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(Circulation. 1996;94:3155-3160.)
© 1996 American Heart Association, Inc.

Articles

Influence of Isoproterenol and Ouabain on Excitation-Contraction Coupling, Cross-Bridge Function, and Energetics in Failing Human Myocardium

Gerd Hasenfuss, MD; Louis A. Mulieri, PhD; Paul D. Allen, MD, PhD; H. Just, MD; N.R. Alpert, PhD

the Medizinische Klinik III (G.H., H.J.), Universitat Freiburg, FRG; Department of Physiology and Biophysics (L.A.M., N.R.A.), University of Vermont, Burlington; and Department of Anesthesiology (P.D.A.), Brigham and Women's Hospital, Boston, Mass.

Correspondence to Gerd Hasenfuss, MD, Medizinische Klinik III, Universitat Freiburg, Hugstetter Str 55, 79106 Freiburg, FRG. E-mail hasenfus@ruf.uni-freiburg.de.

	Abstract

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Background In patients with heart failure, long-term treatment with catecholamines and phosphodiesterase inhibitors, both of which increase cyclic AMP levels, may be associated with increased mortality, whereas mortality may not be increased with glycoside treatment. Differences in clinical benefit between cyclic AMP–dependent inotropic agents and cardiac glycosides may be related to differences of these drugs on calcium cycling and myocardial energetics.

Methods and Results Isometric heat and force measurements were used to investigate the effects of isoproterenol and ouabain on myocardial performance, cross-bridge function, excitation-contraction coupling, and energetics in myocardium from end-stage failing human hearts. Isoproterenol (1 µmol/L) increased peak twitch tension by 55% and decreased time to peak tension and relaxation time by 30% and 26%, respectively (P<.005). Ouabain (0.38±0.11 µmol/L) increased peak twitch tension and relaxation time by 41% and 20%, respectively, and decreased time to peak tension by 12% (P<.05). With isoproterenol, the amount of excitation-contraction coupling–related heat evolution (tension-independent heat) increased by 246% (P<.05) and the economy of excitation-contraction coupling decreased by 61% (P<.05). Ouabain increased tension-independent heat by only 61% (P<.05) and did not significantly influence economy of excitation-contraction coupling. The effects of isoproterenol on excitation-contraction coupling resulted in a 21% (P<.005) decrease of overall contraction economy, which was not significantly changed with ouabain. Neither isoproterenol nor ouabain influenced energetics of cross-bridge cycling or recovery metabolism.

Conclusions Major differences between the effects of isoproterenol and ouabain in failing human myocardium are related to calcium cycling with secondary effects on myocardial energetics.

Key Words: myocardium • calcium • heart failure • inotropic agents • catecholamines

	Introduction

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Several recent clinical studies have indicated that inotropic agents that act primarily by increasing cAMP may not be suited for the long-term treatment of patients with congestive heart failure.^{1 2 3} In contrast, there is considerable evidence that treatment with ß-adrenoceptor blockers, which decrease cAMP production, may be beneficial with respect to myocardial function, clinical symptoms, and, potentially, survival of patients.^{4 5 6} In view of the advantage of blocking the effects of catecholamines and the unfavorable effects of phosphodiesterase inhibitors, the only clinically available positive inotropic agents for long-term treatment of congestive heart failure are the cardiac glycosides, which act independently of cAMP and even exhibit sympatholytic effects. In fact, there is good evidence that glycosides improve both left ventricular function and exercise capacity of patients with heart failure.⁷

At the myocardial level, the unfavorable long-term effects of ß-adrenoceptor stimulation and phosphodiesterase inhibition may be due to a cAMP-mediated increase in myocardial energy consumption and thus an impaired energy balance of the failing heart.^{8 9 10 11} In addition, cAMP, by its effects on calcium cycling, may trigger arrhythmias. On the other hand, energy turnover as well as calcium cycling may be more favorable with cardiac glycosides.

