Milrinone inhibits contractility in skinned skeletal muscle fibers

C. Y. Seow, L. Morishita, and B. H. Bressler

Departments of Anatomy and Pharmacology and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Direct action of the cardiotonic bipyridine milrinone on the cross bridges of single fibers of skinned rabbit skeletal muscle was investigated. At 10°C and pH 7.0, milrinone reduced isometric tension in a logarithmically concentration-dependent manner, with a 55% reduction in force at 0.6 mM. Milrinone also reduced Ca2+ sensitivity of skinned fibers in terms of force production; the shift in the force-pCa curve indicated a change in the pCa value at 50% maximal force from 6.10 to 5.94. The unloaded velocity of shortening was reduced by 18% in the presence of 0.6 mM milrinone. Parts of the transient tension response to step change in length were altered by milrinone, so that the test and control transients could not be superimposed. The results indicate that milrinone interferes with the cross-bridge cycle and possibly detains cross bridges in low-force states. The results also suggest that the positive inotropic effect of milrinone on cardiac muscle is probably not due to the drug's direct action on the muscle cross bridges. The specific and reversible action of the bipyridine on muscle cross bridges makes it a potentially useful tool for probing the chemomechanical cross-bridge cycle.

cardiotonic bipyridine; force-pCa relations; tension transients; shortening velocity

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

MILRINONE (1,6-dihydro-2-methyl-6-oxo-3,4'-bipyridine-5-carbonitrile) and the related bipyridine amrinone, 5-amino-(3,4'-bipyridine)-6(1H)-one, are positive cardiac inotropic agents with vasodilator properties (1, 2). The cardiovascular effects are thought to be partly due to the bipyridines' ability to inhibit phosphodiesterase III and therefore increase cAMP level (2, 6, 8). Other mechanisms that increase Ca2+ influx into myocardium are also believed to be involved (1). It has been shown that direct binding of amrinone on skeletal muscle cross bridges potentiates isometric force (4, 12, 13). The bipyridine's inotropic property, however, is probably not due to the compound's direct interaction with the contractile proteins, because the binding also results in decreased shortening velocity and hence decreased power output of the muscle (4). One interesting observation made in these studies is that amrinone appears to be able to detain cross bridges in high-force states in the ATPase cycle. Very few compounds are known to have this property. The bipyridines are therefore potentially useful tools for studying the chemomechanics of the actomyosin cross-bridge interaction. Direct effects of milrinone on muscle cross-bridge cycle have not been studied. One objective of the present study is to determine whether milrinone's direct action on the cross bridges could contribute to the inotropic effect of the drug. Another objective of this study is to evaluate milrinone as a potential probe for investigating the chemomechanics of muscle cross-bridge cycle.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Muscle preparation. Adult New Zealand White rabbits weighing 2-3 kg were killed in a carbon monoxide chamber. Bundles of muscle fibers were then dissected from the psoas muscle, skinned as previously described (17). Skinned single fibers were dissected from a bundle. The ends of the fiber segment were fixed with glutaraldehyde and held by aluminum clips.

Solutions. All solutions contain 56 g/l dextran T-70, sufficient KCl to bring the ionic strength to 200 mM, sufficient Mg2+ to yield a free ion concentration of 1 mM, and 10 mM PIPES to buffer the solution to pH 7.0. Dithiothreitol (0.5 mM) was also added to the solutions. The ATP-regenerating system was provided by 20 mM creatine phosphate and the endogenous phosphocreatine kinase. Activating solution contained Ca2+ buffered to the desired pCa with 5 mM EGTA. Relaxing solution contained 5 mM EGTA, and a rinse solution of 0.1 mM EGTA was used to lower the EGTA concentration immediately before each activation. Skinning solution was the same as the relaxing solution except that the free Mg2+ concentration was 0.1 mM, and the fiber was briefly (5 min) exposed to 1% Triton X-100 immediately before the experiment. Milrinone (purchased from Sigma, St. Louis, MO) was made into a 60 mM stock solution by dissolving the chemical into 150 mM HCl solution. The milrinone stock was later diluted to desired concentrations in relaxing, rinse, and activating solutions. The same amount of HCl (without milrinone) was added to the corresponding control solutions, and the pH of both control and test solutions was then adjusted back to 7.0 with KOH.

