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Effects of postconditioning of adenosine and acetylcholine on the ischemic isolated rat ventricular myocytes
European Journal of Pharmacology 549 (2006) 133 – 139 www.elsevier.com/locate/ejphar
Effects of postconditioning of adenosine and acetylcholine on the ischemic isolated rat ventricular myocytes Jun Lu a , Wei-Jin Zang a,b,⁎, Xiao-Jiang Yu a , Bing Jia a , Alzbeta Chorvatova c,d , Lei Sun a a
b
Department of Pharmacology, School of Medicine, Xi'an Jiaotong University, Xi'an, 710061, PR China Key Laboratory of Environment and Genes Related to Diseases of Ministry of Education, School of Medicine, Xi'an Jiaotong University, Xi'an, 710061, PR China c Research Centre, Sainte-Justine Hospital, University of Montreal, Montreal, Canada d Department of Pediatrics, University of Montreal, Montreal, Canada Received 26 July 2006; received in revised form 5 August 2006; accepted 14 August 2006 Available online 26 August 2006
Abstract In this study, protective effects of adenosine and acetylcholine-induced postconditioning were investigated on the contractile function of the ischemic isolated rat ventricular myocytes. A video-based edge-detection system was used to monitor single ventricular myocytes contraction. Adenosine and acetylcholine were administrated for 6 min before ischemia as preconditioning, or 15 min after ischemia as postconditioning. Adenosine and acetylcholine receptor antagonists and mitoKATP inhibitor were used to analyze pathways underlying the effects on postconditioning. Results: (1) The peak shortening of ischemic heart cells was improved by both adenosine and acetylcholine during preconditioning (84.72 ± 5.34% and 68.61 ± 8.10% vs. control: 8.43 ± 5.35% of the pre-ischemia value), as well as postconditioning (76.47 ± 7.87% and 57.48 ± 6.97% vs. control: 8.43 ± 5.35% of the pre-ischemia value) and the effects of preconditioning and postconditioning were comparable. More datum in the normal text. (2) Observed effects of adenosine and acetylcholine postconditioning were missing in the presence of adenosine A1 receptor and muscarinic M2 receptor antagonists, respectively. (3) Adenosine and acetylcholine-induced postconditioning was also blocked by mitoKATP antagonist. These results suggest that both adenosine and acetylcholine protect the contractile function of ischemic heart cells to a similar extent during preconditioning and postconditioning. The postconditioning of adenosine and acetylcholine is relative to the adenosine A1 and muscarinic M2 receptors, respectively. MitoKATP is implicated in the postconditioning of both acetylcholine and adenosine. © 2006 Elsevier B.V. All rights reserved. Keywords: Acetylcholine; Adenosine; Ischemia; Myocyte; Postconditioning
1. Introduction Ischemic heart disease, a common cardiovascular disease, severely endangers health and is therefore a major focus of medical studies (Lazzarino et al., 1994; Pierce and Czubryt, 1995). There are two important concepts, preconditioning and postconditioning, refer to the topic. Ischemic preconditioning was first introduced by Murry et al. (1986) as a potent endogenous form of cardioprotection against ischemic–reperfusion injury. It enhances the recovery of cardiac function after global ischemia, reduces infarct size and the ap⁎ Corresponding author. Department of Pharmacology, School of Medicine, Xi'an Jiaotong University, Xi'an, 710061, PR China. Tel.: +86 29 82655150; fax: +86 29 82655003. E-mail address: [email protected] (W.-J. Zang). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.08.030
pearance of apoptosis in hearts subjected to ischemia–reperfusion injury (Lott et al., 1996). In 2003, Zhao et al. reported another endogenous form of cardioprotection similar to that observed with ischemic preconditioning–ischemic postconditioning, which is defined as a series of brief interruptions of reperfusion applied at the very onset of reperfusion (Zhao et al., 2003). Ischemic preconditioning/postconditioning is a powerful endogenous phenomenon in which brief periods of a sub-toxic ischemic insult induce robust protection against lengthy, even lethal ischemia. Manifestation of the cardioprotective effects of ischemic preconditioning was found in many species, including rat. The ability to reproduce the cardioprotective effects of ischemic preconditioning/postconditioning with pharmacological agents raises the possibility that a drug may ultimately be introduced into clinical practice to treat human hearts undergoing ischemia/reperfusion (Baker, 2005).
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Adenosine and acetylcholine-induced preconditioning process involves adenosine A1 and muscarinic M2 receptors, respectively, and mitochondrial ATP-sensitive potassium channels (mitoKATP) were proposed to play an important role in this process (Zaugg and Schaub, 2003). Both adenosine and muscarinic receptors are the Gαi-coupled receptors that can open the KACh channel. However, it is still unclear whether adenosine and acetylcholine can also induce postconditioning process and hence affect cardiac contractility following ischemia and/or what are the underlying mechanisms (Critza et al., 2005). In this study, we therefore questioned possible protective effects of acetylcholine and adenosine postconditioning on the contractile function of isolated rat ventricular myocytes and underlying mechanisms. 2. Materials and methods 2.1. Cell isolation Adult Sprague–Dawley rats of both sexes, supplied by the Experimental Animal Center of Xi'an Jiaotong University, China, and weighing 250–300 g were used in accordance with the Guidelines on the Care and Use of Laboratory Animals issued by the Chinese Council on Animal Research and the Guidelines of Animal Care. The study was approved by the ethical committee of Xi'an Jiaotong University. Ventricular myocytes were isolated enzymatically from rats using a conventional method (Zang et al., 1993). Briefly, heparinized rats were anaesthetized with sodium pentobarbitone (17 mg/kg, i.p.) (Zang et al., 2003). The heart was rapidly excised, placed in ice-cold Ca2+-free modified Tyrode solution (in mmol/l: NaCl, 143; KCl, 5.4; MgCl2, 0.5; NaH2PO4, 0.33; Hepes, 5.0; and glucose, 5.0; pH titrated to 7.35 with NaOH), cannulated and then perfused retrogradely with Ca2+-free Tyrode solution via the aorta on a Langendorff perfusion apparatus for about 5 min until spontaneous contractions ceased. The heart was then perfused with Ca2+-free Tyrode solution containing 0.7 mg/ml collagenase (type I, Sigma, St Louis, MO, USA) and 1 mg/ml bovine serum albumin (Sigma) for 20 min. Finally, the enzymes were washed out with a high-K+, low-Cl− solution (KB solution, in mmol/l: KCl, 25; taurine, 20; L-glutamic acid, 70; KH2PO4, 10; MgCl2, 3; EGTA, 0.5; glucose, 10; and Hepes, 10; pH 7.35) for 5 min. All of the solutions used during perfusion were bubbled with 100% O2 and maintained at 37 °C. Following perfusion, the ventricle was placed in a beaker filled with KB solution and then minced. The ventricular cells were dispersed by shaking the beaker gently and the undigested tissue was removed by filtration through a 250 μm nylon mesh. The cells were kept in Tyrode solution (in mmol/l: NaCl, 136.9; KCl, 5.4; MgCl2, 0.5; NaH2PO4, 0.33; Hepes, 5.0; glucose, 10.0; and CaCl2, 1.8; pH 7.4; bubbled with 100% O2) at room temperature for at least 1 h before use. 2.2. Protocols The ischemia-mimetic behaviour of single cardiomyocytes was simulated using a modified ischemia-mimetic solution (in
