Limiting cardiac ischemic injury by pharmacologically augmenting the AMPK signaling pathway
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Myocardial ischemia is the leading cause of morbidity and mortality worldwide and has become a global burden on healthcare. Myocardial ischemia is accompanied by significant suppression of glucose and fatty acid metabolism due to oxygen deprivation, leading to impaired mitochondrial oxidative phosphorylation and energy crisis in cardiomyocytes. One of the strategies to limit cardiac injury is targeting the energy salvage pathway. AMP-activated protein kinase (AMPK) is a key regulator in metabolic and stress pathways in response to ischemia. Previous studies in our group and other groups have demonstrated that AMPK is cardioprotective in both ex vivo and in vivo models of ischemia/reperfusion. The major mechanisms underlying the protective effects of AMPK are the stimulation of glucose uptake and acceleration of glycolysis for ATP generation, and inhibition of ATP-consuming pathways such as protein and fatty acid synthesis. Given the reported findings that AMPK regulates myocardial glucose metabolism to protect against ischemic injury, we investigated whether pharmacologically targeting AMPK by two potential activators could prevent and limit myocardial ischemic injury in the heart. In the first study in chapter three, activated protein C (APC), a natural plasma serine protease that down-regulates the clotting and inflammatory pathways, was examined for protective effects in the ischemic heart. Intravenous infusion of APC during 20 min ischemia via left coronary artery occlusion in vivo significantly reduced myocardial infarction. This cardioprotective effect of APC is due to its signaling activity since infarct size was decreased in mouse hearts treated with APC-2Cys (protein derivative only possessing signaling activity), but not in mouse hearts treated with APC-E170A (protein derivative only possessing anticoagulant activity). APC or APC-2Cys, but not APC-E170A, stimulated AMPK activation in both ischemic hearts and isolated cardiomyocytes. The activation of cardiac AMPK by APC was mediated through the APC receptors EPCR and PAR-1, and was dependent on CaMKKbeta. The APC-AMPK cascade can modulate substrate metabolism in the ischemic heart. As investigated in an ex vivo perfusion system, APC initiated AMPK signaling, augmented glucose uptake, and significantly improved the post-ischemic left ventricular contractile function in isolated WT hearts subjected to 20 min global ischemia followed by 30 min reperfusion. Moreover, APC decreased inflammatory signaling pathways JNK and NF-κB, and down-regulated the expression of inflammatory cytokines in ischemic hearts. These cardioprotective effects of APC, including cardiac repair, cardiac function restoration and anti-inflammation, were abrogated in AMPKalpha2-KO mice. The work in chapter three demonstrated that APC protected against myocardial ischemic injury by triggering AMPK signaling pathway. The second study in chapter four focused on the role of small MIF agonists MIF20 in cardiac protection during ischemia/reperfusion. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that is released by ischemic cardiomyocytes and exerts a protective effect in the heart by activating AMPK. While developing small molecule antagonists of the MIF receptor for immunomodulatory applications, we uncovered the compound MIF20 that had the property of increasing MIF's affinity for its cell surface receptor. An in vitro assay applying recombinant MIF and MIF20 to isolated cardiomyocytes showed that MIF20, in the presence of MIF, significantly augmented AMPK phosphorylation when compared to either MIF or MIF20 alone. The addition of MIF plus MIF20 increased by 50% the cell surface expression of glucose transporter (GLUT4) and led to a 25% increase in cellular glucose uptake when compared to MIF treatment alone. Murine WT hearts perfused with MIF20 for 15 min prior to a standardized protocol of 25 min no-flow ischemia and 30 min reperfusion, demonstrated an improved post-ischemic left ventricular function, an increased cardiac MIF-AMPK activation and augmented glucose uptake. By contrast, the cardioprotective action of MIF20 during ischemia and reperfusion was not observed in mouse hearts lacking MIF or CD74 receptor. Finally, the cardioprotection of MIF20 was verified in an in vivo regional ischemia model by reduced myocardial infarction in WT mice. The work in chapter four revealed that MIF receptor agonism offered protection against cardiac ischemic injury by augmenting the MIF-AMPK signaling pathway. Chapter five contains overall conclusions from this work. An overall discussion composed of the metabolic and non-metabolic downstream effects of AMPK activation by pharmacological factors during ischemia/reperfusion is included. Moreover, a comparison of current AMPK activators and these two novel AMPK activators as a potential therapy for myocardial ischemia is considered.