Experimental procedure. The clipped fibers were attached to a force transducer and servomotor horizontally in a trough containing experiment solutions. A circulating coolant underneath the trough maintained the temperature of the solution at 10°C. The photoelectric force transducer had a resonance frequency of 18 kHz, a sensitivity of 10 mV/mg, and a signal-to-noise ratio of ~20. The transducer was of the same type used in experiments by Seow and Ford (17). The servomotor (Cambridge Technology model 308) was modified to achieve a faster step speed. The modification involved replacing the aluminum motor arm with a much lighter carbon fiber arm strengthened by a thin layer of epoxy. The step duration of the modified motor was 160 µs for steps as large as 100 µm. Sarcomere length of resting fiber was determined with the use of a videocamera mounted on an inverting microscope. Displaying of the images and calculation of the sarcomere spacing were facilitated by the OPTIMAS computer program (Optimas). The resting sarcomere length was set to 2.6 µm. The isometrically contracted sarcomere length was ~2.5 µm. The sarcomere spacing was checked after each contraction, and, if the spacing changed significantly, the fiber was discarded.

Test and control conditions were always studied in pairs. Fibers were relaxed after each activation. Between contractions, several minutes were allowed for the complete diffusion of milrinone. For tension transient experiments, a length step was applied to the fiber at the plateau of contraction. A large step release was applied at the end of the transient for two purposes: one to make the fiber go slack, thus setting zero baseline for the force transducer, and the other to determine maximal fiber shortening velocity with the "slack test." The size of the large end steps was varied to allow the fiber to take up the slack after a brief period of time, and the time of tension upstroke was determined. A plot of the step size vs. time required to take up the slack gave maximal velocity of shortening.

Hill's equation was used to fit the force-pCa curves: y = a/[1 + 10(x-b) · n], where y was the normalized force value, x was the pCa value, and a, b, and n were fitting constants that represented the maximal force, the pCa value corresponding to 50% of maximal force (pCa50), and the Hill's coefficient, respectively.

High-speed and high-resolution recording was achieved through the use of a Tektronix TDS 420 digitizing oscilloscope. The maximal sampling rate used to record the T1 and T2 phase of a tension transient was 40 µs/sample.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Milrinone reduced Ca2+-activated (pCa 4.5) isometric force of skinned fibers in a logarithmically concentration-dependent manner, as shown in the plot of force vs. the logarithm of milrinone concentration (Fig. 1). The milrinone concentration ranged from 0.05 to 1.2 mM, and a linear relation was observed within the range.


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Fig. 1.   Semilog plot of isometric force vs. milrinone concentration (mM) of skinned fibers at 10°C and pCa 4.5. Means ± SE bars are plotted. A straight line is fitted to data through linear regression. Numbers on data points indicate number of fibers used.

In addition to reducing isometric force, milrinone also reduced Ca2+ sensitivity of the fiber in terms of force generation, as shown in the right shift of the force-pCa curve (Fig. 2). Solid symbols are the controls, solid circles representing data obtained before addition of milrinone and solid triangles representing data obtained after removal of milrinone; open circles represent data obtained with 0.6 mM of milrinone. The pCa50 points were 6.12 ± 0.030 (SE) and 6.08 ± 0.028 for controls before and after addition of milrinone, respectively. The pCa50 value for 0.6 mM milrinone was 5.94 ± 0.025. Values were obtained from nonlinear curve fitting of the Hill equation described in METHODS. The Hill's coefficients were 3.01 ± 0.54 for data obtained before addition of milrinone, 2.80 ± 0.43 after removal of milrinone, and 3.39 ± 0.56 with milrinone. The goodness of fit (r2) for the three curves was >0.97, and the shift due to milrinone was significant (P < 0.05).


