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Cardiovascular Research 2006 70(2):354-363; doi:10.1016/j.cardiores.2006.01.004
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Copyright © 2006, European Society of Cardiology

Dystrophin is a possible end-target of ischemic preconditioning against cardiomyocyte oncosis during the early phase of reperfusion

Shiori Kyoi, Hajime Otani*, Ayako Hamano, Seiji Matsuhisa, Yuzo Akita, Hiroyoshi Fujiwara, Reiji Hattori, Hiroji Imamura, Hiroshi Kamihata and Toshiji Iwasaka

Cardiovascular Center, Kansai Medical University, Moriguchi City, Japan

* Corresponding author. Tel.: +81 6 6992 1001; fax: +81 6 6994 7022. Email address: otanih{at}takii.kmu.ac.jp

Received 30 August 2005; revised 15 December 2005; accepted 2 January 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective Dystrophin is a sarcolemmal membrane protein that prevents the myocyte from oncosis induced by physical stress. Because ischemic preconditioning (IPC) protects mitochondria and prevents oncosis during reperfusion, we hypothesized that dystrophin is an end-target of IPC distal to mitochondrial protection.

Methods and results Isolated rat hearts were subjected to 30 min ischemia followed by reperfusion. IPC was introduced by 3 cycles of 5 min ischemia and 5 min reperfusion. The loss of sarcolemmal dystrophin and myocardial ATP during ischemia was comparable between the control and the IPC heart. Similar changes in sarcolemmal dystrophin and myocardial ATP were observed when the heart was treated with 2,4-dinitrophenol (DNP), an uncoupler of mitochondrial respiration, or oligomycin, an inhibitor of mitochondrial F1F0-ATPase. However, the IPC heart increased sarcolemmal dystrophin during reperfusion associated with an increase in tetramethylrhodamine ethylester (TMRE) uptake, an indicator of mitochondrial membrane potential ({delta}{Psi}m), and myocardial ATP and inhibited myocyte oncosis. The increase in myocardial ATP and relocalization of dystrophin to the sarcolemma mediated by IPC was inhibited by treatment with DNP or oligomycin during reperfusion. In vitro experiments demonstrated that mitochondria isolated from the ischemic IPC heart increased ATP generation and facilitated relocalization of dystrophin from the insoluble to the soluble fractions in a manner sensitive to DNP and oligomycin.

Conclusions These results suggest that enhanced relocalization of dystrophin to the sarcolemma during reperfusion may be a mechanistic link between IPC-mediated improvement of mitochondrial function and its protection against oncosis during the early phase of reperfusion.

KEYWORDS Ischemic preconditioning; Dystrophin; Mitochondria; Reperfusion; Oncosis


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Ischemic preconditioning (IPC) is an endogenous form of cardioprotection triggered by a sublethal period of ischemia. The mechanism of cardioprotection afforded by IPC has been extensively investigated during the last decade. Accumulating evidence indicates that cardioprotective signal transduction derived from IPC converges on protection of mitochondria [1–4]. Nevertheless, the molecular link between mitochondrial protection and cardioprotection afforded by IPC has not been clarified.

The most salient cardioprotective effect of IPC is the limitation of infarct size. Myocardial infarction during an early stage of reperfusion is produced by myocyte oncosis which is defined as a primary increase in plasma membrane permeability and is distinguished from necrosis which occurs as a result of secondary plasma membrane disintegration associated with apoptosis [5]. Oncosis is characteristically introduced in myocytes upon reperfusion following a relatively brief period of ischemia [6,7]. It has long been realized that sarcolemmal fragility is a cause of oncosis during reperfusion [8]. Therefore, reversal from sarcolemmal fragility during reperfusion was considered as a primary mechanism of cardioprotection conferred by IPC against oncosis [9].

