Mitochondrial DNA Copy Research Articles

Nuclear Respiratory Factor 2 Induces the Expression of Many but Not All Human Proteins Acting in Mitochondrial DNA Transcription and Replication

In mammals, NRF-2 (nuclear respiratory factor 2), also named GA-binding protein, is an Ets family transcription factor that controls many genes involved in cell cycle progression and protein synthesis as well as in mitochondrial biogenesis. In this paper, we analyzed the role of NRF-2 in the regulation of human genes involved in mitochondrial DNA transcription and replication. By a combination of bioinformatic and biochemical approaches, we found that the factor binds in vitro and in vivo to the proximal promoter region of the genes coding for the transcription termination factor mTERF, the RNA polymerase POLRMT, the B subunit of the DNA polymerase-gamma, the DNA helicase TWINKLE, and the single-stranded DNA-binding protein mtSSB. The role of NRF-2 in modulating the expression of those genes was further established by RNA interference and overexpression strategies. On the contrary, we found that NRF-2 does not control the genes for the subunit A of DNA polymerase-gamma and for the transcription repressor MTERF3; we suggest that these genes are under regulatory mechanisms that do not involve NRF proteins. Since NRFs are known to positively control the expression of transcription-activating proteins, the novelty emerging from our data is that proteins playing antithetical roles in mitochondrial DNA transcription, namely activators and repressors, are under different regulatory pathways. Finally, we developed a more stringent consensus with respect to the general consensus of NRF-2/GA-binding protein when searching for NRF-2 binding sites in the promoter of mitochondrial proteins.

Early long-term L-T3 replacement rescues mitochondria and prevents ischemic cardiac remodelling in rats

3,5,3′-Levo-triiodothyronine (L-T3) is essential for DNA transcription, mitochondrial biogenesis and respiration, but its circulating levels rapidly decrease after myocardial infarction (MI). The main aim of our study was to test whether an early and sustained normalization of L-T3 serum levels after MI exerts myocardial protective effects through a mitochondrial preservation. Seventy-two hours after MI induced by anterior interventricular artery ligation, rats were infused with synthetic L-T3 (1.2 μg/kg/day) or saline over 4 weeks. Compared to saline, L-T3 infusion restored FT3 serum levels at euthyroid state (3.0 ± 0.2 versus 4.2 ± 0.3 pg/ml), improved left ventricular (LV) ejection fraction (39.5 ± 2.5 versus 65.5 ± 6.9%), preserved LV end-systolic wall thickening in the peri-infarct zone (6.34 ± 3.1 versus 33.7 ± 6.21%) and reduced LV infarct-scar size by approximately 50% (all P < 0.05). Moreover, L-T3 significantly increased angiogenesis and cell survival and enhanced the expression of nuclear-encoded transcription factors involved in these processes. Finally, L-T3 significantly increased the expression of factors involved in mitochondrial DNA transcription and biogenesis, such as hypoxic inducible factor-1α, mitochondrial transcription factor A and peroxisome proliferator activated receptor γ coactivator-1α, in the LV peri-infarct zone. To further explore mechanisms of L-T3 protective effects, we exposed isolated neonatal cardiomyocytes to H2O2 and found that L-T3 rescued mitochondrial biogenesis and function and protected against cell death via a mitoKATP dependent pathway. Early and sustained physiological restoration of circulating L-T3 levels after MI halves infarct scar size and prevents the progression towards heart failure. This beneficial effect is likely due to enhanced capillary formation and mitochondrial protection.

Nitric oxide synthase-2 regulates mitochondrial Hsp60 chaperone function during bacterial peritonitis in mice

