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Cardioprotection mediated by urocortin is dependent on PKC activation

K. M. Lawrence^*,1, A. M. N. Kabir, M. Bellahcene, S. Davidson^*, X. B. Cao, J. McCormick^*, R. A. Mesquita, C. J. Carroll^*, A. Chanalaris^*, P. A. Townsend^*, M. Hubank^*, A. Stephanou^*, R. A. Knight, M. S. Marber and D. S. Latchman^*

^* Medical Molecular Biology Unit, Institute of Child Health, University College, London;
The Rayne Institute, St. Thomas’s Hospital, London; and
National Heart and Lung Institute, Royal Brompton Hospital, London, UK

¹ Correspondence: Medical Molecular Biology Unit, Institute of Child Health, University College, 30 Guilford St., London WC1N 1EH, UK. E-mail:k.lawrence{at}ich.ucl.ac.uk

SPECIFIC AIMS

Urocortin (Ucn) is an endogenous cardioprotective agent that protects against the damaging effects of ischemia and reperfusion injury in vitro and in vivo. The mechanism of action of Ucn involves acute activation of target molecules and, as revealed using affymetrix gene chip technology, altered gene expression of different end effector molecules. Here we report an increase in mRNA and protein levels of protein kinase C epsilon (PKC) produced by 24 h exposure to Ucn in rat primary cardiomyocytes and Langendorff-perfused ex vivo hearts. A 10 min exposure to Ucn in both systems caused specific translocation/activation of PKC from a cytosolic to a membrane-rich fraction. The importance of the PKC isoform in cardioprotection and its relationship to cardioprotection produced by urocortin was revealed using PKC-specific inhibitor peptides. In the presence of these inhibitors, Ucn lost its cardioprotective effect. This loss of cardioprotection by Ucn was also seen in whole hearts from PKC knockout mice. These findings indicate that the cardioprotective effect of urocortin is dependent on PKC activation.

PRINCIPAL FINDINGS

1. PKC protein levels increase when exposed to Ucn in primary cardiomyocytes and whole heart
We confirmed our gene chip data at the protein level by performing Western blots from extracts derived from primary cardiomyocytes exposed to Ucn for 4 and 24 h and Langendorff-perfused ex vivo hearts exposed to Ucn for 3 h. In both model systems, Ucn produced an increase in PKC protein compared with untreated cells or hearts. A related isozyme, PKC, was unaffected by Ucn (n=3 observations).

2. Ucn causes the specific translocation/activation of PKC in primary cardiomyocytes and whole heart
When primary cardiomyocytes were treated for 10 min with Ucn, there was a significant increase in translocation of PKC from a cytosolic to a membrane-rich fraction compared with untreated cells. This translocation was blocked by the Ucn receptor antagonist helical CRH (Fig. 1 a, c). No differences in translocation were seen when fractions were probed with an antibody specific for PKC (Fig. 1b, c ) (n=3 observations). Ucn-induced translocation of PKC was blocked by a PKC-specific inhibitor peptide (Fig. 1d, e ) (n=3 observations).

View larger version (30K): [in this window] [in a new window]   Figure 1. Primary cardiomyocytes untreated (Con) or exposed to urocortin for 10 min (Ucn) or Ucn + helical CRH were fractionated into cytosolic (c) and membrane (m) fractions, then probed with a) PKC antibody or b) PKC antibody. c) Mean density of membrane-to-cytosol signal ratio (translocation ratio) (n=3). Data presented as mean ±SE. *P < 0.05. Primary cardiomyocytes untreated (Con) or exposed to urocortin (Ucn) or PKC inhibitor, urocortin (Ucn+1), or control scramble peptide, then urocortin (Ucn+S), were fractionated into cytosolic (c) and membrane (m) fractions and probed with d) PKC antibody. e) Mean density of membrane-to-cytosol signal ratio (translocation ratio) (n=3). Data presented as mean ±SE. *P < 0.05. Langendorff-perfused ex vivo heats were perfused with buffer (Con) or Ucn for 10 min, fractionated into cytosolic (c), membrane (m), and particulate (p) fractions, and probed with f) PKC or g) PKC antibody. h) Mean density of membrane-to-cytosolic signal ratio (translocation ratio) (n=3). Data are mean ±SE.

Urocortin produced a specific translocation of PKC in the Langendorff-perfused ex vivo heart (Fig. 1f, h ); no Ucn-induced translocation of PKC was seen (Fig. 1g, h ) (n=3 observations).

3. Urocortin causes PKC to localize with mitochondria selective dye
Cardiomyocytes were stained with the mitochondria selective dye mitotracker green; the same cells were probed with a PKC-specific antibody and their subcellular distribution was examined. We found a high degree of overlap of PKC with mitotracker after cells had been treated with Ucn for 10 min compared with untreated control cardiomyocytes.

4. Translocation of PKC is attenuated by specific RACK inhibitors
When cardiomyocytes were incubated with an epsilon-specific inhibitor peptide for 3 or 5 min, there was a significant reduction in the PMA-induced translocation of PKC compared with cells exposed to the control scramble peptide (n=3 observations).

5. PKC is necessary for cardioprotection in primary cardiomyocytes and is crucial for the cardioprotective effect of Ucn
As we have demonstrated here and earlier, Ucn potently protects cardiomyocytes from ischemia and reperfusion injury (Fig. 2 a). When the PKC inhibitor peptide was introduced into primary cardiomyocytes before exposure to ischemia and reperfusion, there was a significant increase in cell death compared with ischemia and reperfusion alone (Fig. 2a ). When cells were treated with Ucn in the presence of the inhibitor peptide, then exposed to ischemia and reperfusion, Ucn lost its cardioprotective effect (Fig. 2a ).

