© 2001 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Direct activation of mitochondrial KATP channels mimics preconditioning but protein kinase C activation is less effective in middle-aged rat hearts
Department of Geriatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-Ku, Tokyo 160-8582, Japan
* Corresponding author. Tel.: +81-3-3353-1211; fax: +81-3-5269-2468 masatota{at}mc.med.keio.ac.jp
Received 23 August 2000; accepted 21 September 2000
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
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Objectives: This study is aimed to determine whether loss of preconditioning (IP) effects in the middle-aged hearts (MA) is due to the failure of protein kinase C (PKC) activation and, if so, whether direct activation of mitochondrial ATP-sensitive potassium channels (m-KATP) or PKC mimics IP. Background: PKC is a mediator and m-KATP may be its downstream effector of IP in young adult hearts (YA), but we have demonstrated that IP is not effective in MA. Methods and results: Isolated hearts from YA (12-week) and MA (50-week) Fischer 344 rats were preconditioned by three cycles of ischemia and reperfusion (5 min each), and the translocation of PKC isoforms and the effects on reperfusion injury were assessed. In some hearts activation of m-KATP or PKC by diazoxide or 1,2-dioctanoyl glycerol (DOG) was performed before 25 min of global ischemia/30 min of reperfusion. IP could improve the recovery of LV function and resulted in higher content of ATP after reperfusion in YA but these beneficial effects of IP was not found in MA. The effects of IP in YA were abolished by 5-hydroxydecanoate. In YA but not in MA, immunohistochemical analysis revealed that IP translocated PKC-
and 
from the cytosolic or membrane to the perinuclear region but immunoblotting analysis showed translocation of PKC-, and 
to the membrane fraction. Pretreatment with diazoxide or DOG mimicked IP and decreased the creatine kinase release in YA. Diazoxide was also effective but effects of DOG were less in MA as compared with in YA. Conclusions: IP is not effective in MA hearts partly due to failure of translocation of PKC isoforms. Moreover, less efficacy of PKC activation by DOG as compared with activities of m-KATP by diazoxide in MA may suggest that defect(s) of cell signaling downstream to PKC may also be involved in the loss of IP effects in MA.
KEYWORDS ATP, adenosine triphosphate; CK, creatine kinase; DOG, 1,2-dioctanoyl-sn-glycerol; dP/dt, the first derivative of left ventricular pressure; 5HD, 5-hydroxydecanoate; IP, ischemic preconditioning; KATP, ATP-sensitive potassium channel(s); LVDP, left ventricular developed pressure; LVEDP, left ventricular end-diastolic pressure; PKC, protein kinase C
This article is referred to in the Editorial by S. Pepe (pages 11–14) in this issue.
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Ischemic (IP) or hypoxic preconditioning does not improve postischemic performance in rat hearts of the senescent [1], although both protect hearts of young adult rats. Recently, we found that IP failed to protect the middle-aged rat hearts [2], although ischemic heart disease in the middle-aged is a most crucial problem clinically. Numerous mechanisms have been proposed to be responsible for IP. Several of these mechanisms may play important roles and can interact in the setting of IP. Recently, several reports have suggested that protein kinase C (PKC) is a mediator of IP via stimulation by endogenous transmitters [3].
Cardiac sarcolemmal ATP-sensitive potassium channels (KATP) have been proposed to be a downstream mediator or an effector of PKC because the effects of IP are reversed by KATP blockers and KATP openers can mimic IP in many species including humans [4,5]. However, recent studies have demonstrated that the IP mimicking effects of KATP openers are not due to the shortening of the action potential duration caused by opening of sarcolemmal KATP [6]. Moreover, both IP and KATP openers are protective in both unstimulated cardiac myocytes [7] and globally ischemic isolated hearts arrested with high K+ cardioplegic solutions [8]. Another isoform of KATP was found in the inner mitochondrial membrane of cardiac myocytes [9]. The responses of mitochondrial KATP to pharmacological agents are similar to those of sarcolemmal KATP, although with different sensitivities and specificities [10,11]. Therefore, the mitochondrial but not the sarcolemmal KATP can be downstream effectors of PKC during IP.
The objectives of the present study were to determine whether loss of IP effects in middle-aged hearts is attributable to failure of PKC isoform activation and, if so, whether direct activation of PKC or mitochondrial KATP can mimic IP effects in middle-aged hearts.
