Cardioprotective Role of Endogenous UrocortinIn the Human Heart Exposed to Cardioplegic Arrest and Reperfusion

As the prevalence of coronary heart disease grows, there is an increasing need for revascularization, including surgical revascularization on cardiopulmonary bypass during which the heart is exposed to iatrogenic ischemia / reperfusion injury (IRI) as a result of cardioplegic arrest and subsequent reperfusion. Subsequent cell death by various mechanisms (apoptosis, oncosis, and autophagy, all of which may result in necrosis) may occur, with some features of each type observed in IRI. Although cardioplegic solutions remain the cornerstone of myocardial protection, identification and utilization of endogenous elements such as Urocortin (Ucn), may provide additional protection. Urocortin is an endogenous polypeptide comprised of forty amino acids. Originally isolated from the rat midbrain and subsequently isolated in other organs including the heart, Ucn is released by cardiac cells exposed to ischemia and provides cardioprotection with an autocrine and paracrine mechanism. Our thesis work consists of three experimental phases. In the first set of experiments, we explored the potential clinical utility of Ucn as a biomarker of myocardial ischemia. We performed assays of Ucn on the perfusate of Langendorff-perfused Sprague–Dawley rat hearts exposed to increasing periods of zero-flow global ischemia followed by reperfusion, as well as Ucn assays in the circulation of Sprague–Dawley rats exposed in vivo to identical periods of regional ischemia and reperfusion. The highest release of Ucn into both perfusate and plasma was documented after 5 and 10 minutes of ischemia, when the post-ischemic functional recovery was complete and there was neither myocyte apoptosis nor release of conventional biomarkers of cardiac necrosis, such as CPK, as compared to longer ischemic intervals of 20 and 30 minutes when myocyte apoptosis and/or necrosis occurred along with a concurrent decline in Ucn levels, suggesting that Ucn expression and release are mainly sustained by metabolically challenged, though still viable myocytes. In addition, unlike the current biochemical markers, such as cardiac troponin and CPK, which may be elevated as a consequence of iatrogenic injury from the surgery itself, serum Ucn was unaffected either by anaesthesia or surgery. Thus, serum Ucn levels may help in the identification of peri- and post-operative cardiac injury. In the second set of experiments, we investigated the cardioprotective role of Ucn in patients undergoing cardiac surgery, and its mechanism of action with respect to protein kinase C epsilon (PKC ε) expression, activation, and relocation. 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 minutes after release of the aortic clamp. In hearts exposed to iatrogenic ischemia/reperfusion injury, Ucn induction was documented at both the mRNA and the protein levels, along with a selective increase of protein kinase C ε mRNA, over-expression of total protein kinase C ε, and increase in protein kinase C ε phosphorylation. Mitochondrial translocation of activated protein kinase C ε was observed only in post cardioplegic samples, using both subcellular fractionation and immunostaining techniques. Enhanced protein kinase C ε/mitochondria colocalization was observed in viable myocytes, which also stained positive for Ucn. Finally, using coimmunoprecipitation, an iatrogenic ischemia/reperfusion injury-enhanced physical interaction of phosphorylated protein kinase C ε with the 6.1 inwardly rectifying potassium channel subunit (Kir 6.1) of the KATP channels. After iatrogenic ischemia/reperfusion injury, Ucn expression in viable cells selectively co-localized with enhanced phosphorylation and mitochondrial relocation of protein kinase C ε, suggesting a cardioprotective role for endogenous Ucn, via physical interaction of activated protein kinase C ε with Kir6.1 KATP channel. In the third set of experiments, we explored the role of Ucn, and PKC isoforms epsilon and delta, in diabetic patients undergoing coronary bypass surgery In addition to inducing oxidative stress and negative inotropism in the heart, hyperglycemia, a powerful activating signal for cardiac PKC isozymes, results in mitochondrial dysfunction, cytochrome-c release and apoptosis in cardiac myocytes. Activation of PKC δ has been reported in ventricular myocytes under hyperglycemic conditions. We performed assays of Ucn, PKC ε and PKC δ from tissue specimens obtained from two sequential biopsies taken from the right atrium of diabetic and non-diabetic patients undergoing on pump coronary artery bypass graft surgery at the start of grafting (internal control) and 10 minutes after release of the aortic clamp. Ucn induction was documented both at the mRNA and protein level in non-diabetic hearts, compared to diabetic hearts in which pre-cardioplegic Ucn levels were 50% lower and post-cardioplegic Ucn induction was not observed. In diabetic hearts, pre-cardioplegic levels of PKC-ε were 30% lower than those of non-diabetic hearts, and cardioplegic arrest was not associated with increased expression of PKC-ε mRNA. After cardioplegic arrest, there was no upregulation of total PKC-ε nor increase in PKCε phosphorylation. Instead, there was overexpression of total PKC-δ and enhanced PKC-δ phosphorylation. Mitochondrial relocation of PKC-ε was mainly detected in postcardioplegic samples from non-diabetic hearts whereas nuclear and mitochondrial translocation of PKC-δ was mainly documented in post-cardioplegic samples from diabetic hearts. Apoptosis, assessed by TUNEL staining, was over 2-fold higher in post-cardioplegic samples from diabetic than non-diabetic hearts. Enhanced PKCε/mitochondria co-localization was observed in viable myocytes showing Ucn-positive staining, with prevention of myocyte apoptosis by mitochondrial relocation of PKC-ε. Cardiac cells showing cytosolic positive staining for Ucn never exhibited nuclear relocation of PKC-δ. In contrast, myocytes exhibiting nuclear relocation of PKC-δ were TUNEL-positive and systematically Ucn-negative, with induction of myocyte apoptosis by nuclear/mitochondrial relocation of PKC-δ. In summary, absence of cardioplegic arrest-induced myocyte overexpression of Ucn I in diabetic hearts was associated with induction, activation, nuclear and mitochondrial relocation of PKCδ, finally resulting in apoptosis. The failure to overexpress Ucn may render the diabetic heart more susceptible to apoptosis and contribute to adverse outcomes.