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(Circulation Research. 1996;79:447-454.)
© 1996 American Heart Association, Inc.

Articles

Inhomogeneous Disappearance of Myofilament-Related Cytoskeletal Proteins in Stunned Myocardium of Guinea Pig

Yasushi Matsumura, Eijiro Saeki, Michitoshi Inoue, Masatsugu Hori, Takenobu Kamada, Hideo Kusuoka

the Department of Medical Information Science (Y.M., E.S., M.I.), The First Department of Medicine (M.H., T.K.), and the Division of Tracer Kinetics, Biomedical Research Center (H.K.), Osaka (Japan) University Medical School.

Correspondence to Hideo Kusuoka, MD, PhD, Division of Tracer Kinetics, Biomedical Research Center, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka, 565 Japan. E-mail kusuoka@tracer.med.osaka-u.ac.jp.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The decrease in Ca²⁺ responsiveness of myofilaments in stunned myocardium implies that there may be structural changes in proteins composing the contractile machinery. To elucidate the lesion in stunned myocardium, isolated guinea pig hearts were subjected to global ischemia at 37°C and reperfused. SDS-PAGE revealed that the contents of desmin, -actinin, and spectrin decreased in the myofibrillar fraction isolated from hearts reperfused after 60-minute ischemia compared with nonischemic control hearts. To examine the change of cytoskeletal proteins in stunned myocardium, immunohistochemical studies with antibodies against these proteins were performed after 15 minutes of ischemia. In stunned myocardium, the staining was largely intact, but there were some lesions where desmin was not stained and -actinin and spectrin were only weakly identified. The percentage of normally stained areas in the myocardium (percent stained area), quantified by image processing, was significantly lower in stunned myocardium (79.6±3.6%, mean±SEM) than in nonischemic control myocardium (96.5±0.7%). Percent recovery of developed pressure significantly correlated with percent stained area (r=.82, P<.001). In hearts subjected to 15-minute ischemia but not reperfused, or in hearts reperfused with Ca²⁺-free solution after 15-minute ischemia, staining by the antibodies remained intact, suggesting that the change of the cytoskeletal proteins is mediated by Ca²⁺ overload during reperfusion. In hearts treated with the protease inhibitor leupeptin (50 µmol/L) or calpain inhibitor I (100 µmol/L), both developed pressure and staining were well preserved. These results indicate that contractile dysfunction in stunned myocardium has a strong correlation with the disappearance of cytoskeletal proteins that may be mediated by a Ca²⁺-dependent intracellular protease activated during reperfusion. The disruption of cytoskeletal proteins is a possible mechanism for stunning, although it may be a secondary effect of protease activation.

Key Words: -actinin • desmin • spectrin • calpain • leupeptin • calpain inhibitor I

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
When hearts are exposed to a brief period of ischemia and then reperfused, contractility remains depressed for days to weeks, despite the absence of necrosis. This contractile dysfunction has been called "stunning."¹ Several findings now indicate that the mechanism of stunning involves changes in myofilament function. In isolated ferret hearts reperfused after 15-minute global ischemia, Ca²⁺ transients were not smaller, despite the decline of the developed pressure.^{2 3} On the other hand, the Ca²⁺ responsiveness of the contractile machinery is definitely reduced in stunned myocardium.^{3 4 5 6} These results indicate that the cellular lesion responsible for excitation-contraction uncoupling in stunned myocardium resides at the level of the myofilaments.

Intracellular Ca²⁺ overload during ischemia and/or reperfusion has been proposed as a major mechanism for contractile dysfunction in stunned myocardium.⁷ Free radicals are also involved, but the two mechanisms are not mutually exclusive.⁸ The Ca²⁺ hypothesis is supported by three lines of evidence: First, [Ca²⁺]_i increases during ischemia and just after reperfusion.^{9 10 11 12} Second, reperfusion with low-calcium solution or treatments antagonizing Ca²⁺ overload protect myocardium against stunning.^{4 13 14} Third, transient Ca²⁺ overload without ischemia produces contractile dysfunction that mimics stunning.^{15 16} Although these results clearly indicate that the intracellular Ca²⁺ overload during ischemia and/or reperfusion plays an important role, the mechanism of contractile dysfunction induced by Ca²⁺ overload has not been clarified.

