The purpose of this study was to determine if decreased heart function after hypoxia followed by reoxygenation (H-R) is associated with increased degradation of cardiac myosin light chain 1 (MLC1) by matrix metalloproteinase-2 (MMP-2), and to investigate the effects of the increased level of peroxynitrite in the hearts of H-R animals on MLC1 degradation by MMP-2. Total of 12 newborn piglets were acutely instrumented to monitor cardiac function as assessed by stroke volume. Anesthetized piglets were block randomized to the normoxic group (n = 6), which received ventilation with room air for 6 h, or to the H-R group (n = 6), which received ventilation with 10-15% oxygen for 2 h, followed by reoxygenation with 100% oxygen for 1 h and then with 21% oxygen for 3 h. Hearts were removed and snap frozen for subsequent biochemical analyses. At the end of the 2-h hypoxia period, cardiac output, mean arterial pressure and stroke volume were significantly decreased in the H-R group. After 1 h of 100% oxygen, these parameters had increased slightly, but remained significantly lower than the normoxic controls throughout the reoxygenation period. Compared to normoxic animals, cardiac MLC1 levels were decreased and MMP-2 activity was increased in H-R animals. MMP-2 was co-localized with MLC1, and the amount of MLC1 associated with MMP-2 was higher in the hearts of H-R animals. In normoxic animals, cardiac MLC1 level was negatively, and cardiac MMP-2 activity was positively, strongly correlated with stroke volume index. This relationship was not seen in the H-R group. However, in both the normoxic group and the H-R group, the activity of cardiac MMP-2 was negatively correlated with the level of cardiac MLC1. There was a more than twofold increase in the level of nitrates, a marker for peroxynitrite formation, in the hearts of H-R animals. Mass spectrometric analyses detected peroxynitrite-induced nitration and S-nitrosylation of MLC1 protein in the hearts of H-R animals. These peroxynitrite-induced modifications of MLC1 were localized directly adjacent to the site at which MMP-2 cleaves MLC1. Peroxynitrite, formed during cardiac reoxygenation following a period of hypoxia, modifies the structure of cardiac MLC1 by nitrating and nitrosylating amino acids adjacent to the site where MMP-2 cleaves MLC1. This facilitates the degradation of MLC1 by MMP-2 and may contribute to cardiac dysfunction induced by H-R and other forms of oxidative stress. The high correlation between MMP-2 activity and MLC1 level in control animals suggests that MMP-2 may play an important role in regulating MLC1 turnover under normal physiological conditions. Determining the optimal parameters for controlled reoxygenation after hypoxia, together with pharmacological treatment with MMP-2 inhibitors and/or inhibitors of nitration/nitrosylation of MLC1, could reduce heart injury during the resuscitation of asphyxiated newborns and improve their long-term prognosis by reducing MLC1 degradation. Since the degradation of MLC1 by MMP-2 appears to be a common feature of oxidative stress, these pharmacological interventions may be useful in reducing tissue damage in other oxidative stress-related disorders as well.