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(Circulation Research. 1995;76:1036-1048.)
© 1995 American Heart Association, Inc.

Articles

Relationship Between Intracellular Calcium and Contractile Force in Stunned Myocardium

Direct Evidence for Decreased Myofilament Ca²⁺ Responsiveness and Altered Diastolic Function in Intact Ventricular Muscle

Wei Dong Gao, Dan Atar, Peter H. Backx, Eduardo Marban

From the Division of Cardiology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Md, and the Centre for Cardiovascular Research (P.H.B.), University of Toronto (Canada).

Correspondence to Eduardo Marban, MD, PhD, Room 844, Ross Bldg, 720 Rutland Ave, Baltimore, MD 21205.

	Abstract

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Abstract To elucidate the abnormalities of excitation-contraction coupling in stunned myocardium, we measured [Ca²⁺]_i and force in thin fura 2–loaded ventricular trabeculae from control or stunned (20 minutes ischemia followed by 20 minutes reflow at 37°C) rat hearts. At any given [Ca²⁺]_o, force development was significantly lower in the stunned trabeculae than in control trabeculae. In contrast, there was no difference in the amplitude of Ca²⁺ transients between the two groups. The steady state force-[Ca²⁺]_i relationship, assessed by tetanization in the presence of ryanodine, revealed both a decrease in maximal Ca²⁺-activated force and an increase in the [Ca²⁺]_i required for 50% activation in stunned trabeculae. Postischemic myocardium also exhibited an accelerated rate of diastolic relaxation that was not due to changes in the rate of Ca²⁺ transient decay. Destabilization of attached cross-bridges in a quantitative model of cardiac myofibrils accurately reproduced the salient systolic and diastolic features of the stunned phenotype, suggesting an abnormality of the thin filaments. In response to supraphysiological increases in [Ca²⁺]_o, diastolic [Ca²⁺]_i and diastolic tone increased much more in stunned trabeculae than in controls, with the frequent occurrence of aftercontractions. This novel experimental model lends further support to the hypothesis that the primary lesion of excitation-contraction coupling resides at the level of the contractile proteins. The finding of enhanced susceptibility to calcium overload helps to rationalize the functional deterioration of stunned myocardium during intense inotropic stimulation and additionally suggests that stunned myocardium may represent a favorable substrate for triggered arrhythmias.

Key Words: intracellular calcium • myofilament Ca²⁺ sensitivity • myocardial ischemia/reperfusion • diastolic relaxation • aftercontraction

	Introduction

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For days to weeks following a severe but brief ischemic insult, myocardial contractility is decreased. This reversible form of dysfunction, known as stunning,^{1 2} has been subjected to extensive investigation at various levels of integration. Although there is a general consensus that free radicals are involved in the pathogenesis of stunning,³ the site of the lesion in excitation-contraction coupling is still a matter of some debate. Electrical activation is normal,⁴ so that the basis for stunned myocardium must lie in either of two broad mechanistic categories: first, the availability of activator Ca²⁺ might be restricted. Such an effect could be mediated by abnormal Ca²⁺ entry into (or removal from) the cytosol due to lesions in one or more cellular Ca²⁺-handling pathways. Alternatively, the responsiveness of the contractile machinery to Ca²⁺ might be blunted such that the myocardium generates less force for any given Ca²⁺ transient; in this case, the availability of Ca²⁺ need not be the limiting factor.

Direct measurements of [Ca²⁺]_i in stunned perfused hearts have revealed that [Ca²⁺]_i is not reduced during twitch contractions.^{5 6} In addition, estimates of maximal Ca²⁺-activated force in perfused hearts have shown a reduction that accounts for roughly half of the decrease in developed pressure observed after stunning.⁷ Although compelling, the results have yet to be confirmed using intact experimental preparations devoid of complications from the superimposed effects of vascular turgor and loading. Studies in skinned preparations have yielded variable results, one showing a reduction in steady state myofilament Ca²⁺ sensitivity⁸ and another showing no changes,⁹ suggesting that at least part of the dysfunction may reflect alterations in soluble cytoplasmic factors. This idea has not been tested, since steady state myofilament properties have yet to be characterized in intact stunned myocardium.

Diastolic function has also been reported to be abnormal in the stunned myocardium: in regional models, relaxation times are prolonged as reflected either by measurements of pressure-length loops¹⁰ or diastolic thinning.^{11 12} Furthermore, isovolumic perfused hearts exhibit a prominent elevation of end-diastolic ventricular pressure in the stunned condition.^{13 14} While much importance has been attached to these apparent abnormalities of diastolic relaxation, it is not yet clear whether any of the observations reflect intrinsic changes in myocardial contractile function. Changes in chamber pressure can influence and distort the function of a relatively hypocontractile segment,¹⁵ and alterations of preload are often difficult to exclude. Both the regional and the global models are susceptible to the superimposed effects of coronary vascular turgor,¹⁶ which may be potentiated by postischemic edema.¹⁷

Diastolic relaxation is mediated by several cellular processes, including Ca²⁺ reuptake by the sarcoplasmic reticulum (SR), Ca²⁺ extrusion via Na⁺-Ca²⁺ exchange, dissociation of Ca²⁺ from troponin C, and cross-bridge detachment. There are good reasons to expect that at least two of these processes are abnormal in the stunned myocardium. Maximal Ca²⁺ uptake capacity has been reported to be decreased in SR vesicles isolated from stunned heart,¹⁸ hinting at a possible impairment of the ability to regulate Ca²⁺-dependent diastolic tone. Superimposed metabolic alterations in the stunned myocardium, including changes in glycolytic flux¹⁹ and a decrease in cytosolic ATP levels,⁵ may in turn influence the proteins that mediate ion homeostasis even if the molecules themselves are normal.

To clarify the nature of both the systolic and the diastolic dysfunctions, we have measured [Ca²⁺]_i and force in ventricular trabeculae from stunned rat hearts. The results confirm that Ca²⁺ transients are not reduced in stunned myocardium and demonstrate that the alterations in steady state Ca²⁺ activation consist of both a decrease in maximal force and desensitization (ie, a rightward shift of the [Ca²⁺]_i-force relationship). In contradiction to previous conclusions from vascularly perfused preparations, we find that diastolic relaxation is faster in trabeculae from stunned hearts than in nonischemic controls. Simple changes of cross-bridge attachment and detachment rates in a quantitative model of myofilament interaction reproduced the salient features of the contractile dysfunction of stunned myocardium. The stunned myocardium also exhibits an increased susceptibility to cellular Ca²⁺ overload, consistent with the reported decrease in the Ca²⁺ uptake capacity of the SR.¹⁸

	Materials and Methods

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Whole Rat Hearts
Rats of either sex (LBN-F1 strain, 200 to 250 g; Harlan, Indianapolis, Ind) were anesthetized with ether. A midsternal thoracotomy was performed to expose the heart, and 0.3 to 0.5 mL heparin (1000 U/mL; Elkins-Sinn, Inc) was injected into the left atrium. The aorta was then clamped and the heart was rapidly excised. The aorta was cannulated and the heart was perfused retrogradely (15 mL/min) with Krebs-Henseleit (K-H) solution equilibrated with 95% O₂/5% CO₂. The K-H solution was composed of (mmol/L) NaCl 120, NaHCO₃ 20, KCl 5, MgCl₂ 1.2, glucose 10, and CaCl₂ 1.0, pH 7.35 to 7.40. Except as indicated below, the hearts were paced at 275 beats per minute via two electrodes placed over the aorta and right ventricle. Isovolumic left ventricular pressure was measured with a custom-made balloon (TSC) filled with water and connected to a pressure transducer (model P23Db, Gould). The volume of the balloon was adjusted to a diastolic pressure of 10 mm Hg, after which the balloon volume was kept constant. The heart was placed in a water-jacketed container. An implantable temperature probe (model 3441, Physitemp) was put inside the ventricle and the temperature was kept at 37°C. After 10 to 15 minutes during which pressure development was allowed to stabilize, the hearts were subjected to 20 minutes of no-flow global ischemia at 37°C. Pacing was stopped after 3 minutes of ischemia and was resumed after 3 minutes of reperfusion. The hearts were removed from the perfusion apparatus after 20 minutes of reperfusion and subsequently perfused with high-K⁺ (20 mmol/L) K-H solution in a dissection dish at room temperature (20°C to 22°C). Control hearts were perfused continuously and paced at 37°C for 60 minutes and then placed in the dissection dish.

Rat Trabeculae
Trabeculae from the two groups of hearts were quickly dissected from the right ventricle and mounted between a force transducer and a micromanipulator according to the methods described previously.^{20 21} The dimensions of the unstretched trabeculae measured under a microscope (model 212219, Nikon; x40 magnification) were (in mm): 2.13±0.5 long, 0.16±0.12 wide, and 0.11±0.05 thick (mean±SD, n=15). Selection of trabeculae was based on the homogeneity of the muscles (uniform thickness, width, and sarcomere length), the absence of branches, and the presence of minimal damage at both ends. Cross-sectional area was calculated by multiplying the thickness and width and was corrected by multiplying a factor of 0.75 (assuming an ellipsoidal shape and an 5% reduction in muscle thickness due to stretching to a sarcomere length of 2.2 µm). The cross-sectional area for control trabeculae was 0.025±0.015 mm² (n=7) and for stunned trabeculae was 0.027±0.024 mm² (n=8) (mean±SD, P=NS). The trabeculae were superfused with K-H solution (except for a reduction of CaCl₂ to 0.5 mmol/L) at a rate of 10 mL/min and stimulated at 0.5 Hz. All experiments were performed at room temperature (20°C to 22°C). Force was measured by a silicon strain gauge (model AEM 801, SensoNor)^{20 21} and was expressed in mN/mm² of cross-sectional area. Sarcomere length was measured as described previously.^{20 21}

Measurement of [Ca²⁺]_i
[Ca²⁺]_i was measured by use of the free acid form of fura 2 as described.^{20 21 22} Fura 2 potassium salt was microinjected iontophoretically into one cell and allowed to spread throughout the whole muscle via gap junctions. The tip of the microelectrode (0.2 µm in diameter) was filled with fura 2 salt (1 mmol/L), and the remainder of the electrode was back-filled with 150 mmol/L KCl. Electrodes had resistances of 200 to 270 M when placed in K-H buffer. The microelectrode was successfully impaled into a superficial cell in the unstimulated muscle, and a hyperpolarizing current of 5 to 8 nA was passed continuously for 20 to 35 minutes. After the injection, fura 2 was initially localized around the site of injection. The preparation was stimulated at 0.5 to 1 Hz for 40 to 60 minutes, after which the preparation was uniformly loaded with fura 2. After loading, a stimulation rate of 0.5 Hz was used to characterize Ca²⁺ transients and twitch contractions. The epifluorescence of fura 2 was measured by exciting at 380, 360, and 340 nm. The fluorescent light was collected at 510 nm by a photomultiplier tube (model R1527, Hamamatsu). The output of the photomultiplier tube was filtered at 100 Hz, collected by an A/D converter, and stored in the computer for later analysis. [Ca²⁺]_i was given by the following equation (after subtraction of the autofluorescence of the muscle):

where R is the observed ratio of fluorescence (340/380), K'_d is the apparent dissociation constant, R_max is the ratio of 340/380 nm at saturating [Ca²⁺]_o, and R_min is the ratio of 340/380 nm at zero [Ca²⁺]_o.^{21 23} The values for K'_d, R_max, and R_min were determined by in vivo calibrations as follows.

In vivo calibrations were carried out using a previously described method.^{20 21} After loading fura 2, the muscle was poisoned with a solution containing (mmol/L) KCl 140, HEPES 25, MgCl₂ 0.75, NaCN 2, and iodoacetic acid (IAA) 0.5, pH 7.4 at room temperature. About 10 minutes after full rigor development, solutions containing (mmol/L) K₂EGTA 10, KCl 100, HEPES 25, MgCl₂ 0.75, NaCN 2, IAA 0.5, and 50 µmol/L ionomyocin (Calbiochem) with varied [Ca²⁺] were applied to the bath. [Ca²⁺] was varied by mixing K₂EGTA and CaEGTA proportionally to obtain various [Ca²⁺]s. The R_max and R_min were 7.5 and 0.50, respectively. The apparent K_d, K'_d, was 3.2 µmol/L. This value is the product of the true K_d of fura 2 for Ca²⁺ multiplied by the ratio of the fluorescence of the Ca²⁺-free to the Ca²⁺-bound forms of fura 2 at 380 nm (S_f2/S_b2, see Reference 23²³ ). The value of S_f2/S_b2 was 12 in our setup, and the true K_d, estimated in vivo, equaled 267 nmol/L.

Tetanization of Trabeculae
Ryanodine was used to obtain steady state activation in the trabeculae. After exposure to ryanodine (5 µmol/L) for 20 minutes, the muscles were tetanized briefly (3 to 5 seconds) by stimulation at 10 Hz. Different tetanized forces were achieved with varied [Ca²⁺]_os (0.25 to 20 mmol/L).^{20 21}

Measurement of Diastolic Tone
Absolute resting force was quantified as follows: the muscle was released to a sarcomere length of 1.7 µm to define the baseline at 0.25 mmol/L [Ca²⁺]_o. At this sarcomere length and [Ca²⁺]_o, neither active force nor restoring force was seen.²⁴ The muscle was then stretched to a sarcomere length of 2.2 µm. The increase in force generated above the baseline level was measured and defined as the resting force. Throughout the experiments, end-diastolic sarcomere length was consistently kept at 2.2 µm. Both twitch force and Ca²⁺ transients were stored in a computer for later analysis.