The pathogenesis of sarcolemmal fragility during reperfusion has been a target of extensive research for many years. The alteration of proteins that link sarcolemma and cytoskeleton has been implicated in this pathogenesis [10,11]. Contractile force generated by actin–myosin interaction is transmitted to the sarcolemma at the lateral costamere junction through the Z-band. Therefore, the physical stress imposed on the fragile sarcolemmal membrane causes its breakage at the site of Z-band attachment. The lateral costamere junction contains a number of structural proteins which link the Z-band and the sarcolemma. Of these, dystrophin serves as a mechanically strong physical linkage between the sarcolemma and the costameric cytoskeleton in the cardiac muscle [12] that stabilizes the sarcolemma from shear stresses imposed during eccentric muscle contraction [13,14]. The absence of the dystrophin gene is associated with vulnerability of the sarcolemma to mechanical force [15,16]. We have recently demonstrated that sarcolemmal dystrophin is translocated from the sarcolemma to the intracellular pool during ischemia and was subsequently lost during reperfusion [17–19]. Reintroduction of contractile activity during reperfusion produced oncosis in myocytes depleted of sarcolemmal dystrophin but not in myocytes replenished with sarcolemmal dystrophin, suggesting that loss of sarcolemmal dystrophin is causally related to reperfusion injury.

There is general agreement that the cardioprotective effect of IPC against ischemia/reperfusion injury is predominantly derived from protection of mitochondria [1–4]. Protection of mitochondria and preservation of mitochondrial function is of prime importance in generating ATP during the post-ischemic reperfusion period. The inability of mitochondria to generate ATP during reperfusion leads to oncosis as a result of sarcolemmal fragility. IPC appears to reverse sarcolemmal fragility during reperfusion by increasing ATP generation by mitochondria. Therefore, we have hypothesized that mitochondrial protection and enhanced generation of ATP mediated by IPC culminates in restoration of sarcolemmal dystrophin and stabilization of the membrane during reperfusion, thereby preventing oncosis induced by the recovery of contractile function upon reperfusion.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Animal preparation and perfusion technique
Male Sprague–Dawley rats weighing 250–300 g were used in the present study. All experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals (NIH publication No. 85-23, revised 1996) and were approved by the Institutional Animal Care Committee of Kansai Medical University (Moriguchi, Osaka).

The isolated and buffer-perfused rat heart preparation was instituted as described [20]. When the buffer contained the contractile blocker 2,3-butanedione monoxime (BDM; 20 mM), an equimolar concentration of NaCl was reduced in the buffer.

2.2 Measurements of left ventricular function
Isovolumic left ventricular (LV) function and coronary flow were measured as described [20].

2.3 Experimental protocol
The experimental protocol was shown in Fig. 1. In the control group, the hearts were subjected to 30 min of normothermic global ischemia followed by reperfusion for 120 min. In the BDM group, BDM was administered for the first 20 min during reperfusion. In the IPC group, IPC was performed by 3 cycles of 5 min ischemia and 5 min reperfusion before 30 min of ischemia. In the IPC/BDM group, IPC was performed as described and BDM was administered for the first 20 min during reperfusion. In the experiments when mitochondrial membrane potential ({delta}{Psi}m) was to be analyzed, tetramethylrhodamine ethylester (TMRE; 0.5 µM, Molecular Probes, Eugene, OR) was administered to the buffer for the first 20 min during reperfusion and excluded for 5 min to washout the dye. TMRE is a red-fluorescing fluorophore that accumulates electrophoretically into mitochondria in response to the negative {delta}{Psi}m and remains bound to the inner mitochondrial membrane until it is gradually lost from the membrane after {delta}{Psi}m is dissipated [21]. In the experiments when myocyte oncosis was to be detected, 0.1% Evans Blue (Sigma, Tokyo, Japan) was administered to the buffer for 20 min at the time of reperfusion or at 20 min after reperfusion and excluded for 5 min to washout the dye. Evans Blue is the membrane impermeable red-fluorescing dyes that accumulate into the cells when the plasma membrane permeability increases. The uncoupler of mitochondrial respiration, 2,4-dinitrophenol (DNP; 0.1 mM, Sigma), and the mitochondrial F1F0-ATPase inhibitor, oligomycin (5 µM, Sigma), were employed for the indicated duration.


Figure 1
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  		Fig. 1 Experimental protocol. Control, ischemia (filled boxes) for 30 min followed by 120 min of reperfusion; IPC, ischemic preconditioning by 3 cycles of 5 min of ischemia and 5 min of reperfusion, BDM, reperfusion with 20 mM 2,3-butanedione monoxime; TMRE, treatment with 0.5 µM tetramethylrhodamine ethylester; EB, treatment with 0.1% Evans blue; WD, withdrawal of BDM.