mmol/l: NaCl, 135; KCl, 8; MgCl2, 0.5; NaH2PO4, 0.33; Hepes, 5.0; CaCl2, 1.8; and Na+-lactate, 20; pH 6.80; bubbled with 100% N2 for N 45 min before the experiment was started; this reduced the oxygen tension by 75%) (Lu et al., 2005). Coverslips to which cardiomyocytes were adhered were placed in a flow-through (1 ml/min, 25 °C) perfusion chamber (the surface of which was surrounded with N 2 to prevent reoxygenation of the bath ischemia-mimetic solution) that was positioned on the stage of an inverted microscope and the chamber was continuously superfused with Tyrode solution. Cells were field stimulated at 0.5 Hz by two Pt electrodes connected to a MyoPacer field stimulator (IonOptix Corporation, Milton, MA, USA). The polarity of the stimulating electrodes was reversed periodically to avoid the potential build up of electrolysis byproducts (Wold et al., 2001). In all cells, the normal Tyrode solution was perfused for 6 min, then the ischemia-mimetic solution for 15 min to mimic ischemia, finally reperfused by the normal Tyrode solution for 15 min. Cells were assigned to one of the following 9 groups based upon the intervention (n = 6 in each group): (1) Control: there was no intervention either before or after the ischemiamimetic solution perfusion; (2) Adenosine preconditioning: adenosine (100 μmol/l) was administrated for 6 min before ischemia; (3) Adenosine postconditioning: adenosine was administrated for 15 min after ischemia; (4) DPCPX (8cyclopentyl-1,3-dipropylxanthine, adenosine A1 receptor antagonist) + adenosine postconditioning: adenosine and DPCPX (1 μmol/l) were administrated for 15 min after ischemia; (5) 5HD (5-hydroxydecanoate, mitoKATP antagonist) + adenosine postconditioning: adenosine and 5-HD (100 μmol/l) were administrated for 15 min after ischemia; (6) Acetylcholine preconditioning: acetylcholine (0.1 μmol/l) was administrated for 6 min before ischemia; (7) Acetylcholine postconditioning: acetylcholine was administrated for 15 min after ischemia; (8) Methoctramine (Muscarinic M2 receptor antagonist) + acetylcholine postconditioning: acetylcholine and Methoctramine (0.1 μmol/l) were administrated for 15 min after ischemia; (9) MitoKATP antagonist 5-HDs + acetylcholine postconditioning: acetylcholine and 5-HD were administrated for 15 min after ischemia. In a subset of myocytes the effects of DPCPX, Methoctramine and 5-HD alone were tested and these agents did not exert any effects alone statistically. 2.3. Myocyte shortening and lengthening measurements Cardiomyocytes were viewed with the aid of an inverted microscope (Olympus X-70, Olympus Optical, Tokyo, Japan) and imaged using an IonOptix MyoCam camera (IonOptix Corporation, Milton, MA, USA) (Zang et al., 2005). Myocyte motion was measured using a video-based edge-detection system (IonOptix Corporation). Changes in cell contractile function were quantified using the following parameters: peak shortening (representing the amplitude of myocyte contraction, viz. contractility) and cell length (resting cell length, representing the extent of contracture). Data were acquired and analysed using IonOptix software.
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2.4. Statistical analysis Results are expressed as means ± S.E.M. One-way factorial ANOVA was used to evaluate statistical significance. A probability value of P b 0.05 was considered indicative of statistical significance. 3. Results Cells were first superfused with Tyrode solution for 6 min to establish a steady state. Ischemia was then induced ex-vivo by exposing isolated myocytes to the ischemia-mimetic solution for 15 min before reperfusion. After 15 min reperfusion (at the later stage of reperfusion), peak shortening and cell length were check to evaluate the contractile function of cells. The values were expressed by percentage relative to pre-ischemic value.
Fig. 2. Effects of preconditioning and postconditioning of acetylcholine (ACh) on peak shortening (A, B) and cell length (C) at the later stage of reperfusion in ischemic rat ventricular myocytes (n = 6, *P b 0.05 vs. control group).
In the control, after 15 min reperfusion, peak shortening was 8.43 ± 5.35%, cell length was 57.06 ± 10.34% of the pre-ischemia value, which indicates that the contractility of the cardiomyocytes was severely reduced by ischemia and reperfusion. 3.1. The protective effects of adenosine preconditioning and postconditioning on the contractility of isolated rat ventricular myocytes
Fig. 1. Effects of preconditioning (precon) and postconditioning (postcon) of adenosine (Ado) on peak shortening (A, B) and cell length (C) at the later stage of reperfusion in ischemic rat ventricular myocytes (n = 6, *P b 0.05 vs. control group).
Fig. 1A shows a rapid-scan image of myocyte contraction illustrating comparative effects of preconditioning and postconditioning of adenosine on peak shortening at the later stage of reperfusion in ischemic rat ventricular myocytes. Peak shortening of adenosine preconditioning group and adenosine postconditioning group was 84.72 ± 5.34% and 76.47 ± 7.87% of the pre-ischemia value, respectively (Fig. 1B, n = 6, P b 0.05 vs. control group: 8.43 ± 5.35%). The effects of adenosine preconditioning and postconditioning on peak shortening were similar (P N 0.05; n = 6).
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Cell length of adenosine preconditioning group and adenosine postconditioning group was 93.00 ± 2.54% and 96.91 ± 1.74% of the pre-ischemia value, respectively (Fig. 1C, n = 6, P b 0.05 vs. control group: 57.06 ± 10.34%). The effects of adenosine preconditioning and postconditioning on cell length have similar extent (P N 0.05; n = 6). These results indicate that both adenosine preconditioning and postconditioning increase the contractility and reduce the contracture of ischemic rat ventricular myocytes to a similar extent. 3.2. The protective effects of acetylcholine preconditioning and postconditioning on the contractility of isolated rat ventricular myocytes Fig. 2A shows a rapid-scan image of myocyte contraction illustrating comparative effects of preconditioning and postconditioning of acetylcholine on peak shortening at the later stage of reperfusion in ischemic rat ventricular myocytes. Peak shortening of acetylcholine preconditioning group and acetylcholine postconditioning group was 68.61 ± 8.10% and 57.48 ± 6.97% of the pre-ischemia value, respectively (Fig. 2B, n = 6, P b 0.05 vs. control group: 8.43 ± 5.35%). The effects of acetylcholine preconditioning and postconditioning on peak shortening were similar (P N 0.05; n = 6). Cell length of acetylcholine preconditioning group and acetylcholine postconditioning group was 86.41 ± 3.50% and 85.67 ± 3.56% of the pre-ischemia value, respectively (Fig. 2C, n = 6, P b 0.05 vs. control group: 57.06 ± 10.34%). The effects of acetylcholine preconditioning and postconditioning on cell length were similar (P N 0.05; n = 6). These results indicate that both acetylcholine preconditioning and postconditioning increase the contractility and reduce the contracture of ischemic rat ventricular myocytes to a similar extent. 3.3. Inhibition of DPCPX and 5-HD on the protective effects of adenosine postconditioning on the contractility of isolated rat ventricular myocytes Next, we investigated the effect of DPCPX, the antagonist of adenosine A1 receptor on adenosine-induced postconditioning. Once we identified that the effect of adenosine on postconditioning was mediated through adenosine A1 receptor, we questioned possible implication of mitoKATP in this action. Specific inhibitor of these channel 5-HD was used. Fig. 3A shows a rapid-scan image of myocyte contraction illustrating the comparative effects of DPCPX and 5-HD on adenosine postconditioning on peak shortening at the later stage of reperfusion in ischemic rat ventricular myocytes. Peak shortening of adenosine postconditioning + DPCPX group and adenosine postconditioning + 5-HD group was 7.15 ± 4.81% and 13.41 ± 5.19% of the pre-ischemia value, respectively (Fig. 3B, n = 6, P b 0.05 vs. adenosine postconditioning group). The effects of control, adenosine postconditioning + DPCPX and adenosine postconditioning + 5-HD on peak shortening were similar (P N 0.05; n = 6).
