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(Circulation Research. 1995;77:603-610.)
© 1995 American Heart Association, Inc.

Articles

Reperfusion of Rat Heart After Brief Ischemia Induces Proteolysis of Calspectin (Nonerythroid Spectrin or Fodrin) by Calpain

Ken-ichi Yoshida, Makoto Inui, Kazuki Harada, Takaomi C. Saido, Yoshihide Sorimachi, Tokuhiro Ishihara, Sei-ichi Kawashima, Kenji Sobue

From the Departments of Legal Medicine (K.Y., K.H., Y.S.) and Pathology (T.I.), Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan; the Department of Neurochemistry and Neuropharmacology, Biomedical Research Center, Osaka University Medical School (M.I., K.S.), Suita, Osaka, Japan; and the Department of Molecular Biology, Tokyo (Japan) Metropolitan Institute of Medical Science (T.C.S., S.K.).

Correspondence to Ken-ichi Yoshida, MD, Department of Legal Medicine, Yamaguchi University School of Medicine, Ube, Yamaguchi 755, Japan.

	Abstract

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Abstract Rat myocardium expresses the 240- and 235-kD polypeptides antigenically related to - and ß-subunits of brain calspectin (nonerythroid spectrin or fodrin), respectively. In the subcellular fractions of the myocardium, -calspectin was found in the 600g, 10 000g, and 100 000g pellets, whereas ß-calspectin was localized to the 10 000g pellet. On the basis of the Na⁺,K⁺-ATPase activity and the contents of a gap junction protein, the sarcolemma was distributed to the 10 000g and 100 000g pellets, and the intercalated disks were enriched in the 10 000g pellet. Both - and ß-calspectin were proteolyzed by calpain in vitro. The two subunits were also proteolyzed in vivo, when the rat hearts underwent 10 to 60 minutes of global ischemia followed by 30 minutes of reperfusion. The reperfusion following the ischemia induced the proteolysis of -calspectin in the 10 000g and 100 000g pellets, producing the 150-kD fragment. A synthetic calpain inhibitor, calpain inhibitor-1, suppressed the degradation of calspectin in vivo, which indicates that calpain is responsible for the reperfusion-induced proteolysis of calspectin. The inhibitor also improved myocardial stunning. Immunohistochemical study revealed that the proteolysis of -calspectin occurs at the intercalated disks and the sarcolemma after postischemic reperfusion, in accord with the biochemical data. These results suggest that degradation of calspectin partly accounts for the contractile failure of the myocardium after postischemic reperfusion by disrupting the membrane skeleton and the intercalated disks.

Key Words: ischemia • calspectin • fodrin • calpain • stunning

	Introduction

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Spectrin is a major constituent of the membrane skeleton, forming a two-dimensional meshwork under the erythrocyte membranes.^{1 2 3} A family of spectrinlike proteins, which is also a prominent component of the membrane skeleton, was identified in most of nonerythroid cells. The latter protein is referred to as calspectin,^{4 5 6 7} fodrin,⁸ or nonerythroid (or brain) spectrin.⁹ Calspectin, as well as spectrin, is a rod-shaped protein and consists of - and ß-subunits. In mammalian hearts, it has been shown by immunofluorescent staining that calspectin is localized to the sarcolemma, the intercalated disks, and Z bands.¹⁰

In the rat brain, postischemic reperfusion was shown to degrade calspectin, which is considered to initiate plasma membrane disruption, resulting in irreversible cell injury.^{11 12 13 14} Calpain, a Ca²⁺-activated neutral protease, has been considered to be involved in the postischemic proteolysis of calspectin. In the canine heart, ischemia or postischemic reperfusion was shown to diminish the immunostaining for vinculin and tubulin.^{15 16 17} Recently, Iizuka et al¹⁸ reported that calspectin was proteolyzed in anoxic cardiomyocytes. We recently showed that ischemia and reperfusion enhanced calpain activity in the perfused rat heart and proteolyzed several proteins in the particulate fractions by increasing Ca²⁺ influx.^{19 20}

In the present study, we found that calpain proteolyzed calspectin at the sarcolemma and intercalated disks in the perfused rat heart after postischemic reperfusion and showed that calspectin was proteolyzed after a much shorter duration of ischemia followed by reperfusion than other cytoskeletal proteins that have been reported to be degraded. We also found that a calpain inhibitor suppressed calspectin proteolysis and myocardial stunning.

