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Cardiovascular Research 2008 77(2):398-405; doi:10.1016/j.cardiores.2007.07.015
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©European Society of Cardiology 2007. All rights reserved.For permissions please email: journals.permissions@oxfordjournals.org

Infarct size limitation by adrenomedullin: protein kinase A but not PI3-kinase is linked to mitochondrial KCa channels

Hirofumi Nishida1,2, Toshiaki Sato1,*, Masaru Miyazaki2 and Haruaki Nakaya1

1 Department of Pharmacology, Chiba University Graduate School of Medicine, Chiba, Japan
2 Department of General Surgery, Chiba University Graduate School of Medicine, Chiba, Japan

* Corresponding author. Department of Pharmacology, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. Tel: +81 43 226 2051; fax: +81 43 226 2052.E-mail address: tsato{at}faculty.chiba-u.jp (T. Sato).

Received 27 March 2007; revised 25 July 2007; accepted 26 July 2007

Time for primary review: 29 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 Acknowledgments
 References
 
Aim: Adrenomedullin (ADM) has been shown to protect the heart against ischaemic injury, but little is known of the underlying mechanism. Mitochondrial Ca2+-activated K+ (mitoKCa) channels play a key role in cardioprotection. This study examined whether mitoKCa channel is involved in the protection afforded by ADM.

Methods: Flavoprotein fluorescence in rabbit ventricular myocytes was measured to assay mitoKCa channel activity. Infarct size in the isolated perfused rabbit hearts subjected to 30-min global ischaemia and 120-min reperfusion was determined by triphenyltetrazolium chloride staining.

Results: The mitoKCa channel opener NS1619 (30 µM) partially oxidized flavoprotein. ADM (10 nM) augmented the NS1619-induced flavoprotein oxidation when applied after the effect of NS1619 had reached steady state. This potentiating effect of ADM was prevented by the protein kinase A (PKA) inhibitor KT5720 (200 nM), but not by the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 (5 µM). The mitoKCa channel blocker paxilline (PX, 2 µM) completely blocked the oxidative effects of NS1619 in the presence of ADM. Treatment with ADM for 10 min before ischaemia significantly reduced infarct size after ischaemia/reperfusion from 63 ± 3% in controls to 32 ± 4% (P < 0.01). This infarct size-limiting effect of ADM was abolished by PX (61 ± 2%), as well as by KT5720 (62 ± 3%). ADM treatment for the first 10 min of reperfusion significantly reduced infarct size compared with controls (42 ± 3%, P < 0.01). This cardioprotective effect of ADM was unaffected by PX (38 ± 4%), but was abolished by LY294002 (60 ± 4%).

Conclusions: ADM augments the opening of mitoKCa channels by PKA activation, but not by PI3-K activation. ADM treatment prior to ischaemia reduces infarct size via PKA-mediated activation of mitoKCa channels. On the other hand, ADM treatment upon reperfusion reduces infarct size via a PI3-K-mediated pathway without activating mitoKCa channels.

KEYWORDS Adrenomedullin; Ca2+-activated K+ channel; Cardioprotection; Mitochondria


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 Acknowledgments
 References
 
Adrenomedullin (ADM) is a potent vasodilator peptide that was originally isolated from human pheochromocytoma tissue by monitoring cyclic 3',5'-adenosine monophosphate (cAMP) levels in platelet.1 Later, the peptide was shown to increase the level of cAMP in a wide variety of cells, including smooth muscle cells,2 endothelial cells,3 and cardiac cells.4 Thus, it was initially postulated that cAMP might be the primary second messenger involved in ADM-induced vasorelaxation. However, it has been shown that ADM induces endothelium-dependent vasodilatation through a phosphatidylinositol 3-kinase (PI3-K)-dependent pathway.5 Recently, evidence has been accumulating that ADM has more pleiotropic effects than initially expected as a vasodilator.6,7 The plasma ADM levels increase in patients with various cardiovascular diseases, including hypertension,8 heart failure,9,10 and myocardial infarction.11,12 Therefore, endogenous ADM, which is ubiquitously distributed, is thought to be involved in the regulation of cardiovascular functions.13 Furthermore, exogenous ADM administration exerts beneficial cardiovascular effects. It has been reported that ADM inhibits apoptosis of rat cardiac myocytes via a cAMP-dependent pathway.14 More recently, Okumura et al.15 reported that intravenous administration of ADM attenuated myocardial ischaemia/reperfusion injury via a PI3-K-dependent pathway. However, the mechanisms that underlie the cardioprotective effects of ADM remain obscure.

Large conductance Ca2+-activated K+ channels of cardiac myocytes are present in the mitochondrial inner membrane (mitoKCa channels) and have been implicated in cardioprotection.16,17,18 Our previous study demonstrated that, with the use of the flavoprotein fluorescence method, mitoKCa channel opening could be potentiated by cAMP-dependent protein kinase (PKA).19 It is therefore reasonable to assume that ADM (through the activation of PKA) acts on mitoKCa channels and may confer cardioprotection. Besides PKA, ADM has been shown to activate PI3-K in the heart.15,20 PI3-K/Akt is one of the pro-survival kinases which have been termed the reperfusion injury salvage kinases (RISK), and the activation of the PI3-K/Akt pathway promotes cell survival during early reperfusion.21 Accordingly, it would be of great interest to know the link between the activation of PI3-K/Akt and the opening of mitoKCa channels. However, it remains unclear whether activation of PI3-K by ADM opens mitoKCa channels and thereby leads to cardioprotection. Therefore, the present study was undertaken to characterize the effect of ADM on mitoKCa channels and to determine the contribution of PKA- and/or PI3-K-mediated pathways.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 Acknowledgments
 References
 
All procedures complied with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, 1996 revision), and were approved by the Institutional Animal Care and Use Committee of Chiba University.

