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Original Article

Chronic Angiotensin II Receptor Blockade Induces Cardioprotection During Ischemia by Increased PKC-ε Expression in the Mouse Heart

Lange, Stefan A; Wolf, Benita; Schober, Kristin; Wunderlich, Carsten; Marquetant, Rainer; Weinbrenner, Christof; Strasser, Ruth H

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Journal of Cardiovascular Pharmacology: January 2007 - Volume 49 - Issue 1 - p 46-55
doi: 10.1097/FJC.0b013e31802c2f77
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Heart protection by ischemic preconditioning has been shown to protect the myocardium from ischemia/reperfusion injury in a variety of species, including humans.1,2 Different signaling pathways are known to be involved in the preconditioning process.3 It has been shown that activation of various receptors (eg, adenosine, angiotensin, bradykinin, opioids, and adrenergic receptors mimicked ischemic preconditioning).3-7 Isoforms of protein kinase C (PKC) play a pivotal role in ischemic preconditioning.8-10 In particular, translocation of PKC-epsilon (PKC-ε ) to the membrane protects cells during infarction by interacting with the permeability transition pore in cardiac mitochondria.11-13

The role of angiotensin II and bradykinin in ischemic preconditioning is still discussed controversially. Most of the experiments with angiotensin II type 1 (AT1) receptor antagonist (ARB) and angiotensin converting enzyme inhibitor (ACE-I) showed a reduction of infarct size and an improvement in LV function.14-16 On the contrary, some authors described an infarct size-reducing and preconditioning effect as a result of angiotensin II receptor stimulation itself 17,18 and an opposite effect of AT1-receptor blockade.19 In most experimental studies agonists or antagonists of the renin-angiotensin-aldosterone system (RAAS) were given as an acute treatment. Little is known about the effect of chronic treatment with ACE-I) or AT1-receptor blocker (ARB) prior to myocardial infarction or ischemic preconditioning. This is an important issue because most of the patients with coronary heart disease chronically receive ACE-I, ARB, or both, for the treatment of hypertension and cardiac failure. Some of them develop unstable angina or infarction during this therapy. Preinfarction angina has been demonstrated to improve the outcome of patients suffering from myocardial infarction and, therefore, has been proposed to represent the phenomenon of ischemic preconditioning.20

The aim of this study was to determine the effect of chronic treatment with the ACE-I ramipril and the ARB candesartan cilexetil on both infarct size and ischemic preconditioning.

Moreover, the subcellular signaling pathway systems, especially the expression of PKC isoforms, belonging to the key enzymes of ischemic preconditioning were characterized.


All procedures were in conformance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and were approved by the local Animal Care and Use Committee.

Pre treatment of Mice With Candesartan Cilexetil and Ramipril

Male C57/BL6 mice (Charles River, Germany), 8 weeks old, were randomly assigned to placebo, pretreatment with ramipril (1.25 mg/kg on days 1 and 2, 2.5 mg/kg on days 3 to 14, Sanofi-Aventis21) or pretreatment with candesartan cilexetil (10 mg/kg on days 1 to 14, AstraZeneca22). The drugs were added to drinking water.

Morphologic Features

The weight of each mouse was measured daily during treatment and before heart explantation. The weight of explanted mice hearts was determined at the end of the reperfusion cycle. Each ratio for heart to body weight was calculated in milligrams per gram of body weight and statistically utilized.

Blood Pressure Measurements

Systolic blood pressure was measured daily during the 14 days of treatment with ramipril and candesartan by the tail-cuff method using an ADInstruments® Blood Pressure analysis system (Model No. ML125/M). All animals were habituated to the blood pressure measurement device for 7 days. They all underwent 1 cycle of 10 measurements recorded by Power Lab Chart software (modified according to Yang et al23).

Langendorff Perfusion

After 2 weeks of placebo or drug-pretreatment, mice were anesthetized with thiopental [Sigma-Aldrich, 200 mg/kg intraperitoneally (ip)]. Heparin (250 IE/kg) was given ip. The chest was opened and the heart was rapidly excised, mounted on a Langendorff apparatus, and retrogradely perfused via the aorta at a constant hydrostatic pressure (100 cm H2O) with 37°C modified Krebs-Henseleit buffer (in mmol/L: NaCl 117.5; KCl 4.7; CaCl2 2.5; MgSO4 1.2; KH2PO4 1.2; NaHCO3 24.8; glucose 10.0, 95% O2/5% CO2). Ventricular function was assessed by a force transducer that was fixed onto the apex of the heart and connected to a bridge amplifier (ADInstruments). The signal of the systolic contraction was digitized. Hearts were paced with 500 beats per minute (A-M System, 120 ms, 70-90 mA). Regional ischemia was induced by total occlusion of the left anterior descending (LAD) artery. The coronary flow (CF) was measured by collecting the coronary venous effluent for 1 minute every 5 minutes during equilibration and reperfusion and every 10 minutes during regional ischemia. Myocardial ischemia was confirmed by regional paleness, a decrease in coronary flow, and a reduction in systolic contraction.