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Fig. 2.   Isometric force vs. pCa curve of skinned fibers. Data are fitted with 3-parameter Hill's equation as described in text. bullet , Initial controls before addition of milrinone; black-down-triangle , final control after removal of milrinone; open circle , 0.6 mM milrinone. Means ± SE are plotted. Number of fibers used per data point ranges from 4 to 9.

Maximal shortening velocity was estimated using the slack test. Large length steps were applied to the fiber making the fiber go slack, and the duration for the fiber shortening under zero load was determined by marking the moment the step was applied and the moment when force deviated from zero, as shown in Fig. 3A. The slack durations, Delta t1 and Delta t2, corresponded to two step releases. The size of the step release and the slack duration were then plotted in Fig. 3B for all fibers used in the experiment. A linear regression was used to determine the slope of the line fitted to the data. The slope represents unloaded shortening velocity. The y-intersects for control (0 milrinone) and test (0.6 mM milrinone) were 16.1 ± 16.9 (SD) and 14.2 ± 15.6 nm/half-sarcomere, respectively, and they were not different statistically. The slopes (maximal shortening velocities, nm · half-sarcomere-1 · ms-1) were, however, significantly different (P < 0.05), and they were 3.53 ± 0.42 and 2.90 ± 0.32, respectively, for 0 and 0.6 mM milrinone.


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Fig. 3.   A: example of step releases in a slack test. Both steps are control steps (0 milrinone). Sizes of length steps are 89.9 and 194.2 nm/half-sarcomere, and corresponding slack durations, Delta t1 and Delta t2, are 23.5 and 54.4 ms, respectively. B: Delta t vs. step release size. bullet , Controls; open circle , tests (0.6 mM milrinone). Straight lines are fitted through data with linear regression. Slope of lines indicates shortening velocity under 0 load.

A tension transient was obtained by applying a rapid length step to a maximally activated fiber. Figure 4, A and B, shows three length steps for control and test, respectively, one stretch and two releases, that were used to obtain the tension transients shown in Fig. 4, C and D. The transients were first described by Huxley and Simmons (10). The extreme tension reached during the step is termed T1; the rapid tension recovery after the step is termed T2; the pause or sometimes reversal of tension recovery after T2 is termed T3; and the slow recovery toward the prestep tension level is termed T4. The traces were averages of nine traces from nine fibers. Data averaging was used to reduce noise and simplify data presentation. High step speed was essential for these experiments because tension recovery after the step was mostly over after 2 ms, as evidenced in Fig. 4, C and D. The length steps applied to the fibers in these experiments were completed in ~200 µs, as shown in Fig. 4A. In Fig. 4, tension traces were normalized to their respective isometric force level; therefore, the control and test traces superimposed at both the isometric and zero force levels. Because the step sizes were the same for both control and test, the greater change in force after the length step observed under test condition (0.6 mM milrinone) indicated that the relative stiffness was higher in the presence of milrinone. This is more clearly illustrated in Fig. 5. Relative stiffness was represented by the slope of the relative T1 curve. There was a slight bent in the T1 curves, the slopes were greater in stretch than in release, and this was more pronounced for test (milrinone) than control. The relative stiffness increased significantly (P < 0.05) by 12.0 ± 2.0% (SE) due to 0.6 mM milrinone in stretch (Fig. 5). With release, the increase in relative stiffness was not significant. The T2 force recovery under the test condition paralleled the control, although within the recording period the relative force never recovered to the control level during the T3 and T4 phases of the transient response (Fig. 4). The force deficit was ~5% of isometric force. The time course of the phases in the transients was not identical in test and control conditions. For example, there was a more pronounced tension reversal after a step release in the T3 phase in the presence of milrinone (Fig. 4). The rate of T2 force recovery was step size dependent, slowest in stretch and fastest in the largest release, characteristic of tension transients first described by Huxley and Simmons (10).


