Mitochondrial Mutations in Atherosclerosis: New Solutions in Research and Possible Clinical Applications

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5942 Current Pharmaceutical Design, 2013, 19, 5942-5953

Mitochondrial Mutations in Atherosclerosis: New Solutions in Research and Possible Clinical Applications

Igor A. Sobenin1,2,*, Dimitry A. Chistiakov3, Yuri V. Bobryshev2,4, Anton Y. Postnov1 and Alexander N. Orekhov2,5

1Laboratory of Medical Genetics, Russian Cardiology Research and Production Complex, 15-a 3rd Cherepkovskaya Str., 121552 Moscow, Russia; 2Laboratory of Cellular Mechanisms of Atherogenesis, Institute of General Pathology and Pathophysiology, Russian Academy of Medical Sciences, 8 Baltiyskaya Str. 8, 125315 Moscow, Russia; 3Department of Medical Nanobiotechnology, Pirogov Russian State Medical University, 1 Ostrovityanova Str., 117997 Moscow, Russia; 4Faculty of Medicine, School of Medical Sciences, University of New South Wales, Kensington, NSW 2052, Sydney, Australia; 5Institute for Atherosclerosis Research, Skolkovo Innova-tive Center, 100 Novaya Str., 143025 Skolkovo, Moscow, Russia

Abstract: Cardiovascular diseases are the leading causes of morbidity and mortality in many industrialized societies. Atherosclerosis is the major risk factor for the development of cardiovascular disease based on arterial endothelial dysfunction caused by the impairment of endothelial-dependent dilation. Atherosclerosis is a complex vascular disease resulted from the harmful interactions between genetic and environmental factors. There is a growing body of evidence in support of a non-redundant role of mitochondrial factors in the pathogene-sis of atherosclerosis. Impaired mitochondrial function and structural and qualitative changes in mitochondrial components such as mito-chondrial DNA (mtDNA) may be directly involved in the development of multiple atherogenic mechanisms including advanced oxida-tive stress, abnormalities in glucose and fat metabolism, and altered energy homeostasis. Recent findings showed that the heteroplasmy level of some somatic mtDNA is associated with coronary atherosclerosis. Although this field should further widely elaborated, hetero-plasmic mtDNA mutations could represent a new promising molecular biomarker of genetic susceptibility to atherosclerosis and related pathologic conditions. In this review, we critically consider the contribution of mitochondria-related factors to the pathogenesis of the ar-terial vascular pathology.

Keywords: Atherosclerosis, atherogenesis, mitochondrial DNA, mutations, heteroplasmy, mitochondrial dysfunction, oxidative stress, cell functions.

INTRODUCTION Cardiovascular diseases are the leading causes of morbidity and mortality in many industrialized societies. Atherosclerosis is the major risk factor for the development of cardiovascular disease based on arterial endothelial dysfunction caused by the impairment of endothelial-dependent dilation [1]. Atherosclerosis is a complex vascular disease resulted from the harmful interactions between genetic and environmental factors. There are many recent reports evaluating a role of candidate genes and polymorphisms of the nuclear genome in susceptibility to atherosclerosis (for review, see [2-4]). However, there is also a growing body of evidence in sup-port of a non-redundant role of mitochondrial factors in the patho-genesis of atherosclerosis. Impaired mitochondrial function and structural and qualitative changes in mitochondrial components such as mitochondrial DNA (mtDNA) may be directly involved in the development of multiple atherogenic mechanisms including advanced oxidative stress, abnormalities in glucose and fat metabo-lism, and altered energy homeostasis [5]. In this review, we criti-cally consider the contribution of mitochondria-related factors to the pathogenesis of the arterial vascular pathology.

MITOCHONDRIAL STRUCTURE AND FUNCTION Mitochondria are semi-autonomous organelles found in every cell in the human body. A single cell can contain from 200 to 2,000 mitochondria [6]. The mitochondria are the source of at least 90% of the energy generated in the cell [7]. The majority of this energy (80%) is produced as ATP by oxidative phosphorylation

*Address correspondence to this author at the Laboratory of Medical Genet-ics, Russian Cardiology Research and Production Complex, 15-a 3rd Che-repkovskaya Str., 121552 Moscow, Russia; Tel: +7-926-359-0050; Fax: +7-495-415-9594; E-mail: [email protected]

(OXPHOS). A series of enzyme-catalyzed redox reactions leads to the production of ATP along with reactive oxygen species (ROS). ATP is produced by a series of five multisubunit enzymes or complexes (Fig. 1). Complexes I, III, IV, and V represent trans-membrane multienzyme assemblies in the inner mitochondrial membrane. Complex I is responsible for the oxidation of NADH, pumping four protons (H+) into the intermembrane space while reducing ubiquinone. Complex II (succinate dehydrogenase) oxi-dizes metabolites such as succinate into malate releasing reducing equivalents (electrons) that are transferred to complex III through ubiquinone. Complex II is unique in that it is not a transmembrane protein and has no mitochondrial genetic coding. Complex III (ubiquinol-cytochrome-c reductase) receives electrons shuttled by ubiquinone liberating two protons (H+) in the process. Complex IV (cytochrome-c oxidase) is a transmembrane complex that receives electrons reducing oxygen from water and producing two protons H+ in the process. As protons are pumped out of the matrix, each complex moves electrons along the chain. The ultimate phosphory-lation of ADP to ATP occurs because of a proton gradient created by the oxidation of various compounds by the first four complexes. The proton gradient creates a transmembrane potential used by complex V (ATP synthase, F1F0 ATPase) to drive the synthesis of ATP via the three-step rotation F1 around F0 causing conforma-tional changes in F1 that activate catalytic domains producing ATP. Mitochondria are referred to as semi-autonomous because, un-like any other organelle, they have their own mtDNA that encodes the production of four of the five enzyme complexes critical for OXPHOS. The human mitochondrial genome is comparatively small consisting of only 16,569 base pairs. The mitochondrial ge-nome encodes for 13 proteins involved with oxidative phosphoryla-tion as well as 22 tRNAs and 2 rRNAs involved in synthesis of these mitochondrial complexes. Each mitochondrion contains 2-10

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Mitochondrial Mutations in Atherosclerosis Current Pharmaceutical Design, 2013, Vol. 19, No. 33 5943

copies of the circular, supercoiled, double-stranded DNA found unprotected within the inner mitochondrial membrane. This circular DNA is attached, at least transiently, to the inner mitochondrial membrane [8].

