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(Circulation Research. 1996;78:455-465.)
© 1996 American Heart Association, Inc.

Articles

Intrinsic Myofilament Alterations Underlying the Decreased Contractility of Stunned Myocardium

A Consequence of Ca²⁺-Dependent Proteolysis?

Wei Dong Gao, Yongge Liu, Ronald Mellgren, Eduardo Marban

From the Division of Cardiology (W.D.G., Y.L., E.M.), Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Md, and the Department of Pharmacology (R.M.), Medical College of Ohio, Toledo.

Correspondence to Eduardo Marban, MD, PhD, Room 844, Ross Building, 720 Rutland Ave, Baltimore, MD 21205. E-mail marban@welchlink.welch.jhu.edu.

	Abstract

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Abstract We investigated the mechanism of the decreased myofilament Ca²⁺ responsiveness in stunned myocardium. The steady state force-[Ca²⁺] relationship was measured before and after skinning in thin ventricular trabeculae from control or stunned (20 minutes of ischemia, 20 minutes of reperfusion) rat hearts. [Ca²⁺]_i was determined using microinjected fura 2 salt in intact muscles, whereas the myofilaments of chemically skinned trabeculae were activated directly with solutions of varied [Ca²⁺]. Maximal Ca²⁺-activated force (F_max) before and after skinning was identical within either the control or stunned groups but was markedly depressed in both groups of stunned trabeculae (P<.001). After ischemia and reperfusion, the [Ca²⁺] required for 50% of maximal activation (Ca₅₀) was increased in both intact (control, 0.60±0.09 µmol/L; stunned, 0.85±0.09 µmol/L; P<.001) and skinned (control, 1.13±0.24 µmol/L; stunned, 1.39±0.21 µmol/L; P=.0025) trabeculae. These data indicate that the decreased Ca²⁺ responsiveness of stunned myocardium is due to intrinsic alterations of the myofilaments. Therefore, we tested the hypothesis that activation of proteases by reperfusion-induced Ca²⁺ overload decreases the Ca²⁺ responsiveness of the cardiac myofilaments. Force-[Ca²⁺] relations were compared before and 5 to 30 minutes after direct exposure of skinned trabeculae to calpain I (18 µg/mL, 20 minutes at [Ca²⁺]=10.8 µmol/L), a Ca²⁺-activated protease that is present in myocardium. Calpain I reduced F_max from 94.3±8.3 to 56±8.5 mN/mm² while increasing Ca₅₀ from 0.94±0.11 to 1.36±0.21 µmol/L (P<.01). Calpastatin, a specific calpain inhibitor, prevented the effects of calpain I on skinned trabeculae. The results show that the reduced Ca²⁺ responsiveness of stunned myocardium reflects alteration of the myofilaments themselves, not of soluble cytosolic factors, which can be faithfully reproduced by exposure to Ca²⁺-dependent protease.

Key Words: force-[Ca²⁺] relation • contractile proteins • calpain I • myocardial ischemia/reperfusion

	Introduction

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Myocardial "stunning" describes the profound but eventually reversible decrease of myocardial contractility that follows a brief ischemic insult.¹ Several recent studies have shown that stunning is at least partially due to decreased Ca²⁺ responsiveness of the contractile proteins.^{2 3 4} Nevertheless, the factors responsible for the decrease in myofilament Ca²⁺ responsiveness in stunned myocardium are not yet clear. There may be structural injury to one or more contractile proteins, as suggested by Kusuoka and Marban.⁵ These investigators proposed that elevated [Ca²⁺]_i during ischemia and during the initial reperfusion period may have long-lasting aftereffects by activating Ca²⁺-dependent proteases, which could then partially degrade the contractile proteins. Alternatively, changes in cytosolic factors may predominate. In the intact cell, the myofilaments are bathed in a cytosolic milieu that contains many "soluble" factors known to influence the ability of the myofilaments to generate force. Force development in intact muscle depends on both the concentrations of these factors and the intrinsic properties of the myofilaments. Chemical or mechanical removal of the surface membrane ("skinning") enables direct access to the myofilaments, with cytosolic factors equalized. Studies in skinned preparations of stunned myocardium have yielded variable results, one showing a reduction in steady state Ca²⁺ sensitivity⁶ and another showing no changes,⁷ suggesting that at least part of the dysfunction may reflect alterations in soluble cytoplasmic factors.

In the present study, we investigated the relative roles of soluble cytosolic factors versus structural alterations in the decreased Ca²⁺ responsiveness of stunned myocardium. We used thin trabeculae from control and stunned hearts and determined the force-[Ca²⁺] relation in the intact condition. We then quantified the Ca²⁺ responsiveness of the myofilaments after skinning in the same muscles, under conditions that equalized the soluble factors bathing the myofilaments. Our results show that alterations in the myofilaments themselves are entirely responsible for the observed decrease in maximal Ca²⁺-activated force and for most, if not all, of the decrease in Ca²⁺ sensitivity. As an initial test of the hypothesis that Ca²⁺-activated proteases produce the myofilament alterations, we examined the direct effect of calpain I, a Ca²⁺-activated neutral protease widely distributed in many tissues including the myocardium,⁸ on skinned cardiac muscle. Both maximal Ca²⁺-activated force and Ca²⁺ sensitivity were decreased by calpain I, and the effects were prevented by calpastatin (a specific calpain inhibitor⁹ ). Thus, Ca²⁺-dependent proteolysis mimics the changes in contractile protein function that have been documented in stunned myocardium.

