**2.1. Mitochondrion – organelle with two faces**

According to mitochondrial theory of aging, mitochondria are both the main source and targets of detrimental reactions initiated in association with age-dependent deterioration of the cellular functions. Reactions leading to increased ROS generation, mtDNA mutations, oxidation of mitochondrial proteins and lipids result in subsequent induction of apoptotic events, impaired oxidative phosphorylation capacity, mitochondrial dynamics, and autophagy [22]. In addition, mitochondrial function may be affected by subject parameters like physical activity history [23], caloric restriction [21], drugs [24] and various comorbidities including obesity [25], insulin resistance and hypertension [26]. The primary function of mitochondria is to produce adenosine triphosphate (ATP) by the process of oxidative phosphorylation. In fact, about 90–95% of cellular oxygen is used up in oxidative phosphorylation and 3% from that pool can be converted to superoxide anion radical (O2 •−). This is a very strong argument to mitochondria as a main source of this oxygen radical [27]. Among the most relevant ROS sources in heart belong NADPH oxidases (NOX) and mitochondria [28]. Recent studies demonstrate that mitochondrial ROS play a critical role in mediating the cellular effects of angiotensin II in the cardiovascular system [29]. Angiotensin II binds to angiotensin receptor 1, thereby activating NOX isoform 2 and 4 leading to increased mitochondrial ROS production in vascular endothelial cells as well as in cardiac myocytes [30].

Two principal scenarios can be envisioned that favor increased mitochondrial ROS-formation: increased formation of O2 •− at the electron transport chain (ETC) and decreased elimination of O2 •− or hydrogen peroxide (H2 O2 ) in the mitochondrial matrix. In heart failure, the first scenario occurs when modifications of ETC complexes like disturbed stoichiometry and PTMs hamper electron flux along the ETC to provoke excessive O<sup>2</sup> •− formation [31] mostly by NADH dehydrogenase (complex I) and Cytochrome c reductase (complex III) causing functional uncoupling of the respiratory chain. The extent and way of individual ETC complexes inhibition is different. According to **Figure 1**, the decline in activity of complex I and cytochrome c oxidase (complex IV) was more obvious during aging when compared to the succinate dehydrogenase (complex II) and complex III activities. Literature data are inconsistent among the studies, mainly due to differences in the experimental age groups and animal models, isolation/purity of mitochondria or enzyme substrate/inhibitor used for study. Complex I is considered to be the most important player in the game of ROS production and/ or proton-motive cascade. Loss of its activity has been attributed to the mutations in mitochondrial DNA (mtDNA) in aging animals and recently was linked to the apoptotic cell death pathway [32].

However, the protein content in homogenates as well as in isolated mitochondrial fraction was maintained during overall aging process comparing the samples from old (14 months

Further, senescent cells exhibit enlarged nuclei and lysosomes, which possess elevated senescence-associated β-galactosidase activity (SA-β-Gal), the most widely used marker [17]. They also enter a proliferative arrest state, detected by cell cycle inhibitor levels such as p53/p21, tumor suppressor p16INK4a [18, 19] and markers of proliferation like Ki-67 and 5-bromodeoxyuridine [20]. Other factors secreted during senescence are cytokines, chemokines, growth factors, proteases, fibronectin as well as ROS and reactive nitrogen species (RNS). Additionally, proteostatic changes during senescence accompanied by an increase in modified proteins, accumulation of protein aggregates and reduced functionality of the proteasomal and autophagy systems [3] will be discussed in following chapters. Only two parameters currently correlate with species longevity in the right sense: the mitochondrial rate of ROS production and the degree of fatty acid unsaturation of tissue membranes. Their basal level is in both cases low in long-lived animals. In addition, the best-known manipulation that extends longevity, dietary restriction, also decreases the rate of mitochondrial ROS production and oxidative damage to mtDNA [21]. The available information supports a mitochondrial free radical theory of aging focused on low generation of endogenous damage and low sensitivity

