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Am J Physiol Heart Circ Physiol 287: H81-H90, 2004. First published March 4, 2004; doi:10.1152/ajpheart.01140.2003
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Ischemic preconditioning-mediated restoration of membrane dystrophin during reperfusion correlates with protection against contraction-induced myocardial injury

Masakuni Kido,1 Hajime Otani,1 Shiori Kyoi,2 Tomohiko Sumida,1 Hiroyoshi Fujiwara,1 Takayuki Okada,1 and Hiroji Imamura1

Departments of 1Thoracic and Cardiovascular Surgery and 2Cardiology, Kansai Medical University, Moriguchi City 570-8507, Japan

Submitted 2 December 2003 ; accepted in final form 1 March 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Dystrophin is an integral membrane protein involved in the stabilization of the sarcolemmal membrane in cardiac muscle. We hypothesized that the loss of membrane dystrophin during ischemia and reperfusion is responsible for contractile force-induced myocardial injury and that cardioprotection afforded by ischemic preconditioning (IPC) is related to the preservation of membrane dystrophin. Isolated and perfused rat hearts were subjected to 30 min of global ischemia, followed by reperfusion with or without the contractile blocker 2,3-butanedione monoxime (BDM). IPC was introduced by three cycles of 5-min ischemia and 5-min reperfusion before the global ischemia. Dystrophin was distributed exclusively in the membrane of myocytes in the normally perfused heart but was redistributed to the myofibril fraction after 30 min of ischemia and was lost from both of these compartments during reperfusion in the presence or absence of BDM. The loss of dystrophin preceded uptake of the membrane-impermeable Evans blue dye by myocytes that occurred after the withdrawal of BDM and was associated with creatine kinase release and the development of contracture. Although IPC did not alter the redistribution of membrane dystrophin induced by 30 min of ischemia, it facilitated the restoration of membrane dystrophin during reperfusion. Also, myocyte necrosis was not observed when BDM was withdrawn after complete restoration of membrane dystrophin. These results demonstrate that IPC-mediated restoration of membrane dystrophin during reperfusion correlates with protection against contractile force-induced myocardial injury and suggest that the cardioprotection conferred by IPC can be enhanced by the temporary blockade of contractile activity until restoration of membrane dystrophin during reperfusion.

reperfusion injury; 2,3-butanedione monoxime

IT HAS LONG BEEN ESTABLISHED that a predominant form of cardiomyocyte death after temporary ischemia is contraction band necrosis, which is characterized by hypercontracture of the contractile apparatus due to massive Ca2+ influx through disrupted sarcolemmal membranes (11, 17, 41). Studies using the contractile blocker 2,3-butanedione monoxime (BDM) have suggested that restoration of mechanical force during reperfusion is responsible for the occurrence of contraction band necrosis (30, 39).

It has been suggested that the fragility of the sarcolemmal membrane is involved in the mechanism of contractile force-induced membrane disruption during reperfusion. Contractile force generated by actin-myosin interaction is transmitted to the sarcolemmal membrane at the sites of Z-band attachment, termed lateral costamere junctions. The mechanical stress imposed on the fragile membrane causes its breakage at the sites of Z-band attachment, leading to subsarcolemmal bleb formation (9), which is a characteristic feature of irreversible myocyte injury after reoxygenation or reperfusion (11, 17, 41). Sarcolemmal fragility has been attributed to loss of the structural proteins localized in the cytoskeleton or the membrane skeleton (9). The lateral costamere junctions contain many structural proteins such as vinculin, talin, and paxillin. However, alterations in these proteins were not consistently correlated with irreversible injury, and it is thought that the loss of other structural proteins that link the Z-band and the sarcolemma, and the sarcolemma and the extracellular matrix, are involved in membrane fragility (2, 9).

Dystrophin is a member of the integral membrane proteins known as the dystrophin-glycoprotein complex in the skeletal muscle, which includes extracellular {alpha}-dystroglycan, transmembrane {beta}-dystroglycan, and {alpha}-, {beta}-, {gamma}-, and {delta}-isoforms of sarcoglycans. {alpha}-Dystroglycan links the extracellular matrix protein laminin-2 with {beta}-dystroglycan (27). Intracellularly, the COOH-terminus of dystrophin links to {beta}-dystroglycan and the NH2-terminus to {gamma}-actin and talin, connecting the extracellular matrix and the cytoskeleton (10). Dystrophin provides a mechanically strong physical linkage between the sarcolemmal membrane and the costameric cytoskeleton in cardiac muscle (29), thereby stabilizing the sarcolemmal membrane against the shear stresses imposed during eccentric muscle contraction (28, 37). The absence of dystrophin causes severe progressive weakness and degeneration of both skeletal and cardiac muscles in patients with Duchenne muscular dystrophy (16). Dystrophin is also absent in X-linked muscular dystrophy (mdx) mice (33). Studies using mdx mice revealed that the sarcolemma of the muscle is vulnerable to mechanical force (21, 38). Despite the crucial importance of dystrophin in maintaining the sarcolemmal membrane integrity in the heart, the role of dystrophin in the pathogenesis of myocardial reperfusion injury is poorly understood. We have recently demonstrated that sarcolemmal membrane dystrophin is translocated to the costameric cytoskeleton and the intercalated disks during ischemia and is subsequently lost upon reperfusion (18). Our previous study showed that necrosis did not occur in myocytes depleted of membrane dystrophin when contractile activity was abolished by reperfusion with BDM but occurred in these myocytes after withdrawal of BDM. Therefore, we hypothesized that the loss of membrane dystrophin may be involved in the pathogenesis of contractile force-induced reperfusion injury.