	Materials and Methods

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Whole Rat Hearts
As previously described,¹⁰ rats of either sex (LBN-F1 strain, 200 to 250 g, Harlan Sprague Dawley, Inc, 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 bpm via electrodes placed over the insertion of the aorta and the right ventricle. Isovolumic left ventricular pressure was measured with a custom-made balloon (TSC) filled with water and connected to a pressure transducer (Gould P23Db, Statham). 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 placed inside the right 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 restarted 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 these two groups of hearts (stunned and control) were quickly dissected from the right ventricle and mounted between a force transducer and a micromanipulator according to the methods described previously.^{11 12} The dimensions of the unstretched trabeculae measured under a microscope (Nikon 212219; magnification, x40) were as follows (mm): control group, 1.92±0.20 long, 0.13±0.04 wide, and 0.09±0.03 thick (n=12); stunned group, 2.03±0.22 long, 0.13±0.05 wide, and 0.10±0.04 thick (n=13). The cross-sectional areas of the trabeculae were as follows (mm²): control, 0.011±0.011; stunned, 0.013±0.009 (P=.6). The trabeculae were superfused with K-H solution (except for 0.5 mmol/L of CaCl₂) at a rate of 10 mL/min and stimulated at 0.5 Hz. All isolated muscle experiments were performed at room temperature (20°C to 22°C).

Force Measurements
Force was measured by a custom-made force transducer from a silicon strain gauge (AEM 801, SensoNor)^{11 12} and was expressed in millinewtons per square millimeter of cross-sectional area.

Sarcomere Length Measurements
Sarcomere length was measured by laser diffraction^{11 12} and monitored routinely by the video system. In a subset of the muscles (n=3 in each group), an electronic measuring and computing system was used for on-line documentation of the changes in sarcomere length during contraction. Briefly, light diffracted by the central region of the muscle was detected by a reticon diode linear array system (RC0100-RG512, EG&G Reticon). The light intensity of the first order of diffraction was integrated, and sarcomere length was determined from the median of the light intensity distribution using a custom-made sarcomere length detection system (Biomedical Technical Support Centre, University of Calgary, Alberta, Canada). Diastolic sarcomere length was set at 2.20 to 2.30 µm.

Measurement of [Ca²⁺]_i
[Ca²⁺]_i was measured using the free acid form of fura 2 as described previously.^{11 12 13} 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 backfilled with 150 mmol/L KCl. After a successful impalement into a superficial cell in the unstimulated muscle, a hyperpolarizing current of 5 to 8 nA was passed continuously for 20 minutes. If necessary to achieve a good signal-to-noise ratio, multiple injections (up to three or four) were applied at different sites, with the duration of current injection limited to <10 minutes at each site. The loading did not affect force development. The epifluorescence of fura 2 was measured by exciting at 380 and 340 nm. The fluorescent light was collected at 510 nm by a photomultiplier tube (R1527, Hamamatsu). The output of the photomultiplier tube was filtered at 100 Hz, collected by an A/D converter, and stored digitally 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 nm/380 nm at saturating [Ca²⁺], and R_min is the ratio of 340 nm/380 nm at zero [Ca²⁺]. The values for K'_d, R_max, and R_min were determined by in vivo calibrations as previously described.^{11 12} R_max and R_min were 9.55 and 0.75, respectively. The apparent K'_d was 2.9 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 Ca²⁺-bound forms of fura 2 at 380 nm (S_f2/S_b2, where S_f2 is for the fluorescence of free fura at 380 nm, and S_b2 is for the fluorescence of the Ca²⁺-bound form of fura 2 at 380 nm; see Reference 14). The value of S_f2/S_b2 was 10.5 in our setup, and the true K_d, estimated in vivo, equaled 276 nmol/L.