	Materials and Methods

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Materials
A synthetic calpain inhibitor, calpain inhibitor-1, was obtained from Nakarai Tesque. The antibody to connexin43 (produced by Drs Laird and Revel²¹ ) was a generous gift of Dr Fujikura (Department of Anatomy, Yamaguchi University School of Medicine). µ-Calpain was partially purified from the hemolysate of human erythrocytes by DEAE cellulose and phenyl-Sepharose chromatography as described previously (specific activity, 1 U/mg protein).¹⁹ Polyclonal antibodies against the -subunit of bovine brain calspectin (-calspectin) were raised in rabbits and purified as previously described.⁷ Polyclonal antibodies against the proteolytic 150-kD fragment of human brain -calspectin were raised in rabbits by using the antigenic 5-mer peptide designed to match the N-terminal sequence of the 150-kD fragment¹⁴ and purified as described previously. Monoclonal antibody against the ß-subunit of bovine brain calspectin (ß-calspectin) was a generous gift from Nippon-Shinyaku.

Perfusion Procedure
Wistar rats weighing 180 to 250 g were used, except for the functional study, in which rats of 350 g were used. The hearts, after having been excised under anesthesia with sodium pentobarbital (1 mg/kg IP), were perfused with modified Krebs-Henseleit (KH) buffer (mmol/L: NaCl 124, NaHCO₃ 24.5, KH₂PO₄ 1.2, KCl 4.7, CaCl₂ 2.5, MgSO₄ 1.2, glucose 5.5, and sodium pyruvate 2.0, pH 7.4) gassed with 95% O₂/5% CO₂ at 80 cm H₂O pressure at 37°C by the Langendorff procedure, as reported previously.¹⁹ After initial perfusion for 10 to 20 minutes, global ischemia was produced by incubating the hearts at 37°C in plastic bags with a small amount of KH buffer without oxygenation. The hearts were reperfused with the oxygenated KH buffer. Calpain inhibitor-1 was dissolved in dimethyl sulfoxide (DMSO) at 100 mmol/L. Calpain inhibitor-1 at 10 to 100 µmol/L (in 0.01% to 0.1% DMSO) was perfused during the last 5 minutes of preischemic perfusion and throughout the postischemic reperfusion. In the experiments to assess the effect of calpain inhibitor-1, the same concentration of DMSO was perfused as a control. DMSO at 0.1% had no significant effect on cardiac function and on the proteolysis of calspectin. In some experiments, CaCl₂ was omitted or KCl was increased to 20 mmol/L in the buffer during reperfusion. In the functional study, the heart rate, ventricular pressure, and its first derivative (dP/dt) were continuously monitored through a latex balloon connected to a pressure transducer that was attached to multichannel polygraph (RMP-6004, Nihon-Koden). The balloon was filled with water to an end-diastolic pressure of 10 mm Hg. The three parameters were recorded with a direct-writing recorder (Thermal Arraycorder, Graphtec).

Subcellular Fractionation
The hearts were homogenized in 2 vol TE buffer (mmol/L: Tris-HCl 20 [pH 7.4], EGTA 1, NaN₃ 5, and ß-mercaptoethanol 10) containing 50 mmol/L NaCl, 20 µmol/L leupeptin, 0.2 mmol/L phenylmethylsulfonyl fluoride, and 150 nmol/L pepstatin A with a Polytron homogenizer four times each for 30 seconds at maximum speed. Leupeptin (20 µmol/L) and calpain inhibitor-1 (10 µmol/L) were included throughout the fractionation procedure. In some experiments, frozen hearts were used. The homogenate was mixed with 4 vol of the same buffer and centrifuged at 600g for 15 minutes at 4°C. The supernatant was centrifuged at 10 000g for 20 minutes, and then the supernatant was further centrifuged at 100 000g for 90 minutes at 4°C. The pellet fractions were suspended in the electrophoresis buffer (125 mmol/L Tris-HCl [pH 6.8], 1 mmol/L EGTA, 2% SDS, and 5% ß-mercaptoethanol) containing the protease inhibitors by brief sonication. The proteolysis of calspectin was analyzed in the 600g, 10 000g, and 100 000g pellets. The rat brain 100 000g pellet was isolated by the same procedure. The human erythrocyte ghost was obtained as previously described.^{1 2}