Accordingly, it was the goal of the present study to compare the effects of the ß-adrenoceptor stimulator isoproterenol and the glycoside ouabain on myocardial energetics and excitation-contraction coupling processes in isolated failing human myocardium. The experiments were performed with the use of a myothermal technique.¹²

	Methods

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Patients and Muscle Preparation
Experiments were performed in left ventricular myocardium from hearts obtained from 10 patients with end-stage failing dilated (n=7) or ischemic (n=3) cardiomyopathy undergoing cardiac transplantation surgery. The mean age of the patients was 50±4 years; 1 patient was female. The average ejection fraction was 14±2%. Previous medication included cardiac glycosides (n=10), ACE inhibitors (n=8), diuretics (n=10), diltiazem (n=2), isosorbide dinitrate (n=2), dobutamine (n=2), dopamine (n=1), quinidine (n=3), milrinone (n=1), and warfarin (n=1).

Left ventricular myocardium was dissected from the endocardial surface of the ventricle within 20 minutes after cardiectomy. The study protocol was reviewed and approved by the committees on human research of the University of Vermont and the Brigham and Women's Hospital.

Muscle Strip Preparation
The excised myocardium was immediately submerged in "protective solution" at room temperature and oxygenated by bubbling with 95% O₂, 5% CO₂.¹³ For preparation of the experimental intact muscle strip, the excised myocardium was transferred to a dissection chamber, and strips were dissected parallel to muscle cell length as described recently.^{13 14 15} To perform the heat and mechanical measurements, the muscle was mounted in contact with the active region of a thermopile and connected to the force gauge. The muscle and thermopile were then submerged in normal oxygenated Krebs-Ringer solution (37°C) to wash out the protective solution, and isometric twitches were evoked at 1-second intervals thereafter. The Krebs-Ringer solution contained (in mmol/L): Na⁺ 152, K⁺ 3.6, Cl^- 135, HCO₃^- 25, Mg²⁺ 0.6, H₂P0₄^- 1.3, SO₄^2- 0.6, Ca²⁺ 2.5, and glucose 11.2, as well as insulin 10 IU/L. After an equilibration period of 60 to 90 minutes, the muscle was stretched gradually (0.05- to 0.1-mm steps) to the length at which maximal steady-state twitch force was reached (l_max). All measurements were performed at this length under steady-state conditions. At the end of each experiment, muscle length at l_max was measured and the blotted weight of this segment was obtained. The cross-sectional area for normalization of force values was calculated as the ratio of blotted weight to muscle length (l_max). The effect of isoproterenol on heat and force was investigated in 11 muscle strip preparations from 7 hearts (dilated cardiomyopathy, n=5; ischemic cardiomyopathy, n=2) with an average cross-sectional area of 0.42±0.05 mm². The effect of ouabain was investigated in 8 muscle strip preparations from 7 hearts (dilated cardiomyopathy, n=5; ischemic cardiomyopathy, n=2) with an average cross-sectional area of 0.41±0.07 mm². In 3 muscle strips, both drugs were sequentially investigated (see "Experimental Protocol").

Experimental Protocol
To perform the measurements, the chamber was drained and thermal and tension signals were recorded during steady-state repetitive stimulation as described previously.¹⁶ After measurements were made under control conditions, isoproterenol or ouabain was applied to the bath and measurements were repeated. In three muscle strips in which both drugs were studied, isoproterenol was investigated first, control measurements were repeated after washout, and then the effects of ouabain were compared with the second set of control measurements. Isoproterenol was applied at 10^-6 mol/L, a concentration that increases tension by 50% without any aftercontractions, extra twitches, or rise in diastolic tension. The dose of ouabain was titrated to get a similar increase in contractile force without an increase in diastolic tension. The average concentration of ouabain was 0.38±0.11 µmol/L (range, 0.1 to 1 µmol/L). Twitch tension is reported as total active tension values minus diastolic tension.