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2 Methods |
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All the procedures in this investigation conformed to the principles outlined in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). We obtained male Fischer 344 rats from Charles River Japan Inc. Thirty percents of the colony of rats are expected to die in 110 weeks. One hundred eighteen young adult (12 weeks; weight: 190–230 g) and 118 middle-aged (50 weeks; weight: 330–360 g) rats were anesthetized by an intraperitoneal injection of sodium pentobarbital (40 mg/kg).
2.1 Preparation and instrumentation of perfused hearts
Hearts were removed and perfused with a modified Krebs–Henseleit buffer (118 mmol/l NaCl, 25 mmol/l NaHCO3, 4.7 mmol/l KCl, 1.2 mmol/l MgSO4, 1.2 mmol/l KH2PO4, 1.75 mmol/l CaCl2, 0.5 mmol/l EDTA, 11 mmol/l glucose, 5 mmol/l pyruvate) gassed with 95% O2:5% CO2 at 37°C according to the Langendorff technique (coronary perfusion pressure: 70 mmHg) [12]. The left ventricular end-diastolic pressure (LVEDP) was adjusted to 10 mmHg by filling the latex balloon with water during atrial pacing at 5 Hz. An epicardial electrocardiogram was recorded. Indices of left ventricular function (systolic pressure, LVSP; developed pressure, LVDP: LVSP–LVEDP; and peak positive dP/dt and peak negative dP/dt) were recorded under control conditions.
2.2 Perfusion protocols (Fig. 1)
2.2.1 Analysis of PKC translocation by IP or DOG
In five hearts from each age group, IP (three cycles of ischemia and reperfusion, 5 min each), or 10 min pretreatment with 1,2-dioctanoyl glycerol (DOG), a PKC activator, and 10 min washout were performed (A2, A3). Another five hearts were perfused for 30 min and served as control (A1).
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) Normoxic perfusion without any agents; (
) normoxic perfusion with vehicle and/or agents; (
) total global ischemia.2.2.2 Effect of diazoxide, DOG or 5-hydroxydecanoate (5HD) on non-ischemic hearts
The effects of diazoxide (20 µmol/l), an mitochondrial KATP opener, DOG (10 µmol/l), or 5HD (100 µmol/l), a specific mitochondrial KATP blocker, on left ventricular function, energy metabolites and the electrocardiogram were assessed in non-ischemic hearts from each age group. We used diazoxide because the K1/2 of diazoxide for mitochondrial KATP activation is 2000 times lower than for sarcolemmal KATP [10,11]. Diazoxide or 5HD were freshly dissolved in methanol while DOG was dissolved in DMSO. The final concentration of vehicles in the perfusate was 0.1 or 0.01%, respectively. Hearts were perfused with vehicle (methanol), diazoxide or 5HD for 5 min (B1) or with vehicle (DMSO) or DOG for 10 min (B2), followed by 55 min perfusion with no agents (n = 3 for each).
2.2.3 Effects of diazoxide, DOG or 5HD on ischemic-reperfused hearts
Eighty-eight hearts from each age group were used for the ischemia–reperfusion protocol. Eight hearts were perfused with vehicle or 5HD for 5 min and then subjected to 25 min of ischemia followed by 30 min of reperfusion (C1 or C2, n = 4 for each). IP was performed in eight hearts and 25 min of ischemia and 30 min of reperfusion were followed (C3 or C4). During the final 5 min of reperfusion before sustained ischemia, 5HD was administered in four hearts (C4). After 30 min of reperfusion following ischemia the hearts were frozen with Wollenberger clamps.
The remaining 72 hearts were pretreated with methanol, diazoxide, 5HD or diazoxide+5HD for 5 min (D1) or with DOG for 10 min (D2) before the institution of sustained ischemia. Four hearts from each age group undergoing each pretreatment were frozen at the end of 25 min of ischemia, and the other eight hearts were frozen after 30 min of reperfusion.
Electrical pacing was discontinued during ischemia and was reinstated after 25 min of reperfusion. After recording the elevation of the LVEDP, the postischemic recovery of LV function at 10 mmHg of LVEDP was measured.