 
2.4 Creatine kinase assay
Coronary effluent was collected at the indicated time point, and creatine kinase (CK) activity was measured as described [18].

2.5 Infarct size measurements
Infarct size measurements were performed by a triphenyltetrazolium chloride (TTC) staining method as described [18].

2.6 Immunofluorescence microscopy for dystrophin and TMRE
At the end of experiments, myocardial tissue samples were excised and immediately frozen in liquid nitrogen. Immunofluorescence microscopy of dystrophin in the frozen myocardial section was performed as described [18]. To quantify sarcolemmal dystrophin and mitochondrial TMRE fluorescence density, 100 myocytes were randomly selected in the mid LV free wall and the mid myocardial layer by a blind observer. The sarcolemma was identified by immunofluorescence analysis of tetrarhodamine isothiocyanate-conjugated wheat germ agglutinin (Molecular Probes) as described [19]. Then, separated images for dystrophin and TMRE as well as merged images were obtained under the same background intensity. The fluorescence intensity of dystrophin and TMRE was quantified using an image analyzing software (Win Roof, Mitani Co., Fukui, Japan).

2.7 Immunofluorescence microscopy for dystrophin and EB
Immunofluorescence microscopy of dystrophin and Evans Blue was performed as described [17,18]. The number of Evans Blue-positive and -negative myocytes was counted on 60 high power fields (magnification x 600) from the endocardium through the epicardium of the mid LV free wall and the percentage of Evans Blue-positive myocytes were calculated.

2.8 Measurement of myocardial ATP content
The heart was quickly frozen in liquid nitrogen and ATP content of the LV muscle was measured as described [19].

2.9 Measurement of mitochondrial generation of ATP and distribution of dystrophin in vitro
The LV muscle obtained from the non-ischemic control heart and the control and the IPC hearts subjected to 30 min ischemia were homogenized, mitochondria were isolated and ATP generation was measured as described [19]. To examine the role of mitochondrial oxidative phosphorylation and ATP generation in the localization of dystrophin, mitochondria were resuspended in 1 ml of mitochondria-free subcellular fractions obtained from the 30 min ischemic control heart. After removing 0.5 ml of the reconstituted subcellular fractions for the baseline measurement of dystrophin, the fractions were supplemented with 5 mM KH2PO4 and 5 mM MgCl2, 10 mM succinate, and 5 mM ADP for 30 min at 37 °C. The incubation mixture was centrifuged at 100,000 x g for 60 min at 4 °C. The supernatant was designated as a cytosol fraction. The sediment was treated with the extraction buffer containing 250 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 1% Triton X-100, a protease inhibitor cocktail (Complete, Roche Diagnostic, Mannheim, Germany), and 10 mM PIPES, pH 6.8 for 30 min at 4 °C. The supernatant was designated as a soluble fraction, while the sediment designated as an insoluble fraction was treated with RIPA buffer for 30 min at 4 °C. These fractions were applied for immunoblot assay to quantify dystrophin as described [18].