Tetanization of Trabeculae
Ryanodine was used to enable steady state activation in the trabeculae.^{11 12} After 15 minutes of exposure to ryanodine (1 µmol/L), tetanization was induced briefly (4 to 8 seconds) by stimulating the muscles at 10 Hz. Different tetanized forces were achieved with varied external [Ca²⁺]s (0.25 to 20 mmol/L).^{11 12}

Skinning of Trabeculae
After determination of the intact force-[Ca²⁺] relation, the trabeculae were skinned in the same bath by 15 to 25 minutes of exposure to 1% Triton X-100 in relaxing solution containing (mmol/L) KCl 80, HEPES 25, K₂EGTA 10, creatine phosphate sodium salt (Na₂CrP) 15, Na₂ATP 5, MgCl₂ 5.15, and leupeptin 0.5 (pH 7.2 with KOH). Varied [Ca²⁺]s were achieved by mixing the relaxing solution and activating solution (mmol/L: Ca²⁺-EGTA 10, KCl 80, HEPES 25, Na₂CrP 15, Na₂ATP 5, MgCl₂ 4.75, and leupeptin 0.5, pH 7.2) in various ratios. [Ca²⁺] was calculated by a computer program that was based on the stability constants and the enthalpy values for the various reactions from Martell and Smith,¹⁵ except values for Mg²⁺-ATP and Ca²⁺-ATP reactions from Pettit and Siddiqui.¹⁶ The ionic strength of the solutions was 181 mmol/L, and the free [Mg²⁺] was 0.5 mmol/L. The muscles were activated with solutions of varied [Ca²⁺] while diastolic sarcomere length was kept the same as before skinning.

Except for the omission of prior [Ca²⁺]_i measurements, identical methods were used for skinning and activating additional unpaired stunned (n=2) and control (n=5) trabeculae. The latter were the same trabeculae used in the control phase of the calpain experiments. Calpain I was prepared from human red blood cells¹⁷ and stored in 50 mmol/L MOPS, 0.2 mmol/L EGTA, and 1 mmol/L dithiothreitol in 50% glycerol (-20°C). Leupeptin was omitted from calpain-containing solutions and from the isochronal control experiments.

Analysis of Steady State Activation
Both intact and skinned steady state force-[Ca²⁺] relations were fit with a function of the following form ("Hill equation"):

where F_max is the maximal Ca²⁺-activated force, Ca₅₀ is the [Ca²⁺] required for 50% of maximal activation, and n is the Hill coefficient.^{11 12} The data were also analyzed with a linearized Hill plot (log-log plot):

where P_r is a fraction of F_max, pCa is -log[Ca²⁺], and k is pCa₅₀. In previous work in skinned muscle,^{18 19} the linearized Hill plot often yields two straight lines with different slopes or Hill coefficients, n₁ and n₂, above and below pCa₅₀.

Statistics
Paired Student's t test, one-way ANOVA, or multivariate ANOVA was used for statistical analysis of the data.^{20 21} A value of P<.05 was considered to indicate significant differences between groups. Unless otherwise indicated, pooled data are expressed as mean±SD.

	Results

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Effects of Ischemia and Reperfusion on Myofilament Properties in Intact and Skinned Trabeculae
Hearts were mounted on a Langendorff apparatus and perfused for 60 minutes (control) or for 15 minutes, followed by 20 minutes of ischemia and 20 minutes of reperfusion (stunned). Table 1 summarizes the left ventricular pressure data in both groups. At baseline, the two groups were similar. The control hearts exhibit no time-dependent changes in ventricular pressure. In contrast, ventricular developed pressure decreased significantly after 20 minutes of reflow. These changes in ventricular pressure are typical of stunning.^{2 4 10}

View this table: [in this window] [in a new window]   Table 1. Left Ventricular Pressure of Control and Stunned Hearts

Trabeculae from both control and stunned hearts were dissected quickly and mounted in the experimental chamber on the stage of an inverted microscope. After mounting, each trabecula was carefully inspected for any visible damage. Trabeculae from stunned hearts looked entirely normal; there were no contracture bands, and the sarcomeres were well aligned. In a previous study,¹⁰ we found that F_max was reduced and that Ca₅₀ was increased (ie, Ca²⁺ sensitivity was reduced) in intact stunned trabeculae. In the present study, we investigated what factors are responsible for the decrease of Ca²⁺ responsiveness in stunned myocardium. We first compared the values of F_max before and after skinning in control and stunned trabeculae. Fig 1A shows paired recordings of maximal force before and after skinning of a control muscle. Two traces of tetani are superimposed (left); the fact that two different saturating levels of [Ca²⁺]_i result in the same level of force demonstrates that F_max was indeed achieved in the intact muscle. The right side of Fig 1A shows the force of the same trabecula after skinning during activation by high (27 µmol/L) [Ca²⁺]. The F_max values under both situations are virtually identical: 96 mN/mm² (intact) and 98 mN/mm² (skinned). Fig 1B shows analogous results from a representative stunned trabecula. The F_max values are distinctly lower than in the control muscle, both before and after skinning. Nevertheless, F_max remained unchanged after skinning in the stunned condition (intact, 73 mN/mm²; skinned, 69 mN/mm²) compared with the control condition.