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Fig. 4.   Averaged records (n = 9) for tension transients. A: averaged control length records. B: averaged test (0.6 mM milrinone) length records. Three steps, one stretch and two releases, are shown in both A and B. Only a small portion of length records are displayed to show details during step. C: superimposed tension transients of control (thick traces) and test (thin trace). Three pairs of transients correspond to 3 length steps shown in A and B. Test transients are normalized to control isometric tension (Po). D: traces are the same as in C, except time scale.


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Fig. 5.   Curves of relative tension reached during step (T1/T0) for control (bullet ) and test (0.6 mM milrinone; open circle ). Means ± SE are plotted.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Direct action of milrinone on skeletal muscle cross bridges has resulted in reduced isometric force and shortening velocity of the fiber. It is therefore unlikely that the positive inotropic effect of the drug on intact cardiac muscle is due to direct interaction of the bipyridine with the cross bridges. Ca2+ handling and other aspects of the excitation-contraction coupling of the muscle are likely targets for milrinone's contractility-enhancing action (14). Although cardiac muscle cross bridges are similar to those of skeletal muscle, it is not known that they will respond to milrinone intervention in exactly the same way. A conclusion regarding milrinone's direct action on cardiac cross bridges has to be derived from experiments on cardiac muscle.

Milrinone's specific interaction with skeletal muscle cross bridge makes it a useful tool for probing the cross-bridge cycle. The bipyridine has many similar properties as inorganic phosphate and 2,3-butanedione monoxime (BDM) in terms of inhibiting force generation in skinned skeletal muscle fibers (15, 18). Both phosphate and BDM detain cross bridges in low-force states. Energy for cross-bridge power stroke is derived from the hydrolysis of MgATP. The free energy of MgATP hydrolysis decreases with the logarithm of metabolite concentration (15). The observation that milrinone inhibits isometric force in a logarithmically dose-dependent manner (Fig. 1) suggests that the drug may interfere with the cross-bridge cycle by decreasing the free energy of MgATP hydrolysis. A more intuitive interpretation is that milrinone binds to a cross bridge in a low-force state and prevents the bridge from going through the force-generating power stroke. Due to the stochastic nature of the cross-bridge transition (9), detaining a bridge before the phosphate-releasing step would have a similar effect as raising the phosphate concentration and preventing phosphate release.

A possible mechanism for the positive inotropic effect of milrinone could be that the drug increases Ca2+ sensitivity of the actomyosin interaction. The present finding that milrinone reduces Ca2+ sensitivity of the skinned fiber in terms of force generation (Fig. 2) excludes that mechanism. It is not clear how milrinone decreases the Ca2+ sensitivity, but it is possible that the drug interferes with the troponin complex.

The 18% decrease in unloaded shortening velocity (Fig. 3B) in the presence of 0.6 mM milrinone supports the interpretation that milrinone detains a fraction of the cross bridges from going through a certain transition. These detained bridges act as internal loads and slow the shortening velocity in much the same way as H+ and BDM molecules do in skinned fibers (16, 18).

