition, BES (100 mM). Relaxing solutions contained 7 mM BAPTA andactivating solutions contained 7 mM CaBAPTA. CaBAPTA was made by mixing equimolar amounts ofCaCO3 and BAPTA. The ionic equivalent of the solutions was adjusted to 133 mM with 1,6-diaminohexane-N,N,N‚,N‚-tetraacetic acid. pH was adjusted with KOH.

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with two different values of Ki: 0.2 mM, determined in skeletalmuscle (Cooke & Pate, 1985), and 0.012 mM, estimated on thebasis of the Vo values measured in this study. [MgATP] (and[MgADP] = 5 mM – [MgATP]) was calculated by numericallysolving Eqn (1) from r = 0 (the centre of the myocyte) to an outerradius (rW), set at 150 mm, with a maximal step size of 0.2 mm. Thistakes the possible influence of an unstirred layer around themyocyte into account. As a boundary condition at t = 0, theintracellular concentrations were considered to be equal tothe nominal concentrations of the surrounding medium. At t = 0,ATPase activity started and consequently MgATP and MgADPconcentration gradients developed, which became approximatelystationary in less than 60 s. The rate of ATP hydrolysis and thediffusion constant for MgADP in solution were estimated asdescribed by Ebus et al. (2001) and Yoshizaki et al. (1987),respectively, after appropriate correction for temperature. Thesesimulations, as well as the model calculations described in theAppendix, were performed using Mathematica 4 (WolframReseach, Inc., Champaign, IL, USA).

Data analysisThe relation between force and pCa was fitted to a modified Hillequation:

PCa = Po([Ca2+]nH/(Ca50nH + [Ca2+]nH)) + P_Ca, (2)

where PCa is the steady-state force, Po is the steady isometric forceat saturating Ca2+ concentration, the Hill coefficient (nH) is ameasure of the steepness of the relationship, Ca50 (or pCa50) is themidpoint of the relation, and P_Ca is defined as the isometric forcerecorded at pCa 10.

Values are given as means ± S.E.M. for n myocytes obtained fromat least five different hearts. Differences were tested by means ofStudent’s unpaired t test at a 0.05 level of significance (P < 0.05).

RESULTSMgADP promotes force production undersimulated ischaemic conditions in the absence andpresence of Ca2+

The effect of MgADP on myocardial force production was

studied under simulated ischaemic conditions in skinned

myocardial cells (Fig. 1) at a sarcomere length of 2.2 mm

and 15 °C. Peak isometric force recorded at pH 7.0 in the

absence of Pi (control conditions) at saturating [Ca2+]

(pCa 4.73) was defined as control Po. This control Po served

as a reference (Fig. 1a) for the effects of simulated

ischaemia (pH 6.2, 30 mM Pi; Fig. 1b) and subsequent

alterations in MgADP concentrations (Fig. 1c and d).

The first two contractures in Fig. 1 reveal a marked reduction

in peak isometric force from a control of 41.1 kN m_2 to

1.9 kN m_2 under simulated ischaemic conditions at

saturating [Ca2+] in the presence of 5 mM [MgATP].

Subsequent activation in the presence of 1 mM MgATP

and 4 mM MgADP demonstrated (as illustrated by Fig. 1c)

that the ischaemic force production was greatly enhanced,

to 23.6 kN m_2, in this case. When the myocyte was

exposed to the same mixture at pCa 10 (i.e. in the relaxing

solution; Fig. 1d) the force development was relatively

small (4.1 kN m_2). This force component (P_Ca) was

obtained after subtraction of the passive force level as

indicated in the Methods. Hence, it was most probably a

consequence of the altered [MgATP] : [MgADP] ratio.

Figure 1e illustrates that P_Ca was further increased (i.e. to

11 kN m_2) at 4.5 mM MgADP and 0.5 mM MgATP.


