Peroxynitrite-induced alpha-actinin nitration and contractile alterations in isolated human myocardial cells.
OBJECTIVE
Peroxynitrite-mediated myocardial protein nitration has been associated with a depressed cardiac pump function. In the present study, an attempt was made to elucidate the molecular background of peroxynitrite-evoked alterations in the human myocardium.


METHODS
Isometric force generation was measured in permeabilized human ventricular myocytes and biochemical methods were employed to identify the proteins affected by peroxynitrite-induced nitrotyrosine formation.


RESULTS
The maximal Ca(2+)-activated isometric force (pCa=4.75) decreased to zero with increasing concentrations of peroxynitrite in a concentration-dependent manner (IC50: 55+/-4 microM; based on a total of 75 myocytes). However, there were no differences before and after the application of 50 microM peroxynitrite in the Ca(2+)-sensitivity of force production (pCa50: 5.89+/-0.02 and 5.86+/-0.04), in the steepness of the Ca(2+)-force relationship (nHill: 2.22+/-0.11 and 2.42+/-0.25), and in the actin-myosin turnover kinetics (k(tr) at saturating [Ca2+]: 1.14+/-0.03 1/s and 1.05+/-0.07 1/s) (P>0.05). Nevertheless, 50 muM peroxynitrite greatly deteriorated the cross-striation pattern and induced a slight, but significant, increase in the passive force component (from 2.1+/-0.1 to 2.5+/-0.2 kN/m2; n=57 cells), reflecting ultrastructural alterations. Western immunoblots revealed that 50 microM peroxynitrite selectively induced the nitration of a protein with an apparent molecular mass of about 100 kDa. Subsequent immunoprecipitation assays identified this nitrated protein as alpha-actinin, a major Z-line protein.


CONCLUSIONS
These results suggest alpha-actinin as a novel target for peroxynitrite in the human myocardium; its nitration induces a contractile dysfunction, presumably by decreasing the longitudinal transmission of force between adjacent sarcomeres.
Borbély
Attila
A
Division of Clinical Physiology, Institute of Cardiology, UD MHSC, P.O. Box 1, H-4004 Debrecen, Hungary.
Tóth
Attila
A
Edes
István
I
Virág
László
L
Papp
Julius Gy
JG
Varró
András
A
Paulus
Walter J
WJ
van der Velden
Jolanda
J
Stienen
Ger J M
GJ
Papp
Zoltán
Z
D000185:Actinin / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003864:Depression, Chemical; D004305:Dose-Response Relationship, Drug; D006352:Heart Ventricles; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D009200:Myocardial Contraction / Q000187:drug effects*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000378:metabolism / Q000494:pharmacology*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D000185:Actinin; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; D002118:Calcium