Reperfusion injury was reduced by the induction of brief periods of ischemia-reperfusion before lethal sustained ischemia, and this intervention was termed ischemic preconditioning (IPC) (25). If cardiomyocyte necrosis occurs upon reperfusion through loss of sarcolemmal membrane dystrophin and subsequent vulnerability to contractile force, cardioprotection afforded by IPC should be mediated by the preservation of sarcolemmal integrity and stability. We hypothesized, therefore, that the cardioprotective effects of IPC correlate with the preservation of membrane dystrophin during ischemia-reperfusion. The results of the present study demonstrate that IPC does not prevent the ischemia-induced redistribution of dystrophin but prevents its degradation during reperfusion and facilitates its restoration in the membrane. The present study also demonstrates that temporary blockade of contractile activity until restoration of membrane dystrophin during reperfusion enhances the cardioprotection conferred by IPC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Perfusion technique. Male Sprague-Dawley rats weighing 250–300 g were used in the present study. All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 86-23, Revised 1996). The rats were anesthetized intraperitoneally with pentobarbital sodium (100 mg/kg). The hearts were excised and perfused as described previously (19) at a constant mean pressure of 70–75 mmHg using a Krebs-Henseleit bicarbonate (KHB) buffer solution of the following composition (in mM): 118 NaCl, 4.7 KCl, 1.2 MgSO4, 25 NaHCO3, 1.2 KH2PO4, 1.8 CaCl2, and 11 glucose; pH 7.4 at 37°C when equilibrated with a mixture of 95% O2-5% CO2 gas.

Isovolumic left ventricular (LV) function was measured as described previously (19). A latex balloon was inserted into the LV through the left atrium and filled with saline to produce a LV end-diastolic pressure (LVEDP) of 5–10 mmHg at baseline. The balloon volume was kept constant throughout the experiment. Hearts producing a LV developed pressure (LVDP) of <80 mmHg or a heart rate of <240 beats/min at baseline were excluded from the study.

Experimental protocol. Thirty minutes after measurements of the baseline data, the control heart was subjected to 30 min of global ischemia, followed by 120 min of reperfusion (Fig. 1). In the experimental hearts, IPC was introduced by three cycles of 5 min of ischemia followed by 5 min of reperfusion. The hearts were then subjected to 30 min of global ischemia, followed by 120 min of reperfusion. When BDM was introduced upon reperfusion, the reperfusion buffer contained 20 mM BDM with an equimolar reduction of NaCl for a duration of 5 or 30 min. This concentration of BDM abolished contractile force (LVDP < 5% of baseline).



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  		Fig. 1. Experimental protocols. Thirty minutes after measurements of the baseline data, the control hearts were subjected to 30 min of global ischemia followed by 120 min of reperfusion. Ischemic preconditioning (IPC) was introduced by 3 cycles of 5 min of ischemia each intervened with 5 min of reperfusion. The hearts were then subjected to 30 min of global ischemia followed by 120 min of reperfusion. 2,3-Butanedione monoxime (BDM) was administered upon reperfusion for a duration of 5 min (BDM R5) or 30 min (BDM R30) in the non-IPC and IPC hearts.

 
Creatine kinase release. Coronary effluent was collected at the indicated time points after reperfusion, and creatine kinase (CK) activity was measured using an enzymatic assay method (19). Heart weight was measured at the end of the experiments, and CK release was corrected on the basis of heart weight and coronary flow.

Infarct size measurements. Upon the termination of experiments, the heart was sliced transversely in a plane perpendicular to the apical-basal axis into {approx}1 mm thickness. The slices were immersed in PBS containing 2% triphenyltetrazolium chloride (TTC) for 15 min at 37°C and fixed with 10% formaldehyde in 0.1 M phosphate buffer (pH 7.2) at room temperature. The brick red area was traced using NIH 1.61 Image processing software, and each digitized image was subjected to equivalent degrees of background subtraction, brightness, and contrast enhancement for improved clarity and distinctness. The areas at risk (equivalent to total LV mass) as well as the infarct zones of each slice were calculated in terms of pixels. The infarct volume was calculated, and the sum of all slices used to compute a ratio of percent infarct to total LV mass.

