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Circulation Research. 2000;86:e1-e6

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(Circulation Research. 2000;86:e1.)
© 2000 American Heart Association, Inc.

UltraRapid Communications

Effects of MgADP on Length Dependence of Tension Generation in Skinned Rat Cardiac Muscle

Norio Fukuda, Hidetoshi Kajiwara, Shin’ichi Ishiwata, Satoshi Kurihara

From the Department of Physiology (II) (N.F., H.K., S.K.), The Jikei University School of Medicine, Nishishinbashi, Minato-ku, Tokyo, Japan; Department of Physics (S.I.), School of Science and Engineering, Advanced Research Institute for Science and Engineering (S.I.), and Materials Research Laboratory for Bioscience and Photonics (S.I.), Waseda University, Okubo, Shinjuku-ku, Tokyo, Japan.


*    Abstract
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 *Abstract
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Abstract—The effect of MgADP on the sarcomere length (SL) dependence of tension generation was investigated using skinned rat ventricular trabeculae. Increasing SL from 1.9 to 2.3 µm decreased the muscle width by {approx}11% and shifted the midpoint of the pCa-tension relationship (pCa50) leftward by about 0.2 pCa units. MgADP (0.1, 1, and 5 mmol/L) augmented maximal and submaximal Ca2+-activated tension and concomitantly diminished the SL-dependent shift of pCa50 in a concentration-dependent manner. In contrast, pimobendan, a Ca2+ sensitizer, which promotes Ca2+ binding to troponin C (TnC), exhibited no effect on the SL-dependent shift of pCa50, suggesting that TnC does not participate in the modulation of SL-dependent tension generation by MgADP. At a SL of 1.9 µm, osmotic compression, produced by 5% wt/vol dextran (molecular weight {approx}464 000), reduced the muscle width by {approx}13% and shifted pCa50 leftward to a similar degree as that observed when increasing SL to 2.3 µm. This favors the idea that a decrease in the interfilament lattice spacing is the primary mechanism for SL-dependent tension generation. MgADP (5 mmol/L) markedly attenuated the dextran-induced shift of pCa50, and the degree of attenuation was similar to that observed in a study of varying SL. The actomyosin-ADP complex (AM.ADP) induced by exogenous MgADP has been reported to cooperatively promote myosin attachment to the thin filament. We hereby conclude that the increase in the number of force-generating crossbridges on a decrease in the lattice spacing is masked by the cooperative effect of AM.ADP, resulting in depressed SL-dependent tension generation. The full text of this article is available at http://www.circresaha.org.


Key Words: MgADP • pimobendan • Ca2+ sensitivity • cardiac muscle • sarcomere length


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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An alteration in ventricular end-diastolic volume results in a marked change in cardiac output.1 2 This intrinsic ability of the heart to alter cardiac output forms the basis for the Frank-Starling law of the heart. It is well established that twitch tension and Ca2+ responsiveness in cardiac muscle preparations are enhanced as muscle length (ie, sarcomere length [SL]) is increased within the normal physiological range (SL from {approx}1.8 to {approx}2.3 µm).1 2 3 4 5 Although a number of studies have been conducted to account for the SL dependence of tension generation in living myocardium, its mechanism has not been completely elucidated.6 However, at the myofilament level, there is an increasing amount of evidence suggesting that the SL dependence is primarily due to a change in the interfilament lattice spacing that accompanies the SL change.7 8 9 A possible consequence of the decreased lattice spacing is an increase in the probability of myosin attachment to the thin filament, resulting in an increase in the number of force-generating crossbridges.7 10 11 Ishiwata and Oosawa12 proposed a model based on the Ca2+-dependent flexibility of the thin filament, in which they assumed that (1) the muscle volume (ie, the lattice volume) remains constant on a change in SL and that (2) there is a critical distance between the thick and thin filaments for tension generation. This model quantitatively explains both the stretch-induced increase in the steady isometric tension and the slower (or faster) rate of tension development (or decline) at a shorter SL,13 14 supporting the hypothesis that a change in the lattice spacing plays a pivotal role in determining the SL-dependent Ca2+ sensitivity of tension. Alternatively, it was proposed that the length-dependent change in myofilament activation is caused by cardiac troponin C (TnC), which acts as a "length sensor" in the cardiac muscle contractile system.15 16 However, this idea has attained little experimental evidence from other groups, and it was challenged by McDonald et al,17 who reported that the expression of skeletal TnC in ventricular myocytes of transgenic mice did not alter the SL dependence of Ca2+ sensitivity of tension in skinned myocytes. Thus, it is unlikely that TnC alone acts as a "length sensor" in cardiac muscle.