STRUCTURAL CHANGES OF MITOCHONDRIA IN ATHEROSCLEROSIS Although intimal cells in atherosclerotic lesions have been stud-ied in a number of electron-microscopic studies [9-11], the informa-tion about the structural changes of mitochondria in atherosclerosis is practically absent. Earlier we reported that mitochondria in leu-cocytes circulating in the blood of patients with atherosclerosis are structurally more heterogeneous than mitochondria in leucocytes in the blood of healthy volunteers [12]. Our recent electron micro-scopic study of human atherosclerotic lesions revealed the existence of profound structural alterations of mitochondria in cells populat-ing atherosclerotic lesions (unpublished data). This investigation showed that whereas in some mitochondria, cristae were well-defined (Fig. 2A), other mitochondria displayed different degrees of the structural alterations of cristae which were manifested as in a reduction of the number of cristae as in changes in the crista shape. In mitochondria with disrupted crista architectonics, the formation of membrane-surrounded structures, which perhaps represented degenerating cristae, was also observed (Fig. 2B, C). The zones of the mitochondrial matrix with the membrane-surrounded structures were usually characterized by oedema (Fig. 2B, C). Even though some mitochondria were observed in direct contact with lysosomes that resulted in the formation of autophagosomal bodies (Fig. 2D),the vast majority of altered mitochondria did not prompt the process of authophagocytos (Fig. 2B, C). The study also revealed that some cells that displayed a dra-matic reduction in the number of cristae and profound oedema pos-sessed well-defined surrounding membranes (Fig. 3A-C) whereas others displayed focal damages of the integrity of surrounding membranes (Fig. 3D). As both intact mitochondria and mitochon-dria with structural alterations were present in the same tissue specimens, the observed changes of mitochondria cannot be consid-ered to be a result of autolysis degeneration which potentially could occur in autopsy material but reflected existing in situ morphologi-cal heterogeneity of mitochondria. It is worth to noting here that tissue specimens used in this study were obtained during urgent autopsies carried out within 1.5-3 hours after death. The observa-tion that the surrounding membranes in some mitochondria were damaged (Fig. 3D) can be indicative of that some components of

Fig. (2). Different ultrastructural appearances of mitochondria in the human atherosclerotic lesions. (A): “Intact” appearances of mitochondria with well-defined cristae and well-preserved surrounding membranes. (B, C): Altera-tions of cristae and the formation of vacuole-like structures (shown by ar-rows) in zones of oedematous matrix of mitochondria. (D): Autophagosomal body which contains mitochondrion surrounded by concentric myelin-like membranes (shown by arrows) that are associated with a lysosome (marked by star). Electron microscopy; Scales = 150 nm (A-D).

mitochondrial matrix could enter to the cytoplasm. It is well estab-lished that a large number of cells in atherosclerotic lesions undergo

Fig. (1). Mitochondrial respiration chain. For explanations, please see the text. Uncoupling proteins (UCP) reside in the inner membrane of mitochondria. UCPs represent proton channels responsible for draining protons from the intermembrane space directly into the mitochondrial matrix bypassing ATP syn-thase. This results in a decreased membrane potential, which is defined as the electrochemical potential difference of protons across the inner mitochondrial membrane.

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Fig. (3). Destructive alterations of mitochondria in intimal cells in human atherosclerotic lesions. (A-C): Formation of round-shaped membrane-surrounded structures (shown by arrows) in mitochondria with reduced number of cristae and swollen mitochondrial matrix. (D): Destruction of the integrity of the surrounding membrane (shown by arrow) in a swollen mito-chondrion. Electron microscopy; Scales = 200 nm (A-D).

cell death [13, 14], with many of them undergoing necrotic mode of death which is accompanied by the release of cytoplasmic content in the extracellular space [14, 15]. Even though a possibility of the presence of antibodies against mitochondrial components in athero-sclerosis have not yet been investigated, it is known that antibodies to other structural components of atherosclerotic lesions and im-mune complexes circulate in the blood and can also be identified in atherosclerotic lesions [16]. Mitochondria play a role in immunity [17] but the contribution of mitochondria in immune reactions in atherosclerosis has not received attention so far.

OXIDATIVE STRESS-INDUCED IMPAIRMENT IN MITO-CHONDRIAL DYNAMICS ENHANCES MITOCHONDRIAL DYSFUNCTION The mitochondria are among the chief producers of oxygen free radicals within the cell. The electron transport chain constantly produces superoxide radical anions, which, in the case of mito-chondrial dysfunction, cause the escape of electrons that readily form hydroxyl radicals and hydrogen peroxide from superoxide. These extremely reactive ROS are risk factors for atherosclerosis associated with lipid and protein oxidation in the vascular wall. ROS formation triggers a cascade of events such as oxidative modi-fication of low density lipoproteins, inflammation, cellular apopto-sis and endothelium injury. In atherosclerotic vessels, advanced ROS production positively correlates with multiple mitochondrial abnormalities including impaired mitochondrial dynamics, mito-chondrial dysfunction, altered OXPHOS capacity, induction of the mitochondrial-dependent apoptosis, and other properties [18]. The dynamic nature of mitochondria is a concept that includes the movement of mitochondria along the cytoskeleton, the regula-tion of mitochondrial architecture (morphology and distribution), and connectivity mediated by tethering and fusion/fission events [19]. This dynamic network is essential to maintain normal mito-chondrial functions and participates in fundamental processes in-cluding development, metabolic efficiency, apoptosis, and aging [20].

Mitochondrial fission occurs when mitochondria are dysfunc-tional, probably caused by the mitochondrial optic atrophy 1 protein (OPA1) cleavage [21]. OPA1 plays a key role in organizing mito-chondrial morphology. This dynamin-like GTPase resides in mito-chondrial inner membranes and forms oligomers that are responsi-ble for the formation of the cristae junction ultimately resulting in mixing of matrix contents [22]. Proteolytic cleavage of OPA1 re-sults in increased mitochondrial fragmentation and fission [23]. Excessive mitochondrial fission and a lack of fusion results in breakdown of the mitochondrial network, loss of mtDNA, respira-tory defects and an increase in ROS [24]. Breaking dynamic bal-ance between mitochondrial fusion and fission maintains mitochon-drial functions, but when cells are affected by excessive oxidative damage that results in apoptosis, the rate of mitochondrial fission outpaces that of fusion and stimulates fission protein DRP1-mediated release of cytochrome c [25].

