Copyright © 2006, European Society of Cardiology
Attenuated cardioprotection by ischemic preconditioning in coronary stenosed heart and its restoration by carvedilol
First Department of Internal Medicine, Fukushima Medical University, Hikarigaoka 1, Fukushima 960-1295, Japan
* Corresponding author. Tel.: +81 24 547 1190; fax: +81 24 548 1821. Email address: maruyama{at}fmu.ac.jp
Received 15 November 2005; revised 13 April 2006; accepted 5 May 2006
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Abstract |
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 Top Abstract 1. Introduction2. Methods3. Results4. DiscussionReferences |
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Objective: Infarct size (IS) reduction by ischemic preconditioning (IPC) has been assessed in the heart in which coronary stenosis (CS)-induced chronic ischemia was not present. The aim of this study is to assess whether and how IS reduction by IPC is modified in the heart in which CS has occurred, and how further therapeutics, if any, modify it.
Methods We assessed the IS produced by a 30-minute acute coronary occlusion and a 24-hour reperfusion (COR) in rat hearts in which CS had developed for 1–12 weeks. Modifications of IS by IPC and the mitochondrial KATP channel (mitoKATP) opener and blocker, and the effects of daily β-blocker treatment with carvedilol on them, were also assessed. Myocardial protein kinase C (PKC)-
activities in the risk areas were measured by Western blotting.
Results: One to 4 weeks after CS induction, myocardial PKC- was activated in the risk area of CS even without IPC, but such CS-induced PKC activation as well as that by IPC was attenuated 8–12 weeks after CS. The IS reductions by the mitoKATP opener as well as by IPC were attenuated 8–12 and 4–12 weeks after CS, respectively. Daily carvedilol treatment after inducing CS restored the malfunctioning PKC-mitoKATP signal cascade and the attenuated IPC and mitoKATP effect on IS.
Conclusion CS activates the PKC-mitoKATP signal cascade, mimicking the IPC effect, whereas this cardioprotective effect is attenuated late after CS via their downregulation. Restoration of these changes may be a novel mechanism for cardioprotection by carvedilol in the CS-induced ischemic heart.
KEYWORDS Ischemia; Infarction; Preconditioning; Protein kinase C; Adrenergic antagonist; K-ATP channel
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1. Introduction |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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Ischemic preconditioning (IPC) is a powerful cardioprotective tool in acute ischemia–reperfusion. In most reports on IPC, brief ischemic insults to induce IPC were given the normal (non-ischemic) heart [1,2]. However, about 30% of myocardial infarctions occur in patients who have already had coronary stenosis (CS) [3]. It is of interest to see whether and how IPC exerts a cardioprotective effect on infarct size in the risk area of chronic CS with maintained myocardial blood flow but diminished coronary flow reserve, as often occurs in patients with chronic coronary artery disease [4].
Protein kinase C (PKC) activation and subsequent opening of mitochondrial ATP-sensitive potassium channel (mitoKATP) are involved in the mechanisms of the IPC-induced infarct size reduction [2,5–7]. If CS mimics the effect of IPC, presumably via repetitive ischemic attacks triggered by daily activities, PKC activation may be repeated in chronic CS. In previous reports, chronic hypoxia increased myocardial mitoKATP channel activity implicating the compensation for mitochondrial bioenergetics [8], and the IPC effect was abolished in hypertrophied myocardium via PKC downregulation [9]. However, there are no reports how chronic CS-induced ischemia modifies activation of the PKC-mitoKATP cascade and interferes with the infarct formation upon acute myocardial ischemia–reperfusion. Accordingly, it is questioned whether such chronic CS-induced activation of the PKC-mitoKATP cascade, if any, always guarantees a cardioprotective effect by IPC.
Carvedilol is known to improve the prognosis of ischemic heart disease [10]. This may be ascribed to an adrenergic receptor-blocking action which results in the attenuation of daily exercise-induced ischemic attacks as well as reduction of the myocardial catecholamine burden. If the PKC-mitoKATP cascade is modified in CS-induced chronic ischemic heart as mentioned above, such effect by carvedilol [11] may work for cardioprotection.
