Dongchon Kang

Mitochondrial DNA Damage and Dysfunction Associated With Oxidative Stress in Failing Hearts After Myocardial Infarction

Mitochondria are one of the enzymatic sources of reactive oxygen species (ROS) and could also be a major target for ROS-mediated damage. We hypothesized that ROS may induce mitochondrial DNA (mtDNA) damage, which leads to defects of mtDNA-encoded gene expression and respiratory chain complex enzymes and thus may contribute to the progression of left ventricular (LV) remodeling and failure after myocardial infarction (MI). In a murine model of MI and remodeling created by the left anterior descending coronary artery ligation for 4 weeks, the LV was dilated and contractility was diminished. Hydroxyl radicals, which originated from the superoxide anion, and lipid peroxide formation in the mitochondria were both increased in the noninfarcted LV from MI mice. The mtDNA copy number relative to the nuclear gene (18S rRNA) preferentially decreased by 44% in MI by a Southern blot analysis, associated with a parallel decrease (30% to 50% of sham) in the mtDNA-encoded gene transcripts, including the subunits of complex I (ND1, 2, 3, 4, 4L, and 5), complex III (cytochrome b), complex IV (cytochrome c oxidase), and rRNA (12S and 16S). Consistent with these molecular changes, the enzymatic activity of complexes I, III, and IV decreased in MI, whereas, in contrast, complex II and citrate synthase, encoded only by nuclear DNA, both remained at normal levels. An intimate link among ROS, mtDNA damage, and defects in the electron transport function, which may lead to an additional generation of ROS, might play an important role in the development and progression of LV remodeling and failure.

Architectural role of mitochondrial transcription factor A in maintenance of human mitochondrial DNA.

ABSTRACT Mitochondrial transcription factor A (TFAM), a transcription factor for mitochondrial DNA (mtDNA) that also possesses the property of nonspecific DNA binding, is essential for maintenance of mtDNA. To clarify the role of TFAM, we repressed the expression of endogenous TFAM in HeLa cells by RNA interference. The amount of TFAM decreased maximally to about 15% of the normal level at day 3 after RNA interference and then recovered gradually. The amount of mtDNA changed closely in parallel with the daily change in TFAM while in organello transcription of mtDNA at day 3 was maintained at about 50% of the normal level. TFAM lacking its C-terminal 25 amino acids (TFAM-ΔC) marginally activated transcription in vitro. When TFAM-ΔC was expressed at levels comparable to those of endogenous TFAM in HeLa cells, mtDNA increased twofold, suggesting that TFAM-ΔC is as competent in maintaining mtDNA as endogenous TFAM under these conditions. The in organello transcription of TFAM-ΔC-expressing cells was no more than that in the control. Thus, the mtDNA amount is finely correlated with the amount of TFAM but not with the transcription level. We discuss an architectural role for TFAM in the maintenance of mtDNA in addition to its role in transcription activation.

Overexpression of Mitochondrial Transcription Factor A Ameliorates Mitochondrial Deficiencies and Cardiac Failure After Myocardial Infarction

Background—Mitochondrial DNA (mtDNA) copy number is decreased not only in mtDNA-mutation diseases but also in a wide variety of acquired degenerative and ischemic diseases. Mitochondrial transcription factor A (TFAM) is essential for mtDNA transcription and replication. Myocardial mtDNA copy number and TFAM expression both decreased in cardiac failure. However, the functional significance of TFAM has not been established in this disease state. Methods and Results—We have now addressed this question by creating transgenic (Tg) mice that overexpress human TFAM gene and examined whether TFAM could protect the heart from mtDNA deficiencies and attenuate left ventricular (LV) remodeling and failure after myocardial infarction (MI) created by ligating the left coronary artery. TFAM overexpression could ameliorate the decrease in mtDNA copy number and mitochondrial complex enzyme activities in post-MI hearts. Survival rate during 4 weeks of MI was significantly higher in Tg-MI than in wild-type (WT) littermates (WT-MI), although infarct size was comparable. LV cavity dilatation and dysfunction were significantly attenuated in Tg-MI. LV end-diastolic pressure was increased in WT-MI, and it was also reduced in Tg-MI. Improvement of LV function in Tg-MI was accompanied by a decrease in myocyte hypertrophy, apoptosis, and interstitial fibrosis as well as oxidative stress in the noninfarcted LV. Conclusions—Overexpression of TFAM inhibited LV remodeling after MI. TFAM may provide a novel therapeutic strategy of cardiac failure.