We previously hypothesized that Ca²⁺ overload activates intracellular proteases such as calpain and that activated proteases hydrolyze the proteins of contractile machinery, resulting in contractile dysfunction. This hypothesis was partially supported by the fact that a protease inhibitor, leupeptin, protected myocardium against stunning.¹⁷ Although this result suggests the participation of proteases in the mechanism of stunning, the target of activated proteases has not been clarified. In the present study, we identify myofilament proteins susceptible to injury during ischemia and reperfusion and elucidate the relation between the change of these proteins and the decreased contractility after reperfusion.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
The experimental preparation has been described previously.¹⁷ Briefly, male Hartley guinea pigs (300 to 350 g of body weight, Charles River Japan Inc, Yokohama, Japan) were anesthetized with sodium pentobarbital (50 mg/kg IP, Abbott Laboratories) and heparinized (1000 U IP, Shimizu Co). After rapid excision of the heart, the aorta was cannulated and retrogradely perfused with HEPES-buffered Tyrode's solution (mmol/L: NaCl 130, KCl 5, MgCl₂ 1, HEPES 5, CaCl₂ 2, glucose 10, and sodium acetate 20, pH 7.4) bubbled with 100% O₂ at 37°C. Heart rate was maintained at 200 to 240 bpm by right ventricular pacing with an electrode connected to a stimulator (SEN-3301, Nihon-Koden). A latex balloon tied to the end of a polyethylene tube was passed into the left ventricle through the mitral valve and connected to a pressure transducer (TP-300T, Nihon-Koden). The balloon was filled with water to an end-diastolic pressure of 2 to 10 mm Hg and then kept isovolumic throughout the experiment. Perfusion pressure was monitored at the cannulation point of the aorta. Left ventricular pressure and perfusion pressure were recorded with a direct-writing recorder (WR3001, Watanabe Instruments Co). Developed pressure was defined as the difference between peak-systolic pressure and end-diastolic pressure. After 20 to 30 minutes of stabilization, the coronary flow rate, controlled by a peristaltic pump, was adjusted such that perfusion pressure equaled 75 to 85 mm Hg. Once adjusted, the coronary flow rate was kept constant throughout the experiment except during the ischemic period. During ischemia, aortic inflow was totally interrupted by shutting off the peristaltic pump and by cross-clamping the perfusion line. After the period of global ischemia, hearts were reperfused for 20 minutes, which was sufficient for developed pressure to recover to a new steady state. Pacing was discontinued during ischemia and for the first 10 minutes of reperfusion.

To reperfuse myocardium with 0 mmol/L [Ca]_o solution, a perfusate containing EGTA (0.3 mmol/L) substituted for CaCl₂ was used. For treatment with leupeptin, hearts were perfused with solution containing leupeptin (50 µmol/L, Sigma Chemical Co) for 10 minutes before ischemia and during reperfusion. Leupeptin was first dissolved into dimethyl sulfoxide (DMSO) to 10% (wt/wt); thus, the perfusate contains 0.025% (wt/vol) DMSO. Although DMSO can scavenge free radicals, this concentration of DMSO had no effect against stunning.¹⁷ A cell-permeant protease inhibitor (calpain inhibitor I, 100 µmol/L, Suntory Institute for Biomedical Research) was also used in a similar protocol. All animal experiments were approved by the Committee for Animal Experimental Research of Osaka University Medical School.

Purification of Myofibrils
After the measurement of hemodynamic parameters, hearts were excised from the cannula and placed on buffer at 4°C. The left ventricle was cut off from the heart and weighed. The myofibrillar fraction was purified by the method of Solaro et al.¹⁸ The muscle of the left ventricle was immersed in 4 vol of 0.3 mol/L sucrose containing 10 mmol/L imidazole (pH 7.0) and protease inhibitors (30 µmol/L leupeptin, 15 µmol/L pepstatin, and 1 mmol/L phenylmethylsulfonyl fluoride), homogenized with a Potter-Elvehjem homogenizer set at 4000 rpm for 60 seconds, and then centrifuged at 18 000g for 20 minutes. The pellet from this spin was resuspended in standard buffer solution (mmol/L: KCl 60, imidazole 30, and MgCl₂ 2, along with protease inhibitors, pH 7.0) and centrifuged for 15 minutes at 750g. This procedure was repeated four times. The pellet was washed with standard buffer containing 2 mmol/L EGTA after two washes with standard buffer containing 1% Triton X-100. The pellet was then washed with standard buffer four times. The final precipitate was suspended in standard buffer. The protein concentration of each sample was measured by the Lowry method.