In the present study, we hypothesized that 1) chronic CS, which limits coronary flow reserve critically, may cause PKC activation and may mimic the IPC effect when acute coronary occlusion–reperfusion is newly added to the same risk area, but that the infarct size reduction by IPC as well as by such IPC-mimetics by CS, if present, may be restricted for a time after CS, and 2) carvedilol may ameliorate such abnormalities for the reasons mentioned above, leading to its therapeutic effects. To test these hypotheses, we assessed infarct size after acute coronary occlusion–reperfusion in the presence or absence of fixed CS, and its possible modification via the PKC-mitoKATP cascade related to the time course after CS, and finally the therapeutic effects of carvedilol.
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2. Methods |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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The investigation conforms to the Guideline on Animal Experiments of Fukushima Medical University, Japanese Government Animal Protection and Management Law (No. 115) and the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85–23, revised 1996).
2.1 Animal model for coronary stenosis
Male Sprague–Dawley rats at 10 weeks of age were anesthetized with an intraperitoneal administration (i. p.) of sodium pentobarbital 45 mg/kg, then at 1–2 mm just below the origin from the ascending aorta, the left coronary artery and a wire were occluded together with a tie (confirmed by transient ST elevation which exceeded R wave on limb lead II electrocardiogram) for 
10 s, followed by quick wire removal [12,13]. Among the 402 rats in which CS was created (CS+ groups), 20 rats died within 24 h (Fig. 1). There were no deaths among rats with sham surgery (CS– groups: n=93).
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2.2 Experimental groups
The experimental designs are summarized in Figs. 1 (including the animal number) and 2. At each time point (1 day after sham surgery, and 1, 4, 8 and 12 weeks after CS creation), acute myocardial infarction was induced by coronary occlusion for 30 min at the site of CS or sham surgery (the tie was the marker of the CS site), followed by reperfusion for 24 h by release of the tie. The success in coronary occlusion was confirmed by ST elevation on electrocardiogram. As the pretreatments just before acute coronary occlusion–reperfusion, aqua as an inert vehicle (CS–-vehicle and CS+-vehicle groups, respectively), diazoxide (a mitoKATP opener: CS–-diazoxide and CS+-diazoxide groups), 5-hydroxydecanoate (5HD, a putative mitoKATP blocker: CS–-5HD and CS+-5HD groups), or diazoxide in combination with 5HD (CS–-diazoxide-5HD and CS+-diazoxide-5HD groups) were administered intravenously for 10 min (at a dose of 16 mg/kg, each equivalent to
100 µM in vivo). Also, two episodes of coronary occlusion for 5 min with an interval of 5 min [14] were performed before coronary occlusion in the sham or CS rats as the IPC procedure (CS–-IPC and CS+-IPC groups).
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Twenty-four hours after acute coronary occlusion–reperfusion, the rats were again anesthetized, and cardiac catheterization was performed using a polyethylene tube, and left ventricular (LV) peak systolic and end-diastolic pressures, and +/ – LVdP/dt were recorded. Then, under artificial ventilation, the chest was opened. The left coronary artery at the previous coronary occlusion–reperfusion site was quickly occluded by a tie, and the risk area was determined by 10% Evans blue solution [15]. Then, the heart was excised and sliced into 3 short-axial layers. The upper layer slices in a part of the groups were used for assessment of myocardial fibrosis as described later. The middle and apical layer slices were incubated with a 2% triphenyltetrazolium chloride (TTC) solution [15]. Using the point-counting method of Weibel, we assessed the extent of the risk and the non-risk areas (Evans blue unstained and stained, respectively) and infarct area (TTC unstained) on the slices stereoscopically under 40 x magnification. Then, the risk area size [%: (the risk area/total LV area) x 100] on the slices and the infarct size [%: (the infarct area/the risk area) x 100] were calculated.
To assess the effects of carvedilol on these parameters [Figs. 1 and 2
(Protocol 2)], carvedilol 10 mg/kg/day (carvedilol groups: total n=102) was administered orally using a cannula once daily after the induction of CS [11]. In these rats, acute coronary occlusion–reperfusion with (CS+-carvedilol-IPC group) or without (CS+-carvedilol group) the IPC procedures was performed 4 or 12 weeks after CS was induced. The effect of diazoxide on infarct size was also assessed in the carvedilol group 12 weeks after CS (CS+-carvedilol-diazoxide group). In these groups, experiments were performed 24 h after the last administration of carvedilol to minimize its acute effects on myocardial infarct size.