Nitric oxide synthase-2 (NOS2) plays a critical role in reactive nitrogen species generation and cysteine modifications that influence mitochondrial function and signaling during inflammation. Here, we investigated the role of NOS2 in hepatic mitochondrial biogenesis during Escherichia coli peritonitis in mice. NOS2 −/− mice displayed smaller mitochondrial biogenesis responses than Wt mice during E. coli infection according to differences in mRNA levels for the PGC-1α coactivator, nuclear respiratory factor-1, mitochondrial transcription factor-A (Tfam), and mtDNA polymerase (Polγ). NOS2 −/− mice did not significantly increase mitochondrial Tfam and Polγ protein levels during infection in conjunction with impaired mitochondrial DNA (mtDNA) transcription, loss of mtDNA copy number, and lower State 3 respiration rates. NOS2 blockade in mitochondrial-GFP reporter mice disrupted Hsp60 localization to mitochondria after E. coli exposure. Mechanistically, biotin-switch and immunoprecipitation studies demonstrated NOS2 binding to and S-nitros(yl)ation of Hsp60 and Hsp70. Specifically, NOS2 promoted Tfam accumulation in mitochondria by regulation of Hsp60–Tfam binding via S-nitros(yl)ation. In hepatocytes, site-directed mutagenesis identified 237Cys as a critical residue for Hsp60 S-nitros(yl)ation. Thus, the role of NOS2 in inflammation-induced mitochondrial biogenesis involves both optimal gene expression for nuclear-encoded mtDNA-binding proteins and functional regulation of the Hsp60 chaperone that enables their importation for mtDNA transcription and replication.

Dynamic Regulation of Genes Involved in Mitochondrial DNA Replication and Transcription during Mouse Brown Fat Cell Differentiation and Recruitment

BackgroundBrown adipocytes are specialised in dissipating energy through adaptive thermogenesis, whereas white adipocytes are specialised in energy storage. These essentially opposite functions are possible for two reasons relating to mitochondria, namely expression of uncoupling protein 1 (UCP1) and a remarkably higher mitochondrial abundance in brown adipocytes.Methodology/Principal FindingsHere we report a comprehensive characterisation of gene expression linked to mitochondrial DNA replication, transcription and function during white and brown fat cell differentiation in vitro as well as in white and brown fat, brown adipose tissue fractions and in selected adipose tissues during cold exposure. We find a massive induction of the majority of such genes during brown adipocyte differentiation and recruitment, e.g. of the mitochondrial transcription factors A (Tfam) and B2 (Tfb2m), whereas only a subset of the same genes were induced during white adipose conversion. In addition, PR domain containing 16 (PRDM16) was found to be expressed at substantially higher levels in brown compared to white pre-adipocytes and adipocytes. We demonstrate that forced expression of Tfam but not Tfb2m in brown adipocyte precursor cells promotes mitochondrial DNA replication, and that silencing of PRDM16 expression during brown fat cell differentiation blunts mitochondrial biogenesis and expression of brown fat cell markers.Conclusions/SignificanceUsing both in vitro and in vivo model systems of white and brown fat cell differentiation, we report a detailed characterisation of gene expression linked to mitochondrial biogenesis and function. We find significant differences in differentiating white and brown adipocytes, which might explain the notable increase in mitochondrial content observed during brown adipose conversion. In addition, our data support a key role of PRDM16 in triggering brown adipocyte differentiation, including mitochondrial biogenesis and expression of UCP1.

Does mtDNA nucleoid organization impact aging?

Somatic cells in tissue culture package several copies of mitochondrial DNA (mtDNA) in aggregates known as nucleoids that appear to be remarkably stable. The clustering of multiple mtDNA genomes in a single nucleoid complex may promote the progressive age-related accumulation of deletion and point mutations in mtDNA in many somatic tissues, particularly in post-mitotic cells. In contrast, oocytes appear to have the ability to select against deleterious mutations in mtDNA, at least in mice. This fundamental difference suggests that oocytes may be better able to detect and remove defective mtDNA genomes than somatic cells, possibly due in part to the simpler organization of the mtDNA in smaller nucleoids. These observations suggest the hypothesis that a complex nucleoid structure containing several mtDNA molecules may impair the ability of the cell to select against deleterious mtDNA mutations, thereby contributing to age-related mitochondrial dysfunction.

β-Catenin is involved in alterations in mitochondrial activity in non-transformed intestinal epithelial and colon cancer cells