Study Groups and Experimental Protocols

Fourteen groups were investigated in 2 different protocols (Figure 1). According to the first protocol, the control protocol (CON), hearts received 60 minutes of regional ischemia after a 30 minute equilibrium period. Ischemia was followed by a 30 minute reperfusion period. Regional ischemia was induced by torch ligature of the LAD. In the second protocol, the ischemic preconditioning protocol (iPC), 3 cycles of 5 minutes of global ischemia, each followed by 5 minutes of reperfusion, were applied just prior to the regional ischemia. To study the effects of chronic treatment with ramipril or candesartan cilexetil on infarct size and iPC, 2 groups for each of them were fed either with ramipril or candesartan cilexetil and equally divided into CON and iPC groups (CON-RAM, n = 12, iPC-RAM, n = 12), (CON- CAND, n = 12, iPC-CAND, n = 12). Two groups, receiving aqua (drinking water ad libidum) only, served as control (CON, n = 11), (iPC, n = 11).

Experimental protocol for Langendorff perfusion.

PD123.319 (0,3 μM, Sigma Aldrich), a potent angiotensin II subtype 2 (AT2)-receptor antagonist, was used to determine a possible involvement of the AT2-receptor on iPC and regional ischemia. It was added to the perfusate 5 minutes prior to regional ischemia or iPC and remained in the perfusate until the end of the infarct ischemia. Two groups without (CON-PD123.319, n = 12; iPC-PD123.319, n = 12) and 2 groups with candesartan cilexetil pretreatment (CON-CAND-PD123.319, n = 12; iPC-CAND-PD123.319, n = 11) were compared. Four additional groups were perfused with the PKC-inhibitor chelerythrine (5 μmol/L, Sigma Aldrich) to investigate the role of PKC in candesartan-mediated signaling of cardioprotection during iPC and regional ischemia (CON-CHEL, n = 6; iPC-CHEL, n = 6; CON-CAND-CHEL, n = 11; iPC-CAND-CHEL, n = 12). Chelerythrine was administered 5 minutes prior to regional ischemia or iPC and was washed out 5 minutes before the end of regional ischemia.

Determination of Infarct Size

After 30 minutes of reperfusion, hearts were perfused with propidium iodide at a concentration of 1 mg/mL for 5 minutes.24 The coronary artery was reoccluded, and 1-10 μm of zinc cadmium sulfide fluorescent particles (Duke Scientific Corp, Palo Alto, Calif) were infused to demarcate the risk zone as the area without fluorescence. The hearts were weighed, frozen, and cut into 1 mm-thick slices. The area of infarct (bright-red fluorescent under ultraviolet light) and the area of risk (nonfluorescent under ultraviolet light) were determined by planimetry. Infarct size was expressed as percentage of the risk zone.

Western Blot Analyses of PKC Isozymes in Cytosol and Particulate Fraction

After 2 weeks of placebo or drug-pretreatment, the mice were anesthetized with thiopental. Heparin was given ip; chests were opened; and hearts were quickly excised, rapidly mounted on the Langendorff apparatus via aorta, and perfused retrogradely at a constant hydrostatic pressure with 37°C modified KHB (method described earlier in detail). After an equilibration period of 30 minutes, mice hearts received global ischemia for 3 minutes and were freeze clamped and stored at −80°C.

Tissue Preparation

Hearts were homogenized in buffer A (1M Tris, 200 mM EDTA [ethylenediamine tetraacetic acid], 200 mM EGTA [ethylene glycol-bis-(2-aminoethyl)-N,N,N′, N′-tetraacetic acid], protease inhibitor, Roche, pH 7.4) by using a polytron (3 × 6 seconds, 19,000 rpm, 0°C).

The homogenates were then centrifuged at 3000g (10 minutes). The resulting supernatants were centrifuged with a high-speed centrifuge, Beckmann, at 41,399g (20 minutes) to separate the soluble fraction from the particulate fraction. Pellets corresponding to the membrane fraction were resuspended in buffer A. The protein content was determined according to the method of Bradford25 using bovine serum albumin as a standard.

