INTRODUCTION

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REPERFUSION AFTER BRIEF MYOCARDIAL ISCHEMIA leaves behind a sustained contractile dysfunction despite the absence of irreversible structural damage and despite complete restoration of coronary flow (5). This condition, which has been termed "stunned myocardium" (7), may persist for hours to up to several days but is, eventually, fully reversible. With the development of methods aimed at achieving prompt reperfusion in patients with acute myocardial ischemia, stunned myocardium is now recognized to be of considerable clinical relevance (4).

Intracellular Ca²⁺ homeostasis in stunned myocardium appears to be unimpaired (19). Depressed contractile function despite normal Ca²⁺ cycling is indicative of a reduced Ca²⁺ responsiveness of contraction, which was, indeed, shown in isolated intact trabeculae from ischemic-reperfused rat hearts (13). This pinpoints the critical lesion to the myofilaments, which translate the intracellular Ca²⁺ signal into force development during the distal steps of excitation-contraction (E-C) coupling. On the basis of reports showing that the activity of the Ca²⁺-dependent proteolytic enzyme calpain is enhanced in postischemic-reperfused myocardium (30, 31), Gao et al. (13) used an immunoblot analysis to probe myofibrillar homogenates from rat stunned myocardium for proteolysis products of a variety of myofilament proteins. The thin-filament regulatory protein troponin I (TnI) uniquely displayed a low-molecular-weight band in addition to the parent protein, indicating specific partial proteolytic degradation. The primary TnI degradation product was subsequently identified as TnI_1-193, which lacks the 17 COOH-terminal residues of full-length cardiac TnI (22).

TnI in striated muscles is a key component of the ternary troponin complex, where it, in concert with the Ca²⁺-binding subunit troponin C (TnC) and the tropomyosin-binding subunit troponin T, Ca²⁺ dependently switches on and off the contractile machinery. This critical involvement in the regulation of thin-filament activation raises the possibility that proteolysis of even a small fraction of TnI may suffice to cause the contractile dysfunction typical of stunned myocardium (13). We (23) generated a transgenic (TG) animal model in which mice cardiac specifically expressed TnI_1-193 at a fraction of total TnI comparable with that found in ischemic-reperfused rat hearts and reported that the myocardium of TG mice at baseline exhibits contractile dysfunction. In that initial report, the response of tension development during twitches to different Ca²⁺ concentrations was not investigated nor was signal transduction probed. Furthermore, precise analysis of the Ca²⁺ sensitivity of contraction was impaired by the low overall Ca²⁺ sensitivity of the intact mouse myocardium.

The present work adds to our recent study by demonstrating that, despite impaired E-C coupling, isolated intact trabeculae from TG mice maintain a contractile reserve that can be recruited by positive inotropic interventions. We also verify that, during twitches, TG mouse trabeculae exhibit both reduced Ca²⁺ sensitivity and Ca²⁺ responsiveness, thereby recapitulating key features of genuine stunned myocardium. A preliminary report has been previously published (see Ref. 18).

	METHODS

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Transgenic mouse model. Transgenic mice with cardiac expression of the equivalent of the major proteolytic product of TnI (TnI_1-193) in stunned myocardium were produced as described (23). Transgene status was assessed by PCR from genomic DNA prepared from tail clips using a Gentra puregene kit and transgene-specific oligonucleotides. Non-TG control mice were either littermates of TG mice or were obtained from the same parental strain (C57BL/6).