Electrophoresis and Immunoblotting
Myofibrillar proteins were separated with SDS and 12% or 5% polyacrylamide gels by the method of Laemmli.¹⁹ The gels were stained with Coomassie blue. Duplicate gels were transferred to polyvinylidene difluoride sheets (Millipore) at 2 mA/cm² for 60 minutes. After protein transfer, the sheets were divided into two sections: One was stained with amido black to reveal total protein content. Another was subjected to staining by antibody according to Towbin et al²⁰ ; the section of the sheet was incubated in Tris-buffered saline (10 mmol/L Tris-HCl, 150 mmol/L NaCl, and 0.05% Tween-20, pH 8.0) containing 5% nonfat dry milk to prevent subsequent nonspecific antibody binding. The sheet was then incubated for 2 hours with buffer containing monoclonal antibody against -actinin (EA-53 clone, diluted 1:2000, Sigma), desmin (DE-U-10 clone, diluted 1:6000, Sigma), or nonerythroid -spectrin (diluted 1:250, Chemicon International Inc). Alkaline phosphatase–linked anti-mouse IgG (H+L) antibody (diluted 1:3000, Bio-Rad Laboratories) was used to localize the primary antibody. Color development was carried out in development buffer (mmol/L: Tris-HCl 100, NaCl 100, and MgCl₂ 5, pH 9.5) containing nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.

Immunofluorescence Microscopy
For immunohistochemical study, the hearts were cut into pyramidal sections. Sections of the mid left ventricle were mounted on stubs with O.T.C. compound (Tissue-Tek, Miles Inc) and rapidly frozen by hexane cooled to -80°C. The tissues were sectioned in cross-sectional planes (2 µm) with a cryostat (model HM500MOK, Microm Co) and placed on gelatin-coated slides. The sections were incubated with antibodies against desmin (diluted 1:100), -actinin (diluted 1:100), -spectrin (diluted 1:50), or -sarcomeric actin (5C5 clone, diluted 1:200, Sigma) for 2 hours, followed by washing with PBS three times and incubation with fluorescein isothiocyanate–labeled anti-mouse IgG antibody (diluted 1:50, Wako Pure Chemical Industries, Ltd) for 1 hour. After further washes, the sections were visualized on fluorescence microscopy (models BX50 and BX-FLA, Olympus Optical Co Ltd). For quantification of the area stained with these antibodies, image analysis was performed on a laser scanning microscope equipped with an image-processing system (LSM 410, Zeiss).

The area stained was quantified as follows: The section was observed at x100 magnification. Each view was transferred to the image-processing system. The intensity of the unstained area was lower than that of normally stained area but higher than in the nonmyocardial space. First, the cutoff level of intensity was set to exclude the nonmyocardial space, and the area of myocardium in the view was measured. Then, the unstained area was excluded by elevation of the cutoff level, and the normally stained area in the view was measured. The prepared specimen was then slid a little to obtain another view without overlap with the previous view and quantified. Fifteen to 30 views were obtained in each section. The sum of the normally stained area in each view was normalized by the sum of the total area of myocardium. This value was defined as percent stained area.