2.1 Isolation of rabbit ventricular myocytes
Rabbit ventricular myocytes were isolated by collagenase digestion. Female rabbits (Japanese White) weighing 2.5–3.5 kg were anesthetized with an intravenous injection of 30 mg/kg pentobarbital sodium. Excised hearts were mounted on a Langendorff apparatus and perfused with modified HEPES-buffered Tyrode's solution composed of (in mM) NaCl 143, KCl 5.4, CaCl2 1.8, NaH2PO4 0.33, MgCl2 0.5, glucose 5.5, and HEPES 5 (pH 7.4). The perfusate was bubbled with 100% O2 and maintained at 37°C. After 5 min perfusion, hearts were perfused without Ca2+ for another 10 min. The perfusion solution was then switched to one containing collagenase (0.25 mg/ml, Wako type I). After 25–30 min of digestion, the heart was perfused with the high-K+ low-Cl– (modified KB) solution containing (in mM) KOH 70, L-glutamic acid 50, KCl 40, taurine 20, KH2PO4 20, MgCl2 3, glucose 10, EGTA 1, and HEPES–KOH buffer 10 (pH 7.4). Ventricular tissue was cut into small pieces in the modified KB solution, and the pieces were gently agitated to dissociate cells. Cells were then filtered through nylon mesh. Once isolated, the cells were suspended in Dulbecco's modified Eagle's medium containing 10% fetal calf serum at room temperature ({approx}22°C) until use.

2.2 Flavoprotein fluorescence measurement
To index mitoKCa or mitochondrial ATP-sensitive K+ (mitoKATP) channel activity, the autofluorescence of mitochondrial flavoprotein was measured by a modification of method described by Sato et al.19,22 Briefly, the cells were superfused with glucose-free Tyrode's solution containing (in mM) NaCl 143, KCl 5.4, CaCl2 1.8, NaH2PO4 0.33, MgCl2 0.5 and HEPES 5 (pH 7.4) at room temperature ({approx}22°C). Flavoprotein fluorescence was excited at 480 nm (for 200 ms every 10 s) and emitted at 520 nm. Relative fluorescence was calibrated with signals recorded after application of the mitochondrial uncoupler 2,4-dinitrophenol (DNP, 100 µM). Emitted fluorescence was monitored with cooled charge-coupled device digital camera (C4742-95, Hamamatsu Photonics, Hamamatsu, Japan). The imaging of flavoprotein was analyzed for average pixel intensities of regions of interest drawn to include whole cell and expressed as a percentage of the DNP-induced maximal oxidation, using an Aquacosmos image-processing system (Hamamatsu Photonics).

2.3 Langendorff perfused rabbit hearts
Female rabbits (Japanese White, 2.5–3.5 kg) were anesthetized by intravenous injection of 30 mg/kg pentobarbital sodium. The hearts were then dissected out, and mounted on a Langendorff apparatus for perfusion with a modified Krebs–Henseleit solution composed of (in mM) NaCl 119, KCl 4.8, KH2PO4 1.2, MgSO4 1.2, CaCl2 2.5, NaHCO3 24.9, glucose 10, and gassed with 95% O2/5% CO2 (pH 7.4, 36°C). Krebs–Henseleit solution was delivered at a constant rate of 25–30 ml/min that established an initial coronary perfusion pressure (CCP) of {approx}40 mm Hg. To measure left ventricular pressure, a fluid-filled balloon was inserted via the left atrium and positioned in the left ventricle. The balloon was expanded with distilled water to achieve an initial baseline left ventricular end-diastolic pressure (LVEDP) between 4 and 8 mm Hg. Hemodynamic parameters, i.e., CCP, heart rate (HR), left ventricular developed pressure (LVDP; difference between left ventricular end-systolic pressure and LVEDP), velocity of contraction (+dP/dt) were monitored continuously using a PowerLab data acquisition system (AD Instruments, Castle Hill, Australia).

2.4 Experimental protocols
Langendorff perfused hearts were stabilized and subjected to 30 min of normothermic global ischaemia followed by 120 min of reperfusion. Global ischaemia was achieved by complete interruption of coronary perfusion. As illustrated in Figure 1, hearts were divided into 7 experimental groups. ADM was administered before ischaemia in protocol 1 (Groups 2–4) and upon reperfusion in protocol 2 (Groups 5–7), respectively. Group 1, CONT (n = 8): no treatment. Group 2, ADM (n = 7): ADM (10 nM) was added directly to the perfusate 10 min before ischaemia, and it was perfused until the onset of ischaemia. Group 3, ADM + PX (n = 7): treatment with the mitoKCa channel blocker paxilline (PX, 2 µM) during ADM. Group 4, ADM + KT (n = 7): treatment with the PKA inhibitor KT5720 (KT, 200 nM) 5 min prior to and during ADM. Group 5, ADM (n = 7): ADM (10 nM) was administered for the first 10 min of reperfusion. Group 6, ADM + PX (n = 7): treatment with PX during ADM. Group 7, ADM + LY (n = 7): treatment with the PI3-K inhibitor LY294002 (LY, 5 µM) during ADM.


Figure 1
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  		Figure 1 Experimental protocols. All hearts were stabilized for at least 30 min and subjected to 30 min of global ischaemia followed by 120 min of reperfusion. Timing of interventions is shown in relation to the 30 min of ischaemia (filled boxes). CONT indicates control hearts (Group 1). ADM, adrenomedullin (10 nM, shaded boxes) was administered either before ischaemia in protocol 1 (Groups 2–4) or upon reperfusion in protocol 2 (Groups 5–7), respectively. PX, treatment with 2 µM paxilline; KT, treatment with 200 nM KT5720; LY, treatment with 5 µM LY294002. Solid lines indicate periods when hearts were exposed to each drug.