ACTIVATION OF MITOCHONDRIAL PATHWAY OF APOPTOSIS The major consequence of atherosclerosis in humans is mainly caused by apoptosis of vascular smooth muscle cells, endothelial cells, and macrophages possibly leading to the promotion of plaque growth and procoagulation and induction of rupture [26]. Mito-chondrial dynamics plays a unique role in cell apoptosis. An imbal-ance of mitochondrial fusion/fission results in excessive fragmenta-tion or tubulation, with pathological consequences [27]. Mitochon-drial fission-related proteins also appear to participate in apoptosis and proteins associated with the regulation of apoptosis have been shown to affect mitochondrial ultrastructure [28]. Two proapoptotic Bcl-2 family members, Bax and Bak, play a key role in the induc-tion of the mitochondrial pathway of apoptosis through regulating mitochondrial outer membrane permeability (MOMP). Apoptosis-related MOMP requires the involvement of Bax and/or Bak that reside on the mitochondrial outer membrane or tranlocate there in response to proapoptotic stimuli [29]. Bax then associates with dynamin-related protein 1 (DRP1), mitofusins, and Bak resulting in mitochondrial fragmentation and release of cytochrome c into cyto-plasm [30]. In the cytoplasm, cytochrome c binds apoptotic prote-ase-activating factor 1 (APAF1) activating the assembly of the apoptosome that activates caspase-9 and subsequently the effectors caspase-3 and caspase-7 [31]. It has been proposed that Bax/Bak-induced mitochondrial fis-sion may be required for release of cytochrome c from the mito-chondrial intermembrane space. However, Sheridan et al. [32] showed that Bcl-xL and other members of Bcl-2 family are able to antagonize Bax and/or Bak-induced cytochrome c release but failed to block mitochondrial fragmentation associated with Bax/Bak activation. Indeed, these data suggest that Bax/Bak-initiated mito-chondrial fission and cytochrome c release are distinct events and that Bcl-2 family proteins can be considered as novel regulators of mitochondrial morphogenesis [33].

DISRUPTION BIOGENESIS UNDER ATHEROSCLEROSIS-INDUCED STRESS: A ROLE OF PARP-1 HYPERACTIVA-TION A chronic oxidative stress promotes atherogenesis since ateros-lerosis is an inflammatory disease accompanied with the lipid and protein oxidation in the vascular wall [34]. The role of oxidative damage in the cardiovascular risk factor-induced mitochondrial dysfunction in atherosclerosis such as aging, diabetes, dyslipidemia, hypertension, homocysteinemia, and cigarette smoking has been extensively reviewed [35, 36]. However, the regulatory mecha-nisms of those factors in mitochondrial functions are still unclear, especially in pathophysiological processes. In this section, we will discuss the related regulatory mechanisms of mitochondrial physi-ology (biogenesis and fusion/fission) during the development of atherosclerosis.


Poly(ADP-ribose) polymerase-1 (PARP-1), the most abundant isoform of the PARP enzyme family, is a nuclear enzyme involved in modulating chromatin structure, regulating gene transcription, and sensing and repairing DNA damage [37]. PARP-1 is activated by single- and double-stranded DNA breaks. When activated, the enzyme forms homodimers and catalyzes the cleavage of NAD+ into nicotinamide and ADP-ribose to form long tails of poly(ADP-ribose) (PAR) polymers on the glutamic acid residues of several target proteins including PARP-1 itself. However, the extensive activation of PARP-1 leads to the rapid depletion of cellular energy sources such as NAD+ and its precursor ATP and may cause ne-crotic cell death [38]. Furthermore, PARP-1 is also involved in apoptotic cell death by mediating the translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus in oxi-datively injured cells (Fig. 4) [39]. The PAR formation serves as a cell death signal that induces release of AIF from mitochondria to cytosol [40]. AIF, an ubiquitous mitochondrial oxidoreductase, itself contributes to the cell death via involvement to the nuclear DNA fragmentation [41]. Hossain et al. [42] reported that nuclear respiratory factor 1 (NRF-1), a key transcriptional activator for nuclear-coded genes involved in mitochondrial biogenesis, can directly interact with PARP-1 associated with DNA-PK/Ku80/Ku70/topoisomerase II�-containing protein complex. The interaction results in the recruit-ment of this complex to the promoter. In contrast, PARP-1 can PARylate NRF-1 that prevents PARP-1/NRF-1 interaction. Since NRF-1 is implicated in the control of expression of multiple nuclear genes required for mitochondrial respiratory functions [43], the ability of PARP-1 to modify this transcription factor suggests on a direct role of PARP-1 in the regulation of mitochondrial biogenesis.

Recent studies revealed broader functions of NRF-1 in relation to mitochondria including regulation of expression of key compo-nents of the protein import and assembly machinery in mitochon-dria [44, 45]. In addition, NRF-1 is able to induce mitochondrial biogenesis via interplay with the peroxisome proliferator-activated receptor � coactivator-1� (PGC-1�), a transcriptional coactivator [46]. PGC-1� was shown to stimulate production of two mitochon-drial transcription activators, NRF-1 and NRF-2, and coactivate NRF-1-mediated transcription of mitochondrial transcription factor A, a direct regulator of mitochondrial DNA replication/trans-cription. Indeed, PGC-1� and PGC-1� may participate in the induc-tion of mitochondrial biogenesis and activation of oxidative phos-phorylation by utilizing NRF-1 or other transcription factors [47]. PGC-1� could contribute to mitochondrial fusion/fission through induction of transcription of mitofusin 2 (Mnf2), a protein located in the outer mitochondrial membrane and responsible for tethering adjacent mitochondria by forming homo- and heterodi-mers [48]. PGC-1�, a second member of the PGC family of tran-scriptional coactivators, is also able to increase mitochondrial fis-sion by enhancement of Mfn2 gene transcription through co-activated nuclear receptor estrogen related receptor � [44]. Thus, these results prove that transcriptional regulators such as NRF-1 not only affect mitochondrial biofunction but also shift the balance between mitochondrial fusion and fission events through coordina-tion of the PGC family to selectively control gene expressions (Fig. 4). Briefly, what is the role of PARP-1 in oxidatively injured cells during atherogenesis? As shown in (Fig. 4), in the early stage of ROS-induced oxidative stress, cells can protect themselves from oxidative damage by activation of PARP-1-related signaling to