Heat Terms, Myothermal Measurements, and Thermal Analysis
Definition of Heat Terms
Under steady-state isometric conditions, all of the energy turned over by the muscle is liberated as heat by the end of the stimulus interval. The total activity-related heat is divisible into activity-related initial and recovery heat.^{15 16} Recovery heat is the energy liberated during oxidative high-energy phosphate resynthesis. Activity-related initial heat is composed of tension-dependent heat and tension-independent heat. The latter is the portion of initial heat remaining after chemical or mechanical inhibition of cross-bridge cycling reflecting high-energy phosphate hydrolysis by excitation-contraction coupling processes.¹⁷ Tension-dependent heat is the difference between activity-related initial heat and tension-independent heat and reflects high-energy phosphate hydrolysis by cycling cross-bridges.

Thermal Measurements and Analysis
Changes in muscle temperature were measured with 14-junction, Hill-type thermopiles with a temperature sensitivity between 1.08 and 1.32 mV/°C and an average heat loss coefficient of 0.54 mcal/°C·s.¹² Thermopile output was amplified by an Ancom chopper amplifier (model 15C-3A, Ancom, Ltd). Isometric force was measured by a cantilever-beam force transducer.¹⁸ Temperature and force signals were displayed on an oscilloscope and simultaneously recorded on a chart recorder (Gould-Brush, model 2400). Measurements and calculations of myothermal data were performed as described recently.^{15 16 19} Partitioning of initial heat into tension-dependent heat and tension-independent heat was performed in seven muscle strips treated with isoproterenol and in eight muscle strips treated with ouabain. The procedure has been described in detail recently.^{16 19} Average tension-independent heat rate was obtained as the ratio of tension-independent heat to the time required for its evolution, which is equal to the twitch time.

Interpretation of Myothermal Data
Several ion transport systems contribute to tension-independent heat: (1) sarcoplasmic reticulum calcium pumps, (2) sarcolemmal calcium pumps, and (3) sodium-potassium pumps and other ATP-using pumps. The sarcolemmal sodium-calcium exchanger is energetically linked to sodium-potassium ATPases.^{20 21} Assuming that contribution of active removal of sodium that enters the cell during depolarization is small (<15%), tension-independent heat results predominantly from calcium transport.^{17 20} Tension-independent heat therefore mainly reflects calcium removal, which during steady-state conditions equals the total amount of calcium cycling. For sarcoplasmic reticulum calcium transport, one high-energy phosphate bond is hydrolyzed per two calcium ions cycled, and for transsarcolemmal calcium transport, the stoichiometry is 1:1.²¹ Because energy stoichiometry is different for sarcoplasmic reticulum and sarcolemmal calcium transport, use of tension-independent heat underestimates the effects on calcium cycling if isoproterenol or ouabain increases the proportion of sarcoplasmic reticulum calcium transport relative to sarcolemmal calcium transport. The potential error should be <15%. During each cross-bridge cycle, there is tight coupling to high-energy phosphate hydrolysis, and one high-energy phosphate is assumed to be hydrolyzed for each cross-bridge cycle.²² Therefore, tension-dependent heat resulting from high-energy phosphate hydrolysis by cycling cross-bridges reflects the total number of cross-bridge interactions during the isometric twitch. From tension-dependent heat and the muscle force-time integral, we can calculate the average cross-bridge force-time integral.^{15 20} Economy of excitation-contraction coupling was calculated as the ratio of tension-dependent heat (number of cross-bridge interactions) to tension-independent heat (number of calcium ions cycling). Overall economy of isometric contraction was calculated as the ratio of isometric tension-time integral to total activity-related heat. The ratio of initial heat to total activity-related heat is an index of the efficiency of metabolic recovery processes.¹⁶

Statistical Analysis
Data are expressed as mean±SEM. Differences between control measurements taken before application of each drug and measurements after application of isoproterenol or ouabain were determined by use of the paired t test. A value of P<.05 was accepted as statistically significant.