In a preliminary study (total n=48), we confirmed that infarct size modulation by IPC or diazoxide was not affected by aging of animals for 12 weeks. Namely, among the CS–-vehicle, CS–-IPC, and CS–-diazoxide groups at 1 to 12 weeks after sham surgery (n=4 each), the risk areas did not differ (54±9% to 58±8% of total LV area), and the infarct size did not differ between the different weeks of age (CS–-vehicle; 65±10, 67±11, 68±11, and 68±10%: CS–-IPC; 33±9, 35±11, 34±10, and 36±10%: CS–-diazoxide; 31±10, 34±10, 32±9, and 34±9% of risk area at 1, 4, 8, and 12 weeks after sham surgery, respectively).
2.3 Assessment of protein kinase C activation
Rats in 19 (n=7–11 each in groups of 1 day after sham surgery, and 1, 4, 8 and 12 weeks after CS; n=3 each in groups of 1 to 12 weeks after sham surgery: corresponding groups shown by 
in Fig. 1) of 34 subgroups were anesthetized, artificially ventilated, and then the chest was opened. The beating heart was quickly excised and the left ventricle was divided into 3 slices as mentioned above for assessment of PKC- activation state.
In the anterior wall (the risk area) of the middle and apical slices in all of 19 groups and the posterior one-third portion of the interventricular septum (the non-risk area) only in the CS+-vehicle group at 4 weeks (n=7 each group), PKC- isoform of the cytosolic and particulate fractions of these samples were assessed by Western blotting and densitomery as described previously [13]. After obtaining the particulate and cytosolic fractions, each protein concentration in the supernatant was assessed by the Bio-Rad protein assay kit. Twenty µg of the tissue supernatant was electrophoresed on 10% SDS/PAGE and blotted onto nitrocellulose membrane. The PKC- translocation ratios were expressed by the formula [(densities of PKC band in the particulate fraction/densities of PKC band in the cytosolic fraction) x 100 (%)] [13]. In the carvedilol treatment groups, myocardial PKC was assessed 24 h after its last administration.
In 18 of 19 subgroups mentioned above (except CS+-carvedilol-diazoxide at 12 weeks after CS), we performed Western blotting of phosphorylated PKC- [16] using its antibody [ab5811 (PKC-phospho S729), Abcam] in myocardial samples of the risk area (n=3 each in groups of 1, 4, 8, and 12 weeks after sham surgery; n=4 each in other groups) in order to confirm PKC- activation. The myocardial tissue specimens were taken and then quickly frozen in liquid nitrogen at – 80 °C until analysis. After homogenization, the myocardial samples were centrifuged at 10,000 x g for 30 min, then, the supernatant was analyzed for protein concentration using a Bio-Rad protein assay kit. Fifty µg of the tissue supernatant was then electrophoresed on 10% SDS/PAGE and blotted onto nitrocellulose membrane. After Western blotting for phosphorylated PKC-, the blot was stripped, then, reprobed with PKC- antibody. The PKC- phosphorylation ratios were expressed by the formula [(densities of phosphorylated PKC- band/densities of total PKC- band) x 100 (%)] using the NIH image.
2.4 Myocardial fibrosis
In 7 groups after acute coronary occlusion–reperfusion [CS–-vehicle 1 day (n=8), CS+-vehicle 1 to 12 weeks after CS, and CS+-carvedilol 4 and 12 weeks after CS and daily carvedilol treatment (n=7–10), Fig. 1] and in a group 12 weeks after sham surgery (n=3), 5 µm-thick paraffin-embedded sections of the upper one of the three myocardial slices mentioned above, which was fixed with a 4% paraformaldehyde solution, were prepared. Then, sections were stained with Azan Mallory, and myocardial fibrosis in the whole risk area [%, the area of collagen (blue-stained)/the whole risk area (Evans blue unstained)] was measured by the point-counting method. In a group 12 weeks after sham surgery which lacked the risk area assessment with Evans blue, the LV anterior wall was considered to be the risk area.
