Peroxynitrite-induced alpha-actinin nitration and contractile alterations in isolated human myocardial cells.

Cardiovascular Research

PubMedID: 16005301

Borbély A, Tóth A, Edes I, Virág L, Papp JG, Varró A, Paulus WJ, Van der Velden J, Stienen GJ, Papp Z. Peroxynitrite-induced alpha-actinin nitration and contractile alterations in isolated human myocardial cells. Cardiovasc Res. 2005;67(2):225-33.
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.