Background:Alteration in respiratory activity and mitochondrial DNA (mtDNA) transcription seems to be an important feature of cancer cells. Leukotriene D4 (LTD4) is a proinflammatory mediator implicated in the pathology of chronic inflammation and cancer. We have shown earlier that LTD4 causes translocation of β-catenin both to the mitochondria, in which it associates with the survival protein Bcl-2 identifying a novel role for β-catenin in cell survival, and to the nucleus in which it activates the TCF/LEF transcription machinery.Methods:Here we have used non-transformed intestinal epithelial Int 407 cells and Caco-2 colon cancer cells, transfected or not with wild type and mutated (S33Y) β-catenin to analyse its effect on mitochondria activity. We have measured the ATP/ADP ratio, and transcription of the mtDNA genes ND2, ND6 and 16 s in these cells stimulated or not with LTD4.Results:We have shown for the first time that LTD4 triggers a cellular increase in NADPH dehydrogenase activity and ATP/ADP ratio. In addition, LTD4 significantly increased the transcription of mtDNA genes. Overexpression of wild-type β-catenin or a constitutively active β-catenin mutant mimicked the effect of LTD4 on ATP/ADP ratio and mtDNA transcription. These elevations in mitochondrial activity resulted in increased reactive oxygen species levels and subsequent activations of the p65 subunit of NF-κB.Conclusions:The present novel data show that LTD4, presumably through β-catenin accumulation in the mitochondria, affects mitochondrial activity, lending further credence to the idea that inflammatory signalling pathways are intrinsically linked with potential oncogenic signals.

P53 Improves Aerobic Exercise Capacity and Augments Skeletal Muscle Mitochondrial DNA Content

Exercise capacity is a physiological characteristic associated with protection from both cardiovascular and all-cause mortality. p53 regulates mitochondrial function and its deletion markedly diminishes exercise capacity, but the underlying genetic mechanism orchestrating this is unclear. Understanding the biology of how p53 improves exercise capacity may provide useful insights for improving both cardiovascular as well as general health. The purpose of this study was to understand the genetic mechanism by which p53 regulates aerobic exercise capacity. Using a variety of physiological, metabolic, and molecular techniques, we further characterized maximum exercise capacity and the effects of training, measured various nonmitochondrial and mitochondrial determinants of exercise capacity, and examined putative regulators of mitochondrial biogenesis. As p53 did not affect baseline cardiac function or inotropic reserve, we focused on the involvement of skeletal muscle and now report a wider role for p53 in modulating skeletal muscle mitochondrial function. p53 interacts with Mitochondrial Transcription Factor A (TFAM), a nuclear-encoded gene important for mitochondrial DNA (mtDNA) transcription and maintenance, and regulates mtDNA content. The increased mtDNA in p53(+/+) compared to p53(-/-) mice was more marked in aerobic versus glycolytic skeletal muscle groups with no significant changes in cardiac tissue. These in vivo observations were further supported by in vitro studies showing overexpression of p53 in mouse myoblasts increases both TFAM and mtDNA levels whereas depletion of TFAM by shRNA decreases mtDNA content. Our current findings indicate that p53 promotes aerobic metabolism and exercise capacity by using different mitochondrial genes and mechanisms in a tissue-specific manner.

DNA conformation‐dependent activities of human mitochondrial RNA polymerase

Mitochondrial RNA polymerase (POLRMT) is a core protein for mitochondrial DNA (mtDNA) transcription. In addition, POLRMT is assumed to be involved in replication, although its exact role is not yet clearly elucidated. We have found novel properties of human POLRMT using a reconstituted transcription system. Various lengths of RNA molecules were synthesized from templates even without a defined promoter sequence, when we used supercoiled circular double-stranded DNA as a template. This promoter-independent activity was as strong as the promoter-dependent one. Promoter-independent DNA conformation-dependent transcription required TFB2M. On supercoiled templates, the promoter-independent activity was strongly suppressed by a putatively physiological amount of TFAM, while promoter-dependent transcription was inhibited to a lesser extent. These different inhibition patterns by TFAM may be important for prevention of random RNA synthesis in vivo. Promoter-independent activity was also observed on relaxed circular single-stranded DNA, where its activity no longer required TFB2M. RNA synthesis on single-stranded DNA was weakly suppressed by a putatively physiological amount of TFAM but restored by the addition of mitochondrial single-stranded DNA binding protein. We suggest that these properties of POLRMT could explain the characteristic features of mammalian mtDNA transcription and replication.

The difference in oxidative stress of the blood between using 5% glucose water and distilled water as the irrigant for BPH patients undergoing transurethral resection of the prostate