2.10 Statistical analysis
All numerical data are expressed as mean±S.E. Statistical analysis of data between groups was performed by one-way ANOVA followed by the Bonferroni post hoc test. The differences within groups and between groups were analyzed by combination of repeated measures ANOVA with one-way ANOVA followed by the Dunnet and the Tukey post hoc test, respectively. The differences were considered significant at a p value of <0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Sarcolemmal localization of dystrophin depends on mitochondrial oxidative phosphorylation and ATP generation
We have previously shown that IPC had no effect on sarcolemmal localization of dystrophin before the index ischemia nor did it inhibit translocation of sarcolemmal dystrophin during the index ischemia [18], suggesting that translocation of sarcolemmal dystrophin is independent of IPC-mediated alteration of signal transduction [3]. Therefore, metabolic changes were considered as a likely mechanism for the translocation of sarcolemmal dystrophin during the index ischemia. The decline of intracellular pH is one of the major consequences of ischemia. However, when the heart was perfused with KHB buffer at pH 6.4 for 30 min, sarcolemmal localization of dystrophin was not changed (not shown). Then, we thought that the cessation of oxidative phosphorylation and subsequent decline of intracellular ATP may be responsible for dystrophin translocation from the sarcolemma. To test this hypothesis, we used the mitochondrial uncoupling agent DNP to abrogate mitochondrial oxidative phosphorylation, and the mitochondrial F1F0-ATPase inhibitor oligomycin to block ATP synthesis. Dystrophin was lost from the sarcolemma and reciprocally increased in the cytoplasmic region 30 min after ischemia (Fig. 2A) associated with the loss of myocardial ATP (Fig. 2B). Translocation of sarcolemmal dystrophin and the loss of ATP during ischemia were not significantly different between the control ischemic and the IPC ischemic heart. When the heart was perfused with DNP or oligomycin for 30 min, dystrophin was translocated from the sarcolemma to the intracellular pool associated with the decline of myocardial ATP. These changes in the distribution of dystrophin and myocardial ATP content were comparable to those observed in the heart subjected to 30 min of ischemia with or without IPC.


Figure 2
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  		Fig. 2 Ischemic preconditioning (IPC) does not prevent ischemic loss of sarcolemmal dystrophin and the loss of sarcolemmal dystrophin is induced by the cessation of mitochondrial oxidative phosphorylation and ATP generation. (A) Immunohistochemical analysis of sarcolemmal (filled boxes) and cytoplasmic (open boxes) dystrophin. The sarcolemma was identified by tetrarhodamine isothiocyanate-conjugated wheat germ agglutinin (not shown). (B) Myocardial ATP content. Control, non-ischemic control heart; IPC, ischemic preconditioning heart; Control isch, control ischemic heart; IPC isch, IPC ischemic heart; DNP, treatment of non-ischemic heart with 0.1 mM 2,4-dinitrophenol for 30 min; Oligo, treatment of non-ischemic heart with 5 µM oligomycin for 30 min. Bar indicates 20 µm. Each bar graph represents mean±S.E. of 5 experiments. *p<0.05 compared to Control.

 
3.2 IPC increases {delta}{Psi}m and myocardial ATP content and facilitates restoration of sarcolemmal dystrophin during reperfusion
IPC is known to protect mitochondria from ischemia/reperfusion injury [1–4]. Therefore, we investigated whether enhanced restoration of sarcolemmal dystrophin during reperfusion in the IPC heart is associated with enhanced recovery of {delta}{Psi}m and increased myocardial ATP. TMRE was accumulated in mitochondria as a granular pattern throughout the myocytes and dystrophin was exclusively localized in the sarcolemma in the non-ischemic control heart (Fig. 3A). However, TMRE uptake by mitochondria was abolished during reperfusion in the control heart associated with the loss of sarcolemmal dystrophin. TMRE uptake and sarcolemmal localization of dystrophin was increased in the IPC heart during reperfusion, although the recovery of TMRE uptake and sarcolemmal dystrophin was inhomogeneous among the myocytes. Reperfusion with BDM significantly increased TMRE uptake and sarcolemmal localization of dystrophin in the control heart. The effect of BDM on TMRE uptake and sarcolemmal localization of dystrophin was more pronounced in the IPC heart where the recovery of TMRE uptake and sarcolemmal dystrophin was homogeneous among the myocytes. DNP abolished the increase in TMRE uptake and redistribution of dystrophin to the sarcolemma in the IPC heart treated with BDM during reperfusion, while oligomycin maintained TMRE uptake but inhibited relocalization of dystrophin to the sarcolemma. IPC significantly increased myocardial ATP content 20 min after reperfusion and reperfusion with BDM further increased the ATP level in the IPC heart (Fig. 3B). The increase in myocardial ATP after reperfusion with BDM in the IPC heart was abolished by treatment with DNP or oligomycin, suggesting that mitochondrial ATP generation rather than {delta}{Psi}m by itself is a critical determinant for sarcolemmal relocalization of dystrophin during reperfusion.


