Activated protein C protects against myocardial ischemic/reperfusion injury through AMP‐activated protein kinase signaling

Summary.  Background: Activated protein C (APC) is a vitamin K‐dependent plasma serine protease that down‐regulates clotting and inflammatory pathways. It is known that APC exerts a cardioprotective effect by decreasing apoptosis of cardiomyocytes and inhibiting expression of inflammatory mediators after myocardial ischemia. Objectives: The objective of this study was to understand the mechanism of the APC‐mediated cardioprotection against ischemic injury. Methods: Cardioprotective activities of wild‐type APC and two derivatives, having either dramatically reduced anticoagulant activity or lacking signaling activity, were monitored in an acute ischemia/reperfusion injury model in which the left anterior descending coronary artery (LAD) was occluded. Results: APC reduced the myocardial infarct size by a mechanism that was largely independent of its anticoagulant activity. Thus, the non‐anticoagulant APC‐2Cys mutant, but not the non‐signaling APC‐E170A mutant, attenuated myocardial infarct size by EPCR and PAR‐1‐dependent mechanisms. Further studies revealed that APC acts directly on cardiomyocytes to stimulate the AMP‐activated protein kinase (AMPK) signaling pathway. The activation of AMPK by APC ameliorated the post‐ischemic cardiac dysfunction in isolated perfused mouse hearts. Moreover, both APC and APC‐2Cys inhibited production of TNFα and IL‐6 in vivo by attenuating the ischemia/reperfusion‐induced JNK and NF‐κB signaling pathways. Conclusions: APC exerts a cardioprotective function in ischemic/reperfusion injury through modulation of AMPK, NF‐κB and JNK signaling pathways.

[1]  P. Schjerling,et al.  Possible CaMKK-dependent regulation of AMPK phosphorylation and glucose uptake at the onset of mild tetanic skeletal muscle contraction. , 2007, American journal of physiology. Endocrinology and metabolism.

[2]  L. Leng,et al.  Macrophage migration inhibitory factor stimulates AMP-activated protein kinase in the ischaemic heart , 2008, Nature.

[3]  Jerry A Nick,et al.  Recombinant human activated protein C reduces human endotoxin-induced pulmonary inflammation via inhibition of neutrophil chemotaxis. , 2004, Blood.

[4]  Jun Ren,et al.  A newly synthetic chromium complex-chromium (D-phenylalanine)3 activates AMP-activated protein kinase and stimulates glucose transport. , 2009, Biochemical pharmacology.

[5]  C. Esmon.  Molecular Events that Control the Protein C Anticoagulant Pathway , 1993, Thrombosis and Haemostasis.

[6]  J. Bae,et al.  Identification of a Specific Exosite on Activated Protein C for Interaction with Protease-activated Receptor 1* , 2007, Journal of Biological Chemistry.

[7]  J. Swantek,et al.  Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) is required for lipopolysaccharide stimulation of tumor necrosis factor alpha (TNF-alpha) translation: glucocorticoids inhibit TNF-alpha translation by blocking JNK/SAPK , 1997, Molecular and cellular biology.

[8]  A. Means,et al.  The Ca2+/Calmodulin-dependent Protein Kinase Kinases Are AMP-activated Protein Kinase Kinases* , 2005, Journal of Biological Chemistry.

[9]  A. Edelman,et al.  Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase. , 2005, Cell metabolism.

[10]  L. Nelin,et al.  MAPK phosphatases — regulating the immune response , 2007, Nature Reviews Immunology.

[11]  W. Ruf,et al.  Activation of Endothelial Cell Protease Activated Receptor 1 by the Protein C Pathway , 2002, Science.

[12]  José A Fernández,et al.  Endogenous EPCR/aPC-PAR1 signaling prevents inflammation-induced vascular leakage and lethality. , 2009, Blood.

[13]  Margaret S. Wu,et al.  Role of AMP-activated protein kinase in mechanism of metformin action. , 2001, The Journal of clinical investigation.

[14]  J. Fischer,et al.  Human Vascular Smooth Muscle Cells Express Functionally Active Endothelial Cell Protein C Receptor , 2007, Circulation research.

[15]  C. Esmon,et al.  The endothelial cell protein C receptor aids in host defense against Escherichia coli sepsis. , 2000, Blood.

