The oxygen-rich postnatal environment induces cardiomyocyte cell-cycle arrest through DNA damage response.

The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary postnatal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen-rich postnatal environment is the upstream signal that results in cell-cycle arrest of cardiomyocytes. Here, we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, whereas hyperoxemia and ROS generators shorten it. These findings uncover a protective mechanism that mediates cardiomyocyte cell-cycle arrest in exchange for utilization of oxygen-dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be an important component of cardiomyocyte proliferation-based therapeutic approaches.

Catalase-dependent H2O2 consumption by cardiac mitochondria and redox-mediated loss in insulin signaling.

We have recently demonstrated that catalase content in mouse cardiac mitochondria is selectively elevated in response to high dietary fat, a nutritional state associated with oxidative stress and loss in insulin signaling. Catalase and various isoforms of glutathione peroxidase and peroxiredoxin each catalyze the consumption of H2O2 Catalase, located primarily within peroxisomes and to a lesser extent mitochondria, has a low binding affinity for H2O2 relative to glutathione peroxidase and peroxiredoxin. As such, the contribution of catalase to mitochondrial H2O2 consumption is not well understood. In the current study, using highly purified cardiac mitochondria challenged with micromolar concentrations of H2O2, we found that catalase contributes significantly to mitochondrial H2O2 consumption. In addition, catalase is solely responsible for removal of H2O2 in nonrespiring or structurally disrupted mitochondria. Finally, in mice fed a high-fat diet, mitochondrial-derived H2O2 is responsible for diminished insulin signaling in the heart as evidenced by reduced insulin-stimulated Akt phosphorylation. While elevated mitochondrial catalase content (∼50%) enhanced the capacity of mitochondria to consume H2O2 in response to high dietary fat, the selective increase in catalase did not prevent H2O2-induced loss in cardiac insulin signaling. Taken together, our results indicate that mitochondrial catalase likely functions to preclude the formation of high levels of H2O2 without perturbing redox-dependent signaling.

High dietary fat selectively increases catalase expression within cardiac mitochondria.

Obesity is a predictor of diabetes and cardiovascular disease. One consequence of obesity is dyslipidemia characterized by high blood triglycerides. It has been proposed that oxidative stress, driven by utilization of lipids for energy, contributes to these diseases. The effects of oxidative stress are mitigated by an endogenous antioxidant enzyme network, but little is known about its response to high fat utilization. Our experiments used a multiplexed quantitative proteomics method to measure antioxidant enzyme expression in heart tissue in a mouse model of diet-induced obesity. This experiment showed a rapid and specific up-regulation of catalase protein, with subsequent assays showing increases in activity and mRNA. Catalase, traditionally considered a peroxisomal protein, was found to be present in cardiac mitochondria and significantly increased in content and activity during high fat feeding. These data, coupled with the fact that fatty acid oxidation enhances mitochondrial H(2)O(2) production, suggest that a localized catalase increase is needed to consume excessive mitochondrial H(2)O(2) produced by increased fat metabolism. To determine whether the catalase-specific response is a common feature of physiological conditions that increase blood triglycerides and fatty acid oxidation, we measured changes in antioxidant expression in fasted versus fed mice. Indeed, a similar specific catalase increase was observed in mice fasted for 24 h. Our findings suggest a fundamental metabolic process in which catalase expression is regulated to prevent damage while preserving an H(2)O(2)-mediated sensing of diet composition that appropriately adjusts insulin sensitivity in the short term as needed to prioritize lipid metabolism for complete utilization.

Redox regulation of insulin sensitivity due to enhanced fatty acid utilization in the mitochondria.

Obesity enhances the risk for the development of type 2 diabetes and cardiovascular disease. Loss in insulin sensitivity and diminished ability of muscle to take up and use glucose are characteristics of type 2 diabetes. Paradoxically, regulatory mechanisms that promote utilization of fatty acids appear to initiate diet-induced insulin insensitivity. In this review, we discuss recent findings implicating increased mitochondrial production of the prooxidant H2O2 due to enhanced utilization of fatty acids, as a signal to diminish reliance on glucose and its metabolites for energy. In the short term, the ability to preferentially use fatty acids may be beneficial, promoting a metabolic shift that ensures use of available fat by skeletal muscle and heart while preventing intracellular glucose accumulation and toxicity. However, with prolonged consumption of high dietary fat and ensuing obesity, the near exclusive dependence on fatty acid oxidation for production of energy by the mitochondria drives insulin resistance, diabetes, and cardiovascular disease.

