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Pivotal role of NOX-2-containing NADPH oxidase in early ischemic preconditioning

Robert M. Bell^*, Alison C. Cave, Sofian Johar, David J. Hearse^*, Ajay M. Shah and Michael J. Shattock^*,1

^* King’s College London, Cardiac Physiology, Cardiovascular Division, The Rayne Institute, St. Thomas’ Hospital, London. SE1 7EH, UK; and
King’s College London, Department of Cardiology, Cardiovascular Division, Guy’s, King’s and St. Thomas’ School of Medicine, Bessemer Road, London. SE5 9PJ, UK

¹Correspondence. King’s College London, Cardiac Physiology, The Rayne Institute, 4th Floor, Lambeth Wing, St. Thomas’ Hospital, London. SE1 7EH, UK. E-mail: Robert.m.bell{at}kcl.ac.uk

SPECIFIC AIMS

Preconditioning is a powerful, inducible endogenous adaptive mechanism that protects the heart against injurious ischemia/reperfusion injury. A role for reactive oxygen species (ROS) has been identified in the myocardial preconditioning signal transduction pathway, though the source of these ROS remains elusive. Cardiac myocytes have recently been demonstrated to contain a phagocyte-type NADPH oxidase, a potent source of ROS. Pharmacological preconditioning triggers, such as angiotensin II, are known to up-regulate NADPH oxidase activity and ROS synthesis in the context of cardiac hypertrophy, a response abrogated by the targeted deletion of the NADPH oxidase core NOX2 (gp91^phox) subunit. However, not all preconditioning mimetics are mediated through ROS synthesis, a notable exception being adenosine and adenosine A₁ receptor agonists.

Our principal aims in this study were to determine whether ROS-dependent preconditioning relies on NOX2-containing NADPH oxidase in a Langendorff-perfused, isolated NOX2 knockout mouse heart model of ischemia/reperfusion injury, and whether ROS-independent signaling is also independent of the NADPH oxidase/ROS pathway in this model.

PRINCIPAL FINDINGS

1. ROS generation and NOX2 containing NADPH oxidase are essential for ischemic preconditioning
Ischemic preconditioning (IPC), using a 2 cycle, 5 min ischemia/5 min reperfusion regime, resulted in significant attenuation of necrotic injury in wild-type (WT) hearts (Fig. 1 A: 26±2% vs. control, 38±2%, P<0.05) but failed to protect NOX2 knockout (KO) hearts (Fig. 1A : 34±3% vs. control, 33±3%). Concomitant with protection, IPC elicited an increase in NADPH oxidase activity in WT left ventricle (Fig. 1B : +41±13%, P<0.05). In contrast, there was no increase in NADPH oxidase activity in NOX2 KO hearts concurrent with the lack of protection in this group of hearts (Fig. 1B : +1.0±28%, P=NS). These data indicate that ischemic preconditioning requires the presence of a NOX2-containing NADPH oxidase.

View larger version (17K): [in this window] [in a new window]   Figure 1. A) Preconditioning (PC) results in significant attenuation of infarct size in wild-type (WT) hearts, protection that is not reciprocated in the NOX2 knockout (KO) hearts (P<0.05, n=6–8/group). B) Concomitant with the significant reduction of infarct size seen in the WT hearts, PC results in a 0.4-fold increase in ROS generation (*P<0.05, n=10/group), an increase that was absent in the KO hearts (n=4/group).

To confirm the hypothesis that ROS are the mediators of NADPH oxidase-dependent protection during IPC, we bracketed the preconditioning regimen with the ROS scavenger N-mercaptopropionylglycine (MPG, 300 µM). MPG blocked IPC in WT type hearts (39±2% vs. MPG control, 33±1%), lending support to the premise of the importance of NADPH oxidase-derived ROS.

2. Adenosine A₁ receptor activation triggers preconditioning independently of NADPH oxidase and ROS
Adenosine A₁ receptor agonists have recently been proposed as putative ROS-independent preconditioning mimetics, and adenosine has been shown (via A₃, and to a lesser extent the A₁ receptor) to inhibit human monocyte NADPH oxidase activity. Therefore, we hypothesized that the adenosine A₁ receptor agonist 2-chloro N6 cyclopentyl adenosine (CCPA, 200 nM), administered as a 10 min infusion with 10 min washout prior to index ischemia, would trigger significant protection in all groups studied: WT, KO, and MPG-treated WT hearts. CCPA preconditioning did indeed result in significant attenuation of infarct size in each of these groups (Fig. 2 A: 24±3%, 20±3%, and 23±1%, respectively, P<0.05 vs. respective controls, 38±2%, 34±3%, and 33±1%). Moreover in WT hearts CCPA did not increase NADPH oxidase activity relative to untreated controls (Fig. 2B : +22±11%, P=NS). These data demonstrate that CCPA-triggered protection is via a NOX2-NADPH oxidase- and ROS-independent pathway.