According to mitochondrial theory of aging, mitochondria are both the main source and targets of detrimental reactions initiated in association with age-dependent deterioration of the cellular functions. Reactions leading to increased ROS generation, mtDNA mutations, oxidation of mitochondrial proteins and lipids result in subsequent induction of apoptotic events, impaired oxidative phosphorylation capacity, mitochondrial dynamics, and autophagy [22]. In addition, mitochondrial function may be affected by subject parameters like physical activity history [23], caloric restriction [21], drugs [24] and various comorbidities including obesity [25], insulin resistance and hypertension [26]. The primary function of mitochondria is to produce adenosine triphosphate (ATP) by the process of oxidative phosphorylation. In fact, about 90–95% of cellular oxygen is used up in oxidative phosphorylation and 3% from that

mitochondria as a main source of this oxygen radical [27]. Among the most relevant ROS sources in heart belong NADPH oxidases (NOX) and mitochondria [28]. Recent studies demonstrate that mitochondrial ROS play a critical role in mediating the cellular effects of angiotensin II in the cardiovascular system [29]. Angiotensin II binds to angiotensin receptor 1, thereby activating NOX isoform 2 and 4 leading to increased mitochondrial ROS production

Two principal scenarios can be envisioned that favor increased mitochondrial ROS-formation:

scenario occurs when modifications of ETC complexes like disturbed stoichiometry and

•− at the electron transport chain (ETC) and decreased elimination

) in the mitochondrial matrix. In heart failure, the first

•−). This is a very strong argument to

old) and adult (6 months old) rat hearts.

64 Mitochondrial Diseases

of membranes to oxidation in long-lived animals.

**2.1. Mitochondrion – organelle with two faces**

pool can be converted to superoxide anion radical (O2

increased formation of O2

•− or hydrogen peroxide (H2

of O2

in vascular endothelial cells as well as in cardiac myocytes [30].

O2

Reduced complex I activity was seen in the rat heart [33], brain synaptic mitochondria [34] as well as continual decrease in the frontal cortex of Parkinson disease patients [35]. It is important to note that the activity and stability of this respiratory complex are determined by its abundance, PTMs and/or specific protein–protein interactions. In contrary, other studies have reported no age-related decrease in complex I activity [36]. Most of the inconsistencies are related to the complexes II, III and IV, where activities of respiratory complexes have been also shown to decline [37], remain unchanged or increased during aging [38]. Our data show age-dependent decrease in all ETC enzyme activities, although the extent of inhibition is different (**Figure 1**). Among them, complex IV was most affected throughout aging and reached 63.4% of adult respiring mitochondria. While complexes I, II and III maintained activities in 14 months old rat mitochondria, cytochrome c oxidase showed deprivation by

**Figure 1.** Activities of ETC complexes in heart mitochondria during aging (yet unpublished data). Values are expressed as Mean ± SEM of 5 individual experiments, \*\*p < 0.01, \*\*\*p < 0.001; significantly different in comparison to 6 months old rats.

21.6% (p < 0.01) when compared to the adults. Unique role in both, Krebs cycle and ETC, has in inner mitochondrial membrane (IMM) embedded complex II. Its function is cardiolipinindependent and is not taking part in respiratory supercomplexes but has been identified as an isolated entity in mildly solubilized mitochondrial membranes [39]. This confirms the least damage by 13.6% in senescent mitochondria when compared to the other respiratory complexes. Interestingly, recent works described the direct connection of the O2 •− formation and respiratory chain complex II [40] when lack of succinate was present. At saturated succinate concentration and high membrane potential complex II is tightly bound with reverse transfer of electrons to O2 •− -producing complex I [41].

Several factors have impact on ETC with specific role of mtDNA. ETC complexes are composed of both nuclear DNA-encoded (more than 80 proteins subunits) and 13 mtDNA-encoded subunits proteins. Respiratory chain and F<sup>1</sup> F0 -ATPase deficits are adverse effects on a variety of cellular and tissue functions, causing a wide range of complex clinical phenotypes. The incidence of inherited mitochondrial diseases is estimated to be about 1 in 5000 but a much larger population may be affected when somatic genetic defects, such as mtDNA mutation and deletions accompanying normal aging, are considered. The frequency of the common 4977-bp mtDNA deletion, a typical consequence of oxidative stress [42], increases with age in the human heart and is estimated to be 5- to 15-fold higher in people over 40 years of age relative to younger individuals [43]. This deletion affects genes encoding 7 polypeptide components of the mitochondrial ETC. Bioenergetic consequences of 4977-bp deletions will be reflected when the proportion of deleted mtDNA exceeds 50–55% of total mtDNA. The involvement of mtDNA mutations in cardiac aging is supported by findings in mice that express a proof reading deficient version of mtDNA polymerase (PolG) [44]. A high load of mtDNA mutations and deletions accumulate in the heart of these mice, in conjunction with the early onset of several age-associated changes including cardiac enlargement, fibrosis, impairment of systolic and diastolic function [30], and reduced activity of ETC complexes [44].