Mouse intact muscle preparation. All experimental procedures were approved by the Johns Hopkins University Animal Care and Use Committee and were carried out essentially as described (14). Adult mice (age 3-8 mo) of either gender were euthanized by intraperitoneal injection of pentobarbitol sodium (~7.5-15 mg), and their hearts were excised. Intact trabeculae or thin papillary muscles were isolated from the right ventricles and mounted in a superfusion bath between a force transducer (type AE 801, SensoNor; Horten, Norway) and a rigid hook connected to a micromanipulator for length adjustment. Thin unbranched preparations that were suitable for experiments were found in ~20% of mouse hearts. The muscles were superfused at 10 ml/min with a modified Krebs-Henseleit solution equilibrated with 95% O₂-5% CO₂, which contained (in mM): 142 Na⁺, 5 K⁺, 1.2 Mg²⁺, 1.0 Ca²⁺, 127 Cl, 2 PO, 1.2 SO, 20 HCO, and 10 glucose; pH 7.37-7.43. The preparations were excited by bipolar electrical field stimulation, with an amplitude of 8-15 V and a pulse width of 5 ms (Grass SD9 stimulator, Grass Instruments; Quincy, MA). After equilibration, muscles were stretched to the length at which the amplitude of developed twitch tension was maximal, corresponding to a sarcomere length of 2.1-2.2 µm (14). Tetanic contractions were elicited by stimulating the preparations at 10 Hz in the presence of 5 µM ryanodine at various extracellular Ca²⁺ concentrations ([Ca²⁺]_o; 0.5-20.0 mM) to achieve an equilibrium between the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and myofilament activation.

Measurement of [Ca²⁺]_i using fura 2. Fura 2 pentapotassium salt (Molecular Probes; Eugene, OR) was microinjected iontophoretically into two to four cells and allowed to spread evenly throughout the entire preparation via gap junctions (2). Fura 2 fluorescence was excited at 340 and 380 nm, and the emitted light was collected at 510 nm by a photomultiplier tube (R2693, Hamamatsu; Bridgewater, NJ). The light signal was filtered at 100 Hz, collected by an analog-to-digital converter, and stored on a computer for later analysis. The autofluorescence of the muscle was subtracted from the raw fluorescence data, and [Ca²⁺]_i was calculated using the following equation (15): [Ca²⁺]_i = K(R  R_min)/(R_max  R), where R is the observed fluorescence ratio (340/380 nm), K is the apparent dissociation constant of fura 2, R_max is the fluorescence ratio (340/380 nm) at saturating [Ca²⁺], and R_min is the ratio (340/380 nm) at 0 [Ca²⁺]. Values for K, R_max, and R_min were obtained during in vivo calibration experiments on sentinel mouse muscles (14). All experiments were carried out at room temperature (22-23°C), which is an established procedure to circumvent the rapid loss of fura 2 from intact preparations that regularly occurs at higher temperatures (20). Accordingly, when measuring twitch contractions, we reduced the stimulation rate to 0.5 Hz to take into consideration the slowing of Ca²⁺ cycling processes that is associated with lower than physiological temperature.

Myofibrillar ATPase activity measurement. Myofibrils were prepared as described (24) with careful attention paid to the use of protease inhibitors. Assays were performed with the use of incubation conditions established by varying the total concentration of metals, salts, and ligands, maintaining ionic strength using the stability constants compiled by Fabiato (9), and were performed at pH 7.0 with 50 mM imidazole, 50 mM KCl, and 2 mM MgATP. Inorganic phosphate liberation was measured using a microtiter plate version of the standard assay as described by Rarick et al. (25), and the ATPase activity was calculated in nanomoles of inorganic phosphate liberated per milligram of myofibrillar protein per minute.

-Adrenergic stimulation. l-Isoproterenol (Sigma; St. Louis, MO) was added to the superfusate from a 1 mM stock solution in distilled H₂O to a final concentration of 300 nM.

Analytical methods. Measured force was converted into tension by normalization with respect to the cross-sectional area of each muscle. To characterize the steady-state tension-[Ca²⁺]_i relationship, tension and [Ca²⁺]_i data obtained from each muscle during tetanic contractions were fitted to a Hill equation as follows: F_X  F_min = (F_max  F_min)[Ca_X]/ [(Ca₅₀) + [Ca_X]], where F_X is actual tension, F_min is resting tension, F_max is maximal Ca²⁺-activated tension, [Ca_X] is actual [Ca²⁺]_i, Ca₅₀ is the [Ca²⁺]_i required for development of half-maximal tension, and n_H is the Hill coefficient. To provide an additional estimate of the Ca²⁺ sensitivity of the preparations, tension was plotted versus [Ca²⁺]_i during twitches, and the descending limb of these phase-plane loops during tension decline was fitted to the Hill equation to calculate Ca₅₀ values for individual twitches. Statistical significance of data was analyzed using a multivariate ANOVA to independently assess the effects of [Ca²⁺]_o and groups. The effects of isoproterenol were analyzed using Student's paired t-test. P < 0.05 was considered statistically significant. Pooled data are expressed as means ± SE.