[16]  B. Culver,et al.  Impaired cardiac contractile function in ventricular myocytes from leptin-deficient ob/ob obese mice. , 2006, The Journal of endocrinology.

[17]  P. Fay,et al.  Regulation of blood coagulation by the protein C system , 1992, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[18]  Xiaoping Yang,et al.  Metallothionein alleviates glutathione depletion-induced oxidative cardiomyopathy in murine hearts , 2008, Critical care medicine.

[19]  José A Fernández,et al.  Activated Protein C Prevents Neuronal Apoptosis via Protease Activated Receptors 1 and 3 , 2004, Neuron.

[20]  Javier Corral,et al.  Endothelial protein C receptor polymorphisms and risk of myocardial infarction , 2008, Haematologica.

[21]  R. Russell,et al.  AMP-activated protein kinase: a key stress signaling pathway in the heart. , 2005, Trends in cardiovascular medicine.

[22]  J. Griffin,et al.  The cytoprotective protein C pathway. , 2007, Blood.

[23]  A. Ayala,et al.  Recombinant human activated protein C inhibits integrin-mediated neutrophil migration. , 2009, Blood.

[24]  A. Chang,et al.  Protein C prevents the coagulopathic and lethal effects of Escherichia coli infusion in the baboon. , 1987, The Journal of clinical investigation.

[25]  F. Castellino,et al.  A cardioprotective role for the endothelial protein C receptor in lipopolysaccharide-induced endotoxemia in the mouse. , 2005, Blood.

[26]  J. Bae,et al.  Engineering a Disulfide Bond to Stabilize the Calcium-binding Loop of Activated Protein C Eliminates Its Anticoagulant but Not Its Protective Signaling Properties* , 2007, Journal of Biological Chemistry.

[27]  L. Young.  AMP-activated protein kinase conducts the ischemic stress response orchestra. , 2008, Circulation.

[28]  José A Fernández,et al.  Activated protein C and ischemic stroke , 2004, Critical care medicine.

[29]  Nigel Mackman,et al.  Endotoxemia and sepsis mortality reduction by non-anticoagulant–activated protein C , 2007, The Journal of experimental medicine.

[30]  T. Noguchi,et al.  Activated protein C reduces ischemia/reperfusion-induced renal injury in rats by inhibiting leukocyte activation. , 2000, Blood.

[31]  O. Benzakour,et al.  Activated protein C alters cytosolic calcium flux in human brain endothelium via binding to endothelial protein C receptor and activation of protease activated receptor-1. , 2003, Blood.

[32]  A. Yıldırır,et al.  Recombinant human-activated protein C inhibits cardiomyocyte apoptosis in a rat model of myocardial ischemia–reperfusion , 2007, Coronary artery disease.

[33]  C. Feistritzer,et al.  Endothelial barrier protection by activated protein C through PAR1-dependent sphingosine 1-phosphate receptor-1 crossactivation. , 2005, Blood.

[34]  J. Ninomiya-Tsuji,et al.  AMP-Activated Protein Kinase Activates p38 Mitogen-Activated Protein Kinase by Increasing Recruitment of p38 MAPK to TAB1 in the Ischemic Heart , 2005, Circulation research.

[35]  R. Heath,et al.  Ca2+/calmodulin-dependent protein kinase kinase-beta acts upstream of AMP-activated protein kinase in mammalian cells. , 2005, Cell metabolism.

[36]  C. Esmon,et al.  Activated Protein C Protects Against Myocardial Ischemia/ Reperfusion Injury via Inhibition of Apoptosis and Inflammation , 2009, Arteriosclerosis, thrombosis, and vascular biology.

[37]  José A Fernández,et al.  Activated protein C blocks p53-mediated apoptosis in ischemic human brain endothelium and is neuroprotective , 2003, Nature Medicine.

[38]  B. Grinnell,et al.  Recombinant human activated protein C attenuates the inflammatory response in endothelium and monocytes by modulating nuclear factor-&kgr;B , 2002, Critical care medicine.

[39]  José A Fernández,et al.  Hyperantithrombotic, noncytoprotective Glu149Ala-activated protein C mutant. , 2009, Blood.

[40]  J Ean,et al.  Efficacy and safety of recombinant human activated protein C for severe sepsis. , 2001, The New England journal of medicine.

[41]  José A Fernández,et al.  Neuroprotective activities of activated protein C mutant with reduced anticoagulant activity , 2009, The European journal of neuroscience.