Evans blue dye perfusion. The following experiments were performed to assess sarcolemmal membrane permeability in myocytes by perfusion with the membrane-impermeable dye Evans blue (EB; Sigma). First, the nonischemic control hearts were perfused with KHB buffer containing 0.1% EB for 15 min. Second, the non-IPC hearts or the IPC hearts subjected to 30 min of ischemia were reperfused with KHB buffer containing EB for 15 min. Third, the non-IPC hearts subjected to 30 min of ischemia were reperfused with BDM containing EB for 15 min. Finally, the non-IPC or the IPC hearts subjected to 30 min of ischemia were reperfused with BDM for 30 min, followed by perfusion with KHB buffer containing EB for 15 min. In all experiments, EB was washed away by perfusion with the same buffer without EB for 5 min.

Immunofluorescence microscopy. Frozen sections were cut at ~6 µm onto glass slides, incubated in acetone and hydrogen peroxide, rinsed with PBS, and blocked with 10% normal rabbit serum. The sections were incubated for 1 h at room temperature with mouse monoclonal anti-dystrophin antibodies (MANDRA-1, Sigma) at a dilution of 1:100 and washed with PBS. They were then incubated for 2 h at room temperature with a fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin at a dilution of 1:100. To visualize the sarcolemma, sections primarily stained for dystrophin were secondary stained with tetrarhodamine isothiocyanate (TRITC)-conjugated wheat germ agglutinin (WGA; Sigma). Double staining was also performed to detect dystrophin as described above alongside the cytoskeletal proteins vinculin and desmin using mouse monoclonal antibodies (Sigma) and TRITC-conjugated rabbit anti-mouse immunoglobulin. Correlation between the loss of membrane dystrophin and the sarcolemmal membrane permeability was examined in EB-perfused heart sections by staining for dystrophin using mouse monoclonal anti-dystrophin antibodies and FITC-conjugated rabbit anti-mouse immunoglobulin. The fluorescence staining was visualized using a confocal laser microscope (Fluoview, Olympus) at an excitation wavelength of 488 nm for FITC and 568 nm for TRITC and EB.

Tissue sample preparation and immunoblot assay. Subcellular fractionation of the heart and immunoblot assays were performed as described previously (20) with some modifications. Briefly, the frozen samples were powdered under liquid nitrogen and homogenized in 10 volumes of buffer (pH 7.4) containing (in mM) 303 sucrose, 20 sodium pyrophosphate, 20 sodium phosphate monohydrate, 1 MgCl2, 0.5 EDTA, 1 EGTA, 1 sodium orthovanadate, and 0.25 PMSF and a protease inhibitor cocktail (Complete, Roche Molecular Biochemicals) using a Teflon-glass hand-held homogenizer. The homogenates were centrifuged at 1,000 g for 15 min, and the resulting pellet was resuspended in buffer (pH 7.0) containing (in mM) 60 KCl, 30 imidazole, and 2.5 MgCl2 and 1% Triton X-100. The resulting suspension was centrifuged at 4,000 g for 10 min. The Triton X-100 insoluble pellet, referred to as the myofibril fraction, was solubilized with RIPA buffer (pH 7.5) containing (in mM) 150 NaCl, 20 Tris·HCl, 10 NaH2PO4, 5 EDTA, 1 dithiothreitol, 1 sodium orthovanadate, and 0.25 PMSF (0.25) with 10% glycerol, 1% sodium deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate, and the protease inhibitor cocktail. The 1,000-g supernatant was centrifuged at 142,000 g for 35 min. The pellet was designated the membrane fraction, and the supernatant was designated the cytosol fraction. Protein concentrations were determined using the DC protein assay kit (Bio-Rad Laboratories).

Protein samples were separated by SDS-PAGE, transferred to nitrocellulose paper in buffer containing 20 mM Tris·HCl and 192 mM glycine, and incubated in blocking buffer containing 5% milk protein, 20 mM Tris·HCl, 137 mM NaCl, and 0.05% Tween-20 for 2 h at room temperature. Blots were then incubated with mouse monoclonal anti-dystrophin (MANDRA-1, Sigma) at a dilution of 1:1,500 for 1 h at room temperature. After two rinses and after being washed with blocking buffer, blots were transferred to blocking buffer containing a peroxidase-conjugated rabbit anti-mouse immunoglobulin (Dako) at a dilution of 1:1,500 for 1 h at room temperature followed by rinses in buffer without milk protein. Blots were developed using the enhanced chemiluminescence method (Amersham) according to the manufacturer's instructions. Relative levels of dystrophin were quantified by densitometric analysis using NIH 1.61 Image processing software.