	RESULTS

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Effect of [Ca²+]_o on Ca²⁺ transients and twitch tension. We simultaneously examined Ca²⁺ cycling and tension development during twitches of TG and non-TG mouse intact right ventricular trabeculae at various [Ca²⁺]_o. We carried out the experiments in a blinded fashion such that the investigator did not know the group identity of a mouse until after analysis of the data. Figure 1 shows the time course of [Ca²⁺]_i and tension of representative non-TG and TG TnI_1-193 preparations at 2.0 and 3.0 mM [Ca²⁺]_o. The [Ca²⁺]_i transients were very similar, whereas twitch tension was greatly reduced in the TG preparation. Considering the generally low responsiveness of the isolated intact mouse myocardium to external Ca²⁺ (14), we explored whether this contractile dysfunction can be overcome by high [Ca²⁺]_o or is preserved over a broader range of inotropic intervention by raising [Ca²⁺]_o incrementally from 1.0 to 6.0 mM. This intervention resulted in a stepwise increase in peak systolic [Ca²⁺]_i in the non-TG control group with a quasilinear relationship (Fig. 2A), whereas diastolic [Ca²⁺]_i remained unaltered, indicating that diastolic Ca²⁺ overload did not occur. The elevation in peak [Ca²⁺]_i was paralleled by an increase in developed twitch tension (i.e., peak systolic tension minus resting tension) from 2.8 ± 1.5 to 40.3 ± 5.8 mN/mm² at 1.0 and 6.0 mM [Ca²⁺]_o, respectively (n = 6; Fig. 2B).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Comparison of intracellular free Ca2+ concentration ([Ca2+]i) transients (top) and twitch tension (bottom) in a nontransgenic (non-TG) control and TG troponin I (TnI) fragment (TnI1-193) preparation. Original records of representative preparations from non-TG control (left) and TG TnI1-193 (right) hearts at 2.0 and 3.0 mM extracellular Ca2+ concentration ([Ca2+]o). Whereas the [Ca2+]i transients were similar, twitch tension was greatly reduced in the TG preparation.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Excitation-contraction coupling in TG muscles and non-TG controls. A: dependence of peak systolic (squares) and diastolic (circles) [Ca2+]i on [Ca2+]o. B: dependence of developed twitch tension on [Ca2+]o. Filled symbols, non-TG controls; open symbols, TG TnI1-193 preparations. Despite similar peak systolic [Ca2+]i values, twitch tension in TG muscles was significantly reduced compared with non-TG controls.