2.5 Assessment of cardiac function and myocardial perfusion
LV function was assessed by echocardiography (Aspen Advanced,TM Acuson) in an anesthetized condition before and 1 to 12 weeks after sham surgery (group, n=13) or CS induction (CS+-vehicle group, n=29) denoted by the asterisk (*) in Fig. 1, respectively. LV end-diastolic and end-systolic diameters were measured, and LV ejection fraction was calculated by the Pombo method [11–13]. In the carvedilol groups (CS+-carvedilol, CS+-carvedilol-IPC, and CS+-carvedilol-diazoxide groups, 12 weeks after CS, n=54 of 62), echo measurements were also done 24 h after the last administration of the drug. Then, in the CS–-vehicle (n=13), CS+-vehicle (n=13) or CS+-carvedilol (n=7 each) group at 12 weeks, myocardial blood flow and coronary flow reserve by intravenous dipyridamole infusion (10 mg/kg/min) were assessed by the colored microsphere method [11–13] (Fig. 1).
2.6 Pharmaceuticals
Diazoxide, TTC, and anti-PKC- were purchased from Sigma Chemical Co., Ltd., and 5HD from ICN Biomedicals Inc., respectively. Carvedilol was a gift from Daiichi Pharmaceutical Co., Ltd.
2.7 Statistical analysis
Data are expressed as mean±SE. For multiple comparisons, factorial ANOVA with repeated measure (Fig. 3) or two-factor factorial ANOVA (Figs. 4–6
) were performed. If F test results were <0.05, Bonferroni's post-hoc comparisons were performed. For analyses of survival rates, the Kruskal–Wallis test was used. The p value<0.05 was considered significant.
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3. Results |
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3.1 Survival
There were no significant differences of the survival rates after adding acute coronary occlusion–reperfusion among the CS+ rats at 1, 4, 8 and 12 weeks or among the 5 groups of CS+-vehicle, CS+-IPC, CS+-diazoxide, CS+-diazoxide-5HD and CS+-5HD (Fig. 1).
3.2 Myocardial perfusion in anesthetized condition
In the CS+-vehicle group, myocardial blood flow was similar to the CS–-vehicle group, whereas coronary flow reserve was lower (p<0.01, Table 1).
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3.3 Echocardiography
Compared to the CS–-vehicle group (Fig. 3), the LV end-diastolic (p<0.01 each at 4 to 12 weeks) and LV end-systolic diameter (p<0.01 each at 1 to 12 weeks) increased, causing the LV ejection fraction to decrease in the CS+-vehicle group (p<0.01 each at 1–12 weeks).
3.4 Risk areas and infarct sizes
The risk area did not differ among the groups at any time point (Fig. 4A). When coronary occlusion–reperfusion was performed in the CS– groups 1 day after sham surgery, the infarct size was 67±4% (the CS–-vehicle group, Fig. 4B), while the infarct size was decreased (p<0.01 each) in the CS–-IPC and CS–-diazoxide groups.
One week after CS, the infarct size was decreased in the CS+-vehicle (p<0.05 versus CS–-vehicle), and further in the CS+-IPC and CS+-diazoxide groups (p<0.01 each versus CS+-vehicle, Fig. 4B). The infarct sizes of the CS+-IPC, and CS+-diazoxide groups were similar to the corresponding CS–IPC and CS–-diazoxide groups, respectively. The infarct size reduction in the CS+-diazoxide group was reversed by the co-administration of 5HD (CS+-diazoxide-5HD), although 5HD administration alone (CS+-5HD) did not modify infarct size of the CS–-vehicle group (asterisks not shown in Fig. 4B).
Four weeks after CS, the infarct size in the CS+-vehicle group was similar to the control value of the CS–-vehicle group (1 day after sham surgery) (Fig. 4B). The infarct size decreased (p<0.05) in the CS+-IPC group compared with the CS+-vehicle group, whereas it was larger (p<0.01) than in the CS–-IPC group (1 day after sham surgery). The infarct size also decreased in the CS+-diazoxide-group (p<0.01 versus CS+-vehicle) more than (p<0.05) in the CS+-IPC group.
Eight and 12 weeks after CS, the infarct sizes in the CS+-vehicle group were similar to those of the CS–-vehicle group (1 day after sham surgery). The infarct size was decreased (p<0.05 each) in the CS+-IPC and CS+-diazoxide groups compared with the CS+-vehicle group, whereas the sizes were larger (p<0.01 each) than the corresponding CS–-IPC and CS–-diazoxide groups (1 day after sham surgery). There was no difference in infarct size between the CS+-IPC and CS+-diazoxide groups (Fig. 4B).
