title: Toll-like receptor 4 knockout protects against anthrax lethal toxin-induced cardiac contractile dysfunction: role of autophagy.
abstract: OBJECTIVE
  Anthrax lethal toxin (LeTx) is known to induce circulatory shock and death, although the underlying mechanisms have not been elucidated. This study was designed to evaluate the role of toll-like receptor 4 (TLR4) in anthrax lethal toxin-induced cardiac contractile dysfunction.
  
  
  METHODS
  Wild-type (WT) and TLR4 knockout (TLR⁻/⁻) mice were challenged with lethal toxin (2 µg·g⁻¹, i.p.), and cardiac function was assessed 18 h later using echocardiography and edge detection. Small interfering RNA (siRNA) was employed to knockdown TLR4 receptor or class III PI3K in H9C2 myoblasts. GFP-LC3 puncta was used to assess autophagosome formation. Western blot analysis was performed to evaluate autophagy (LC3, Becline-1, Agt5 and Agt7) and endoplasmic reticulum (ER) stress (BiP, eIF2α and calreticulin).
  
  
  RESULTS
  In WT mice, lethal toxin exposure induced cardiac contractile dysfunction, as evidenced by reduced fractional shortening, peak shortening, maximal velocity of shortening/re-lengthening, prolonged re-lengthening duration and intracellular Ca²⁺ derangement. These effects were significantly attenuated or absent in the TLR4 knockout mice. In addition, lethal toxin elicited autophagy in the absence of change in ER stress. Knockdown of TLR4 or class III PI3 kinase using siRNA but not the autophagy inhibitor 3-methyladenine significantly attenuated or inhibited lethal toxin-induced autophagy in H9C2 cells.
  
  
  CONCLUSIONS
  Our results suggest that TLR4 may be pivotal in mediating the lethal cardiac toxicity induced by anthrax possibly through induction of autophagy. These findings suggest that compounds that negatively modulate TLR4 signalling and autophagy could be used to treat anthrax infection-induced cardiovascular complications.
authors.affiliation: Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, College of Health Sciences, Laramie, WY, USA.
authors.forename: Machender R
authors.initials: MR
authors.lastname: Kandadi
authors.forename: Arthur E
authors.initials: AE
authors.lastname: Frankel
authors.forename: Jun
authors.initials: J
authors.lastname: Ren
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000633:toxicity*; D001343:Autophagy / Q000187:drug effects*; D001427:Bacterial Toxins / Q000633:toxicity*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D058546:Class III Phosphatidylinositol 3-Kinases / Q000235:genetics; D004452:Echocardiography; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects; D055785:Gene Knockdown Techniques; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D032386:Myoblasts, Cardiac / Q000187:drug effects / Q000378:metabolism; D009200:Myocardial Contraction / Q000187:drug effects*; D034741:RNA, Small Interfering / Q000008:administration & dosage; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D051197:Toll-Like Receptor 4 / Q000235:genetics* / Q000502:physiology
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D034741:RNA, Small Interfering; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; C030325:anthrax toxin; D058546:Class III Phosphatidylinositol 3-Kinases; D002118:Calcium