Molecular Cardiology Cardioprotection Through S-Nitros(yl)ation of Macrophage Migration Inhibitory Factor

Background—Macrophage migration inhibitory factor (MIF) is a structurally unique inflammatory cytokine that controls cellular signaling in human physiology and disease through extra-and intracellular processes. Macrophage migration inhibitory factor has been shown to mediate both disease-exacerbating and beneficial effects, but the underlying mechanism(s) controlling these diverse functions are poorly understood. Methods and Results—Here, we have identified an S-nitros(yl)ation modification of MIF that regulates the protective functional phenotype of MIF in myocardial reperfusion injury. Macrophage migration inhibitory factor contains 3 cysteine (Cys) residues; using recombinant wtMIF and site-specific MIF mutants, we have identified that Cys-81 is modified by S-nitros(yl)ation whereas the CXXC-derived Cys residues of MIF remained unaffected. The selective S-nitrosothiol formation at Cys-81 led to a doubling of the oxidoreductase activity of MIF. Importantly, S-nitrosothiol-MIF formation was measured both in vitro and in vivo and led to a decrease in cardiomyocyte apoptosis in the reperfused heart. This decrease was paralleled by a S-nitrosothiol-MIF– but not Cys81 serine (Ser)–MIF mutant– dependent reduction of infarct size in an in vivo model of myocardial ischemia/reperfusion injury. Conclusions—S-nitros(yl)ation of MIF is a pivotal novel regulatory mechanism, providing enhanced activity resulting in increased cytoprotection in myocardial reperfusion injury. M yocardial inhibitory factor (MIF) is a structurally unique inflammatory cytokine that plays an important role as an upstream regulator of innate and acquired immunity as well as in cellular redox signaling. Because of its inflammatory activities, MIF is a mediator of a number of acute and chronic inflammatory diseases. 1– 6 This includes an exacerbating role in atherosclerosis, a major underlying cause of cardiovascular disease in the Western world. 7–10 However, accumulating evidence indicates that the role of MIF in cardiovascular pathology is more complex. MIF regulates key functions in myocardial ischemia/reperfusion (I/R) injury with an overall cardioprotective activity profile. 11–14 Mecha-nistically, this property involves protection against c-Jun N-terminal kinase (JNK)–mediated apoptosis and against oxidative cell stress which prominently occurs during the early phase of reperfusion in myocardial infarction. Protection conferred by MIF against redox stress has been attributed to its intrinsic thiol-protein oxidoreductase (TPOR) activity. MIF's role in redox regulation is due to its structural properties which are unique within the protein family of cytokines and plays a critical role in cellular redox homeo-stasis and apoptosis inhibition. 1,11,15 The TPOR activity of MIF is mediated through a conserved Cys 57-Ala-Leu-Cys 60 motif 15 resembling the CXXC motif of TPORs like thiore-doxin (TRX). Moreover, residue Cys-60 is …