2.3 Immunohistochemical and immunoblotting analysis of PKC isoforms
Control and preconditioned hearts (Fig. 1A, n = 5 for each group) were blotted and the ventricles were separated and divided into two pieces. One half of the ventricle was immersed in OCT compound (Sakura Finetechnical Co., Ltd., Tokyo) and rapidly frozen in liquid nitrogen. After sectioning (5 µm) with a cryostat, the specimens were fixed on slides for 10 min at –20°C with 70% acetone/30% methanol, washed with PBS (pH 7.4) three times for 5 min, and blocked with 1.5% normal serum in PBS for 20 min. The specimens were incubated overnight at 4°C with specific rabbit polyclonal IgG antibodies raised against PKC isoforms (, , or 
, 1:1000 dilution) suspended in 1% BSA in PBS and then immunostained using the Vectastatin ABC AP kit (AK-5001) and vector-red alkaline phosphatase substrate kit (SK-5100) (Vector Lab. Inc., Burlingame, CA). The specimens were dehydrated with ethanol and xylene and mounted on a slide glass for fluorescent microscopic analysis (Axiovert S100 microscope, Carl Zeiss Inc., Tokyo).
The other half of the ventricle was minced and homogenized at 4°C in 2 ml of isolation buffer containing 320 mmol/l sucrose, 1 mol/l Tris–HCl (pH 7.4), 1 mmol/l EGTA, 2 mmol/l EDTA, 5 mmol/l NaN3, 50 mmol/l NaF, 10 mmol/l β-mercaptoethanol, 20 µmol/l leupeptin, 0.2 mmol/l phenylmethane sulfonylfluoride, 0.15 µmol/l Pepstatin A using a Polytron homogenizer at the maximum speed (PT1200, Kinematica AG, Littau) for 15-s bursts. The same amount of buffer was added to the homogenate and was then centrifuged at 1000xg for 10 min and the pellet was designated the nuclear fraction (N). The supernatant was sonicated and centrifuged at 100 000xg for 60 min. The resulting supernatant was designated the cytosolic fraction (C) while the pellet was designated the membrane fraction (M). Protein concentrations were determined by the method of Bradford [13].
The cellular fractions were diluted with a buffer (pH 6.8) containing 100 mmol/l Tris–HCl, 4% SDS, 20% glycerol, 10% mercaptoethanol, and 0.2% bromophenol blue. The samples were subjected to 7.5% SDS–polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. The blots were blocked overnight with 5% skim milk in buffer containing 137 mmol/l NaCl, 20 mmol/l Tris–HCl (pH 7.4), and 0.2% Tween-20, and then incubated with one of the rabbit polyclonal antibodies raised against PKC isoforms (, and , 1:1000 dilution) for 2.5 h at room temperature. The PKC isoforms were detected using the Vistra ECF Western blotting kit (Amersham Inc., Tokyo). The amounts of PKC isoforms in immunoblots were measured by densitometry (Densitograph AE-6900, Atto) and were corrected for the variation in protein recovery. Total PKC activity was measured at 25°C using a non-radioactive protein kinase assay kit (Calbiochem-Novabiochem Corp., San Diego, CA).
2.4 Analysis of energy metabolites and collection of released creatine kinase (CK)
The neutralized perchloric acid extracts of ventricles were obtained for assay of ATP, creatine phosphate, and lactate using standard enzymatic procedures [12]. The coronary effluents obtained during the 10 min of preischemic perfusion and the 30 min of reperfusion were assayed for the release of CK from the myocytes (n = 8).
2.5 Materials and chemicals
All chemicals were obtained from Sigma–Aldrich Japan Inc. (Tokyo) and Research Biochemicals International (Natick, MA) unless stated otherwise. Rabbit polyclonal IgG antibodies directed against PKC isoforms were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
2.6 Statistical analysis
Data are expressed as the mean±S.E. Comparisons between groups or between time points in a group were performed using two-way analysis of variance (ANOVA) followed by Tukey's test. A value of P<0.05 was considered as statistically significant.
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3 Results |
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3.1 Effects of vehicles, 5HD, diazoxide, or DOG on perfused hearts
Preliminary studies demonstrated that diazoxide, 5HD or vehicles did not change LV function, heart rate, or electrocardiographic morphology, although creatine phosphate content was lower in normoxic hearts pretreated with 5HD (data not shown).
The baseline values of ATP, creatine phosphate, lactate or the release of CK before ischemia did not differ among all groups of hearts in young adult and middle-aged hearts (data not shown).
3.2 Effects of IP and/or 5HD on recovery of LV function, energy metabolite contents and release of CK in reperfused hearts
In the ischemia–reperfusion experiments, the two age groups in any protocols did not differ significantly with respect to indices of left ventricular function during pre-ischemic perfusion but the recoveries of all the functional indices after 25 min of reperfusion in the non-preconditioned hearts were significantly lower in middle-aged than in young adult hearts (Table 1). IP improved the functional recovery in young adult hearts, although it didn't change, or even impaired in middle-aged hearts. 5HD did not change left ventricular function before ischemia nor did it impair the functional recovery in both age groups. However, 5HD reversed the IP effects on the functional recovery in young adult hearts.