Figure 1. Substitution of MgADP for MgATP enhances isometric force production undersimulated ischaemic conditionsThe maximal isometric force (Po) recorded in an isolated myocyte at pH 7.0 without added Pi (control) atsaturating Ca2+ concentration (pCa 4.73; a) dropped after lowering of the pH to 6.2 and elevation of Pi to30 mM (simulated ischaemia, indicated by a bold horizontal line) at pCa 3.4 in the presence of 5 mM MgATP(b). Subsequent partial substitution of MgADP for MgATP enhanced Po (c) and induced force generation inthe relaxing solution (at pCa 10, d and e). The total [MgATP] + [MgADP] was kept constant at 5 mM. Duringprotocol a, the CP concentration was 15 mM; CP was not present during measurements b–d. The short-dashed line indicates zero force level. The long-dashed line at ~7 % of the control Po indicates the passiveforce level.

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A decrease in [MgATP] in combination with a rise in[MgADP] promotes ischaemic force developmentmore effectively than low [MgATP] aloneThe adenine nucleotide concentration dependences of

ischaemic Po and ischaemic P_Ca were obtained by using a

range of MgATP concentrations (Fig. 2). In one series

of measurements, a range of MgATP concentrations

(between 5 and 0.1 mM) was applied, while the total

adenine nucleotide concentration ([MgADP] + [MgATP])

was kept constant at 5 mM (Fig. 2A). Another series of

measurements was performed in which only [MgATP]

was varied (from 5 to 0.01 mM) (Fig. 2B) in order to isolate

the pure [MgATP] dependence of the isometric force. In

these experiments, intracellular MgADP accumulation

due to actomyosin ATPase was avoided by the inclusion of

15 mM phosphocreatine.

Figure 2 demonstrates that the originally minute ischaemic

force values present at 5 mM MgATP gradually increased

towards the nonischaemic reference value (i.e. towards 1)

at low [MgATP]. It is also apparent that, in the presence of

MgADP, the isometric force increased to higher levels

than in its virtual absence. Comparison of the means of

the ischaemic force values at identically high Ca2+

concentrations (Po; 0 vs. •, pCa 3.4) and low Ca2+

concentrations (P_Ca; 1 vs. ª, pCa 10) indicated that, in the

presence of MgADP, Po and P_Ca were both enhanced at

intermediate MgATP concentrations. Comparison of the

forces in the absence and presence of Ca2+ revealed that Po

is more sensitive than P_Ca to a decrease in MgATP.

Furthermore, in the presence of MgADP the force

levels became elevated at significantly higher MgATP

concentrations than in its absence. In summary, MgADP

contributed greatly to the increase in ischaemic force when

[MgATP] was less than 5 mM (up to 0.1 mM MgATP) at

pCa 3.4. This increase in ischaemic force up to a [MgATP]

of 0.5 mM was chiefly due to enhancement of the Ca2+-

activated force.

Figure 2 also presents computer simulations based on the

cross-bridge model described in the Appendix. It can be

seen that the simulations agree well with the data at

saturating Ca2+ concentrations. Hence the model provides

an adequate explanation of the enhanced force-promoting

role of MgADP under mimicked ischaemic conditions.

MgADP partly restores the Ca2+ sensitivity of forceproduction under ischaemic conditionsTo gain further insight into the MgADP-associated recovery

of ischaemic force, the Ca2+ sensitivities of the isometric force

(Fig. 3) under control and simulated ischaemic conditions

(at [MgATP] = 5 mM) were compared with the Ca2+

sensitivity observed under ischaemic conditions in the

presence of MgADP. To assess the effect of MgADP on the

Ca2+ sensitivity, test solutions containing 4 mM MgADP and

1 mM MgATP were chosen because P_Ca was relatively small

(3 ± 1 % of the control Po at pCa 10) at these nucleotide

concentrations. Moreover, a number of reports (Allen et al.1985; Kingsley et al. 1991) have suggested that ischaemic

contracture might develop at intracellular MgATP

concentrations of ~1 mM. The isometric force values

expressed relative to the control (Fig. 3A) illustrate that

MgADP partly restored the isometric force not only at

saturating (to 49 ± 2 % of the control) but also at

intermediate Ca2+ concentrations. Partial recovery of the

Ca2+ sensitivity is illustrated in Fig. 3B, which shows force

values normalized to the maximal isometric force levels and

the curves obtained by fitting the data to the Hill equation.