Fig. 3. Effects of DPCPX and 5-HD on adenosine postconditioning on peak shortening (A, B) and cell length (C) at the later stage of reperfusion in ischemic rat ventricular myocytes (n = 6, *P b 0.05 vs. control group; #P b 0.05 vs. postconditioning group).
Cell length of adenosine postconditioning + DPCPX group and adenosine postconditioning + 5-HD group was 49.36 ± 8.16% and 49.78 ± 9.53% of the pre-ischemia value, respectively (Fig. 3C, n = 6, P b 0.05 vs. adenosine postconditioning group). The effects of control, adenosine postconditioning + DPCPX and adenosine postconditioning + 5-HD on cell length were similar (P N 0.05; n = 6). These results indicate that the effect of adenosine postconditioning to the contractility and contracture of ischemic rat ventricular myocytes is mediated by adenosine A1 receptor and involved mitoKATP.
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Fig. 4A shows a rapid-scan image of myocyte contraction illustrating the comparative effects of Methoctramine and 5-HD on acetylcholine postconditioning on peak shortening at the later stage of reperfusion in ischemic rat ventricular myocytes. Peak shortening of acetylcholine postconditioning + Methoctramine group and acetylcholine postconditioning + 5-HD group was 12.07 ± 5.47% and 11.66 ± 7.39% of the pre-ischemia value, respectively (Fig. 3B, n = 6, P b 0.05 vs. acetylcholine postconditioning group). The effects of control, acetylcholine postconditioning + Methoctramine and acetylcholine postconditioning + 5HD on peak shortening were similar (P N 0.05; n = 6). Cell length of acetylcholine postconditioning + Methoctramine group and acetylcholine postconditioning + 5-HD group was 60.09 ± 7.48% and 54.27 ± 9.91% of the pre-ischemia value, respectively (Fig. 3C, n = 6, P b 0.05 vs. acetylcholine postconditioning group). The effects of control, acetylcholine postconditioning + Methoctramine and acetylcholine postconditioning + 5HD on cell length were similar (P N 0.05; n = 6). These results indicate that the effect of acetylcholine postconditioning to the contractility and contracture of ischemic rat ventricular myocytes can be inhibited by Methoctramine and 5-HD. The results show that: (1) The contractile function of ischemic heart cells was improved by both adenosine and acetylcholine during preconditioning, as well as postconditioning and the effects of the two drugs on the contractility of ischemic rat ventricular myocytes were comparable. (2) Observed effects of adenosine and acetylcholine were missing in the presence of adenosine A1 receptor and muscarinic M2 receptor antagonists, respectively. (3) Adenosine and acetylcholine-induced pre- and postconditioning were also blocked by mitoKATP antagonist. 4. Discussion
Fig. 4. Effects of Methoctramine and 5-HD on acetylcholine postconditioning on peak shortening (A, B) and cell length (C) at the later stage of reperfusion in ischemic rat ventricular myocytes (n = 6, *P b 0.05 vs. control group; #P b 0.05 vs. postconditioning group).
3.4. Inhibition of Methoctramine and 5-HD on the protective effects of acetylcholine postconditioning on the contractility of isolated rat ventricular myocytes In Section 3.2, we demonstrated protective effect of acetylcholine on pre- and postconditioning. We therefore wanted to further investigate receptors implicated in the acetylcholine action on postconditioning. Specific antagonist of muscarinic M2 receptors, Methoctramine was used. Furthermore, implication of mitoKATP was also tested in acetylcholine action.
Cardiac preconditioning and postconditioning represent a potent and reproducible method to render the myocardium more resistant against irreversible structural and functional damage induced by a variety of noxious stimuli (Zaugg and Schaub, 2003). However, sometimes, postconditioning is more efficient than preconditioning (Yang et al., 2005). Contractile function is one of the important indexes of cardiac function. Injury of contractile function can be reflected by an increased diastolic force (contracture) and a decreased systolic force (Vassalle and Lin, 2004). The diastolic force and systolic force can be represented by resting cell length and peak shortening in single ventricular myocytes, respectively. Our datum suggests that: (1) Both acetylcholine and adenosine protect the contractile function of ischemic heart cells to a similar extent during preconditioning and postconditioning. (2) The postconditioning of adenosine and acetylcholine is relative to the adenosine A1 and muscarinic M2 receptors, respectively. MitoKATP is implicated in the postconditioning of both acetylcholine and adenosine. 4.1. Myocardial ischemic preconditioning/postconditioning and that by adenosine and acetylcholine Myocardial ischemic preconditioning is a phenomenon by which a brief episode(s) of myocardial ischemia increases the
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ability of the heart to tolerate a subsequent prolonged period of ischemic injury. This phenomenon and term was first described by Murry et al. (1986) in the canine infarct model. Preconditioning has been shown to improve post-ischemic recovery of function, slow energy metabolism during the early stages of ischemia, as well as reduce reperfusion arrhythmias (Eisen et al., 2004). Opening of mitoKATP was described to be crucial steps in the mechanism of ischemic preconditioning (Fryer et al., 2001; Wang et al., 2001). Adenosine and acetylcholine-induced preconditioning process was already demonstrated to be mediated by adenosine A1 and muscarinic M2 receptors, respectively (Zaugg and Schaub, 2003). Here, we confirmed this finding and we further demonstrated that postconditioning process is also mediated by these receptors. Ischemic postconditioning was first described by (Zhao et al. (2003), in which brief intermittent repetitive interruptions to reperfusion at the onset of reperfusion after a prolonged period of ischemia reduced myocardial injury to an extent comparable to IPC, offers a novel approach to myocardial protection (Tsang et al., 2004). Postconditioning initiates some responses such as modulation of endogenous autacoid release during early reperfusion and has many features in common with preconditioning, such as involving specific activation of the mitoKATP (Vinten-Johansen et al., 2005). We also demonstrated in this work that mitoKATP is implicated in postconditioning. However, despite possible more important therapeutical applications, postconditioning was so far much less studied. Here, we demonstrate that adenosine and acetylcholine can induce postconditioning process by adenosine A1 and muscarinic M2 receptors, respectively too. These results strongly indicate that acetylcholine and adenosine have protective effects on ischemic heart. Both of their main receptors are the Gαi-coupled receptors and can open the KACh channel (Zaugg and Schaub, 2003). Base on our result, both acetylcholine and adenosine can induce preconditioning and postconditioning to protect the contractility of ischemic heart cells. The postconditioning of acetylcholine and adenosine to the contractility of ischemic heart cells is relative to the mitoKATP. 4.2. MitoKATP and contractile function of myocardium MitoKATP was proposed to be a crucial mechanism implicated in cardioprotection. Several possible implications have been proposed: Opening of mitoKATP might lead to a depolarization of the mitochondrial membrane, causing dissipation of the mitochondrial potential and reducing the force for Ca2+ uptake and preservation of mitochondrial function to improve mitochondrial energy production during ischemia and reperfusion (Obal et al., 2005). These results of Iwai et al. (2002) suggest that the ability of mitochondria to produce ATP may be a crucial determinant for post-ischemic contractile recovery of the perfused rat heart. Murata et al. (2001) demonstrated that mitochondrial Ca2+ accumulation during simulated ischemia was attenuated by mitoKATP opening and this mechanism reduced the magnitude of mPTP (mitochondrial permeability transition pore) opening on reperfusion. The protective effect