Statistics
Student's t test or one-way ANOVA was used for statistical analysis of the data.²⁵ A P value of <.05 was considered to indicate significant differences between the groups. Unless otherwise indicated, pooled data are expressed as mean±SD.

	Results

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Effects of Ischemia on Pressure Development in Isolated Rat Hearts
The initial step was to produce stunned hearts and nonischemic isochronal controls. The stunning protocol, which consisted of total global ischemia followed by reperfusion, was similar to that used in previous studies.^{5 7 13} Fig 1A (left panel) shows a typical recording of left ventricular pressure during normal perfusion, 20 minutes of ischemia, and 20 minutes of reperfusion. Left ventricular pressure decreased to very low levels quickly after the cessation of flow. By the end of 20 minutes of global ischemia, there was usually a small contracture (10 mm Hg increase in diastolic left ventricular pressure). Upon reperfusion, diastolic left ventricular pressure increased, accompanied by a period of arrhythmias and potentiated contractions that subsided in a few minutes. During the following 20 minutes of reperfusion, diastolic left ventricular pressure gradually decreased, and systolic left ventricular pressure reached a level lower than that before the insult. The right panel in Fig 1A shows pooled data for left ventricular pressure before ischemia and after 20 minutes of reperfusion in all the hearts from which trabeculae were dissected. Peak systolic pressure and left ventricular developed pressure (peak systolic pressure minus end-diastolic pressure) decreased significantly (developed pressure fell from 80±5 to 40±2 mm Hg, P=.001). Fig 1B shows left ventricular pressure recorded from a control heart, which was simply perfused for 60 minutes (left panel); neither systolic pressure nor diastolic pressure changed (right panel).

View larger version (12K): [in this window] [in a new window]   Figure 1. Left ventricular pressure development in stunned (upper panels) and control (lower panels) rat hearts. Hearts were perfused (15 mL/min) with Krebs-Henseleit buffer saturated with 95% O2/5% CO2 at 37°C and pH 7.35. Pacing rate was 275 beats per minute. A, left, Representative recording of left ventricular pressure development during control, 20-minute ischemia, and 20-minute reperfusion periods. Global ischemia was achieved by turning off the flow pump. Pacing was stopped 3 minutes after ischemia and 3 minutes before reperfusion; right, mean data for diastolic () and systolic () pressures of hearts subjected to ischemia/reperfusion. B, left, Typical recording of left ventricular pressure from a control heart that was perfused continuously for 60 minutes; right, pooled systolic () and diastolic () pressure data from hearts that were perfused for 60 minutes.

Force Development and Ca²⁺ Transients in Stunned Trabeculae
Trabeculae from both stunned and control hearts were dissected rapidly and mounted in the experimental chamber on the stage of an inverted microscope. After mounting the trabecula, it was inspected for any visible damage. Trabeculae from the stunned hearts looked entirely normal: there were no contracture bands, and the sarcomeres were well aligned. We first determined whether these trabeculae were stunned in a fashion representative of the hearts as a whole. Fig 2 shows Ca²⁺ transients and force of typical trabeculae from control and stunned hearts. Despite a remarkable similarity of the Ca²⁺ transients, force was approximately 60% lower in the stunned trabecula than in the control trabecula, which had never been subjected to ischemia. In contrast to end-diastolic pressure in the intact hearts, diastolic force was not increased in the stunned trabeculae relative to controls. This is evident from the examples in Fig 2 and from pooled data (absolute end-diastolic force=2.87±2.75 mN/mm² in controls vs 3.44±2.08 mN/mm² in stunned at 1.0 mmol/L [Ca²⁺]_o; P=NS). Nevertheless, latent abnormalities in diastolic Ca²⁺ handling were unmasked at higher [Ca²⁺]_o. Diastolic function will be explored in detail after we first investigate the basis of the systolic dysfunction.

View larger version (14K): [in this window] [in a new window]   Figure 2. Intracellular Ca2+ transients and force development in representative trabeculae from a control heart (left) and a stunned heart (right). The trabeculae were bathed in Krebs-Henseleit solution and 1 mmol/L [Ca2+]o at room temperature. Peak values of force were 39.9 mN/mm2 in the control and 18.8 mN/mm2 in the stunned trabecula. Peak [Ca2+]i transients equaled 0.81 µmol/L in the control and 0.88 µmol/L in the stunned trabecula.

As an initial assessment of excitation-contraction coupling, Fig 3 shows the peak [Ca²⁺]_i and peak force of the control and stunned trabeculae measured at various [Ca²⁺]_os. Although there is a small degree of overlap between the two groups, force development at any given [Ca²⁺]_i tends to be lower in the stunned muscles. If we assume a linear relationship between peak [Ca²⁺]_i and peak force, the slope of this relation is markedly decreased after stunning (P<.001 compared with control muscles). These data agree with and extend the findings from intact heart studies,^{5 6} which concluded that the availability of activator Ca²⁺ is not reduced in stunned myocardium.

View larger version (18K): [in this window] [in a new window]   Figure 3. Pooled data for peak force and peak [Ca2+]i from all the control () and stunned () muscles in this study at various [Ca2+]os. A linear relation between force and [Ca2+]i was assumed and fit by least-squares minimization. The slope of the relation was 71.7±14.2 (mN/mm2) · (µmol/L)-1 in control muscles and 29.3±7.6 (mN/mm2) · (µmol/L)-1 in stunned muscles (P<.001).

Maximal Ca²⁺-Activated Force and Ca²⁺ Sensitivity of Stunned Trabeculae
The results above suggest that the responsiveness of the myofilaments to Ca²⁺ is depressed in stunned trabeculae. The responsiveness of the myofilaments to Ca²⁺, however, cannot be reliably assessed based on peak Ca²⁺ and peak force.²⁶ Furthermore, it is impossible to distinguish whether the changes reflect an underlying decrease in maximal Ca²⁺-activated force and/or a decreased Ca²⁺ sensitivity of the myofilaments without determining the complete force-[Ca²⁺]_i relation. We therefore tetanized the same trabeculae to achieve steady state activation of the myofilaments^{20 21 27} over a broad range of [Ca²⁺]_i. Fig 4 shows individual tetanized forces and the corresponding [Ca²⁺]_i (upper panels) as well as the steady state force-[Ca²⁺]_i relations (lower panels) from representative control and stunned trabeculae. The control muscle exhibits steady state Ca²⁺-activation properties typical of those described previously in acutely dissected trabeculae^{20 21} : the force-[Ca²⁺]_i relationship is steep, with near saturation by 1 µmol/L [Ca²⁺]_i and a robust maximal force. In contrast, the stunned muscle shows a rightward shift of the force-[Ca²⁺]_i relationship and a depressed maximal Ca²⁺-activated force as well.