Figure 2. The isometric force is greatly enhanced by[MgADP] under simulated ischaemic conditionsA, the peak isometric force (expressed as a fraction of the controlPo) increased both at saturating [Ca2+] (pCa 3.4, 0) and in therelaxing solution (pCa 10, 1) when the reduction of [MgATP] wasaccompanied by an increase in [MgADP] (from a [MgATP] of2.5 mM downwards). B, in the absence of MgADP the isometricforce was enhanced only at lower [MgATP], either in the presenceof Ca2+ (pCa 3.4, •, below [MgATP] = 0.25 mM) or in the absenceof Ca2+ (pCa 10, ª, below [MgATP] = 0.1 mM). Symbols indicatemeans ± S.E.M. (n = 6–20). All data resulted from simulatedischaemia experiments. Data in A were recorded in the absence ofCP. In contrast, data in B were obtained in the presence of CP(15 mM). The continuous lines indicate the cross-bridgesimulations based on the model described in the Appendix.

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Under the control conditions, pCa50 was 5.62 ± 0.03. It

decreased to 4.10 ± 0.07 during simulated ischaemia and

recovered to 4.74 ± 0.05 when the 4 mM MgATP was

replaced by 4 mM MgADP (these changes were highly

significant, P < 0.0001). The Hill coefficient (nH) decreased

from the control value of 3.1 ± 0.2 to 1.9 ± 0.3 (P < 0.01) due

to simulated ischaemia, and did not change significantly

when 4 mM MgADP (and 1 mM MgATP) was applied

(nH = 2.0 ± 0.2).

Increasing MgADP concentration decreases Vo andktr under ischaemic conditionsThe effects of the different [MgADP] and [MgATP]

combinations on ischaemic Vo and ktr were studied at

saturating Ca2+ concentrations. The Vo was determined from

the force signals obtained when the amplitude of shortening

during slack tests was varied. Figure 4A demonstrates that

the same increases in the amplitude of the length changes

resulted in more pronounced elongations in slack time when

[MgADP] was increased from 2.5 to 4 mM ([MgATP]

decreased from 2.5 to 1 mM). Vo decreased from 0.38 to

0.13 Lo s_1 as a result of this change in adenine nucleotide

composition.The magnitude of ktr was obtained by fitting an

exponential to the force transients after restretching of the

myocytes to Lo. These measurements were also performed

under control conditions (5 mM MgATP). The example in

Fig. 4B illustrates that the time course of force recovery was

greatly slowed in an ischaemic solution containing 4 mM

MgADP and 1 mM MgATP (ktr = 0.71 s_1) in comparison

with the control (nonischaemic) value (ktr = 9.78 s_1). The

means of the measured Vo and ktr values, together with an

estimate of 2 Lo s_1 for Vo under the control conditions

(Ricciardi et al. 1994; McDonald et al. 1998), are shown in

Fig. 4C. The marked and gradual reduction in ktr on elevation

of the [MgADP] (and decrease of the [MgATP]), together

with the similar concentration dependence of Vo, suggests

that a reduction in the cross-bridge cycling rate is involved in

the observed [MgADP]-dependent increase in force under

the simulated ischaemic conditions.

The measurements of Vo at different MgADP and MgATP

concentrations were also used to estimate the inhibition

constant for MgADP (Ki) under ischaemic conditions.

Ki was obtained from the curve fit shown in Fig. 4C, with

the assumption of the competitive inhibition of MgADP

and MgATP for the same myosin binding site (Cooke &

Pate, 1985):

Vo,max[MgATP]Vo = —————————————, (3)

Km(1 + [ADP]/Ki) + [MgATP]

where Vo,max denotes the velocity of unloaded shortening at

5 mM MgATP and Km (10 mM) is the Michaelis-Menten

constant for MgATP binding (Ebus et al. 2001). The curve

fit resulted in a Vo,max (± S.D.) of 0.66 ± 0.10 Lo s_1 (which is

similar to the value obtained by Ricciardi et al. (1994) at

pH 6.2) and a Ki of 0.012 ± 0.003 mM.