3.5 PKC activation
Each of the PKC- translocation ratios and phosphorylation ratios 1, 4, 8, and 12 weeks after sham surgery (data not shown) did not differ from the corresponding values at 1 day. The examples of PKC- bands in cytosolic and particulate fractions, and of PKC- phosphorylation status by Western blotting are shown in Fig. 5A.
As shown in Fig. 5B, compared with the sham (CS–), myocardial PKC- translocation ratios in the risk area increased 1 (p<0.01) and 4 (p<0.05) weeks after CS in the CS+-vehicle groups even without IPC [IPC (–) in Fig. 5B], but not at 8 and 12 weeks after CS. The ratios were larger in the CS+-IPC group [i.e., at 1–12 weeks, p<0.05 versus each corresponding group without IPC, and at 1 and 4 weeks (p<0.01, each) and at 8 and 12 weeks (p<0.05, each) versus CS– group without IPC] as well as in the CS–-IPC group (p<0.01 versus the CS–-vehicle group). However, increases in the ratios became smaller at 8 and 12 weeks in the CS condition (p<0.05 versus CS– group with IPC). Thus, increases in the translocation ratios (both spontaneous and IPC-induced) were attenuated 8 and 12 weeks after CS occurred. The PKC- translocation ratios of the non-risk area were 109±9%, and these ratios were not changed by the various interventions performed in this study (data not shown).
Compared with the sham (CS–-vehicle), myocardial PKC- phosphorylation ratios in the risk area (Fig. 5C) increased (p<0.05) 1 and 4 weeks after CS induction in the CS+-vehicle groups even without IPC, but not at 8 and 12 weeks. The ratios were larger (p<0.05) in 4 CS+-groups with IPC than the corresponding CS+ groups without IPC. However, increases in the ratios became smaller at 8 and 12 weeks in the CS condition (p<0.05 versus CS– group with IPC).
3.6 Effects of carvedilol
Compared to the CS+-vehicle group, the CS+-carvedilol groups reduced LV end-diastolic and end-systolic diameters, and increased LV ejection fraction 4 to 12 weeks after CS, respectively (Fig. 3, p<0.05 or 0.01 each). In the CS+-carvedilol group 12 weeks after CS, compared with the corresponding CS+-vehicle group, myocardial blood flow was similar but coronary flow reserve was higher (p<0.05 in Table 1), whereas LV peak systolic pressure tended to be lower and heart rate was lower (p<0.05).
The PKC- translocation and phosphorylation ratios, and infarct size in relation to the effects by carvedilol are shown in Fig. 6A–C, respectively. Four and 12 weeks after CS in the CS+-carvedilol group, the infarct size by coronary occlusion–reperfusion was similar to the corresponding value of the CS+-vehicle group (Fig. 6C). However, when IPC was added prior to coronary occlusion–reperfusion under the daily carvedilol treatment in the CS rats (CS+-carvedilol-IPC group), the infarct size was reduced compared with the CS+-IPC (p<0.05) or CS+-carvedilol (without IPC) (p<0.01) group 4 and 12 weeks after CS occurred, indicating that daily carvedilol treatment restored the attenuated IPC effect on infarct size 4 and 12 weeks after CS. In addition, daily carvedilol treatment restored (p<0.01) the attenuation of diazoxide-induced infarct size reduction 12 weeks after CS (Figs. 4B and 6C).
The PKC- translocation (Fig. 6A) and phosphorylation ratios (Fig. 6B) decreased (p<0.05 each) in the CS+-carvedilol group compared with that of the CS+-vehicle group 4 weeks after CS, indicating the relatively early restoration by daily carvedilol treatment of the CS-induced PKC- activation. PKC- translocation and phosphorylation ratios after IPC procedures with daily carvedilol treatment (CS+-carvedilol-IPC group) increased 4 and 12 weeks (p<0.01 or <0.05) after CS, compared with the CS+-carvedilol group. Moreover, each value in the CS+-carvedilol-IPC group 12 weeks after CS was also greater (p<0.05 each) than the corresponding CS+-IPC group without the carvedilol treatment, indicating daily carvedilol treatment restored impairment of IPC-induced PKC activation.