To characterize the particulate fractions, the contents of connexin43 antigen (a marker for intercalated disks)²¹ was determined by densitometric scans of the immunoblot (see below). The Na⁺,K⁺-ATPase activity (a marker for sarcolemma) was measured spectrophotometrically as described by Jones et al.²² After having been exposed to various concentrations of SDS, the samples were incubated at 37°C in the mixture containing (mmol/L) imidazole-HCl 100, MgCl₂ 6, EGTA 1, NaCl 150, KCl 20 [pH 7.4], and ATP 6 either in the presence or absence of 1 mmol/L ouabain. The Na⁺,K⁺-ATPase activity was expressed as the ouabain-sensitive enzyme activity per milligram protein at the optimum SDS concentration.

In Vitro Proteolysis
The myocardial 10 000g pellet was prepared as described above without proteinase inhibitors for calpain. The pellet (1.5 mg/mL) in 20 mmol/L Tris-HCl (pH 7.4), 0.2% Triton X-100, and 10 mmol/L ß-mercaptoethanol was incubated with erythrocyte µ-calpain (0.04 U/mL) at 25°C for 3, 10, 30 and 120 minutes in the presence of 2 mmol/L CaCl₂ and 0.8 mmol/L EGTA. At the end of incubation, leupeptin and EGTA were added at final concentrations of 20 µmol/L and 10 mmol/L, respectively, and then mixed with the electrophoresis buffer, followed by heating for 2 to 3 minutes in boiling water.

Electrophoresis and Immunoblotting
SDS-PAGE (polyacrylamide gel, 6.5%) and immunoblotting were performed by the methods of Laemmli²³ and Towbin et al,²⁴ respectively. In the experiments for the resolution of calspectin subunits (Fig 1) and for the immunoblotting of connexin43, 5% and 12.5% gels were used, respectively. Nonfat dry milk (5%) was included in the Tris-buffered saline (150 mmol/L NaCl, 10 mmol/L Tris-HCl [pH 7.4], and 0.05% Tween 20) when it was used for blocking and antibody incubation. Some gels were stained with Coomassie brilliant blue. The antigens were detected by the luminescence method (ECL Western blotting detection kit, Amersham) with peroxidase-labeled protein A. To determine the amounts of calspectin or connexin43, various amounts of each fractions were applied to a gel. After immunoblotting, the film was scanned by densitometric scanner (PAN-802, Johkoh Co) at 570 nm. The amounts of the proteins in each fraction were quantified from the intensity of the bands, which had a linearity to the amounts of the fraction applied to the gel. The intensity of bands was expressed as an arbitrary unit. The statistical difference was evaluated by ANOVA with Fisher's posthoc analysis.

View larger version (18K): [in this window] [in a new window]   Figure 1. Immunoreactive polypeptides in the myocardial subfractions with anti-calspectin antibodies. The rat hearts were fractionated as described in "Materials and Methods." The 600g pellet (lane 1), 10 000g pellet (lane 2), 100 000g pellet (lane 3), and 100 000g supernatant (lane 4) of rat myocardium and 5 µg of 100 000g pellet of rat brain (lane 5) and human erythrocyte ghost (lane 6) were subjected to SDS-PAGE and stained with Coomassie brilliant blue (CBB) (left) or immunostained either with a polyclonal antibody against -calspectin (middle) or with a monoclonal antibody against ß-calspectin (right). Equal amounts of the fractions per wet weight tissue were applied, except for the 600g pellet (1/20), for comparison. The 240- and 235-kD polypeptides of the myocardium reacted with the antibodies against - and ß-calspectin, respectively.