It is known that the degree of activation of the thin filament is regulated not only by the binding of Ca2+ to TnC but also by the formation of strong-binding crossbridges, such as the rigor18 19 and crossbridges that bind ADP.20 21 22 Fukuda et al22 reported that the actomyosin-ADP complex (AM.ADP) induced by exogenous MgADP can "turn on" adjacent actin molecules in a cooperative manner so that the actin and myosin interaction becomes possible, just as if Ca2+ were bound to TnC. Consequently, upon the addition of MgADP, the pCa-tension relationship for skinned cardiac muscle is shifted to the left, showing greater Ca2+ sensitivity of tension.21 22 23

To investigate the influence of the formation of strong-binding crossbridges on SL-dependent tension generation in cardiac muscle, we measured the SL-dependent shift of the pCa-tension relationship in the presence of varying concentrations of MgADP using skinned rat ventricular trabeculae. The formation of strong-binding crossbridges is known to increase the affinity of TnC for Ca2+.3 5 24 Thus, to clarify whether the effect of MgADP is based on the increased affinity of TnC for Ca2+, we examined the effect of pimobendan, a Ca2+ sensitizer, which promotes Ca2+ binding to TnC.25 26 A preliminary report has been published in abstract form.27


*    Materials and Methods
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 up arrowIntroduction
 *Materials and Methods
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Experimental Procedure
The heart was removed from male Wistar rats (250 to 300 g) anesthetized with sodium pentobarbital (50 mg/kg IP). The rats were supplied by Saitama Experimental Animals Supply (Saitama, Japan), and the present study conforms with the Guiding Principles for the Care and Use of Animals approved by the Council of the Physiological Society of Japan. Thin trabecula muscles with the diameter of 100 to 150 µm were dissected from the right ventricle in oxygenated Tyrode solution without Ca2+ at 30°C. The preparations were skinned by superfusion with 1% vol/vol Triton X-100 in the relaxing solution (in mmol/L: MgATP 4, MOPS 10, EGTA 10, free Mg2+ 1, and ionic strength 180 [pH 7.0]) for 60 minutes at {approx}2°C. The ionic strength (IS) was adjusted with KCl. The preparations were then washed with the relaxing solution to remove Triton X-100 and stored at -20°C in the relaxing solution containing 50% vol/vol glycerol and 2 mmol/L leupeptin for 1 week or less.

Both ends of the preparation were tied to thin tungsten wires with a silk thread. One end was attached to a tension transducer (BG-10; Kulite Semiconductor Products, Inc, Leonia, NJ) and the other to a micromanipulator (Narishige, Tokyo, Japan). The SL was adjusted to either 1.9 or 2.3 µm by measuring laser light diffraction in the relaxing solution. Ca2+-activated isometric tension was measured in activating solutions containing 4 mmol/L MgATP, 10 mmol/L MOPS, 1 mmol/L free Mg2+, a varying concentration of free Ca2+ (adjusted with Ca/[10 mmol/L EGTA]), 0.1 mmol/L P1,P5-di(adenosine-5')pentaphosphate (AP5A), 15 mmol/L creatine phosphate (CP), 15 U/mL creatine phosphokinase (CPK), and 180 mmol/L IS [pH 7.0], at the two SLs with/without MgADP or pimobendan (donated by Nippon Boehringer Ingelheim; Kawanishi, Hyogo, Japan).

The control pCa-tension relationship without MgADP or pimobendan was first obtained at a SL of 1.9 µm and then at 2.3 µm. By using the same preparation, the pCa-tension relationships in the presence of MgADP or pimobendan were obtained at the two SLs. Each pCa-tension relationship was obtained by cumulatively raising the Ca2+ concentration from the relaxing condition. Because we noted a variation in nH (and related parameters), depending on the preparation, paired experiments were carried out on the same preparation. Finally, maximal Ca2+-activated tension (at pCa 4.8) was measured at the two SLs in the control condition without MgADP or pimobendan to examine the reproducibility of tension development. We only used the data in which the final tension values were greater than 70% of those measured at the beginning of the experiment.