 
2.5 Infarct size measurement
After 120 min of reperfusion, the heart was removed from the Langendorff apparatus, and then cut into six to eight transverse slices from apex to base. The slices were incubated for 5 min at 37°C in a 1% solution of triphenyltetrazolium chloride to visualize infarcts. All slices were weighed and photographed after staining. The areas of infarct and the ventricles in each slice were measured by computed planimetry (Aquacosmos; Hamamatsu Photonics). Infarct weight was determined with the following equation: % infarct area x weight of each slice, as described previously,23 and expressed as a percentage of the total tissue weight.

2.6 Chemicals
ADM, diazoxide, NS1619, paxilline, KT5720, 8-bromoadenosine 3'5'-cyclic monophosphate (8Br-cAMP), and LY294002 were purchased from Sigma-Aldrich (St. Louis, MO, USA). DNP was purchased from Wako Pure Chemicals (Osaka, Japan). Erythropoietin was a kind gift from Chugai Pharmaceutical (Tokyo, Japan). Diazoxide, NS1619, paxilline, KT5720, 8Br-cAMP, and LY294002 were dissolved in dimethyl sulfoxide (DMSO) before they were added to the experimental solution, and the final concentration of DMSO was ≤0.1%. ADM, DNP, and erythropoietin were dissolved in the perfusate.

2.7 Statistical analysis
Data are expressed as mean ± SEM, and the number of cells or experiments is shown as n. Statistical comparisons were made using Student's t-test or ANOVA combined with Fisher post hoc test, as appropriate. A value of P < 0.05 was regarded as significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 Acknowledgments
 References
 
3.1 Effects of ADM on flavoprotein oxidation
It has been shown that in guinea pig ventricular myocytes the flavoprotein fluorescence is a useful index to assay not only mitoKATP but also mitoKCa channel activity.19 We therefore examined the effects of ADM on flavoprotein oxidation in rabbit ventricular cells. Figure 2A shows the time course of changes in flavoprotein fluorescence in a cell exposed to diazoxide and ADM. Opening of mitoKATP channels by diazoxide (100 µM) oxidized flavoprotein and increased the fluorescence intensity. Subsequent exposure to ADM (10 nM) did not affect the oxidative effect of diazoxide. As summarized in Figure 2B, diazoxide alone increased flavoprotein oxidation to 29.0 ± 5.6% of the DNP value (n = 4). Although ADM alone did not affect the flavoprotein fluorescence (3.6 ± 2.8% of the DNP value, n = 5, data not shown), the diazoxide-induced flavoprotein oxidation was not affected by ADM (27.5 ± 5.7% of the DNP value, n = 4). The lack of potentiating effect of ADM was not due to the maximum oxidation by 100 µM diazoxide because ADM did not increase the flavoprotein oxidation during application of 30 µM diazoxide (18.3 ± 3.3% vs. 16.4 ± 2.8% of the DNP value, n = 5, data not shown). Furthermore, the mitoKCa channel blocker paxilline (2 µM) did not inhibit the flavoprotein oxidation during the application of diazoxide and ADM (26.2 ± 3.1%, n = 4). Figure 2C shows the time course of changes in flavoprotein fluorescence in a cell exposed to NS1619 and ADM. The mitoKCa channel opener NS1619 (30 µM) partially oxidized flavoproteins, as we have reported previously.19 ADM augmented the NS1619-induced flavoprotein oxidation when applied after the effect of NS1619 had reached steady state. As summarized in Figure 2D, NS1619 alone increased flavoprotein oxidation to 20.7 ± 2.3% of the DNP value (n = 6). ADM significantly increased the NS1619-induced flavoprotein oxidation to 41.5 ± 4.7% of the DNP value (n = 6, P < 0.05). Paxilline completely blocked the oxidative effects of NS1619 in the presence of ADM (2.7 ± 2.4% of the DNP value, n = 5). These results indicate that ADM augments the opening of mitoKCa but not mitoKATP channels.


Figure 2
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  		Figure 2 Effects of ADM on flavoprotein (FP) oxidation. (A and C) Representative data showing effects of ADM (10 nM) on diazoxide (DIAZ, 100 µM)- and NS1619 (30 µM)-induced FP oxidation. Bars indicate periods when the cells were exposed to each drug. FP fluorescence was calibrated by exposing the cells to 2,4-dinitrophenol (DNP, 100 µM) at the end of experiments. (B and D) Summarized data for percentage of FP oxidation. Neither ADM nor paxilline (PX, 2 µM) affected the DIAZ-induced FP oxidation. ADM potentiated the NS1619-induced FP oxidation, and PX completely inhibited oxidative effect of NS1619 in the presence of ADM. Values are expressed as percent relative to those obtained with DNP. *P < 0.05 vs. NS1619 alone.

