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

Abstract

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 (IC 50 : 55T 4 AM; based on a total of 75 myocytes). However, there were no differences before and after the application of 50 AM peroxynitrite in the Ca 2+ -sensitivity of force production (pCa 50 : 5.89 T 0.02 and 5.86 T 0.04), in the steepness of the Ca 2+ -force relationship (nHill: 2.22 T 0.11 and 2.42 T 0.25), and in the actin -myosin turnover kinetics (k tr at saturating [Ca 2+ ]: 1.14 T 0.03 1/s and 1.05 T 0.07 1/s) ( P > 0.05). Nevertheless, 50 AM peroxynitrite greatly deteriorated the cross-striation pattern and induced a slight, but significant, increase in the passive force component (from 2.1 T 0.1 to 2.5 T 0.2 kN/m 2 ; n = 57 cells), reflecting ultrastructural alterations. Western immunoblots revealed that 50 AM 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 a-actinin, a major Z-line protein.Conclusions: These results suggest a-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.

Cellular senescence in honey bee brain is largely independent of chronological age

“…2, lane 7) demonstrates that this unidentified protein can be selectively nitrated. Similar selective nitration has been reported previously for p130(cas) (130 kDa) from human neuroblastoma SH-SY5Y cells (Saeki and Maeda, 1999), alpha-tubulin (50-55 kDa) from human gliomas and rat brain (Dremina et al, 2005;Fiore et al, 2006), alpha-actinin (100 kDa) from human myocardium (Borbely et al, 2005), and prostaglandin endoperoxide synthase-2 (72 kDa) from RAW 264.7 macrophages (Schildknecht et al, 2006). Yet, although our data indicate that selective nitration by peroxynitrite targets one protein product also in honey bee brain, the cumulative data from the immunohistochemical (Fig.…”

Protein Kinase C Contributes to the Maintenance of Contractile Force in Human Ventricular Cardiomyocytes

“…Myocyte Preparation and Measurement of the Mechanical Properties-Left ventricular tissue samples (about 0.2 g of wet weight) were treated and isolated as described earlier (26). In short, the tissue was mechanically disrupted in isolating solution (containing 4 mmol/liter Na 2 ATP, 1 mmol/liter Mg 2ϩ , 145 mmol/liter KCl, 2 mmol/liter EGTA, 10 mmol/liter imidazole, pH 7.0), and the particulate fraction was separated from the cytosol by centrifugation (1,000 ϫ g, 5 min; supernatant is referred as cytosol).…”

“…One needle was attached to a force transducer element (SensoNor, Horten, Norway), and the other was attached to an electromagnetic motor (Aurora Scientific Inc., Aurora, Canada). The measurements were performed at 15°C, and the average sarcomere length was adjusted to 2.2 m as described previously (26). A mounted cardiomyocyte is shown on Fig.…”

Activation of the Poly(ADP-Ribose) Polymerase Pathway in Human Heart Failure

“…proteins (such as alfa-actinin) could be nitrated by peroxynitrite treatment, in vitro, with a consequent decrease in the contractile force (10). Via the same approach, failing and donor hearts were tested for nitrated proteins: no difference was found (Figure 2).…”