was associated with strong depolarization of ΔΨm (mitochondrial membrane potential) under anoxic conditions and a consequent decrease in mitochondrial Ca2+ loading, which prevented a mitochondrial permeability transition on reoxygenation. Mitochondrial function was improved and more energy was available for contractile function of myocardium, resulting in enhanced contractility and reduced the contracture of ischemic heart cells and hence improved post-ischemic function. Both preconditioning and postconditioning activate the same key pathways, which include mitochondrial: They protect the heart through mitoKATP opening and the inhibition of mPTP opening (Hausenloy et al., 2004; Argaud et al., 2005). Some studies show that adenosine receptor activation during reperfusion affords cardioprotection via adenosine A2 and A3 receptors-mediated mechanisms mainly, but not adenosine A1 receptor (Kin et al., 2005). However, this evidence has largely been accumulated via determinating the infarct size of heart but not contractile function, and there is recent evidence of adenosine A1 receptor-mediated protection during reperfusion (Peart and Headrick, 2000). Reductions in post-ischemic contractile function reflect Ca2+ overload during reperfusion (Hampton et al., 1998) with impaired myofibrillar sensitivity to Ca2+ (Gao et al., 1995). Adenosine A1 receptor activation by endogenous adenosine may modify sarcolemmal Ca2+ fluxes to inhibit Ca2+ overload by activation of mitoKATP (Kim et al., 1997), then improve mitochondrial function and myocardial energy state (Headrick et al., 1998). Some studies were unable to mimic or abolish ischemic preconditioning with adenosine and adenosine antagonists in rat hearts (Ganote and Armstrong, 2000; Vasara et al., 2003). The inability to abolish ischemic preconditioning with adenosine antagonists could be related to the fact that: (1) 1. Interstitial adenosine is much higher in rat than, e.g., rabbit heart, which may require more adenosine receptor antagonist. (2) In the rat, sub-optimal adenosine A receptor activation could be due to active endothelial 1 adenosine degrading enzymes, the endothelial barrier for adenosine transport, and the short halflife of blood adenosine (De Jonge et al., 2002). We conclude that both acetylcholine and adenosine can induce preconditioning and postconditioning to protect the contractile function of ischemic heart cells and that these preconditioning and postconditioning effects are comparable. The postconditioning of adenosine and acetylcholine is mediated by the adenosine A1 and muscarinic M2 receptors, respectively, and both implicate the mitoKATP. In some circumstance, postconditioning is more useful than preconditioning. Preconditioning was generally assumed as a salvage of ischemic myocardium. But it has not yet been possible to apply this knowledge to the clinical arena. This was in large part because of a mismatch between the almost universal presentation of patients with acute myocardial infarction following coronary occlusion and hence the onset of ischemia and the need to apply a preconditioning intervention or pharmacologic mimetic agent prior to the onset of ischemia. For this impediment, attention has more recently turned to drugs and interventions that could be used after the onset of ischemia to salvage ischemic myocardium (Yang et al., 2005). So our
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results have some clinical significance, since they suggest that adenosine and acetylcholine can be administrated after reperfusion to protect the function of ischemic heart. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (No. 30470633) and external intercourse and cooperation item (No. 30511120267), the Research Fund for the Doctoral Program (No. 20050698012), the Cultivation Fund of the Key Scientific and Technical Innovation Project (No.705045) and “The Spring Bud Program” Cooperation project (No. Z2005-271001) of Ministry of Education of China, as well as FRSQ-NSFC support (No. 5540) to WeiJin Zang and Alzbeta Chorvatova of Canada. References Argaud, L., Gateau-Roesch, O., Raisky, O., Loufouat, J., Robert, D., Ovize, M., 2005. Postconditioning inhibits mitochondrial permeability transition. Circulation 111, 194–197. Baker, J.E., 2005. Erythropoietin mimics ischemic preconditioning. Vasc. Pharmacol. 42, 233–241. Critza, S.D., Cohenb, M.V., Downey, J.M., 2005. Mechanisms of acetylcholineand bradykinin-induced preconditioning. Vasc. Pharmacol. 42, 201–209. De Jonge, R., Out, M., Maas, W.J., De Jonge, J.W., 2002. Preconditioning of rat hearts by adenosine A1 or A3 receptor activation. Eur. J. Pharmacol. 441, 165–172. Eisen, A., Fisman, E.Z., Rubenfire, M., Freimark, D., McKechnie, R., Tenenbaum, A., Motro, M., Adler, Y., 2004. Ischemic preconditioning: nearly two decades of research. A comprehensive review. Atherosclerosis 172, 201–210. Fryer, R.M., Hsu, A.K., Gross, G.J., 2001. Mitochondrial KATP channel opening is important during index ischemia and following myocardial reperfusion in ischemic preconditioned rat hearts. J. Mol. Cell. Cardiol. 33, 831–834. Ganote, C.E., Armstrong, S.C., 2000. Adenosine and preconditioning in the rat heart. Cardiovasc. Res. 45, 134–140. Gao, W.D., Atar, D., Backx, P.H., Marban, E., 1995. Relationship between intracellular calcium and contractile force in stunned myocardium: direct evidence for decreased myofilament Ca2+ responsiveness and altered diastolic function in intact ventricular muscle. Circ. Res. 76, 1036–1048. Hampton, T.G., Amende, I., Travers, K.E., Morgan, J.P., 1998. Intracellular calcium dynamics in mouse model of myocardial stunning. Am. J. Physiol., Heart Circ. Physiol. 274, H1821–H1827. Hausenloy, D., Wynne, A., Duchen, M., Yellon, D., 2004. Transient mitochondrial permeability transition pore opening mediates preconditioning-induced protection. Circulation 109, 1714–1717. Headrick, J.P., Gauthier, N.S., Berr, S.S., Matherne, G.P., 1998. Transgenic A1 adenosine receptor overexpression improves myocardial energy state during ischemia–reperfusion. J. Mol. Cell. Cardiol. 30, 1059–1064. Iwai, T., Tanonaka, K., Inoue, R., Kasahara, S., Kamo, N., Takeo, S., 2002. Mitochondrial damage during ischemia determines post-ischemic contractile dysfunction in perfused rat heart. J. Mol. Cell. Cardiol. 34, 725–738. Kim, E., Han, J., Ho, W., Earm, Y.E., 1997. Modulation of ATP sensitive K+ channels in rabbit ventricular myocytes by adenosine A1 receptor activation. Am. J. Physiol., Heart Circ. Physiol. 272, H325–H333. Kin, H., Zatta, A.J., Lofye, M.T., Amerson, B.S., Halkos, M.E., Kerendi, F., Zhao, Z.Q., Guyton, R.A., Headrick, J.P., Vinten-Johansen, J., 2005. Postconditioning reduces infarct size via adenosine receptor activation by endogenous adenosine. Cardiovasc. Res. 67, 124–133.