 
3.2 Effects of PKA and PI3-K on mitoKCa channel activation
In the following experiments we tested whether ADM augmented the oxidative effect of NS1619 via activation of PKA and/or PI3-K. As shown in Figure 3A and B, when the PKA inhibitor KT5720 (200 nM) was administered 5 min prior to and during ADM, ADM failed to augment the oxidative effect of NS1619 (26.3 ± 4.9% vs. 31.1 ± 7.1% of the DNP value, n = 4, P = NS). Furthermore, consistent with our earlier findings,19 the cell-permeable cAMP analogue 8Br-cAMP (0.5 mM) mimicked the potentiating effect of ADM and significantly increased the NS1619-induced flavoprotein oxidation from 23.3 ± 3.5% to 43.0 ± 2.2% of the DNP value (n = 5, P < 0.05). These results suggest that ADM-induced potentiation of mitoKCa channels is mediated by cAMP/PKA pathway. Contrarily, as shown in Figure 3C and D, ADM significantly increased the oxidative effect of NS1619 even in the presence of LY294002 (5 µM), a potent inhibitor of PI3-K (23.8 ± 2.0% vs. 42.9 ± 3.4% of the DNP value, n = 7, P < 0.05). Moreover, erythropoietin (10 units/ml), a potent activator of PI3-K,24,25 did not augment the NS1619-induced flavoprotein oxidation (24.0 ± 4.4% vs. 30.7 ± 6.6% of the DNP value, n = 4, P = NS). These results suggest that mitoKCa channel is not regulated by PI3-K.


Figure 3
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  		Figure 3 Summarized effects of PKA and PI3-K on mitoKCa channels. (A) In the presence of KT5720 (200 nM), ADM (10 nM) failed to augment NS1619 (30 µM)-induced FP oxidation. (B) 8-Bromoadenosine 3',5'-cyclic monophosphate (8Br-cAMP, 0.5 mM) mimicked the potentiating effect of ADM. (C) ADM augmented the oxidative effect of NS1619 even in the presence of LY294002 (5 µM). (D) Erythropoietin (EPO, 10 units/ml) could not mimic the oxidative effect of ADM. Values are expressed as percent relative to those obtained with DNP. *P < 0.05 vs. NS1619 alone.

 
3.3 ADM reduces infarct size when given before ischaemia (protocol 1)
We next examined the effect of ADM on infarct size in the isolated perfused rabbit hearts subjected to ischaemia/reperfusion. ADM alone produced no significant effects on CCP, heart rate, LVDP, and +dP/dtmax. We also found no significant differences in hemodynamic parameters among the groups before the start of ischaemia (Table 1). As shown in Figure 4, administration of ADM to the heart for 10 min prior to ischaemia significantly reduced infarct size compared with the control group (31.8 ± 4.4% for Group 2 vs. 63.4 ± 3.2% for Group 1, P < 0.01). Moreover, ADM treated hearts showed improved recovery of postischaemic LVDP and +dP/dtmax compared with control hearts (Table 1). To test whether the infarct size-limiting effect of ADM is dependent on PKA-mediated activation of mitoKCa channels, we treated the hearts with paxilline, a blocker of mitoKCa channels, or KT5720, a cell-permeable inhibitor of PKA. Infarct size in the group treated with paxilline alone (63.7 ± 2.1%, n = 4) or KT5720 alone (62.1 ± 4.3%, n = 4) was not significantly different from that of the control group (data not shown). Treatment with paxilline blocked the ADM-induced reduction in infarct size (60.6 ± 1.8% for Group 3) and improvement in postischaemic LVDP and +dP/dtmax (Table 1). Similarly, treatment with KT5720 abolished the infarct size-limiting effect of ADM (62.0 ± 3.1% for Group 4) and blocked the ADM-induced improvement in postischaemic contractile function (LVDP and +dP/dtmax). However, treatment with the PI3-K inhibitor LY294002 (5 µM) 5 min prior to and during ADM did not abolish the infarct size-limiting effect of ADM (36.6 ± 4.0%, n = 4, data not shown). These results indicate that ADM treatment prior to ischaemia provides cardioprotection, and that the protection is mediated by PKA-dependent activation of mitoKCa channels.


Figure 4
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  		Figure 4 Summarized effects of ADM on myocardial infarct size in protocol 1. ADM was administered before ischaemia in protocol 1. Pretreatment with paxilline (Group 3) or KT5720 (Group 4) significantly blocked infarct size-limiting effect of ADM (Group 2). Each open circle represents infarct size in an individual heart, and closed circles with error bars are group means ± SEM. *P < 0.01 vs. control (Group 1).

 


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  		Table 1 Hemodynamic Parameters

 
3.4 ADM reduces infarct size when given at reperfusion (protocol 2)
The cardioprotective effect of ADM at reperfusion was examined. There was no significant difference in hemodynamic parameters among the groups before ischaemia (Table 1). As shown in Figure 5, administration of ADM for the first 10 min of reperfusion significantly reduced infarct size compared with the control group (41.5 ± 3.2% for Group 5 vs. 63.4 ± 3.2% for Group 1, P < 0.01). However, LVDP and dP/dtmax after ischaemia/reperfusion were not significantly different between groups (Table 1). In contrast to the results seen in protocol 1, infarct size limitation by ADM was not attenuated by paxilline (38.0 ± 3.8% for Group 6). These results indicate that opening of mitoKCa channels is not involved in the ADM-mediated cardioprotection when given at the onset of the reperfusion. Next, we examined the involvement of PI3-K in the cardioprotective effect of ADM upon reperfusion using the PI3-K inhibitor LY294002. Infarct size in the group treated with LY294002 alone for the first 10 min of reperfusion was not significantly different from that of the control group (61.2 ± 4.3%, n = 4, data not shown). Treatment with LY294002 blocked the ADM-induced reduction in infarct size (59.7 ± 4.0% for Group 7), suggesting the cardioprotective effect of ADM at reperfusion is dependent on PI3-K pathway.