Statistical analysis. All data are expressed as means ± SE. Statistical analysis was performed by one-way ANOVA, followed by the Bonferroni post hoc test for comparison of infarct size and quantitative immunoblot data or repeated-measures ANOVA followed by unpaired t-test for comparison of CK release data. The differences were considered significant at a P value of <0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
IPC does not prevent redistribution of membrane dystrophin during ischemia. Immunofluorescence confocal laser microscopy revealed that dystrophin was distributed throughout the sarcolemmal membrane of myocytes, and there was no visible immunostaining of dystrophin in the cytoplasmic region in the normally perfused heart (Fig. 2, A and inset). IPC alone had no appreciable effect on the distribution of dystrophin (Fig. 2, B and inset). However, the immunofluorescence intensity of dystrophin in the membrane decreased after 30 min of ischemia associated with a reciprocal increase in immunofluorescence intensity in the cytoplasmic region (Fig. 2C). In these ischemic hearts, the immunofluorescence staining of dystrophin showed a costameric pattern in the longitudinal section (Fig. 2C, inset), suggesting that dystrophin had translocated to the costameric cytoskeleton such as Z-disks and/or T-tubules. Our previous study (18) showed that there was a transmural gradient of redistribution of dystrophin during ischemia, which was greater in the endocardial layer of the LV. This pattern of dystrophin redistribution induced by 30 min of ischemia was not altered in IPC hearts (Fig. 2, D and inset).



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  		Fig. 2. Effect of IPC on the localization and abundance of dystrophin during ischemia. A–D: immunofluorescence analysis of dystrophin (Dys). Insets, longitudinal sections. A: control perfusion heart before ischemia; B: IPC heart before ischemia; C: control heart after 30 min of ischemia; D: IPC heart after 30 min of ischemia. Bars = 20 µm. E: immunoblot assay for dystrophin. Control, control perfusion heart before ischemia; IPC, IPC heart before ischemia; control I, control heart after 30 min of ischemia; IPCI, IPC heart after 30 min of ischemia. Each bar represents the mean ± SE of 7 experiments. *P < 0.01 compared with control.

 
Only the inner half of the LV myocardial samples were used for immunoblot analysis of dystrophin because of a spatial difference in the loss of dystrophin. Immunoblot analysis demonstrated that the dystrophin content decreased in the membrane fraction and reciprocally increased in the myofibril fraction after 30 min of ischemia (Fig. 2E). Only a trivial amount of dystrophin was present in the cytosol fraction before and after 30 min of ischemia (not shown). IPC alone did not cause significant redistribution of dystrophin, nor did it prevent the redistribution of dystrophin from the membrane to the myofibril fractions induced by 30 min of ischemia. Total myocardial dystrophin levels were not changed during ischemia in either group of hearts.

IPC restores membrane dystrophin during reperfusion. The sarcolemmal dystrophin was lost with a variable degree in myocytes localized in the middle myocardial layer in the non-IPC hearts 30 min after reperfusion (Fig. 3A). The cytoplasmic dystrophin decreased toward the basal level in the majority of myocytes, although the immunostaining was not completely abolished. The endocardial layer always showed uniform loss of the sarcolemmal dystrophin, whereas the epicardial layer showed uniform preservation of the sarcolemmal dystrophin (not shown). IPC restored the sarcolemmal dystrophin in the majority of myocytes (Fig. 3B). Immunoblot analysis demonstrated that the dystrophin content in the membrane fraction remained depleted 30 min after reperfusion in the non-IPC hearts (Fig. 3C). The dystrophin content in the myofibril fraction decreased from the end-ischemic levels during reperfusion in these hearts, although it remained significantly higher than the basal level. This was associated with the loss of total dystrophin content. IPC significantly but not completely restored membrane dystrophin and inhibited the loss of total dystrophin content compared with the non-IPC hearts during reperfusion. These results demonstrate that the loss of dystrophin from the membrane and the total cellular pool occurs during reperfusion in the non-IPC hearts as well as in the IPC hearts with a smaller degree. However, it is not clear whether the loss of dystrophin is the cause rather than the consequence of contraction-induced myocyte necrosis.



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  		Fig. 3. Effect of IPC on the localization and abundance of dystrophin after reperfusion. A and B: immunofluorescence analysis of dystrophin. A: control heart after 30 min of reperfusion; B: IPC heart after 30 min of reperfusion. Bars = 20 µm. C: immunoblot assay of dystrophin. Control, control perfusion heart before ischemia; control R30, non-IPC heart after 30 min of reperfusion; IPC R30, IPC heart after 30 min of reperfusion. Each bar represents the mean ± SE of 7 experiments. *P < 0.05 and **P < 0.01 compared with Control; {dagger}P < 0.05 and {dagger}{dagger}P < 0.01 compared with control R30.

 
Therefore, we investigated the effect of reperfusion with BDM on the localization and abundance of dystrophin to clarify the relationship between the loss of sarcolemmal dystrophin and the contraction-induced myocyte necrosis. Dystrophin was lost from the sarcolemmal membrane in myocytes with a variable degree 5 min after reperfusion with BDM in the non-IPC hearts, whereas these myocytes still retained visible cytoplasmic dystrophin (Fig. 4A). In the non-IPC hearts, sarcolemmal dystrophin was almost completely lost in some myocytes but was almost normally restored in others 30 min after reperfusion with BDM (Fig. 4B). In the IPC hearts, cytoplasmic dystrophin returned to the sarcolemma 5 min after reperfusion with BDM in some myocytes, although a considerable amount of dystrophin remained in the cytoplasmic region in others (Fig. 3C). Reperfusion with BDM for 30 min in the IPC hearts completely restored membrane dystrophin and decreased cytoplasmic dystrophin to the basal level in virtually all myocytes (Fig. 3D).