Fig. (4). The molecular regulation of nuclear and mitochondrial cross-talk signaling on ROS-triggered atherosclerosis pathogenesis. ROS activate PARP-1 and primarily cause atherosclerosis by increasing endothelial and smooth muscle cell death (mitochondria-dependent or not) and triggering inflammatory reactions. In addition, proinflammatory cytokines and chemokines induce plaque formation and plaque vulnerability that leads to deterioration in the later stages of athe-rosclerosis. However, PARP-1 can also participate in NRF-1 regulation or indirectly activate PGC-1 to improve mitochondrial biofunctions such as biogenesis and fusion. This mechanism defends against ROS-induced progression of AST by inhibiting mitochondrial-mediated apoptosis and against the occurrence of AST at an early stage. ROS: Reactive oxygen species; PARP-1: Poly(ADP-ribose) polymerase-1; NRF-1: Nuclear respiratory factor 1; PGC-1: Peroxisome proliferator-activated receptor � coactivator-1; mtDNA: Mitochondrial DNA; AIF: Apoptosis-inducing-factor; NF-�B: Nuclear factor �-light-chain-enhancer of activated B cells; TNF-�: Tumor necrosis factor-�; IL-6: Interleukin-6.

Plaqueformation

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MITOCHONDRIONmtDNA transcriptionHeme byosynthesisProtein import and assembly

activitydynamics

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support mitochondrial biofunctions and prevent mitochondrial-mediated cell apoptosis. When this is not enough to sustain the extensive ROS production, PARP-1 plays the opposite role by pro-moting the progression of atheroslerosis.

ROS-INDUCED OXIDATIVE STRESS AND MITOCHON-DRIAL BIOGENESIS As shown, ROS-mediated mitochondrial biogenesis may be induced by stimulation of NRF-1 and NRF-2. Furthermore, ROS can also induce mitochondrial biogenesis through direct regulation of PGC-1� transcription [49]. Kim et al. [50] reported that overex-pression of the PGC-1� gene in human aortic smooth muscle and endothelial cells leads to a significant reduction in intracellular and mitochondrial-dependent ROS production as well as NAD(P)H oxidase activity, which is induced by tumor necrosis factor � (TNF-�). Therefore, PGC-1�-mediated activation of mitochondrial bio-genesis in the vasculature in response to oxidative stress should benefit a better protection against atherosclerosis. Recently, Won et al. [51] suggested for the presence of another mechanism, by which PGC-1� is able to contribute to enhanced defense against vascular disease. They showed that adenoviral overexpression of PGC-1� prevented linoleic acid-induced in-creases in ROS generation and cell apoptosis in human aortic endo-thelial cells by increasing fatty acid oxidation, decreasing cytosolic fat metabolites and increasing ATP/ADP translocase activity. Fatty acid oxidation facilitates the development of metabolic and vascular disease through dysregulation of adiponectin, a cytokine secreted into blood by adipocytes [52]. Adiponectin was found to protect vessels against oxidative stress by several ways. One of these ways involves adiponectin-dependent activation of PGC-1� expression and activity via stimulating Ca2+ signaling, AMP-activated protein

kinase, and Sirtuin type 1 that leads to increased mitochondrial biogenesis [53]. However, this finding was observed in skeletal muscles, and it is necessary to prove whether a similar mechanism exists in the vascular endothelium or vasculature. Adiponectin was shown to influence nitric oxide (NO) produc-tion by endothelial cells via activation of phosphatidylinositol 3-kinase-dependent phosphorylation of endothelial NO synthase (eNOS) [54]. It is well known that NO possess a variety of antia-therogenic affects including the maintenance of vascular structure integrity and preventing the proliferation of vascular smooth muscle cells, the aggregation of platelets, and the adherence and infiltration of inflammatory cells [55]. Besides multiple protective actions to blood vessels, NO is still a reactive radical, and NO overproduction could induce a nitrosative stress [56], which in turn enhances devas-tating action of oxidative stress. NO reacts to ROS and produces peroxynitrite anion (ONOO-), which is a potent oxidizing agent capable of causing oxidative damage [36]. Under advanced oxida-tive stress, the peroxynitrite anion can oxidize tetrahydrobiopterin (BH4), an essential cofactor of eNOS, leading to a reduction in NO bioactivity and a further increase in ROS production (Fig. 5) [57]. The reduction in NO production can negatively influence the permeability of mitochondrial KATP channels [58]. Recently, a car-dioprotective role of opening mitochondrial KATP channels was shown in a rat model of heart ischemia-reperfusion injury [59]. The activation of KATP channels causes inhibition of the permeability transition pore in mitochondria thereby preventing the release of cytochrome c and AIF [60] and triggering ROS production by mi-tochondria [61]. The protective role of NO against ROS-induced acute ischemia was also supported by Wajima et al. [62] who showed a cardioprotective effect of intravenous administration of BH4 in rats with myocardial infarction following ische-

Fig. (5). Activation of atheroslerosis progression via the opening of mitoKATP channels. Decreased nitric oxide (NO) production by endothelial NO synthase (eNOS) and increased expression of reactive oxygen species (ROS) induce atherosclerosis. Excessive ROS with affinity for NO bind with it to produce the peroxynitrite anion (ONOO.), which inhibits tetrahydrobiopterin (BH4), an essential cofactor of eNOS, leading to further reduction of NO. Significantly re-duced NO not only increases ROS production, but also induces the opening of mitochondrial ATP-sensitive K+ (mitoKATP) channels, followed by activation of the permeability transition pore (PTP), leading to mtROS release and atheroslerosis development.

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mia/reperfusion. Its protective effect appears to be involved in the opening of mitochondrial KATP channels through increased NO production. Although the activation of mitoKATP channels by NO was observed in cardiomyocytes [62, 63], the existence of a similar mechanism in endothelial or smooth muscle cells requires further research.

ROS-INDUCED OXIDATION AND DEPLETION OF MTDNA The close proximity of mtDNA to the harmful ROS by-products of OXPHOS makes mtDNA more vulnerable to attack. During oxidative phosphorylation, 5% of the oxygen used in respi-ration is converted to superoxide anions or other ROS (Fig. 6) [64]. It is estimated mtDNA has a mutation rate 10-20 times higher than nuclear DNA [65]. Two factors contribute to the vulnerability of mtDNA to mutation as compared to nuclear DNA. First, coupled with the close proximity to ROS, mtDNA is also lacking the protec-tive strategies associated with nuclear DNA such as protective his-tones, chromatin structure, and introns [66] and second, the proof reading apparatus for mtDNA is much less efficient than that of nuclear DNA [67]. Mutations in mtDNA can have a significant detrimental effect due to decreased noncoding regions. Mitochon-drial DNA has two noncoding areas – a control region characterized by three hypervariable areas as well a displacement region (D-loop). The D-loop region controls replication of the mtDNA. Increased oxidative stress induced by ROS was shown to di-rectly damage mtDNA [68]. 8-hydroxy-2’-deoxyguanosine (8-OHdG), a product of DNA oxidation, was found to be a good bio-marker of oxidative stress-induced DNA damage since its amount in the urine or blood serum/plasma of patients well correlates with the level of DNA oxidation [69]. Furthermore, there was a signifi-cant correlation between the severity of coronary artery disease and 8-OHdG content [70, 71]. 8-OHdG results from the attack of DNA nucleosides by hydroxyl radicals. Although 8-OHdG can be pro-

duced from nuclear DNA, this product is generated from oxidized mtDNA at rates 3-23 fold higher [72]. In several studies, a correlation between the severity of oxida-tive stress and reduced copy number of mtDNA in peripheral blood mononuclear cells (PMBCs) was observed [73-75]. In addition, Liu et al. [73] reported that a lower copy number of mtDNA was asso-ciated with increased levels of 8-OHdG thereby reflecting the rela-tionship with advanced rate of DNA damage. Interestingly, de-creased copy number of mtDNA was found to correlate with sever-ity of independent risk factors of atherosclerosis such as hyperlipi-demia [76] and diabetic hyperglycemia [61, 77] suggesting on the insufficient mitochondrial biogenesis and capability to scavenge oxidized lipids and glucose. Studies of healthy young men showed that mtDNA content in PMBCs have a direct relationship to fat and carbohydrate oxidation rate [78]. In macrophages, reduced ability of mitochondria to utilize oxidized lipids can accelerate transforma-tion of macrophages to ‘foam cells’ and therefore speed up athero-genesis. Reduced mitochondrial biogenesis is associated with a less efficient energy intake from sugars and fats through OXPHOS and increased formation of by-products such as ROS, which further intensifies oxidative stress [79].

SOMATIC MUTATIONS OF MTDNA: MUTATIONAL THRESHOLD AND HETEROPLASMY When damage to mtDNA is not repaired, it can result in a cas-cade of events ultimately leading to a number of diseases. A mito-chondrial disorder can result from the substitution, deletion, and duplication of mtDNA bases and depletion of mtDNA copies. To add to the complexity of mitochondrial disorders, they can arise from one mtDNA mutation or from a number of independent muta-tions that in turn can lead to more than one disease type. The natu-rally occurring circle of mtDNA is also referred to as wild-type mtDNA. The number of mutations can increase in a particular tis-sue, while not being reflected in other parts of the body. The mix-

Fig. (6). Schematic model of mitochondrial ROS production. During mitochondrial respiration, a small amount of the molecular oxygen consumed by cells is converted into superoxide anion (O2.� ) by complexes I and III as toxic by-products of OXPHOS. Superoxide dismutase (SOD) enzymes (MnSOD and CuZn SOD) convert O2.� to hydrogen peroxide (H2O2), which can be sequentially converted into H2O by glutathione peroxidase (GPx) or peroxiredoxin enzymes. Also, H2O2 can react with Fe2+ to generate a hydroxyl radical (. OH). This radical can attack all molecules including mtDNA and consequently cause a decrease in mitochondrial mRNA and altered expression of mitochondrial proteins essential for ETC and ATP synthesis. Defects in mitochondrial proteins affect elec-tron transport chain (ETC) activity culminating in a vicious cycle of ROS production.

O2

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tures of wild-type and mutant mtDNA coexisting in the same mito-chondria are referred to as heteroplasmic mutations. Mitochondrial mutations can also be homoplasmic in nature when cellular mito-chondria contain all of the same mutant mtDNA. Repeated cell division leads to the separation of heteroplasmic and homoplasmic cell lines in a phenomenon of random segrega-tion. Mutant mtDNA increases with aging and the cellular energy capacity can decrease. This decrease in turn affects the threshold of a minimal cell function [80]. Although cells may harbor mutant mtDNA, the expression of disease is dependent on the percent of mutations. Modeling confirms that an upper threshold level might exist for mutations beyond which the mitochondrial population collapses with a concomitant decrease in ATP [81]. This decrease in ATP results in the phenotypic expression of disease [82]. It is estimated that in many patients with clinical manifestations of mi-tochondrial disorders the proportion of mutant DNA exceeds 50% [83]. The concept of a threshold level of mutation can be illustrated in a mutation of base pair 8993 in the ATPase gene. This hetero-plasmic mutation can result in the phenotypic expression of diabe-tes, neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP), or Leigh Disease [84]. The phenotypic expression seems to be contingent on the level of mitochondrial mutation. If the mu-tation rate is below 60% it can express itself as diabetes. When the mutation rate is 60-90% the phenotypic expression results in the diagnosis of NARP. And, when expression of this mitochondrial mutation is over 90% the condition of Leigh Disease manifests in the patient, a condition characterized by psychomotor retardation, dysphagia, hypotonia, ataxia, weakness, external ophthalmoplegia, convulsions, blindness, and deafness, with a usual prognosis of less than two years [85]. Regardless of the type of mutation or the amount of hetero-plasmy in affected mitochondria, unrepaired damage leads to a decrease in ATP, which in turn causes the phenotypic manifestation of disease. The manifestation of disease not only depends on the ATP level but also the tissue affected. Various tissues have differ-ing levels of demand on OXPHOS capacity. For example, the tissue threshold for muscle or neuronal tissue is thought to be as high as or higher than 90% for damaged mtDNA [86]. This means the propor-tions of damaged mtDNA in the total cellular pool must exceed 90% before onset of biochemical abnormalities appear. Another example illustrating the relative thresholds of various tissues was reported in the classic mitochondrial disorder of Leber’s hereditary optic neuropathy, in which mitochondrial respiratory demand from highest to lowest was neurons, skeletal muscle, cardiac muscle, kidney, and liver, respectively [87]. Thus, the threshold of malfunc-tion in a given tissue is dependent on the energy demand and the sensitivity of the tissue to mitochondrial dysfunction. For biochemical malfunctions to occur, threshold values for complex I dysfunction have been calculated to be 70-80% for mus-cle, liver, and kidney, 64% for the heart, and 50% for the brain. For complex III dysfunction, threshold values were found to be high and showed little tissue variation. For complex IV dysfunction, threshold values for the heart and skeletal muscle were calculated to be 67% while kidney and brain were 86%. The sensitivity of threshold levels to demand can be illustrated with a complex IV example. An 80% decrease in activity of complex IV will lead to a small decrease in liver function, while the heart will experience a 40% decrease in function [88].

HETEROPLASMIC MTDNA MUTATIONS IN ATHERO-SCLEROSIS During ageing, mtDNA accumulates more and more somatic mutations because of the aggregation of DNA damage due to the limited efficiency of DNA repair machinery. In experiments in-volved mice deficient for mitochondrial DNA polymerase (so called mtDNA-mutator mice), Trufinovic et al. [89] showed that

premature aging is unlikely to result from advanced oxidative stress but from increase in mtDNA mutations. The mutations primarily altered protein-coding genes affecting the function of the respira-tory chain and thereby causing reduced steady-state levels of com-plex I, III, and IV [90]. Therefore, insufficient mitochondrial bio-genesis (i.e. respiratory function decline) may be the primary in-ducer of premature aging in mtDNA mutator mice [91]. Further-more, Vermulst et al. [92] showed the clonal pattern of the distribu-tion of mtDNA mutations in different tissues of mtDNA mutator mice. Due to the large number of mitochondrial genome copies within each cell, it is the ratio of mutated to wild-type mtDNA that is a significant determinant of phenotype [93]. Indeed, accumula-tion of heteroplasmic somatic point mutations in mtDNA may be primarily responsible for mitochondrial respiratory function de-cline. Atherosclerosis is an age-related pathology. Similarly with observations of Vermulst et al. [92] in mtDNA mutator mice, sev-eral studies showed the monoclonality of amplification of vascular smooth muscle cells in atherosclerotic lesions [94, 95]. Thus, the monoclonal origin of atherosclerotic plaques may represent a likely mechanism of expansion of cells containing mtDNA with a high content of mutated mtDNA (i.e. with higher heteroplasmy levels) within an atherosclerotic plaque. To date, few studies have evaluated only whether heteroplasmic mutations of mtDNA are associated with atherosclerosis. The ‘common’ mtDNA mutation del4977 resulted in a deletion of a nearly 5-kb mtDNA region between nucleotides 8470 and 13459 (contains five genes for tRNA and seven genes for components of the respiration chain) was assessed in two studies but no significant difference in heteroplasmy levels was found between healthy con-trols and patients with coronary atherosclerosis [96, 97]. However, the frequency of this mutation was 5-fold higher in affected patients than in controls [97]. Nomiyama et al. [98] found significantly higher (4-fold) heteroplasmy levels of mutation A3243G located in the mitochondrial tRNALeu 1(UUA/G) gene in Japanese diabetic pa-tients with atherosclerosis compared to age-matched non-diabetic controls. Recently, we performed a comprehensive analysis of somatic mtDNA mutations in atherosclerotic tussues. Of 40 analyzed muta-tions, we found ten whose heteroplasmy levels were significantly differed in atherosclerotic lesions compared to those in normal aor-tas (Table 1) [99, 100]. The atherosclerosis-associated mutations were located in mitochondrial genes MT-RNR1 (encodes rRNA 12S); MT-TL1 (tRNA-Leu, recognizes UUR); MT-TL2 (tRNA-Leu, recognizes CUN); MT-ND1, MT-ND2, MT-ND5, and MT-ND6 (subunits 1, 2, 5 and 6, respectively, of NADH dehydro-genase), and MT-CYB (cytochrome b) were potentially associated with atherosclerosis [99]. For each of those mutations, a range of 29-86% of aortic samples displayed a significant difference in the heteroplasmy levels between atherosclerotic plaques and unaffected tissues. Further studies of those ten mtDNA mutations involving the analysis of increased numbers of aortic biopsies, revealed five mu-tations, the mean level of heteroplasmy of which was significantly different in atherosclerotic intimal homogenates in comparison with the unaffected tissue. These mutations were A1555G, C3256T, T3336C, G13513A, and G15059A [100]. Thus, the association with atherosclerosis was confirmed at least for five heteroplasmic mtDNA mutations occurring in MT-RNR1, MT-TL1, MT-ND2, MT-ND5, and MT-CYB [100]. Furthermore, we assessed association between the level of het-eroplasmy of the mtDNA mutation C3256T in human white blood cells, extent of carotid atherosclerosis, and presence of coronary heart disease (CHD), the major clinical manifestation of atheroscle-rosis. The highly significant relationship between the C3256T het-eroplasmy level and susceptibility to atherosclerosis was observed. In individuals with low predisposition to atherosclerosis (according to high-resolution B-mode ultrasonography), the mean level of


C3256T heteroplasmy was significantly lower as compared to mod-erately predisposed subjects, and further to highly predisposed sub-jects [12]. The level of C3256T heteroplasmy of mitochondrial genome in human white blood cells was considered to be a bio-marker of mitochondrial dysfunction and risk factor for atheroscle-rosis; therefore, it was suggested as an informative marker of ge-netic susceptibility to atherosclerosis, CHD, and myocardial infarc-tion [12]. The mutation C3256T is situated in the MT-TL1 gene encoding tRNALeu(UUR). The mutation causes the MELAS syndrome, a neu-rodegenerative mitochondrial disease phenotypically expressed as the presence of mitochondrial-related myopathy, encephalopathy, lactic acidosis, and stroke-like episodes [101]. The mutation C3256T is also associated with mitochondrial diabetes [102]. The pathophysiological mechanism by which this mutation could be involved in atherogenenesis is unknown and needs in turn to be evaluated. It should be noted that level of heteroplasmy of mitochondrial mutations associated with atherosclerosis is significantly lower then that usually required for the phenotypic expression of a mitochon-drial disease (Table 1). A vast majority of mitochondrial disorders affects myocardium, skeletal muscles, and brain, i.e. tissues with a greater metabolic activity and minimal cell turnover compared to other tissues [81]. Indeed, mtDNA mutations accumulate much faster in brain and heart tissue. For example, mitochondrial muta-tions and DNA damage in the left ventricular cardiac muscle, which is responsible for pumping blood flow across a whole body except for lungs, arise at a 5-10-fold rate higher then in the less active right ventricle [103]. Compared to the skeletal muscle and neuronal tissues, the vas-cular endothelium and vasculature do not require the extremely high energy consumption. Indeed, significantly less heteroplasmy levels of somatic mtDNA mutations at the vascular wall appear to be needed for inducing endothelial dysfunction and atherogenesis. For example, the average heteroplasmy level of the mutation A3243G was found to be very low in both normal and affected vascular wall (0.004% and 0.016%) [98]. The A3243G mutation in the leucine-specific tRNA corresponding to the codon UUR (tRNALeu(UUR)) induces a lack of modification of uridine at the first position of anticodon [104]. This suggests that such mutant tRNALeu(UUR) can recognize all four nucleotides at the third position

of the codon giving rise to the translation of not only the usual UUR (R=A or G) leucine codons but also UUY (Y=C or U) phenyla-lanine codons, which could eventually lead to the incorporation of leucine into phenylalanine sites at a certain rate [105]. The resulting synthesis of premature proteins due to this mistranslation could affect cells considerably, even if there is only a minor amount of the A3243G mutant mtDNA. Of interest, mtDNA A3243G itself in-creases the intracellular production of ROS, which could in turn cause secondary somatic mutations [106].

CONCLUDING REMARKS: POSSIBLE CLINICAL APPLI-CATIONS AND FUTURE PERSPECTIVES Atherogenesis is a complex multi-step pathogenic process. Al-terations in many biological pathways related to vascular function substantially contribute to the pathogenesis of atheroslerosis at different stages of lesion formation. Among those, oxidative stress is probably the most significant factor occurring in atherosclerotic disease. Advanced oxidative stress is tightly linked to mitochondrial dysfunction causing increased damage and mutagenesis of mtDNA. Impaired mitochondrial function and alterations in mtDNA could in turn enhance ROS production and oxidative stress. At present, it is clear that mitochondrial factor may play a piv-otal role in susceptibility, induction and progression of arterial athe-rosclerosis. However, the impact of certain somatic mtDNA muta-tions to atherogenesis is widely unknown. More studies focused on the assessment of new heteroplasmic somatic mtDNA mutations and their influences on the mitochondrial function in relation to atherosclerosis are definitely required. It is also necessary to inves-tigate, in which cell population (endothelial cells, smooth muscle cells, macrophages, dendritic cells, and/or T lymphocytes) resided in an atherosclerotic lesion, mitochondrial mutations play a key role in vascular disease. Concerning monocytes, the role of each cell subset in human atherosclerosis remains unknown, although there are some clues. In vivo, human coronary artery lesions contain macrophage subpopulations with different gene-expression pat-terns, which points to the existence of heterogeneity (107). This finding in turn complicates the precise studying of a role of mito-chondrial dysfunction in each monocyte cell subset in relation to atherogenesis. The deep knowledge about the pathophysiological relevance of each coronary artery disease-associated mtDNA muta-tion to atherosclerosis is of great importance.

Table 1. Heteroplasmic mtDNA Mutations Associated with Atherosclerosis.

Heteroplasmy, % [99, 100] Gene Mutation

Unaffected intima Atherosclerotic intima

12S RNA 652insG 0 9

12S RNA A1555G 26 8

tRNALeu(UUR) C3256T 11 25

NADH dehydrogenase subunit 1 T3336C 2 12

NADH dehydrogenase subunit 2 C5178A 17 10

tRNALeu(CUN) G12315A 10 35

NADH dehydrogenase subunit 5 G13513A 29 43

NADH dehydrogenase subunit 6 G14459A 2 7

Cytochrome B G14846A 4 10

Cytochrome B G15059A 34 53


Our preliminary results showed that heteroplasmic mtDNA mutations may contribute to atherogenesis, and the mean level of mtDNA mutation heteroplasmy in the plaque is correlated with the atherosclerosis progression. Typically, the higher the heteroplasmy level of a disease-associated mutation in non-affected vascular tis-sue the higher predisposition to atherosclerosis. However, some mutations such as C5178A displayed reduced levels of hetero-plasmy in affected tissue compared to the normal tissue [99, 108, 109] that could suggest about their protective effect [110]. The heteroplasmy of mtDNA mutations can be quantified in blood cells such as PMBCs [109] or peripheral lymphocytes [12], and the levels of heteroplasmy of certain mtDNA mutations in white blood cells were shown to well correlate with the mean in-tima-media thickness in different sites of the bilateral carotid arter-ies [12, 109] and the plaque size [12]. These findings suggest for a potential clinical value of heteroplasmic mtDNA mutations as mo-lecular biomarkers of atherogenesis. However, before clinical ap-plication, it is necessary to evaluate how well mtDNA heteroplasmy in blood correlates with mtDNA heteroplasmy in the aortic and carotid arterial wall. In the field of clinical and epidemiological studies of athero-sclerotic disease, the good portion of efforts is aimed to the devel-opment of novel tools for more precise risk prediction to allow preventive treatment targeted at high-risk individuals. These tools usually include direct visualization of preclinical atherosclerotic disease as well as an evaluation of a variety of risk factors, includ-ing markers of the activity of atherosclerotic disease, thrombogenic risk, and genetic polymorphisms. Within this aspect the search of novel risk factors and markers of atherosclerosis is highly encour-aged. Among the potential biomarkers of the activity of atheroscle-rosis, blood serum atherogenicity may play a significant role, hith-erto not established well. The phenomenon of blood serum athero-genicity may be generally described as the ability of human serum to induce lipid accumulation in cultured cells [111]. It is well known that the deposition of intracellular cholesterol and subse-quent foam cell formation is a key step for atherosclerosis devel-opment [112]. Therefore, serum atherogenicity may play a signifi-cant role in pathogenesis of atherosclerosis, but this association remained unclear for a long time. By present, several achievements may give a good theoretical and clinical background for further investigations of the phenomenon of serum atherogenicity, espe-cially with the respect of its diagnostic and prognostic significance. By now, it is known that blood sera from the majority of patients with established coronary atherosclerosis, unlike sera from healthy subjects, are capable of inducing lipid accumulation in cultured cells, i.e. possess atherogenic properties [111, 113]. The corre-sponding cellular models based on the primary cultures of human intimal aortic cells and human blood-derived monocytes/macro-phages had been developed to demonstrate this phenomenon and to characterize it in both quantitative and qualitative manner [113-115]. Serum atherogenicity correlates well with the extent of athe-rosclerotic disease in coronary bead, and the prevalence of serum atherogenicity is increased in persons with subclinical atherosclero-sis and high-risk patients (e.g., Type 2 diabetics, lupus erythemato-sis patients, severe hyperlipidemics) [116-118]. On the other hand, in samples taken from general population, serum and LDL athero-genicity looks like stand-alone factor, independent of conventional risk factors, such as serum lipid levels, arterial blood pressure, age and possibly gender. It is also known that serum atherogenicity is mainly due to the presence of modified low density lipoprotein in circulation [113], and at certain extent to non-lipid atherogenic factors [119, 120]. Therefore, blood serum atherogenicity revealed in cell culture test can be suggested as the integral characteristic of intracellular lipid deposition, the primary step in atherogenesis. Certainly, intracellular lipid deposition occurred during atherogene-sis acts as a trigger event followed by other pathologic changes in vascular wall, such as the enhanced synthesis of connective tissue

matrix components, migration of hematogenic cells, cellular prolif-eration and, possibly, the development of local inflammatory reac-tions [121]. The mechanisms of interaction of these quite different processes remain obscure. The same could be said about the factors regulating or modulating the extent of atherogenic effect. Recently we have performed an experimental study, in which we have meas-ured the level of intracellular cholesterol accumulation by cultured blood-derived monocytes-macrophages from 68 apparently healthy subjects aged 21-65 (28 men, 40 women) (unpublished data). The cells were incubated for 24 h in the presence of the same prepara-tion of atherogenic low-density lipoprotein (100 �g/ml) isolated from the blood of CHD patients. Baseline level of intracellular cho-lesterol varied from 21 to 152 �g/mg cell protein; the mean value was 46 �g/mg cell protein (SD=23). An absolute increase in intra-cellular cholesterol level varied from 20 to 355% from baseline level; the mean value accounted for 84% (SD=61). So, LDL from CHD patients induced statistically significant intracellular choles-terol accumulation in all 68 cases, thus demonstrating atherogenic effect. However, all conventional cardiovascular risk factors taken together (age, gender, systolic and diastolic blood pressure, body mass index, blood cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, smoking, family history of CHD) could explain only 9% variability of intracellular cholesterol accumulation caused by atherogenic LDL. Moreover, the same combination of risk factors explained only 2% variability of baseline intracellular cholesterol level. The last parameter was considered very important, since the baseline level of intracellular cholesterol could predict the subse-quent cholesterol accumulation caused by atherogenic LDL: the higher was baseline cholesterol content in cultured monocytes-macrophages, the higher additional amount of cholesterol the cells were able to accumulate (r=0.64, p<0.001). Since conventional cardiovascular risk factors failed to explain variability of intracellu-lar cholesterol accumulation, we have supposed that genetic back-ground may play a leading role. Our preliminary data demonstrate that integral mutation burden of mitochondrial genome can explain not less than 65% variability of intracellular cholesterol accumula-tion caused by atherogenic LDL. The further important areas of research should include the stud-ies evaluating the mechanistic role of mtDNA mutations in cellular and molecular mechanisms of atherogenesis, the changes in levels of oxidative DNA damage by means of specific drugs as well as chemopreventive agents (i.e. antioxidants), and the effects of such interventions on the initiation and progression of atherosclerosis. This understanding is of direct clinical relevance because increased mtDNA damage can be an important pathogenic factor, an addi-tional prognostic predictor, and a potential target of therapeutic strategies in atherosclerosis.

CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest.

ACKNOWLEDGEMENTS This study was supported by the Ministry of Education and Science of the Russian Federation, contract No. 11.519.11.2044.

ABBREVIATIONS AIF = Apoptosis-inducing factor APAF1 = Apoptotic protease-activating factor 1 BH4 = Tetrahydrobiopterin CHD = Coronary heart disease DRP1 = Dynamin-related protein 1 eNOS = Endothelial NO synthase KATP channel = Potassium ATP-dependent channel Mnf2 = Mitofusin 2


MOMP = Mitochondrial outer membrane permeability mtDNA = Mitochondrial DNA NARP = Neurogenic muscle weakness, ataxia and

retinitis pigmentosa (mitochondrial disease) NO = Nitric oxide NRF-1 = Nuclear respiratory factor 1 8-OHdG = 8-hydroxy-2’-deoxyguanosine ONOO- = Peroxynitrite anion OPA1 = Optic atrophy 1 protein OXPHOS = Oxidative phosphorylation PAR = Poly(ADP-ribose) PARP-1 = Poly(ADP-ribose) polymerase-1 PGC-1� = Peroxisome proliferator-activated receptor �

coactivator-1�PMBC = Peripheral blood mononuclear cell ROS = Reactive oxygen species TNF-� = Tumor necrosis factor �

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Received: January 28, 2013 Accepted: February 14, 2013

Mitochondrial Mutations in Atherosclerosis: New Solutions in Research and Possible Clinical Applications

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