Date of Award

January 2012

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Lawrence H. Young

Subject Area(s)

Medicine, Physiology

Abstract

THE ROLE OF D-DOPACHROME TAUTOMERASE IN ISCHEMIA-REPERFUSION IN THE HEART.

Kwame Atsina, Dake Qi, Richard Bucala, Lawrence Young, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT.

AMP-activated protein kinase (AMPK) is a well-known energy sensor that regulates cardiac metabolism. In the heart, it is also regulated by the pleiotropic cytokine, macrophage migration inhibitory factor (MIF) to protect the myocardium from ischemia-reperfusion injury. D-dopachrome tautomerase (DDT) is a novel protein that binds with high affinity to the MIF receptor, CD74, through which MIF regulates AMPK-mediated cardioprotection.

However, the role of DDT in regulating the cardiac response to ischemia reperfusion is unknown. We investigated the role of DDT in regulating AMPK activation and cardiac injury during experimental ischemia/reperfusion in mouse hearts, rat papillary muscle and rat cardiomyocytes. To investigate the effect of CD74 and MIF deficiency on the heart during ischemia reperfusion, hearts were subjected to ischemia for 15 minutes (Langendorff perfusion) or mice were subjected to 20 minutes of coronary ligation. CD74-/- hearts exposed to ischemia-reperfusion injury had greater myocardial necrosis and contractile dysfunction, and decreased AMPK activation than Mif-/- hearts. To determine DDT expression in cardiac tissue, mouse hearts received baseline perfusion or were exposed to ischemia (perfused heart model) for 15 minutes. Hearts were fixed and immunohistochemistry staining with anti-MIF and anti-DDT antibody revealed that MIF and DDT are released from the heart in response to ischemia. To investigate the effect of antibody neutralization of MIF and DDT on AMPK activation and cardiac injury following ischemia-reperfusion, (1) rat papillary muscle and rat cardiomyocytes were subjected to 15 and 30 minutes of hypoxia respectively, and (2) mouse hearts were subjected to 15 minutes of ischemia via Langendorff perfusion mode. 100 ìg/mL of non-specific IgG, anti-DDT or anti-MIF antibody was administered to the medium or buffer prior to hypoxia or ischemia. Antibody neutralization of DDT and MIF impaired

AMPK activation in all three animal models, and exacerbated myocardial necrosis and LV contractile functional recovery in the mouse hearts following ischemia. These findings demonstrate that DDT plays an important role in regulating AMPK activity and protecting the heart from ischemia-reperfusion.

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