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  		Fig. 4. Effect of IPC on the localization and abundance of dystrophin after reperfusion with BDM. A–D: immunofluorescence analysis of dystrophin. A: control heart after reperfusion with BDM for 5 min; B: control heart after reperfusion with BDM for 30 min; C: IPC heart after reperfusion with BDM for 5 min; D: IPC heart after reperfusion with BDM for 30 min. Bars = 20 µm. E: immunoblot assay for dystrophin. Each bar represents the mean ± SE of 7 experiments. *P < 0.01 compared with control.

 
Immunoblot analysis (Fig. 4E) demonstrated that the dystrophin content in the membrane fraction remained depleted 5 min after reperfusion with BDM in the non-IPC hearts. The dystrophin content in the myofibril fraction decreased from the end-ischemia levels, resulting in the loss of total myocardial dystrophin in these hearts (Fig. 3E). Membrane dystrophin and total myocardial dystrophin continued to decrease 30 min after reperfusion with BDM. In the IPC hearts, membrane dystrophin was partially restored but a significant amount of dystrophin remained in the myofibril fraction 5 min after reperfusion with BDM. Total myocardial dystrophin in these hearts remained unchanged. Dystrophin was completely restored in the membrane fraction 30 min after reperfusion with BDM in the IPC hearts associated with a decrease in dystrophin content in the myofibril fraction but with no change in total myocardial dystrophin levels.

Gross structure of the sarcolemma and the cytoskeleton in membrane dystrophin-depleted myocytes. We then investigated the temporal relationship between loss of membrane dystrophin and alteration in the gross structure of the sarcolemma and the cytoskeleton. WGA binds to the membrane lipid and glycoprotein components by cross-linking terminally linked N-acetyl-D-glucosamine and/or sialic acid in the sarcolemma (7). Vinculin is a major membrane-associated component localized in the costameric junctions and fascia adherence junctions of intercalated disks (5). Loss of vinculin has been implicated in membrane fragility to osmotic stress and contractile force during reoxygenation or reperfusion (12, 36). Desmin is the intermediate filament protein involved in cytoskeletal integrity (9). The immunostaining pattern for WGA, vinculin, and desmin in the normally perfused heart is shown in Fig. 5, A, D, and G. No visible alteration of immunostaining of these proteins was noted 30 min after ischemia (not shown). Loss of dystrophin immunostaining after reperfusion with BDM was not associated with loss of WGA staining (Fig. 5B). Similarly, these myocytes retained vinculin and desmin immunostaining (Fig. 5, E and H), suggesting that the gross structure of the sarcolemma and the cytoskeleton was preserved in these dystrophin-depleted myocytes. However, withdrawal of BDM resulted in loss of WGA, vinculin, and desmin staining (Fig. 5, C, F, and I). The staining pattern of WGA, vinculin, and desmin was not altered in the IPC hearts after reperfusion with BDM and after its withdrawal (not shown).



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  		Fig. 5. Representative images of double immunofluorescence staining for dystrophin and wheat germ agglutinin (WGA; A–C), dystrophin and vinculin (D–F), and dystrophin and desmin (G–I). A, D, and G: control perfusion heart. B, E, and H: control heart after reperfusion with BDM for 30 min. Note that immunofluorescence staining for WGA, vinculun, and desmin remains preserved when dystrophin is lost from the sarcolemmal membrane. C, F, and I: control heart after reperfusion with BDM for 30 min followed by perfusion with Krebs-Henseleit bicarbonate (KHB) buffer solution for 15 min. Although membrane-bound WGA is lost, extracelluar WGA remains abundant. Also note that, whereas sarcolemmal membrane-located vinculin is lost, intercalated disk-located vinculin remains preserved. Bars = 20 µm.

 
IPC prevents EB accumulation in myocytes after withdrawal of BDM. To further our understanding of the relationship between loss of membrane dystrophin and contractile force-induced myocyte injury, the IPC and the non-IPC hearts subjected to 30 min of ischemia were treated with the membrane-impermeable dye EB in the presence or absence of BDM. In the normally perfused heart, dystrophin was distributed throughout the sarcolemmal membrane in myocytes, and no accumulation of EB in these myocytes was observed (Fig. 6A). However, in the non-IPC hearts, EB accumulated in myocytes depleted of membrane dystrophin during reperfusion in the absence of BDM (Fig. 6B). The number of myocytes depleted of membrane dystrophin and containing EB was reduced in the IPC hearts (Fig. 6C). EB did not accumulate in myocytes depleted of membrane dystrophin after reperfusion with BDM in the non-IPC hearts (Fig. 6D). However, when these hearts were treated with EB after the withdrawal of BDM, accumulation of the dye occurred in myocytes depleted of membrane dystrophin (Fig. 6E). In the IPC hearts, reperfusion with BDM for 30 min, followed by its removal in the presence of EB resulted in no EB uptake by myocytes associated with the normal membrane distribution of dystrophin (Fig. 6F).