View larger version (12K): [in this window] [in a new window]   Figure 1. Maximal Ca2+-activated force (Fmax) in control (A) and stunned (B) trabeculae. A, Fmax of an intact trabecula (top left) and the corresponding [Ca2+]i (bottom left). Fmax was obtained by stimulating the muscle at 10 Hz in the presence of ryanodine. The two traces were obtained at [Ca2+]o values of 15 and 20 mmol/L, respectively. Note the saturation of force. On the right, Fmax and [Ca2+]i are shown for the same trabecula after skinning when exposed to 27 µmol/L [Ca2+]. B, Fmax of a stunned trabecula (top left) and the corresponding [Ca2+]i values at [Ca2+]o values of 15 and 20 mmol/L (bottom left). On the right, Fmax and [Ca2+]i are shown for the same trabecula after skinning.

Fig 2 summarizes the paired data for F_max in both control and stunned trabeculae. F_max values of the stunned trabeculae were significantly decreased compared with control trabeculae (control, 114±30 mN/mm² [intact] and 115±29 mN/mm² [skinned]; stunned, 64±21 mN/mm² [intact] and 71±22 mN/mm² [skinned]) (P<.001). In both the control and stunned groups, however, F_max values were not different before and after skinning. Since skinning equalizes the soluble cytosolic factors, these data indicate that the decrease of F_max in stunned myocardium reflects intrinsic alterations of the myofilaments.

View larger version (16K): [in this window] [in a new window]   Figure 2. Pooled data of paired maximal Ca2+-activated force (Fmax) values of all control (, ) and stunned (, ) trabeculae in the present study. The filled symbols are the Fmax values of the skinned counterparts. The mean±SD for each group is also plotted. *P<.001 vs control Fmax values; **P<.001 vs control Fmax values.

Trabeculae contracting isometrically are known to undergo some internal shortening due to the presence of a series elastance (attributable to connective tissue and damaged ends).²² If present, major differences in the extent of internal shortening could theoretically accentuate the differences between control and stunned myocardium. The results in Fig 3 show that this is not the case. The changes of sarcomere length during both twitches and steady state contractions are roughly equivalent in representative control trabeculae (Fig 3A, left) and stunned trabeculae (Fig 3A, right). Fig 3B shows the mean data for sarcomere length in both control and stunned trabeculae. In control trabeculae, the diastolic sarcomere length was 2.29±0.01 µm, end-systolic sarcomere length was 1.97±0.11 µm, and the degree of shortening was 0.33±0.11 µm. The values in stunned trabeculae were virtually identical (diastolic, 2.27±0.03 µm; end-systolic, 1.98±0.08 µm; and shortening, 0.30±0.07 µm).

View larger version (14K): [in this window] [in a new window]   Figure 3. Changes in sarcomere length during twitches and steady state activations in control and stunned trabeculae. A, Raw records of force (top) and sarcomere length (bottom) from control (left) and stunned (right) trabeculae. The twitch was obtained at [Ca2+]o of 1.0 mmol/L, and the maximal activation was obtained at [Ca2+]o of 20.0 mmol/L. Note the similar end-systolic sarcomere lengths in both control and stunned trabeculae. B, Bar graphs of mean±SD diastolic and systolic sarcomere length (SLdias and SLsys, respectively) data during maximal activations. The degree of sarcomere shortening of control and stunned trabeculae is 14.4% and 13.2%, respectively.

The steady state results in Figs 1 and 2 focus on one fundamental parameter of myofilament Ca²⁺ activation, the maximal force. Two other parameters characterize the Ca²⁺ sensitivity of the contractile proteins: Ca₅₀ and the steepness of the force-[Ca²⁺] relation (the Hill coefficient n). Previous studies have demonstrated either a decrease in Ca²⁺ sensitivity of the myofilaments^{2 10} or no changes.^{3 7} We determined whether the Ca²⁺ sensitivity is decreased in intact stunned myocardium and, if so, whether the changes are due to alterations of the myofilaments or to soluble cytosolic factors. The latter merits particular attention, especially given that some of these factors (eg, Mg²⁺) have been shown to be elevated in stunned myocardium^{23 24} and are known to affect Ca²⁺ sensitivity of the myofilaments without affecting maximal force.^{11 25} In order to measure Ca₅₀ reliably, a complete force-[Ca²⁺] relation has to be obtained before and after skinning. Complete paired force-[Ca²⁺] relations were obtained from six muscles in each group. Fig 4 shows normalized force-[Ca²⁺] relations of the control (Fig 4A) and stunned (Fig 4B) experiments. Each muscle in each group has its own symbol, open in the intact preparation and filled after skinning. The force-[Ca²⁺] relation is shifted to the right after skinning, as previously reported.¹¹ Paired t tests revealed significant differences in Ca₅₀ between the intact and skinned muscles in both groups (P=.001). Fig 4C summarizes the Ca₅₀ values of both the intact and the skinned muscles, including results from several additional experiments in which the force-[Ca²⁺] relation was determined only after skinning (see "Materials and Methods"); intact data are depicted as open bars; skinned data, as filled bars. The Ca₅₀ values were significantly different in the respective control and stunned muscles (intact, 0.60±0.09 µmol/L versus 0.85±0.09 µmol/L, n=6, P=.001; skinned, 1.13±0.23 µmol/L, n=11 versus 1.39±0.21 µmol/L, n=8, P=.025). The absolute values of the differences in Ca₅₀ in control versus stunned myocardium were comparable before and after skinning (250 nmol/L [intact] versus 260 nmol/L [skinned]). Thus, both of the distinctive changes in myofilament function (the decrease in F_max and the increase in Ca₅₀) are comparable in skinned muscle and in intact muscle.