	Results

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Mechanical Parameters
Fig 1 shows typical recordings of isometric tension during control conditions and after application of isoproterenol and ouabain. Table 1 summarizes the effects of ouabain and isoproterenol on mechanical parameters. Ouabain increased peak twitch tension by 41% and the maximal rate of tension rise by 72% and did not significantly increase the maximal rate of tension fall. Isoproterenol increased peak twitch tension by 55% (not significantly different from the effect of ouabain), maximal rate of tension rise by 152%, and maximal rate of tension fall by 138%. The isometric tension-time integral increased with ouabain by 40% and did not significantly change with isoproterenol. There was no rise in diastolic tension with ouabain or isoproterenol. Both agents reduced time to peak tension (ouabain by 12% and isoproterenol by 30%). Time from peak twitch tension to 50% relaxation did not significantly change with ouabain but decreased by 30% with isoproterenol, whereas total relaxation time increased by 20% with ouabain and decreased by 26% with isoproterenol.

View larger version (13K): [in this window] [in a new window]   Figure 1. Original recordings showing the influence of isoproterenol (ISO) and ouabain (OUA) on isometric twitches in muscle strips from end-stage failing human hearts. Note that complete relaxation occurs during all conditions.

View this table: [in this window] [in a new window]   Table 1. Influence of Isoproterenol and Ouabain on Isometric Twitch Parameters

Energetic Parameters
Initial heat (high-energy phosphate hydrolysis by cross-bridges and excitation-contraction coupling processes) increased by 43% with ouabain and by 21% with isoproterenol (Table 2). The ratio of initial heat to total activity-related heat (an index of the efficiency of recovery metabolism) did not significantly change with either intervention (Table 2). This indicates that with both interventions, the observed increases in total activity-related heat resulted primarily from increased high-energy phosphate hydrolysis (initial heat) with a normal amount of associated increase in recovery metabolism.

View this table: [in this window] [in a new window]   Table 2. Influence of Isoproterenol and Ouabain on Myothermal Parameters

Initial heat was partitioned into its two components: tension-dependent heat (cross-bridge cycling) and tension-independent heat (calcium cycling). Tension-dependent heat was increased with ouabain by 41% whereas it was not changed with isoproterenol (Table 2). Cross-bridge force-time integral was not significantly changed with ouabain (0.179±0.021 versus control value of 0.169±0.016 piconewtons times seconds [pNs]) or isoproterenol (0.169±0.021 versus control value of 0.178±0.016 pNs). Tension-independent heat increased by 61% with ouabain and by 246% with isoproterenol (Fig 2). The rate of tension-independent heat evolution, reflecting the rate of calcium removal, did not significantly increase with ouabain but increased by 422% with isoproterenol (Fig 2). To evaluate the economy of excitation-contraction coupling processes, tension-dependent heat was related to tension-independent heat. The ratio of tension-dependent heat to tension-independent heat, indicating the number of cross-bridges activated per number of calcium ions cycling, was not changed with ouabain but was reduced considerably (by 61%) with isoproterenol (Fig 3).

View larger version (31K): [in this window] [in a new window]   Figure 2. Amount and rate of tension-independent heat evolution during control and after application of ouabain (OUA) or isoproterenol (ISO). Tension-independent heat reflects high-energy phosphate hydrolysis associated with excitation-contraction coupling and thus is an index of the amount of calcium cycling. Tension-independent heat rate is a reflection of the rate of calcium removal from the cytosol.

View larger version (24K): [in this window] [in a new window]   Figure 3. Influence of ouabain (OUA) and isoproterenol (ISO) on economy of excitation-contraction coupling processes as calculated by the ratio of tension-dependent heat to tension-independent heat. This ratio reflects the number of cross-bridge interactions per number of calcium ions cycling during one contraction-relaxation cycle.