View larger version (16K): [in this window] [in a new window]   Figure 4. Typical examples of tetani and steady state force-[Ca2+]i relationships of control (left panels) and stunned (right panels) trabeculae. Upper left, Representative tracings of tetanus and [Ca2+]i in a control muscle at 2.0 and 12.0 mmol/L [Ca2+]o, respectively. Tetanization of the muscles was achieved by stimulating the muscles at 10 Hz in the presence of 5 µmol/L ryanodine, and different levels of activation were obtained by varying [Ca2+]o (see text for detailed protocol). Lower left, Steady state force-[Ca2+]i relationship of the control trabecula. The relation was fit with the Hill equation: F=Fmax[Ca2+]in/K1/2n+[Ca2+]in, where Fmax is the maximal force, K1/2 is [Ca2+]i at half Fmax, and n is the Hill coefficient. In this control muscle, Fmax=112 mN/mm2, K1/2=0.54 µmol/L, and n=4.7. Upper right, Representative tracings of tetanus and [Ca2+]i in the stunned trabecula at 2.0 and 16.0 mmol/L [Ca2+]o, respectively. Note the high [Ca2+]i and low force achieved. Lower right, Steady state force-[Ca2+]i relationship of this stunned muscle: Fmax=50 mN/mm2, K1/2=0.79 µmol/L, and n=2.34.

Fig 5 shows pooled data for all the force-[Ca²⁺]_i relations in control (n=7) and stunned (n=8) trabeculae. The data were normalized to their respective maximal values and plotted with respect to the means of the absolute maximal value in each group. In control trabeculae, maximal Ca²⁺-activated force was 102.4±24.1 mN/mm², and the [Ca²⁺]_i required for 50% activation was 0.60±0.11 µmol/L. In stunned trabeculae, maximal force equaled 56.2±15.3 mN/mm² (P<.01 vs control), and [Ca²⁺]_i required for 50% activation was 0.83±0.16 µmol/L (P<.05 vs control). The Hill coefficient was 5.85±2.06 for control muscles and 3.78±1.94 for stunned muscles (P>.05). Hence, both the maximal Ca²⁺-activated force and Ca²⁺ sensitivity of the myofilaments were decreased in stunned trabeculae.

View larger version (15K): [in this window] [in a new window]   Figure 5. Pooled data for the steady state force-[Ca2+]i relationship of control () and stunned () trabeculae. Forces were normalized with respect to the maximal value of each muscle. The means±SEM of the absolute maximal forces from both groups of muscles are also plotted at the respective highest [Ca2+]is.

Time Courses of Twitch Force and Ca²⁺ Transients
The recordings of twitch force and Ca²⁺ transients in trabeculae have thus far been interpreted only in terms of their peak values, but these recordings contain much additional information which can yield insights into the diastolic properties of stunned myocardium. Fig 6 shows representative recordings of force and Ca²⁺ transients from control and stunned trabeculae at two different [Ca²⁺]_os. In spite of the similarity of the Ca²⁺ transients (left panels), force development was lower in the stunned trabecula (center panels). Comparison of the superimposed and normalized forces (right panels) reveals a surprising change in the time course of contraction in the stunned trabecula: both the time to peak force and the time from the peak to 50% decay are accelerated, resulting in an abbreviation of overall force development. Ca²⁺ transients, on the other hand, showed only small changes in the time to peak and no significant change in the time to 50% decay. Fig 7, which shows the pooled data for these indices, verifies that the findings in Fig 6 are indeed typical. While the time to peak of Ca²⁺ transients increased over the whole range of [Ca²⁺]_o (P=.017), time to peak force decreased (P=.005). The time to 50% decay of force was also shortened (P=.001), with no change in the decay of the underlying Ca²⁺ transients (P=.57). It is worth emphasizing that the changes in Ca²⁺ transients, while modest, are in the wrong direction to explain the concomitant acceleration of relaxation.

View larger version (15K): [in this window] [in a new window]   Figure 6. Representative recordings of Ca2+ transients and forces of stunned and control trabeculae at two different [Ca2+]os (0.5 and 1.0 mmol/L). Force recordings are superimposed in absolute terms (center) and normalized (right) in order to highlight differences in the time course of the contractions. The muscles were stimulated at 0.5 Hz and end-diastolic sarcomere lengths were set at 2.2 µm. ST indicates stunned trabecula and CT, control trabecula.

View larger version (21K): [in this window] [in a new window]   Figure 7. Pooled data for time to peak (left panels) and time from peak to 50% relaxation (right panels) of Ca2+ transients (upper panels) and twitch force (lower panels) at varied [Ca2+]os. , indicates control trabeculae and , , stunned trabeculae.

Modeling of Cross-Bridge Cycling in Stunned Myocardium
In order to determine whether the observed decrease in Ca²⁺ responsiveness suffices to explain the decrease of contractility and the changes of relaxation in stunned muscles, we used the quantitative cross-bridge model depicted in Fig 8C. We have previously used this scheme to analyze the basis of the negative inotropic effect of 2,3-butanedione monoxime (BDM).²¹ The biochemical states, rate constants, and equilibrium constants were determined from empirical studies whenever possible (see Backx et al²¹ for details). When a control Ca²⁺ transient is provided as the input, this model predicts a twitch contraction that is very similar in phase relations and kinetics to those recorded experimentally (Fig 8A, left panel). We then determined whether simple changes in cross-bridge attachment and detachment rates, which reflect the modulatory effects of the thin filaments, would suffice to reproduce the contractile phenotype of the stunned myocardium. Fig 8A (right panel) shows the force predicted by the model with a stunned Ca²⁺ transient as the input when k₅₁, k₆₁, and k₇₁ were slowed twofold, k₁₅, k₁₆, and k₁₇ were accelerated twofold, and k₁₂ was increased from 5 sec^-1 to 15 sec^-1. All of these changes are consistent with a destabilization of the AMDP state of the cross-bridge (state 1 in Fig 8C), implying a reduced ability of Ca²⁺ binding to the thin filament to appropriately expose the myosin binding site on actin. The changes in simulated twitch force in Fig 8A are quite similar to those measured empirically (Fig 2). Fig 8B shows the steady state force-[Ca²⁺]_i relationship generated by the model for the "control" (ie, no change in rate constants) and for the "stunned" situation based on the same kinetic changes as in Fig 8A (right panel). The simulated steady state force-[Ca²⁺]_i relationships agree very well with the experimental data: both maximal force and sensitivity were decreased, without major changes in the Hill coefficient.

View larger version (15K): [in this window] [in a new window]   Figure 8. Simulated twitch forces (A) and steady state force-[Ca2+]i relations (B) for control and stunned cardiac muscle, predicted by a quantitative cross-bridge model (C). A, The superimposed Ca2+ transients were recorded from a control (left) and stunned trabecula (right) and used as inputs. B, The force-[Ca2+]i relations predicted by the model at steady levels of [Ca2+]i. The kinetic cross-bridge model used to simulate force is represented schematically in panel C. A indicates actin; M, myosin; T, adenosine triphosphate; D, adenosine diphosphate; P, phosphate; Ca, calcium; Ki, equilibrium constants; and kij, rate constants for transitions between states (see Reference 21 for detailed information).