Characterization of the tension-[Ca²+]_i relationship. We measured isometric tension and [Ca²⁺]_i during the plateau phase of tetani elicited by 10-Hz stimulation in the presence of ryanodine to characterize the steady-state tension-[Ca²⁺]_i relationship (Fig. 3, A and B; data partially included in Ref. 23). Non-TG control muscles attained a substantially higher maximum Ca²⁺-activated tension than TG muscles (48.14 ± 7.23, n = 5, vs. 28.70 ± 0.88 mN/mm², n = 4, P = 0.0036). Resting tensions and Hill coefficients were similar in both groups. From Fig. 3B, which illustrates the normalized tension-[Ca²⁺]_i relationships, it is obvious that the midpoint of the relation was shifted toward higher [Ca²⁺]_i in TG muscles by ~340 nM, but this effect was not significant. However, a problematic feature of these steady-state tension-[Ca²⁺]_i relationships is the low Ca²⁺ sensitivity of mouse intact preparations compared, e.g., with the rat myocardium. The Ca₅₀ for isometric tension falls in a range (~1.3-1.6 µM) that is relatively far off the K_d of fura 2 for Ca²⁺ (224 nM; see Ref. 15) such that subtle changes in fluorescence properties reflect large differences in [Ca²⁺]. Therefore, to obtain a more accurate estimate for the Ca²⁺ sensitivity of the preparations, we analyzed the relationship of tension and [Ca²⁺]_i during the relaxation phase of regular twitches. In Fig. 4, A and B, developed tension is plotted versus [Ca²⁺]_i during twitches at three different [Ca²⁺]_o for representative non-TG and TG_1-193 muscles (phase-plane loops). The descending limb of these loops, due to the phase lag between [Ca²⁺]_i and tension, is shifted toward lower [Ca²⁺]_i compared with the steady-state tension-[Ca²⁺]_i relationship of the same muscle (1) and therefore falls in a range of [Ca²⁺]_i that is more accurately reflected by the fura 2 fluorescence properties. In TG TnI_1-193 muscles, half-maximal tension during relaxation was attained at significantly higher [Ca²⁺]_i than in non-TG control muscles (0.53 ± 0.04 vs. 0.42 ± 0.03 µM, respectively, P = 0.035; Fig. 4C), indicating reduced Ca²⁺ sensitivity.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Steady-state tension-[Ca2+]i relationship. A: absolute values of tension during tetani. B: tension values normalized to the maximum tension under the respective condition. Straight line and , non-TG controls; dashed line and , TG TnI1-193 preparations. Maximum Ca2+-activated tension is significantly reduced in TG preparations. After normalization, a rightward shift of the relationship in TG preparations became apparent, which was, however, not significant.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Tension-[Ca2+]i relationship derived from twitch data. A: phase-plane loops of a representative non-TG control preparation at 3.0, 4.0, and 6.0 mM [Ca2+]o. B: phase-plane loops of a representative TG TnI1-193 preparation at 4.0, 5.0, and 7.0 mM [Ca2+]o. C: comparison of [Ca2+]i required for development of half-maximal tension (Ca50) values during the relaxation phase of twitches. Hill curves have been fitted to the descending limbs of the loops in A and B. During relaxation, 50% of peak tension was reached at significantly higher [Ca2+]i in TG TnI1-193 preparations compared with control (Con), indicating Ca2+ desensitization.

Myofibrillar ATPase activity. We measured the ATPase activity of myofibrils extracted from non-TG control and TG TnI_1-193 mouse hearts (Fig. 5). The Ca²⁺-independent basal myofibrillar ATPase activity was similar in both groups. Non-TG control myofibrils exhibited a maximum Ca²⁺-activated ATPase activity of 193 ± 9 nmol · mg¹ · min¹ (n = 8). In TG myofibrils, this ATPase activity was reduced by 38% to 119 ± 5 nmol · mg¹ · min¹ (n = 7, P = 0.002). This reduction closely corresponds to the 40% lower maximum Ca²⁺-activated tension observed in intact muscle preparations.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Myofibrillar ATPase activity. Maximum Ca2+-activated myofibrillar ATPase activity was reduced by 38% in TG TnI1-193 compared with non-TG control preparations, which is equivalent to the reduction in maximum Ca2+-activated tension observed in intact TG TnI1-193 muscle preparations.