Statistical Analysis
Data are presented as mean±SEM. Statistical analysis was performed by ANOVA with Tukey's test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Changes in Myofibrillar Protein in Myocardium Reperfused After Prolonged Ischemia
To find myofibrillar proteins susceptible to ischemia and reperfusion, we first hypothesized that proteins that remain intact in myocardium reperfused after a long period of ischemia could be excluded from the candidates. Thus, we examined myocardium reperfused after 60-minute global ischemia at 37°C. Fig 1 shows the myofibrillar proteins separated by polyacrylamide gel (12% and 5%) electrophoresis (SDS-PAGE). In 12% gel (Fig 1c), the bands corresponding to 55 and 100 kD were reduced in reperfused hearts (lane I) compared with nonischemic control (lane C), whereas the band corresponding to 255 kD was reduced in 5% gel (Fig 1a). As shown in Fig 1b and 1d, Western blot analysis demonstrated that the bands corresponding to 55, 100, and 255 kD reacted with antibodies to desmin, -actinin, and -spectrin, respectively. These bands in Western blots were also reduced in the reperfused hearts (lane I) compared with the nonischemic control hearts (lane C). In reperfused hearts, there are several bands corresponding to molecular weights between 100 and 200 kD that were reduced, and a new band appeared near desmin. However, these bands were unidentifiable. In contrast, in hearts reperfused after 60-minute ischemia, major bands of myofibril, including actin, myosin heavy chain, myosin light chain I and II, troponin T and I, and tropomyosin, showed no significant change. These results suggest that desmin, -actinin, and spectrin are more susceptible to disruption during ischemia and reperfusion than other major myofibrillar proteins. However, it should be noted that this approach could not detect the changes in proteins that are present in only small amounts nor target the proteins not in the myofibril.

View larger version (42K): [in this window] [in a new window]   Figure 1. SDS-PAGE and immunoblotting of myofibrillar fraction of the nonischemic control heart (C) and the heart reperfused after 60-minute ischemia (I). a, 5% polyacrylamide gel stained with Coomassie blue. b, The transferred sheet stained by anti–-spectrin antibody (Sp) and by amido black (AB). c, 12% polyacrylamide gel. d, The transferred sheet stained by anti–-actinin antibody (A), anti-desmin antibody (Des), and AB.

Localization of Desmin, -Actinin, and Spectrin in Nonischemic Control Heart
The previous results obtained from 60-minute experiments suggest that desmin, -actinin, or spectrin is susceptible to ischemia and reperfusion. However, stunned myocardium (ie, myocardium reperfused after 15-minute ischemia) did not show any clear change in the bands of desmin, -actinin, or spectrin after SDS-PAGE (data not shown). Thus, we used a more sensitive method to detect possible changes, namely, an immunohistochemical approach. First, we checked the localization of these cytoskeletal proteins in normal myocardium. In normal hearts, desmin, -actinin, and spectrin were stained homogeneously in the myocardium. Desmin stained in a striped pattern in the longitudinally sectioned myocyte (Fig 2a). The boundary lines of the myocyte also stained well, indicating that desmin is located in the Z bands and the intercalated disks (Fig 2b). -Actinin was also identified as stripes in the longitudinally sectioned myocyte (Fig 2c and 2d), consistent with the previous reports that -actinin is located in Z bands.²¹ Spectrin was stained in stripes like -actinin and was also visualized at the membrane (Fig 2e and 2f). Spectrin is known to be a membrane-associated cytoskeletal protein. The striped staining in the myocardium by anti-spectrin antibody indicates that spectrin is located in the T tubules and/or Z bands of myofilaments, as reported by Isayama et al.²²

View larger version (160K): [in this window] [in a new window]   Figure 2. Immunofluorescent microscopic observation of nonischemic control myocardium at original magnifications of x100 (a, c, and e) and x400 (b, d, and f). Panels show staining with antibody against desmin (a and b), -actinin (c and d), and spectrin (e and f), respectively.