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  		Fig. 6. Representative images of dystrophin and Evans blue (EB). Immunofluorescence staining for dystrophin and EB was performed as described in the text. A: control heart undergoing perfusion with KHB buffer solution containing 0.1% EB for 15 min; B: control heart undergoing reperfusion with KHB containing EB for 15 min; C: IPC heart undergoing reperfusion with KHB containing EB for 15 min; D: control heart undergoing reperfusion with BDM and EB for 15 min; E: control heart undergoing reperfusion with BDM for 30 min, followed by perfusion with KHB containing EB for 15 min; F: IPC heart undergoing reperfusion with BDM for 30 min, followed by perfusion with KHB containing EB for 15 min. Bars = 50 µm in A–C and F and 20 µm in D and E.

 
Temporary reperfusion with BDM enhances cardioprotection afforded by IPC. We then investigated the correlation between the preservation of membrane dystrophin and protection against contractile force-induced myocardial injury in the IPC hearts. Appreciable CK release was observed 5 min after reperfusion in the absence of BDM in the control hearts (Fig. 7A). CK release in these hearts reached a maximum level 30 min after reperfusion. IPC in the absence of BDM after reperfusion significantly reduced CK release and infarct size compared with the control hearts. No CK release was observed after reperfusion while BDM was present, even in the non-IPC hearts. However, marked CK release occurred after the withdrawal of BDM. Accordingly, infarct size in the non-IPC hearts in the presence of BDM after reperfusion was not significantly different from the control hearts (Fig. 7B). IPC-induced reduction of CK release and infarct size was significantly enhanced by reperfusion with BDM for 5 min. Furthermore, CK release and infarction was virtually absent when the IPC hearts were reperfused with BDM for the first 30 min after ischemia.



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  		Fig. 7. A: effect of reperfusion with BDM on creatine kinase release [in IU·min–1·g dry wt (gDW)–1]. Control hearts ({bullet}) underwent 30 min of global ischemia and 120 min of reperfusion with KHB buffer solution. IPC hearts ({circ}) underwent 30 min of ischemia and 120 min of reperfusion with KHB. Non-IPC hearts subjected to 30 min of ischemia underwent reperfusion with BDM for 5 min, followed by perfusion with KHB ({blacktriangleup}) or for 30 min, followed by perfusion with KHB ({blacksquare}). IPC hearts subjected to 30 min of ischemia underwent reperfusion with BDM for 5 min, followed by perfusion with KHB ({triangleup}) or for 30 min, followed by perfusion with KHB ({square}). Each symbol represents the mean ± SE of 7 experiments. *P < 0.05 and **P < 0.01 compared with control hearts; {dagger}P < 0.05 and {dagger}{dagger}P < 0.01 compared with IPC hearts without BDM. B: effect of reperfusion with BDM on infarct size. Each bar graph represent the mean ± SE of 7 experiments. *P < 0.01 compared with control; {dagger}P < 0.01 compared with IPC.

 
The correlation between the abundance of membrane dystrophin and LV function was also investigated. Control reperfusion after 30 min of ischemia was associated with the development of contracture (Fig. 8A). IPC inhibited the increase in LVEDP during reperfusion (Fig. 8B). Reperfusion in the presence of BDM abolished contractile activity (Fig. 8C). However, withdrawal of BDM resulted in development of contracture shortly after restoration of normal contractility. In contrast, no increase in LVEDP was observed in the IPC heart after withdrawal of BDM (Fig. 8D). Table 1 summarizes LV function after reperfusion, and Fig. 8E shows a comparison of LV pressure 45 min after reperfusion, at which maximum recovery of peak systolic pressure was observed in all groups of hearts. Peak systolic pressure was not significantly different in all groups of hearts 45 min after reperfusion. A major difference was noted in LVEDP at this stage. LVEDP was significantly lower after reperfusion in the absence of BDM in the IPC hearts compared with the non-IPC hearts giving rise to significant differences in LVDP. In the non-IPC hearts, LVDP, heart rate, and LVEDP after withdrawal of BDM were not significantly different between reperfusion with or without BDM. In the IPC hearts, the increase in LVEDP during reperfusion in the absence of BDM was significantly inhibited and LVDP was significantly improved by reperfusion with BDM for 30 min.



