Intracellular sources of peroxynitrite formation and potential targets for this powerful oxidant and nitrating agent have not been identified after acetaminophen (AAP) overdose. Therefore, we tested the hypothesis that peroxynitrite generated in mitochondria may be responsible for mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage. C3Heb/FeJ mice were treated with 300 mg/kg AAP and monitored for up to 12 h. Loss of mtDNA (assayed by slot blot hybridization) and substantial nDNA fragmentation (evaluated by anti-histone enzyme-linked immunosorbent assay, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and agarose gel electrophoresis) were observed as early as 3 h after AAP overdose. Analysis of nitrotyrosine protein adducts in subcellular fractions established that peroxynitrite was generated predominantly in mitochondria beginning at 1 h after AAP injection. Delayed treatment with a bolus dose of glutathione (GSH) accelerated the recovery of mitochondrial glutathione, which then effectively scavenged peroxynitrite. However, mtDNA loss was only partially prevented. Despite the absence of nitrotyrosine adducts in the nucleus after AAP overdose, nDNA damage was almost completely eliminated with GSH administration. A direct comparison of nDNA damage after AAP overdose with nDNA fragmentation during tumor necrosis factor receptor-mediated apoptosis showed similar DNA ladders on agarose gels but quantitatively different results in three other assays. We conclude that peroxynitrite may be partially responsible for mtDNA loss but is not directly involved in nDNA damage. In contrast, nDNA fragmentation after AAP overdose is not caused by caspase-activated DNase but most likely by other intracellular DNase(s), whose activation is dependent on the mitochondrial oxidant stress and peroxynitrite formation.

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Mitochondrial dysfunction and internucleosomal DNA fragmentation are well-recognized features of acetaminophen (AAP)-induced hepatocyte cell death. However, the endonucleases responsible for this effect have not been identified. Apoptosis-inducing factor (AIF) and endonuclease G are nucleases located in the intermembrane space of mitochondria. AIF is thought to trigger chromatin condensation and induce cleavage of DNA into high molecular weight fragments (50-300 kb), and endonuclease G can produce oligonucleosomal DNA fragments. Therefore, the objective of this investigation was to test the hypothesis that endonuclease G and AIF could be involved in AAP-induced nuclear DNA fragmentation. Using immunofluorescence microscopy, it was shown that in primary cultured mouse hepatocytes, endonuclease G and AIF translocated to the nucleus between 3 and 6 h after exposure to 5 mM AAP. In contrast, other mitochondrial intermembrane proteins such as cytochrome c or the second mitochondria-derived activator of caspases (Smac) did not accumulate in the nucleus. The translocation of AIF and endonuclease G correlated with mitochondrial dysfunction as indicated by the progressive loss of the mitochondrial membrane potential (measured with the JC-1 assay) and the appearance of nuclear DNA fragments in the cytosol (determined by an anti-histone ELISA). Pretreatment with 20mM N-acetylcysteine prevented mitochondrial dysfunction, the nuclear translocation of endonuclease G and AIF, and the nuclear DNA fragmentation. The data support the conclusion that endonuclease G and AIF translocate to the nucleus in response to AAP-induced mitochondrial dysfunction and may be responsible, at least in part, for the initial DNA fragmentation during AAP hepatotoxicity.

Leiomodin 2 (Lmod2) is an actin-binding protein that has been implicated in the regulation of striated muscle thin filament assembly; its physiological function has yet to be studied. We found that knockout of Lmod2 in mice results in abnormally short thin filaments in the heart. We also discovered that Lmod2 functions to elongate thin filaments by promoting actin assembly and dynamics at thin filament pointed ends. Lmod2-KO mice die as juveniles with hearts displaying contractile dysfunction and ventricular chamber enlargement consistent with dilated cardiomyopathy. Lmod2-null cardiomyocytes produce less contractile force than wild type when plated on micropillar arrays. Introduction of GFP-Lmod2 via adeno-associated viral transduction elongates thin filaments and rescues structural and functional defects observed in Lmod2-KO mice, extending their lifespan to adulthood. Thus, to our knowledge, Lmod2 is the first identified mammalian protein that functions to elongate actin filaments in the heart; it is essential for cardiac thin filaments to reach a mature length and is required for efficient contractile force and proper heart function during development.actin-thin filaments | cardiomyopathy | cytoskeletal dynamics S triated muscle cells contain arrays of protein filaments assembled into contractile units that are nearly crystalline in structure. Efficient contraction at the molecular level is predicated upon accurate overlap of actin-containing thin and myosin-containing thick filaments. Therefore, proper control of filament assembly is absolutely critical.In striated muscle it is currently thought that the thin-filament pointed end capping protein tropomodulin (Tmod) is the predominant regulator of thin filament length, with Tmod1 being the sole isoform expressed in cardiomyocytes (1). Extensive in vitro work has revealed that Tmod1 uses two actin-and two tropomyosin-binding sites to associate with the end of the thin filament and to prevent addition or loss of actin monomers, thereby controlling length of the thin filament (2-7). Tmod1 is essential for life; Tmod1-KO mice are embryonic lethal because of cardiac defects (8-11).Identification of additional but structurally different members of the Tmod family of proteins, the leiomodins (Lmods), raises the possibility that thin filament lengths are not regulated solely by Tmod at thin filament pointed ends (12). Although there are three Lmod genes (Lmod1-3), Lmod2 and 3 are expressed in striated muscle with Lmod2 being the predominant isoform in cardiac muscle and Lmod3 the predominant isoform in skeletal muscle (12-16). The Lmods share ∼40% sequence identity at the protein level with the Tmods but do not contain a recognizable second tropomyosin-binding domain and have an additional C-terminal extension that includes a proline-rich region and an actin-binding Wiskott-Aldrich syndrome protein homology 2 (WH2) domain (12, 17). Lmod2 has been proposed to be the long-sought muscle actin filament nucleator because it robustly nucleates actin filament formation in ...

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