Endoplasmic reticulum stress-mediated mitochondrial dysfunction in aged hearts

Aging impairs the mitochondrial electron transport chain (ETC), especially in interfibrillar mitochondria (IFM). Mitochondria are in close contact with the endoplasmic reticulum (ER). Induction of ER stress leads to ETC injury in adult heart mitochondria. We asked if ER stress contributes to the mitochondrial dysfunction during aging. Subsarcolemmal mitochondria (SSM) and IFM were isolated from 3, 18, and 24 mo. C57Bl/6 mouse hearts. ER stress progressively increased with age, especially in 24 mo. mice that manifest mitochondrial dysfunction. OXPHOS was decreased in 24 mo. IFM oxidizing complex I and complex IV substrates. Proteomic analysis showed that the content of multiple complex I subunits was decreased in IFM from 24 mo. hearts, but remained unchanged in in 18 mo. IFM without a decrease in OXPHOS. Feeding 24 mo. old mice with 4-phenylbutyrate (4-PBA) for two weeks attenuated the ER stress and improved mitochondrial function. These results indicate that ER stress contributes to the mitochondrial dysfunction in aged hearts. Attenuation of ER stress is a potential approach to improve mitochondrial function in aged hearts.     

Age-related decline in mitochondrial bioenergetics: Does supercomplex destabilization determine lower oxidative capacity and higher superoxide production?

Mitochondrial decay plays a central role in the aging process. Although certainly multifactorial in nature, defective operation of the electron transport chain (ETC) constitutes a key mechanism involved in the age-associated loss of mitochondrial energy metabolism. Primarily, mitochondrial dysfunction affects the aging animal by limiting bioenergetic reserve capacity and/or increasing oxidative stress via enhanced electron leakage from the ETC. Even though the important aging characteristics of mitochondrial decay are known, the molecular events underlying inefficient electron flux that ultimately leads to higher superoxide appearance and impaired respiration are not completely understood. This review focuses on the potential role(s) that age-associated destabilization of the macromolecular organization of the ETC (i.e. supercomplexes) may be important for development of the mitochondrial aging phenotype, particularly in post-mitotic tissues.     

Mitochondrial effectors of cellular senescence: beyond the free radical theory of aging

Cellular senescence is a process that results from a variety of stresses, leading to a state of irreversible growth arrest. Senescent cells accumulate during aging and have been implicated in promoting a variety of age‐related diseases. Mitochondrial stress is an effective inducer of cellular senescence, but the mechanisms by which mitochondria regulate permanent cell growth arrest are largely unexplored. Here, we review some of the mitochondrial signaling pathways that participate in establishing cellular senescence. We discuss the role of mitochondrial reactive oxygen species (ROS), mitochondrial dynamics (fission and fusion), the electron transport chain (ETC), bioenergetic balance, redox state, metabolic signature, and calcium homeostasis in controlling cellular growth arrest. We emphasize that multiple mitochondrial signaling pathways, besides mitochondrial ROS, can induce cellular senescence. Together, these pathways provide a broader perspective for studying the contribution of mitochondrial stress to aging, linking mitochondrial dysfunction and aging through the process of cellular senescence.     

Induction of mitochondrial oxidative stress in astrocytes by nitric oxide precedes disruption of energy metabolism

Inhibition of the mitochondrial electron transport chain (ETC) ultimately limits ATP production and depletes cellular ATP. However, the individual complexes of the ETC in brain mitochondria need to be inhibited by approximately 50% before causing significant depression of ATP synthesis. Moreover, the ETC is the key site for the production of intracellular reactive oxygen species (ROS) and inhibition of one or more of the complexes of the ETC may increase the rate of mitochondrial ROS generation. We asked whether partial inhibition of the ETC, to a degree insufficient to perturb oxidative phosphorylation, might nonetheless induce ROS production. Chronic increase in mitochondrial ROS might then cause oxidative damage to the ETC sufficient to produce prolonged changes in ETC function and so compound the defect. We show that the exposure of astrocytes in culture to low concentrations of nitric oxide (NO) induces an increased rate of O2*- generation that outlasts the presence of NO. No effect was seen on oxygen consumption, lactate or ATP content over the 4-6 h that the cells were exposed to NO. These data suggest that partial ETC inhibition by NO may initially cause oxidative stress rather than ATP depletion, and this may subsequently induce irreversible changes in ETC function providing the basis for a cycle of damage.     

Lipid peroxidation associated cardiolipin loss and membrane depolarization in rat brain mitochondria

Oxidative stress induced by Fe2+ (50 microM) and ascorbate (2 mM) in isolated rat brain mitochondria incubated in vitro leads to an enhanced lipid peroxidation, cardiolipin loss and an increased formation of protein carbonyls. These changes are associated with a loss of mitochondrial membrane potential (depolarization) and an impaired activity of electron transport chain (ETC) as measured by MTT reduction assay. Butylated hydroxytoluene (0.2 mM), an inhibitor of lipid peroxidation, can prevent significantly the loss of cardiolipin, the increased protein carbonyl formation and the decrease in mitochondrial membrane potential induced by Fe2+ and ascorbate, implying that the changes are secondary to membrane lipid peroxidation. However, iron-ascorbate induced impairment of mitochondrial ETC activity is apparently independent of lipid peroxidation process. The structural and functional derangement of mitochondria induced by oxidative stress as reported here may have implications in neuronal damage associated with brain aging and neurodegenerative disorders.     