Figure 5
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  		Figure 5 Summarized effects of ADM on myocardial infarct size in protocol 2. ADM was administered upon reperfusion in protocol 2. Infarct size-limiting effect of ADM (Group 5) was not attenuated by paxilline (Group 6). Treatment with LY294002 (Group 7) abolished the protective effect of ADM. Each open circle represents infarct size in an individual heart, and closed circles with error bars are group means ± SEM. *P < 0.01 vs. control (Group 1).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 Acknowledgments
 References
 
Our previous study has shown that mitochondrial oxidation induced by opening of mitoKATP and mitoKCa channels occurs independently of each other.19 In the present study, the effects of ADM on mitoKATP and mitoKCa channels in rabbit ventricular myocytes were investigated by measuring flavoprotein fluorescence. The mitochondrial redox potential is an indirect way of detecting mitoKATP or mitoKCa channel activity and may be affected even by the K+ channel-independent uncoupling of mitochondria. Despite this limitation, the mitochondrial flavoprotein oxidation state has provided the only convenient measure of mitoKATP or mitoKCa channel opening in intact cells.18

The concentration of ADM (10 nM) used in this study was based on previous studies which showed that the concentration of ≥10 nM ADM is needed to activate both cAMP/PKA and PI3-K/Akt pathways in cardiac myocytes.15,26,27 We found that flavoprotein oxidation induced by the mitoKATP channel opener diazoxide was not affected by ADM (Figure 2A and B). Therefore, it seems unlikely that activation of cAMP and/or PI3-K by ADM is linked to mitoKATP channels. The salient experimental observations were that ADM potentiated the flavoprotein oxidation induced by the mitoKCa channel opener NS1619 (Figure 2C). The effects of ADM and NS1619 reflected mitoKCa channel activation, because they could be blocked by the mitoKCa channel blocker paxilline (Figure 2D). The PKA inhibitor KT5720 prevented the ability of ADM to augment the oxidative effect of NS1619 (Figure 3A). Moreover, the NS1619-induced flavoprotein oxidation was similarly potentiated by the cell-permeable cAMP analogue 8Br-cAMP (Figure 3B), as we have seen in previous study.19 These results suggest that ADM modulates mitoKCa channels through cAMP/PKA, although ADM itself can not open the mitoKCa channels directly. Previous studies have shown that PKC activators, phorbol 12-myristate 13-acetate and adenosine, do not suffice to open mitoKATP channels, they shift channels into the primed state, from which channels can be opened much more readily by endogenous stimuli during ischaemia.22,28 It is reasonable to suppose that, as in the case of mitoKATP channel, ADM activates PKA and primes the mitoKCa channel to open earlier and more intensely in response to elevation of mitochondrial Ca2+ during ischaemia. Therefore, ADM can be cardioprotective without direct mitoKCa channel opening.

It has been demonstrated that PKA potentiates mitoKCa channel activation,19 whereas PKC and PKG potentiate mitoKATP channel activation.22,29 However, the possible role of PI3-K in the regulation of mitoKCa as well as mitoKATP channels has not yet been investigated. In the present study, we found that ADM augmented the NS1619-induced flavoprotein oxidation even in the presence of the PI3-K inhibitor LY294002 (Figure 3C). These results suggest that activation of PI3-K does not contribute to the potentiating effect of ADM. Further support for this notion comes from the observation that the NS1619-induced flavoprotein oxidation was not affected by erythropoietin, a potent activator of PI3-K.24,25 PI3-K/Akt is known to stimulate nitric oxide synthase to produce nitric oxide (NO).30 It has also been reported that NO activates sarcolemmal BKCa channels in smooth muscle cells.31 Although NO has been shown to activate mitoKATP channels,32 the possible role of NO in the regulation of mitoKCa channels remains to be clarified.

In this study, we found that in isolated rabbit hearts administration of ADM for 10 min before ischaemia significantly reduced infarct size after ischaemia/reperfusion (Figure 4). Since the coronary flow rate was maintained constant throughout the experiments using a roller pump, cardioprotective effects of ADM are not due to vasodilation in our experimental conditions. ADM-induced cardioprotection was attenuated by administration of the mitoKCa channel blocker paxilline and the PKA inhibitor KT5720. These results suggest that ADM treatment prior to ischaemia reduced infarct size via PKA-mediated activation of mitoKCa channels. There is support for the concept that activation of PKA prior to ischaemia can protect hearts against ischaemia/reperfusion injury. It has been reported that brief exposure to β-adrenergic receptor agonist isoproterenol prior to ischaemia is protective against ischaemic injury in rat and murine hearts.33,34 Furthermore, activation of cAMP/PKA is required for ischaemic preconditioning,35,36 in which lethal injury to the heart can be dramatically blunted by brief conditioning periods of ischaemia.37 Recent study by Cao et al.38 demonstrated that in rat hearts cardioprotection conferred by ischaemic preconditioning was abolished by paxilline administered before ischaemia. Considering above findings together with the effects of ADM on mitoKCa channels, we conclude that cAMP/PKA-mediated priming of mitoKCa channels is involved in the cardioprotection that observed when ADM was administered before ischaemia. It has been proposed that PI3-K is involved in mediating ischaemic preconditioning.39,40 Contrary to previous reports, however, the infarct size-limiting effect of ADM was not abolished by LY294002 administered before ischaemia. Although the reason for this discrepancy in experimental results is not clear, our results suggest that activation of PI3-K/Akt pathway before ischaemia may not be involved in the preconditioning-like effect of ADM. In agreement with this suggestion, Hanlon et al.41 reported that LY294002 did not block the cardioprotection afforded by treatment with erythropoietin prior to ischaemia.