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  		Fig. 8. Effect of reperfusion with BDM on left ventricular (LV) function. A–D: representative traces of LV pressure (LVP). A: control hearts subjected to 30 min of global ischemia (Isc) underwent reperfusion with KHB buffer solution; B: IPC hearts subjected to 30 min of ischemia underwent reperfusion with KHB; C: non-IPC hearts subjected to 30 min of ischemia underwent reperfusion with BDM for 30 min, followed by perfusion with KHB; D: IPC hearts subjected to 30 min of ischemia underwent reperfusion with BDM for 30 min, followed by perfusion with KHB; E: quantitative analysis of LVP. Control, time-matched control perfusion group; KHB R45, control group of hearts underwent 45 min of reperfusion with KHB; IPC+KHB R45, IPC group of hearts underwent 45 min of reperfusion with KHB; BDM R30+KHB15, non-IPC group of hearts underwent reperfusion with BDM for 30 min followed by perfusion with KHB for 15 min; IPC+BDM R30+KHB15, IPC group of hearts underwent reperfusion with BDM for 30 min followed by perfusion with KHB for 15 min. The top and bottom of the bars in E indicate peak systolic pressure and end-diastolic pressure, respectively. Data are expressed as means ± SE of 7 experiments. *P < 0.01 compared with control; {dagger}P < 0.01 compared with KHB R45; #P < 0.01 compared with IPC+KHB R45.

 

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  		Table 1. Effect of IPC and reperfusion with BDM on LV function

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Despite extensive research, the mechanism of reperfusion injury has not been completely elucidated. Excess generation of reactive oxygen species and intracellular Ca2+ overload have been implicated in the pathophysiology of reperfusion injury (26). These cellular stress events either alone or in conjunction weaken the sarcolemmal membrane through as-yet-unknown mechanisms and cause myocyte necrosis upon reintroduction of contractile force (17). Therefore, preservation of the structural proteins responsible for sarcolemmal membrane integrity has been a target of the investigation of strategies to prevent reperfusion injury. Of the structural proteins associated with the sarcolemmal membrane, we have focused on dystrophin because this membrane protein forms a mechanically strong link between the sarcolemma and the costameric cytoskeleton (29). Selective loss of dystrophin in patients with Duchenne muscular dystrophy and mdx mice predisposes the vulnerability of skeletal and cardiac myocytes to mechanical force. Despite circumstantial evidence suggesting the involvement of dystrophin in the pathogenesis of reperfusion injury, little attention has been paid to this protein until recently.

We have previously demonstrated a unique change in intracellular distribution and abundance of dystrophin during ischemia and reperfusion in the rat heart. A significant portion of membrane dystrophin was translocated to the myofibril fraction during ischemia and was lost upon reperfusion (18). The present study further demonstrated that the loss of dystrophin was an early event after reperfusion that preceded any evidence of myocyte necrosis. First, loss of dystrophin occurred during reperfusion with BDM when disruption of the gross structure of the sarcolemmal membrane and the cytoskeleton had not occurred, as evidenced by the normal immunostaining pattern of WGA-cross linking proteins, vinculin, and desmin. Second, EB uptake by membrane dystrophin-depleted myocytes and CK release from the heart did not occur after reperfusion until withdrawal of BDM and restoration of contractile activity. Moreover, these hearts transiently regained normal contractile activity before the development of contracture after withdrawal of BDM, suggesting that myocardial function was maintained during reperfusion with BDM. These observations are consistent with the hypothesis that loss of membrane dystrophin may be causally related to the pathogenesis of contractile force-induced reperfusion injury.

It has long been known that IPC delays the onset of lethal ischemic damage and prevents reperfusion injury (25). The beneficial effect of IPC on myocardial salvage from contraction band necrosis during reperfusion prompted us to hypothesize that the end effect of IPC is stabilization of the sarcolemmal membrane against mechanical force. Thus we investigated whether the cardioprotection afforded by IPC correlates with the localization and abundance of dystrophin. We showed that 30 min of ischemia resulted in the redistribution of dystrophin from the membrane to the myofibril fraction, which contains the costameric cytoskeleton, in both the non-IPC and the IPC hearts. A similar translocation of dystrophin in isolated ischemic cardiomyocytes has been reported by Armstrong and associates (3). In their study, IPC did not prevent ischemia-induced redistribution of dystrophin and subsarcolemmal bleb formation after hypoosmotic challenge in ischemic cardiomyocytes, although it prevented osmotic fragility. Thus the role of membrane dystrophin in the protection of myocytes against osmotic fragility conferred by IPC was not proven in that study. It was also not clear whether IPC could protect myocytes from mechanical force-induced injury during reperfusion in intact hearts because hypoosmotic challenge was employed under continuing ischemia and the fate of dystrophin during reoxygenation was not investigated. The present study demonstrated for the first time that dystrophin is lost from both the membrane and the myofibril compartments during reperfusion in the non-IPC hearts but is restored in the IPC hearts. The restoration of membrane dystrophin during reperfusion in the IPC hearts was associated with significant inhibition of CK release and infarction compared with the non-IPC hearts. Treatment of the ischemic hearts with BDM for the first 5 or 30 min after reperfusion did not restore membrane dystrophin and did not provide significant cardioprotection in the non-IPC hearts. However, reperfusion with BDM for 5 min in the IPC hearts partially replenished membrane dystrophin and partially reduced CK release and infarct size after withdrawal of BDM compared with those not treated with BDM. Reperfusion with BDM for 30 min in the IPC hearts completely restored membrane dystrophin, abrogated CK release and EB uptake by myocytes, and completely restored LV function after withdrawal of BDM. Thus the present study provides evidence of a close correlation between the restoration of membrane dystrophin during reperfusion and cardioprotection conferred by IPC. Moreover, reperfusion of the IPC hearts in the absence of BDM, when dystrophin remained translocated to the myofibril fraction of the myocytes, resulted in appreciable CK release and EB uptake in myocytes. This indicates that a significant number of potentially viable myocytes deteriorate to necrosis by the sudden reintroduction of contractile activity during reperfusion in these hearts.

