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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 16, 2003 as doi:10.1096/fj.02-0832fje. |
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Medical Molecular Biology Unit, Institute of Child Health, University College London, 30 Guilford St., London WC1N 1EH, UK; and
* National Heart and Lung Institute, Royal Brompton Hospital, London, UK
2Correspondence: Medical Molecular Biology Unit, Institute of Child Health, University College London, 30 Guilford St., London WC1N 1EH, UK. E-mail: k.lawrence{at}ich.ucl.ac.uk
SPECIFIC AIMS
We recently demonstrated that the 40 amino acid corticotrophin-releasing hormone family member urocortin (Ucn) is protective to cardiac myocytes during ischemia and at reperfusion. Because this protective effect of Ucn requires altered protein synthesis, we used Affymetrix gene chips to identify genes whose expression is modulated by treating cardiac myocytes with Ucn. We report on the mechanisms by which one of these altered gene products, calcium-insensitive phospholipase A2 (iPLA2), mediates Ucn-induced cardioprotection.
PRINCIPAL FINDINGS
1. iPLA2 mRNA and protein levels are attenuated by Ucn
To confirm gene changes from the Affymetrix gene chip, RT-PCR was performed on RNA from cardiac myocytes exposed for 24 h to Ucn. A single band of the predicted size for iPLA2 was seen in PCR reactions from RNA derived from untreated and Ucn-treated cells. The intensity of this band was 3.2-fold lower (n=3 observations) in Ucn-treated cells vs. controls. Expression of the housekeeping gene cyclophilin showed no difference among samples (Fig. 1
a). RNA slot blots confirm those of RT-PCR. The intensity of the signal from the RNA slot probed with labeled iPLA2 cDNA was 2.8-fold lower (n=3 observations) in Ucn-treated cells compared with untreated cells. No differences were seen with a cyclophilin probe (Fig. 1a
). Using an antibody to iPLA2, protein levels reflected those of RNA, Ucn again causing a down-regulation in protein levels by 2.3-fold (n=3 observations). No differences were seen with an actin antibody (Fig. 1a
).
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2. Attenuation of iPLA2 protein levels during Ucn treatment is time dependent; during ischemia/ reperfusion, this process is caused by Ucn binding to its receptor
Pretreatment of cardiac myocytes for various times caused a maximum decrease in iPLA2 protein levels after 16 h (Fig. 1b
).
The effect of ischemia and reperfusion on the protein expression of iPLA2 was investigated. Exposure to 4 h ischemia and 24 h of reperfusion lowered the levels of iPLA2. When the CRH receptor antagonist 
-helical CRH was applied to cells before exposure to ischemia and reperfusion, the reduction in iPLA2 protein levels was reversed (Fig. 1c
). This suggests that the lower iPLA2 levels are caused by endogenous Ucn being released during ischemia/reperfusion and binding to its cardiac receptor in an autocrine/paracrine fashion.
3. The ischemia-induced increase in myocyte lysophosphatidylcholine (LPC) concentration is lowered by Ucn
To test whether the lower levels of iPLA2 produced by Ucn treatment had a functional effect, we measured LPC levels. Pretreatment of cardiac myocytes for 24 h with Ucn caused a 30% decrease in the concentration of LPC generated compared with control cells. A greater effect (50% reduction in LPC concentration) was produced by 24 h pretreatment with the specific iPLA2 inhibitor bromoenol lactone (BEL). Ischemia and ischemia/reperfusion both caused a large increase in cardiac myocyte LPC concentration, which was significantly lowered by pretreatment with Ucn (Fig. 2
a).
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4. The effect of LPC, alone and in combination with Ucn and BEL, on myocyte survival
Exogenously applied LPC is highly toxic to our primary myocyte preparations, causing a 2.5-fold increase in apoptotic cell death compared with untreated controls (Fig. 2b
). When Ucn or BEL is applied to myocytes together with LPC, the toxic effect of LPC is reversed, close to control values. A combination of Ucn and BEL did not produce greater protection than either agent alone, suggesting they are working via a common mechanism (Fig. 2b
).
