Peroxynitrite-induced α-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 Ca2+-activated isometric force (pCa=4.75) decreased to zero with increasing concentrations of peroxynitrite in a concentration-dependent manner (IC50: 55 +/- 4 mu M; based on a total of 75 myocytes). However, there were no differences before and after the application of 50 mu M peroxynitrite in the Ca2+-sensitivity of force production (pCa(50): 5.89 +/- 0.02 and 5.86 +/- 0.04), in the steepness of the Ca2+-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 mu M 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/m(2); n=57 cells), reflecting ultrastructural alterations. Western immunoblots revealed that 50 mu M 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 myocardiurn; its nitration induces a contractile dysfunction, presumably by decreasing the longitudinal transmission of force between adjacent sarcomeres. (c) 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.