Regulation of mitochondrial D‐loops by transcription factor A and single‐stranded DNA‐binding protein

During replication, mitochondrial DNA (mtDNA) takes on a triple‐stranded structure called a D‐loop. Although their physiological roles are not understood, D‐loops are implicated in replication and transcription of mtDNA. Little is known about the turnover of D‐loops. We investigated the effects of mitochondrial transcription factor A (TFAM) and single‐stranded DNA‐binding protein (mtSSB) on D‐loops. In human HeLa cells, TFAM and mtSSB are, respectively, 1700‐ and 3000‐fold more abundant than mtDNA. This level of TFAM is two orders of magnitude higher than reported previously and is sufficient to wrap human mtDNA entirely. TFAM resolves D‐loops in vitro if added in similar stoichiometries. mtSSB inhibits the resolution of mtDNA by TFAM but enhances resolution by RecG, a junction‐specific helicase from Escherichia coli. Hence, mtSSB functions in both stabilization and resolution. We propose that TFAM and mtSSB are cooperatively involved in stabilizing D‐loops and in the maintenance of mtDNA.

Mitophagy Plays an Essential Role in Reducing Mitochondrial Production of Reactive Oxygen Species and Mutation of Mitochondrial DNA by Maintaining Mitochondrial Quantity and Quality in Yeast

Background: The physiological importance of mitophagy in yeast has been largely unexplored. Results: Mitochondrial DNA deletion frequently occurs in mitophagy-deficient cells during nitrogen starvation because of overproduction of the reactive oxygen species from unregulated mitochondria. Conclusion: Mitophagy prevents excess reactive oxygen species production and mitochondrial DNA mutation. Significance: Our findings provide insight into mitophagy-related disorders such as Parkinson disease. In mammalian cells, the autophagy-dependent degradation of mitochondria (mitophagy) is thought to maintain mitochondrial quality by eliminating damaged mitochondria. However, the physiological importance of mitophagy has not been clarified in yeast. Here, we investigated the physiological role of mitophagy in yeast using mitophagy-deficient atg32- or atg11-knock-out cells. When wild-type yeast cells in respiratory growth encounter nitrogen starvation, mitophagy is initiated, excess mitochondria are degraded, and reactive oxygen species (ROS) production from mitochondria is suppressed; as a result, the mitochondria escape oxidative damage. On the other hand, in nitrogen-starved mitophagy-deficient yeast, excess mitochondria are not degraded and the undegraded mitochondria spontaneously age and produce surplus ROS. The surplus ROS damage the mitochondria themselves and the damaged mitochondria produce more ROS in a vicious circle, ultimately leading to mitochondrial DNA deletion and the so-called “petite-mutant” phenotype. Cells strictly regulate mitochondrial quantity and quality because mitochondria produce both necessary energy and harmful ROS. Mitophagy contributes to this process by eliminating the mitochondria to a basal level to fulfill cellular energy requirements and preventing excess ROS production.

Increased 8-oxo-dGTPase in the mitochondria of substantia nigral neurons in Parkinson's disease

There is growing evidence for the involvement of oxidative stress and mitochondrial respiratory failure in nigral neuronal death in Parkinsons disease (PD). We report increased immunoreactivity of 8‐oxo‐dGTPase (8‐oxo‐7, 8‐dihydrodeoxyguanosine triphosphatase [hMTH1]), an enzyme known to play an important role in controlling spontaneous mutagenesis, and 8‐oxo‐7, 8‐deoxyguanosine (8‐oxo‐dG) in the mitochondria of the substantia nigra of 6 PD patients. Our results suggest that hMTH1 might be a useful marker of oxidative stress and can be used to explore the relation between such oxidative stress and genomic instability.

Phosphorylation of Serine 114 on Atg32 mediates mitophagy

Mitophagy, which selectively degrades mitochondria via autophagy, has a significant role in mitochondrial quality control. When autophagy selects mitochondria as a cargo, Atg32 is bound by Atg11. It is shown that the phosphorylation of Atg32, especially phosphorylation of Ser-114 on Atg32, mediates the Atg11–Atg32 interaction and mitophagy.

Reverse of Age-Dependent Memory Impairment and Mitochondrial DNA Damage in Microglia by an Overexpression of Human Mitochondrial Transcription Factor A in Mice

Mitochondrial DNA (mtDNA) is highly susceptible to injury induced by reactive oxygen species (ROS). During aging, mutations of mtDNA accumulate to induce dysfunction of the respiratory chain, resulting in the enhanced ROS production. Therefore, age-dependent memory impairment may result from oxidative stress derived from the respiratory chain. Mitochondrial transcription factor A (TFAM) is now known to have roles not only in the replication of mtDNA but also its maintenance. We herein report that an overexpression of TFAM in HeLa cells significantly inhibited rotenone-induced mitochondrial ROS generation and the subsequent NF-κB (nuclear factor-κB) nuclear translocation. Furthermore, TFAM transgenic (TG) mice exhibited a prominent amelioration of an age-dependent accumulation of lipid peroxidation products and a decline in the activities of complexes I and IV in the brain. In the aged TG mice, deficits of the motor learning memory, the working memory, and the hippocampal long-term potentiation (LTP) were also significantly improved. The expression level of interleukin-1β (IL-1β) and mtDNA damages, which were predominantly found in microglia, significantly decreased in the aged TG mice. The IL-1β amount markedly increased in the brain of the TG mice after treatment with lipopolysaccharide (LPS), whereas its mean amount was significantly lower than that of the LPS-treated aged wild-type mice. At the same time, an increased mtDNA damage in microglia and an impaired hippocampal LTP were also observed in the LPS-treated aged TG mice. Together, an overexpression of TFAM is therefore considered to ameliorate age-dependent impairment of the brain functions through the prevention of oxidative stress and mitochondrial dysfunctions in microglia.

Alterations of mitochondrial DNA in common diseases and disease states: aging, neurodegeneration, heart failure, diabetes, and cancer.

It has long been considered that mitochondrial DNA disease is a rare genetic disorder causing neuromyopathy. However, alterations of mitochondrial DNA recently have been recognized to play an important role in the pathogenesis of so-called common diseases such as heart failure, diabetes, and cancer. Although some of these alterations are inherited, more and more attention is being focused on the accumulation of mitochondrial DNA mutations in somatic cells, particularly terminally differentiated cells such as cardiomyocytes and neurons that occurs with age. Mitochondrial DNA is more vulnerable to alteration than nuclear DNA, mainly for two reasons. First, mitochondria are a major source of intracellular reactive oxygen species (ROS). Therefore mitochondrial DNA is under much stronger oxidative stress than is nuclear DNA. Second, mitochondria have a matrix-side negative membrane potential for oxidative phosphorylation. This membrane potential concentrates lipophilic cations inside mitochondria up to approximately 1,000-fold. Unfortunately, some therapeutic reagents are lipophilic cations, and such exogenously added chemicals are prone to damage mitochondria. AZT, an anti-HIV drug, causes mitochondrial myopathy as a side effect, which is a typical example of how chemotherapeutics adversely affect metabolism of mitochondrial DNA. In this review, we focus on ROS and chemical damage of mitochondrial DNA in common diseases.

Mitochondrial Oxidative Stress and Mitochondrial DNA

Abstract Mitochondria produce reactive oxygen species (ROS) under physiological conditions in association with activity of the respiratory chain in aerobic ATP production. The production of ROS is essentially a function of O2 consumption. Hence, increased mitochondrial activity per se can be an oxidative stress to cells. Furthermore, production of ROS is markedly enhanced in many pathological conditions in which the respiratory chain is impaired. Because mitochondrial DNA, which is essential for execution of normal oxidative phosphorylation, is located in proximity to the ROS-generating respiratory chain, it is more oxidatively damaged than is nuclear DNA. Cumulative damage of mitochondrial DNA is implicated in the aging process and in the progression of such common diseases as diabetes, cancer, and heart failure. Clin Chem Lab Med 2003; 41(10):12811288