cycling. Therefore regeneration of NADPH is very important and depends on Krebs cycle enzymes, especially ICDH [46]. Interestingly, the activity of ICDH was affected only in senescent mitochondria but by a significant 42.5% decrease (from 246.52 ± 1.90 to 144.04 ± 5.58) μmol/ min/mg protein. Almost equivalent to ICDH activity in senescent hearts was drop in KGDH activity but this enzyme has shown gradual decline during whole aging process (**Figure 2**).

**Figure 2.** Activities of Krebs cycle enzymes in heart mitochondria during aging (yet unpublished data). Values are expressed as Mean ± SEM of 5 individual experiments, \*\*p < 0.01, \*\*\*p < 0.001; significantly different in comparison to

Interplay Between Mitochondrial Proteins and Age-Associated Risk of Cardiovascular Diseases

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67

drial Ca2+ uptake. Therefore, mitochondrial Ca2+ uptake critically regulates mitochondrial ROS production/removal [28] in cooperation with Krebs cycle enzymes and other ions to maintain mitochondrial homeostasis. Activation of ion channels in the IMM such as mitochondrial permeability transition pore (MPTP), the inner membrane anion channel (IMAC) and ATP-

electron flux required to maintain ATP production. Opening of these channels might promote ROS production, but interestingly they can be activated by ROS themselves. Moreover, O<sup>2</sup>

can be released from mitochondria via MPTP and IMAC. ROS in such a situation trigger oscillations of membrane potential leading to higher incidence of arrhythmias during reperfusion in the heart [47]. It seems that oxidative stress is one of the key events in myocardial senescence progression and development; however, during evolution cells were equipped with antioxidant defense mechanisms that can prevent/recover cells from an oxidative to a reductive state.

Mitochondria play important role in generation of ROS and RNS but they are themselves players in different signaling pathways, in which the mitochondrial oxidative defense system


promotes ROS-formation by reducing mitochon-

•−

In heart failure, elevated intracellular Na+

**3. Redox homeostasis in mitochondria**

dependent K+

6 months old rats.

To maintain proton-motive force and electron transfer through ETC, reduced equivalents are required. In mitochondria, the Krebs cycle generates NADH, which delivers electrons to the ETC inducing translocation of protons across the inner mitochondrial membrane. This establishes a membrane potential that fuels the F1 F0 -ATPase to generate ATP. At the ETC, electrons can leak to produce O2 • which is dismutated to H2 O2 by Mn-dependent superoxide dismutase (Mn-SOD) and, in turn, is detoxified by enzymes that require NADPH. Accordingly, equilibrium exists between reduced and oxidized forms of equivalents NADH/NAD<sup>+</sup> and NADPH/ NADP<sup>+</sup> [45].

In experiments, citrate synthase is frequently used as basal mitochondrial activity marker to avoid possible effects of different yield of mitochondria. Increase in its activity shown in **Figure 2** led to more pronounced activity changes in ETC complexes with age. Starting from citrate synthase, some of the Krebs cycle enzymes play inevitable role in reduced equivalents machinery, such as NAD(P)<sup>+</sup> -isocitrate dehydrogenase (ICDH) and α-ketoglutarate dehydrogenase (KGDH). Both of the enzymes share some common features as NADH production and they are regulated by calcium (Ca2+). Calcium uptake dynamically controls the redox state of NAD(P)H in working cardiac myocytes [28] and availability of NADPH together with reduced glutathione is required for H2 O2 removal by glutathione peroxidase/glutathione reductase

Interplay Between Mitochondrial Proteins and Age-Associated Risk of Cardiovascular Diseases http://dx.doi.org/10.5772/intechopen.71789 67