The muscle width was measured under a microscope (Nikon SMZ645) at a magnification of x225. The concentrations of chemicals in solutions were estimated by computer calculation.28 All experiments were carried out at 20±0.2°C.

Data and Statistical Analyses
The pCa-tension relationship was fitted to the Hill equation: log[P/(100-P)]=nH[pCa50-pCa], where P is the relative tension expressed as a percentage of the maximum (+Ca2+, pCa 4.8), nH is the Hill coefficient, and pCa50 is -log[Ca2+] at P=50. All data are expressed as mean±SEM. Paired Student’s t test was used, and statistical significance was verified at P<0.05.


*    Results
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 *Results
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Effect of MgADP on the Length Dependence of Ca2+ Sensitivity of Tension
Figure 1Down shows the effect of MgADP on the SL dependence of Ca2+ sensitivity of tension. In the control condition without MgADP or pimobendan, maximal absolute Ca2+-activated tension values were 52.5±4.7 and 77.9±4.1 mg (n=17; P<0.001) at SL 1.9 and 2.3 µm, respectively, and pCa50 was shifted leftward by about 0.2 pCa units by increasing SL from 1.9 to 2.3 µm. The degree of the SL-dependent shift of pCa50 was consistent with the result of a previous study using rat ventricular muscle strips.29 In the absence of MgADP, the muscle width was reduced from 132±6 to 118±4 µm (n=4; P<0.001) on extension of SL during relaxation (ie, {approx}11% reduction): The addition of MgADP (up to 5 mmol/L) did not change the muscle width at either SL (131±7 and 117±5 µm [n=4; P<0.001] at SL 1.9 and 2.3 µm, respectively, in the presence of 5 mmol/L MgADP).



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  		Figure 1. Effects of MgADP on pCa-tension relationships at different SLs. Solvent conditions: 4 mmol/L MgATP, 10 mmol/L MOPS (pH 7.0), 1 mmol/L free Mg2+, varying [Ca2+] (pCa adjusted by Ca/EGTA), 0.1 mmol/L AP5A, 15 mmol/L CP, 15 U/mL CPK, and IS maintained at 180±1 mmol/L. MgADP was added to the above solution at a concentration of 0.1, 1, or 5 mmol/L. The ATP regenerating system (CP-CPK) was not added for experiments that used MgADP. Panels A, B, and C show the effects of MgADP at concentrations of 0.1, 1, and 5 mmol/L, respectively, on the pCa-tension relationships at SLs of 1.9 µm (circles) and 2.3 µm (squares); note that different muscle preparations were used. Solid lines and open symbols indicate pCa-tension relationships in the absence of MgADP; dotted lines and closed symbols, those in the presence of MgADP. Data obtained for each preparation were fitted to the Hill equation, and the results were simulated by the Hill equation with the mean values of pCa50 and nH. Each inset represents {Delta}pCa50 (ie, difference between the values of pCa50 at long and short SLs) in the absence (control) and presence of MgADP. pCa-tension relationships were normalized by the maximum tension at pCa 4.8. Temperature was maintained at 20±0.2°C. Vertical bars indicate SEM of 4 to 5 data points.

Consistent with our previous studies using skinned bovine cardiac muscle,22 23 MgADP shifted the pCa-tension relationship to the left in a concentration-dependent manner (Figure 1Up). Concomitantly, the SL-dependent shift of the pCa-tension relationship was diminished in a concentration-dependent manner; in the presence of 5 mmol/L MgADP, {Delta}pCa50 was decreased to {approx}20% of the control value (Figure 1CUp, inset).

The TableDown summarizes the nH values of the pCa-tension relationships shown in Figure 1Up. MgADP at a concentration of 5 mmol/L significantly decreased nH at both SLs,23 whereas no significant changes were observed for 0.1 and 1 mmol/L MgADP.