Histochemical Study
Rat hearts were fixed with 2% paraformaldehyde buffered with 0.1 mol/L sodium phosphate (pH 7.4) overnight at 4°C, embedded in paraffin, and sectioned. After having been deparaffinized, the sections were incubated either with antibody against -calspectin, ß-calspectin, or the 150-kD product of -calspectin in Tris-buffered saline for 1 to 3 hours at room temperature and then immunostained by the avidin-biotin-peroxidase complex (ABC) method of Hsu et al²⁵ with a Vectastain ABC kit (Vector Laboratories). The peroxidase label was visualized by exposing the sections to diaminobenzidine. Other sections of the same specimens were stained with hematoxylin and eosin.

	Results

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The distribution of calspectin in the rat myocardium was examined with the subcellular fractions by using antibodies against - and ß-calspectin. The polyclonal antibody against -calspectin reacted with a 240-kD band in the 600g, 10 000g, and 100 000g pellets but not in the 100 000g supernatant; ß-calspectin was detected as a 235-kD band by the monoclonal antibody only in the 10 000g pellet (Fig 1). The 600g, 10 000g, and 100 000g pellets contained 79%, 11.5%, and 6.5% of the total -calspectin in the heart, respectively. No immunoreactivity was observed in the rat myocardium with our polyclonal antibodies against human erythrocyte spectrin (data not shown). In our subcellular fractions, the sarcolemma was distributed in both the 10 000g and 100 000g pellets on the basis of Na⁺,K⁺-ATPase activity (Table). From the contents of a gap junction protein (connexin43), the gap junctions (intercalated disks) were enriched in the 10 000g pellet (Table). The 600g pellet contained myofibrils, nuclei, and few unbroken cells (not shown). In the following experiments, we analyzed calspectin in the pellet fractions from frozen hearts, since freezing and thawing made no significant difference in the antigen distribution.

View this table: [in this window] [in a new window]   Table 1. Na+,K+-ATPase Activity and the Contents of Connexin43 in Myocardial Subfractions

To determine whether calspectin in the rat myocardium can be a substrate of calpains, the in vitro proteolysis of calspectin was examined. When the 10 000g pellet fraction was incubated with µ-calpain, both - and ß-calspectin decreased with time (Fig 2). The antibody against -calspectin revealed a decrease in the 240-kD band with a concomitant increase in the 150-kD band (sometimes doublets), which was also recognized by the antibody against the 150-kD fragment of -calspectin (Fig 2). The latter antibody did not recognize the intact -calspectin (240-kD). m-Calpain showed almost the same time course of proteolysis as did µ-calpain, when the same activity of the enzymes was added to the fraction (data not shown).

View larger version (30K): [in this window] [in a new window]   Figure 2. In vitro proteolysis of calspectin in 10 000g pellet by µ-calpain. The 10 000g pellet fraction (1.5 mg/mL) was incubated at 25°C with 0.04 U/mL of µ-calpain and then subjected to SDS-PAGE. The proteolysis of calspectin was analyzed by immunoblotting with antibodies against -calspectin (left), the 150-kD fragment of -calspectin (middle), and ß-calspectin (right). The antibody against -calspectin recognized both 240- and 150-kD polypeptides, whereas that against the 150-kD fragment recognized the 150- but not the 240-kD polypeptide.

The in vivo proteolysis of calspectin was studied by using an ischemia/reperfusion model. The rat hearts underwent various durations of ischemia followed by reperfusion for 30 minutes. In the 10 000g pellet, both - and ß-calspectin decreased with the time of ischemia (Fig 3, top). The rate of the proteolysis of ß-calspectin seemed to be faster than that of -calspectin both in vitro (Fig 2) and in vivo (Fig 3, top). The 150-kD fragment of -calspectin was produced after 10 minutes of ischemia followed by reperfusion. The quantitative analysis of the 150-kD product is shown in Fig 3, bottom. It should be noted that a short period of ischemia, such as 10 minutes, followed by reperfusion produced the proteolytic fragment (150 kD) of calspectin. Essentially, the same results were obtained with the 100 000g pellet (Fig 4). The proteolysis of calspectin in the 600g pellet was negligible after ischemia/reperfusion (not shown), so that we did not further analyze the fraction in the following experiments.