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In hearts treated with vehicle or 5HD, neither the content of ATP, creatine phosphate, nor lactate at the end of ischemia differed significantly between both age groups (data not shown).
IP resulted in higher CP content at the end of reperfusion in young adult hearts (17.7±2.9 to 25.6±2.9 µmol/g dry, P<0.05) and decreased release of CK (267±53 to 143±26 U/g, P<0.05) during 30 min of reperfusion when compared to young adult hearts without IP. However, IP did not change the contents of metabolites or the release of CK in middle-aged hearts (data not shown). Pretreatment with 5HD did not affect the content of metabolites or the release of CK in non-preconditioned hearts from either age group but reversed the beneficial effects of IP in young adult hearts (CP; 17.3±2.9 µmol/g dry, CK release; 256±72 U/g).
3.3 Effects of diazoxide, or DOG on recovery of LV function, energy metabolite contents and release of CK in reperfused hearts
Diazoxide improved the functional recovery in both young adult and middle-aged hearts. although left ventricular function was still lower in middle-aged hearts (Table 2). In addition, DOG was less effective than diazoxide, especially in middle-aged rat hearts. Pretreatment with 5HD did not change the functional recovery, although it reversed the beneficial effects of diazoxide in both age groups.
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In hearts treated with vehicle, 5HD, diazoxide, 5HD+diazoxide or DOG, neither the content of ATP, creatine phosphate, nor lactate at the end of ischemia differed significantly between the various groups (data not shown).
The content of energy metabolites during the 30 min of reperfusion was slightly higher with diazoxide, and was associated with a reduction of the release of CK in both young adult and middle-aged hearts (young adult: ATP 9.3±3.2 to 15.2±2.1, CP 16.1±3.5 to 29.8±3.1 µmol/g dry, CK release 278±67 to 116±32 U/g; middle-aged: ATP 7.8±1.3 to 14.2±4.1, creatine phosphate 14.3±3.1 to 29.6±2.7, lactate 28.3±9.1 to 10.7±2.3 µmol/g dry, CK release 416±38 to 186±33 U/g, P<0.05). The addition of 5HD to diazoxide reversed these beneficial effects (young adult: ATP 8.9±1.4, creatine phosphate 15.1±3.0 µmol/g dry, CK release 267±56 U/g; middle-aged: ATP 8.3±1.9, creatine phosphate 15.6±3.3, lactate 26.4±4.7 µmol/g dry, CK release 398±57 U/g), although the administration of 5HD alone did not have harmful effect (data not shown). DOG also resulted in higher content of metabolites (young adult: creatine phosphate 15.6±3.3 to 23.8±3.1 µmol/g dry; middle-aged: creatine phosphate 13.6±3.3 to 21.7±2.6 µmol/g dry, P<0.05) and decreased release of CK (young adult: 304±42 to 212±28 U/g; middle-aged: 436±44 to 306±38 U/g, P<0.05) but the effects were modest in both age groups.
3.4 Effects of IP or DOG on the translocation of PKC isoforms
The main staining site was determined as membrane (Fig. 2B), cytosol (Fig. 2C) or nucleus (actually stained in perinuclear region but also usually sarcolemma) (Fig. 2D) as compared with negative control (Fig. 2A).
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PKC-
and localized primarily to the cytosol, while PKC- and localized predominantly to the sarcolemmal membranes (Table 3, Fig. 2B) in young adult hearts without IP. IP translocated PKC- from the cytosol to the sarcolemmal membranes and PKC- to the perinuclear and membrane regions (Fig. 2D). In contrast, PKC- and were diffusely distributed in the cytosol. In middle-aged hearts, PKC-, , and were present predominantly in the cytosol and PKC- was present in the membrane (Fig. 3A,B). IP didn't translocate PKC isoforms, except for PKC-, in middle-aged hearts. DOG translocated PKC-, and to the perinuclear or membrane regions in both young adult and middle-aged hearts (Fig. 3C–F).
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IP or DOG didn't change total PKC activity of each subcellular fraction in both young adult and middle-aged hearts (data not shown).