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  		Table 1. Change in Hill Coefficient With MgADP

Because the ATP regenerating system (CP-CPK) was not used in the presence of MgADP, we estimated the concentration of contaminating MgADP inside the preparation. In the absence of MgADP, the pCa50 values obtained with and without CP-CPK were 5.45±0.05 and 5.53±0.05, respectively (n=4; P<0.001), at SL 1.9 µm. Thus, we estimated the contaminant MgADP to be {approx}0.1 mmol/L under our experimental condition (see Figure 1AUp). This estimation is within the range of reported values for skinned cardiac and skeletal muscles.30 31

Effect of Pimobendan on the Length Dependence of Ca2+ Sensitivity of Tension
Pimobendan was reported to shift the pCa-tension relationship to the left with little influence on maximal Ca2+-activated tension in skinned porcine ventricular muscle.32 In the present study, 10-4 mol/L pimobendan substantially shifted the pCa-tension curve to the left at SLs of 1.9 and 2.3 µm, whereas in contrast to MgADP, pimobendan did not diminish the SL-dependent shift of the pCa-tension relationship (Figure 2Down).



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  		Figure 2. Effects of pimobendan on pCa-tension relationships at different SLs. Conditions are the same as in Figure 1Up except that MgADP was absent and 10-4 mol/L pimobendan was present as well as CP and CPK. Pimobendan was initially dissolved in DMSO and diluted with the individual solutions. The final concentration of DMSO was 1%, having no effect on tension development. Symbols are the same as in Figure 1Up except that dotted lines and closed symbols represent the pCa-tension relationships in the presence of pimobendan at SL 1.9 µm (circles) and at SL 2.3 µm (squares). Inset represents {Delta}pCa50 in the absence (control) and presence of pimobendan. NS indicates not significant compared with control. pCa-tension relationships were normalized with respect to the maximum tension at pCa 4.8. Vertical bars indicate SEM of 4 data points.

The nH values in the absence and presence of pimobendan were 4.01±0.27 and 3.93±0.15 (n=4; P>0.1), respectively, at SL 1.9 µm and 4.45±0.32 and 3.75±0.12 (n=4; P>0.1), respectively, at SL 2.3 µm. Pimobendan did not significantly change nH at either SL.

Effect of MgADP or Pimobendan on Maximal Tension
Figure 3Down summarizes the effect of MgADP or pimobendan on maximal Ca2+-activated tension (pCa 4.8) at SLs of 1.9 and 2.3 µm. It has been reported that MgADP significantly potentiates maximal Ca2+-activated tension in cardiac22 23 33 and skeletal muscles.20 33 We also found that MgADP augmented maximal tension in a concentration-dependent manner, and this potentiating effect was significantly less pronounced at the longer SL (P<0.01). In contrast, pimobendan only minimally increased maximal tension at both SLs, and there was no significant difference in the increases in maximal tension between the two SLs.



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  		Figure 3. Effects of MgADP or pimobendan on maximal Ca2+-activated tension. Conditions are the same as in Figures 1Up and 2Up for MgADP and pimobendan, respectively, except that pCa was fixed at 4.8. Open columns show SL 1.9 µm, and filled columns show SL 2.3 µm. NS indicates not significant. The degree of increase in tension was determined with respect to the maximal tension obtained without MgADP or pimobendan at each SL. Vertical bars indicate SEM of 4 to 5 data points for MgADP and 4 data points for pimobendan.

Effect of MgADP on Dextran-Induced Shift of the pCa-Tension Relationship
Dextran (5% wt/vol) reduced the width of muscle from 135±5 to 117±4 µm (n=7; P<0.001) under the relaxing condition at SL 1.9 µm. The degree of reduction ({approx}13%) was similar to that observed when increasing SL to 2.3 µm without dextran ({approx}11%, see above).