The mechanisms by which opening of mitoKCa channels could protect the hearts remain to be elucidated. It seems that mitoKCa channel activation confers cardioprotection in a manner similar to but independent of mitoKATP channel activation. We previously reported that opening of mitoKCa channels attenuates the mitochondrial Ca2+ overload with accompanying depolarization of mitochondrial membrane.19 Recent study by Stowe et al.42 reported that opening of mitoKCa channels by NS1619 generates reactive oxygen species (ROS) to initiate pharmacological preconditioning, as in the case of mitoKATP channel activation.43 It has been shown that ROS per se increases the open probability of mitoKATP channel,44 and activates the downstream kinases that feed back in a positive manner and keep the channel open.37 However, there is a need to determine whether such a mechanism could offer explanations for our findings, because conflicting data can be found in the literature. It has been reported that ROS production is attenuated by ADM,45 and ROS rather inhibits the opening of sarcolemmal BKCa channels.46

Another important finding of this study was that ADM given at the onset of reperfusion results in a significant reduction in infarct size (Figure 5). This finding is consistent with the previous study in rat hearts.47 We further found that the cardioprotective effect of ADM at reperfusion was not affected by the mitoKCa channel blocker paxilline, but was completely abolished by the PI3-K inhibitor LY294002. These observations reinforce our notion that PI3-K is not linked to mitoKCa channels. Furthermore, it is concluded that activation of PI3-K, but not mitoKCa channel, is involved in the cardioprotective effect of ADM at reperfusion. PI3-K/Akt pathway is a component of the RISK pathway,21 and much attention has been paid in recent years to the cardioprotective role of PI3-K at the time of reperfusion. Like ADM, it has been shown that cardioprotective effect of erythropoietin given at the onset of reperfusion is mediated by activation of PI3-K.41 Although the present study did not investigate the downstream effectors of PI3-K, recent study suggest that the activation of RISK pathway confers its cardioprotection through the inhibition of mitochondrial permeability transition pore (PTP).21 PTP promotes the release of proapoptotic signaling molecules from the mitochondria and triggers apoptosis.48 Indeed, ADM has been shown to reduce myocardial apoptotic death through the PI3-K/Akt-dependent pathway.15,20,49 However, previous reports have not provided evidence that ADM exerts its antiapoptotic effect by the inhibition of PTP. Recent evidence suggests that ADM exerts its cardioprotective action during early reperfusion through a NO-dependent mechanism.47 Although NO has been shown to inhibit PTP opening,50 it remains unclear whether NO generation by ADM inhibits PTP in the reperfused myocardium.

In summary, we have shown for the first time that ADM links to mitoKCa channels and augments channel openings through cAMP/PKA-dependent pathway. ADM treatment prior to ischaemia reduces infarct size via PKA-mediated activation of mitoKCa channels (Figure 6). Based on present and previous results,19 we postulate that mitoKCa channel acts as a common downstream effectors of cAMP/PKA-mediated cardioprotection. Importantly, we show that ADM treatment at reperfusion reduces infarct size via a PI3-K-mediated pathway. However, PI3-K is not linked to mitoKCa channels, and further experiments are required to investigate how activation of PI3-K by ADM induces cardioprotection at the time of reperfusion.


Figure 6
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  		Figure 6 Schematic representation of the mechanism of cardioprotection afforded by ADM. ADM administered prior to ischaemia potentiates the opening of mitoKCa channels via cAMP/PKA pathway and confers cardioprotection. ADM administered upon reperfusion confers cardioprotection through PI3-K/Akt-mediated pathway. However, PI3-K/Akt is not linked to mitoKCa channels. It has been demonstrated that PI3-K/Akt pathway is involved in cardioprotection through the inhibition of mitochondrial permeability transition pore (PTP).

 

   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
Acknowledgments
 References
 
This study was supported in part by Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science, K. Watanabe Research Foundation, and the Vehicle Racing Commemorative Foundation.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 Acknowledgments
References
 
We thank M. Tamagawa and I. Sakashita for excellent technical and secretarial assistance.

Conflict of interest: none declared.


   Notes
 
{dagger} This article was published online by Elsevier on 2 August, 2007. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 Acknowledgments
 References
 