Tension transients obtained in the presence of milrinone have been scaled to match the isometric tension of the control. This normalization procedure implicitly assumes that the force recovery after a length step is the sum of stochastic responses of individual bridges. If milrinone merely reduces the number of active cross bridges and has no effect on the transitions of the cross-bridge cycle, one would expect the normalized transients to be parallel to each other. Figure 4 shows that this is not the case and that milrinone appears to interfere with the transitions among the attached states. The results can best be explained by a model in which some of the bridges respond to the step length change normally, whereas others are affected by the presence of milrinone. If a drug intervention detains bridges in low-force states, the isometric force is mainly attributed to attached bridges that have moved beyond the inhibited transition. These bridges are assumed to be able to respond normally. This could explain the time course of the T2 tension recovery in the first few milliseconds, which is relatively unaltered by milrinone (Fig. 4); i.e., the traces are largely parallel to each other. This suggests that the site of intervention of milrinone is probably not the power stroke itself. Note that, as tension response progresses, some of the normally responding bridges will move to detained states, whereas detained bridges will move past the inhibited transition. T3 and T4 phases of recovery therefore provide information on the subsequent movements of the bridges after the initial power stroke. The more prominent T3 tension reversal (negative slope) after a step release suggests that milrinone facilitates detachment of the bridges after the power stroke. The inhibitory effect of milrinone on shortening velocity is therefore probably not due to the drug's inhibition of cross-bridge detachment after power stroke, as with ADP (17), but instead an inhibition of transition between low-force states before power stroke.

Stiffness of muscle fiber is related to the number of attached cross bridges (5, 7). More recently, it has been found that the nonoverlap portion of the thin filaments contribute significantly to the fiber compliance (3, 11, 19). Stiffness of the fiber is therefore not a straightforward function of the number of attached cross bridges. Because of the lack of sarcomere length control in this study and also the lack of knowledge about the stress-strain relations of the myofilaments in our preparation, the relationship between fiber stiffness and the number of attached cross bridges cannot be described accurately. Therefore no attempt is made to use relative stiffness change as an indicator of shifting of bridges between high- and low-force states.

In the presence of milrinone, the fiber appears to be stiffer during stretch than during release. We have previously identified a low-force state in the cross-bridge cycle that possesses such a characteristic. High ionic strength appears to cause accumulation of bridges in a low-force state from which the bridges can easily detach after a step release (16) but not a stretch. This explains the fact that high ionic strength inhibits isometric force but not shortening velocity. The existence of such a state has been predicted by A. F. Huxley (9) to accommodate the finding that energy consumption rate decreases when the muscle is shortening near its maximal velocity. The detachment from this state of initial attachment prevents the bridges from going through the power stroke and hydrolyzing ATP. The present results indicate that milrinone causes accumulation of bridges in the initial state of attachment from which the bridges can detach easily without impeding shortening velocity. The inhibitory effect of milrinone on velocity further suggests that accumulation of the bridges must occur in other low-force states as well. This also explains why milrinone causes a much greater force decrease (55%) than velocity decrease (18%). Our previous finding (16, 18) of a comparable force inhibition by H+ and BDM accompanies a much greater depression of velocity.

Milrinone and its closely related analog amrinone are both positive cardiac inotropic agents. The small variation in the structure of these bipyridines, however, has led to a large difference in the way the compounds interact with the muscle cross bridges. Amrinone has been found to increase isometric force and drastically decrease shortening velocity (4; also our own unpublished observation), whereas milrinone in the same preparation causes a decrease in force and slight decrease in velocity. It appears that amrinone detains muscle cross bridges in a high-force state and that milrinone detains them in a low-force state. Understanding the nature of the interactions of these molecules with the actomyosin complex will undoubtedly yield insights into the transitions in a cross-bridge cycle.

    FOOTNOTES

Address for reprint requests: C. Y. Seow, Dept. Pharmacology and Therapeutics, University of British Columbia, 2176 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3.

Received 2 September 1997; accepted in final form 5 February 1998.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

1.  Alousi, A. A., and D. C. Johnson. Pharmacology of the bipyridines: amrinone and milrinone. Circulation 73, Suppl. III: III-10-III-24, 1986.

2.   Alousi, A. A., G. P. Stankus, J. C. Stuart, and L. H. Walton. Characterization of the cardiotonic effects of milrinone, a new and potent cardiac bipyridine, on isolated tissues from several animal species. J. Cardiovasc. Pharmacol. 5: 804-811, 1983[Medline].