To evaluate the difference in oxidative stress in the blood between using 5% glucose water and using distilled water as the irrigant for BPH patients receiving transurethral resection of the prostate (TURP), we conducted this prospective study, on a total of 38 patients with symptomatic BPH. All the patients were randomly divided into 2 groups. Distilled water in group A and 5% glucose water in group B were used as the irrigant. The oxidative stress and antioxidant capacity were evaluated by measuring malondialdehyde (MDA), mitochondrial DNA (mtDNA) copy number and total capacity of antioxidants (TOA) in the blood. The data were correlated with serum creatinine and sodium, which were measured before and immediately after TURP in all patients. Serum creatinine increased from 0.86 +/- 0.17 to 1.02 +/- 0.20 mg/dL while serum sodium decreased from 139.7 +/- 2.3 to 136.4 +/- 4.0 mEq/L immediately after surgery in patients of group A (p < 0.05). MDA was increased and TOA decreased in all the patients immediately after TURP. Patients in group A had significantly higher MDA, lower mtDNA copy number, higher degree of oxidative mtDNA damage and lower TOA than those in group B immediately after TURP. In conclusion, we observed an increase of MDA and decrease of TOA in blood in all the patients immediately after TURP, which might induce renal damage and decrease serum sodium. Moreover, the oxidative stress levels in blood of patients in group A were significantly higher than those in group B immediately after TURP.

Mitochondrial gene expression in diabetes mellitus: effect of nutrition.

Diabetes mellitus is a collection of genetic diseases that share a common phenotype: glucose intolerance. The genetic origins of this disease are being widely investigated. An estimated 0.19% of the population with diabetes has the disorder owing to one or more mutations in the mitochondrial genome. Diet can affect the expression of the genome as well as the function of its gene products. The antioxidant nutrients serve to protect this very vulnerable genome from oxidative damage. These nutrients may affect mitochondrial DNA transcription and nutrients that affect membrane fluidity affect the function of the gene products.

Region-specific changes in mitochondrial D-loop in aged rat CNS

Impaired mitochondrial oxidative phosphorylation (OXPHOS) is considered a cause of aging. A reduction in mitochondrial DNA (mtDNA) replication and/or transcription may contribute to this OXPHOS diminution. Impairments in the displacement (D) loop, or non-coding, region of the mitochondrial genome, or accumulation of mtDNA mutations, may affect mtDNA replication and transcription. We determined the effects of age on the D-loop and on mtDNA deletion mutations in the spinal cord, medulla, midbrain, cerebellum, striatum, and cerebral cortex of Fischer 344 rats. D-loop, 7S DNA levels were reduced by 3-fold in striatum, 2.5-fold in cortex, and 2-fold in the spinal cord of older animals. We did not detect a population of mtDNA affected by the most prevalent known (ND4-containing) deletions, indicating they do not comprise a significant portion of total mtDNA. However, we detected an age-related and region-specific increase in the common deletion, which comprised 0.0003–0.0007% of total mtDNA. Mitochondrial genome copy number varied between regions, in addition to an overall 18% decrease with age across the whole brain. These results suggest the age-related decline in OXPHOS may be related to a reduction in D-loop function.

Genetic association analysis of 13 nuclear-encoded mitochondrial candidate genes with type II diabetes mellitus: the DAMAGE study

Mitochondria play an important role in many processes, like glucose metabolism, fatty acid oxidation and ATP synthesis. In this study, we aimed to identify association of common polymorphisms in nuclear-encoded genes involved in mitochondrial protein synthesis and biogenesis with type II diabetes mellitus (T2DM) using a two-stage design. In the first stage, we analyzed 62 tagging single nucleotide polymorphisms (SNPs) in the Hoorn study (n=999 participants) covering all common variation in 13 biological candidate genes. These 13 candidate genes were selected from four clusters regarded essential for correct mitochondrial protein synthesis and biogenesis: aminoacyl tRNA synthetases, translation initiation factors, tRNA modifying enzymes and mitochondrial DNA transcription and replication. SNPs showing evidence for association with T2DM were measured in second stage genotyping (n=10164 participants). After a meta-analysis, only one SNP in SIRT4 (rs2522138) remained significant (P=0.01). Extending the second stage with samples from the Danish Steno Study (n=1220 participants) resulted in a common odds ratio (OR) of 0.92 (0.85-1.00), P=0.06. Moreover, in a large meta-analysis of three genome-wide association studies, this SNP was also not associated with T2DM (P=0.72). In conclusion, we did not find evidence for association of common variants in 13 nuclear-encoded mitochondrial proteins with T2DM.