Distribution of Cytoskeletal Proteins in Stunned Myocardium
In isolated perfused guinea pig hearts, reperfusion after 15- or 20-minute ischemia increased the end-diastolic pressure and decreased the systolic pressure compared with initial control, resulting in a decrease of developed pressure (71.0±5.1% of initial control developed pressure in the group with 15-minute ischemia, n=9, P<.05; 38.9±6.2% of control in the group with 20-minute ischemia, n=4, P<.05). Staining with hematoxylin and eosin demonstrated no distinguishable feature in stunned myocardium compared with nonischemic control myocardium. Nevertheless, immunohistochemical examination revealed inhomogeneous displacement of the cytoskeletal proteins in stunned myocardium. In hearts subjected to 15-minute ischemia and then reperfused, the staining by antibodies against desmin (Fig 3a), -actinin (Fig 3b), or spectrin (Fig 3c) was normal in most of the myocardium, but there were several small areas unstained by these antibodies. Furthermore, the area not stained by one antibody was also resistant to staining by other antibodies, as shown in Fig 3. These results indicate that the cytoskeletal proteins had disappeared in some of myocytes in stunned myocardium and that such a disappearance of cytoskeletal protein occurred in the same myocytes. In the unstained myocytes, anti-desmin antibody stained neither Z bands nor the intercalated disks in the longitudinal section (Fig 4b). On the other hand, Z bands were stained lightly with the anti–-actinin antibody (Fig 4d), and the Z bands and membrane were also stained weakly with the anti-spectrin antibody (Fig 4f), indicating that the structure of Z bands and membrane was maintained even in the damaged myocyte. The percent stained area evaluated using the anti-desmin antibody was 79.6±3.6%.

View larger version (96K): [in this window] [in a new window]   Figure 3. Immunofluorescent microscopic observation with antibody against desmin (a), -actinin (b), and spectrin (c) of the consecutive sections of the heart reperfused after 15-minute ischemia (original magnification x100).

View larger version (142K): [in this window] [in a new window]   Figure 4. Immunofluorescent microscopic observation of the same sections shown in Fig 3 at higher magnification (original magnification x400). Panels a, c, and e are normally stained area, whereas panels b, d, and f are unstained area, with antibody against desmin (a and b), -actinin (c and d), and spectrin (e and f), respectively.

To elucidate whether or not these changes were artifactual, six hearts were perfused for 45 minutes with normal and well-oxygenated perfusate. No significant change was observed in systolic and diastolic pressure after 45 minutes of perfusion; the developed pressure at the end of 45-minute perfusion was 104.3±6.4% of the initial control. The sections from the normally perfused hearts demonstrated that a very small part of the myocardium was unstained with these antibodies. However, the unstained area was limited to the papillary muscles; this lack of staining may have been caused by damage induced by the balloon inserted into the left ventricle. The percent stained area was 96.5±0.7% in nonischemic control hearts.

In the hearts reperfused after 20-minute ischemia, similar changes in the staining with antibody against desmin, -actinin, or spectrin were observed, but the percent stained area (61.0±8.2%, n=4, P<.05) was even lower than that in the hearts reperfused after 15-minute ischemia.

Change of Stained Area in Different Procedures
Fig 5 summarizes the percent stained area in the different experimental conditions. The percent stained area was significantly decreased in the groups of 15-minute ischemia (P<.05) and 20-minute ischemia (P<.01) compared with the nonischemic control group. To elucidate the protective effect of a potent protease inhibitor, leupeptin, against the change in staining, six hearts were perfused with a solution containing 50 µmol/L leupeptin for 10 minutes before 15-minute ischemia and during 20-minute reperfusion. The percent recovery of developed pressure was significantly higher than that of untreated stunned hearts (94.9±3.7%, P<.05). The percent stained area in leupeptin-treated hearts was also significantly greater than that in untreated stunned hearts (94.2±2.6%, P<.05). Similar results were obtained in the hearts treated by calpain inhibitor I; the percent recovery of developed pressure (89.4±3.7%) and the percent stained area (89.2±1.4%) were significantly higher than those in untreated stunned hearts (P<.05).

View larger version (20K): [in this window] [in a new window]   Figure 5. The percent stained area in each experimental group.

To elucidate whether the change of these cytoskeletal proteins is caused by ischemia itself or by reperfusion, three hearts were subjected to 15-minute ischemia but not reperfused. In the sections from these hearts, the distributions of desmin, -actinin, and spectrin were the same as in nonischemic control hearts, and the percent stained area (99.0±1.0%) was identical to that in nonischemic control hearts. These results indicate that the disappearance of these cytoskeletal proteins occurs during reperfusion and not during ischemia.