Role of transcript and interplay between transcription and replication in triplet-repeat instability in mammalian cells.

Triplet-repeat expansions cause several inherited human diseases. Expanded triplet-repeats are unstable in somatic cells, and tissue-specific somatic instability contributes to disease pathogenesis. In mammalian cells instability of triplet-repeats is dependent on the location of the origin of replication relative to the repeat tract, supporting the 'fork-shift' model of repeat instability. Disease-causing triplet-repeats are transcribed, but how this influences instability remains unclear. We examined instability of the expanded (GAA•TTC)(n) sequence in mammalian cells by analyzing individual replication events directed by the SV40 origin from five different locations, in the presence and absence of doxycycline-induced transcription. Depending on the location of the SV40 origin, either no instability was observed, instability was caused by replication with no further increase due to transcription, or instability required transcription. Whereas contractions accounted for most of the observed instability, one construct showed expansions upon induction of transcription. These expansions disappeared when transcript stability was reduced via removal or mutation of a spliceable intron. These results reveal a complex interrelationship of transcription and replication in the etiology of repeat instability. While both processes may not be sufficient for the initiation of instability, transcription and/or transcript stability seem to further modulate the fork-shift model of triplet-repeat instability.

E. coli mismatch repair acts downstream of replication fork stalling to stabilize the expanded (GAA.TTC)(n) sequence.

Expanded triplet repeat sequences are known to cause at least 16 inherited neuromuscular diseases. In addition to short-length changes, expanded triplet repeat tracts frequently undergo large changes, often amounting to hundreds of base-pairs. Such changes might occur when template or primer slipping creates insertion/deletion loops (IDLs), which are normally repaired by the mismatch repair system (MMR). However, in prokaryotes and eukaryotes, MMR promotes large changes in the length of (CTG.CAG)(n) sequences, the motif most commonly associated with human disease. We tested the effect of MMR on instability of the expanded (GAA.TTC)(n) sequence, which causes Friedreich ataxia, by comparing repeat instability in wild-type and MMR-deficient strains of Escherichia coli. As expected, the prevalence of small mutations increased in the MMR-deficient strains. However, the prevalence of large contractions increased in the MMR mutants specifically when GAA was the lagging strand template, the orientation in which replication fork stalling is known to occur. After hydroxyurea-induced stalling, both orientations of replication showed significantly more large contractions in MMR mutants than in the wild-type, suggesting that fork stalling may be responsible for the large contractions. Deficiency of MMR promoted large contractions independently of RecA status, a known determinant of (GAA.TTC)(n) instability. These data suggest that two independent mechanisms act in response to replication stalling to prevent instability of the (GAA.TTC)(n) sequence in E. coli, when GAA serves as the lagging strand template: one that is dependent on RecA-mediated restart of stalled forks, and another that is dependent on MMR-mediated repair of IDLs. While MMR destabilizes the (CTG.CAG)(n) sequence, it is involved in stabilization of the (GAA.TTC)(n) sequence. The role of MMR in triplet repeat instability therefore depends on the repeat sequence and the orientation of replication.

Deficiency of RecA-dependent RecFOR and RecBCD pathways causes increased instability of the (GAA*TTC)n sequence when GAA is the lagging strand template.

The most common mutation in Friedreich ataxia is an expanded (GAA*TTC)n sequence, which is highly unstable in human somatic cells and in the germline. The mechanisms responsible for this genetic instability are poorly understood. We previously showed that cloned (GAA*TTC)n sequences replicated in Escherichia coli are more unstable when GAA is the lagging strand template, suggesting erroneous lagging strand synthesis as the likely mechanism for the genetic instability. Here we show that the increase in genetic instability when GAA serves as the lagging strand template is seen in RecA-deficient but not RecA-proficient strains. We also found the same orientation-dependent increase in instability in a RecA+ temperature-sensitive E. coli SSB mutant strain (ssb-1). Since stalling of replication is known to occur within the (GAA*TTC)n sequence when GAA is the lagging strand template, we hypothesized that genetic stability of the (GAA*TTC)n sequence may require efficient RecA-dependent recombinational restart of stalled replication forks. Consistent with this hypothesis, we noted significantly increased instability when GAA was the lagging strand template in strains that were deficient in components of the RecFOR and RecBCD pathways. Our data implicate defective processing of stalled replication forks as a mechanism for genetic instability of the (GAA*TTC)n sequence.