View larger version (25K): [in this window] [in a new window]   Figure 4. A and B, Force-[Ca2+]i relations of control (A) and stunned (B) trabeculae before (open symbols) and after (filled symbols) skinning. Each symbol represents an individual muscle in each group. All data were normalized to their maximal Ca2+-activated force (Fmax) values for comparison, and the force-[Ca2+]i relations were fitted by the Hill equation (see "Materials and Methods"). C, Bar plot of [Ca2+] required for 50% of maximal activation (Ca50) values of the force-[Ca2+] relations from control and stunned trabeculae before (open bars) and after (solid bars) skinning. Data are mean±SD. *P<.001 vs intact control; **P<.001 vs control intact; and #P<.001 vs stunned intact. D, Linearized Hill plot of the same force-[Ca2+]i relations of the intact muscles from control () and stunned () muscles. Data within various bins of pCa were pooled for clarity. Pr is a fraction of Fmax.

In contrast to F_max and Ca₅₀, the Hill coefficients, derived from the best fits to the Hill equation, were no different in control versus stunned trabeculae (P=.5). Because this parameter quantifies the steepness of the force-[Ca²⁺] relation and is crucial in shaping the overall myofilament responsiveness, we performed linearized Hill plot analysis to check these results. Fig 4D shows log-log plots of control (open circles) and stunned (solid circles) force-[Ca²⁺] relations in the intact muscles. The averages of the best fits to these linearized data are again statistically indistinguishable (control, 4.99±1.22; stunned, 4.16±1.33; P=.2). Thus, the Hill coefficient is not changed in stunned myocardium. Interestingly, these intact muscle data do not require two different Hill coefficients to obtain adequate fits above or below pCa₅₀, unlike published studies in skinned muscle.^{18 19} This feature will be considered again below, when the skinned muscle results are analyzed in detail.

Fig 5, left, summarizes the results of the intact muscle experiments, whereas Fig 5, right, shows the pooled results after skinning. The data were first normalized to their respective maximal values and then scaled according to the absolute values for maximal Ca²⁺-activated force in the control and stunned conditions. Multivariate ANOVA revealed significant differences between the force-[Ca²⁺] relations of intact control and stunned muscles (P=.01). The difference was attributable solely to differences in F_max and Ca₅₀, and not the Hill coefficient. The force-[Ca²⁺] relations between skinned control and stunned muscles were also significantly different (P=.007), and once again, the differences were due to F_max as well as Ca₅₀. Possible changes in the Hill coefficient in skinned muscle are explored in depth in the log-log analysis of Fig 9C and in the corresponding text.

View larger version (14K): [in this window] [in a new window]   Figure 5. Force-[Ca2+] relations of intact (left) and skinned (right) trabeculae from control (, ) and stunned (, ) groups. The absolute maximal Ca2+-activated force (Fmax) values (mean±SEM) from all groups are shown as well. All the submaximal values were normalized to their respective Fmax values for comparison. In skinned muscles, only the averaged values (mean±SEM) at each particular [Ca2+] are shown. The dashed lines indicate the best fits to the Hill equation in which the averaged values for Fmax, [Ca2+] required for 50% of maximal activation, and Hill coefficients (n) were used (see text for details). Note the parallel changes of the force-[Ca2+] relation before and after skinning.

View larger version (15K): [in this window] [in a new window]   Figure 9. Comparison of maximal Ca2+-activated force (Fmax) (A and B) and the [Ca2+] required for 50% of maximal activation (C and D) for control and stunned trabeculae after skinning (A and C) and skinned trabeculae before and after calpain I exposure (B and D). Data are reproduced from Figs 2, 4, and 6. A and B, Bar graphs of Fmax. In panel A, open bar indicates control trabeculae, and solid bar indicates stunned trabeculae (*P<.001). In panel B, open bar indicates before calpain I exposure, and solid bar indicates after calpain I exposure (**P<.001). C and D, Hill log-log plots (mean±SEM). Panel C shows Hill log-log plot (where Pr is a fraction of Fmax) of skinned control () and stunned () muscles. Panel D shows Hill log-log plot of skinned muscles before () and after () calpain I exposure. Note the linearization of the Hill plots in stunned muscles and in muscles after calpain I treatment.