Figure 3
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  		Fig. 3 Enhanced relocalization of dystrophin to the sarcolemma during reperfusion in the ischemic preconditioning (IPC) heart is not only dependent on {delta}{Psi}m but also dependent on mitochondrial F1F0-ATPase to generate ATP. (A) Immunohistochemical analysis of sarcolemmal dystrophin and tetramethylrhodamine ethylester (TMRE) uptake. The sarcolemma was identified by tetrarhodamine isothiocyanate-conjugated wheat germ agglutinin (not shown). (B) Myocardial ATP content. Control, non-ischemic control heart; Control-R, control heart after reperfusion with normal Krebs–Henseleit bicarbonate (KHB) buffer; IPC-R, IPC heart after reperfusion with KHB; Control-R BDM, control heart after reperfusion with 2,3-butanedione monoxime (BDM); IPC-R BDM, IPC heart after reperfusion with BDM; IPC-R BDM+DNP, IPC heart after reperfusion with BDM and 2,4-dinitrophenol (DNP); IPC-R BDM+Oligo, IPC heart after reperfusion with BDM and oligomycin. Bar indicates 20 µm. Each bar graph represents mean±S.E. of 5 experiments. *p<0.05 compared to Control, {dagger}p<0.05 compared to Control-R, #p<0.05 compared to IPC-R BDM.

 
3.3 Increased membrane localization of dystrophin by ischemic IPC heart mitochondria in vitro is dependent on mitochondrial oxidative phosphorylation and ATP generation
Then, we investigated the role of oxidative phosphorylation and ATP generation of mitochondria in localization of dystrophin in vitro. The non-ischemic control heart mitochondria generated the greatest amount of ATP (Fig. 4A). ATP generation was markedly reduced in mitochondria isolated from the control ischemic heart but IPC significantly improved mitochondrial ATP generation in the ischemic heart. The increased ATP generation of mitochondria isolated from the IPC ischemic heart was abolished by treatment with DNP or oligomycin.


Figure 4
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  		Fig. 4 Mitochondria isolated from the ischemic preconditioning (IPC) heart after 30 min of ischemia increases ATP generation and relocalization of dystrophin from the insoluble to the soluble fractions. (A) Mitochondrial ATP generation. (B) Immunoblot analysis for dystrophin. Baseline, before ATP generation; Control, non-ischemic control mitochondria; Control isch, control ischemic mitochondria; IPC isch, IPC ischemic mitochondria; IPC isch+DNP, IPC ischemic mitochondria treated with 2,4-dinitrophenol; IPC isch+Oligo, IPC ischemic mitochondria treated with oligomycin. Each bar graph represents mean±S.E. of 5 experiments. In panel A, *p<0.05 compared to Control, {dagger}p<0.05 compared to Control isch, #p<0.05 compared to IPC isch. In panel B, *p<0.05 compared to Baseline, {dagger}p<0.05 compared to Control, #p<0.05 compared to Control isch, §p<0.05 compared to IPC isch.

 
The reconstituted subcellular fraction contained dystrophin in the insoluble fraction at an amount comparable to that in the soluble fraction at the baseline (Fig. 4B). Under the ATP-generating condition, the non-ischemic control heart mitochondria increased dystrophin in the soluble fraction at the greatest amount. The increase in dystrophin in the soluble fraction was associated with a reciprocal decrease in dystrophin in the insoluble fraction, indicating that dystrophin was translocated from the insoluble to the soluble fractions. Although the control ischemic heart mitochondria showed no significant increase in dystrophin in the soluble fraction under the ATP-generating condition, the ischemic IPC heart mitochondria significantly increased dystrophin in the soluble fraction and decreased dystrophin in the insoluble fraction compared to the control ischemic heart mitochondria. The enhanced redistribution of dystrophin to the soluble fraction conferred by the ischemic IPC heart mitochondria was abolished by DNP or oligomycin.