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Lazzarino, G., Raatikainen, P., Nuutinen, M., Nissinen, J., Tavazzi, B., Di Pierro, D., 1994. Myocardial release of malondialdehyde and purine compounds during coronary bypass surgery. Circulation 90, 291–297. Lott, F.D., Guo, P., Toombs, C.F., 1996. Reduction in infarct size by ischemic preconditioning persists in a chronic rat model of myocardial ischemia– reperfusion injury. Pharmacology 52, 113–118. Lu, J., Zang, W.J., Yu, X.J., Chen, L.N., Zhang, C.H., Jia, B., 2005. Effects of ischaemia-mimetic factors on isolated rat ventricular myocytes. Exp. Physiol. 90, 497–505. Murata, M., Akao, M., O'Rourke, B., Marbán, E., 2001. Mitochondrial ATP sensitive potassium channels attenuate matrix Ca2+ overload during simulated ischemia and reperfusion: possible mechanism of cardioprotection. Circ. Res. 89, 891–898. Murry, C.E., Jennings, R.B., Reimer, K.A., 1986. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74, 1124–1136. Obal, D., Dettwiler, S., Favoccia, C., Scharbatke, H., Preckel, B., Schlack, W., 2005. The influence of mitochondrial KATP-channels in the cardioprotection of preconditioning and postconditioning by sevoflurane in the rat in vivo. Anesth. Analg. 101, 1252–1260. Peart, J., Headrick, J.P., 2000. Intrinsic A1 adenosine receptor activation during ischemia or reperfusion improves recovery in mouse hearts. Am. J. Physiol., Heart Circ. Physiol. 279, H2166–H2175. Pierce, G.N., Czubryt, M.P., 1995. The contribution of ionic imbalance to ischemia/reperfusion injury. J. Mol. Cell. Cardiol. 27, 53–65. Tsang, A., Hausenloy, D.J., Mocanu, M.M., Yellon, D.M., 2004. Postconditioning: a form of "modified reperfusion” protects the myocardium by activating the phosphatidylinositol 3-kinase-Akt pathway. Circ. Res. 95, 230–232. Vasara, E., Katharou, I., Lazou, A., 2003. Myocardial adenosine does not correlate with the protection mediated by ischaemic or pharmacological preconditioning in rat heart. Clin. Exp. Pharmacol. Physiol. 30, 350–356. Vassalle, M., Lin, C.I., 2004. Calcium overload and cardiac function. J. Biomed. Sci. 11 (5), 542–565. Vinten-Johansen, J., Zhao, Z.Q., Zatta, A.J., Kin, H., Halkos, M.E., Kerendi, F., 2005. Postconditioning a new link in nature's armor against myocardial ischemia–reperfusion injury. Basic Res. Cardiol. 100, 1–16. Wang, S., Cone, J., Liu, Y., 2001. Dual roles of mitochondrial KATP channels in diazoxide-mediated protection in isolated rabbit hearts. Am. J. Physiol., Heart Circ. Physiol. 280, H246–H256. Wold, L.E., Saari, J.T., Ren, J., 2001. Isolated ventricular myocytes from copper-deficient rat hearts exhibit enhanced contractile function. Am. J. Physiol., Heart Circ. Physiol. 281, H476–H481. Yang, X.M., Philipp, S., Downey, J.M., Cohen, M.V., 2005. Postconditioning's protection is not dependent on circulating blood factors or cells but involves adenosine receptors and requires PI3-kinase and guanylyl cyclase activation. Basic Res. Cardiol. 100 (1), 57–63. Zang, W.J., Yu, X.J., Honjo, H., Kirby, M.S., Boyett, M.R., 1993. On the role of G protein activation and phosphorylation in desensitization to acetylcholine in guinea-pig atrial cells. J. Physiol. 464, 649–679. Zang, W.J., Yu, X.J., Zang, Y.M., 2003. Ca2+ sparks evoked by depolarization of rat heart cells involve multiple release sites. Acta Pharmacol. Sinica 24, 555–562. Zang, W.J., Chen, L.N., Yu, X.J., Fang, P., Lu, J., Sun, Q., 2005. Comparison of effects of acetylcholine on electromechanical characteristics in guinea-pig atrium and ventricle. Exp. Physiol. 90, 123–130. Zaugg, M., Schaub, M.C., 2003. Signaling and cellular mechanisms in cardiac protection by ischemic and pharmacological preconditioning. J. Muscle Res. Cell Motil. 24, 219–249. Zhao, Z.Q., Corvera, J.S., Halkos, M.E., Kerendi, F., Wang, N.P., Guyton, R.A., Vinten-Johansen, J., 2003. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am. J. Physiol., Heart Circ. Physiol. 285, H579–H588.
Effects of postconditioning of adenosine and acetylcholine on the ischemic isolated rat ventricular myocytes Jun Lu a , Wei-Jin Zang a,b,⁎, Xiao-Jiang Yu a , Bing Jia a , Alzbeta Chorvatova c,d , Lei Sun a a
b
Department of Pharmacology, School of Medicine, Xi'an Jiaotong University, Xi'an, 710061, PR China Key Laboratory of Environment and Genes Related to Diseases of Ministry of Education, School of Medicine, Xi'an Jiaotong University, Xi'an, 710061, PR China c Research Centre, Sainte-Justine Hospital, University of Montreal, Montreal, Canada d Department of Pediatrics, University of Montreal, Montreal, Canada Received 26 July 2006; received in revised form 5 August 2006; accepted 14 August 2006 Available online 26 August 2006
Abstract In this study, protective effects of adenosine and acetylcholine-induced postconditioning were investigated on the contractile function of the ischemic isolated rat ventricular myocytes. A video-based edge-detection system was used to monitor single ventricular myocytes contraction. Adenosine and acetylcholine were administrated for 6 min before ischemia as preconditioning, or 15 min after ischemia as postconditioning. Adenosine and acetylcholine receptor antagonists and mitoKATP inhibitor were used to analyze pathways underlying the effects on postconditioning. Results: (1) The peak shortening of ischemic heart cells was improved by both adenosine and acetylcholine during preconditioning (84.72 ± 5.34% and 68.61 ± 8.10% vs. control: 8.43 ± 5.35% of the pre-ischemia value), as well as postconditioning (76.47 ± 7.87% and 57.48 ± 6.97% vs. control: 8.43 ± 5.35% of the pre-ischemia value) and the effects of preconditioning and postconditioning were comparable. More datum in the normal text. (2) Observed effects of adenosine and acetylcholine postconditioning were missing in the presence of adenosine A1 receptor and muscarinic M2 receptor antagonists, respectively. (3) Adenosine and acetylcholine-induced postconditioning was also blocked by mitoKATP antagonist. These results suggest that both adenosine and acetylcholine protect the contractile function of ischemic heart cells to a similar extent during preconditioning and postconditioning. The postconditioning of adenosine and acetylcholine is relative to the adenosine A1 and muscarinic M2 receptors, respectively. MitoKATP is implicated in the postconditioning of both acetylcholine and adenosine. © 2006 Elsevier B.V. All rights reserved. Keywords: Acetylcholine; Adenosine; Ischemia; Myocyte; Postconditioning
1. Introduction Ischemic heart disease, a common cardiovascular disease, severely endangers health and is therefore a major focus of medical studies (Lazzarino et al., 1994; Pierce and Czubryt, 1995). There are two important concepts, preconditioning and postconditioning, refer to the topic. Ischemic preconditioning was first introduced by Murry et al. (1986) as a potent endogenous form of cardioprotection against ischemic–reperfusion injury. It enhances the recovery of cardiac function after global ischemia, reduces infarct size and the ap⁎ Corresponding author. Department of Pharmacology, School of Medicine, Xi'an Jiaotong University, Xi'an, 710061, PR China. Tel.: +86 29 82655150; fax: +86 29 82655003. E-mail address: [email protected] (W.-J. Zang). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.08.030
pearance of apoptosis in hearts subjected to ischemia–reperfusion injury (Lott et al., 1996). In 2003, Zhao et al. reported another endogenous form of cardioprotection similar to that observed with ischemic preconditioning–ischemic postconditioning, which is defined as a series of brief interruptions of reperfusion applied at the very onset of reperfusion (Zhao et al., 2003). Ischemic preconditioning/postconditioning is a powerful endogenous phenomenon in which brief periods of a sub-toxic ischemic insult induce robust protection against lengthy, even lethal ischemia. Manifestation of the cardioprotective effects of ischemic preconditioning was found in many species, including rat. The ability to reproduce the cardioprotective effects of ischemic preconditioning/postconditioning with pharmacological agents raises the possibility that a drug may ultimately be introduced into clinical practice to treat human hearts undergoing ischemia/reperfusion (Baker, 2005).
