Prostaglandin EP4 receptor mediated attenuation of hypoxia-induced cell death in H9c2 cardiomyoblasts depends on activation of mitochondrial KATP channels

Treml, Marcel , Prof. Dr. Schrör, Karsten , Prof. Dr. Hohlfeld, Thomas , Dr. Meyer-Kirchrath, Jutta. University of Duesseldorf Pharmacology, Universitaetsstr.1, Geb.22.1, 40225 Duesseldorf, Germany.

Background and purpose: In the heart, the generation of prostaglandin E2 (PGE2) is significantly increased in acute myocardial ischemia and is regarded as an intrinsic mechanism of protection against ischemia/reperfusion injury. PGE2 acts through specific EP receptor-subtypes, namely EP1-4, all of which belong to the family of G-protein coupled receptors. The involvement of the EP4 receptor subtype in antiischemic PGE2 actions has been postulated.

Experimental approach: To investigate the role of the EP4 receptor subtype in PGE2-mediated cell survival we generated H9c2 rat cardiomyoblasts expressing the human EP4 receptor. Cells were starved and subjected to 24 h of hypoxia with subsequent reoxygenation for 60 min. LDL activity in supernatants was measured and the number of dead cells was determined by SYTOX® Green fluorescence.

Key results: Specific EP4 stimulation reduced LDH release from 124 ± 10 to 42 ± 8 U/L (p<0.001) and markedly decreased the number of dead cells by 45 ± 11 % (p<0.001). This effect could not be abrogated by specific inhibition of either PI3 kinase, ERK1/2 or EGFR transactivation. Taking into consideration exchange protein directly activated by cAMP (Epac) as an alternative target, we confirmed its expression in the cells and verified EP4-dependent activation of the downstream effector Rap1. However, the application of a specific and direct activator of Epac (8-pCPT-2’-OMe-cAMP, 250 µM) had no effect on cell survival. Interestingly, inhibition of mitochondrial KATP channels with hydoxydecanoate (100µM) completely abrogated EP4 mediated effects on cell survival.

Conclusions and implications: EP4 receptor stimulation improves cell survival of cardiomyocytes under hypoxic conditions by activating mitochondrial KATP channels.