View larger version (68K): [in this window] [in a new window]   Figure 3. Effect of ischemia on the proteolysis of calspectin in 10 000g pellet. Top, The rat hearts were subjected to various durations (0 to 60 minutes) of ischemia followed by reperfusion for 30 minutes. The 10 000g pellet fraction was analyzed by immunoblotting with antibodies against -calspectin (left), the 150-kD fragment of -calspectin (middle), and ß-calspectin (right). Bottom, The amounts of the 150-kD fragment of -calspectin (mean±SEM) were determined by densitometric scans of the immunoblots as described in "Materials and Methods." *P<.05 vs control.

View larger version (59K): [in this window] [in a new window]   Figure 4. Effect of ischemia on the proteolysis of calspectin in 100 000g pellet. The 100 000g pellet was prepared from the same hearts used in Fig 3. The proteolysis of calspectin was analyzed by the antibodies against -calspectin (left) and the 150-kD fragment of -calspectin (right).

The proteolysis of calspectin was also examined after various durations of reperfusion following 20 minutes of ischemia. When the 10 000g pellet was analyzed, 20-minute ischemia (also ischemia up to 60 minutes [not shown]) without reperfusion generated only a small amount of the 150-kD fragment of -calspectin (Fig 5, top). However, postischemic reperfusion even for 1 or 3 minutes induced significant increase in the 150-kD product. Fig 5, bottom, depicts the densitometric determination of the amounts of the 150-kD fragment produced. Significant decrease in - and ß-calspectin was observed after 30 minutes of reperfusion (Fig 5, top).

View larger version (41K): [in this window] [in a new window]   Figure 5. Effect of reperfusion after ischemia on the proteolysis of calspectin. Top, The rat hearts underwent various durations (0 to 30 minutes) of reperfusion after 20 minutes of ischemia. The 10 000g pellet fraction was analyzed by immunoblotting with antibodies against - and ß-calspectin. Bottom, The amounts of the 150-kD fragment of -calspectin (mean±SEM) were determined by densitometric scans of the immunoblots with an antibody against the 150-kD fragment of -calspectin as described in "Materials and Methods." *P<.05 vs control.

To determine whether calpain is responsible for in vivo proteolysis of calspectin, we examined the effect of a synthetic inhibitor for calpain, calpain inhibitor-1, on the proteolysis of -calspectin. We chose calpain inhibitor-1 because it is more specific for calpain and more permeable to the cells than leupeptin²⁶ and because it is less deleterious to the perfused heart than other inhibitors with higher specificity (eg, calpeptin and E64d) (authors' unpublished data, 1994). When the amounts of the 150-kD fragment produced were compared in the 10 000g pellet fraction, calpain inhibitor-1 at concentrations of 10 and 100 µmol/L significantly inhibited the proteolysis of calspectin after 20 minutes of ischemia followed by reperfusion for 30 minutes (Fig 6). We further examined the effects of no Ca²⁺ or high KCl (20 mmol/L) during reperfusion on the proteolysis of calspectin, both of which arrested the heart. Reperfusion without Ca²⁺ diminished the -calspectin proteolysis, whereas reperfusion with high KCl did not (Fig 6).

View larger version (35K): [in this window] [in a new window]   Figure 6. Effect of a calpain inhibitor, reperfusion with no Ca2+, or high KCl on the proteolysis of calspectin after postischemic reperfusion. The rat hearts underwent 20 minutes of ischemia followed by reperfusion for 30 minutes. A synthetic calpain inhibitor, calpain inhibitor-1 (CAI) at 10 or 100 µmol/L (in 0.01% or 0.1% dimethyl sulfoxide [DMSO]), was perfused during the last 5 minutes of preischemic perfusion and throughout the postischemic reperfusion. To examine the direct effect of DMSO, 0.1% DMSO was perfused without CAI. When the hearts were reperfused with no Ca2+ or high KCl, CaCl2 was omitted or KCl was increased to 20 mmol/L during reperfusion. The proteolysis of calspectin was analyzed by immunoblotting of the 10 000g pellet with the antibody against the 150-kD fragment of -calspectin. The amounts of the 150-kD fragment were determined by densitometric scans and expressed as percentage of the 150-kD fragment (mean±SEM) in the sample after postischemic reperfusion without treatments such as CAI, no Ca2+, or high KCl. *P<.05 vs control.