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Adenosine and acetylcholine-induced preconditioning process involves adenosine A1 and muscarinic M2 receptors, respectively, and mitochondrial ATP-sensitive potassium channels (mitoKATP) were proposed to play an important role in this process (Zaugg and Schaub, 2003). Both adenosine and muscarinic receptors are the Gαi-coupled receptors that can open the KACh channel. However, it is still unclear whether adenosine and acetylcholine can also induce postconditioning process and hence affect cardiac contractility following ischemia and/or what are the underlying mechanisms (Critza et al., 2005). In this study, we therefore questioned possible protective effects of acetylcholine and adenosine postconditioning on the contractile function of isolated rat ventricular myocytes and underlying mechanisms. 2. Materials and methods 2.1. Cell isolation Adult Sprague–Dawley rats of both sexes, supplied by the Experimental Animal Center of Xi'an Jiaotong University, China, and weighing 250–300 g were used in accordance with the Guidelines on the Care and Use of Laboratory Animals issued by the Chinese Council on Animal Research and the Guidelines of Animal Care. The study was approved by the ethical committee of Xi'an Jiaotong University. Ventricular myocytes were isolated enzymatically from rats using a conventional method (Zang et al., 1993). Briefly, heparinized rats were anaesthetized with sodium pentobarbitone (17 mg/kg, i.p.) (Zang et al., 2003). The heart was rapidly excised, placed in ice-cold Ca2+-free modified Tyrode solution (in mmol/l: NaCl, 143; KCl, 5.4; MgCl2, 0.5; NaH2PO4, 0.33; Hepes, 5.0; and glucose, 5.0; pH titrated to 7.35 with NaOH), cannulated and then perfused retrogradely with Ca2+-free Tyrode solution via the aorta on a Langendorff perfusion apparatus for about 5 min until spontaneous contractions ceased. The heart was then perfused with Ca2+-free Tyrode solution containing 0.7 mg/ml collagenase (type I, Sigma, St Louis, MO, USA) and 1 mg/ml bovine serum albumin (Sigma) for 20 min. Finally, the enzymes were washed out with a high-K+, low-Cl− solution (KB solution, in mmol/l: KCl, 25; taurine, 20; L-glutamic acid, 70; KH2PO4, 10; MgCl2, 3; EGTA, 0.5; glucose, 10; and Hepes, 10; pH 7.35) for 5 min. All of the solutions used during perfusion were bubbled with 100% O2 and maintained at 37 °C. Following perfusion, the ventricle was placed in a beaker filled with KB solution and then minced. The ventricular cells were dispersed by shaking the beaker gently and the undigested tissue was removed by filtration through a 250 μm nylon mesh. The cells were kept in Tyrode solution (in mmol/l: NaCl, 136.9; KCl, 5.4; MgCl2, 0.5; NaH2PO4, 0.33; Hepes, 5.0; glucose, 10.0; and CaCl2, 1.8; pH 7.4; bubbled with 100% O2) at room temperature for at least 1 h before use. 2.2. Protocols The ischemia-mimetic behaviour of single cardiomyocytes was simulated using a modified ischemia-mimetic solution (in
mmol/l: NaCl, 135; KCl, 8; MgCl2, 0.5; NaH2PO4, 0.33; Hepes, 5.0; CaCl2, 1.8; and Na+-lactate, 20; pH 6.80; bubbled with 100% N2 for N 45 min before the experiment was started; this reduced the oxygen tension by 75%) (Lu et al., 2005). Coverslips to which cardiomyocytes were adhered were placed in a flow-through (1 ml/min, 25 °C) perfusion chamber (the surface of which was surrounded with N 2 to prevent reoxygenation of the bath ischemia-mimetic solution) that was positioned on the stage of an inverted microscope and the chamber was continuously superfused with Tyrode solution. Cells were field stimulated at 0.5 Hz by two Pt electrodes connected to a MyoPacer field stimulator (IonOptix Corporation, Milton, MA, USA). The polarity of the stimulating electrodes was reversed periodically to avoid the potential build up of electrolysis byproducts (Wold et al., 2001). In all cells, the normal Tyrode solution was perfused for 6 min, then the ischemia-mimetic solution for 15 min to mimic ischemia, finally reperfused by the normal Tyrode solution for 15 min. Cells were assigned to one of the following 9 groups based upon the intervention (n = 6 in each group): (1) Control: there was no intervention either before or after the ischemiamimetic solution perfusion; (2) Adenosine preconditioning: adenosine (100 μmol/l) was administrated for 6 min before ischemia; (3) Adenosine postconditioning: adenosine was administrated for 15 min after ischemia; (4) DPCPX (8cyclopentyl-1,3-dipropylxanthine, adenosine A1 receptor antagonist) + adenosine postconditioning: adenosine and DPCPX (1 μmol/l) were administrated for 15 min after ischemia; (5) 5HD (5-hydroxydecanoate, mitoKATP antagonist) + adenosine postconditioning: adenosine and 5-HD (100 μmol/l) were administrated for 15 min after ischemia; (6) Acetylcholine preconditioning: acetylcholine (0.1 μmol/l) was administrated for 6 min before ischemia; (7) Acetylcholine postconditioning: acetylcholine was administrated for 15 min after ischemia; (8) Methoctramine (Muscarinic M2 receptor antagonist) + acetylcholine postconditioning: acetylcholine and Methoctramine (0.1 μmol/l) were administrated for 15 min after ischemia; (9) MitoKATP antagonist 5-HDs + acetylcholine postconditioning: acetylcholine and 5-HD were administrated for 15 min after ischemia. In a subset of myocytes the effects of DPCPX, Methoctramine and 5-HD alone were tested and these agents did not exert any effects alone statistically. 2.3. Myocyte shortening and lengthening measurements Cardiomyocytes were viewed with the aid of an inverted microscope (Olympus X-70, Olympus Optical, Tokyo, Japan) and imaged using an IonOptix MyoCam camera (IonOptix Corporation, Milton, MA, USA) (Zang et al., 2005). Myocyte motion was measured using a video-based edge-detection system (IonOptix Corporation). Changes in cell contractile function were quantified using the following parameters: peak shortening (representing the amplitude of myocyte contraction, viz. contractility) and cell length (resting cell length, representing the extent of contracture). Data were acquired and analysed using IonOptix software.