Inotropic response to -adrenergic receptor stimulation. A typical feature of stunned myocardium is its maintained contractile reserve when stimulated with positive inotropic agents, e.g., catecholamines (3, 8). We therefore treated non-TG control and TG muscles with 300 nM l-isoproterenol to examine whether this property is preserved in mouse muscles expressing TnI_1-193. In non-TG control muscles, isoproterenol induced a clear positive inotropic response: developed twitch tension at [Ca²⁺]_o of 1.0, 2.0, and 3.0 mM was markedly and significantly increased 15-, 6-, and 3.2-fold, respectively, compared with twitch contractions at the same [Ca²⁺]_o in the absence of isoproterenol (Fig. 6B). At 3.0 mM [Ca²⁺]_o in the presence of isoproterenol, twitch tension approached a maximal level (56.66 ± 18.16 mN/mm²), which was not significantly different from either maximum Ca²⁺-activated tension in tetanic contractions or the greatest twitch tension attained at 6.0 mM [Ca²⁺]_o in the absence of isoproterenol (48.11 ± 7.23 and 44.48 ± 6.44 mN/mm², respectively). Increasing [Ca²⁺]_o to values >3.0 mM in the presence of isoproterenol invariably caused diastolic Ca²⁺ overload and irregular spontaneous contractions. The elevation of twitch tension was associated with a pronounced increase in peak systolic [Ca²⁺]_i (Fig. 6A), consistent with the known pharmacological property of -adrenergic receptor agonists to exert their positive inotropic action by increasing calcium availability.

View larger version (41K): [in this window] [in a new window]   Fig. 6.   Effect of isoproterenol on [Ca2+]i transients and twitch tension. A: peak systolic [Ca2+]i. B: developed twitch tension. Left: Non-TG controls (filled symbols); right: TG1-193 preparations (open symbols). Squares, no isoproterenol; circles, 300 nM isoproterenol. Insets: time courses of [Ca2+]i transients (A) and twitch tension (B), respectively. Gray trace, no isoproterenol; solid trace, 300 nM isoproterenol. *Significant difference between untreated and isoproterenol-treated condition. In both groups, isoproterenol recruited a contractile reserve by increasing peak systolic [Ca2+]i. Developed twitch tension in TG TnI1-193 preparations remained reduced compared with non-TG controls.

Kinetic effects of isoproterenol on twitch tension and Ca²⁺ transients. We compared twitch contractions at 3.0 mM [Ca²⁺]_o before and after -adrenergic receptor stimulation. Figure 6, A and B (insets), shows normalized records of [Ca²⁺]_i and tension versus time from representative muscles in each group in the absence and presence of isoproterenol. Isoproterenol exerted a similar lusitropic action in both non-TG control and TG muscles (Table 1). The time to peak systolic [Ca²⁺]_i was not significantly altered. In contrast, the time from peak to half-decay of [Ca²⁺]_i, the time to peak tension, and the time from peak tension to half relaxation were significantly reduced by isoproterenol in both groups to a similar extent.

	DISCUSSION

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The present study extends our previous findings (23) in several major ways. Here, we show that intracellular Ca²⁺ transients, sampled over a wide range of [Ca²⁺]_o, are indistinguishable in trabeculae from TG mouse hearts and control muscles, consistent with unimpaired Ca²⁺ cycling. In marked contrast, twitch tension in TG muscles is greatly decreased, indicating uncoupling of excitation and contraction. The contractile dysfunction in TG TnI_1-193 preparations is due to both a reduced overall tension-generating capacity of TG muscles, reflected by the lower maximum Ca²⁺-activated tension (which is paralleled by an equivalent reduction in maximum Ca²⁺-activated myofibrillar ATPase activity), and a decrease in Ca²⁺ sensitivity, reflected by the increase in Ca₅₀ during the relaxation phase of twitches. The proportionately similar inotropic responses to increases in [Ca²⁺]_o and to isoproterenol reveal that muscles from both groups have a sizable contractile reserve that can be recruited by positive inotropic intervention and that the -adrenergic signaling cascade is intact. In both the TG TnI_1-193 and non-TG control myocardium, the maximum twitch tension attainable is limited by the maximum Ca²⁺-activated tension, as found in tetanic contractions at saturating [Ca²⁺]_o.