**Figure 2.** Activities of Krebs cycle enzymes in heart mitochondria during aging (yet unpublished data). Values are expressed as Mean ± SEM of 5 individual experiments, \*\*p < 0.01, \*\*\*p < 0.001; significantly different in comparison to 6 months old rats.

cycling. Therefore regeneration of NADPH is very important and depends on Krebs cycle enzymes, especially ICDH [46]. Interestingly, the activity of ICDH was affected only in senescent mitochondria but by a significant 42.5% decrease (from 246.52 ± 1.90 to 144.04 ± 5.58) μmol/ min/mg protein. Almost equivalent to ICDH activity in senescent hearts was drop in KGDH activity but this enzyme has shown gradual decline during whole aging process (**Figure 2**). In heart failure, elevated intracellular Na+ promotes ROS-formation by reducing mitochondrial Ca2+ uptake. Therefore, mitochondrial Ca2+ uptake critically regulates mitochondrial ROS production/removal [28] in cooperation with Krebs cycle enzymes and other ions to maintain mitochondrial homeostasis. Activation of ion channels in the IMM such as mitochondrial permeability transition pore (MPTP), the inner membrane anion channel (IMAC) and ATPdependent K+ -channel causes depolarization of IMM. This is accompanied by an increase in electron flux required to maintain ATP production. Opening of these channels might promote ROS production, but interestingly they can be activated by ROS themselves. Moreover, O<sup>2</sup> •− can be released from mitochondria via MPTP and IMAC. ROS in such a situation trigger oscillations of membrane potential leading to higher incidence of arrhythmias during reperfusion in the heart [47]. It seems that oxidative stress is one of the key events in myocardial senescence progression and development; however, during evolution cells were equipped with antioxidant defense mechanisms that can prevent/recover cells from an oxidative to a reductive state.

#### **3. Redox homeostasis in mitochondria**

21.6% (p < 0.01) when compared to the adults. Unique role in both, Krebs cycle and ETC, has in inner mitochondrial membrane (IMM) embedded complex II. Its function is cardiolipinindependent and is not taking part in respiratory supercomplexes but has been identified as an isolated entity in mildly solubilized mitochondrial membranes [39]. This confirms the least damage by 13.6% in senescent mitochondria when compared to the other respiratory com-

respiratory chain complex II [40] when lack of succinate was present. At saturated succinate concentration and high membrane potential complex II is tightly bound with reverse transfer

Several factors have impact on ETC with specific role of mtDNA. ETC complexes are composed of both nuclear DNA-encoded (more than 80 proteins subunits) and 13 mtDNA-encoded sub-

cellular and tissue functions, causing a wide range of complex clinical phenotypes. The incidence of inherited mitochondrial diseases is estimated to be about 1 in 5000 but a much larger population may be affected when somatic genetic defects, such as mtDNA mutation and deletions accompanying normal aging, are considered. The frequency of the common 4977-bp mtDNA deletion, a typical consequence of oxidative stress [42], increases with age in the human heart and is estimated to be 5- to 15-fold higher in people over 40 years of age relative to younger individuals [43]. This deletion affects genes encoding 7 polypeptide components of the mitochondrial ETC. Bioenergetic consequences of 4977-bp deletions will be reflected when the proportion of deleted mtDNA exceeds 50–55% of total mtDNA. The involvement of mtDNA mutations in cardiac aging is supported by findings in mice that express a proof reading deficient version of mtDNA polymerase (PolG) [44]. A high load of mtDNA mutations and deletions accumulate in the heart of these mice, in conjunction with the early onset of several age-associated changes including cardiac enlargement, fibrosis, impairment of sys-

To maintain proton-motive force and electron transfer through ETC, reduced equivalents are required. In mitochondria, the Krebs cycle generates NADH, which delivers electrons to the ETC inducing translocation of protons across the inner mitochondrial membrane. This estab-

F0

(Mn-SOD) and, in turn, is detoxified by enzymes that require NADPH. Accordingly, equilib-

In experiments, citrate synthase is frequently used as basal mitochondrial activity marker to avoid possible effects of different yield of mitochondria. Increase in its activity shown in **Figure 2** led to more pronounced activity changes in ETC complexes with age. Starting from citrate synthase, some of the Krebs cycle enzymes play inevitable role in reduced equivalents

genase (KGDH). Both of the enzymes share some common features as NADH production and they are regulated by calcium (Ca2+). Calcium uptake dynamically controls the redox state of NAD(P)H in working cardiac myocytes [28] and availability of NADPH together with reduced

O2

F0

tolic and diastolic function [30], and reduced activity of ETC complexes [44].

