Abstract 1552: Cardioplegic Arrest And Subsequent Reperfusion Induce Urocortin Expression In Non-apoptotic Cardiac Cells Selectively Associated With Mitochondrial Relocation Of PKCϵ
BACKGROUND: Cardioplegic arrest and subsequent reperfusion inevitably expose the heart to an iatrogenic ischemia/reperfusion injury (iIRI). We previously reported that iIRI caused mitochondria-initiated myocyte apoptosis, but also induction of urocortin (Ucn), an endogenous cardioprotective peptide. We also showed that Ucn induced PKCϵ-mediated opening of mitochondrial KATP channels in isolated heart mitochondria.
AIM: To investigate, in patients exposed to iIRI, the cardioprotective role and the mechanism of action of Ucn, with respect to PKCϵ expression, activation and relocation.
METHODS AND RESULTS: Two sequential biopsies were obtained from the right atrium of 25 patients undergoing coronary artery bypass grafting at the start of grafting (internal control) and 10 mins after release of the aortic clamp. Mean values of ejection fraction, aortic cross-clamping time and number of grafts were 51±8; 48±8 mins; and 3.6±0.5 respectively. In hearts exposed to iIRI, RT-PCR and immunostaining showed Ucn induction at the mRNA (255% of basic levels, p<0.05) and protein level (28±2.1% positive myocytes vs 3.1±0.6% of internal control; p<0.01) respectively. iIRI also induced a selective increase of PKC-ϵ mRNA (225% of internal control; p<0.05) and a two-fold overexpression of total PKCϵ isoform (assessed by Western blotting; p<0.05), which paralleled a 2.9 fold increase in PKCϵ phosphorylation (p<0.01). TUNEL positivity (<0.1% and 2.9±0.7% positive myocytes pre- and post-iIRI respectively; p<0.01) was only seen in Ucn-negative cells, and, of note, Ucn-positive myocytes showed concurrent mitochondrial relocation of phosphorylated PKCϵ, as documented by mitochondrial-activated PKCϵ colocalization, calculated by confocal microscopy with an image analyzer software (% overlap: 57±5 vs 11±2 in Ucn-negative cells; p<0.01). Western blotting carried out in pools of cytosolic and mitochondrial fractions confirmed a 2.5 fold increase in mitochondrial localization of phosphorylated PKC-ϵ following iIRI (p<0.05).
CONCLUSIONS: In patients exposed to iIRI, Ucn expression in viable cells was selectively associated with phosphorylation and mitochondrial relocation of PKCϵ, suggesting a cardioprotective role for endogenous Ucn in the human heart.