In some experiments (n = 5 myocytes) P1,P5-di(adenosine-

5‚) pentaphosphate (0.2 mM) was included in the test

solutions in order to determine the potential effect of the

myokinase activity on the intracellular [MgADP] and the

subsequent force production during ischaemic conditions.

Previous experimental efforts that followed the same

approach led to controversial results (Ventura-Clapier &

Veksler, 1994; Veksler et al. 1997; Stapleton & Allshire,

1998). In our experiments no effect of the myokinase

inhibitor P1,P5-di(adenosine-5‚) pentaphosphate on the

mechanical data was observed under the simulated

ischaemic conditions (data not shown).


Figure 3. The relations between force and pCa indicatethe partial recovery of the Ca2+ sensitivity of isometricforce production under ischaemic conditions in thepresence of MgADPAveraged force vs. pCa relations were obtained by fitting means offorce values to the Hill equation under control conditions (1) orsimulated ischaemic conditions in the presence of 5 mM

MgATP + 0 mM MgADP (ª) and in the presence of 1 mM

MgATP + 4 mM MgADP (•). A, means of force values areexpressed relative to the control Po (relative force vs. pCa relation).B, means are normalized to the reconstructed Po values resultingfrom curve fittings on A (normalized force vs. pCa relation).Symbols depict means ± S.E.M. (n = 8–13) in A; error bars areomitted from B.

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Reconstruction of intra- and extracellular MgATPand MgADP concentration gradients for myocytesCooke & Pate (1985) calculated the intracellular

concentration gradients of adenine nucleotides in skinned

skeletal muscle fibres activated in the absence of CP. We

performed similar calculations for preparations of

myocytes with significantly smaller diameters (20 mm in

our calculations; Fig. 5). During our experiments, we did

not routinely stir the solutions, so extracellular diffusion

and an unstirred layer were therefore taken into account.

The extracellular adenine nucleotide diffusion constant

(270 mm2 s_1) was derived in accordance with Yoshizaki etal. (1987). The ratio of this value for the extracellular

diffusion (270 mm2 s_1) and that used by Cooke & Pate

(1985) for the intracellular diffusion (20 mm s_1) is

significantly higher than the ratio found by Kushmerick &

Podolsky (1969). Therefore, model calculations were also

performed for cases in which the intracellular adenine

nucleotide diffusion constant was raised to 110 mm2 s_1 in

accordance to this latter study (1, 0). Figure 5 reveals that


Figure 4. Reduction in cross-bridge cycling by [MgADP] under simulated ischaemicconditionsA, the slack times increased more with increasing slack amplitudes (expressed as percentages of Lo) in thepresence of 4 mM MgADP (and 1 mM MgATP, right) than in the presence of 2.5 mM MgADP (and 2.5 mM

MgATP, left) at pCa 3.2. This reflects a decrease in the rate of unloaded shortening velocity (Vo). Lengthchanges are shown schematically at the top and an expanded view of the corresponding force signals at thebottom. The zero force levels reached at different amplitudes of length changes are indicated by dashed lines,and are shifted vertically for illustrative purposes. End-points of slack times are connected by continuousstraight lines. B, rates of tension redevelopment (ktr) were determined by the exponential fitting of tensiontraces after restretching of the myocyte (as depicted at the top) to Lo at the end of slack tests from 80 % Lo.1, control data obtained at pCa 4.73; ª, recordings in the presence of 4 mM MgADP (and 1 mM MgATP) atpCa 3.4 under ischaemic conditions. Differences in ktr are illustrated by superimposed results of exponentialfits (dashed lines). A dash on the left indicates a zero force level. C, Vo and ktr values (means ± S.E.M.) forvarious MgATP and MgADP concentration pairs (n = 5–15). An estimate for Vo (2 Lo s_1) under controlconditions is indicated by 6. The curve fitted to Vo data (dashed line) resulted a Ki of 0.012 mM for MgADP.