• which is dismutated to H2

O2

rium exists between reduced and oxidized forms of equivalents NADH/NAD<sup>+</sup>

•− formation and




removal by glutathione peroxidase/glutathione reductase

by Mn-dependent superoxide dismutase

and NADPH/

plexes. Interestingly, recent works described the direct connection of the O2

•− -producing complex I [41].

units proteins. Respiratory chain and F<sup>1</sup>

lishes a membrane potential that fuels the F1

can leak to produce O2

[45].

machinery, such as NAD(P)<sup>+</sup>

glutathione is required for H2

NADP<sup>+</sup>

of electrons to O2

66 Mitochondrial Diseases

Mitochondria play important role in generation of ROS and RNS but they are themselves players in different signaling pathways, in which the mitochondrial oxidative defense system contributes to maintain redox homeostasis. Cellular redox state is determined by the reduction potentials and reducing capacities of the redox couples, such as GSH/GSSG, NAD(P) H/NAD(P)<sup>+</sup> , thioredoxin (reduced/oxidized), glutaredoxin (reduced/oxidized) and cysteine/ cystine, From these the GSH/GSSG system is considered to be the most abundant among endogenous antioxidants with 2 to 4-fold higher abundance than other redox systems.

Aerobic respiration may result in an increase of H2

cal (GS•) has deleterious effects. It is able to take H<sup>+</sup>

O2

H2 O2

of H2 O2

responses.

ins (Prx). They react with H<sup>2</sup>

has Prx2 which traps almost all H<sup>2</sup>

O2

Interplay Between Mitochondrial Proteins and Age-Associated Risk of Cardiovascular Diseases

peroxidase (GPx), while GSH is recycled by the action of glutathione reductase (GR). Since both of the enzymes were affected in old as well as senescent rat hearts, overproduction of

thione redox potential, but also increase in amount of protein mixed disulfides. Addition of GSH to protein cysteine residues results to post-translational modification known as S-glutathionylation. It is a reversible process with potential to activate or inactivate protein function by modulating different cellular pathways. It is able to influence gene expression by affecting different transcription factors such as Nrf2 (nuclear factor erythroid 2-related factor 2) or NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) [52]. The Nrf2-Keap1 (Kelch-like ECH-associated protein 1) pathway is the major regulator of cytoprotective responses to oxidative and electrophilic stress. In the presence of ROS, critical cysteine residues in Keap1 become oxidized leading to a conformational change, which prevents its binding to Nrf2. As a consequence, Nrf2 degradation is stopped and its nuclear translocation promoted [53]. Recently was shown that S-glutathionylation of endothelial NO synthase (eNOS) at Cys689 and Cys908 leads to eNOS uncoupling, diminished NO production and enhanced oxidative stress linked with superoxide overproduction [54]. S-glutathionylation as well as GSH alone interacts with earlier mentioned nuclear factor Nrf2. This factor is key transcription factor of 4-hydroxy-2-nonenal (HNE). HNE is highly reactive aldehyde product of lipid peroxidation with potential to modulate function of proteins and lipids. The Nrf2 under stress conditions activates HNE-mediated antioxidant protection, when at a sub-lethal concentration 5 μmol/l HNE stimulates biosynthesis of GSH in cardiomyocytes. In contrary, glutathione after oxidation to the glutathione radi-

saturated fatty acids or to induce lipid peroxidation [55]. Altogether, S-glutathionylation may be a double-edged sword in the sense that it promotes antioxidant or pro-oxidant