The biochemical analyses in the present study suggest that calspectin in the rat myocardium is susceptible to reperfusion-induced proteolysis by calpain. To identify the sites of proteolysis, we performed an immunohistochemical study using the same antibodies. The antibody against -calspectin immunostained the intercalated disks, the sarcolemma, and the striations in the longitudinal section of the control myocardium (Fig 7A). Absorption of the antibody with the low-salt extract from the 600g pellet (rich in calspectin) before incubation with the sections diminished the staining (not shown). Reperfusion after the 20 minutes of ischemia altered neither the morphology nor the immunostaining for -calspectin (Fig 7B). After ischemia for 60 minutes followed by reperfusion, the myocytes became swollen and dissociated, whereas the immunostaining remained almost unchanged. It should be noted that our antibodies against -calspectin react with the 150-kD fragment as well as the 240-kD polypeptide. On the other hand, the antibody against the 150-kD fragment of -calspectin did not significantly stain the control myocardium (Fig 7D). It specifically reacted with the intercalated disks, the sarcolemma, and the striations after 20 minutes of ischemia followed by 30 minutes of reperfusion (Fig 7E). The transverse section, when it was stained with the anti–150-kD product antibody (Fig 7F), delineated the localization of proteolyzed calspectin at the cell circumference corresponding to the sarcolemma and the intercalated disks. Under the conditions used in the present study, the monoclonal antibody against ß-calspectin did not stain the specific structure of the myocardium (not shown), probably because the antigenicity was lost during the fixation and embedding.

View larger version (0K): [in this window] [in a new window]   Figure 7. Localization of -calspectin and the 150-kD product of -calspectin in the heart after postischemic reperfusion. The control heart (A and D) and the heart that underwent ischemia for 20 minutes (B, E, and F) or for 60 minutes (C) followed by reperfusion for 30 minutes were immunostained by an antibody against -calspectin (A, B, and C) or against the 150-kD product of -calspectin (D, E, and F). The antibody against -calspectin stained the intercalated disks, the sarcolemma, and the striations in the control heart (A). Ischemia for 20 minutes (B) or 60 minutes (C) followed by reperfusion for 30 minutes did not change immunostaining, whereas the cells became swollen and dissociated after 60 minutes of ischemia followed by reperfusion (C). The antibody against the 150-kD product of -calspectin did not stain a specific structure in the control heart (D) but reacted with the intercalated disks, the sarcolemma, and the striations after 20 minutes of ischemia followed by reperfusion (30 minutes) (E, longitudinal section; F, transverse section). Bar=10 µm.

To explore whether the proteolysis of calspectin alters the myocardial contractility during reperfusion, we examined the effect of calpain inhibitor-1 on left ventricular developed pressure (LVDP), its first derivative (dP/dt), and heart rate during preischemia, ischemia, and postischemic reperfusion (Fig 8). Calpain inhibitor-1 (50 µmol/L) and its solvent, DMSO (0.05%), had no significant effect on these parameters during preischemic perfusion. During reperfusion following ischemia for 20 minutes, calpain inhibitor-1 significantly improved the recovery of these parameters (Fig 8). When the recovery of LVDP after reperfusion for 30 minutes was compared with the extent of the -calspectin proteolysis in the 10 000g pellet, the correlation was observed between the recovery of LVDP and the inhibition of the proteolysis in the groups treated or not with calpain inhibitor-1 (Fig 9).