These results implicate the contractile proteins themselves as the locus for the decreased myofilament Ca²⁺ responsiveness of stunned heart muscle. Since it has been established that cytosolic factors must play a relatively minor role, it is logical to wonder whether Ca²⁺-activated proteolysis is responsible for the modification of the contractile proteins in reperfused myocardium. We tested this idea by quantification of the force-[Ca²⁺] relations in skinned muscles before and after direct exposure to calpain I, a Ca²⁺-activated neutral protease that is plentiful in the myocardium.⁸

Effect of Calpain I on the Force-[Ca²⁺] Relation in Skinned Trabeculae
Fig 6 shows the changes in the force-[Ca²⁺] relation before and after 20 minutes of exposure to calpain I (18 µg/mL). The top panel shows the experimental protocol in these experiments. After skinning, the muscles were bathed in relaxing solution, and sarcomere length was set to 2.2 µm. Different levels of force were elicited by rapidly exposing the muscle to activating solutions. After the control force-[Ca²⁺] relation was obtained, the muscle was activated with 10.8 µmol/L [Ca²⁺]; during this activation, calpain I was applied for 20 minutes. The muscle was then bathed again in relaxing solution, and another force-[Ca²⁺] relation was measured. Sarcomere length was kept identical to that before exposure to calpain I. The bottom panel shows the force-[Ca²⁺] relation before and after exposure to calpain I in this particular trabecula. Maximal force decreased after calpain I treatment, and Ca₅₀ doubled.

View larger version (15K): [in this window] [in a new window]   Figure 6. Effect of calpain I on the force-[Ca2+] relation in a skinned rat trabecula. Top, Contraction responses (lower trace, F) of skinned trabecula to changes in [Ca2+] (upper trace). The changes in [Ca2+] are separated by 2- to 3-minute intervals, during which sarcomere length was checked to ascertain that it remain unchanged. The arrow indicates addition of calpain I (18 µg/mL). Bottom, Force-[Ca2+] relation of this trabecula before () and after () exposure to calpain I. The relations were fitted to the Hill equation. The [Ca2+] required for 50% of maximal activation increased from 0.94 to 1.28 µmol/L, and the Hill coefficient n decreased from 3.64 to 3.15 after treatment with calpain I. Maximal Ca2+-activated force is decreased by 44%.

Fig 7 shows pooled force-[Ca²⁺] relations before and after exposure to calpain I in four different muscles. Before exposure to calpain I, Ca₅₀ was 0.94±0.11 µmol/L. Exposure to calpain I resulted in an increase of Ca₅₀ to 1.36±0.21 µmol/L (P=.006 versus before exposure). Maximal Ca²⁺-activated force was also decreased from 94.3±8.3 to 56±8.5 mN/mm² (P=.01, paired t test). Thus, exposure to calpain I and high [Ca²⁺] decreased the Ca²⁺ responsiveness of the myofilaments.

View larger version (13K): [in this window] [in a new window]   Figure 7. Pooled data for the force-[Ca2+] relations from four trabeculae before () and after () treatment with calpain I in the presence of 10.8 µmol/L [Ca2+]. Data are mean±SEM.

Effect of Calpain I on Myofilaments in the Presence of Calpastatin
To determine whether the results shown in Fig 7 represent a specific effect of calpain I, we performed another series of experiments in which calpastatin was present when the muscles were treated with calpain I. Calpastatin is a specific endogenous calpain inhibitor.⁹ In this series of experiments, calpastatin (from bovine heart, 24 µg/mL), purified as described previously,²⁶ was added to the activating solution containing 10.8 µmol/L [Ca²⁺]. The trabeculae were then exposed to this solution, and after force had reached a steady level, calpain I (18 µg/mL) was added to the bath. After 20 minutes, the activating solution was rapidly replaced by relaxing solution free of calpain and calpastatin. Fig 8 shows the average force-[Ca²⁺] relations before and after calpain I exposure in the presence of calpastatin in three muscles. In spite of the presence of calpain I, Ca₅₀ did not change (1.02±0.42 versus 1.12±0.34 µmol/L, P=.2) when calpastatin was also present. The small decrease in F_max (15%) was comparable to that observed in four parallel experiments in which the force-[Ca²⁺] relation was compared before and after 20 minutes of exposure to 10.8 µmol/L Ca²⁺ without calpain I or calpastatin (data not shown). Thus, calpastatin prevented the decreases in Ca²⁺ responsiveness caused specifically by exposure to calpain I.

View larger version (12K): [in this window] [in a new window]   Figure 8. Force-[Ca2+] relations before () and after () exposure to calpain I in the presence of calpastatin in three trabeculae. Calpastatin (24 µg/mL) was added to the activating solution with 10.8 µmol/L Ca2+. After full activation of the muscles, calpain I was added, and after 20 minutes, the muscles were bathed in relaxing solution. Data are mean±SEM.