Peroxidase activity is found in GPx and in the thiol-specific proteins called peroxiredox-

residue in the active site. Peroxiredoxins can also reduce and detoxify peroxynitrite anion and a wide range of organic hydroperoxides. The highest reaction rate and abundance

protein was maintained until the age of 14 months, but senescent mitochondria lost 35.1% of Prx2 amount (**Figure 3**). Oxidized cysteine residues of Prx are specifically reduced by Trx. Oxidized Trx as well as other oxidized cellular proteins can be reversibly reduced by TrxR in a NADPH-dependent manner. The Trx/TrxR system appears to have a protective function against oxidative stress, e.g. supports the activity of ribonucleotide reductase and inhibits apoptosis signal-regulated kinase-1 [57]. Thioredoxin reductase has been severely affected during aging process by 41.9% in group of 14 months old hearts with an extension until the age of 27 months (**Table 2**). The same scenario was observed in Trx protein level where the amount of protein has decreased to 62.4% in 14 months old as well as 27 months

or lack of reduced NADPH was present. Moreover, peroxidase-mediated elimination

will also augment the level of GSSG. This in turn, may not only lower the gluta-

, which is metabolized by glutathione

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69

from lipid side chains and polyun-

at a very high rate and their activity depends on cysteine

O2 *in vivo* [56]. The level of this powerful thiol-specific

#### **3.1. Thiol–disulfide redox state of mitochondria**

Reduced form of GSH (γ-L-glutamyl-L-cysteinyl glycine) is two electron donating molecule and in humans is almost uniquely present in a quite high concentration (1–10 mmol/L). This allows GSH to scavenge ROS either directly or indirectly. As an antioxidant, it reacts with reactive forms and radicals produced in association with electron transport, xenobiotic metabolism and inflammatory responses [48]. GSH homeostasis is not only regulated by its *de novo* synthesis, but also by other factors such as utilization, recycling and cellular export. Cooperation with other antioxidant, redox-related enzymes is important for recycling and maintenance of the optimal redox environment. It was reported that GSH level decreases over time in heart mitochondria [49] and several brain regions [50]. This phenomenon was confirmed by GSH measurement in three age groups (**Table 2**). Rapid decline of GSH level was accompanied by increase in oxidized GSSG form in senescent rat hearts. Interestingly, 14 months old hearts were able to maintain basal concentrations of that present in adult ones. The relative GSH/GSSG ratio indicates a decrease in GSH levels leading to more oxidized environment in senescent cardiomyocytes and experimentally dilated cardiomyopathy in mice [51]. The total content of thiol-containing compounds (R-SH), in contrary to GSH, decreases very slowly (by 19.2% in senescent) during aging process.


Values are expressed as means ± SEM of 5 individual experiments. \* p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001; significantly different in comparison to 6 months old rats.

**Table 2.** The content and activities of GSH cycle-related molecules in heart during aging (yet unpublished data).

Aerobic respiration may result in an increase of H2 O2 , which is metabolized by glutathione peroxidase (GPx), while GSH is recycled by the action of glutathione reductase (GR). Since both of the enzymes were affected in old as well as senescent rat hearts, overproduction of H2 O2 or lack of reduced NADPH was present. Moreover, peroxidase-mediated elimination of H2 O2 will also augment the level of GSSG. This in turn, may not only lower the glutathione redox potential, but also increase in amount of protein mixed disulfides. Addition of GSH to protein cysteine residues results to post-translational modification known as S-glutathionylation. It is a reversible process with potential to activate or inactivate protein function by modulating different cellular pathways. It is able to influence gene expression by affecting different transcription factors such as Nrf2 (nuclear factor erythroid 2-related factor 2) or NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) [52]. The Nrf2-Keap1 (Kelch-like ECH-associated protein 1) pathway is the major regulator of cytoprotective responses to oxidative and electrophilic stress. In the presence of ROS, critical cysteine residues in Keap1 become oxidized leading to a conformational change, which prevents its binding to Nrf2. As a consequence, Nrf2 degradation is stopped and its nuclear translocation promoted [53]. Recently was shown that S-glutathionylation of endothelial NO synthase (eNOS) at Cys689 and Cys908 leads to eNOS uncoupling, diminished NO production and enhanced oxidative stress linked with superoxide overproduction [54]. S-glutathionylation as well as GSH alone interacts with earlier mentioned nuclear factor Nrf2. This factor is key transcription factor of 4-hydroxy-2-nonenal (HNE). HNE is highly reactive aldehyde product of lipid peroxidation with potential to modulate function of proteins and lipids. The Nrf2 under stress conditions activates HNE-mediated antioxidant protection, when at a sub-lethal concentration 5 μmol/l HNE stimulates biosynthesis of GSH in cardiomyocytes. In contrary, glutathione after oxidation to the glutathione radical (GS•) has deleterious effects. It is able to take H<sup>+</sup> from lipid side chains and polyunsaturated fatty acids or to induce lipid peroxidation [55]. Altogether, S-glutathionylation may be a double-edged sword in the sense that it promotes antioxidant or pro-oxidant responses.