3.7 Myocardial fibrosis
Histological myocardial fibrosis was 0.2±0.1, 0.2±0.1, 3.1±1.2, *12.6±3.5, *13.4±3.2, *14.1±4.2, *5.6±2.1, * and 6.2±3.1%* of the whole risk area in the CS–-vehicle (1 day and 12 weeks after sham surgery), CS+-vehicle (1, 4, 8 and 12 weeks after CS), and CS+-carvedilol (4 and 12 weeks after CS and daily carvedilol treatment) groups (*p<0.01 versus CS–-vehicle 1 day after surgery, p<0.01 versus the corresponding CS+-vehicle group).
3.8 Hemodynamics 24 h after coronary occlusion–reperfusion
The +LVdP/dt (mmHg/s) was higher (p<0.05 each) in the CS–-IPC and CS–-diazoxide groups (1 day after sham surgery), the CS+-IPC and CS+-diazoxide groups (1 week after CS), and in the CS+-carvedilol-IPC group 4 weeks after CS than the CS–-vehicle group (Table 2). LV systolic pressure was lower (p<0.05) in the 5 carvedilol treated groups than the CS–-vehicle group.
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4. Discussion |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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The results of the present study are summarized as follows. First, the infarct size-reducing effect appeared only 1 week after CS induction, and IPC or diazoxide augmented the infarct size reduction. However, the infarct size-reducing effects by diazoxide as well as by IPC were attenuated late (8 and 12 weeks) after CS. These results were not attributed to the aging of animals. CS-induced PKC-
activation followed by its downregulation in a time course after CS creation appeared to be partially involved in these phenomena. Second, daily carvedilol treatment restored such PKC- activation and impairment of IPC-induced PKC- activation 4 and 12 weeks after CS, leading to recovery of the IPC effect. The present results also may warrant usage of carvedilol for alleviating CS-induced cardiac dysfunction and may postulate a new mechanism for the reduction of infarct size by IPC on carvedilol treatment in coronary artery disease.
4.1 PKC activation by chronic coronary stenosis and modification by IPC and mitoKATP opener
There are reports which observed PKC activation in chronic hypoxic conditions [17–19] whereas there have been no reports which assessed PKC modulation in chronic ischemic settings. In the risk area, there was activation of PKC- 1–4 weeks after CS, whereas there was no such activation after 8 and 12 weeks (CS+ groups without IPC versus CS– group without IPC, Fig. 5B and C). The IPC enhanced PKC- activation 1–12 weeks after CS induction. In this process 4 weeks after CS, the attenuation of infarct size reduction was observed despite increased PKC- translocation and phosphorylation ratios by IPC. Since infarct size reduction by the mitoKATP opener was not impaired at this time point (Fig. 4B), it is assumed that the process between PKC and mitoKATP (post-PKC and pre-mitoKATP) may have been impaired 4 weeks after CS. Thus, the activation downstream of PKC rather than the PKC activation itself might have been affected at first after chronic CS, leading to a smaller reduction of infarct size. Then, the effect of IPC on PKC- activation was attenuated 8 and 12 weeks after CS induction (p<0.05 each versus CS– group with IPC, Fig. 5B and C), suggesting downregulation of PKC- 8 and 12 weeks after CS. In accordance with such downregulation, the infarct size reduction by IPC was attenuated. Since the infarct size reduction by diazoxide was also attenuated 8 and 12 weeks after CS (Fig. 4B), the malfunction at the mitoKATP level for cardioprotection, as well as possible PKC- downregulation, is probable. Although the detailed mechanisms of these processes remain to be determined, we speculate that CS-induced PKC- activation (not continuous but possibly recurrent) might cause the downregulation at the mitoKATP level. Also, it is likely that mild infarct size reduction 1 week after CS creation might be due to the IPC-mimetic PKC- activation induced by CS.
Overstimulation of the angiotensin II receptor-coupled G protein signals can cause PKC- downregulation in hypertrophic but non-ischemic myocardium [9]. However, considering that myocardial hypertrophy is not evident in our CS model [11–13], and that PKC activation was not triggered 4 weeks after CS in the non-risk area of our model, the involvement of this mechanism seems unlikely in our chronic ischemia model.
4.2 Effects of daily carvedilol treatment
Carvedilol application (the last one 24 h prior to onset of coronary occlusion–reperfusion) did not reduce infarct size following coronary occlusion–reperfusion (Fig. 6B). This may imply that infarct size-reducing effect by β blockade may have been lost by the 24-h washout period before the intervention.