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the intracellular MgATP concentrations are significantly

reduced compared to the nominal ones only in the event of

no stirring, with the lower diffusion constant. This

might imply that our experimentally obtained relationship

between the nominal MgATP (and MgADP) concentrations

and force could have been distorted by the combination of

MgATP hydrolysis and limited diffusion. In three

myocytes, however, where vigorous stirring was switched

on and off in a repetitive fashion, no appreciable difference

in force could be discerned at various MgATP and MgADP

levels, suggesting that the model calculations illustrated by1, 0 or • (Fig. 5B) reflect our experimental conditions

more faithfully. This indicates that small vibrations in the

set-up provide adequate stirring of the solution and/or

that the rate of intracellular diffusion of the adenine

nucleotides is greater than 20 mm2 s_1. Furthermore, it may

be noted that a reduction in the Ki for MgADP of ATPase

activity from 0.2 mM (Cooke & Pate, 1985) to the Ki value

obtained in this study on the basis of Vo measurements,

0.012 mM, would reduce the deviation between the mean

and nominal MgATP concentrations to less than 25 %.

DISCUSSIONThis paper characterizes the influence of MgADP on Ca2+-

dependent and Ca2+-independent force in the presence of

Pi and at low pH in isolated myocyte preparations. Kinetic

data and model calculations of cross-bridge cycling

demonstrate the key role of MgADP in promoting force

generation during advanced stages of cardiac ischaemia.

MgADP increases isometric force under ischaemicconditionsMgADP promoted both the active and the Ca2+-

independent force under simulated ischaemic conditions

(Fig. 2). Between MgADP concentrations of 2.5 and

4.5 mM, the increase in the active, Ca2+-activated force was

chiefly responsible for the gradual recovery in iso-

metric force. Above 4.5 mM MgADP, however, the Ca2+-

independent component became dominant. In the

presence of 1 mM MgATP and 4 mM MgADP the isometric

force recovered to almost 50 % of the nonischaemic

control value. Quantification of the active component at

this force level is complicated because of the cooperative

interaction between the MgADP-bound cross-bridges and

the Ca2+ activation process (Lu et al. 2001). However, the

low force value in the absence of Ca2+ (3 % of the control)

implies that this cooperative interaction plays a minor role

in the presence of 1 mM MgATP and 4 mM MgADP under

the ischaemic conditions.

In our experiments, 4 mM MgADP partly restored the Ca2+

sensitivity of isometric force production (from pCa50 4.1 to

4.74, DpCa50 = 0.64, which corresponds to a 61.2 mM

difference in free [Ca2+]) in the presence of 1 mM MgATP.

Godt & Nosek (1989), using 0.7 mM MgADP, could not

prevent the reduction in isometric force and in its Ca2+

sensitivity when 17.38 mM Pi was added and pH was

reduced from 7.0 to 6.65, either in skeletal or in cardiac

muscle preparations from the rabbit. Hence, our findings

indicate that higher MgADP concentrations than applied

by Godt & Nosek (1989) are required to effectively oppose

the depressant effects of Pi and low pH on the maximal

Ca2+-activated force and its Ca2+ sensitivity in cardiac

muscle.


Figure 5. The extent of possible intracellular MgADPconcentration changes due to an interplay betweenactomyosin ATPase and adenine nucleotide diffusion inthe absence of CPA, calculations of MgATP concentration profiles inside andoutside the preparations under ischaemic conditions according toEqn (1). The initial [MgATP] and [MgADP] were 1 and 4 mM,respectively. Concentration profiles illustrate results ofcalculations with low intracellular adenine nucleotide diffusion(20 mm2 s_1). B, log–log plot of averaged [MgATP] for a myocytecross-section with a diameter of 20 mm as a function of thenominal [MgATP] in the bathing solution. Symbols illustrate thecalculated mean [MgATP] for different intracellular diffusionconstants and the presence or absence of stirring (0, 110 mm2 s_1,stirred; 1, 110 mm2 s_1, unstirred; •, 20 mm2 s_1, stirred;ª, 20 mm2 s_1, unstirred). Calculations involving low intracellularadenine nucleotide diffusion (20 mm2 s_1) in the absence of stirringresulted in a significant deviation from the nominal [MgATP].Data points illustrate results of calculations with Ki = 0.2 mM.Model calculations with a lower inhibitory constant(Ki = 0.012 mM) resulted in smaller (< 25 %) concentrationdifferences.