The contribution of Ca²⁺ overload to the change of these cytoskeletal proteins was also evaluated. Three hearts were subjected to 15-minute ischemia but reperfused with a solution containing 0.3 mmol/L EGTA and no Ca²⁺. There were no distinguishable changes in the distribution of desmin, -actinin, and spectrin compared with the distribution in nonischemic control hearts, and the percent stained area (99.6±0.4%) was also identical to that in nonischemic control hearts. Coupled with the previous results, Ca²⁺ overload during reperfusion appears to mediate these changes in cytoskeletal proteins.

Relation Between Contractile Recovery and Percent Stained Area
The relation between the percent stained area and the percent recovery of developed pressure obtained from nonischemic control hearts and the hearts reperfused after 15- or 20-minute ischemia is depicted in Fig 6. There was a significant correlation between these two parameters (r=.82, P<.001). Furthermore, when we plotted the percent recovery of developed pressure and the percent stained area in the hearts treated with protease inhibitors, the data agreed well with the regression line obtained above (Fig 6). These results suggest the strong correlation between the functional recovery and the disappearance of cytoskeletal proteins in the stunned myocardium.

View larger version (15K): [in this window] [in a new window]   Figure 6. The relation between the percent stained area and the percent recovery of developed pressure (DP) obtained from nonischemic control hearts and the hearts reperfused after 15- or 20-minute ischemia (). The data obtained from the hearts treated with leupeptin () or calpain inhibitor I () are superimposable in the regression line.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Change of Cytoskeletal Proteins in Stunned Myocardium
Despite the profound postischemic depression in contractile function, the structure of stunned myocardium has been reported not to be damaged histologically, as assessed by light microscopy.²³ Furthermore, electron microscopic examination revealed only subtle changes, ie, relaxation of myofibrils as manifested by wide I bands, glycogen depletion, mild intermyofibrillar edema, rare cytoplasmic vacuoles, chromatin clumping of the nuclei, and twisted or unfolded configuration of mitochondrial cristae.²⁴ In contrast, immunohistochemical approach in the present study revealed the damages in cytoskeleton near Z bands, ie, changes in desmin, -actinin, and spectrin in 20% of myocytes in stunned myocardium. However, the myocardium that exhibited cytoskeletal damage could not be distinguished from normally staining myocardium by routine light microscopy.

The areas unstained with anti-desmin antibody always coincided with the areas unstained with anti–-actinin antibody or anti-spectrin antibody, suggesting that the susceptibility of these cytoskeletal proteins to ischemia and reperfusion is localized in the same lesion. The hearts subjected to ischemia without reperfusion displayed minimal changes in cytoskeletal proteins, as did hearts reperfused with Ca²⁺-free solution. Furthermore, staining by anti-actin antibody showed little change in the area where anti-desmin antibody did not stain (Fig 7). These results indicate that the change of these cytoskeletal proteins is specific for stunning and is mediated by Ca²⁺ overload during reperfusion; intracellular enzymes activated by Ca²⁺ overload during reperfusion may degrade -actinin, desmin, and spectrin themselves or binding proteins that bind and localize these cytoskeletal components. Partial inhibition of the degradation by protease inhibitor also supports this hypothesis.

View larger version (163K): [in this window] [in a new window]   Figure 7. Immunofluorescent microscopic observation (original magnification x100) with antibodies against actin (a) or desmin (b) of the consecutive sections of stunned heart.

Enzymes Participating in the Change of Cytoskeletal Proteins During Reperfusion
Although our results strongly indicate that Ca²⁺ overload triggers the change of the cytoskeletal proteins, it is not clarified which enzyme participates in this reaction. We previously reported that the protease inhibitor leupeptin protected the myocardium against stunning.¹⁷ In the present study, leupeptin or calpain inhibitor I partially prevented not only the decline of contractility but also the damage of cytoskeletal proteins. The relation between the percent recovery of developed pressure and the percent stained area was identical to that obtained from the untreated hearts. Recent study directly indicates that not only protease inhibitor I but also leupeptin applied extracellularly permeate into cytosol.²⁵ Thus, our results suggest that the activation of intracellular protease, which can be inhibited by leupeptin and calpain inhibitor I, may be primarily responsible for the change in proteins in stunned myocardium. Calpain is a protease activated by Ca²⁺ located in the cytosol and localizes especially near the Z bands in myocytes.²⁶ It has been reported that calpain dislocates -actinin from Z bands and digests desmin and spectrin.^{27 28 29} It is also reported that exogenously administered calpain shifts the Ca²⁺ responsiveness of the myofilaments.³⁰ Thus, calpain is one prominent candidate among the proteases that may be responsible for the change of cytoskeletal proteins during reperfusion.