SDS PAGE, Western Blotting, and Ponçeau Staining

Proteins were separated on 8% SDS-polyacrylamide gel26 and transferred to polyvinylidene fluoride (PVDF) membrane (Immobilion™ P, Millipore27). Detection of PKC isoforms was performed using polyclonal peptide antibodies (Santa Cruz, Calif). After being washed with 0.3% Tris-buffered saline Tween 20 (TBST; 1:1000) for 3 × 10 minutes, the PVDF membrane was then incubated with antirabbit immunoglobulin G (DAKO) goat serum (Santa Cruz) 1:1200 and finally washed with 0.3% TBST 3 times for 10 minutes each. Proteins were detected by using chemiluminescence (BioRad).

Membranes were incubated with Ponçeau S red-dye solution (Sigma Aldrich, Munich, Germany) to detect the protein range of protein staining. This range that was used for normalization of protein account (between 20 and 250 kD) was performed densitometrically.

Drug Solution

Candesartan cilexetil is insoluble in water and was used as a suspending preparation in 5% gum arabic solution (TCV- 116). PD123.319, ramipril, and chelerythrine were dissolved in water.


Values are means ± standard error of means (SEM). Student t test and analysis of variance (ANOVA) with Bonferroni post hoc test were used to test for differences between infarct sizes and hemodynamic parameters. For blood pressure, body, and heart weight measurements the Turkey-Kramer test was used. A probability value of less than 0.05 was considered to be statistically significant.


Morphologic Features

There were no different morphologic features in heart weight between the ramipril and placebo pretreated groups (5.53 ± 0.1 vs 5.52 ± 0.1 mg/g heart weight/body weight ratio). The candesartan cilexetil pretreated group versus the ramipril and the placebo pretreated groups revealed a significant decrease of the heart/body weight ratio (4.88 ± 0.1 mg/g; P < 0.0001). This observation was in accordance with Tanaka et al in 1994.28

Hemodynamic Data

All hearts were paced with 500 beats per minute. Coronary flow was comparable in all groups at baseline. Co-perfusion with chelerythrine increased coronary flow significantly during the period of iPC, as already described.29,30 Coronary artery occlusion resulted in a significant and marked reduction in CF in all of the experimental groups (Table 1).

Mean Coronary Flow (CF) Measured During the Equilibrium Period, in the Last Minute of the Reperfusion Cycle After Each iPC, During the Regional Ischemia and Reperfusion Period.

Systolic contraction parameters, measured by the force transducer, were not significantly different between any of the groups throughout the experiment (data not shown).

Systolic Blood Pressure In Situ

Control mice and ramipril- and candesartan-treated mice were subjected to systolic blood pressure measurement using a tail-cuff computerized system. As shown in Figure 2, blood pressure was significantly reduced in ramipril- and candesartan-treated mice versus control mice, as expected (102.18 ± 1.94 vs 101.41 ± 1.94 vs 115.07 ± 1.94 mm Hg, P < 0.0001). The systolic blood pressure in the ramipril- and candesartan-treated groups did not differ significantly (P = 0.96).

Blood pressure measurement. Systolic blood pressure was significantly reduced in ramipril- and candesartan-treated mice (RAM 102.18 ± 1.94 vs CAND 101.41 ± 1.94 vs CON 115.07 ± 1.94 mm Hg,P < 0.0001). The systolic blood pressure in the ramipril- and candesartan-treated groups did not differ significantly (P = 0.96). Turkey-Kramer test.

Infarct Size Data

Infarct sizes were normalized as a percentage of the ischemic (risk) zone. No significant differences were detected between the risk areas of all groups (55.5 ± 1.5%). Control hearts (CON) had an infarct size of 59.8 ± 4.2%, which was reduced to 24.5 ± 1.7% by iPC, as expected (Figure 3, left side).

Infarct size of control and iPC hearts in untreated and ramipril pretreated groups. Chronic treatment with the ACE-I ramipril did not reduce infarct size as candesartan did (CON-RAM) but blocked iPC completely (IPC-RAM). One-way ANOVA.

Infarct Sizes After Chronic Treatment With an AT1-Receptor Blocker

Chronic treatment with the ARB candesartan cilexetil significantly reduced infarct sizes after MI from 59.8 ± 4.2% (CON) to 38.1 ± 3.1% (CON-CAND, P < 0,001). Ischemic preconditioning had no further protective effect on chronically candesartan cilexetil pretreated hearts (CON-CAND: 38.1 ± 3.1% vs iPC-CAND: 40.3 ± 3.4%) (Figure 4).