View larger version (23K): [in this window] [in a new window]   Figure 2. Apoptotic cell death assay in primary cardiomyocytes during normoxia (norm) or ischemia and reperfusion in the presence of Ucn, control scramble peptide (con pep), and inhibitor peptide (inhib pep) (n=3). Data are presented as mean ±SE. *P < 0.05. b) Langendorff-perfused homozygous PKC knockout mice PKC–/– (n=5) or wild-type littermates PKC+/+ (n=5). Both groups were treated with Ucn for 30 min (10–8 mol/L) prior to 40 min index ischemia, then reperfusion with Ucn for 30 min; individual infarct sizes were measured. Data are presented as mean ±SE. *P < 0.05 was considered significant. Normalized infarct sizes (infarct size:risk volume ratio) were compared by 1- and 2-way ANOVA. Linear regression was carried out using the Sigma Stat statistical package. Infarct volume with respect to total myocardial volume was compared by ANCOVA using an Excell plug-in (Ferris State University).

6. PKC is necessary for the cardioprotective effect of Ucn in Langendorff-perfused ex vivo mouse heart
When we exposed PKC knockout mice (PKC^–/–) to ischemia and reperfusion but in the presence of Ucn (n=5 hearts), we noted a very large infarct size. However, when we performed the same experiment on their wild-type littermates (PKC^+/+), infarct size was reduced by almost 50% (Fig. 2c ). There was no statistical difference in baseline weights, volume, or hemodynamics between the two groups.

CONCLUSIONS

We previously showed that gene expression levels of a Katp channel subtype Kir6.1 and an iPLA₂ enzyme are altered by Ucn. Here we demonstrate that PKC protein levels are increased by Ucn in primary cardiomyocytes and Langendorff-perfused ex vivo hearts. No differences were seen in a related cardiac isozyme, PKC.

We tested whether Ucn has a specific acute effect on PKC activation. We assayed PKC translocation from a cytosolic fraction to a membrane-rich fraction. In primary cardiomyocytes and whole heart, Ucn enhanced PKC translocation after 10 min exposure (Fig. 1a, c, d, e, f, h ). This translocation was removed by the Ucn receptor antagonist -helical CRH (Fig. 1a, c ). No activation of PKC was seen in either model system (Fig. 1b, c, g, h ).

Inhibitor peptides have been developed recently that block the interaction of a specific isozyme of PKC with its specific RACK, inhibiting its translocation/activation. Our method successfully produced a specific inhibition of the phorbol ester PMA stimulated translocation of PKC by close to 50% vs. translocation seen with the control scramble peptide. When we exposed our cardiomyocytes to a PKC-specific inhibitor peptide, the specific Ucn-induced translocation of PKC was removed (Fig. 1d, e ),

Using a mitochondria selective dye as marker (mitotracker green), we found a large increase in colocalization of PKC with the dye after Ucn treatment, suggesting that Ucn causes the translocation of PKC to the cardiomyocte mitochondria. Other studies report a mitochondrial localization for PKC; some even demonstrate an interaction with mitochondrial proteins.

Activation of PKC by Ucn is a significant finding, as studies overexpressing PKC-specific pseudo RACK as a means of enhancing PKC activity demonstrate increased cardioprotection. Activation of PKC in some studies has been reported to be damaging during ischemia and reperfusion and may cause harmful cardiac hypertrophy upon activation. Significantly, Ucn had no effect on the gene expression or activation of this isozyme.

When PKC activation was attenuated by specific inhibitor peptides, significantly greater cell death was produced by ischemia and reperfusion than with ischemia and reperfusion alone or in the presence of the control scramble peptide, suggesting that PKC activity is indeed important for cardiomyocyte survival during ischemia and reperfusion injury (Fig. 2a ). When Ucn was applied to cardiomyocytes, its usual cardioprotection was lost during ischemia and reperfusion when the PKC inhibitor peptide was present (Fig. 2a ). These findings suggest that Ucn exerts its cardioprotective mechanism of action via PKC activation. We extended this finding to the whole heart model and found that, as in primary cardiomyocytes, Ucn lost its ability to protect against ischemia and reperfusion injury when PKC was knocked out (Fig. 2b ).

This study demonstrates that PKC activation is necessary for cardioprotection against ischemia and reperfusion injury and is the first study to demonstrate that the cardioprotective mechanism of action of Ucn is dependent on PKC activation. Furthermore, we have demonstrated that Ucn treatment translocates PKC from a cytosolic to a membrane fraction, and others have reported that PKC is localized to the mitochondria. From our gene chip data, the two other gene products modulated by Ucn are the ATP-sensitive Katp channel and the iPLA₂ enzyme, both thought to be localized to cardiomyocyte mitochondria.

Thus, a scenario may be envisaged for the cardioprotective mechanism of action of Ucn whereby initially PKC would become activated and translocate to the mitochondria. Regulation of the mitochondrial Katp channel by PKC could occur, resulting in channel modulation, thereby enhancing cardioprotection. Indeed, some reports support such a relationship. At the same time, a reduction in the activity of mitochondrially localized iPLA₂ by PKC would lower the concentration of its toxic metabolite lysophosphatidylcholine, which we have shown to be damaging during ischemia and reperfusion. Soon after Ucn treatment, PKC activation could regulate protective and damaging target proteins, affording early protection before beneficial effects of Ucn could occur, such as inducing de novo protein synthesis of additional PKC end effectors such as the Katp channel and down-regulating damaging gene products such as iPLA₂ (Fig. 3 ).

View larger version (38K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2506fje;

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