NHR-14 loss of function couples intestinal iron uptake with innate immunity in C. elegans through PQM-1 signaling.

Iron is essential for survival of most organisms. All organisms have thus developed mechanisms to sense, acquire and sequester iron. In C. elegans, iron uptake and sequestration are regulated by HIF-1. We previously showed that hif-1 mutants are developmentally delayed when grown under iron limitation. Here we identify nhr-14, encoding a nuclear receptor, in a screen conducted for mutations that rescue the developmental delay of hif-1 mutants under iron limitation. nhr-14 loss upregulates the intestinal metal transporter SMF-3 to increase iron uptake in hif-1 mutants. nhr-14 mutants display increased expression of innate immune genes and DAF-16/FoxO-Class II genes, and enhanced resistance to Pseudomonas aeruginosa. These responses are dependent on the transcription factor PQM-1, which localizes to intestinal cell nuclei in nhr-14 mutants. Our data reveal how C. elegans utilizes nuclear receptors to regulate innate immunity and iron availability, and show iron sequestration as a component of the innate immune response.

A quantitative proteomic profile of the Nrf2-mediated antioxidant response of macrophages to oxidized LDL determined by multiplexed selected reaction monitoring.

The loading of macrophages with oxidized low density lipoprotein (LDL) is a key part of the initiation and progression of atherosclerosis. Oxidized LDL contains a wide ranging set of toxic species, yet the molecular events that allow macrophages to withstand loading with these toxic species are not completely characterized. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master regulator of the cellular stress response. However, the specific parts of the Nrf2-dependent stress response are diverse, with both tissue- and treatment-dependent components. The goal of these experiments was to develop and use a quantitative proteomic approach to characterize the Nrf2-dependent response in macrophages to oxidized LDL. Cultured mouse macrophages, the J774 macrophage-like cell line, were treated with a combination of oxidized LDL, the Nrf2-stabilizing reagent tert- butylhydroquinone (tBHQ), and/or Nrf2 siRNA. Protein expression was determined using a quantitative proteomics assay based on selected reaction monitoring. The assay was multiplexed to monitor a set of 28 antioxidant and stress response proteins, 6 housekeeping proteins, and 1 non-endogenous standard protein. The results have two components. The first component is the validation of the multiplexed, quantitative proteomics assay. The assay is shown to be fundamentally quantitative, precise, and accurate. The second component is the characterization of the Nrf2-mediated stress response. Treatment with tBHQ and/or Nrf2 siRNA gave statistically significant changes in the expression of a subset of 11 proteins. Treatment with oxidized LDL gave statistically significant increases in the expression of 7 of those 11 proteins plus one additional protein. All of the oxLDL-mediated increases were attenuated by Nrf2 siRNA. These results reveal a specific, multifaceted response of the foam cells to the incoming toxic oxidized LDL.

Epigenetic silencing in Friedreich ataxia is associated with depletion of CTCF (CCCTC-binding factor) and antisense transcription.

BACKGROUND: Over 15 inherited diseases are caused by expansion of triplet-repeats. Friedreich ataxia (FRDA) patients are homozygous for an expanded GAA triplet-repeat sequence in intron 1 of the FXN gene. The expanded GAA triplet-repeat results in deficiency of FXN gene transcription, which is reversed via administration of histone deacetylase inhibitors indicating that transcriptional silencing is at least partially due to an epigenetic abnormality. METHODOLOGY/PRINCIPAL FINDINGS: We found a severe depletion of the chromatin insulator protein CTCF (CCCTC-binding factor) in the 5'UTR of the FXN gene in FRDA, and coincident heterochromatin formation involving the +1 nucleosome via enrichment of H3K9me3 and recruitment of heterochromatin protein 1. We identified FAST-1 (FXNAntisense Transcript - 1), a novel antisense transcript that overlaps the CTCF binding site in the 5'UTR, which was expressed at higher levels in FRDA. The reciprocal relationship of deficient FXN transcript and higher levels of FAST-1 seen in FRDA was reproduced in normal cells via knockdown of CTCF. CONCLUSIONS/SIGNIFICANCE: CTCF depletion constitutes an epigenetic switch that results in increased antisense transcription, heterochromatin formation and transcriptional deficiency in FRDA. These findings provide a mechanistic basis for the transcriptional silencing of the FXN gene in FRDA, and broaden our understanding of disease pathogenesis in triplet-repeat diseases.