Recently, Yoshida et al³¹ reported that calpain is activated in myocardium reperfused after ischemia and that spectrin (calspectin or fodrin) is digested by the protease in reperfused myocardium. Their results are consistent with our observation, but it should be noted that our data indicate that not only spectrin but also other myofilament-related cytoskeletal proteins disappeared in stunned myocardium. As indicated below, it is difficult to relate contractile dysfunction with changes in cytoskeletal proteins other than -actinin.

Role of Cytoskeletal Proteins in Force Generation
The size of the myocardium showing the changes in the cytoskeletal proteins was well correlated with the recovery of the developed pressure after reperfusion. This result suggests that the cytoskeletal changes in myocytes may cause contractile dysfunction and is consistent with previous observations indicating that stunning is caused by impairments in the Ca²⁺ responsiveness of myofilaments^{5 32} rather than the lack of activator Ca²⁺.² However, the role of these cytoskeletal proteins in force generation is still unknown. -Actinin is located in the Z bands connecting the opposite ends of the actin filaments.²² Thus, when -actinin is removed, the force generated by actin and myosin may not be effectively transmitted at the ends of the cell. In vitro study has demonstrated that -actinin enhances the activation of myosin ATPase,³³ suggesting that the loss of -actinin from myofilaments may inhibit the myosin ATPase activity and result in decreased contractility. Desmin is an intermediate filament of myocytes located in the intermyofilament space, contacting with Z disks and linking mitochondria and Z disks to neighboring myofilament.³⁴ Although desmin has been proposed to play a role in maintaining cell structure as a microfilament, it is not clear whether the change of desmin influences myocardial cell function. Spectrin is a membrane cytoskeletal protein localized to the plasma membrane and is supposed to have a role in the maintenance of cell structural integrity.³⁵ In myocardial cells, spectrin localized not only to the sarcolemma but also to the Z bands associated with T tubules.³⁶ Although spectrin may act as a conduit for T-tubule guidance³⁷ in the process of myocardial cell development, the precise role of spectrin in the myocardial cell is still unknown. Thus, under present knowledge, only the loss of -actinin from myofilaments could explain the decline of force generation in stunned myocardium.

Limitation of the Present Study
In the present study, we focused on desmin, -actinin, and spectrin as the candidate proteins in myofibril involved in stunned myocardium. This was based on the observation by SDS-PAGE analysis of myofibril of the hearts reperfused after 60-minute ischemia, demonstrating the decrease in the contents of -actinin, desmin, and spectrin. SDS-PAGE analysis is not enough to detect the slight change in proteins or to analyze the extremely large or small proteins. Thus, it cannot be denied that myofibrillar proteins other than -actinin, desmin, and spectrin are also modified by ischemia and reperfusion. Further work will be necessary to show a causal relationship between stunning and the changes in cytoskeletal proteins.

In conclusion, the present results indicate that protease is activated by Ca²⁺ overload during reperfusion, resulting in the disappearance of desmin, -actinin, and spectrin in stunned myocardium. These changes in cytoskeletal proteins were mild, but the extent of the myocardium exhibiting these changes in the cytoskeleton significantly correlated with the recovery of developed pressure after reperfusion. Activation of calpain is a possible mechanism for the changes in myocardial content of cytoskeletal proteins in stunning.

	Acknowledgments

 
This study was supported by a Grant-in-Aid for Scientific Research B (No. 05454270 to Dr Kusuoka) and Grant-in-Aid for Encouragement of Young Scientists (Nos. 05770465 and 06770492 to Dr Matsumura) of the Ministry of Education, Science, and Culture of Japan. We thank Drs Yoshitane Dohi, Atsushi Hirayama, and Kunimitsu Iwai for their suggestions on technical procedures and Suntory Institute for Biomedical Research for their kind gift of calpain inhibitor I.

Received October 17, 1995; accepted May 16, 1996.

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