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2.4. Statistical analysis Results are expressed as means ± S.E.M. One-way factorial ANOVA was used to evaluate statistical significance. A probability value of P b 0.05 was considered indicative of statistical significance. 3. Results Cells were first superfused with Tyrode solution for 6 min to establish a steady state. Ischemia was then induced ex-vivo by exposing isolated myocytes to the ischemia-mimetic solution for 15 min before reperfusion. After 15 min reperfusion (at the later stage of reperfusion), peak shortening and cell length were check to evaluate the contractile function of cells. The values were expressed by percentage relative to pre-ischemic value.
Fig. 2. Effects of preconditioning and postconditioning of acetylcholine (ACh) on peak shortening (A, B) and cell length (C) at the later stage of reperfusion in ischemic rat ventricular myocytes (n = 6, *P b 0.05 vs. control group).
In the control, after 15 min reperfusion, peak shortening was 8.43 ± 5.35%, cell length was 57.06 ± 10.34% of the pre-ischemia value, which indicates that the contractility of the cardiomyocytes was severely reduced by ischemia and reperfusion. 3.1. The protective effects of adenosine preconditioning and postconditioning on the contractility of isolated rat ventricular myocytes
Fig. 1. Effects of preconditioning (precon) and postconditioning (postcon) of adenosine (Ado) on peak shortening (A, B) and cell length (C) at the later stage of reperfusion in ischemic rat ventricular myocytes (n = 6, *P b 0.05 vs. control group).
Fig. 1A shows a rapid-scan image of myocyte contraction illustrating comparative effects of preconditioning and postconditioning of adenosine on peak shortening at the later stage of reperfusion in ischemic rat ventricular myocytes. Peak shortening of adenosine preconditioning group and adenosine postconditioning group was 84.72 ± 5.34% and 76.47 ± 7.87% of the pre-ischemia value, respectively (Fig. 1B, n = 6, P b 0.05 vs. control group: 8.43 ± 5.35%). The effects of adenosine preconditioning and postconditioning on peak shortening were similar (P N 0.05; n = 6).
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Cell length of adenosine preconditioning group and adenosine postconditioning group was 93.00 ± 2.54% and 96.91 ± 1.74% of the pre-ischemia value, respectively (Fig. 1C, n = 6, P b 0.05 vs. control group: 57.06 ± 10.34%). The effects of adenosine preconditioning and postconditioning on cell length have similar extent (P N 0.05; n = 6). These results indicate that both adenosine preconditioning and postconditioning increase the contractility and reduce the contracture of ischemic rat ventricular myocytes to a similar extent. 3.2. The protective effects of acetylcholine preconditioning and postconditioning on the contractility of isolated rat ventricular myocytes Fig. 2A shows a rapid-scan image of myocyte contraction illustrating comparative effects of preconditioning and postconditioning of acetylcholine on peak shortening at the later stage of reperfusion in ischemic rat ventricular myocytes. Peak shortening of acetylcholine preconditioning group and acetylcholine postconditioning group was 68.61 ± 8.10% and 57.48 ± 6.97% of the pre-ischemia value, respectively (Fig. 2B, n = 6, P b 0.05 vs. control group: 8.43 ± 5.35%). The effects of acetylcholine preconditioning and postconditioning on peak shortening were similar (P N 0.05; n = 6). Cell length of acetylcholine preconditioning group and acetylcholine postconditioning group was 86.41 ± 3.50% and 85.67 ± 3.56% of the pre-ischemia value, respectively (Fig. 2C, n = 6, P b 0.05 vs. control group: 57.06 ± 10.34%). The effects of acetylcholine preconditioning and postconditioning on cell length were similar (P N 0.05; n = 6). These results indicate that both acetylcholine preconditioning and postconditioning increase the contractility and reduce the contracture of ischemic rat ventricular myocytes to a similar extent. 3.3. Inhibition of DPCPX and 5-HD on the protective effects of adenosine postconditioning on the contractility of isolated rat ventricular myocytes Next, we investigated the effect of DPCPX, the antagonist of adenosine A1 receptor on adenosine-induced postconditioning. Once we identified that the effect of adenosine on postconditioning was mediated through adenosine A1 receptor, we questioned possible implication of mitoKATP in this action. Specific inhibitor of these channel 5-HD was used. Fig. 3A shows a rapid-scan image of myocyte contraction illustrating the comparative effects of DPCPX and 5-HD on adenosine postconditioning on peak shortening at the later stage of reperfusion in ischemic rat ventricular myocytes. Peak shortening of adenosine postconditioning + DPCPX group and adenosine postconditioning + 5-HD group was 7.15 ± 4.81% and 13.41 ± 5.19% of the pre-ischemia value, respectively (Fig. 3B, n = 6, P b 0.05 vs. adenosine postconditioning group). The effects of control, adenosine postconditioning + DPCPX and adenosine postconditioning + 5-HD on peak shortening were similar (P N 0.05; n = 6).
Fig. 3. Effects of DPCPX and 5-HD on adenosine postconditioning on peak shortening (A, B) and cell length (C) at the later stage of reperfusion in ischemic rat ventricular myocytes (n = 6, *P b 0.05 vs. control group; #P b 0.05 vs. postconditioning group).
Cell length of adenosine postconditioning + DPCPX group and adenosine postconditioning + 5-HD group was 49.36 ± 8.16% and 49.78 ± 9.53% of the pre-ischemia value, respectively (Fig. 3C, n = 6, P b 0.05 vs. adenosine postconditioning group). The effects of control, adenosine postconditioning + DPCPX and adenosine postconditioning + 5-HD on cell length were similar (P N 0.05; n = 6). These results indicate that the effect of adenosine postconditioning to the contractility and contracture of ischemic rat ventricular myocytes is mediated by adenosine A1 receptor and involved mitoKATP.
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Fig. 4A shows a rapid-scan image of myocyte contraction illustrating the comparative effects of Methoctramine and 5-HD on acetylcholine postconditioning on peak shortening at the later stage of reperfusion in ischemic rat ventricular myocytes. Peak shortening of acetylcholine postconditioning + Methoctramine group and acetylcholine postconditioning + 5-HD group was 12.07 ± 5.47% and 11.66 ± 7.39% of the pre-ischemia value, respectively (Fig. 3B, n = 6, P b 0.05 vs. acetylcholine postconditioning group). The effects of control, acetylcholine postconditioning + Methoctramine and acetylcholine postconditioning + 5HD on peak shortening were similar (P N 0.05; n = 6). Cell length of acetylcholine postconditioning + Methoctramine group and acetylcholine postconditioning + 5-HD group was 60.09 ± 7.48% and 54.27 ± 9.91% of the pre-ischemia value, respectively (Fig. 3C, n = 6, P b 0.05 vs. acetylcholine postconditioning group). The effects of control, acetylcholine postconditioning + Methoctramine and acetylcholine postconditioning + 5HD on cell length were similar (P N 0.05; n = 6). These results indicate that the effect of acetylcholine postconditioning to the contractility and contracture of ischemic rat ventricular myocytes can be inhibited by Methoctramine and 5-HD. The results show that: (1) The contractile function of ischemic heart cells was improved by both adenosine and acetylcholine during preconditioning, as well as postconditioning and the effects of the two drugs on the contractility of ischemic rat ventricular myocytes were comparable. (2) Observed effects of adenosine and acetylcholine were missing in the presence of adenosine A1 receptor and muscarinic M2 receptor antagonists, respectively. (3) Adenosine and acetylcholine-induced pre- and postconditioning were also blocked by mitoKATP antagonist. 4. Discussion
Fig. 4. Effects of Methoctramine and 5-HD on acetylcholine postconditioning on peak shortening (A, B) and cell length (C) at the later stage of reperfusion in ischemic rat ventricular myocytes (n = 6, *P b 0.05 vs. control group; #P b 0.05 vs. postconditioning group).