View larger version (19K): [in this window] [in a new window]   Figure 2. A) Control infarct sizes we equivalent in the WT, MPG-treated WT, and the KO groups. Administration of 200 nmol/L CCPA triggered significant protection in all groups studied (*P<0.05 relative respective controls, n=6–8/group). B) NADPH oxidase activity assay demonstrated that CCPA triggers no increase in NADPH oxidase activity despite the evident protection observed in WT hearts (*P<0.05, n=10/group).

3. It is possible to recruit ROS-independent ischemic preconditioning in NOX2-KO and MPG-treated hearts
Given the evidence with CCPA that there are ROS and NADPH oxidase-independent signaling cascades, we predicted that by increasing the number of preconditioning cycles it would be possible to recruit these pathways in NOX2-KO and WT hearts treated with MPG. In effect, it would be possible to overcome the lack of protection observed after 2-cycle ischemic preconditioning. In this case, 4-cycle ischemic preconditioning resulted in significant attenuation of infarct size in both KO and MPG-WT hearts (22±4% and 21±2%, P<0.05 vs. respective controls), in contrast to that seen after 2-cycle ischemic preconditioning.

4. PKC is upstream of NADPH oxidase-mediated ischemic preconditioning
To determine the role and relationship of PKC with NADPH oxidase in ROS-dependent preconditioning, we administered the PKC inhibitor chelerythrine (2 µM) to bracket the preconditioning stimulus. Consistent with earlier reports, PKC inhibition resulted in complete abrogation of ischemic preconditioning protection in the WT hearts (35±3% vs. chelerythrine alone, 35±2%). Chelerythrine administration, while having not affecting basal NADPH oxidase activity, abrogated the increase in NADPH oxidase activity in response to ischemic preconditioning (+3.4±9.9% P=NS increase over chelerythrine controls). Thus, PKC appears to be upstream of NADPH oxidase.

CONCLUSIONS AND SIGNIFICANCE

The present study demonstrates for the first time that ischemic preconditioning is reliant upon the recruitment of NOX2-containing NADPH oxidase activity to generate ROS to elicit protection. ROS has been the primary candidate for deleterious pro-death processes in ischemia reperfusion injury but, as we and others demonstrate, ROS signaling is a double-edged sword; although excess ROS generation can certainly trigger cell death, more modest ROS generation appears to have clear protective properties, recruiting prosurvival adaptive signaling pathways.

While NADPH oxidase is a novel ROS generating signaling pathway in the paradigm of preconditioning, it does not exclude the potential for ROS release from mitochondria as a potential protective mechanism. Indeed, increasing literature supports the ROS-induced ROS release from mitochondria: it is possible that triggering ROS for mitochondrial ROS release may be derived from NADPH oxidase.

The present data also demonstrate that the activity of an upstream PKC isoform is critical to preconditioning triggered NADPH oxidase activity and to the subsequent protection. This result is interesting in the context of the myocardial preconditioning literature: many earlier studies suggest that PKC downstream of the ROS generating step. However, our data suggest there is an isoform of PKC upstream of NADPH oxidase and, therefore, of ROS generation. Whether different isoforms of PKC play up- and downstream roles in this ROS-dependent pathway remains to be determined.

We found that preconditioning triggered by the adenosine A₁ receptor agonist CCPA resulted in protection that was not only independent of the generation of ROS, but also independent of NADPH oxidase. This confirms the presence of at least two preconditioning pathways—one being ROS dependent, the other ROS independent (Fig. 3 ). This apparent built-in "redundancy" of the signaling pathway in myocardium was further enhanced by the ability to recruit these NADPH oxidase/ROS-independent pathways by increasing the robustness of the preconditioning stimulus. Indeed, the present study shows that whereas no protection is observed in preconditioned hearts treated with MPG or in preconditioned hearts deficient in NOX2, by increasing the number of preconditioning cycles from 2 to 4 protection could be effectively restored.

View larger version (30K): [in this window] [in a new window]   Figure 3. Proposed schema of preconditioning. Two disparate signaling cascades, one being ROS dependent, signaling via PKC and NADPH oxidase, the other (an example being adenosine A1 receptor-mediated protection) being ROS independent.

Thus, we demonstrate the protective properties of a novel NADPH oxidase/ROS signaling cascade in the context of acute myocardial ischemia that may provide a pharmacological target in terms of preserving myocardium in the face of injurious ischemia.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2774fje;

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This Article Full Text (PDF) All Versions of this Article: 19/14/2037 04-2774fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bell, R. M. Articles by Shattock, M. J. Search for Related Content PubMed PubMed Citation Articles by Bell, R. M. Articles by Shattock, M. J.