3.4 IPC prevents contractile force-induced oncosis during reperfusion
To verify that oncosis is indeed produced in myocytes depleted of sarcolemmal dystrophin associated with the recovery of contractile function upon reperfusion, we investigated correlation between the loss of sarcolemmal dystrophin and myocyte oncosis in the presence or absence of myocardial contraction. No accumulation of Evans Blue was observed in myocytes with homogeneous localization of dystrophin in the non-ischemic control heart showing vigorous beating (not shown). Evans Blue was accumulated in myocytes depleted of sarcolemmal dystrophin in the control reperfusion heart (Fig. 5). The number of myocytes that accumulated Evans Blue during reperfusion was significantly reduced in the IPC heart. Reperfusion with BDM in the control heart abrogated accumulation of Evans Blue in myocytes depleted of sarcolemmal dystrophin. However, reintroduction of contractile activity after the withdrawal of BDM gave rise to accumulation of Evans Blue in myocytes depleted of sarcolemmal dystrophin. Little accumulation of Evans Blue was noted after the withdrawal of BDM in the IPC heart associated with restoration of sarcolemmal dystrophin.


Figure 5
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  		Fig. 5 The ischemic preconditioning (IPC) heart replenishes dystrophin in the sarcolemma during reperfusion with 2,3-butanedione monoxime (BDM) and prevents Evans Blue uptake. Control, non-ischemic control heart; Control-R, control heart after reperfusion without BDM; IPC-R, IPC heart after reperfusion without BDM; Control-R BDM, control heart after reperfusion with 2,3-butanedione monoxime (BDM); Control-R BDM/WD, control heart after reperfusion with BDM followed by its withdrawal; IPC-R BDM, IPC heart after reperfusion with BDM; IPC-R BDM/WD, IPC heart after reperfusion with BDM followed by its withdrawal. Bar indicates 50 µm. Each bar graph represents mean±S.E. of 5 experiments. *p<0.05 compared to control, {dagger}p<0.05 compared to Control-R, #p<0.05 compared to Control-R BDM/WD.

 
3.5 Temporary blockade of contraction during reperfusion inhibits CK release and enhances a protective effect of IPC against infarction and LV dysfunction
Finally, we examined whether temporary blockade of contraction during reperfusion enhances cardioprotective effects of IPC against infarction and LV dysfunction. CK release at 10 and 20 min after reperfusion was comparable between the control and the IPC heart but was significantly reduced in the IPC heart thereafter (Fig. 6A). CK release was abolished during reperfusion with BDM in both the control and the IPC heart. Although CK release was increased after the withdrawal of BDM in the control heart, no appreciable CK release was observed in the IPC heart after the withdrawal of BDM. Temporary blockade of contractility during reperfusion with BDM significantly reduced infarct size in the control heart and completely abolished infarction in the IPC heart (Fig. 6B). BDM reduced LV developed pressure <10% of the baseline during reperfusion (Table 1). Temporary blockade of contractility during reperfusion with BDM increased LV developed pressure and reduced LV end-diastolic pressure after the withdrawal of BDM in both the control and the IPC heart but the effect of BDM on the recovery of LV function was more pronounced in the IPC heart.


Figure 6
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  		Fig. 6 Ischemic preconditioning (IPC) and treatment with 2,3-butanedione monoxime (BDM) during reperfusion synergistically inhibits creatine kinase (CK) release and infarction. (A) CK release. Pre-I indicates pre-ischemia and BDM indicated 2,3-butanedione monoxime. Filled circle, control heart; open circle, ischemic preconditioning (IPC) heart; filled triangle, control heart reperfused with BDM; open triangle, IPC heart reperfused with BDM. Each symbol represents mean±S.E. of 6 experiments. *p<0.05 compared to control heart, {dagger}p<0.05 compared to IPC heart. (B) Infarct size. Control, control heart after reperfusion; IPC, IPC heart after reperfusion; BDM, control heart after reperfusion with BDM followed by perfusion with Krebs–Henseleit bicarbonate (KHB) buffer; IPC BDM, IPC heart after reperfusion with BDM followed by perfusion with KHB buffer. *p<0.05 compared to Control, {dagger}p<0.05 compared to IPC. Each bar graph represents mean±S.E. of 6 experiments.