3.   Bagni, M. A., G. Cecchi, F. Colomo, and C. Poggesi. Tension and stiffness of frog muscle fibres at full filament overlap. J. Muscle Res. Cell Motil. 11: 371-377, 1990[Medline].

4.   Bottinelli, R., V. Cappelli, S. E. J. N. Morner, and C. Reggiani. Effects of amrinone on shortening velocity and force development in skinned skeletal muscle fibres. J. Muscle Res. Cell Motil. 14: 110-120, 1993[Medline].

5.  Bressler, B. H., and N. F. Clinch. The compliance of contracting skeletal muscle. J. Physiol. (Lond.) 237: 477-493.

6.   Endoh, M., S. Yamashita, and N. Taira. Positive inotropic effect of amrinone in relation to cyclic nucleotide metabolism in the canine ventricular muscle. J. Pharmacol. Exp. Ther. 221: 775-783, 1982[Abstract].

7.   Ford, L. E., A. F. Huxley, and R. M. Simmons. The relation between stiffness and filament overlap in stimulated frog muscle fibres. J. Physiol. (Lond.) 311: 219-249, 1981[Abstract].

8.   Honerjager, P., M. Schafer-Korting, and M. Reiter. Involvement of cyclic AMP in the direct inotropic action of amrinone: biochemical and function evidence. Naunyn Schmiedebergs Arch. Pharmacol. 318: 112-120, 1981[Medline].

9.   Huxley, A. F. Reflections on Muscle. The Sherrington Lecture XIV. Liverpool, UK: Liverpool Univ. Press, 1980.

10.   Huxley, A. F., and R. M. Simmons. Proposed mechanism of force generation in striated muscle. Nature 233: 533-538, 1971[Medline].

11.   Huxley, H. E., A. Stewart, H. Sosa, and T. Irving. X-ray diffraction measurements of the extensibility of actin and myosin filaments in contracting muscle. Biophys. J. 67: 2411-2421, 1994[Abstract].

12.   Mansson, A., and K. A. P. Edman. Effects of amrinone on the contractile behaviour of frog striated muscle fibres. Acta Physiol. Scand. 125: 481-493, 1985[Medline].

13.   Mansson, A., J. Morner, and K. A. P. Edman. Effects of amrinone on twitch, tetanus and shortening kinetics in mammalian skeletal muscle. Acta Physiol. Scand. 136: 37-45, 1989[Medline].

14.  Morgan, J. P., J. K. Gwathmey, T. T. DeFeo, and K. G. Morgan. The effects of amrinone and related drugs on intracellular calcium in isolated mammalian cardiac and vascular smooth muscle. Circulation 73, Suppl. III: III-65-III-77, 1986.

15.   Pate, E., and R. Cooke. Addition of phosphate to active muscle fibers probes actomyosin states within the powerstroke. Pflügers Arch. 414: 73-81, 1989[Medline].

16.   Seow, C. Y., and L. E. Ford. High ionic strength and low pH detain activated skinned rabbit skeletal muscle crossbridges in a low force state. J. Gen. Physiol. 101: 487-511, 1993[Abstract].

17.   Seow, C. Y., and L. E. Ford. Exchange of ATP for ADP on high-force cross-bridges of skinned rabbit muscle fibers. Biophys. J. 72: 2719-2735, 1997[Abstract].

18.   Seow, C. Y., S. G. Shroff, and L. E. Ford. Detachment of low force bridges contributes to the rapid tension transients of skinned rabbit skeletal muscle fibres. J. Physiol. (Lond.) 501: 149-164, 1997[Abstract].

19.   Wakabayashi, K., Y. Sugimoto, H. Tanaka, Y. Ueno, Y. Takezawa, and Y. Ameniya. X-ray diffraction evidence for the extensibility of actin and myosin filaments during muscle contraction. Biophys. J. 67: 2422-2435, 1994[Abstract].


AJP Cell Physiol 274(5):C1306-C1311
0363-6143/98 $5.00 Copyright © 1998 the American Physiological Society




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