View larger version (35K): [in this window] [in a new window]   Figure 8. Effects of calpain inhibitor-1 (CAI) on the left ventricular developed pressure (LVDP, A), dP/dt (B), and heart rate (C). Each parameter during preischemia, ischemia (20 minutes), and reperfusion (30 minutes) was expressed as percentage of the pretreatment value in each heart . During preischemic perfusion, the three parameters of the hearts perfused with 50 µmol/L CAI (n=6) (hatched bars) for 5 minutes were not different from those perfused with its solvent, dimethyl sulfoxide (DMSO, 0.05%) (n=7) (open bars). The three parameters were suppressed during ischemia for 5 minutes (P<.01) to 20 minutes (not shown). During reperfusion after 20 minutes of ischemia, LVDP, dP/dt, and heart rate of the DMSO-treated hearts were still reduced compared with hearts treated with preischemic perfusion (stunning). CAI significantly improved the LVDP, dP/dt, and heart rate during reperfusion (*P<.05).

View larger version (9K): [in this window] [in a new window]   Figure 9. Relation between calspectin proteolysis and the recovery of left ventricular developed pressure (LVDP) after 30 minutes of reperfusion following 20 minutes of ischemia. The 10 000g pellets from the hearts analyzed in Fig 8 were subjected to immunoblotting, and the amounts of 150-kD proteolytic product were determined as described in "Materials and Methods." The recovery of LVDP (percentage of pretreatment value) was inversely correlated with the proteolysis of the -calspectin (arbitrary unit) in the group treated with 50 µmol/L calpain inhibitor-1 (CAI) (n=6, ) and the group treated with 0.05% DMSO (n=7, ) after 30 minutes of reperfusion (r=-.727, P<.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Rat myocardium expresses the 240- and 235-kD polypeptides antigenically related to - and ß-subunits of brain calspectin (fodrin or nonerythroid spectrin), respectively (Fig 1). The present study demonstrated that the two subunits of calspectin are substrates of calpain in vitro (Fig 2) and that calspectin is proteolyzed in vivo in the perfused heart after postischemic reperfusion (Figs 3 through 5). Since a calpain inhibitor significantly suppressed the degradation of calspectin after postischemic reperfusion (Fig 6), calpain may be responsible for the in vivo proteolysis of calspectin.

Our immunohistochemical study revealed that calspectin was proteolyzed at the subsarcolemmal region and the intercalated disks (Fig 7). A sensitive detection of the proteolysis of calspectin has become possible by an antibody against the 150-kD fragment of -calspectin, which does not recognize the intact -calspectin. Using this antibody, Saido et al¹⁴ have found regional (supracellular) differences in calspectin proteolysis after postischemic reperfusion in the gerbil hippocampus. In the present study, the staining of the sarcolemma and the intercalated disks by this antibody became distinct after postischemic reperfusion (Fig 7), indicating that these subcellular structures are the specific sites of the calspectin proteolysis in the heart. The striations stained by this antibody may also be a site for the proteolysis of calspectin, representing the Z bands or T tubules as Messina and Lemansky¹⁰ suggested. These immunohistochemical data were well supported by the biochemical data. A brief ischemia followed by reperfusion induced the proteolysis of calspectin in the 10 000g and 100 000g pellets (Figs 3 through 5). In our subcellular fractions, the sarcolemma was distributed in both pellets; the intercalated disks were enriched in the 10 000g pellet (Table).

In our subcellular fractionation, 80% of -calspectin was present in the 600g pellet. This was not due to inadequate homogenization for the following reasons: Longer homogenization made no apparent difference in the content of -calspectin in this fraction. Ischemia/reperfusion did not induce the proteolysis of -calspectin in the fraction, whereas -calspectin in the 10 000g and 100 000g pellets was proteolyzed after ischemia/reperfusion, producing the 150-kD fragment (Figs 3 through 5). Therefore, -calspectin recovered in the 600g pellet may be derived from a compartment distinct from that recovered in the 10 000g and 100 000g pellets. At present, we do not know from where the calspectin in the 600g pellet is derived. Ishiyama et al²⁷ reported that the anti-brain -calspectin antibodies stained diffusely throughout the myocardium in addition to the prominent staining of intercalated disks. The diffusely distributed calspectin may be recovered in the 600g pellet, interacting with contractile proteins.