  1. Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, et al. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun (1993) 192:553–560.[CrossRef][Web of Science][Medline]
  2. Ishizaka Y, Tanaka M, Kitamura K, Kangawa K, Minamino N, Matsuo H, et al. Adrenomedullin stimulates cyclic AMP formation in rat vascular smooth muscle cells. Biochem Biophys Res Commun (1994) 200:642–646.[CrossRef][Web of Science][Medline]
  3. Shimekake Y, Nagata K, Ohta S, Kambayashi Y, Teraoka H, Kitamura K, et al. Adrenomedullin stimulates two signal transduction pathways, cAMP accumulation and Ca2+ mobilization, in bovine aortic endothelial cells. J Biol Chem (1995) 270:4412–4417.[Abstract/Free Full Text]
  4. Sato A, Canny BJ, Autelitano DL. Adrenomedullin stimulates cAMP accumulation and inhibits atrial natriuretic peptide gene expression in cardiomyocytes. Biochem Biophys Res Commun (1997) 230:311–314.[CrossRef][Web of Science][Medline]
  5. Nishimatsu H, Suzuki E, Nagata D, Moriyama N, Satonaka H, Walsh K, et al. Adrenomedullin induces endothelium-dependent vasorelaxation via the phosphatidylinositol 3-kinase/Akt-dependent pathway in rat aorta. Circ Res (2001) 89:63–70.[Abstract/Free Full Text]
  6. Hinson JP, Kapas S, Smith DM. Adrenomedullin, a multifunctional regulatory peptide. Endocr Rev (2000) 21:138–167.[Abstract/Free Full Text]
  7. Shimosawa T, Matsui H, Xing G, Itakura K, Ando K, Fujita T. Organ-protective effects of adrenomedullin. Hypertens Res (2003) 26:S109–S112.[CrossRef][Web of Science][Medline]
  8. Ishimitsu T, Nishikimi T, Saito Y, Kitamura K, Eto T, Kangawa K, et al. Plasma levels of adrenomedullin, a newly identified hypotensive peptide, in patients with hypertension and renal failure. J Clin Invest (1994) 94:2158–2161.[Web of Science][Medline]
  9. Nishikimi T, Saito Y, Kitamura K, Ishimitsu T, Eto T, Kangawa K, et al. Increased plasma levels of adrenomedullin in patients with heart failure. J Am Coll Cardiol (1995) 26:1424–1431.[Abstract]
  10. Jougasaki M, Wei CM, McKinley LJ, Burnett JC Jr. Elevation of circulating and ventricular adrenomedullin in human congestive heart failure. Circulation (1995) 92:286–289.[Abstract/Free Full Text]
  11. Yoshitomi Y, Nishikimi T, Kojima S, Kuramochi M, Takishita S, Matsuoka H, et al. Plasma levels of adrenomedullin in patients with acute myocardial infarction. Clin Sci (1998) 94:135–139.[Web of Science][Medline]
  12. Richards AM, Nicholls MG, Yandle TG, Frampton C, Espiner EA, Turner JG, et al. Plasma N-terminal pro-brain natriuretic peptide and adrenomedullin: new neurohormonal predictors of left ventricular function and prognosis after myocardial infarction. Circulation (1998) 97:1921–1929.[Abstract/Free Full Text]
  13. Ishimitsu T, Ono H, Minami J, Matsuoka H. Pathophysiologic and therapeutic implications of adrenomedullin in cardiovascular disorders. Pharmacol Ther (2006) 111:909–927.[CrossRef][Web of Science][Medline]
  14. Tokudome T, Horio T, Yoshihara F, Suga S, Kawano Y, Kohno M, et al. Adrenomedullin inhibits doxorubicin-induced cultured rat cardiac myocyte apoptosis via a cAMP-dependent mechanism. Endocrinology (2002) 143:3515–3521.[Abstract/Free Full Text]
  15. Okumura H, Nagaya N, Itoh T, Okano I, Hino J, Mori K, et al. Adrenomedullin infusion attenuates myocardial ischaemia/reperfusion injury through the phosphatidylinositol 3-kinase/Akt-dependent pathway. Circulation (2004) 109:242–248.[Abstract/Free Full Text]
  16. Xu W, Liu Y, Wang S, McDonald T, Van Eyk JE, Sidor A, et al. Cytoprotective role of Ca2+-activated K+ channels in the cardiac inner mitochondrial membrane. Science (2002) 298:1029–1033.[Abstract/Free Full Text]
  17. Ohya S, Kuwata Y, Sakamoto K, Muraki K, Imaizumi Y. Cardioprotective effects of estradiol include the activation of large-conductance Ca2+-activated K+ channels in cardiac mitochondria. Am J Physiol Heart Circ Physiol (2005) 289:H1635–H1642.[Abstract/Free Full Text]
  18. O'Rourke B. Evidence for mitochondrial K+ channels and their role in cardioprotection. Circ Res (2004) 94:420–432.[Abstract/Free Full Text]
  19. Sato T, Saito T, Saegusa N, Nakaya H. Mitochondrial Ca2+-activated K+ channels in cardiac myocytes: a mechanism of the cardioprotective effect and modulation by protein kinase A. Circulation (2005) 111:198–203.[Abstract/Free Full Text]
  20. Kato K, Yin H, Agata J, Yoshida H, Chao L, Chao J. Adrenomedullin gene delivery attenuates myocardial infarction and apoptosis after ischaemia and reperfusion. Am J Physiol Heart Circ Physiol (2003) 285:H1506–H1514.[Abstract/Free Full Text]
  21. Hausenloy DJ, Yellon DM. New directions for protecting the heart against ischaemia–reperfusion injury: targeting the Reperfusion Injury Salvage Kinase (RISK)-pathway. Cardiovasc Res (2004) 61:448–460.[Abstract/Free Full Text]
  22. Sato T, O'Rourke B, Marbán E. Modulation of mitochondrial ATP-dependent potassium channels by protein kinase C. Circ Res (1998) 83:110–114.[Abstract/Free Full Text]
  23. Suzuki M, Sasaki N, Miki T, Sakamoto N, Ohmoto-Sekine Y, Tamagawa M, et al. Role of sarcolemmal KATP channels in cardioprotection against ischaemia/reperfusion injury in mice. J Clin Invest (2002) 109:509–516.[CrossRef][Web of Science][Medline]
  24. Tramontano AF, Muniyappa R, Black AD, Blendea MC, Cohen I, Deng L, et al. Erythropoietin protects cardiac myocytes from hypoxia-induced apoptosis through an Akt-dependent pathway. Biochem Biophys Res Commun (2003) 308:990–994.[CrossRef][Web of Science][Medline]