The changes in diastolic relaxation that we have observed in the stunned myocardium are also quite similar to those caused by exposure to BDM.²¹ At concentrations of <10 mmol/L, BDM reduced both twitch-force amplitude and duration without affecting the Ca²⁺ transients. We therefore hypothesized that the contractile changes observed in the stunned trabeculae might share a similar mechanism with BDM; we probed this question by simulating the observed changes in force using the same cross-bridge model²¹ and the same rate-constant changes described above for the stunned myocardium. Experimentally measured Ca²⁺ transients from both control and stunned trabeculae were used as inputs to the model. As shown in Fig 9, the simulated forces are strikingly similar to the measured forces (Fig 6) both in their amplitude and in their kinetics. In particular, the acceleration of relaxation in the "stunned" situation is well reproduced. Thus, the same changes in cross-bridge cycling that accounted for the systolic dysfunction of stunned myocardium also predict the abbreviation of the twitch and the acceleration of relaxation.

View larger version (23K): [in this window] [in a new window]   Figure 9. Simulation of force development in stunned cardiac muscle. The experimentally measured Ca2+ transients shown in Fig 6 were used as inputs into the model of Backx et al.21 In stunned muscle simulation, the rate constant k12 was increased from 5 sec-1 to 15 sec-1, rate constants k15, k16, and k17 were increased twofold, and rate constants k51, k61, and k71 were slowed twofold.

Phase-Plane Analysis of Relaxation
To check our interpretation of the modeling results, we sought additional evidence that changes in cross-bridge kinetics underlie the accelerated diastolic relaxation in stunned myocardium. We used phase-plane analysis,²⁸ in which [Ca²⁺]_i and force are linked on a point-to-point basis over the entire trajectory of the twitch. Fig 10A shows phase-plane plots and illustrates the methods of construction of such plots. Twitch force is shown on the left parallel to the y axis, while the corresponding Ca²⁺ transient parallels the x axis. At any [Ca²⁺]_i, the corresponding force was plotted in the force-[Ca²⁺]_i plane. Fig 10B schematically depicts the phase-plane trajectory of a typical twitch and the steady state relationship that would be measured in the same muscle; panel C shows real data from control and stunned muscles. Note that the force-[Ca²⁺]_i relationship during the time course of a twitch does not coincide with the steady state relationship. The underlying basis for this lack of correspondence can be understood by recognizing that the kinetics of force development are slow relative to the kinetics of Ca²⁺ handling. Indeed, since the relaxation phase of the twitch (eg, point 5 in panel A and the corresponding point in panel B) is shifted leftward relative to the steady state relationship, the intrinsic rate of relaxation of the contractile system, and not Ca²⁺ removal from the cytosol, must be rate-limiting. Similarly, during the rising phase of force development (ie, points 2 and 3 in panel A), force development lags behind the changes in [Ca²⁺]_i, accounting for the rightward shift of the force-[Ca²⁺]_i relationship during this phase of the twitch as compared with steady state. Accelerating the kinetics of force development or slowing the dynamics of Ca²⁺ handling would tend to make the force-[Ca²⁺]_i relationship during a twitch coincide more closely with that measured during steady state.^{28 29} If the kinetics of force development were accelerated sufficiently (or the kinetics of the Ca²⁺ transient slowed sufficiently), the relation of force to [Ca²⁺]_i would coincide precisely with the steady state relationship. Indeed, Backx et al²⁸ have shown that slowing the rate of relaxation of Ca²⁺ transients results in precise correspondence of the force-[Ca²⁺]_i relationship during the relaxation phase of twitches and at steady state (see also Spurgeon et al³⁰ ).

View larger version (21K): [in this window] [in a new window]   Figure 10. Phase-plane analysis of relaxation. A, Phase-plane plot of force vs [Ca2+]i during the twitch. Twitch force (upper left) and the corresponding [Ca2+]i (lower right) are shown. The phase-plane plot (upper right) starts at point 1 and follows the numerical sequence of the points as indicated. Finally, as force relaxes, [Ca2+]i returns to its baseline level. B, Schematic phase-plane plot superimposed with the corresponding steady state force-[Ca2+]i relation (broken line). The two points () indicate the same force level in the twitch (left, corresponding to the force level at 50% relaxation) and at steady state (right). The difference in [Ca2+]i between the two different forms of activation is designated as [Ca2+]i. C, Phase-plane plots superimposed with steady state force-[Ca2+]i relations from representative control (left) and stunned (right) trabeculae. The level of twitch force at 50% relaxation and the corresponding force in steady state activation are indicated by points () on the respective curves. Force was normalized with respect to the maximal force in steady state activation. D, Changes in [Ca2+]i in control and stunned trabeculae at varied [Ca2+]os. Data are mean±SEM. Note the statistically significant difference between control and stunned trabeculae.

The force-[Ca²⁺]_i relationship during a twitch coincides most closely with the steady state relationship during the relaxation phase of force, because [Ca²⁺]_i is changing more slowly during this period.^{28 29} As a result, the degree of correspondence of the force-[Ca²⁺]_i relationship during relaxation will be sensitive to changes in the kinetics of force development relative to the dynamics of Ca²⁺ handling. The degree of correspondence can be analyzed as shown in panel B: [Ca²⁺]_i represents the difference between [Ca²⁺]_i quantified at half relaxation and the [Ca²⁺]_i at steady state for the same force. Since the kinetics of Ca²⁺ transients are not markedly altered by stunning, the shift in the force-[Ca²⁺]_i relationship during the relaxation phase of the twitch relative to the steady state should be a sensitive indicator of cross-bridge kinetics. Fig 10C shows phase-plane trajectories and steady state force-[Ca²⁺]_i relationships from a representative control muscle (left panel) and from a stunned trabecula (right panel). The relative shift in the force-[Ca²⁺]_i relationship during the relaxation phase of the twitch compared with the steady state relationship is greater in the control than in the stunned trabecula. This result is representative: Fig 10D shows pooled data quantifying [Ca²⁺]_i at different [Ca²⁺]_os in all control and stunned trabeculae. As can be seen, [Ca²⁺]_i is significantly reduced in the stunned trabeculae for all levels of activation when compared with controls. Again, since the kinetics of Ca²⁺ transient decay were not affected by stunning, these results entirely reflect changes in myofilament properties, bolstering the hypothesis that the kinetics of cross-bridge cycling are altered in stunned hearts.