In the present study, no cross-reactivity was detected in the rat myocardium with the antibodies against erythrocyte spectrin, although previous studies reported the antigenic similarities of erythrocyte spectrin to the spectrinlike proteins of the human or hamster heart.^{10 28} The discrepancy is probably due to either the difference in species or antibody specificity. In the subcellular fractions of the present study, ß-calspectin was exclusively localized to the 10 000g pellet, whereas all the pellet fractions contained -calspectin (Fig 1). This may be because the monoclonal antibody used in the present study did not recognize some ß-calspectin variants in the myocardium. In support of this, Vybiral et al²⁸ detected five immunoreactive polypeptides with anti-human erythrocyte spectrin antibodies and three with anti-rat calspectin in the human myocardium. Despite the discrepancies in immunoreactivity, our immunohistochemical data for control hearts were essentially the same as those described in the previous reports (Fig 7).^{10 27 28}

The degradation of calspectin under the sarcolemma and at the intercalated disks was quite an early event after postischemic reperfusion and preceded the development of histological change for irreversible injury, such as contraction bands. The latter became evident after 30 minutes or more of ischemia followed by reperfusion.²⁹ Ischemia for only 10 minutes was enough for the proteolysis of calspectin, when it was followed by reperfusion for 30 minutes (Figs 3 through 5). Other than calspectin proteolysis, no earlier change after postischemic reperfusion has been found in the previous studies. For example, Steenbergen et al¹⁵ reported that 120 but not 60 minutes of ischemia followed by 60 minutes of reperfusion reduced vinculin immunostaining in the canine heart after coronary artery ligation. Sato et al¹⁷ found that 15 minutes of ischemia followed by 1 hour of reperfusion decreased immunostaining for tubulin in the same model. Iizuka et al¹⁸ showed that sarcolemmal calspectin underwent proteolysis in isolated myocytes after anoxia for 4 hours. Previously, we observed that postischemic reperfusion is essential for the activation of calpain in the particulate fraction.¹⁹ In the present study, we found that reperfusion without Ca²⁺ diminished the calspectin proteolysis, whereas the reflow with high KCl did not suppress the proteolysis (Fig 6), although both interventions arrested the heart. These observations indicate the involvement of a Ca²⁺-dependent process rather than mechanical disruption of the sarcolemma in the proteolysis of calspectin during reperfusion.

Reperfusion after brief ischemia does not induce necrosis but does prolong contractile failure.³⁰ This phenomenon, known as myocardial stunning, has been thought to be a reversible process. However, our observations suggest that an irreversible change, calspectin degradation, which occurred after 10 minutes of ischemia followed by reperfusion, may partly account for the stunning. Recently, Matsumura et al³¹ reported that leupeptin improved the left ventricular function of guinea pig heart during postischemic reperfusion. We showed that calpain inhibitor-1 also improved the recovery in LVDP, dP/dt, and heart rate during reperfusion after 20 minutes of ischemia (Fig 8). Furthermore, we found the correlation between the recovery of left ventricular function and the inhibition of calspectin proteolysis (Fig 9). These observations suggest that the proteolysis of calspectin by calpain is closely related to myocardial stunning, although other calpain-sensitive proteins may also be responsible for it. It has been proposed that calspectin maintains the integrity of the plasma membranes as a constituent of the membrane skeleton.^{2 3} The degradation of sarcolemmal calspectin may alter the properties of ion channels and pumps of the sarcolemma. The proteolysis of calspectin at the intercalated disks may result in the disorganization of the gap junction, which then may disorder the electrical conduction. It remains to be clarified how the proteolysis of calspectin causes myocardial stunning.

	Acknowledgments

 
This study was supported by grants from the Japanese Education Ministry and Kobayashi Magobe Memorial Foundation. The authors are grateful for the kind guidance of Dr Masafumi Kitakaze (The First Department of Internal Medicine, Osaka University School of Medicine) and Dr Michihiro Kohno and Prof Masunori Matsuzaki (The Second Department of Internal Medicine, Yamaguchi University School of Medicine) in the experiments on myocardial contractility.

Received October 24, 1994; accepted May 10, 1995.

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