contributes to maintain redox homeostasis. Cellular redox state is determined by the reduction potentials and reducing capacities of the redox couples, such as GSH/GSSG, NAD(P)

cystine, From these the GSH/GSSG system is considered to be the most abundant among endogenous antioxidants with 2 to 4-fold higher abundance than other redox systems.

Reduced form of GSH (γ-L-glutamyl-L-cysteinyl glycine) is two electron donating molecule and in humans is almost uniquely present in a quite high concentration (1–10 mmol/L). This allows GSH to scavenge ROS either directly or indirectly. As an antioxidant, it reacts with reactive forms and radicals produced in association with electron transport, xenobiotic metabolism and inflammatory responses [48]. GSH homeostasis is not only regulated by its *de novo* synthesis, but also by other factors such as utilization, recycling and cellular export. Cooperation with other antioxidant, redox-related enzymes is important for recycling and maintenance of the optimal redox environment. It was reported that GSH level decreases over time in heart mitochondria [49] and several brain regions [50]. This phenomenon was confirmed by GSH measurement in three age groups (**Table 2**). Rapid decline of GSH level was accompanied by increase in oxidized GSSG form in senescent rat hearts. Interestingly, 14 months old hearts were able to maintain basal concentrations of that present in adult ones. The relative GSH/GSSG ratio indicates a decrease in GSH levels leading to more oxidized environment in senescent cardiomyocytes and experimentally dilated cardiomyopathy in mice [51]. The total content of thiol-containing compounds (R-SH), in contrary to GSH,

, thioredoxin (reduced/oxidized), glutaredoxin (reduced/oxidized) and cysteine/

**6 14 27**

p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001; significantly different

H/NAD(P)<sup>+</sup>

68 Mitochondrial Diseases

**3.1. Thiol–disulfide redox state of mitochondria**

decreases very slowly (by 19.2% in senescent) during aging process.

R-SH content 8.66 ± 0.46 7.12 ± 0.26 7.00 ± 0.09\* GSH content 3.00 ± 0.25 3.74 ± 0.39 1.68 ± 0.19\*\* GSSG content 1.77 ± 0.21 1.61 ± 0.25 3.81 ± 0.31\*\*

GPx activity 9.033 ± 0.860 8.239 ± 0.081\*\*\* 6.489 ± 0.112\*\*\* GR activity 4.790 ± 0.093 4.194 ± 0.306\*\* 3.960 ± 0.124\*\*\* TrxR activity 0.085 ± 0.018 0.050 ± 0.008\*\* 0.052 ± 0.013\*\*

**Table 2.** The content and activities of GSH cycle-related molecules in heart during aging (yet unpublished data).

GSH/GSSG ratio 1.64 2.10 0.51

**(μmol/g of tissue) Age (months)**

Values are expressed as means ± SEM of 5 individual experiments. \*

*Enzyme activity* (μmol/min/mg protein)

in comparison to 6 months old rats.

Peroxidase activity is found in GPx and in the thiol-specific proteins called peroxiredoxins (Prx). They react with H<sup>2</sup> O2 at a very high rate and their activity depends on cysteine residue in the active site. Peroxiredoxins can also reduce and detoxify peroxynitrite anion and a wide range of organic hydroperoxides. The highest reaction rate and abundance has Prx2 which traps almost all H<sup>2</sup> O2 *in vivo* [56]. The level of this powerful thiol-specific protein was maintained until the age of 14 months, but senescent mitochondria lost 35.1% of Prx2 amount (**Figure 3**). Oxidized cysteine residues of Prx are specifically reduced by Trx. Oxidized Trx as well as other oxidized cellular proteins can be reversibly reduced by TrxR in a NADPH-dependent manner. The Trx/TrxR system appears to have a protective function against oxidative stress, e.g. supports the activity of ribonucleotide reductase and inhibits apoptosis signal-regulated kinase-1 [57]. Thioredoxin reductase has been severely affected during aging process by 41.9% in group of 14 months old hearts with an extension until the age of 27 months (**Table 2**). The same scenario was observed in Trx protein level where the amount of protein has decreased to 62.4% in 14 months old as well as 27 months

mechanisms involved in resistance to various types of stress together with maintenance of bioenergetic capacity and redox homeostasis may be critical in the evolution of longevity.