Effects of Stunning on the Myofilaments Compared With Effects of Calpain I
If Ca²⁺-activated proteolysis produces stunning, the phenotype of calpain-treated muscles could reasonably be expected to mimic that of the stunned myocardium. Fig 9 compares stunned muscles (Fig 9A and 9C) with calpain-treated muscles (Fig 9B and 9D) with regard to the changes in F_max and Ca²⁺ sensitivity. In both stunned (Fig 9A) and calpain-treated (Fig 9B) myocardium, F_max is reduced by 40%. To optimize the resolution of changes in Ca²⁺ sensitivity, fractional force was displayed as a function of activator [Ca²⁺] in log-log plots (Fig 9C and 9D). The slopes of the relations yield the Hill coefficients; the x intercept corresponds to pCa₅₀. Both stunning (Fig 9C) and calpain (Fig 9D) produce rightward shifts in pCa₅₀. The changes in the Hill coefficient are also uncannily similar in the two groups. In agreement with previous studies in skinned muscle^{18 19} (but not in intact muscle; compare with Fig 4D), the relations in the control condition are bent and require two linear segments, one above and the other below the abscissa, for an adequate fit. Two Hill coefficients result: that above the abscissa (n₁) is shallower than that below (n₂). It is notable that this characteristic bent shape is lost in both stunned (Fig 9C) and calpain-treated (Fig 9D) muscles; along with the overall shift to the right, the log-log force-[Ca²⁺] relation becomes quite linear. As a result, n₁ increases slightly, while n₂ decreases significantly (Table 2). The mechanistic implications of these changes are discussed below; what merits particular emphasis here is the striking similarity between the effects of stunning and those of calpain, not just the overall changes but also their quantitative details.

View this table: [in this window] [in a new window]   Table 2. Hill Coefficient Data From the Hill Plots in Fig 8

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Muscle skinning enables precise control of the concentrations of the various components of the cytosol including Ca²⁺, Mg²⁺, and pH, thus offering a direct way to study the properties of the myofilaments. By comparing and contrasting the skinned muscle results with the properties of the myofilaments in intact muscle, we investigated the force-[Ca²⁺] relation in a unique and powerful manner.¹¹ This approach enabled us to determine the relative contributions of soluble cytosolic factors, which can be controlled after skinning, and the myofilaments themselves to the decreased Ca²⁺ responsiveness seen in stunned myocardium. The data indicate that alterations of the myofilaments account for the decreased maximal Ca²⁺-activated force and for most, if not all, of the decreased Ca²⁺ sensitivity.

The Role of Alterations of the Myofilaments in the Decreased Ca²⁺ Responsiveness of Stunned Myocardium
Our results showing a fixed decrease in F_max in stunned myocardium are inconsistent with studies that showed no changes in F_max (compared with control values) of skinned cardiac preparations.^{6 7} The inconsistency may be due to the different protocols used to produce stunning: one used low-flow regional ischemia in porcine hearts; the other used 40 minutes of total global ischemia in rat hearts. In addition, it is not clear whether physiological levels of F_max were actually achieved in those studies, especially without parallel comparison to F_max under intact conditions. This concern is heightened by the fact that the absolute values of F_max of both control and stunned preparations reported in these studies were twofold to threefold smaller than the values we obtained in this and previous studies.^{11 12}

The observed changes in cross-bridge cooperativity, as gauged by Hill coefficients, are complex. Two effects merit particular mention. First, log-log analysis reveals that the Hill coefficient, which is known to be altered by skinning itself, changes from a single quantity in intact muscles to two coefficients, n₁ and n₂, after skinning. Second, the effects of stunning are manifested differently before and after skinning. Although we found that the Hill coefficient was not changed in intact muscles, the linearized Hill plots of the skinned muscles revealed that the usual bent shape in control muscles was replaced by a single line in both stunned and calpain-treated muscles (Fig 9). This type of change seems unique to the stunned and calpain-treated muscles, since such changes were not seen with the desensitizing effects of P_i,²⁷ pH,²⁸ ionic strength,²⁹ protein kinase A,³⁰ and length-dependent activation.³¹ When linearized, the data from these studies are parallel to lower pCa₅₀ values. The mechanism for the convergence of the two phases of Hill plots into a single line is not clear at present.

In our experiments, we did not control sarcomere length during activations, and changes in sarcomere length can affect myofilament sensitivity. Kentish et al³² showed that the effect of sarcomere length on the force-pCa relation was dramatic when corrected for sarcomere shortening. However, compared with the force-pCa relation obtained from a muscle without correction (at a resting sarcomere length of 2.10 µm), there was a significant reduction only in the slope of the force-pCa relation, not in pCa₅₀. Given the fact that our experiments were performed at 2.20 to 2.30 µm, the effect of uncontrolled changes in sarcomere length on pCa₅₀ should be even smaller. Sarcomere length does have a significant effect on the slope of the force-pCa relation. Therefore, our values for Hill coefficients may be underestimated. Nevertheless, we found that changes in sarcomere length were identical in both control and stunned muscles, not only during maximal activations but also in submaximal activations (Fig 3). Whatever the effects of uncontrolled changes in sarcomere length may have been, the control and stunned muscles would have been subject to similar errors.