In the absence of MgADP, dextran shifted pCa50 to the left by 0.19±0.03 pCa units (Figure 4Down). The degree of the shift of pCa50 was comparable, albeit slightly smaller, to what was observed when increasing SL to 2.3 µm (see Figures 1Up and 2Up). Maximal tension was also augmented by {approx}15% in the presence of dextran as previously reported by other investigators using skinned skeletal11 and cardiac muscles.34



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  		Figure 4. Effect of 5 mmol/L MgADP on pCa-tension relationships in the absence and presence of 5% wt/vol dextran at SL 1.9 µm. Solvent conditions are the same as in Figure 1Up except that 5% wt/vol dextran (molecular weight {approx}464 000; Sigma) was used to shrink the muscle volume instead of increasing SL to 2.3 µm. Circles indicate in the absence of dextran; squares, in the presence of dextran. Solid lines and open symbols indicate pCa-tension relationships in the absence of MgADP; dotted lines and closed symbols, pCa-tension relationships in the presence of MgADP. Inset represents {Delta}pCa50 (difference between the values of pCa50 with and without dextran) in the absence (control) and presence of MgADP. pCa-tension relationships were normalized with respect to the maximal tension at pCa 4.8. Vertical bars indicate SEM of 7 data points.

It was found that in the presence of 5 mmol/L MgADP, the dextran-induced increase in apparent Ca2+ sensitivity was markedly diminished (Figure 4Up), and the degree of attenuation was similar to what was obtained in a study of varying SL (see Figure 1CUp).

The nH values in the absence and presence of 5 mmol/L MgADP were 6.32±0.61 and 2.52±0.18 (n=7; P<0.05), respectively, without dextran and 5.29±0.31 and 1.68±0.06 (n=7; P<0.05), respectively, with dextran.


*    Discussion
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 *Discussion
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We demonstrated that using skinned rat ventricular trabeculae, MgADP increases the Ca2+ sensitivity of tension and reduces the magnitude of SL-dependent changes in Ca2+ sensitivity. On the other hand, a simple increase in the Ca2+ binding affinity of TnC by pimobendan had no effect on the SL-dependent change in the Ca2+ sensitivity of tension. We discuss the implications of these results, focusing on the role of strong-binding crossbridges in the regulation of Ca2+ sensitivity of tension.

First, an increase in SL results in a decrease in the lateral separation between the thick and thin filaments in living cardiac muscle.35 Similarly, the interfilament lattice spacing is decreased by increasing SL in skinned (glycerinated) muscle.36 Although the latter study was conducted using skeletal muscle, it is reasonable to assume that the result can be extended to skinned cardiac muscle. In the present study, we observed that increasing SL from 1.9 to 2.3 µm produced about an 11% reduction in the width of muscle. Thus, it is suggested that there also occurred a corresponding reduction (ie, {approx}11%) in the lattice spacing on extension of SL from 1.9 to 2.3 µm.

On the other hand, 5% wt/vol dextran decreased the muscle width by {approx}13%. An X-ray diffraction study showed that a reduction in the width of skinned skeletal muscle produced by dextran reflects a proportional change in the lattice spacing.37 Although an X-ray study has not been conducted with cardiac muscle, the conclusion made, on the basis of skeletal muscle, could be applicable to cardiac muscle.38 It can thus be said that in the present study, 5% wt/vol dextran decreased the lattice spacing by {approx}13%. Given the fact that both an increase in SL and osmotic compression produced a similar reduction in the muscle width and that both increased Ca2+ sensitivity of tension to a similar degree (Figures 1Up, 2Up, and 4Up), we consider that the decreased lattice spacing is the primary mechanism for length-dependent tension generation in skinned cardiac muscle.

There was, however, a slight mismatch between the effect of increasing SL and that of osmotic compression on Ca2+ sensitivity of tension and maximal Ca2+-activated tension. Increasing SL to 2.3 µm resulted in about an 11% decrease in the muscle width, whereas osmotic compression decreased the width by {approx}13%, yet the shift of pCa50 and the augmentation of maximal Ca2+-activated tension were somewhat more pronounced by the lengthening. The exact reason(s) for this mismatch is unknown. However, it may be attributable to some direct effect of dextran on the crossbridge cycle39 and/or to the shape of the muscle being altered differently by mechanical stretch compared with osmotic compression.7