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MgADP modulates overall cross-bridge kineticsunder ischaemic conditionsIt is generally accepted that the rate of cross-bridge

detachment limits the unloaded shortening velocity

(e.g. Ferenczi et al. 1984) and that MgADP dissociation

from actomyosin is sufficiently slow to control this process

(Siemankowski et al. 1985; Martin & Barsotti, 1994).

Increase of the level of MgADP in competitive inhibition

with the declining MgATP level (Pate & Cooke, 1989)

would then conserve cross-bridges in strongly bound

force-generating states, resulting in an apparent reduction

in overall cross-bridge cycling rate and an increase in

isometric force. Pi accumulation, however, inhibits the

transition leading to force generation (Hibberd et al. 1985;

Kentish, 1991; Palmer & Kentish, 1994) and may

accelerate transitions between cross-bridge states. In the

presence of ischaemic metabolites (30 mM Pi and pH 6.2),

a decrease in the MgATP concentration reduced the rate of

cross-bridge cycling, but major alterations became

apparent only below 1 mM MgATP (Ebus et al. 2001).

Our Vo and ktr data show that application of MgADP (with

decreasing [MgATP]) in combination with the ischaemic

metabolites resulted in a large and concentration-

dependent reduction in overall cross-bridge cycling rate

even at relatively high [MgATP] (>1 mM). On the basis of

these observations and data in the literature (Ricciardi etal. 1994; McDonald et al. 1998), we postulate that Vo and ktr

report similar relative changes in overall cross-bridge

kinetics between [MgADP] of 2.5 and 4.9 mM. The value of

0.35 Lo s_1 for Vo in the presence of 2.5 mM MgADP (and

2.5 mM MgATP) is considerably less than expected on the

basis of acidosis (Ricciardi et al. 1994), and cannot readily

be explained by the combination of acidosis and Pi either

(Cooke et al. 1988). Thus, the reduction in Vo and ktr,

together with the profound effects on isometric force

production and its Ca2+ sensitivity, strongly suggest that

the overall cross-bridge kinetics and the distribution

between weakly and strongly bound cross-bridges are

effectively influenced by MgADP in the presence of Pi, low

pH and low [MgATP].

Values of the inhibition constant (Ki) of MgADP for

myofibrillar ATPase in skeletal muscle fibres range between

0.1 and 0.2 mM (cf. Sleep & Glyn, 1986). The results of

Cooke & Pate (1985) suggest that the inhibition constants

for shortening velocity and ATPase are similar. Our

measurements of Vo at different MgADP concentrations

suggest that Ki might be considerably less (0.012 ±

0.003 mM) in rat cardiac myocytes. This reduction by an

order of magnitude is surprising but it is consistent

with the previous qualitative observation that cardiac

contractile proteins are more sensitive than skeletal muscle

proteins to [MgADP] (Tian et al. 1997).

It appeared that the [MgATP] dependences of the

ischaemic force with and without Ca2+ diverged more in

the presence of MgADP than in its absence (Fig. 2). The

most likely explanation for this observation is that

alterations in the distribution of cross-bridges due to the

combination of MgADP and low MgATP enhanced the

cooperative process of Ca2+ activation more effectively

than did low MgATP concentration alone. Alternatively,

in the absence of CP, diffusion limitations might have

resulted in higher intracellular MgADP and lower MgATP

concentrations during the mimicked ischaemic conditions.

Our model calculations and experiments with stirring,

however, argue against this latter possibility. The apparent

lack of an appreciable intracellular adenine nucleotide

concentration gradient can be explained by the smaller

diameter of the myocytes compared to skinned skeletal

muscle fibres, a faster intracellular nucleotide diffusion

than used by Cooke & Pate (1985), the low Ki value of

MgADP for myofibrillar ATPase in cardiac tissue, or by a

combination of these factors.

The simulations of the cross-bridge model for cardiac

tissue (described in the Appendix) clearly indicate that the

alterations in metabolite concentrations and their impact

on the free energy change of ATP hydrolysis under

ischaemic conditions are sufficient to explain the change

observed in the MgATP dependence of force development

(Fig. 2). Therefore, it appears that the enhancement of

the force-promoting role of MgADP under ischaemic

conditions is a natural consequence of the thermodynamic

changes in the free energy profiles of the different cross-

bridge states.