Diastolic [Ca²⁺]_i and Diastolic Tone in Stunned Trabeculae
Studies from intact hearts^{31 32} revealed that diastolic [Ca²⁺]_i did not increase in postischemic myocardium, despite an elevated diastolic pressure.¹³ At an [Ca²⁺]_o of 1 mmol/L, we have found a clear-cut dissociation between the elevated end-diastolic pressure in stunned hearts and the absolute diastolic force in trabeculae from such hearts, which is normal. The increase in diastolic pressure can probably be explained by enhanced coronary turgor upon reperfusion.^{16 33} Nevertheless, latent abnormalities of diastolic Ca²⁺ handling were revealed when we measured [Ca²⁺]_i and force in trabeculae at various [Ca²⁺]_os. Whereas the peak of Ca²⁺ transients in stunned muscles followed that in controls at all [Ca²⁺]_os, diastolic [Ca²⁺]_i behaved differently (Fig 11A): in the stunned muscles only, diastolic [Ca²⁺]_i increased as [Ca²⁺]_o was raised. The changes in diastolic [Ca²⁺]_i as [Ca²⁺]_o was increased were statistically significant (Fig 11A, lower panel). The increases in resting force, quantified as the increment of resting force compared with the value at 0.25 mmol/L [Ca²⁺]_o at the same sarcomere length (ie, 2.2 µm), which was arbitrarily defined as the 0% reference level, were likewise significant in stunned trabeculae in the stunned myocardium but not in controls (Fig 11B, lower panel). The increment of resting force in stunned trabeculae was over 150% when [Ca²⁺]_o was increased from 0.5 to 2.0 mmol/L, whereas the increment was only 50% in control muscles.

View larger version (23K): [in this window] [in a new window]   Figure 11. Ca2+-dependent diastolic properties in control and stunned myocardium. A, Peak systolic (upper panels) and end-diastolic (lower panels) [Ca2+]i in control and stunned trabeculae at varied [Ca2+]os. B, upper panel, Changes in peak systolic and diastolic forces as [Ca2+]o was raised in control and stunned trabeculae; lower panel, increment of resting force at varied [Ca2+]os. The increment of resting force in response to [Ca2+]o is expressed as percent of increases over the resting force at 0.25 mmol/L [Ca2+]o at optimal sarcomere length.

Consistent with the elevation of diastolic [Ca²⁺]_i, aftercontractions occurred frequently in stunned trabeculae as [Ca²⁺]_o was raised.³⁴ Fig 12A shows typical recordings of contractile force during and after the transition from 1.5 to 2.0 mmol/L [Ca²⁺]_o. While there was no secondary rise in force during diastole in the control muscle, the stunned trabecula developed clear-cut aftercontractions. Aftercontractions were observed in two of eight stunned muscles at 1.0 mmol/L [Ca²⁺]_o and in six of eight stunned muscles at 2.0 mmol/L [Ca²⁺]_o (Fig 12B). No aftercontractions were recorded in control muscles at any [Ca²⁺]_o. The increases in diastolic [Ca²⁺]_i and in diastolic tone, coupled with the propensity toward aftercontractions, indicate that stunned muscles are less tolerant to Ca²⁺ loading than control muscles.

View larger version (18K): [in this window] [in a new window]   Figure 12. Aftercontractions in stunned myocardium. A, Effect of 2.0 mmol/L [Ca2+]o on force development in control (upper tracing) and stunned (lower tracing) trabeculae. Arrows indicate time of change of [Ca2+]o from 1.5 mmol/L to 2.0 mmol/L. The muscles were stimulated at 0.5 Hz. Note the occurrence of aftercontractions and increases in resting force in the stunned trabecula but not in the control trabecula under these conditions. B, Summary of frequency of occurrence of aftercontractions in control and stunned trabeculae.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ca²⁺ Responsiveness in Stunned Myocardium
Stunning is characterized by depressed contractile performance. Previous studies have shown that the availability of activator Ca²⁺ is not reduced in stunned hearts.^{5 6} Our results confirm and extend those studies. The force of contraction at any given [Ca²⁺]_i was lower in stunned muscles than in controls; this was true during physiological contractions as well as under conditions of steady state activation.

The present study has demonstrated that both maximal Ca²⁺-activated force and the Ca²⁺ sensitivity of the myofilaments are reduced in stunned cardiac muscle (Fig 5). Direct measurements of force and [Ca²⁺]_i in trabeculae offer several important advantages over studies from intact hearts. We have found that the apparent changes in diastolic tone are much greater in the intact heart, where vascular turgor exerts an important influence, than in the myocardium itself. Trabeculae are superior not only for the study of mechanics but also for the quantification of activator Ca²⁺. Measurement of [Ca²⁺]_i using nuclear magnetic resonance (NMR) in intact hearts requires prolonged sampling and signal averaging to achieve useful signal-to-noise ratios. The NMR technique also requires large amounts of tissue and heavy indicator loading; therefore, a much higher [Ca²⁺]_o is needed to generate reasonable pressure.^{2 5} Measurement of [Ca²⁺]_i using aequorin³¹ provides useful real-time Ca²⁺ transients; the limitations of this consumable photoprotein are complementary to those of the ratiometric fluorescent Ca²⁺ indicators. The use of fura 2 salt in trabeculae offers both reliable Ca²⁺ transients and direct measurements of contractile force.^{20 21 22} Nevertheless, to adapt this approach for investigating the stunned myocardium, trabeculae from two groups had to be compared in an unpaired fashion. We were fortunate to find little variability in the steady state force-[Ca²⁺]_i relations within each of the two groups, facilitating a reliable comparison between control and stunned muscles. Another concern was whether trabeculae were stunned to the same extent as the whole heart because they are free-running and thin, which might be expected to render them somewhat resistant to ischemia. We found that the decrease in developed force in stunned trabeculae as compared with controls was similar to the reduction in developed pressure in stunned hearts, confirming that trabeculae in stunned hearts were also stunned. This is not surprising given our choice of a total global ischemia/reperfusion model. Thus, the use of trabeculae from stunned hearts offers a novel and useful approach to investigate excitation-contraction coupling in the stunned myocardium.

Decreased Ca²⁺ responsiveness of the myofilaments in stunned myocardium has been demonstrated recently in both intact hearts and skinned muscle fibers. Whether the effect is due to decreased maximal Ca²⁺-activated force or decreased Ca²⁺ sensitivity, or both, is controversial. Kusuoka et al^{5 7} argued that both of these fundamental aspects of myofilament function are depressed in postischemic hearts. Hofmann et al⁸ observed decreased Ca²⁺ sensitivity but no resolvable change in maximal Ca²⁺-activated force in skinned single cell–sized fragments of porcine stunned myocardium, but there was a large variation in the absolute values of the maximal force (ranging from 55% to 150% of the mean value). Carrozza et al⁶ concluded that only the maximal Ca²⁺-activated force was depressed. One major limitation with regard to the interpretation of their data was uncertainty regarding the actual maximal level of activation, because saturation of force with respect to [Ca²⁺]_i was not clearly observed.