Infarct size of control hearts in untreated and candesartan pretreated groups. In comparison to control hearts (CON) candesartan pretreatment reduced infarct size significantly (CON-CAND). This protection could not be abolished by coadministration of PD123.319, the AT2-receptor antagonist (CON-CAND-PD). PD123.319 itself reduced infarct size without ischemic preconditioning (CON-PD). Chelerythrine completely abolished candesartan-induced infarct size reduction (CON-CAND-CHEL). One-way ANOVA.

To elucidate the molecular mechanisms of ARB- induced cardioprotection and their effects of precondition in more detail, control hearts and pretreated hearts were coperfused with either PD123.319, a selective AT2-receptor antagonist, or with chelerythrine, a PKC inhibitor, during the last 5 minutes of equilibrium, iPC, and regional ischemia.

As shown in Figure 4, acute treatment with PD123.319 reduced infarct size in the non-pre-treated hearts (CON-PD: 39.5 ± 4.3%). Ischemic preconditioning could not enhance this protective effect (iPC-PD: 42.5 ± 3.9%). In contrast to chronically candesartan pretreated hearts, ischemic preconditioning showed no additional protection (CON-CAND: 38.1 ± 3.1%, iPC-CAND: 40.3 ± 3.4%). The acute administration of AT2-A in chronically ARB pretreated hearts did not alter the protective effect of chronic ARB pretreatment (CON-CAND-PD123.319: 40.7 ± 3.7% vs CON-CAND: 38.1 ± 3.1% and iPC-CAND-PD123.319: 38.2 ± 4.1% vs iPC-CAND: 40.3 ± 3.4%).

Coperfusion of isolated hearts with chelerythrine (5 μM) completely blocked the protective effect of ARB pre-treatment (CON-CAND: 38.1 ± 3.1% vs CON-CAND-CHEL: 71.1 ± 6.6%, P < 0.001) (Figure 4). Again, ischemic pre-conditioning had no significant additional effect (CON-CAND-CHEL: 71.7 ± 6.6% vs iPC-CAND-CHEL: 52.9 ± 7.8 %, P = ns).

Infarct Sizes After Chronic Treatment With an ACE Inhibitor

The infarct sizes in the chronically ramipril pretreated group did not differ significantly from infarct sizes in the untreated control group (CON-RAM: 51.5 ± 3.0% vs CON: 59.8 ± 4.2%; P = ns). However, ramipril pre-treatment completely blocked the protective effect of ischemic preconditioning (iPC-RAM = 57.7 ± 3.9% vs iPC = 24.5 ± 1.7%) (Figure 3).

Table 2 summarizes the cardio protective effects of different therapies.

Cardioprotection Depending on Different Therapies

Western Blot Analysis of PKC Isoforms

To address the question as to whether an altered expression of PKC isozymes may play a role in the observed effects of chronic candesartan cilexetil and ramipril pretreatment, PKC isozymes were investigated by isozyme- specific Western blot analysis using subtype-specific polyclonal peptide antibodies in cytosolic and particulate fraction. PKC-ε showed a highly significant increase in particulate fraction of candesartan pre-treated hearts (Figure 5A).

A, PKC-e expression in the particulate fraction of candesartan pretreated hearts was normalized to the percentage of control group (CON). PKC-e expression was increased in both the iPC group (iPC-CAND) and the control group (CON-CAND). Studentt test. B,PKC-e expression in the particulate fraction of ramipril pretreated hearts, which was normalized to the control group (CON). In both pretreated groups, iPC (iPC-RAM) and control (CON-RAM), PKC-e expression decreased compared to the untreated control. Student t test.

Similarly, an increased expression could be found for the isozyme PKC-ζ in iPC protocol (Figure 6A).

A, PKC-z expression in the particulate fraction of candesartan pretreated hearts was normalized to the percentage of control group (CON). PKC-z expression in the particulate fraction of candesartan pretreated hearts was significantly increased only in iPC protocol. Studentt test. B, PKC-z expression in the particulate fraction of ramipril pretreated hearts was normalized to the percentage of control group (CON). PKC-z expression in the particulate fraction of ramipril pretreated hearts was not significantly changed. Student t test.

Expressions of PKC-α (Figure 7A) and PKC-δ (Figure 8A) in the particulate fraction did not differ significantly between control and pre-treated hearts.