Total activity-related heat increased by 41% with ouabain and by 34% with isoproterenol (Table 2). The ratio of tension-time integral to total activity-related heat (overall economy of myocardial contraction) was unchanged with ouabain but decreased significantly by 21% with isoproterenol (Fig 4). There was no significant correlation between the increase in contractile force and the decrease in economy of excitation-contraction coupling processes or overall economy of myocardial contraction within the isoproterenol group.

View larger version (23K): [in this window] [in a new window]   Figure 4. Influence of ouabain (OUA) and isoproterenol (ISO) on overall economy of myocardial contraction as calculated by the ratio of isometric tension-time integral to total activity-related heat (total heat).

	Discussion

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The present study shows that isoproterenol results in a pronounced increase in myocardial calcium turnover with a decreased economy of excitation-contraction coupling processes. The latter causes overall economy of myocardial contraction to decrease. In contrast, for a similar inotropic effect with ouabain, there is a moderate increase in calcium turnover without a change in economy of excitation-contraction coupling or overall contraction economy. Neither isoproterenol nor ouabain influences the energetics of cross-bridge cycling or recovery processes.

Effects on Isometric Contractions and Contractile Protein Function
Isoproterenol did not influence tension-dependent heat and cross-bridge force-time integral. These findings indicate that the inotropic effect of isoproterenol occurred without changes in the total number of cross-bridge interactions during one contraction-relaxation cycle and without changes in the behavior of the force-generating state of the individual cross-bridge cycle. Therefore, the isoproterenol effect resulted from an increased rate of cross-bridge activation with an increased number of cross-bridges recruited and attached per unit of time.

The finding of unaltered cross-bridge force-time integral is intriguing, because in nonfailing human myocardium, cross-bridge force-time integral was shown to be smaller than for failing myocardium and decreased even further with administration of isoproterenol.^{16 19 23} Reduced cross-bridge force-time integral with isoproterenol in nonfailing myocardium was interpreted to reflect an abbreviation of strongly bound cross-bridge attachment time (or an increase in off-rate "g"²⁴ ) associated with an increase in cross-bridge cycling rate.¹⁹ Accordingly, the present data indicate that the isoproterenol effect on cross-bridge behavior is lost in the failing human myocardium. Because unloaded shortening velocity and power development of the myocardium depend on cross-bridge attachment time, this also may suggest that the effect of isoproterenol to increase shortening velocity and power output is attenuated. In addition, the findings of an increased rate of relaxation and decreased relaxation time that occurred without changes in cross-bridge force-time integral indicate that relaxation is controlled by excitation-contraction coupling exclusively. This is in contrast to data from Brutsaert and Sys,^{25 26} who suggested that isometric relaxation would be controlled predominantly by cross-bridge behavior. Of course, findings may be different in mild or moderate degrees of heart failure.

Ouabain increased tension-dependent heat in proportion to contractile force without a change in cross-bridge force-time integral. This indicates that the effect is associated with a recruitment of additional cross-bridge interactions during the contraction-relaxation cycle.

Excitation-Contraction Coupling
With isoproterenol, the increase in the amount of calcium cycled (tension-independent heat) was threefold higher than with ouabain. Accordingly, compared with ouabain, isoproterenol significantly decreased the economy of excitation-contraction coupling processes. This can be seen when tension-dependent heat is related to tension-independent heat (Fig 3). The differences between both agents are even more pronounced when the kinetics of tension-independent heat evolution are analyzed (Fig 2).