Interplay Between Mitochondrial Proteins and Age-Associated Risk of Cardiovascular Diseases

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One of the major goals of gerontology is to understand the comprehensive mechanisms involved in aging at different levels and hopefully to help understand age-related diseases. The complexity of the proteome supersedes that of the genome, due to alternative splicing events and PTMs of proteins. Proteomes are expected to be two to three orders of magnitude more complex than would be predicted from numbers of protein-encoding genes present in the respective genomes [59]. It is widely recognized that cellular aging causes changes in the proteome. However, the nature and targets of these changes and their consequences have not yet been completely identified. In recent years, mass spectrometry (MS) has been recognized as a golden standard tool for the identification and analysis of individual proteins. For further understanding of the molecular changes during heart aging, we have identified several proteins and compared the differences in the mitochondrial protein expression profiles among

Precipitated proteins from mitochondria of 6 and 27 months old rat hearts were separated with two-dimensional electrophoresis (2-DE) to provide a protein profile (**Figure 4**). Interestingly, the change in protein level was statistically significant (1.5-fold change, 95% confidence interval) in only 12 proteins (marked with red circles) from the total protein pool of mitochondria. All the proteins were down-regulated in senescent mitochondria in comparison to the adult ones. Despite the small number of quantitatively modified proteins, these create an interesting protein-protein network. The strength of data support represents line thickness of protein-protein interaction network generated by String

**Figure 4.** Representative 2-DE analysis of mitochondrial proteins in aging rat heart (yet unpublished data).

**3.2. Mitochondrial protein-protein network**

two age groups.

**Figure 3.** Scheme of (A) Thiol-disulfide network and (B) protein level for Prx2, Trx (yet unpublished data). GSH-reduced glutathione, GSSG-oxidized glutathione, GPx-Glutathione peroxidase, GR-Glutathione reductase, Grx-Glutaredoxin, Prx-Peroxiredoxin, Trx-Thioredoxin, TrxR-Thioredoxin reductase, SH-reduced and SS-oxidized form of Protein, ICDHisocitrate dehydrogenase.

old rat hearts (**Figure 3**). Low Trx level is probably result of TrxR malfunctioning due to lack of NADPH, overproduction of ROS and/or RNS. Thioredoxin also plays a role in the reversible S-nitrosylation of cysteine residues in target proteins, and thereby contributes to the response of intracellular nitric oxide (NO). In addition, Trx is able to block caspase-3 activity through nitrosylation of the active cysteine site in response to NO. Therefore, Trx protein deficiency may contribute to the stimulation of caspase-dependent apoptosis. The most studied enzyme in the process of protein S-glutathionylation is glutaredoxin (Grx). The high specificity of Grx to S-glutathionylated proteins is used as a tool for studying and identifying them. In general, it is stated that the main task of Grx is to remove GSH from S-glutathionylated proteins. Thus, reduced thiol-containing protein is restored, which was confirmed in experiments with siRNA (small interfering RNA). The suppression of Grx genes with siRNA is an approach to study not only the "antioxidant" properties of Grx but also the role of protein S-glutathionylation [58]. Grx2 catalyzes S-glutathionylation of IMM proteins in a relatively reduced GSH/GSSG ratio equal to 6. The fact that thiol-disulfide oxidoreductases can catalyze both oxidizing and reducing reactions is not exclusive to Grx. Thioredoxin can act as an oxidant in oxidizing environment. Currently, the biggest challenge of researchers is to identify which enzymes are responsible for (de)glutathionylation, and if it is spontaneous process or catalyzed by enzymes. Existing information suggests that the mechanisms involved in resistance to various types of stress together with maintenance of bioenergetic capacity and redox homeostasis may be critical in the evolution of longevity.