These features closely recapitulate the functional characteristics found in isolated trabeculae from rat hearts subjected to 20-min ischemia/20-min reperfusion (12) and also reproduce a well-established property of stunned myocardium in that the contractile dysfunction can partially be overcome by stimulation with positive inotropic agents (3, 8). This phenotype also finds its equivalent in the more physiological model of regional low-flow ischemia-reperfusion in pigs, where likewise maximum calcium-stimulated contractility was greatly impaired, whereas a reduction in calcium sensitivity appeared to be less prominent (16). Therefore, the presence of even a low amount of TnI_1-193 in the absence of any additional damage suffices to produce the defective E-C coupling with a maintained -adrenergic signaling cascade typical of stunning. These findings, as well as the impairment in hemodynamic function in these mice in vivo (23), strongly support the putative causal role of TnI degradation in the pathogenesis of myocardial stunning. Moreover, because in this TG mouse model the TnI fragment 1-193 is generated independent of proteolysis of the parent protein, the 17-residue COOH-terminal fragment formed as a byproduct of TnI proteolysis in genuine stunned myocardium is not necessary to induce the "stunned" phenotype.

In this and also our previous study (23), the comparative analysis of the steady-state tension-[Ca²⁺]_i relationships did not allow us to make a conclusive statement about differences in the Ca²⁺ sensitivity of TG TnI_1-193 and non-TG control preparations. This was due to the generally low Ca²⁺ sensitivity of tension observed in the mouse intact myocardium (14) in which Ca₅₀ values fall in a range where [Ca²⁺]_i calculated from fura 2 fluorescence data shows considerable dispersion. Because of this inaccuracy, to confirm a statistically significant difference of Ca₅₀ values between groups would require experimental numbers that are both impractical and ethically questionable to achieve, given the low frequency of suitable preparations in mouse hearts and considering that reliable information on the Ca²⁺ sensitivity can be extracted from twitch data using the alternative method we established here. During the relaxation phase of a twitch, myofilament activation (i.e., tension) and [Ca²⁺]_i exhibit a sigmoidal relationship. Because of the phase lag between [Ca²⁺]_i and tension during a twitch, this relationship is shifted toward lower [Ca²⁺]_i compared with steady-state conditions during tetani elicited in the presence of ryanodine, to a range in which fura 2 fluorescence properties allow for calculation of [Ca²⁺]_i with greater accuracy. In the twitch tension-[Ca²⁺]_i plots (phase-plane "loops"), a Hill equation can be fitted to the descending limb and a Ca₅₀ value can be calculated that represents the [Ca²⁺]_i at which active tension has declined to 50% of the peak value.

To calculate Ca₅₀ from the steady-state tension-[Ca²⁺]_i relationship is an accepted procedure to analyze the Ca²⁺ sensitivity of intact cardiac preparations, because during the plateau of tetanic contractions a well-characterized and highly reproducible equilibrium exists between [Ca²⁺]_i and the level of myofilament activation. This equilibrium, however, is generated under unphysiological conditions involving 10-Hz stimulation and disruption of sarcoplasmic reticulum function using ryanodine. In contrast, the experimental conditions during twitches are more physiological. The leftward shift of the descending limb compared with the steady-state tension-[Ca²⁺]_i relationship indicates that relaxation in cardiac muscle is not limited by the decay rate of the [Ca²⁺]_i transient but rather by the myofilaments themselves (1). Thus the Ca₅₀ value calculated from twitch relaxation truly represents properties intrinsic to the myofilaments and therefore can be used to compare the Ca²⁺ sensitivities of different groups. It is clear, however, that Ca₅₀ values calculated from twitch relaxation data must not be compared with Ca₅₀ values calculated from steady-state data. Because of damaged-end compliance (28) and the length dependence of Ca²⁺ sensitivity, sarcomere shortening in the central part of the preparation may theoretically decrease the overall Ca²⁺ sensitivity of a preparation. Additionally, central sarcomere shortening has been observed to result in an increase of the peak of the Ca²⁺ transient (17). Both these effects would be expected to be more pronounced in the group of preparations contracting more strongly, i.e., in non-TG control muscles. If anything, this effect will tend to underestimate the difference in Ca²⁺ sensitivity between control and TG preparations, as detected in this study, and therefore does not affect the conclusions drawn regarding the effect of TnI_1-193 expression on Ca²⁺ sensitivity.