Ca²⁺-Activated Proteases and the Stunned Myocardium
Ca²⁺-activated proteases are widely distributed in myocytes.⁸ Muscle-derived calpains have been well characterized with regard to their physical and chemical properties as well as their substrate specificity in numerous in vitro studies. Their role in muscle physiology, however, remains largely unknown. Previous studies involving only skinned smooth muscle fibers have reported that calpain I decreased the Ca²⁺-activated isometric force.³³ The present study provides the first direct evidence of the effects of calpain I on the Ca²⁺ responsiveness of striated muscle.

Several factors merit consideration in evaluating the physiological relevance of our calpain I study. The K_m of calpain I for Ca²⁺ has been shown to be 1 to 20 µmol/L in vitro.³⁴ The [Ca²⁺] required to activate calpains may be lower in intact cells because of the presence of membrane phospholipids and autolysis of calpains.³⁵ Thus, activation of calpain I, which constitutes 20% of total calpain in rat myocardium, may require much lower [Ca²⁺] in intact cells than in skinned cells. Myocardial stunning is always preceded by short-lived elevation of [Ca²⁺]_i, which occurs during ischemia and early reperfusion,^{36 37 38} and the magnitude of the increase of [Ca²⁺]_i exceeds 1 to 3 µmol/L. Despite being underestimated because of Ca²⁺ buffering, this value nevertheless falls within the range of [Ca²⁺] required for activation of calpain I in vitro.^{8 34} Calpains cause only limited proteolysis (ie, they yield large protein fragments, not individual amino acids).³⁹ Thus, it is not surprising that possible changes caused by calpains in stunned myocardium may not be visualized using conventional histological methods. For all these reasons, calpain I is a physiologically relevant candidate for intracellular myofilament proteolysis after ischemia and reperfusion.

The substrate specificity of calpain I on cardiac myofibrillar proteins is not well understood at present. Most of the information concerning myofilament substrate specificity of calpain derives from studies using calpain II.³⁹ The physiological significance of these findings is unclear, given the fact that calpain II needs millimolar [Ca²⁺] to be activated. A number of cardiac contractile proteins including troponins T and C, tropomyosin, and myosin have been shown to be susceptible to calpain in previous studies^{40 41} in which calpain II was used. Because of the similarity in the molecular structure of the protease domain of the calpains,⁴² it might be expected that calpain I has a similar substrate specificity. Our own preliminary studies^{43 44} point to troponin I as a potential target for proteolysis in the stunned myocardium. Further investigation will be required to ascertain which myofilament proteins are degraded by calpain I and how they compare with those in the stunned heart and to determine the extent of the correlation between the occurrence of such degradation and the stunned phenotype.

To date, direct evidence that calpain I is activated in stunned myocardium is lacking. Recently, Yoshida et al⁴⁵ have provided new evidence that the activity of calpain I was increased after ischemia/reperfusion in rat hearts. Although the duration of ischemia they used was longer than that used in the present study, a later study⁴⁶ from the same group showed that proteolysis of calspectin, a cytoskeletal protein and a specific substrate of calpain I, occurred after periods of ischemia as brief as 10 minutes, followed by 30 minutes of reperfusion. These studies suggest that calpain I may well be activated after brief ischemia followed by reperfusion.

Limitations of the Study and Future Directions
The changes in the Ca²⁺ responsiveness of the myofilaments after calpain I exposure mimic quite faithfully those that we have found in skinned muscles from stunned hearts. Fig 9 reproduces the data of Figs 4B and 5 in linearized Hill plots in order to facilitate direct comparison. Decreases in F_max are seen in both stunned and calpain-treated muscles (Fig 9A and 9B, respectively). Similarly, Ca₅₀ was increased in both stunned and calpain-treated muscles, as shown explicitly by the linearized Hill plots (Fig 9C and 9D, respectively). In addition, the shapes of the plots changed in an identical manner in the stunned and calpain-treated muscles. However, these similarities do not conclusively establish the role of calpains in the pathogenesis of myocardial stunning. The findings reported in the present study support (but do not prove) the "proteolysis" hypothesis for myocardial stunning. The similarities between the effects of stunning and those of direct exposure of the myofilaments to calpain I do not prove a causal role for calpain I in stunning. Several important questions remain to be answered. The myofibrillar substrates of calpain I need to be identified and matched with the putative myofibrillar alterations in stunned myocardium. Activation of calpains needs to be demonstrated in the process of stunning. More important, studies designed to alleviate stunning by preventing proteolysis are necessary to pave the way for clinical application of the proteolysis hypothesis. At least two such strategies have already proved successful in isolated perfused hearts.^{47 48}

Myocardial stunning usually resolves within days, a time course consistent with protein degradation and resynthesis.^{49 50} Thus, the notion that Ca²⁺-activated proteolysis occurs during reperfusion provides a specific rationale for the characteristically slow recovery of function in the stunned myocardium.

	Acknowledgments

 
This study was supported by the National Institutes of Health (R01 HL-44065). We thank Drs Hideo Kusuoka, Peter Backx, and Dan Atar for helpful discussions.

Received August 9, 1995; accepted December 12, 1995.

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