MgADP accumulation during ischaemiaExtrapolation of our results to the intact ischaemic

myocardium is complicated by differences between the

applied experimental and in vivo conditions. While the

force–Pi relationship was independent of temperature in

skeletal myofibrils of the rabbit (Tesi et al. 2002),

intracellular acidification decreased the contractile force

(Pate et al. 1995) much less at 30 than at 10 °C. The effect of

temperature on the Pi, pH and MgADP dependences of

myocardial force has not yet been extensively investigated.

However, based on a postulated decreased myocardial

effect of acidosis at 37 °C, restoration of the contractile

force by MgADP is expected in the presence of lower

MgADP concentrations than shown in this study.

In contrast to the applied isometric conditions, length

changes occur during the physiological cardiac cycle.

Therefore, one may speculate that MgADP may affect

cross-bridges differently during shortenings (i.e. during

isotonic conditions). The resemblance of the [MgADP]

dependences of ktr (measured at a constant 2.2 mm

sarcomere length) and of Vo (measured at 79–83 % of Lo),

however, argues against this possibility.

The physiological myoplasmic MgADP level ([MgADP]i)

is kept low by ATP resynthesis, a process depending


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chiefly on the creatine kinase reaction. However, during

myocardial ischaemia the exact [MgADP]i, and more

precisely [MgADP]i in the myofibrillar compartment, is

unknown. Following the cessation of blood flow, the

intracellular CP pool rapidly declines, leading ultimately

to a decrease in [MgATP]i and to the accumulation of its

breakdown products (Allen & Orchard, 1987), possibly in

a spatially inhomogeneous manner. MgADP might be

degraded further (Allen et al. 1985) by the myokinase

reaction. The calculation of [MgADP]i during ischaemia is

not straightforward (Ingwall, 1987) because creatine

kinase (Ventura-Clapier & Veksler, 1994), myokinase

(Golding et al. 1995) and other enzymes involved in the

catabolic processes (Gustafson et al. 1999) are all sensitive

to ischaemic metabolites. Recent data on intact skeletal

muscle fibres suggest that [MgADP]i may reach levels high

enough to induce marked alterations in cross-bridge

kinetics (Westerblad et al. 1998).

Results of this study indicate that MgADP stabilizes the

force-generating cross-bridge state in the presence of

ischaemic metabolites, causes an increase in Ca2+

sensitivity, facilitates the process of Ca2+ activation and

shifts the MgATP threshold concentration of Ca2+-

independent force development to higher levels. All of

these mechanisms may contribute to the development

of myocardial ischaemic contracture at relatively high

[MgATP]i, provided that MgADP reaches sufficient intra-

cellular levels during ischaemia.

APPENDIXCross-bridge model for cardiac muscleTo simulate the MgATP dependences of isometric force

under the mimicked ischaemic conditions given in the text

(Fig. 2), we used the Pate & Cooke (1989) model with

modifications in the free energy profiles and rate

constants, as described in Tables A1 and A2. The key

features of this five-state cross-bridge model are illustrated

in Fig. A1. It is based on the biochemical scheme for ATP

hydrolysis shown in Fig. A1A. The transitions between the

states depend on the spatial variable x (cross-bridge

distortion). The fractional occupancy of the cross-bridges

in each state (i) is denoted by the function ni(x). For the

definitions of the rate constants governing the transitions

between states, see the legend to Table A2. The modified

free energy profiles under the control and ischaemic

conditions, shown in Fig. A1B, were obtained by adapting

the free energy profiles for fast skeletal muscle (Getz et al.1998) according to the difference in association constants

between cardiac and fast skeletal muscle (Kawai et al.1993). Numerical integration of the set of differential

equations describing the cross-bridge cycle described in

Fig. A1A was performed by using Mathematica (version 4.0)

and a personal computer (G3, Apple), with a range of

cross-bridge distortions (x) from _4 to +10 nm.