 

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  		Table 1 Left ventricular function

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
Our previous studies [17–19] suggested that loss of sarcolemmal dystrophin is causally related to myocardial reperfusion injury. The present study confirmed those earlier findings and suggests further that enhanced relocalization of dystrophin to the sarcolemma during reperfusion may be a mechanistic link between improved mitochondrial function and protection against oncosis conferred by IPC. The salient findings to support this hypothesis were (1) the decrease in sarcolemmal dystrophin during 30 min ischemia was comparable between the control and the IPC heart, (2) however, the IPC heart restored sarcolemmal dystrophin during reperfusion associated with the increase in {delta}{Psi}m and myocardial ATP especially when the heart maintained mechanical arrest by treatment with BDM, (3) the enhanced restoration of sarcolemmal dystrophin in the IPC heart was abolished by DNP, the uncoupler of mitochondrial respiration, or by oligomycin, an mitochondrial ATP synthase inhibitor, (4) when the role of mitochondrial oxidative phosphorylation and ATP generation in the distribution of dystrophin was investigated in vitro, ATP generation and relocalization of dystrophin to the soluble fraction was significantly increased by the ischemic IPC heart mitochondria, (5) this enhanced ATP generation and relocalization of dystrophin to the soluble fraction by the ischemic IPC heart mitochondria was blocked by DNP or oligomycin, (6) finally, the enhanced relocalization of dystrophin to the sarcolemma in the IPC heart was associated with the prevention of contractile force-induced myocyte oncosis. These observations suggest that relocalization of dystrophin to the sarcolemma during reperfusion depends on mitochondrial oxidative phosphorylation and ATP generation and may represent an end-effect of IPC against myocyte oncosis.

Accumulating evidence indicates that cardioprotective signal transduction derived from IPC converges on protection of mitochondria [1–4]. Myocardial ischemia and reperfusion imposes oxidative stress and Ca2+ overload on mitochondria that induces the mitochondrial permeability transition [22,23]. Opening of the mitochondrial permeability transition pore not only induces apoptosis by releasing pro-apoptotic factors such as cytochrome c and apoptosis-inducing factor from the inter-membranous space into the cytosol but also induces necrosis by abolishing {delta}{Psi}m and myocardial ATP generation [24]. It is known that the cardioprotective effect of IPC is manifested via inhibition of the mitochondrial permeability transition [25]. In our experimental model, the fact that IPC increased mitochondrial TMRE uptake in vivo and mitochondrial ATP generation in vitro suggests that IPC-induced improvement of mitochondrial function may be mediated by inhibition of the mitochondrial permeability transition.

The present study demonstrated that the increase in {delta}{Psi}m and myocardial ATP, the restoration of sarcolemmal dystrophin and the inhibition of oncosis conferred by IPC were synergistically enhanced by temporary blockade of contractility by treatment with BDM during reperfusion. Although care must be taken to interpret the results obtained from the experiments using BDM because this agent acts as an organic phosphatase to exert multiple side effects [26], these observations suggest that a combined effect of IPC and the contractile blockade promotes robust protection against oncosis during reperfusion. It should be noted that CK release during reperfusion was peaked at 40 min after reperfusion in the control heart, while it was peaked at 10 min after reperfusion in the IPC heart. Thus, the critical time window of protection against oncosis by reperfusion with BDM in the IPC heart was shifted to earlier reperfusion time than the control heart. This shift of the time to peak CK release may be attributed to enhanced recovery of contractility in the IPC heart that imposed stronger physical stress on the sarcolemma before restoration of sarcolemmal dystrophin. Although IPC is known to reduce ultimate infarct size, temporal differences in the extent of oncosis in the IPC heart have been paid little attention. The fact that myocyte oncosis occurred predominantly for the first 20 min during reperfusion in the IPC heart and virtually no infarction was produced when the IPC heart was treated with BDM during this period suggest that considerable extent of reperfusion injury does exist in the IPC heart during the early phase of reperfusion and it can be prevented by temporary blockade of contractility upon reperfusion. Although it is still premature to conclude that restoration of sarcolemmal dystrophin during reperfusion is an obligatory end-effect of cardioprotection conferred by IPC, temporal relationship between restoration of sarcolemmal dystrophin and prevention of contractile force-induced oncosis during reperfusion in conjunction with the evidence that relocalization of dystrophin to the sarcolemma depends on mitochondrial oxidative phosphorylation and ATP generation lends support to the hypothesis that dystrophin is an end-target of IPC against myocyte oncosis during the early phase of reperfusion.