In our previous study (23), impedance catheter measurements revealed that -adrenergic receptor stimulation increased the maximal rate of pressure development (dP/dt_max) in both TG and non-TG mice. The maximal rate of decay of pressure (dP/dt_min), however, was not affected in vivo by isoproterenol treatment (23). If the -adrenergic pathway is intact, cAMP-dependent phosphorylation of phospholamban and TnI is expected to accelerate relaxation in cardiac muscle. We therefore analyzed the effects of isoproterenol on the kinetics of tension and [Ca²⁺]_i transient during twitches of trabeculae isolated from their environment. Our data confirm that, in isolated muscle preparations from both TG TnI_1-193 and non-TG control mice, isoproterenol treatment significantly enhanced the speed of contraction and relaxation, consistent with the -adrenergic cascade being intact in our transgenic model. The lack of effect of isoproterenol on dP/dt_min in vivo may be explained by prior sympathetic activation due to anesthesia of the animals or by additional effects on the systemic vascular resistance, which can be excluded in the isolated cardiac muscle preparation.

The mechanism by which TnI_1-193 impairs contractility is unknown. Degradation of a certain amount of intact TnI could result in partial "loss of function" of the full-length protein. Alternatively, a "gain of function" of the degraded form of TnI could occur, exposing regulatory properties different from those of the parent protein. A complete loss of function of a fraction of TnI most likely would result in failure of the myocardium to relax fully, analogous to myocardium from which TnI has been partially extracted, e.g., using vanadate (27). This is not likely to be the case in TG preparations, because we did not observe any elevation in resting tension. The concept of altered regulatory properties of the deletion mutant is supported by Rarick et al. (26), who demonstrated that recombinant cTnI_1-188, which is missing only six more residues than the transgenically generated cTnI_1-193 fragment used in our study, displayed impaired ability to fully inhibit myofibrillar ATPase activity in the absence of Ca²⁺ and to fully activate myofibrillar ATPase activity at saturating [Ca²⁺], whereas cTnI_1-199 behaved essentially as the wild-type protein. A recent study (10) mapping cardiac TnI-TnC interactions with synthetic peptides identified an interaction between TnI_191-210 and TnC (10). Furthermore, two point mutations located within this region, Gly²⁰³Ser and Lys²⁰⁶Gln, are associated with familial hypertrophic cardiomyopathy (6), underlining the possible functional relevance of loss of this segment. Whether or not the regulatory properties of TnI_1-193 are qualitatively or quantitatively different from those of the parent protein cannot be resolved on intact isolated muscle preparations of this TG mouse model. Our observation that maximum Ca²⁺-activated myofibrillar ATPase activity in TG preparations is reduced to a similar extent as maximum Ca²⁺-activated tension in intact trabeculae indicates that the effect of TnI truncation on actomyosin interaction does not depend on the strain on the cross-bridges, because a suspension of myofibrils in solution bears no load. Further studies involving the biochemical characterization of purified recombinant troponin subunits are required to clarify this point.

An important question remains as to whether TnI degradation is necessary for the development of stunning, because several studies (21, 29) in larger animal models failed to demonstrate TnI degradation in stunned myocardium. It has, however, been pointed out that to reliably exclude the presence of TnI degradation products by Western blot analysis requires probing of tissue samples with a variety of antibodies directed against different epitopes on TnI (11), and, importantly, TnI degradation has been observed in myocardial biopsies of human ischemic heart disease patients (23).

In summary, we present evidence confirming that TnI_1-193 is capable of producing the stunned phenotype in the absence of any additional ischemia-reperfusion-induced damage, supporting the hypothesis that stunning is caused by proteolytic degradation of TnI. The preservation of upstream inotropic responsiveness implies that calcium and -adrenergic signaling remain intact in the TG mice, as they do in genuine stunned myocardium.