A, PKC-a expression in the particulate fraction of candesartan pretreated hearts was normalized to the percentage of control group (CON). PKC-a expression was not increased significantly in the iPC group (iPC-CAND) and the control group (CON-CAND). Studentt test. B,PKC-a expression in the particulate fraction of ramipril pretreated hearts was normalized to the percentage of control group (CON). PKC-a expression was not significantly changed in both the iPC group (iPC-RAM) and the control group (CON-RAM). Student t test.
A, PKC-d expression in the particulate fraction of candesartan pretreated hearts was normalized to the percentage of control group (CON). PKC-d expression was not increased significantly in the iPC group (iPC-CAND) and the control group (CON-CAND). Studentt test. B, PKC-d expression in the particulate fraction of ramipril pretreated hearts was normalized to the percentage of control group (CON). PKC-d expression was not changed significantly in both the iPC group (iPC-RAM) and the control group (CON-RAM). Student t test.

In contrast to candesartan pre-treatment, in ramipril pre-treated hearts PKC-ε concentration in the particulate fraction clearly decreased in comparison to control hearts (Figure 5B).

PKC-ζ (Figure 6B), -α (Figure 7B) and -δ (Figure 8B) showed similar expressions as control hearts. No significant differences between isozyme concentrations in the cytosolic fractions of all groups could be distinguished (data not shown).


The scientific findings of the present study are that chronic treatment with candesartan cilexetil, an angiotensin II type 1 receptor antagonist, prior to myocardial infarction reduces the infarct size in mouse hearts. Acute AT2-receptor blockade in these chronically pre treated hearts can neither abolish nor modify this protective effect. AT2-receptor inhibition with the selective angiotensin II type 2 receptor antagonist PD123.319 reduces infarct size significantly, even when it was given alone as an acute treatment.

The findings, that chelerythrine, an unselective PKC inhibitor that blocks the protective effect of the angiotensin II-AT1-receptor antagonist completely, led us to further investigations of PKC isozyme expression in soluble and particulate fraction in mouse hearts. The results of these investigations support the hypothesis that an increased content of PKC-ε at the particulate fraction of ARB pre treated hearts is involved in the ARB-induced cardioprotection.

The protection from chronic pretreatment with candesartan cannot be further increased by additional cycles of ischemic preconditioning.

Moreover, it has been shown for the first time that chronic pretreatment with the ACE-I ramipril abolished the cardioprotective effect of ischemic preconditioning completely. The reason for these findings seems to be a reduction of PKC-ε expression in the particulate fraction.

In contrast to candesartan cilexetil pretreatment, chronic treatment with ramipril has no effect on infarct size of control hearts.

In contrast to our findings, it was shown that the protective effect of candesartan could be abolished by AT2- receptor antagonism.31 PD123.319 was therefore given directly into coronary arteries of pig hearts prior to myocardial infarction. In our study, acute AT2-A (PD123.319) application led to infarct size reduction itself. This result is in accordance with findings that describe an acute protective effect of PD123.319 in ischemia reperfusion in rat hearts. PD123.319 resulted in a significant upregulation of AT2-receptor messenger ribonucleic acid (mRNA) and protein.32,33 In the present study, iPC cannot further limit infarct sizes in AT2-A-treated hearts. It cannot be finally told whether the observed differences in the mechanism of AT2 signaling may be a result of the different models (invivo, in vitro) or different species (rat, pig) used.

Chelerythrine, an unselective inhibitor of all PKC isozymes, abolishes the infarct size-reducing effect of ARB pretreatment. This suggests the connection between PKC isozymes and candesartan cilexetil-induced cardioprotection. PKCε translocation especially has been shown to play a pivotal role in the signaling of ischemic preconditioning.12,13,34 IPC-induced translocation of PKC-δ to the membrane fraction also led to improvement in cardiac function.35 In contrast to this, Inagaki showed that PKC-δ inhibition itself caused infarct size reduction in rodents.36 In the present study, PKC-δ translocation is not affected by treatment with ARB or ACE-I. PKC-α, downstream of the mitoKATP channels and PKC-ε signaling, is even involved in myocardial preconditioning in humans.37 In this study, concentration of PKCα does not differ between pretreated and control groups, possibly because there is only a transient increase of PKCα after iPC.35 PKC-ζ is involved in a wide range of physiologic processes including mitogenesis, protein synthesis, cell survival, and transcriptional regulation. Moreover, it has received considerable attention as a target of phosphoinositide 3-kinase (PI3K).38 In the present study, ARB treatment changes expression of PKC-ζ in the particulate fraction. Higher levels of PKC-ζ have been detected in iPC but not in the control protocol. No differences in the expression of PKC-δ and PKC-α have been seen in soluble and particulate fractions of treated and untreated groups, indicating that no activation with translocation occurred.

