title: The H₁-H₂ domain of the α₁ isoform of Na+-K+-ATPase is involved in ouabain toxicity in rat ventricular myocytes.
abstract: The composition of different isoforms of Na+-K+-ATPase (NKA, Na/K pump) in ventricular myocytes is an important factor in determining the therapeutic effect and toxicity of cardiac glycosides (CGs) on heart failure. The mechanism whereby CGs cause these effects is still not completely clear. In the present study, we prepared two site-specific antibodies (SSA78 and WJS) against the H₁-H₂ domain of α₁ and α₂ isoforms of NKA in rat heart, respectively, and compared their influences on the effect of ouabain (OUA) in isolated rat ventricular myocytes. SSA78 or WJS, which can specifically bind with the α₁ or α₂ isoform, were assessed with enzyme linked immunosorbent assay (ELISA), Western blot and immunofluorescent staining methods. Preincubation of myocytes with SSA78 inhibited low OUA affinity pump current but not high OUA affinity pump current, reduced the rise in cytosolic calcium concentration ([Ca²⁺](i)), attenuated mitochondrial Ca²⁺ overload, restored mitochondrial membrane potential reduction, and delayed the decrease of the myocardial contractile force as well as the occurrence of arrhythmic contraction induced by high concentrations (1 mM) but not low concentrations (1 μM) of OUA. Similarly, preincubation of myocytes with WJS inhibited high OUA affinity pump current, reduced the increase of [Ca²⁺](i) and the contractility induced by 1 μM but not that induced by 1 mM OUA. These results indicate that the H₁-H₂ domain of the NKA α₁ isoform mediates OUA-induced cardiac toxicity in rat ventricular myocytes, and inhibitors for this binding site may be used as an adjunct to CGs treatment for cardiovascular disease.
authors.affiliation: The Key Laboratory of Neural and Vascular Biology, Ministry of Education, Hebei Medical University, Shijiazhuang, PR China.
authors.forename: Chen
authors.initials: C
authors.lastname: Xiong
authors.forename: Jun-xia
authors.initials: JX
authors.lastname: Li
authors.forename: Hui-cai
authors.initials: HC
authors.lastname: Guo
authors.forename: Li-nan
authors.initials: LN
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Guo
authors.forename: Jing
authors.initials: J
authors.lastname: Meng
authors.forename: Yong-li
authors.initials: YL
authors.lastname: Wang
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002301:Cardiac Glycosides / Q000008:administration & dosage / Q000633:toxicity*; D002316:Cardiotonic Agents / Q000008:administration & dosage / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D006352:Heart Ventricles / Q000166:cytology / Q000187:drug effects* / Q000473:pathology; D007527:Isoenzymes; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000473:pathology; D010042:Ouabain / Q000008:administration & dosage / Q000633:toxicity*; D051381:Rats; D017207:Rats, Sprague-Dawley; D000254:Sodium-Potassium-Exchanging ATPase / Q000187:drug effects* / Q000378:metabolism
chemical_list: D000906:Antibodies; D002301:Cardiac Glycosides; D002316:Cardiotonic Agents; D007527:Isoenzymes; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium