Mitochondrial DNA integrity and function are critical for endothelium-dependent vasodilation in rats with metabolic syndrome.

Endothelial dysfunction in diabetes is generally attributed to oxidative stress, but this view is challenged by observations showing antioxidants do not eliminate diabetic vasculopathy. As an alternative to oxidative stress-induced dysfunction, we interrogated if impaired mitochondrial function in endothelial cells is central to endothelial dysfunction in the metabolic syndrome. We observed reduced coronary arteriolar vasodilation to the endothelium-dependent dilator, acetylcholine (Ach), in Zucker Obese Fatty rats (ZOF, 34 ± 15% [mean ± standard deviation] 10-3 M) compared to Zucker Lean rats (ZLN, 98 ± 11%). This reduction in dilation occurred concomitantly with mitochondrial DNA (mtDNA) strand lesions and reduced mitochondrial complex activities in the endothelium of ZOF versus ZLN. To demonstrate endothelial dysfunction is linked to impaired mitochondrial function, administration of a cell-permeable, mitochondria-directed endonuclease (mt-tat-EndoIII), to repair oxidatively modified DNA in ZOF, restored mitochondrial function and vasodilation to Ach (94 ± 13%). Conversely, administration of a cell-permeable, mitochondria-directed exonuclease (mt-tat-ExoIII) produced mtDNA strand breaks in ZLN, reduced mitochondrial complex activities and vasodilation to Ach in ZLN (42 ± 16%). To demonstrate that mitochondrial function is central to endothelium-dependent vasodilation, we introduced (via electroporation) liver mitochondria (from ZLN) into the endothelium of a mesenteric vessel from ZOF and restored endothelium-dependent dilation to vasoactive intestinal peptide (VIP at 10-5 M, 4 ± 3% vasodilation before mitochondrial transfer and 48 ± 36% after transfer). Finally, to demonstrate mitochondrial function is key to endothelium-dependent dilation, we administered oligomycin (mitochondrial ATP synthase inhibitor) and observed a reduction in endothelium-dependent dilation. We conclude that mitochondrial function is critical for endothelium-dependent vasodilation.

Mitochondrial DNA: A disposable genome?

In mammalian cells, mitochondria are the only organelles besides the nucleus that house genomic DNA. The mammalian mitochondrial genome is represented by prokaryotic-type, circular, highly compacted DNA molecules. Today, more than a half-century after their discovery, the biology of these small and redundant molecules remains much less understood than that of their nuclear counterparts. One peculiarity of the mitochondrial genome that emerged in recent years is its disposable nature, as evidenced by cells abandoning a fraction of their mitochondrial DNA (mtDNA) in response to various stimuli with little or no physiological consequence. Here, we review some recent developments in the field of mtDNA biology and discuss emerging questions on the disposability and indispensability of mtDNA.

Impaired coronary metabolic dilation in the metabolic syndrome is linked to mitochondrial dysfunction and mitochondrial DNA damage.

Mitochondrial dysfunction in obesity and diabetes can be caused by excessive production of free radicals, which can damage mitochondrial DNA. Because mitochondrial DNA plays a key role in the production of ATP necessary for cardiac work, we hypothesized that mitochondrial dysfunction, induced by mitochondrial DNA damage, uncouples coronary blood flow from cardiac work. Myocardial blood flow (contrast echocardiography) was measured in Zucker lean (ZLN) and obese fatty (ZOF) rats during increased cardiac metabolism (product of heart rate and arterial pressure, i.v. norepinephrine). In ZLN increased metabolism augmented coronary blood flow, but in ZOF metabolic hyperemia was attenuated. Mitochondrial respiration was impaired and ROS production was greater in ZOF than ZLN. These were associated with mitochondrial DNA (mtDNA) damage in ZOF. To determine if coronary metabolic dilation, the hyperemic response induced by heightened cardiac metabolism, is linked to mitochondrial function we introduced recombinant proteins (intravenously or intraperitoneally) in ZLN and ZOF to fragment or repair mtDNA, respectively. Repair of mtDNA damage restored mitochondrial function and metabolic dilation, and reduced ROS production in ZOF; whereas induction of mtDNA damage in ZLN reduced mitochondrial function, increased ROS production, and attenuated metabolic dilation. Adequate metabolic dilation was also associated with the extracellular release of ADP, ATP, and H2O2 by cardiac myocytes; whereas myocytes from rats with impaired dilation released only H2O2. In conclusion, our results suggest that mitochondrial function plays a seminal role in connecting myocardial blood flow to metabolism, and integrity of mtDNA is central to this process.

Mitochondrial DNA ligase is dispensable for the viability of cultured cells but essential for mtDNA maintenance.

Multiple lines of evidence support the notion that DNA ligase III (LIG3), the only DNA ligase found in mitochondria, is essential for viability in both whole organisms and in cultured cells. Previous attempts to generate cells devoid of mitochondrial DNA ligase failed. Here, we report, for the first time, the derivation of viable LIG3-deficient mouse embryonic fibroblasts. These cells lack mtDNA and are auxotrophic for uridine and pyruvate, which may explain the apparent lethality of the Lig3 knock-out observed in cultured cells in previous studies. Cells with severely reduced expression of LIG3 maintain normal mtDNA copy number and respiration but show reduced viability in the face of alkylating and oxidative damage, increased mtDNA degradation in response to oxidative damage, and slow recovery from mtDNA depletion. Our findings clarify the cellular role of LIG3 and establish that the loss of viability in LIG3-deficient cells is conditional and secondary to the ρ(0) phenotype.

Mitochondrial DNA: Consensuses and Controversies.

In the course of its short history, mitochondrial DNA (mtDNA) has made a long journey from obscurity to the forefront of research on major biological processes. mtDNA alterations have been found in all major disease groups, and their significance remains the subject of intense research. Despite remarkable progress, our understanding of the major aspects of mtDNA biology, such as its replication, damage, repair, transcription, maintenance, etc., is frustratingly limited. The path to better understanding mtDNA and its role in cells, however, remains torturous and not without errors, which sometimes leave a long trail of controversy behind them. This review aims to provide a brief summary of our current knowledge of mtDNA and highlight some of the controversies that require attention from the mitochondrial research community.

Oxidative stress induces degradation of mitochondrial DNA.

Mitochondrial DNA (mtDNA) is located in close proximity of the respiratory chains, which are the main cellular source of reactive oxygen species (ROS). ROS can induce oxidative base lesions in mtDNA and are believed to be an important cause of the mtDNA mutations, which accumulate with aging and in diseased states. However, recent studies indicate that cumulative levels of base substitutions in mtDNA can be very low even in old individuals. Considering the reduced complement of DNA repair pathways available in mitochondria and higher susceptibility of mtDNA to oxidative damage than nDNA, it is presently unclear how mitochondria manage to maintain the integrity of their genetic information in the face of the permanent exposure to ROS. Here we show that oxidative stress can lead to the degradation of mtDNA and that strand breaks and abasic sites prevail over mutagenic base lesions in ROS-damaged mtDNA. Furthermore, we found that inhibition of base excision repair enhanced mtDNA degradation in response to both oxidative and alkylating damage. These observations suggest a novel mechanism for the protection of mtDNA against oxidative insults whereby a higher incidence of lesions to the sugar-phosphate backbone induces degradation of damaged mtDNA and prevents the accumulation of mutagenic base lesions.

Mitochondrial DNA damage initiates a cell cycle arrest by a Chk2-associated mechanism in mammalian cells.

Previous work from our laboratory has focused on mitochondrial DNA (mtDNA) repair and cellular viability. However, other events occur prior to the initiation of apoptosis in cells. Because of the importance of mtDNA in ATP production and of ATP in fuel cell cycle progression, we asked whether mtDNA damage was an upstream signal leading to cell cycle arrest. Using quantitative alkaline Southern blot technology, we found that exposure to menadione produced detectable mtDNA damage in HeLa cells that correlated with an S phase cell cycle arrest. To determine whether mtDNA damage was causatively linked to the observed cell cycle arrest, experiments were performed utilizing a MTS-hOGG1-Tat fusion protein to target the hOGG1 repair enzyme to mitochondria and enhance mtDNA repair. The results revealed that the transduction of MTS-hOGG1-Tat into HeLa cells alleviated the cell cycle block following an oxidative insult. Furthermore, mechanistic studies showed that Chk2 phosphorylation was enhanced following menadione exposure. Treatment of the HeLa cells with the hOGG1 fusion protein prior to menadione exposure resulted in an increase in the rate of Chk2 dephosphorylation. These results strongly support a direct link between mtDNA damage and cell cycle arrest.

A retro-lentiviral system for doxycycline-inducible gene expression and gene knockdown in cells with limited proliferative capacity.

Currently, there is no reliable system for regulated gene expression and regulated gene knockdown in cells with finite lifespan. In this manuscript, we describe a vector system, consisting of a retrovirus for the delivery of rtTA, and a lentivirus for the delivery of either a transgene or a miR-shRNA for the modification of primary cells. Primary rat pulmonary microvascular endothelial cells (PMVEC) modified by these vectors for the inducible expression of Gaussia luciferase or DsRed Express demonstrated greater than 100-fold induction of the transgene expression with doxycycline. The system works reliably in both sequential and simultaneous infection modes, with about 95% of the sells selected with two antibiotics being inducible in each mode. The lentiviral vector for gene knockdown allows for the direct cloning of shRNA oligos using alpha-complementation, and for the monitoring of induction of RNA interference with fluorescent reporter, mCherry. The gene knockdown vector was validated by knocking down beta-actin expression in PMVECs, with two of the four constructs showing 59 and 75% knockdown, respectively, compared to uninduced controls. The vectors described here were successfully used for the modification of various primary and established cell lines for regulated gene expression and regulated knockdown.

Targeting repair proteins to the mitochondria of mammalian cells through stable transfection, transient transfection, viral transduction, and TAT-mediated protein transduction.

The mitochondrial genome represents a target for exogenous and endogenous damage. Its necessity for successful electron transport makes its repair valuable to the cell. Previous work from our lab has shown that mitochondrial DNA (mtDNA) can be repaired in mammalian cells, and the use of mitochondrial-targeted repair proteins can augment repair to enhance viability following genotoxic stress. In addition, it has also been shown that other repair enzymes that are targeted to the mitochondria can sensitize the cell to DNA damaging agents, thereby aiding the effectiveness of certain chemotherapeutic agents. The methods herein describe the development of mitochondrial-targeted proteins using plasmids or protein transduction domains. It includes the utilization of these constructs to create stably transfected cell lines, transiently transfected cell lines, viral-mediated transduction, and protein transduction domain-mediated mitochondrial protein localization. The end result will be a mammalian cell that expresses the mitochondrial-targeted protein of interest.

Human mitochondrial transcription factor A possesses multiple subcellular targeting signals.

The mitochondrial transcription factor A (TFAM) is a member of a high-mobility group (HMG) family represented mostly by nuclear proteins. Although nuclear localization of TFAM has been demonstrated in some tumors and after treatment of tumor cells with anticancer drugs, the significance of these observations has not been fully elucidated. Here we report that both TFAM overexpression and impairment of its mitochondrial targeting can result in nuclear accumulation of the protein. Both M1 and M7 methionines of human TFAM (hTFAM) can be used for translation initiation with almost equal efficiency resulting in two polypeptides. The shorter polypeptide, however, is not located in the nucleus, despite truncation in the mitochondrial targeting sequence, and both isoforms are targeted to mitochondria with similar efficiency. We further demonstrate that nuclear TFAM confers significant cytoprotection against the chemotherapeutic drugs etoposide, camptothecin, and cisplatin. Three regions of hTFAM [HMG-like domain 1 (HMG1) and HMG-like domain 2 (HMG2), as well as the tail region] can effect nuclear accumulation of enhanced green fluorescent protein (EGFP) fusions. The HMG1 domain contains a bipartite nuclear localization sequence whose identity is supported by site-directed mutagenesis. However, this bipartite nuclear localization sequence is weak, and both N-terminal and C-terminal flanking sequences enhance the nuclear targeting of EGFP. Finally, several mutations in the HMG1 domain increased the mitochondrial targeting of the EGFP fusions, suggesting that the mitochondrial targeting sequence of hTFAM may extend beyond the cleavable presequence.

Mitochondrial transcription in mammalian cells.

As a consequence of recent discoveries of intimate involvement of mitochondria with key cellular processes, there has been a resurgence of interest in all aspects of mitochondrial biology, including the intricate mechanisms of mitochondrial DNA maintenance and expression. Despite four decades of research, there remains a lot to be learned about the processes that enable transcription of genetic information from mitochondrial DNA to RNA, as well as their regulation. These processes are vitally important, as evidenced by the lethality of inactivating the central components of mitochondrial transcription machinery. Here, we review the current understanding of mitochondrial transcription and its regulation in mammalian cells. We also discuss key theories in the field and highlight controversial subjects and future directions as we see them.

Novel Fusion Protein Targeting Mitochondrial DNA Improves Pancreatic Islet Functional Potency and Islet Transplantation Outcomes.

Long-term graft survival is an ongoing challenge in the field of islet transplantation. With the growing demand for transplantable organs, therapies to improve organ quality and reduce the incidence of graft dysfunction are of paramount importance. We evaluated the protective role of a recombinant DNA repair protein targeted to mitochondria (Exscien I-III), as a therapeutic agent using a rodent model of pancreatic islet transplantation. We first investigated the effect of therapy on isolated rat islets cultured with pro-inflammatory cytokines (interleukin-1 ß, interferon γ, and tumor necrosis factor α) for 48 h and documented a significant reduction in apoptosis by flow cytometry, improved viability by immunofluorescence, and conserved functional potency in vitro and in vivo in Exscien I-III-treated islets. We then tested the effect of therapy in systemic inflammation using a rat model of donor brain death (BD) sustained for a 6-h period. Donor rats were allocated to 4 groups: (non-BD + vehicle, non-BD + Exscien I-III, BD + vehicle, and BD + Exscien I-III) and treated with Exscien I-III (4 mg/kg) or vehicle 30 min after BD induction. Sham (non-BD)-operated animals receiving either Exscien I-III or vehicle served as controls. Islets purified from BD + Exscien I-III-treated donors showed a significant increase in glucose-stimulated insulin release in vitro when compared to islets from vehicle-treated counterparts. In addition, donor treatment with Exscien I-III attenuated the effects of BD and significantly improved the functional potency of transplanted islets in vivo. Our data indicate that mitochondrially targeted antioxidant therapy is a novel strategy to protect pancreas and islet quality from the deleterious effects of cytokines in culture and during the inflammatory response associated with donation after BD. The potential for rapid translation into clinical practice makes Exscien I-III an attractive therapeutic option for the management of brain-dead donors or as an additive to islets in culture after isolation setting.

TAT-mediated protein transduction and targeted delivery of fusion proteins into mitochondria of breast cancer cells.

The protein transduction domain (PTD) from the HIV-1 TAT protein has been widely utilized to deliver biologically active macromolecules, including full-length proteins, into a variety of cell types in vitro and in vivo. Without additional targeting signals, the intracellular localization of the proteins delivered in this fashion appears to be cytoplasmic, nuclear or, as recently reported, endosomal. In this study, we show that the presence of the mitochondrial targeting signal (MTS) from hMnSOD on the N-terminus of TAT-fusion proteins directs them into mitochondria of breast cancer cells. We generated and purified fusion proteins containing GFP (MTS-GFP-TAT) or Exonuclease III (MTS-ExoIII-TAT) from Escherichia coli. The results of Western blots of subcellular fractions and fluorescent microscopic analyses revealed efficient protein transduction and mitochondrial localization of the fusion proteins. Specific exonuclease activity was found in the mitochondrial extracts isolated from MTS-ExoIII-TAT transduced cells. This increased exonuclease activity reduced the repair of mtDNA damage following oxidative stress. This diminished mtDNA repair led to a decrease in survival of breast cancer cells. Thus, the present study demonstrates the applicability of this new approach for intramitochondrial targeting of TAT-fusion proteins capable of modulating mitochondrial function and cell survival.

The "fast" and the "slow" modes of mitochondrial DNA degradation.

In a living cell, oxidative stress resulting from an external or internal insult can result in mitochondrial DNA (mtDNA) damage and degradation. Here, we show that in HeLa cells, mtDNA can withstand relatively high levels of extracellular oxidant H2O2 before it is damaged to a point of degradation, and that mtDNA levels in these cells quickly recover after removal of the stressor. In contrast, mtDNA degradation in mouse fibroblast cells is induced at eight-fold lower concentrations of H2O2, and restoration of the lost mtDNA proceeds much slower. Importantly, mtDNA levels in HeLa cells continue to decline even after withdrawal of the stressor thus marking the "slow" mode of mtDNA degradation. Conversely, in mouse fibroblasts maximal loss of mtDNA is achieved during treatment, and is already detectable at 5 min after exposure, indicating the "fast" mode. These differences may modulate susceptibility to oxidative stress of those organs, which consist of multiple cell types.

The efficiency of the translesion synthesis across abasic sites by mitochondrial DNA polymerase is low in mitochondria of 3T3 cells.

Translesion synthesis by specialized DNA polymerases is an important strategy for mitigating DNA damage that cannot be otherwise repaired either due to the chemical nature of the lesion. Apurinic/Apyrimidinic (abasic, AP) sites represent a block to both transcription and replication, and are normally repaired by the base excision repair (BER) pathway. However, when the number of abasic sites exceeds BER capacity, mitochondrial DNA is targeted for degradation. Here, we used two uracil-N-glycosylase (UNG1) mutants, Y147A or N204D, to generate AP sites directly in the mtDNA of NIH3T3 cells in vivo at sites normally occupied by T or C residues, respectively, and to study repair of these lesions in their native context. We conclude that mitochondrial DNA polymerase γ (Pol γ) is capable of translesion synthesis across AP sites in mitochondria of the NIH3T3 cells, and obeys the A-rule. However, in our system, base excision repair (BER) and mtDNA degradation occur more frequently than translesion bypass of AP sites.

The expression of Exonuclease III from E. coli in mitochondria of breast cancer cells diminishes mitochondrial DNA repair capacity and cell survival after oxidative stress.

The ability to sensitize cancer cells to radiation would be highly beneficial for successful cancer treatment. One mode of action for ionizing radiation is the induction of cell death through infliction of extensive oxidative damage to cellular DNA, including mitochondrial DNA (mtDNA). The ability of cells to repair mtDNA and otherwise maintain the integrity of their mitochondria is vital for protection of the cells against oxidative damage. Because efficient repair of oxidative damage in mtDNA may play a crucial role in cancer cell resistance, interference with this repair process could be an effective way to achieve a radiation sensitive phenotype in otherwise resistant cancer cells. Successful repair of DNA is achieved through a precise and highly regulated multistep process. Expression of excessive amounts of one of the repair enzymes may cause an imbalance of the whole repair system and lead to the loss of repair efficiency. To study the effects of changing mtDNA repair capacity on overall cell survival following oxidative stress, we expressed a bacterial repair enzyme, Exonuclease III (ExoIII) containing the mitochondrial targeting signal of manganese superoxide dismutase, in a human malignant breast epithelial cell line, MDA-MB-231. Following transfection, specific exonuclease activity was found in mitochondrial extracts. In order to examine the effects on repair of oxidative damage in mtDNA, cells were exposed to the enzyme xanthine oxidase and its substrate hypoxanthine. mtDNA repair was evaluated using quantitative Southern blot analysis. The results revealed that cells expressing ExoIII in mitochondria are deficient in mtDNA repair when compared with control cells that express ExoIII without MTS. This diminished mtDNA repair capacity rendered MDA-MB-231 cells more sensitive to oxidative damage, which resulted in a decrease in their long-term survival following oxidative stress.

alpha-complementation-enabled T7 expression vectors and their use for the expression of recombinant polypeptides for protein transduction experiments.

Over the past few years protein transduction has emerged as a powerful means for the delivery of proteins into cultured cells and into whole mice. This method is based on the ability of proteins containing protein transduction domains (PTDs), short stretches of 9-16 predominantly basic amino acids, to traverse the cytoplasmic membrane and accumulate inside cells in a time- and dose-dependent fashion. The number of PTDs, both natural and synthetic, is constantly expanding, as is the need to test newly discovered PTDs for their ability to mediate the internalization of the corresponding fusion proteins. Here we describe a strategy and methodology that can be used for the construction of vectors for the T7 RNA polymerase-driven expression of PTD fusions. The cloning in these vectors is facilitated by alpha-complementation. Also, these vectors are small in size (less than 3 kbp) and express influenza virus hemagglutinin tag as well as His tag as part of the fusion for immunological identification and purification respectively of expressed proteins.

Aging: A mitochondrial DNA perspective, critical analysis and an update.

The mitochondrial theory of aging, a mainstream theory of aging which once included accumulation of mitochondrial DNA (mtDNA) damage by reactive oxygen species (ROS) as its cornerstone, has been increasingly losing ground and is undergoing extensive revision due to its inability to explain a growing body of emerging data. Concurrently, the notion of the central role for mtDNA in the aging process is being met with increased skepticism. Our progress in understanding the processes of mtDNA maintenance, repair, damage, and degradation in response to damage has largely refuted the view of mtDNA as being particularly susceptible to ROS-mediated mutagenesis due to its lack of "protective" histones and reduced complement of available DNA repair pathways. Recent research on mitochondrial ROS production has led to the appreciation that mitochondria, even in vitro, produce much less ROS than previously thought, automatically leading to a decreased expectation of physiologically achievable levels of mtDNA damage. New evidence suggests that both experimentally induced oxidative stress and radiation therapy result in very low levels of mtDNA mutagenesis. Recent advances provide evidence against the existence of the "vicious" cycle of mtDNA damage and ROS production. Meta-studies reveal no longevity benefit of increased antioxidant defenses. Simultaneously, exciting new observations from both comparative biology and experimental systems indicate that increased ROS production and oxidative damage to cellular macromolecules, including mtDNA, can be associated with extended longevity. A novel paradigm suggests that increased ROS production in aging may be the result of adaptive signaling rather than a detrimental byproduct of normal respiration that drives aging. Here, we review issues pertaining to the role of mtDNA in aging.

The maintenance of mitochondrial DNA integrity--critical analysis and update.

DNA molecules in mitochondria, just like those in the nucleus of eukaryotic cells, are constantly damaged by noxious agents. Eukaryotic cells have developed efficient mechanisms to deal with this assault. The process of DNA repair in mitochondria, initially believed nonexistent, has now evolved into a mature area of research. In recent years, it has become increasingly appreciated that mitochondria possess many of the same DNA repair pathways that the nucleus does. Moreover, a unique pathway that is enabled by high redundancy of the mitochondrial DNA and allows for the disposal of damaged DNA molecules operates in this organelle. In this review, we attempt to present a unified view of our current understanding of the process of DNA repair in mitochondria with an emphasis on issues that appear controversial.

Persistent damage induces mitochondrial DNA degradation.

Considerable progress has been made recently toward understanding the processes of mitochondrial DNA (mtDNA) damage and repair. However, a paucity of information still exists regarding the physiological effects of persistent mtDNA damage. This is due, in part, to experimental difficulties associated with targeting mtDNA for damage, while sparing nuclear DNA. Here, we characterize two systems designed for targeted mtDNA damage based on the inducible (Tet-ON) mitochondrial expression of the bacterial enzyme, exonuclease III, and the human enzyme, uracil-N-glyosylase containing the Y147A mutation. In both systems, damage was accompanied by degradation of mtDNA, which was detectable by 6h after induction of mutant uracil-N-glycosylase and by 12h after induction of exoIII. Unexpectedly, increases in the steady-state levels of single-strand lesions, which led to degradation, were small in absolute terms indicating that both abasic sites and single-strand gaps may be poorly tolerated in mtDNA. mtDNA degradation was accompanied by the loss of expression of mtDNA-encoded COX2. After withdrawal of the inducer, recovery from mtDNA depletion occurred faster in the system expressing exonuclease III, but in both systems reduced mtDNA levels persisted longer than 144h after doxycycline withdrawal. mtDNA degradation was followed by reduction and loss of respiration, decreased membrane potential, reduced cell viability, reduced intrinsic reactive oxygen species production, slowed proliferation, and changes in mitochondrial morphology (fragmentation of the mitochondrial network, rounding and "foaming" of the mitochondria). The mutagenic effects of abasic sites in mtDNA were low, which indicates that damaged mtDNA molecules may be degraded if not rapidly repaired. This study establishes, for the first time, that mtDNA degradation can be a direct and immediate consequence of persistent mtDNA damage and that increased ROS production is not an invariant consequence of mtDNA damage.