It was shown that both inhibition and stimulation of the AT2-receptor resulted in an increased PKC expression,39 especially of PKC-ε.40,41

Our Western blot results suggest an increased concentration of PKC-ε in the particulate fraction in hearts pretreated with candesartan cilexetil. These findings support the thesis that cardioprotection after candesartan cilexetil pretreatment is mediated by PKC-ε. An upregulation of PKC-ε caused by chronic treatment with the ARB losartan and a decreased infarct size could also be shown in dog hearts.33

Ischemic preconditioning in hearts pretreated with candesartan cilexetil fails to protect the myocardium from infarction. This is in accordance with data from Diaz et al19 who did not succeed in protecting the rabbit heart with ischemic preconditioning after pretreating it with an intracoronary infusion of the AT1-receptor antagonist losartan. Yet, in contrast to our data, acute treatment with losartan had no infarct size-reducing effect on control hearts, as we have observed after chronic ARB pretreatment.

ACE-I treatment is pivotal in the therapy of coronary heart disease, heart failure, and hypertension. Nozawa et al42 demonstrated in rabbits in vivo that chronic pretreatment with the ACE-I temocapril lowers the threshold for ischemic preconditioning. In our study, chronic pretreatment with the ACE-I ramipril blocks the cardioprotective effect of ischemic preconditioning completely and has no effect on infarct sizes in control hearts.

An increased bradykinin level, a side effect of ACE inhibition, is known as a potent stimulator of preconditioning.43-45 In in vitro Langendorff experiments, as a limitation of this study, the absence of bloodborne kininogens45 could be a reason for decreased bradykinin levels and, as a result, abolish ischemic preconditioning. According to recent findings, this mechanism can be excluded because bradykinin was detected in hearts in vitro and in vivo.46,47

In contrast to ARB pretreatment, ACE-I pretreatment significantly decreases the expression of PKC-ε in the particulate fraction of mouse hearts. These data are concordant with observations that describe a significant decrease of mRNA and protein levels of PKC-ε in ischemic myocardium of ramipril pretreated rat hearts.48 A decreased PKC-ε−expression was also observed after chronic fosinopril treatment.49


Chronic treatment with the ARB candesartan cilexetil prior to myocardial ischemia reduces the infarct size in mouse hearts. IPC cannot enhance this protection. An increased stimulation of the AT2-receptor is not crucial for this effect because the selective blockade of the AT2-receptor does not alter this protective effect of infarct size reduction. One possible mechanism for this protection is an increased expression of PKC-ε.

Furthermore, chronic treatment with the ACE-I ramipril prior to myocardial infarction has no beneficial influence on infarct size. Beyond this, iPC has been abolished completely. PKC-ε expression has been lowered by ramipril pretreatment.

Transferred to the clinical situation, these results suggest that patients with known coronary artery disease and unstable angina preferably should be treated with AT1-antagonists rather than with ACE inhibitors.


1. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986;74:1124-1136.
2. Yellon DM, Downey JM. Preconditioning the myocardium: from cellular physiology to clinical cardiology. Physiol Rev. 2003;83:1113-1151.
3. Yellon DM, Baxter GF, Garcia-Dorado D, et al. Ischaemic preconditioning: present position and future directions. Cardiovasc Res. 1998;37:21-33.
4. Leesar MA, Stoddard M, Ahmed M, et al. Preconditioning of human myocardium with adenosine during coronary angioplasty. Circulation. 1997;95:2500-2507.
5. Fryer RM, Auchampach JA, Gross GJ. Therapeutic receptor targets of ischemic preconditioning. Cardiovasc Res. 2002;55:520-525.
6. Leesar MA, Stoddard MF, Manchikalapudi S, et al. Bradykinin-induced preconditioning in patients undergoing coronary angioplasty. J Am Coll Cardiol. 1999;34:639-650.
7. Schultz JE, Rose E, Yao Z, et al. Evidence for involvement of opioid receptors in ischemic preconditioning in rat hearts. Am J Physiol. 1995;268:H2157-H2161.
8. Mackay K, Mochly-Rosen D. Localization, anchoring, and functions of protein kinase C isozymes in the heart. J Mol Cell Cardiol. 2001;33:1301-1307.
9. Weinbrenner C. Protein kinase C-the key-enzyme in ischemic preconditioning? In Downey JM, ed. Adenosine, cardioprotection and its clinical application. Norwell, MA: Kluwer Academic Publishers; 1997;73-91
10. Ytrehus K, Liu Y, Downey JM. Preconditioning protects ischemic rabbit heart by protein kinase C activation. Am J Physiol. 1994;266:H1145-H1152.
11. Baines CP, Song CX, Zheng YT, et al. Protein kinase Cepsilon interacts with and inhibits the permeability transition pore in cardiac mitochondria. Circ Res. 2003;92:873-880.
12. Liu GS, Cohen MV, Mochly-Rosen D, et al. Protein kinase C-epsilon is responsible for the protection of preconditioning in rabbit cardiomyocytes. J Mol Cell Cardiol. 1999;31:1937-1948.
13. Ping P, Zhang J, Qiu Y, et al. Ischemic preconditioning induces selective translocation of protein kinase C isoforms epsilon and eta in the heart of conscious rabbits without subcellular redistribution of total protein kinase C activity. Circ Res. 1997;81:404-414.
14. Jalowy A, Schulz R, Heusch G. AT1 receptor blockade in experimental myocardial ischemia/reperfusion. Basic Res Cardiol. 1998;93:85-91.
15. Schwarz ER, Montino H, Fleischhauer J, et al. Angiotensin II receptor antagonist EXP 3174 reduces infarct size comparable with enalaprilat and augments preconditioning in the pig heart. Cardiovasc Drugs Ther. 1997;11:687-695.
16. Weidenbach R, Schulz R, Gres P, et al. Enhanced reduction of myocardial infarct size by combined ACE inhibition and AT(1)-receptor antagonism. Br J Pharmacol. 2000;131:138-144.
17. Liu Y, Tsuchida A, Cohen MV, et al. Pretreatment with angiotensin II activates protein kinase C and limits myocardial infarction in isolated rabbit hearts. J Mol Cell Cardiol. 1995;27:883-892.
18. Ford WR, Clanachan AS, Jugdutt BI. Opposite effects of angiotensin AT1 and AT2 receptor antagonists on recovery of mechanical function after ischemia-reperfusion in isolated working rat hearts. Circulation. 1996;94:3087-3089.
19. Diaz RJ, Wilson GJ. Selective blockade of AT1 angiotensin II receptors abolishes ischemic preconditioning in isolated rabbit hearts. J Mol Cell Cardiol. 1997;29:129-139.
20. Kloner RA, Shook T, Przyklenk K, et al. Previous angina alters in-hospital outcome in TIMI 4. A clinical correlate to preconditioning? Circulation. 1995;91:37-45.
21. Yang XP, Liu YH, Mehta D, et al. Diminished cardioprotective response to inhibition of angiotensin-converting enzyme and angiotensin II type 1 receptor in B(2) kinin receptor gene knockout mice. Circ Res. 2001;88:1072-1079.
22. Furukawa Y, Matsumori A, Hirozane T, et al. Angiotensin II receptor antagonist TCV-116 reduces graft coronary artery disease and preserves graft status in a murine model. A comparative study with captopril. Circulation. 1996;93:333-339.
23. Yang T, Huang YG, Ye W, et al. Influence of genetic background and gender on hypertension and renal failure in COX-2-deficient mice. Am J Physiol Renal Physiol. 2005;288:F1125-F1132.
24. Wolff RA, Chien GL, Van Winkle DM. Propidium iodide compares favorably with histology and triphenyl tetrazolium chloride in the assessment of experimentally-induced infarct size. J Mol Cell Cardiol. 2000;32:225-232.
25. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254.
26. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-685.
27. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979;76:4350-4354.
28. Tanaka A, Matsumori A, Wang W, et al. An angiotensin II receptor antagonist reduces myocardial damage in an animal model of myocarditis. Circulation. 1994;90:2051-2055.