The mechanism by which improved oxidative phosphorylation and enhanced ATP generation by mitochondria facilitated relocalization of dystrophin to the sarcolemma is unknown at present. However, the facts that dystrophin localization in the sarcolemma correlates with a myocardial ATP level in vivo and translocation of dystrophin from the insoluble to the soluble fractions depends on mitochondrial ATP generation in vitro suggest that myocardial energy status is a critical determinant of intracellular distribution of dystrophin. Similar ATP-dependent redistribution was reported in the case of connexin 43 which is a phosphoprotein localized in gap junction and plays an important role in regulating electrical and chemical communications between the adjacent myocytes. It has been demonstrated that phosphorylation of connexin 43 was exquisitely sensitive to myocardial ATP levels so that when ATP fell below a critical threshold during ischemia or hypoxia, connexin 43 was dephosphorylated and translocated from the gap junction to the intracellular pool [27,28]. Dystrophin is also a phosphoprotein that has abundant phosphorylation sites [29]. However, because the vast majority of kinases involved in protein phosphorylation have a Km for ATP in the low micromolar range and a cellular ATP level during ischemia and reperfusion is maintained at far above this level, it is unknown whether there is such a kinase of which activity is regulated by physiological fluctuations of intracellular ATP and is involved in phosphorylation and sarcolemmal localization of dystrophin. In addition, phosphatases may be involved in redistribution of dystrophin during ischemia. However, treatment with the protein phosphatase 1/2A inhibitor, calyculin, protein phosphatase 2B inhibitor, FK506, or tyrosine phosphatase inhibitor, sodium vanadate, at concentrations that known to inhibit these phosphatases in the isolated and perfused rat heart did not inhibit ischemic translocation of dystrophin from the sarcolemma in our system (data not shown), suggesting that known phosphatases are not involved in redistribution of dystrophin during ischemia. Because many cellular events are regulated by ATP, the exact mechanism for ATP-dependent sarcolemmal localization of dystrophin remains a subject for future studies.

The present study demonstrated that relocalization of dystrophin to the sarcolemma and prevention of oncosis during reperfusion in the IPC heart is dependent on oxidative phosphorylation and ATP generation by mitochondria. Therefore, it is anticipated that any interventions aimed at protecting mitochondria and improving mitochondrial function would cause the same effect as IPC. Moreover, it is suggested that synergistic protection against oncosis by IPC in combination with BDM treatment during reperfusion is not specific for IPC but may be a general phenomenon when mitochondria are protected and myocardial contraction is temporary blocked during reperfusion until the sarcolemma is replenished with dystrophin. This is because the recovery of contractility upon reperfusion, especially when it is increased, disrupts the sarcolemma which is not yet replenished with dystrophin, leading to oncosis associated with irreversible damage on mitochondria even though they have been protected from the opening of the permeability transition pore and have acquired the potential to perform sufficient oxidative phosphorylation to prevent oncosis. Our previous study [19] demonstrated that treatment with the p38 MAP kinase inhibitor SB203580 alone during reperfusion increased contractility but aggravated oncosis in myocytes depleted of sarcolemmal dystrophin, while temporary blockade of contractility by BDM or the ultra-short-acting β-blocker during treatment with SB203580 unmasked a protective effect of this SB compound against myocyte oncosis by improving mitochondrial function and increasing myocardial ATP associated with enhanced relocalization of dystrophin to the sarcolemma. Taken together, the combination of biochemical interventions that protect mitochondria with mechanical interventions that prevent disruption of the sarcolemma until regaining stability of the sarcolemma may represent a novel paradigm of cardioprotection against myocyte oncosis during the early phase of reperfusion. This hypothesis should be tested by various animal models and proven in the clinical arena.

In conclusion, dystrophin is a possible end-target of IPC and enhanced relocalization of dystrophin to the sarcolemma during reperfusion may be a mechanistic link between improved mitochondrial function and protection against oncosis conferred by IPC.


   Notes
 
Time for primary review 42 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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Bringing preconditioning and postconditioning into focus
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