The localization of PKC isoforms in subcellular fractions of control, preconditioned and DOG-treated hearts also was determined by immunoblot analysis (Fig. 4A–H). PKC isoforms in young adult and middle-aged localized more predominantly to the cytosolic than membrane or nuclear fraction, although PKC- localized less to the cytosolic fraction of both young adult and middle-aged hearts before IP. After IP, location of PKC-, and in the membrane fraction from young adult hearts became more prominent while those in the nuclear fraction decreased (Fig. 4A,C). No significant changes in the distribution of PKC isoforms were found in middle-aged hearts after IP, although PKC- increased while PKC- decreased in the membrane fraction of middle-aged hearts (Fig. 4B,D). DOG translocated PKC- and to the nuclear or membrane fraction in both young adult and middle-aged hearts (Fig. 4E–H) while no significant change was observed in PKC-.
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Others and we have shown that IP could not protect middle-aged or senescent rat hearts against ischemia–reperfusion injury [1,2]. The present study confirmed these previous reports and is the first to demonstrate that loss of IP effects in middle aged hearts can be attributed, in part, to the failure to activate PKC. The present study also demonstrates that inhibition of mitochondrial KATP by 5HD abolishes the beneficial effects of IP in young adult rat hearts in which IP activates PKC isoforms. Moreover, direct activation of PKC or mitochondrial KATP mimics IP effects, although PKC activation is less effective, especially in middle-aged hearts. 5HD also reverses the beneficial effects of mitochondrial KATP activation by diazoxide. These results suggest that the IP cascade downstream and/or upstream from PKC is defective in middle-aged hearts.
4.1 Possible mechanisms responsible for the loss of PKC activation
Among the numerous mechanisms proposed for IP, a number of recent studies have suggested that PKC activation is an important mediator in rats and rabbits via receptor stimulation by endogenous transmitters [3]. These include adenosine, endothelin, bradykinin, and catecholamines. Adenosine and norepinephrine are the most likely candidates, acting through adenosine A1- and/or A3- [14,15], and 1-adrenergic [16] receptors, respectively. However, stimulation of 1-adrenergic receptors and adenosine A1 receptors may activate PKC independently in rat hearts [3]. An age-related decrease in norepinephrine content and/or its production [17] has been reported, which may cause a decrease in norepinephrine release and therefore less activation of PKC during IP. In contrast, an increase in the adenosine content of aging myocardium has been reported [18]. Elevated tissue adenosine concentrations suggest the possibility that downregulation of adenosine A1-receptors may occur. Recently, Cai et al. [19] reported reduced adenosine A1-receptor and G protein coupling in rat ventricular myocardium during aging, although several reports indicate that adenosine A1- or 1-receptors cannot play a role in IP in rats [20,21].
4.2 Role of PKC isoforms in IP
At least nine to 11 isoforms of PKC are present in myocardial tissue [22,23]. Their subcellular localization and/or redistribution by activation can be characterized by substrate specificity in vivo for each isoform [24,25]. The physiologic roles of PKC in the maintenance of cellular ion homeostasis and the regulation of myocardial contractile elements have been characterized. These effects include modification of the Na+/H+ exchanger, L-type Ca2+ channels, phospholamban, troponin I, troponin T, myosin light chain 2, and cytoskeleton. However, the specificity of each PKC isoforms for these substrates remains unclear.
Recently, direct evidence of translocation of PKC isoforms by both ischemia and hypoxia has been reported [23,26], although there are significant differences in isoforms activated by IP. This may be partly due to difference in the methods for detection of translocation because we found some disagreement between immunohistochemical and immunoblotting analysis probably due to preparation procedure of subcellular fractions in the latter during which PKC translocated to the perinuclear region might be detached and creep into the cytosolic fraction. Also, there are species differences in the pattern of isoform expression and/or distribution in the cell [23,26]. In rats, PKC-, or is activated by IP [26], which is consistent with the data in the present study. In contrast, PKC- and translocate in response to IP in rabbits [23]. In addition, sustained PKC translocation may not be necessary for protection in rabbit myocytes [27], suggesting that translocation of PKC isoforms is transient and only a trigger for the cascade of IP.