MTERF2 is a nucleoid component in mammalian mitochondria

The mammalian MTERF family of proteins has four members, named MTERF1 to MTERF4, which were identified in homology searches using the mitochondrial transcription termination factor, mTERF (here denoted MTERF1) as query. MTERF1 and MTERF3 are known to participate in the control of mitochondrial DNA transcription, but the function of the other two proteins is not known. We here investigate the structure and function of MTERF2. Protein import experiments using isolated organelles confirm that MTERF2 is a mitochondrial protein. Edman degradation of MTERF2 isolated from stably transfected HeLa cells demonstrates that mature MTERF2 lacks a targeting peptide (amino acids 1–35) present in the precursor form of the protein. MTERF2 is a monomer in isolation and displays a non sequence-specific DNA-binding activity. In vivo quantification experiments demonstrate that MTERF2 is relatively abundant, with one monomer present per ∼265 bp of mtDNA. In comparison, the mtDNA packaging factor TFAM is present at a ratio of one molecule per ∼10–12 bp of mtDNA. Using formaldehyde cross-linking we demonstrate that MTERF2 is present in nucleoids, and therefore must be located in close proximity to mtDNA. Taken together, our work provides a basic biochemical characterization of MTERF2, paving the way for future functional studies.

Organization and Structural Features of Phosphofructokinase and other Glycolytic Enzymes to Meet their Role in Energy Metabolism

Although glycolytic enzymes can be isolated as cytosolic components, some of them are known to bind to proteins from the cytoplasm and other cellular compartments, such as the mitochondria, the nucleus, or the plasma membrane. In some cases these protein-protein interactions are considered significant for the regulation of energy metabolism, as well as for the modulation of other cell functions. Among glycolytic enzymes, phosphofructokinase (PFK) is thought to play a fundamental role in the control of this pathway, because of the number of metabolic signals that can regulate its complex allosteric behavior. No X-ray structure of a characteristic eukaryotic PFK is available yet. However, sequence data indicated that the eukaryotic enzyme originated by duplication, fusion and divergence of an ancestral prokaryotic gene, such that the duplicated fructose 6-phosphate catalytic site in the C-terminal half became an allosteric site for the activator fructose 2,6-bisphosphate. It has been suggested that both sites are shared in the interface between subunits aligned in an antiparallel orientation. To test the contribution of each terminal domain to these two binding sites, chimeric mammalian PFKs involving exchange of their terminal domains have been found to exhibit affinity properties for fructose 6-phosphate and fructose 2,6-bisphosphate that resembled those of the native isozyme that donated the N-terminal half and the C-terminal half, respectively. Further mutational analyses of muscle PFK led us to investigate the composition of the fructose phosphate binding sites and to gain insight into the structural organization of eukaryotic PFK. Data will also be presented evidencing the relationship between mitochondrial function and the operation of glycolysis, as observed after impairment of mitochondrial DNA transcription.

Nuclear Control of Respiratory Chain Expression by Nuclear Respiratory Factors and PGC‐1‐Related Coactivator

Expression of the respiratory apparatus depends on both nuclear and mitochondrial genes. Although these genes are sequestered in distinct cellular organelles, their transcription relies on nucleus-encoded factors. Certain of these factors are directed to the mitochondria, where they sponsor the bi-directional transcription of mitochondrial DNA. Others act on nuclear genes that encode the majority of the respiratory subunits and many other gene products required for the assembly and function of the respiratory chain. The nuclear respiratory factors, NRF-1 and NRF-2, contribute to the expression of respiratory subunits and mitochondrial transcription factors and thus have been implicated in nucleo-mitochondrial interactions. In addition, coactivators of the PGC-1 family serve as mediators between the environment and the transcriptional machinery governing mitochondrial biogenesis. One family member, peroxisome proliferator-activated receptor gamma coactivator PGC-1-related coactivator (PRC), is an immediate early gene product that is rapidly induced by mitogenic signals in the absence of de novo protein synthesis. Like other PGC-1 family members, PRC binds NRF-1 and activates NRF-1 target genes. In addition, PRC complexes with NRF-2 and HCF-1 (host cell factor-1) in the activation of NRF-2-dependent promoters. HCF-1 functions in cell-cycle progression and has been identified as an NRF-2 coactivator. The association of these factors with PRC is suggestive of a role for the complex in cell growth. Finally, shRNA-mediated knock down of PRC expression results in a complex phenotype that includes the inhibition of respiratory growth on galactose and the loss of respiratory complexes. Thus, PRC may help integrate the expression of the respiratory apparatus with the cell proliferative program.