The mechanism underlying the decreased Ca²⁺ responsiveness is not known at present. The results from skinned myocardium suggest that stunning reflects alterations within the myofilaments themselves.⁸ However, another study showed no changes in the force-[Ca²⁺]_i relationship in skinned trabeculae from rat hearts reperfused after 40 minutes of ischemia.⁹ Although the authors referred to this as "stunned myocardium," 40 minutes of total ischemia, during which contracture developed and was maintained for 20 minutes, probably produced significant irreversible injury rather than stunning.^{35 36} Thus, it is possible that the changes in steady state myofilament Ca²⁺ responsiveness are specific for reversible reperfusion injury. In addition to structural modification of the myofilaments, other mechanisms may influence the Ca²⁺ sensitivity of stunned myocardium in vivo. For example, it has been reported that [Mg²⁺]_i is elevated in stunned myocardium.^{37 38} Mg²⁺ is known to be an important factor affecting the force-[Ca²⁺]_i relationship.^{20 39} There is also abundant evidence implicating oxygen free radicals in the pathogenesis of myocardial stunning.³ In addition to their effects on Ca²⁺ homeostasis, oxygen free radicals also promote the production of hydrogen peroxide, which is then decomposed by the catalase/peroxidase system, resulting in decreases in the content of reduced glutathione and increases in oxidized glutathione.^{40 41} Oxidized glutathione has been shown to decrease Ca²⁺ sensitivity in skinned cardiac muscles, while reduced glutathione has the opposite effect⁴² ; thus, changes in the redox state of cytosolic glutathione may contribute to the desensitization of the stunned myofilaments. Clearly, additional studies are needed to determine the relative importance of "soluble" versus structural factors in the diminished Ca²⁺ responsiveness of stunned myocardium.

Mechanism of Twitch Abbreviation in Stunned Myocardium
Impaired diastolic relaxation in stunned myocardium has been suggested by previous studies in which the diastolic function of intact hearts was investigated. Przyklenk et al¹⁰ showed prolonged diastolic relaxation times after 15 minutes of regional ischemia followed by 3 hours of reperfusion. In intact rat heart subjected to 3 minutes of global ischemia followed by 20 minutes of reperfusion, both the negative and positive derivatives of left ventricular pressure were depressed before returning to normal values.⁴³ More recently, diastolic function in stunned myocardium was examined in more detail in conscious dogs in which occlusion of the left anterior descending coronary artery was performed to produce myocardial stunning.¹¹ While no noticeable changes in systemic hemodynamics occurred, significant impairment of regional wall thickening and decrease in the mean time to half end-diastolic wall thinning were observed. Mosca et al¹⁴ showed that the diastolic stiffness of the heart was increased after stunning, an effect which could not be explained by impaired relaxation. Although this limited experimental evidence tends to suggest abnormal diastolic properties of the stunned myocardium, it does not provide definitive answers. Measurement of relaxation parameters in intact hearts is hampered by complicated geometry, uncertain control of loading conditions, and the superimposed effect of the coronary vasculature.³³

The results from stunned trabeculae in the present study showed that contraction was abbreviated: the time to peak was shortened and relaxation was accelerated, in surprising contrast to the general belief that diastolic relaxation is impaired in stunned myocardium. The underlying Ca²⁺ transients showed a slightly prolonged time to peak and no changes in the rates of decay. The acceleration of relaxation in the absence of changes in the decay of [Ca²⁺]_i is consistent with a decreased Ca²⁺ sensitivity of the myofilaments.²⁶ Since the decay of Ca²⁺ transients is governed predominantly by the SR Ca²⁺ pump and sarcolemmal Na⁺-Ca²⁺ exchange,⁴⁴ the normal decline of the Ca²⁺ transients in the stunned myocardium suggests that Ca²⁺ removal by these two processes is relatively unaffected. The fact that time to peak force and time to peak [Ca²⁺]_i change in opposite directions indicates an uncoupling between these two processes. In fact, these two processes are known not to be at dynamic equilibrium.²⁶ The increased time to peak [Ca²⁺]_i suggests impaired release of Ca²⁺ by the SR,⁴⁵ while we have argued that the mechanism for the decreased time to peak force resides at the cross-bridge level.

Myocytes isolated from regionally stunned rabbit ventricle exhibit abbreviated time to peak shortening and accelerated relaxation reminiscent of the changes in twitch force in the present study.⁴⁶ Thus, two studies in nonperfused myocardial preparations coincide in concluding that diastolic relaxation is accelerated, rather than delayed, in stunned heart muscle. The directionally opposite changes reported in regional models and in perfused hearts suggest, by exclusion, that loading effects and/or superimposed vascular turgor can lead to misrepresentation of the intrinsic myocardial properties.

A Model of Cross-Bridge Cycling for Stunned Myocardium
We implemented a quantitative cross-bridge model²¹ to account for the experimental findings relating to both systolic and diastolic dysfunction. In this model, cooperativity is built in and the rate constants are experimentally constrained. The model is capable of taking an input function (Ca²⁺ transient) and producing twitches with an appropriate time course (Fig 8A, 9). Since compelling experimental evidence supports the notion that stunning occurs at the myofilament level (References 2, 5, and 8^{2 5 8} and the present study), we modeled the contractile dysfunction of stunned myocardium primarily by altering the cross-bridge attachment and detachment rates. The mechanism for such changes can be rationalized as follows. Normally the site for cross-bridge attachment to actin is sterically inhibited by the troponin-tropomyosin complex. Following Ca²⁺ binding to troponin C, a structural alteration occurs within tropomyosin that exposes the myosin binding site on actin. The free energy required for the conformational change of the tropomyosin complex is derived from the energy of Ca²⁺ binding to troponin C and is transduced by troponin T and troponin I. Proteolytic digestion of any of the subunits of the troponin-tropomyosin complex might reasonably be expected to interfere with the ability of Ca²⁺ binding to induce an appropriate conformational change in tropomyosin. As a result of the cooperative nature of Ca²⁺ binding and cross-bridge attachment, a decreased ability of Ca²⁺ binding to evoke tropomyosin movement would result in an increase in the free energy of myosin binding to actin. Thus, it would not be unreasonable to expect the rates of cross-bridge attachment to be slowed and the rates for detachment to be accelerated.⁴⁷ Indeed, the changes in the amplitude of twitch force and the steady state force-[Ca²⁺]_i characteristics predicted by the model when cross-bridge attachment rates were slowed twofold and detachment rates accelerated twofold are very similar to the changes observed in stunned trabeculae. The additional change in k₁₂ is needed primarily to speed up the time to peak of the twitch in the stunned myocardium.

In an attempt to reveal the mechanism underlying the temporal changes in contraction, we also simulated the time course of the twitch by use of the measured Ca²⁺ transients from both control and stunned trabeculae as inputs. The predicted dynamics of contractions under both "control" and "stunned" situations reproduced very well the experimentally recorded twitch contractions in control and stunned trabeculae. These results support the notion that changes in the function of the myofibrillar regulatory proteins are predominantly responsible for the changes in both the amplitude and the kinetics of contraction. In addition, the results of simulations of twitch contraction and steady state activation indicate that changes in twitch dynamics and steady state activation characteristics share a common mechanism that resides at the level of thin filament regulatory contractile proteins (troponins and tropomyosin).