As previously described, 8 and 12 weeks after CS, the dysfunction at the mitoKATP level as well as the downregulation of PKC- activation was assumed. Carvedilol treatment alleviated this malfunction of the PKC-mitoKATP process. Thus, daily carvedilol treatment prevented CS-induced PKC- activation 4 weeks after CS (Fig. 6A and B). In addition, PKC downregulation 12 weeks after CS was restored in the carvedilol-CS+-IPC group. In contrast, there were similar increases in PKC ratios at 4 and 12 weeks (also, p<0.05 versus CS+-IPC group at 12 weeks, Fig. 6A and B) in CS+-carvedilol-IPC groups, and carvedilol treatment resuscitated the blunted diazoxide-induced infarct size reduction 12 weeks after CS (Fig. 6C).
In our previous study [11], daily carvedilol treatment alleviated CS-induced reduction of coronary flow reserve, suggesting alleviation of CS-induced myocardial ischemia. Such increase in coronary flow reserve appeared to be contributed by its anti-oxidant property in addition to the β blocking effect [11]. Although we did not assess myocardial oxidative stresses in the present study, similar mechanisms may have functioned. Thus, by alleviating ischemic burden via increased coronary flow reserve, carvedilol treatment may have attenuated PKC- activation and resultant downregulation of its signal pathways leading to a rebuilding of the blunted IPC effects.
4.3 Effects of myocardial fibrosis on infarct size
The question may arise that a prolongation of coronary stenosis might induce irreversible tissue damage [19], thereby reducing the capability of IPC to protect against the subsequent coronary occlusion–reperfusion. In the present study, changes in myocardial fibrosis were 14% of the risk area 4 to 12 weeks after CS compared with the sham, and carvedilol reduced fibrosis to 6%. On the other hand, control infarct size in the CS– group was 67% of the risk area, and IPC (Fig. 4) or carvedilol (Fig. 6C) reduced it to 40%. It would be virtually impossible to compare the respective absolute values obtained by the different methodologies. Although the magnitude of the changes in myocardial fibrosis seems to be relatively small and does not exceed that in myocardial infarct size, %infarct size would be modulated depending on the time period after CS, and therefore, the present results regarding %infarct size may not directly reflect the acute effect of coronary occlusion–reperfusion alone.
4.4 Severity of myocardial ischemia
To assess whether our CS model provokes demand ischemia, we performed treadmill exercise. By assessing changes in LV wall thickening in a closed chest condition [12], we documented inducible myocardial stunning in our CS model. One may speculate that myocardial stunning reduces myocardial oxygen demand, and resultantly demand ischemia might be attenuated even in the presence of CS. It is quite difficult to validate this assumption in vivo. One can only note that even 12 weeks after CS, active myocardial inflammation is detected histopathologically [13], suggesting the persistence of myocardial ischemia.
4.5 Clinical relevance
In the era of postconditioning [20] for cardioprotection, preconditioning may still have a benefit in some situations such as pre-infarction angina. Our results allow the assumption that, in the presence of CS [4], the cardioprotective effect by pre-infarct angina may be attenuated to some extent [6]. In order to alleviate this morbidity, a new treatment modality must be developed, and carvedilol may indeed be one of the candidates for such therapy.
4.6 Study limitations
There are several limitations in the present study. First, we have not determined the detailed mechanistic insights of PKC downregulation and other possible factors involved [6]. Second, we did not assess PKC other than the isoform [21]. Third, we did not assess the second window of IPC [6]. Fourth, the dispersion of the risk areas in our rats was small as shown by the small error bars in Fig. 4. Therefore, we showed the actually measured infarct size rather than that assessed from the slopes of the linear regression lines of the risk areas and infarct sizes.
4.7 Conclusions
In the heart in which CS has occurred, a tolerance to new exposure to acute ischemia–reperfusion may be acquired via endogenous PKC- activation. However, such transient cardioprotection both by IPC and the mitoKATP opener are attenuated later after CS. Alteration of the intrinsic cardioprotective mechanisms via the PKC-mitoKATP cascade appears to be involved. Daily carvedilol treatment, which restores such morbidity of the cascade, may be an effective therapeutic strategy for a prospective acute coronary occlusive event.
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Notes |
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Time for primary review 28 days
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