Translocation of PKC- from cytosol to sarcolemmal membrane by 1-adrenergic stimulation in rat hearts [28] has been reported to inhibit the decrease in intracellular pH during ischemia, which may contribute to the reduction of intracellular calcium overload via H+/Na+/Ca2+ exchange [12]. PKC- has also been reported to have a regulatory effect on contractility by translocation to the myofilaments [24]. Activation of PKC- by phorbol esters also results in attenuation of changes in intracellular pH and cellular protection while activation of PKC- by adenosine provides protection against myocardial stunning in rat hearts [29]. However, PKC activation by DOG, but not by phorbol esters, changed the intracellular pH in hearts. Chelerythrine attenuates this effect of DOG on intracellular pH but not the IP-induced attenuation in intracellular pH decrease [30]. IP activates PKC isoforms that cannot be translocated by DOG. The present study also showed translocated sites of PKC- and by DOG may be different from those by IP because Western blot analysis revealed that DOG translocated PKC- and/or mainly to the nuclear fraction in both young adult and middle-aged hearts while IP increased those isoforms in the membrane fraction but decreased in the nuclear fraction. We might speculate DOG firmly attached these isoforms to perinuclear region or translocated to intranucleus while IP caused their loose attachment to perinuclear region. Therefore, it is plausible that DOG cannot provide full protection obtained by IP in young adult hearts. In addition, less protective effects in middle-aged than young adult hearts may imply some defects in signal transduction with aging. Recently, Ladilov et al. [31] have demonstrated that stimulation of PKC by DOG before and during anoxia reduced intracellular calcium overload without significant effect on intracellular pH change, probably through altering the influx of calcium across the sarcolemma in rat myocytes. Gwathmey and Hajjar [32] have reported that activation of several PKC isoforms, including PKC- and , by phorbol esters inhibits Ca2+ release from the sarcoplasmic reticulum of human hearts. Inappropriate opening of sarcoplasmic reticulum Ca2+ release channels has been shown to be inhibited not only by IP but also by hypoxic preconditioning [2,33,34].
4.3 Role of mitochondrial KATP in IP
Sarcolemmal KATP has been proposed to be the effector of IP since KATP openers mimic and KATP inhibitors block IP [3]. Shortening of the action potential by opening of sarcolemmal KATP would cause less influx of Ca2+ during the period and depression of contractility, which has been proposed to be the mechanism responsible for protection by IP [3]. However, evidences refuting this hypothesis include dissociation of the degree of action potential shortening from the extent of myocyte protection [6] and occurrence of both IP and the IP mimicking protection of KATP openers in quiescent myocytes [7,8]. The existence of another type of KATP in the inner mitochondrial membrane has been reported [9] and low concentrations (1–100 µmol/l) of diazoxide open mitochondrial KATP selectively in intact cells while sarcolemmal KATP are minimally affected by these concentrations of the drug [10,11]. In the present study, the IP mimicking effects of diazoxide in middle-aged hearts were obtained at a concentration of 20 µmol/l at which the drug had no significant effects on the epicardial electrocardiogram, heart rate, or LV function. These IP mimicking effects of diazoxide were reversed by low concentrations of 5HD (100 µmol/l), a selective blocker of mitochondrial KATP [11]. Although indirect evidence of a mechanistic link between signal transduction of IP via PKC activation and mitochondrial KATP was provided [11], questions remain as to which PKC isoform(s) are responsible for IP. The opening of mitochondrial KATP would dissipate the mitochondrial membrane potential maintained by the proton pump so that activation of mitochondrial KATP should be an important signal in regulating cellular bioenergetics through acceleration of electron transfer by the respiratory chain, promotion of the binding of endogenous mitochondrial ATPase inhibitor IF1, and control of mitochondrial volume changes [35,36]. The dissipation of the mitochondrial membrane potential could decrease the driving force for calcium influx through the calcium uniporter. Several previous reports support this hypothesis because selective inhibition of mitochondrial calcium uptake protects isolated rat hearts against ischemia–reperfusion injury [37,38].
In conclusion, we confirmed the previous reports that IP could protect young but not middle aged rat hearts. However, direct activation of mitochondrial KATP using diazoxide mimicked IP in both age groups and effects of both IP and mitochondrial KATP activation were abolished by mitochondrial KATP blocker 5HD, which supports a pivotal role of mitochondrial KATP in IP. On the other hand, PKC activation with DOG mimicked IP to a lesser degree in middle aged rat hearts, suggesting that defect(s) of cell signaling downstream to PKC may also be involved in the loss of IP effects in MA.
Time for primary review 25 days.
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Acknowledgements |
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This study was supported by grants from the Ministry of Education, Science, Sports and Culture, Japan (Tokyo), from the Keio Health Counseling Center, Tokyo, Japan, and the Sumitomo Marine Welfare Foundation.
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