Although Ca2+ is a physiological activator of myocardium, it has been known that Ca2+ alone does not fully activate the thin filament40 and that strong-binding crossbridges, such as the rigor complex, can further activate the thin filament.18 We have reported that the formation of AM.ADP upon the addition of MgADP regulates the number of force-generating crossbridges, synergistically with Ca2+ binding to TnC.20 21 22 In the present study, we found that MgADP, in addition to its apparent Ca2+ sensitizing effect, diminished the SL-dependent shift of the pCa-tension relationship in a concentration-dependent manner (Figure 1Up). Further, MgADP (5 mmol/L) attenuated the increase in Ca2+ sensitivity of tension produced by osmotic compression, to a similar degree observed when SL was increased (Figure 4Up). As discussed above, SL-dependent tension generation can be largely explained due to a decrease in the lattice spacing, which results in an increase in the number of force-generating crossbridges.7 10 11 Therefore, it is realized that when force-generating crossbridges predominate inside the muscle through the cooperative effect of AM.ADP, the effect of lattice shrinkage becomes relatively small, leading to depressed SL-dependent tension generation. This interpretation is consistent with the fact that the potentiating effect of MgADP was significantly less pronounced at a longer SL (Figure 3Up).

Fitzsimons and Moss9 reported that an application of N-ethylmaleimide–modified myosin subfragment 1 to single skinned rat ventricular myocytes diminishes the SL-dependent shift of the pCa-tension relationship. It is thus safe to conclude that when the number of force-generating crossbridges is increased through the cooperative effect of strong-binding crossbridges, the effect of increasing SL (ie, lattice shrinkage) to produce force-generating crossbridges is offset.

In skinned muscle preparations, strong-binding crossbridges promote Ca2+ binding to TnC.24 If TnC acts as a "length sensor" in the cardiac contractile system,15 16 then it follows that the SL dependence of tension generation would be modulated by a change in the affinity of TnC for Ca2+, and the depressed shift of the pCa-tension relationship seen in the presence of MgADP may have been caused by the increased affinity of TnC for Ca2+. However, pimobendan was found to have no effect on the SL-dependent shift of the pCa-tension relationship (Figure 2Up). Thus, it is unlikely that the increased affinity of TnC for Ca2+ is the major cause of the attenuation of SL-dependent tension generation by MgADP.

McDonald et al41 hypothesized that the activation state of muscle with a higher cooperativity varies more dramatically as a result of length-induced variations in the number of force-generating crossbridges. However, in the presence of 0.1 or 1 mmol/L MgADP, the SL dependence was significantly diminished (Figures 1AUp and 1BUp), whereas nH was not significantly changed (TableUp). Thus, it is unlikely that the attenuation of SL-dependent tension generation by MgADP underlies the decreased cooperative activation of the thin filament.

It should be stressed that MgADP as low as 0.1 mmol/L (or {approx}0.2 mmol/L when contaminating MgADP is taken into account) augmented maximal and submaximal tension and diminished the SL dependence of tension generation (Figures 1AUp and 3Up). Because it has been known that cardiac contractile proteins are more sensitive to MgADP than skeletal muscle proteins,22 42 it is possible that MgADP at {approx}0.1 mmol/L significantly influences cardiac contractile performance, as in the in vitro motility assay system.43 Recently, Tian et al44 demonstrated that {approx}0.1 mmol/L MgADP significantly increased the left ventricular end-diastolic pressure in intact rat heart. It has been pointed out that in the intracellular milieu of ischemic or hypoxic cardiac muscle, the concentration of ADP increases whereas that of ATP decreases.45 Reportedly, an increase in the ratio of the concentration of ADP to that of ATP in the vicinity of crossbridges may elicit ischemic contracture.46 The present results suggest that during ischemia or hypoxia, the accumulation of ADP may impair the Frank-Starling mechanism.


*    Acknowledgments
 
This work was supported in part by Grants-in-Aid for Scientific Research, for Scientific Research on Priority Areas, and for High-Tech Research Center Project from the Ministry of Education, Science, Sports and Culture of Japan, by Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Corporation, and from the Vehicle Racing Commemorative Foundation. We would like to thank Nippon Boehringer Ingelheim for providing pimobendan, Prof Masataka Kawai of the University of Iowa for critical reading of the manuscript, and Naoko Tomizawa for technical assistance.


*    Footnotes
 
Previously published in abstract form (Circulation. 1998;98[suppl I]:I-258).

Address reprint requests to Dr Shin’ichi Ishiwata, Department of Physics, School of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.

Received August 26, 1999; accepted November 17, 1999.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
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 *References
 

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