The different effects of ouabain and isoproterenol on excitation-contraction coupling parameters can be explained by their different modes of action. Ouabain is believed to act by inhibiting the sodium-potassium-ATPase, thereby increasing intracellular sodium, which in turn leads to an increase in intracellular calcium via the activation of the Na⁺-Ca²⁺ exchanger.²⁷ In contrast, isoproterenol results in phosphorylation of various intracellular proteins through cAMP-dependent protein kinase A activation.²⁸ This may result in increased calcium influx across the sarcolemma, increased calcium release from the sarcoplasmic reticulum, increased rate of calcium removal, and decreased calcium affinity of troponin C. The latter can also be the cause of decreased economy of excitation-contraction coupling.

Previous myothermal measurements indicated that the total amount of calcium cycled is decreased in end-stage failing human myocardium.¹⁶ Fura-2 measurements in isolated myocytes from end-stage failing human hearts have consistently suggested decreased calcium transients.²⁹ Furthermore, a continuous decrease in calcium transients and force with increasing stimulation frequency was observed in the failing human myocardium.³⁰ This was attributed to decreased expression of the sarcoplasmic reticulum calcium pump (SR-Ca²⁺-ATPase).^{31 32} In light of these findings, the present data indicate that despite decreased SR-Ca²⁺-ATPase levels, calcium turnover can be stimulated considerably by cAMP-dependent phosphorylation in the failing human myocardium. Interestingly, we recently observed that SR-Ca²⁺-ATPase protein levels were decreased to a greater proportion than protein levels of its inhibitory protein, phospholamban.³³ Therefore, one might speculate that the degree by which phospholamban phosphorylation, relieving SR-Ca²⁺-ATPase inhibition, increases SR-Ca²⁺-ATPase activity is more pronounced in the failing human heart. This may also explain the finding that the depressed force-frequency relation of failing human myocardium can be reversed by small concentrations of forskolin, which is a stimulator of adenylyl cyclase.³⁴

Myocardial Energy Consumption
The influence of inotropic interventions on myocardial energetics has been investigated extensively in animal experiments (for review, see Reference 8). Considerable differences based on species investigated and experimental techniques applied have been observed. The present data show that both inotropic interventions were associated with a comparable increase in total energy consumption (total activity-related heat) due to increased breakdown of high-energy phosphates but with unchanged efficiency of recovery metabolism. With ouabain, the increase in total activity-related heat resulted from an increased number of cross-bridge interactions and a moderate increase in calcium cycling. The mechanical counterpart was an increase in isometric tension and tension-time integral. In contrast, with isoproterenol, the increase in total activity-related heat resulted from a pronounced increase in calcium turnover with increased rates of cross-bridge activation and deactivation but without a change in the total number of cross-bridge interactions. Accordingly, the mechanical counterpart was an increase in isometric tension associated with increased rates of tension rise and fall without a change in tension-time integral.

The ratio of tension-time integral to total activity-related heat is an index of the overall economy of myocardial contraction. With ouabain, total activity-related heat increased in proportion to the tension-time integral. However, with isoproterenol, total activity-related heat increased without a significant change in tension-time integral. Therefore, overall economy of contraction was decreased by 21% with isoproterenol whereas it was unchanged with ouabain.

In summary, the present study shows that for a given increase in contractile force with isoproterenol or ouabain in isolated failing human myocardium, subcellular mechanisms are considerably different. Major differences are related to intracellular calcium handling. The pronounced increase in calcium turnover after ß-adrenoceptor stimulation may be favorable because it accelerates tension development and relaxation. However, it may be a cause of increased arrhythmias in patients with heart failure. In addition, the decrease in economy of myocardial contraction, although moderate, may be harmful in failing hearts with impaired energy balance. Both mechanisms may contribute to the detrimental effects of increased endogenous catecholamine levels or a pharmacologically stimulated cAMP system.

	Acknowledgments

 
This study was supported by NIH grant No. USPHS P01HL-2800-13 and DFG grant HA 1233/3-2. Dr Hasenfuss is an Established Investigator of the German Research Foundation (DFG, Heisenberg-Stipendium HA 1233/4-1).

Received February 21, 1996; revision received August 1, 1996; accepted August 7, 1996.

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