Association between trinucleotide CAG repeats of the DNA polymerase gene (POLG) with age of onset of Iranian Friedreich’s ataxia patients

Friedreich's ataxia (FRDA), an autosomal recessive neurodegenerative disorder, is in most cases due to a homozygous intronic expansion resulting in the loss of function of frataxin. As mitochondrial DNA (mtDNA) copy number has been decreased in FRDA cells and mtDNA polymerase (POLG) is involved in the replication of mtDNA, we searched a trinucleotide CAG repeat length of this enzyme. The POLG CAG repeat length was determined in DNA samples extracted from 20 FRDA patients and 49 control subjects. Our findings showed that the distribution of the POLG CAG repeat length in the patients' samples matched the distribution for control samples, but we found a statistically significant inverse correlation (r=-0.81) between the POLG CAG repeats and age of onset in FRDA patients. Our results suggest POLG CAG repeat instability would constitute a predisposing factor that, in combination with environmental risk factors, affect age of onset and disease progression.

Activation of Mitochondrial Biogenesis by Hormesis

Mitochondria play a major role in oxidative energy production, reduction–oxidation reaction (redox) control and calcium homeostasis. Although mitochondria contain DNA with mitochondrial-specific genes, most mitochondrial proteins are encoded by the nDNA, synthesized in the cytosol, and imported into mitochondria. The expression of nuclear genes that encode mitochondrial proteins that function in metabolic pathways such as the trichloroacetic acid cycle (TCA), oxidative phosphorylation, heme synthesis, and in mitochondrial DNA replication and transcription (eg, mitochondrial transcription factor A [Tfam]), is coordinately regulated by the transcriptional coactivators PPARγ coactivator (PGC)-1α and PGC-1β through activation of nuclear respiratory factor (NRF)-1 and NRF-2.1 In their recent publications, Piantadosi et al provided insight into the mechanisms underlying the interaction between mitochondria-derived reactive oxygen species (ROS) signaling and mitochondrial biogenesis. First, lipid hydroperoxide regulates Tfam expression through phosphorylation of NRF-1 via Akt activation, which promotes nuclear translocation of NRF-1 and binding to the Tfam promoter.2 Second, carbon monoxide (CO) induced mitochondrial biogenesis via activation of Akt1/PKB and guanylate cyclase, which augmented gene and protein expression of NRF-1 and NRF-2, PGC-1α, and TFAM.3 CO-induced mitochondrial ROS result in the activation of AKT. Third, the anthracycline anticancer agent doxorubicin suppresses the nuclear program for mitochondrial biogenesis, and its associated intrinsic antiapoptosis proteins, leading to severe mitochondrial DNA (mtDNA) depletion and apoptosis. CO inhalation or heme oxygenase (Hmo)1 overexpression prevented doxorubicin-induced mtDNA depletion and apoptosis via activation of AKT and guanylate cyclase.4 Lastly, new work in this issue of Circulation Research 5 sheds light on the role of NF-E2–related factor (Nrf)2 as a key transcriptional regulator in mitochondrial …

Abstract 1372: Sirt3 Promotes Survival of Cardiac Myocytes under oxidative stress and Upregulates Mitochondrial Genes Through Nuclear Respiratory Factor

Silent information regulator 1 (Sirt1), the prototype member of the sirtuin family, has been implicated in longevity and stress resistance. Sirt3, another member of the sirtuin family, is localized primarily in mitochondria and involved in metabolism in both white and brown adipose tissues. However, the cardiac role of Sirt3 is not well understood. Here, we examined the cellular function of Sirt3 in cardiac myocytes. Western blot analysis revealed that Sirt3 is highly expressed in cardiac myocytes, at the level comparable to Sirt3 in white and brown adipose tissues. Although Sirt1 in the heart was upregulated in response to glucose deprivation, high-fat diet, or pressure overload, expression of Sirt3 was unchanged in these conditions. Expression of Sirt3 was increased two-fold, however, after H 2 O 2 (100 μ M) treatment in primary cardiac myocytes and two-fold by aging (18 vs 3 months) in the heart. Sirt3 is localized primarily in the mitochondria in myocytes under normal conditions, but it translocates into the nucleus when cells are treated with H 2 O 2 . Adenovirus-mediated overexpression of Sirt3 significantly reduced myocyte cell death in serum free media or in the presence of H 2 O 2 as determined by cytoplasmic accumulation of mono- and oligonucleosomes (p&lt;0.05), suggesting that Sirt3 protects myocytes from apoptosis under oxidative stress. Furthermore, Sirt3 increased expression of NRF1, a transcriptional modulator of metabolic genes and mitochondrial DNA transcription, by 2.5-fold. There was a 3.4-fold increase in Glut4 expression as well as a 4-fold increase in COX IV. On the other hand, Sirt1 failed to upregulate NRF1. Sirt3 neither interacted with PGC-1 α nor stimulated PGC-1 α -mediated transcription. Thus, although both Sirt1 and Sirt3 exert their function by regulating metabolic genes, their mechanism of action is different. In summary, cardiac myocytes abundantly express Sirt3 in mitochondria. Sirt3 is upregulated by oxidative stress and translocates into the nucleus, where Sirt3 regulates expression of mitochondrial genes, such as Glut4 and COX IV, through upregulation of NRF1 and plays a protective role against oxidative stress in cardiac myocytes.