Our interpretation that the results of the cross-bridge model implicate changes at the level of the thin filaments needs to be substantiated in future experiments. There is no published evidence for or against structural changes in the troponin-tropomyosin complex in the stunned myocardium. A recent study using cardiac muscle subjected to 60 minutes of ischemia found decreases in the Ca²⁺ sensitivity of the myofilaments with concomitant degradation of troponin I and troponin T.⁴⁸ Although reflow was not studied, this experimental evidence supports our general reasoning that changes in the molecules of troponin may indeed underlie the functional alterations of the myofilaments in the stunned myocardium.

A Hypothesis
Based on the results of previous studies, Kusuoka and Marban⁴⁹ proposed a hypothesis for the molecular mechanism of stunning: Increases in [Ca²⁺]_i during late ischemia or early reperfusion^{31 32 50} activate endogenous Ca²⁺-dependent proteases that cause limited proteolysis of the contractile proteins. The resultant structural changes cause the decreased Ca²⁺ responsiveness of the myofilaments seen experimentally. The regulatory proteins of the thin filaments are known to be sensitive to proteolysis⁵¹ and merit particular scrutiny given the results of our modeling. Many key features of the stunned myocardium can be explained by this hypothesis. Transient Ca²⁺ overload is known to occur in stunned myocardium.^{31 32 50} Only limited proteolysis occurs when endogenous Ca²⁺-activated protease (calpain) is activated,⁵¹ which need not be visible histologically to alter contractile function if the thin filaments are affected.⁵² Limited proteolysis of the myofilaments would not affect the upstream mechanisms controlling [Ca²⁺]_i, rationalizing why the stunned myocardium remains capable of responding to inotropic stimuli.⁵³ The replacement of the degraded myofilaments by newly synthesized ones would be expected to follow the time course of protein degradation and new protein synthesis,^{54 55} which is roughly consistent with the observed time course of recovery from stunning.¹ In support of this hypothesis, the phenotype of stunning can be reproduced by direct exposure of the myofilaments to activated calpain I (an endogenous Ca²⁺-dependent protease⁵⁶ ) in skinned cardiac muscles.⁵⁷ Nevertheless, the ultimate test of the hypothesis awaits evidence of activation of Ca²⁺-dependent proteases in stunned myocardium and elucidation of the molecular changes in the myofilaments.

Myofilament-Independent Changes in Ca²⁺ Homeostasis: Mechanisms of Increased Diastolic Tone
A novel finding in this study is the increase in diastolic [Ca²⁺]_i with concomitant increases in resting force in stunned muscles and the frequent occurrence of aftercontractions as [Ca²⁺]_o was raised. The muscles became intolerant to Ca²⁺ at high (>1.0 mmol/L) [Ca²⁺]_o, suggesting a potential defect of Ca²⁺ handling in stunned trabeculae. The increases in resting force and diastolic [Ca²⁺]_i may be caused by decreased Ca²⁺ removal from the cytosol either due to decreased extrusion and/or malfunction of the SR. The present data tend to suggest a leaky SR as the underlying mechanism for the increased diastolic [Ca²⁺]_i since relaxation of Ca²⁺ transients was not affected at any [Ca²⁺]_o. Moreover, the occurrence of aftercontractions suggests spontaneous Ca²⁺ release from the SR that results from cytosolic Ca²⁺ overload.⁵⁸ The Ca²⁺ overload is probably due to a decreased maximal capacity for Ca²⁺ reuptake by the SR in stunned myocardium and a leaky SR.¹⁸ Studies using intact hearts did not show a consistent increase in diastolic [Ca²⁺]_i,^{5 31} although increases in diastolic pressure were seen.^{5 7 13} These results suggest that the elevated diastolic pressure seen under basal conditions is due to factors extrinsic to the stunned myocardium rather than to an increase in diastolic [Ca²⁺]_i. The findings from this study suggest a latent defect in Ca²⁺ handling, but this may result in increased diastolic tone only under conditions that tend to favor cellular Ca²⁺ overload.

Pathophysiological and Therapeutic Implications
The last decade has witnessed a major transition, from a supporting role to center stage, in our recognition of the pathophysiological importance of the myofilaments: the contractile proteins have now been implicated in fundamental disease processes ranging from hypertrophy-associated sudden death^{59 60} to the stunned myocardium. Various lines of evidence now point to the cardiac myofilaments as the primary culprits in heritable forms of hypertrophic cardiomyopathy.^{61 62 63} While the first molecular abnormalities to be described were point mutations of the thick filament protein myosin, similar phenotypes can result from lesions of at least two thin filament proteins.⁶⁴ Altered patterns of expression of various contractile protein isoforms accompany ordinary congestive heart failure, but the functional significance of these changes relative to abnormalities of Ca²⁺ handling remains to be clarified.⁶⁵

Our work supports the view that stunned myocardium represents an acquired, reversible disease of the myofilaments. As such, treatment with Ca²⁺ sensitizers is not only effective⁶⁶ but also rational. The acceleration of diastolic relaxation in stunned heart muscle constitutes a built-in margin of safety against the diastolic dysfunction that at least theoretically, may restrict the utility of sensitizer therapy in other forms of contractile failure.⁶⁷

Two other considerations rationalize the effectiveness of moderate positive inotropic therapy⁵³ : First, the upstream mechanisms that control [Ca²⁺]_i appear to be largely intact; second, the stunned myocardium (like control myocardium) recruits only a fraction of its maximal force during physiological contractions (compare Figs 3 and 5). Thus, interventions that increase [Ca²⁺]_i can increase force, even in stunned heart. Nevertheless, our observation that the stunned myocardium copes poorly with an increased calcium load (elevated [Ca²⁺]_o) serves as a caution against excessive inotropic therapy. The susceptibility of stunned myocardium to calcium overload may underlie the functional deterioration that has been reported with high-dose dobutamine echocardiography in many reperfused segments that respond positively to low-dose dobutamine infusion.^{68 69} In addition to such mechanical implications, the finding of relative Ca²⁺ intolerance hints that stunned myocardium might be a favorable substrate for reperfusion arrhythmias related to delayed afterdepolarizations.⁷⁰

	Acknowledgments

 
This study was supported by the National Institutes of Health (R01-HL-44065 to Dr Marban), the American Heart Association (International Research Fellowship to Dr Gao), and The Swiss National Science Foundation (fellowship to Dr Atar). We thank Dr Hideo Kusuoka for many helpful discussions and Dr David Kass for commenting on the manuscript.

Received August 24, 1994; accepted January 9, 1995.

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				W. Dong Gao, Y. Liu, R. Mellgren, and E. Marban Intrinsic Myofilament Alterations Underlying the Decreased Contractility of Stunned Myocardium : A Consequence of Ca2+-Dependent Proteolysis? Circ. Res., March 1, 1996; 78(3): 455 - 465. [Abstract] [Full Text]

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