In our simulations, the size of the power stroke (h) was

increased from 4 to 6 nm. This change proved necessary in

order to simulate the larger changes of isometric force

produced by Pi and MgADP in cardiac muscle compared

to fast skeletal muscle. In addition, use was made of the

distortion dependence suggested by Slawnych et al. (1994)

for the rate constants governing the power stroke and

cross-bridge detachment. This latter modification reduced

the number of parameters in the model.

Finally, relatively minor changes in the rate constants were

made by trial and error so as to improve the correspondence

between the data in Fig. 2 and the computer simulations.

It can be concluded from Fig. 2 that this model provides an

adequate description of the reduction in force under

mimicked ischaemic conditions at 5 mM MgATP, and of

the rise in force when [MgATP] is reduced both in the

absence and in the presence of CP. This model unifies a

robust collection of recent theoretical advances on cross-

bridge action and experimental data in both cardiac and

skeletal muscle but it is certainly not unique.


Table A1. Free energy in units of kT as a function of cross-bridge distortion (x)

G1(x) = 0

G2(x) = _2.3

G3(x) = _1.9 + (k/2)(x _ h)2

G4(x) = _11.5 + (k/2)x2 + ln[Pi]

G5(x) = G4(x) + 8.4 + ln[ADP]

DGATP = _13.1 + ln([ATP]/([ADP][Pi]))

Subscripts correspond to states in Fig. A1A as follows: 1, M.T;2, M.D.P; 3, AM.D.P; 4, AM.D; 5, AM. k = 1.8 kT nm_2, where k isthe elastic force constant, k is the Boltzmann constant and T is the

absolute temperature; h = 6 nm.

Table A2. Rate constants (s_1) as a function of distortion (nm)

Control conditions Ischaemic conditions (pH 6.2)

k1,2 = 50 k1,2 = 50

k2,3 = 150 exp{_(x _ h)/1.1)2} k2,3 = 150 exp{_(x _ h)/1.1)2}

k3,4 = 50/{1 + exp(hk(x _ h))} k3,4 = 25/{1 + exp[hk(x _ h)]}

k4,5 = 250 k4,5 = 450

k1,5 = 5 exp{_(x/1.08)2} k1,5 = 20 exp{_(x/1.08)2}

Subscripts correspond to states in Fig. A1A as indicated in Table A1.Values for k and h as in Table A1. The rate constants from state i tostate j are indicated by ki,j. Reverse rates result from the Gibbsequation ki,j = kj,i exp(Gj _ Gi)/kT, using the free energies listed inTable A1. At pH 6.2, Pi release (k3,4) was assumed to be decreased2-fold (cf. Chase & Kushmerick, 1988), and ADP release (k4,5) wasassumed to be increased almost 2-fold in order to account for thereduction in the maximum force and the increase in tension cost(Potma et al. 1995; Ebus et al. 2001). The 4-fold increase in rate k1,5

at pH 6.2 resulted in the correspondence between the data and thesimulated curves shown in Fig. 2A and B.

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yMgADP during ischaemiaJ. Physiol. 543.1 187

Figure A1. A five-state model for cross-bridge action in cardiac muscleA, biochemical scheme of the cross-bridge cycle (M, myosin; A, actin; AM, actomyosin complex; D, ADP;P, inorganic phosphate; T, ATP). The free energy profiles, forward rate constants and cross-bridgedistributions under control conditions (pH 7, Pi 0.1 mM, MgADP 30 mM, MgATP 5 mM) and mimickedischaemic conditions (pH 6.2, Pi 30 mM, MgADP 4 mM, MgATP 1 mM) are plotted as a function of the cross-bridge distortion (x) in B, C and D, respectively. The subscripts refer to the states defined in Table A1.ni(x) denotes the fractional occupancy of cross-bridges in state i at distortion x. The occupancy of n5 is verysmall and is not visible in D.

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Acknowledgements This work was supported by a Dutch–Hungarian (NWO-OTKA)research collaboration grant and by the Dutch Heart Foundation.The authors would like to thank David Durham for the Englishlanguage edition.


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The mechanism of the force enhancement by mgADP under simulated isChaemic conditions in rat cardiac myocytes