29. Falck G. Hyperosmotic pretreatment reduces infarct size in the rat heart. Physiol Res. 1999;48:331-340.
30. Noguchi T, Chen Z, Bell SP, et al. Activation of PKC decreases myocardial O2 consumption and increases contractile efficiency in rats. Am J Physiol Heart Circ Physiol. 2001;281:H2191-H2197.
31. Jalowy A, Schulz R, Dorge H, et al. Infarct size reduction by AT1-receptor blockade through a signal cascade of AT2-receptor activation, bradykinin and prostaglandins in pigs. J Am Coll Cardiol. 1998;32:1787-1796.
32. Kumar D, Menon V, Ford WR, et al. Effect of angiotensin II type 2 receptor blockade on activation of mitogen-activated protein kinases after ischemia-reperfusion in isolated working rat hearts. J Cardiovasc Pharmacol Ther. 2003;8:285-296.
33. Xu Y, Menon V, Jugdutt BI. Cardioprotection after angiotensin II type 1 blockade involves angiotensin II type 2 receptor expression and activation of protein kinase C-epsilon in acutely reperfused myocardial infarction in the dog. Effect of UP269-6 and losartan on AT1 and AT2-receptor expression and IP3 receptor and PKCepsilon proteins. J Renin Angiotensin Aldosterone Syst. 2000;1:184-195.
34. Gray MO, Karliner JS, Mochly-Rosen D. A selective epsilon-protein kinase C antagonist inhibits protection of cardiac myocytes from hypoxia-induced cell death. J Biol Chem. 1997;272:30945-30951.
35. Kawamura S, Yoshida K, Miura T, et al. Ischemic preconditioning translocates PKC-delta and -epsilon, which mediate functional protection in isolated rat heart. Am J Physiol. 1998;275:H2266-H2271.
36. Inagaki K, Hahn HS, Dorn GW, et al. Additive protection of the ischemic heart ex vivo by combined treatment with delta-protein kinase C inhibitor and epsilon-protein kinase C activator. Circulation. 2003;108:869-875.
37. Hassouna A, Matata BM, Galinanes M. PKC-epsilon is upstream and PKC-alpha is downstream of mitoKATP channels in the signal transduction pathway of ischemic preconditioning of human myocardium. Am J Physiol Cell Physiol. 2004;287:C1418-C1425.
38. Chou MM, Hou W, Johnson J, et al. Regulation of protein kinase C zeta by PI 3-kinase and PDK-1. Curr Biol. 1998;8:1069-1077.
39. Bartunek J, Weinberg EO, Tajima M, et al. Angiotensin II type 2 receptor blockade amplifies the early signals of cardiac growth response to angiotensin II in hypertrophied hearts. Circulation. 1999;99:22-25.
40. Xu Y, Clanachan AS, Jugdutt BI. Enhanced expression of angiotensin II type 2 receptor, inositol 1,4, 5-trisphosphate receptor, and protein kinase cepsilon during cardioprotection induced by angiotensin II type 2 receptor blockade. Hypertension. 2000;36:506-510.
41. Jugdutt BI, Xu Y, Balghith M, et al. Cardioprotection induced by AT1R blockade after reperfused myocardial infarction: association with regional increase in AT2R, IP3R and PKCepsilon proteins and cGMP. J Cardiovasc Pharmacol Ther. 2000;5:301-311.
42. Nozawa Y, Miura T, Tsuchida A, et al. Chronic treatment with an ACE inhibitor, temocapril, lowers the threshold for the infarct size-limiting effect of ischemic preconditioning. Cardiovasc Drugs Ther. 1999;13:151-157.
43. Morris SD, Yellon DM. Angiotensin-converting enzyme inhibitors potentiate preconditioning through bradykinin B2 receptor activation in human heart. J Am Coll Cardiol. 1997;29:1599-1606.
44. Hartman JC, Wall TM, Hullinger TG, et al. Reduction of myocardial infarct size in rabbits by ramiprilat: reversal by the bradykinin antagonist HOE 140. J Cardiovasc Pharmacol. 1993;21:996-1003.
45. Goto M, Liu Y, Yang XM, et al. Role of bradykinin in protection of ischemic preconditioning in rabbit hearts. Circ Res. 1995;77:611-621.
46. Sato M, Engelman RM, Otani H, et al. Myocardial protection by preconditioning of heart with losartan, an angiotensin II type 1-receptor blocker: implication of bradykinin-dependent and bradykinin-independent mechanisms. Circulation. 2007;:III346-III351.
47. Linz W, Wiemer G, Gohlke P, et al. Contribution of kinins to the cardiovascular actions of angiotensin-converting enzyme inhibitors. Pharmacol Rev. 1995;47:25-49.
48. Simonis G, Braun MU, Kirrstetter M, et al. Mechanisms of myocardial remodeling: ramiprilat blocks the expressional upregulation of protein kinase C-epsilon in the surviving myocardium early after infarction. J Cardiovasc Pharmacol. 2003;41:780-787.
49. Kim L, Lee T, Fu J, et al. Characterization of MAP kinase and PKC isoform and effect of ACE inhibition in hypertrophy in vivo. Am J Physiol. 1999;277:H1808-H1816.

angiotensin converting enzyme inhibitors; angiotensin; infarction; preconditioning; protein kinase C

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