Abstract 1371: Overexpression Of Mitochondrial Transcription Factor A Protects Failing Myocardium From Oxidative Stress

Background: Reactive oxygen species (ROS) from mitochondria play a pivotal role in the pathogenesis and progression of heart failure. Mitochondrial transcription factor A (TFAM), a nucleus-encoded protein, in mitochondria promotes transcription of mitochondrial DNA (mtDNA), maintains mtDNA, and increases mtDNA copy number. We previously reported that overexpression of human TFAM ameliorated cardiac remodeling and improved survival by maintaining mitochondrial function. We investigated whether those beneficial effects result from the increase of antioxidative properties both in vivo and in vitro. Methods and Results: [in vivo study] We created myocardial infarction (MI) in wild type (WT) mice and human TFAM transgenic (TG) mice as a model of heart failure. We evaluated free radical generation by in vivo ESR on 28th day after the operation. MI size did not different between WT and MI. Overexpression of TFAM ameliorated MI-induced cardiac hypertrophy (histology) and LV dysfunction (2D echo and cath). Furthermore, the rate of signal decay in in vivo ESR, indicative of oxidative stress, was accelerated in WT-MI (0.10 ± 0.01 vs 0.19 ± 0.02/min, n=4–5, P&lt;0.01) whereas decelerated in TG-MI (0.12 ± 0.02/min; n=4, P&lt;0.01). DNA microarrays analysis of myocardium (8 weeks old, male) indicated that TG overexpressed (double or more) antioxidant relevant genes mostly encoded in nuclear DNA, such as Mthfd2 and Adh1 without affecting mitochondrial respiratory enzyme activities. [in vitro study] We measured superoxide in Hela cells using Dihydroethidium in the presence of rotenone, a complex I inhibitor. Overexpression of TFAM in Hela cells significantly reduced superoxide production (72.8 %). Conclusion: Overexpression of TFAM suppresses ROS. Such an antioxidative property of TFAM may contribute to its powerful anti-remodeling effect in failing heart.

Alterations in mitochondrial function and cytosolic calcium induced by hyperglycemia are restored by mitochondrial transcription factor A in cardiomyocytes

Mitochondrial transcription factor A (TFAM) is essential for mitochondrial DNA transcription and replication. TFAM transcriptional activity is decreased in diabetic cardiomyopathy; however, the functional implications are unknown. We hypothesized that a reduced TFAM activity may be responsible for some of the alterations caused by hyperglycemia. Therefore, we investigated the effect of TFAM overexpression on hyperglycemia-induced cytosolic calcium handling and mitochondrial abnormalities. Neonatal rat cardiomyocytes were exposed to high glucose (30 mM) for 48 h, and we examined whether TFAM overexpression, by protecting mitochondrial DNA, could reestablish calcium fluxes and mitochondrial alterations toward normal. Our results shown that TFAM overexpression increased to more than twofold mitochondria copy number in cells treated either with normal (5.5 mM) or high glucose. ATP content was reduced by 30% and mitochondrial calcium decreased by 40% after high glucose. TFAM overexpression returned these parameters to even higher than control values. Calcium transients were prolonged by 70% after high glucose, which was associated with diminished sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a and cytochrome-c oxidase subunit 1 expression. These parameters were returned to control values after TFAM overexpression. High glucose-induced protein oxidation was reduced by TFAM overexpression, indicating a reduction of the high glucose-induced oxidative stress. In addition, we found that TFAM activity can be modulated by O-linked beta-N-acetylglucosamine glycosylation. In conclusion, TFAM overexpression protected cell function against the damage induced by high glucose in cardiomyocytes.