The mechanism by which dystrophin is translocated from the sarcolemma to the myofibril fraction during ischemia and is subsequently degraded upon reperfusion in some myocytes but is restored in the membrane in others is currently unknown. However, correlation between the restoration of membrane dystrophin and the cardioprotection conferred by IPC suggests that certain cardioprotective machinery activated by IPC is involved in regaining the normal distribution of dystrophin without its degradation. One of the mediators of IPC is activation of mitochondrial ATP-sensitive K+ channels, which protect the mitochondria from oxidative stress- and Ca2+ overload-induced injury (23, 40). Improved oxidative phosphorylation activity of mitochondria may be necessary for the normal redistribution of dystrophin to the membrane during reperfusion, because dystrophin is a phosphoprotein which is phosphorylated in vivo within the COOH-terminal at both serine and threonine residues (22). The signal transduction pathways that converge on mitochondrial protection (24) may also be involved in restoration of membrane dystrophin during reperfusion. Alternatively, IPC-mediated mitigation of intracellular Ca2+ overload (14) might prevent the degradation of dystrophin by Ca2+-dependent proteases. Dystrophin has been shown to be a substrate for calpain, a Ca2+-activated neutral protease (46). Therefore, preservation of mitochondrial function and attenuation of intracellular Ca2+ overload may provide a potential mechanistic link between IPC and restoration of membrane dystrophin during reperfusion.

BDM treatment after reperfusion has produced disparate effects on the recovery of ischemic heart, i.e., protective (13, 15, 30, 32, 39), no effect (15), or harmful (1, 45), depending on the experimental models, duration of BDM treatment, and concentrations of BDM used. The present study employed 20 mM BDM for either 5 or 30 min to obtain temporary contractile arrest after reperfusion. This protocol, however, provided no beneficial effect on ultimate infarct size and recovery of LV function in the non-IPC hearts. The net effect of BDM on the ischemic-reperfused heart depends on the balance between cardioprotective and detrimental actions. The beneficial effect of reperfusion with BDM is not solely attributed to inhibition of contractile activity. It is possible that inhibition of myosin ATPase by BDM results in the preservation of myocardial ATP (6, 42), thereby activating energy-requiring cellular processes necessary for cardioprotection such as phosphorylation of dystrophin as discussed above. On the other hand, it has been demonstrated that BDM at concentrations above 5 mM is toxic (1, 45) presumably because BDM activates type 1 and 2A phosphatases (47). In this context, dystrophin may also be a target of these phosphatases. Weinbrenner and associates (44) indeed demonstrated that inhibition of phosphatase 2A protects the ischemic heart from reperfusion injury. It is, therefore, speculated that the beneficial effect of contractile arrest and preservation of ATP by reperfusion with BDM is mitigated by the phosphatase action in the non-IPC hearts. In contrast, IPC appears to overcome this potent detrimental effect of BDM presumably by increasing the phosphorylation of dystrophin through the preservation of mitochondrial function at the time of reperfusion (8) and the activation of kinases (31). The importance of mitochondrial function in determining the efficacy of reperfusion with BDM is suggested by the previous studies demonstrating that withdrawal of BDM did not induce contracture and enzyme release if the mitochondria had already recovered their capacity to perform oxidative phosphorylation during treatment with BDM (34, 35). In addition, it has been shown that BDM inhibits the Na+/Ca2+ exchanger via the mechanism independent of phosphatase action (43). Inhibition of the Na+/Ca2+ exchanger may ameliorate Ca2+ overload-induced mitochondrial damage (4) that leads to loss of dystrophin. Taken together, it is conceivable that reperfusion with BDM is beneficial when membrane dystrophin is restored by IPC or other cardioprotective techniques that ameliorate mitochondrial function during reperfusion.

In conclusion, the present study demonstrates that loss of membrane dystrophin during ischemia and reperfusion precedes myocyte necrosis and suggests that it may be causally related to contractile force-induced reperfusion injury. Temporary blockade of contractile activity by reperfusion with BDM is not effective at preventing myocyte necrosis when myocytes are unable to restore membrane dystrophin. However, it does potentiate the cardioprotection conferred by IPC which facilitates the restoration of membrane dystrophin during reperfusion with BDM.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan and The Science Research Promotion Fund.


   ACKNOWLEDGMENTS
 
We are grateful for the technical assistance of Rie Yasuda and Chiaki Miyamoto.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Otani, Dept. of Thoracic and Cardiovascular Surgery, Kansai Medical Univ., 10-15 Fumizono-cho, Moriguchi City 570-8507, Japan (E-mail: otanih{at}takii.kmu.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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