3.4. Inhibition of Methoctramine and 5-HD on the protective effects of acetylcholine postconditioning on the contractility of isolated rat ventricular myocytes In Section 3.2, we demonstrated protective effect of acetylcholine on pre- and postconditioning. We therefore wanted to further investigate receptors implicated in the acetylcholine action on postconditioning. Specific antagonist of muscarinic M2 receptors, Methoctramine was used. Furthermore, implication of mitoKATP was also tested in acetylcholine action.
Cardiac preconditioning and postconditioning represent a potent and reproducible method to render the myocardium more resistant against irreversible structural and functional damage induced by a variety of noxious stimuli (Zaugg and Schaub, 2003). However, sometimes, postconditioning is more efficient than preconditioning (Yang et al., 2005). Contractile function is one of the important indexes of cardiac function. Injury of contractile function can be reflected by an increased diastolic force (contracture) and a decreased systolic force (Vassalle and Lin, 2004). The diastolic force and systolic force can be represented by resting cell length and peak shortening in single ventricular myocytes, respectively. Our datum suggests that: (1) Both acetylcholine and adenosine protect the contractile function of ischemic heart cells to a similar extent during preconditioning and postconditioning. (2) The postconditioning of adenosine and acetylcholine is relative to the adenosine A1 and muscarinic M2 receptors, respectively. MitoKATP is implicated in the postconditioning of both acetylcholine and adenosine. 4.1. Myocardial ischemic preconditioning/postconditioning and that by adenosine and acetylcholine Myocardial ischemic preconditioning is a phenomenon by which a brief episode(s) of myocardial ischemia increases the
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ability of the heart to tolerate a subsequent prolonged period of ischemic injury. This phenomenon and term was first described by Murry et al. (1986) in the canine infarct model. Preconditioning has been shown to improve post-ischemic recovery of function, slow energy metabolism during the early stages of ischemia, as well as reduce reperfusion arrhythmias (Eisen et al., 2004). Opening of mitoKATP was described to be crucial steps in the mechanism of ischemic preconditioning (Fryer et al., 2001; Wang et al., 2001). Adenosine and acetylcholine-induced preconditioning process was already demonstrated to be mediated by adenosine A1 and muscarinic M2 receptors, respectively (Zaugg and Schaub, 2003). Here, we confirmed this finding and we further demonstrated that postconditioning process is also mediated by these receptors. Ischemic postconditioning was first described by (Zhao et al. (2003), in which brief intermittent repetitive interruptions to reperfusion at the onset of reperfusion after a prolonged period of ischemia reduced myocardial injury to an extent comparable to IPC, offers a novel approach to myocardial protection (Tsang et al., 2004). Postconditioning initiates some responses such as modulation of endogenous autacoid release during early reperfusion and has many features in common with preconditioning, such as involving specific activation of the mitoKATP (Vinten-Johansen et al., 2005). We also demonstrated in this work that mitoKATP is implicated in postconditioning. However, despite possible more important therapeutical applications, postconditioning was so far much less studied. Here, we demonstrate that adenosine and acetylcholine can induce postconditioning process by adenosine A1 and muscarinic M2 receptors, respectively too. These results strongly indicate that acetylcholine and adenosine have protective effects on ischemic heart. Both of their main receptors are the Gαi-coupled receptors and can open the KACh channel (Zaugg and Schaub, 2003). Base on our result, both acetylcholine and adenosine can induce preconditioning and postconditioning to protect the contractility of ischemic heart cells. The postconditioning of acetylcholine and adenosine to the contractility of ischemic heart cells is relative to the mitoKATP. 4.2. MitoKATP and contractile function of myocardium MitoKATP was proposed to be a crucial mechanism implicated in cardioprotection. Several possible implications have been proposed: Opening of mitoKATP might lead to a depolarization of the mitochondrial membrane, causing dissipation of the mitochondrial potential and reducing the force for Ca2+ uptake and preservation of mitochondrial function to improve mitochondrial energy production during ischemia and reperfusion (Obal et al., 2005). These results of Iwai et al. (2002) suggest that the ability of mitochondria to produce ATP may be a crucial determinant for post-ischemic contractile recovery of the perfused rat heart. Murata et al. (2001) demonstrated that mitochondrial Ca2+ accumulation during simulated ischemia was attenuated by mitoKATP opening and this mechanism reduced the magnitude of mPTP (mitochondrial permeability transition pore) opening on reperfusion. The protective effect
was associated with strong depolarization of ΔΨm (mitochondrial membrane potential) under anoxic conditions and a consequent decrease in mitochondrial Ca2+ loading, which prevented a mitochondrial permeability transition on reoxygenation. Mitochondrial function was improved and more energy was available for contractile function of myocardium, resulting in enhanced contractility and reduced the contracture of ischemic heart cells and hence improved post-ischemic function. Both preconditioning and postconditioning activate the same key pathways, which include mitochondrial: They protect the heart through mitoKATP opening and the inhibition of mPTP opening (Hausenloy et al., 2004; Argaud et al., 2005). Some studies show that adenosine receptor activation during reperfusion affords cardioprotection via adenosine A2 and A3 receptors-mediated mechanisms mainly, but not adenosine A1 receptor (Kin et al., 2005). However, this evidence has largely been accumulated via determinating the infarct size of heart but not contractile function, and there is recent evidence of adenosine A1 receptor-mediated protection during reperfusion (Peart and Headrick, 2000). Reductions in post-ischemic contractile function reflect Ca2+ overload during reperfusion (Hampton et al., 1998) with impaired myofibrillar sensitivity to Ca2+ (Gao et al., 1995). Adenosine A1 receptor activation by endogenous adenosine may modify sarcolemmal Ca2+ fluxes to inhibit Ca2+ overload by activation of mitoKATP (Kim et al., 1997), then improve mitochondrial function and myocardial energy state (Headrick et al., 1998). Some studies were unable to mimic or abolish ischemic preconditioning with adenosine and adenosine antagonists in rat hearts (Ganote and Armstrong, 2000; Vasara et al., 2003). The inability to abolish ischemic preconditioning with adenosine antagonists could be related to the fact that: (1) 1. Interstitial adenosine is much higher in rat than, e.g., rabbit heart, which may require more adenosine receptor antagonist. (2) In the rat, sub-optimal adenosine A receptor activation could be due to active endothelial 1 adenosine degrading enzymes, the endothelial barrier for adenosine transport, and the short halflife of blood adenosine (De Jonge et al., 2002). We conclude that both acetylcholine and adenosine can induce preconditioning and postconditioning to protect the contractile function of ischemic heart cells and that these preconditioning and postconditioning effects are comparable. The postconditioning of adenosine and acetylcholine is mediated by the adenosine A1 and muscarinic M2 receptors, respectively, and both implicate the mitoKATP. In some circumstance, postconditioning is more useful than preconditioning. Preconditioning was generally assumed as a salvage of ischemic myocardium. But it has not yet been possible to apply this knowledge to the clinical arena. This was in large part because of a mismatch between the almost universal presentation of patients with acute myocardial infarction following coronary occlusion and hence the onset of ischemia and the need to apply a preconditioning intervention or pharmacologic mimetic agent prior to the onset of ischemia. For this impediment, attention has more recently turned to drugs and interventions that could be used after the onset of ischemia to salvage ischemic myocardium (Yang et al., 2005). So our
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