  25. Cai Z, Semenza GL. Phosphatidylinositol-3-kinase signaling is required for erythropoietin-mediated acute protection against myocardial ischaemia/reperfusion injury. Circulation (2004) 109:2050–2053.[Abstract/Free Full Text]
  26. Ikeda U, Kanbe T, Kawahara Y, Yokoyama M, Shimada K. Adrenomedullin augments inducible nitric oxide synthase expression in cytokine-stimulated cardiac myocytes. Circulation (1996) 94:2560–2565.[Abstract/Free Full Text]
  27. Ihara T, Ikeda U, Tate Y, Ishibashi S, Shimada K. Positive inotropic effects of adrenomedullin on rat papillary muscle. Eur J Pharmacol (2000) 390:167–172.[CrossRef][Web of Science][Medline]
  28. Sato T, Sasaki N, O'Rourke B, Marbán E. Adenosine primes the opening of mitochondrial ATP-sensitive potassium channels: a key step in ischaemic preconditioning? Circulation (2000) 102:800–805.[Abstract/Free Full Text]
  29. Costa AD, Garlid KD, West IC, Lincoln TM, Downey JM, Cohen MV, et al. Protein kinase G transmits the cardioprotective signal from cytosol to mitochondria. Circ Res (2005) 97:329–336.[Abstract/Free Full Text]
  30. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature (1999) 399:601–605.[CrossRef][Medline]
  31. Mistry DK, Garland CJ. Nitric oxide (NO)-induced activation of large conductance Ca2+-dependent K+ channels (BKCa) in smooth muscle cells isolated from the rat mesenteric artery. Br J Pharmacol (1998) 124:1131–1140.[CrossRef][Web of Science][Medline]
  32. Sasaki N, Sato T, Ohler A, O'Rourke B, Marbán E. Activation of mitochondrial ATP-dependent potassium channels by nitric oxide. Circulation (2000) 101:439–445.[Abstract/Free Full Text]
  33. Frances C, Nazeyrollas P, Prevost A, Moreau F, Pisani J, Davani S, et al. Role of β1- and β2-adrenoceptor subtypes in preconditioning against myocardial dysfunction after ischaemia and reperfusion. J Cardiovasc Pharmacol (2003) 41:396–405.[CrossRef][Web of Science][Medline]
  34. Tong H, Bernstein D, Murphy E, Steenbergen C. The role of β-adrenergic receptor signaling in cardioprotection. FASEB J (2005) 19:983–985.[Abstract/Free Full Text]
  35. Lochner A, Genade S, Tromp E, Podzuweit T, Moolman JA. Ischaemic preconditioning and the β-adrenergic signal transduction pathway. Circulation (1999) 100:958–966.[Abstract/Free Full Text]
  36. Sanada S, Asanuma H, Tsukamoto O, Minamino T, Node K, Takashima S, et al. Protein kinase A as another mediator of ischaemic preconditioning independent of protein kinase C. Circulation (2004) 110:51–57.[Abstract/Free Full Text]
  37. Yellon DM, Downey JM. Preconditioning the myocardium: from cellular physiology to clinical cardiology. Physiol Rev (2003) 83:1113–1151.[Abstract/Free Full Text]
  38. Cao CM, Xia Q, Gao Q, Chen M, Wong TM. Calcium-activated potassium channel triggers cardioprotection of ischaemic preconditioning. J Pharmacol Exp Ther (2005) 312:644–650.[Abstract/Free Full Text]
  39. Tong H, Chen W, Steenbergen C, Murphy E. Ischaemic preconditioning activates phosphatidylinositol-3-kinase upstream of protein kinase C. Circ Res (2000) 87:309–315.[Abstract/Free Full Text]
  40. Robinet A, Hoizey G, Millart H. PI 3-kinase, protein kinase C, and protein kinase A are involved in the trigger phase of β-1 adrenergic preconditioning. Cardiovasc Res (2005) 66:530–542.[Abstract/Free Full Text]
  41. Hanlon PR, Fu P, Wright GL, Steenbergen C, Arcasoy MO, Murphy E. Mechanisms of erythropoietin-mediated cardioprotection during ischaemia–reperfusion injury: role of protein kinase C and phosphatidylinositol 3-kinase signaling. FASEB J (2005) 19:1323–1325.[Abstract/Free Full Text]
  42. Stowe DF, Aldakkak M, Camara AK, Riess ML, Heinen A, Varadarajan SG, et al. Cardiac mitochondrial preconditioning by Big Ca2+-sensitive K+ channel opening requires superoxide radical generation. Am J Physiol Heart Circ Physiol (2006) 290:H434–H440.[Abstract/Free Full Text]
  43. Forbes RA, Steenbergen C, Murphy E. Diazoxide-induced cardioprotection requires signaling through a redox-sensitive mechanism. Circ Res (2001) 88:802–809.[Abstract/Free Full Text]
  44. Zhang DX, Chen YF, Campbell WB, Zou AP, Gross GJ, Li PL. Characteristics and superoxide-induced activation of reconstituted myocardial mitochondrial ATP-sensitive potassium channels. Circ Res (2001) 89:1177–1183.[Abstract/Free Full Text]
  45. Shimosawa T, Shibagaki Y, Ishibashi K, Kitamura K, Kangawa K, Kato S, et al. Adrenomedullin, an endogenous peptide, counteracts cardiovascular damage. Circulation (2002) 105:106–111.[Abstract/Free Full Text]
  46. Tang XD, Garcia ML, Heinemann SH, Hoshi T. Reactive oxygen species impair Slo1 BK channel function by altering cysteine-mediated calcium sensing. Nat Struct Mol Biol (2004) 11:171–178.[CrossRef][Web of Science][Medline]
  47. Hamid SA, Baxter GF. Adrenomedullin limits reperfusion injury in experimental myocardial infarction. Basic Res Cardiol (2005) 100:1–10.[CrossRef][Web of Science][Medline]
  48. Weiss JN, Korge P, Honda HM, Ping P. Role of the mitochondrial permeability transition in myocardial disease. Circ Res (2003) 93:292–301.[Abstract/Free Full Text]
  49. Yin H, Chao L, Chao J. Adrenomedullin protects against myocardial apoptosis after ischaemia/reperfusion through activation of Akt-GSK signaling. Hypertension (2004) 43:109–116.[Abstract/Free Full Text]
  50. Schulz R, Kelm M, Heusch G. Nitric oxide in myocardial ischaemia/reperfusion injury. Cardiovasc Res (2004) 61:402–413.[Abstract/Free Full Text]

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