5. The effect of ischemia/reperfusion on cell death and the effects of Ucn and BEL
When present during ischemia/reperfusion, Ucn is cardioprotective compared with cells exposed to ischemia/reperfusion alone. The selective iPLA2 inhibitor BEL is also cardioprotective. When applied together, their protective effects are not additive; the cardioprotection afforded by Ucn and BEL in combination is equivalent to that produced by either agent alone (Fig. 2c
). Extending our studies to the intact heart, we examined the effect of LPC during ischemia/reperfusion of whole hearts ex vivo. Pretreatment with Ucn or BEL significantly reduced infarct size compared with ischemia/reperfusion alone (Fig. 2d, h
). Preischemic infusion of LPC produced a significant enlargement of the infarcted area compared with control hearts exposed to ischemia/reperfusion (Fig. 2d
). Pretreatment with Ucn or BEL before infusion of LPC significantly reduced the extent of infarct size compared with ischemia/reperfused hearts exposed to LPC alone. This protection was similar to that seen with Ucn and BEL alone during ischemia/reperfusion (Fig. 2d
). We measured left ventricular end diastolic pressure (LVEDP) and left ventricular systolic pressure (LVSP). Ucn and BEL produced an almost complete recovery in both parameters at the end of reperfusion vs. ischemia and reperfusion alone (Fig. 2e-g
).
CONCLUSIONS
Using microarray technology, we have demonstrated for the first time that expression of a specific iPLA2 enzyme is reduced in rat primary neonatal cardiac myocytes exposed to the cardioprotective agent Ucn. Specific decreases in mRNA and enzyme protein of iPLA2 were confirmed (Fig. 1a
). iPLA2 levels decreased in a time-dependent manner after exposure to Ucn for up to 16 h. Moreover, the Ucn receptor antagonist -helical CRH abolished the decrease in iPLA2 protein caused by simulated ischemia/reperfusion, suggesting that endogenous Ucn is responsible for the decrease in iPLA2 protein (Fig. 1b, c
).
One catabolic product of this enzyme is LPC, which has been shown to accumulate in cardiac myocytes during ischemia because of an increase in activity of iPLA2. In our study, Ucn could lower this ischemia-induced increase In LPC concentration (Fig. 2a
). When applied exogenously to cardiac myocytes, LPC induced apoptosis. LPC, a product of iPLA2 activity, is an important mediator of ischemia/reperfusion-induced injury; this injury can be reversed by Ucn.
BEL is a specific and irreversible inhibitor of iPLA2 activity; like Ucn, it reduces LPC concentration (Fig. 2a
) and protects cardiac myocytes from LPC-induced cell death (Fig. 2b
) and the effects of simulated ischemia/reperfusion injury (Fig. 2c
). This confirms the importance of the iPLA2 isoform in LPC generation and in ischemia/reperfusion injury. We have extended this study to the whole heart ex vivo and find LPC enhances cardiac cell death by increasing infarct size when applied before ischemia compared with ischemia alone. Again, both Ucn (Fig. 2h
) and BEL significantly reduce infarct size produced by ischemia and ischemia together with LPC (Fig. 2d, h
). Either Ucn or BEL corrected the altered hemodynamics produced by ischemia and reperfusion (Fig. 2e-g
).
Thus, Ucn, a novel cardioprotective agent, reduces the level of a specific iPLA2 enzyme and its toxic metabolite, LPC. We have demonstrated that reduction of the iPLA2 pool produced by Ucn is one of its cardioprotective mechanisms. These data also suggest that CRH-R2 agonists and and/or iPLA2 antagonists may be useful therapies for minimizing cell loss and cardiac dysfunction after ischemia/reperfusion injury.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0832fje; doi: 10.1096/fj.02-0832fje 
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