The efficiency of mitochondrial electron transport chain is increased in the long‐lived mrg19 Saccharomyces cerevisiae

Integrity of mitochondrial functionality is a key determinant of longevity in several organisms. In particular, reduced mitochondrial ROS (mtROS) production leading to decreased mtDNA damage is believed to be a crucial aspect of longevity. The generation of low mtROS was thought to be due to low mitochondrial oxygen consumption. However, recent studies have shown that higher mitochondrial oxygen consumption could still result in low mtROS and contribute to longevity. This increased mitochondrial efficiency (i.e. low mtROS generated despite high oxygen consumption) was explained as a result of mitochondrial biogenesis, which provides more entry points for the electrons to the electron transport chain (ETC), thereby resulting in low mtROS production. In this study, we provide evidence for the existence of an alternative pathway to explain the observed higher mitochondrial efficiency in the long‐lived mrg19 mutant of Saccharomyces cerevisiae. Although we observe similar amounts of mitochondria in mrg19 and wild‐type (wt) yeast, we find that mrg19 mitochondria have higher expression of ETC components per mitochondria in comparison with the wt. These findings demonstrate that more efficient mitochondria because of increased ETC per mitochondria can also produce less mtROS. Taken together, our findings provide evidence for an alternative explanation for the involvement of higher mitochondrial activity in prolonging lifespan. We anticipate that similar mechanisms might also exist in eukaryotes including human.     

Early mitochondrial dysfunction in long-lived Mclk1+/- mice

Reduced activity of CLK-1/MCLK1 (also known as COQ7), a mitochondrial enzyme that is necessary for ubiquinone biosynthesis, prolongs the lifespan of nematodes and mice by a mechanism that is distinct from that of the insulin signaling pathway. Here we show that 2-fold reduction of MCLK1 expression in mice reveals an additional function for the protein, as this level of reduction does not affect ubiquinone levels yet affects mitochondrial function substantially. Indeed, we observe that the phenotype of young Mclk1(+/-) mutants includes a severe reduction of mitochondrial electron transport, ATP synthesis, and total nicotinamide adenine dinucleotide (NAD(tot)) pool size as well as an alteration in the activity of key enzymes of the tricarboxylic acid cycle. Surprisingly, we also find that Mclk1 heterozygosity leads to a dramatic increase in mitochondrial oxidative stress by a variety of measures. Furthermore, we find that the mitochondrial dysfunction is accompanied by a decrease in oxidative damage to cytosolic proteins as well as by a decrease in plasma isoprostanes, a systemic biomarker of oxidative stress and aging. We propose a mechanism for the conjunction of low ATP levels, high mitochondrial oxidative stress, and low non-mitochondrial oxidative damage in a long-lived mutant. Our model helps to clarify the relationship between energy metabolism and the aging process and suggests the need for a reformulation of the mitochondrial oxidative stress theory of aging.     

Mitochondria and Organismal Longevity

Mitochondria are essential for various biological processes including cellular energy production. The oxidative stress theory of aging proposes that mitochondria play key roles in aging by generating reactive oxygen species (ROS), which indiscriminately damage macromolecules and lead to an age-dependent decline in biological function. However, recent studies show that increased levels of ROS or inhibition of mitochondrial function can actually delay aging and increase lifespan. The aim of this review is to summarize recent findings regarding the role of mitochondria in organismal aging processes. We will discuss how mitochondria contribute to evolutionarily conserved longevity pathways, including mild inhibition of respiration, dietary restriction, and target of rapamycin (TOR) signaling.     

The yeast complex I equivalent NADH dehydrogenase rescues pink1 mutants

Pink1 is a mitochondrial kinase involved in Parkinson's disease, and loss of Pink1 function affects mitochondrial morphology via a pathway involving Parkin and components of the mitochondrial remodeling machinery. Pink1 loss also affects the enzymatic activity of isolated Complex I of the electron transport chain (ETC); however, the primary defect in pink1 mutants is unclear. We tested the hypothesis that ETC deficiency is upstream of other pink1-associated phenotypes. We expressed Saccaromyces cerevisiae Ndi1p, an enzyme that bypasses ETC Complex I, or sea squirt Ciona intestinalis AOX, an enzyme that bypasses ETC Complex III and IV, in pink1 mutant Drosophila and find that expression of Ndi1p, but not of AOX, rescues pink1-associated defects. Likewise, loss of function of subunits that encode for Complex I-associated proteins displays many of the pink1-associated phenotypes, and these defects are rescued by Ndi1p expression. Conversely, expression of Ndi1p fails to rescue any of the parkin mutant phenotypes. Additionally, unlike pink1 mutants, fly parkin mutants do not show reduced enzymatic activity of Complex I, indicating that Ndi1p acts downstream or parallel to Pink1, but upstream or independent of Parkin. Furthermore, while increasing mitochondrial fission or decreasing mitochondrial fusion rescues mitochondrial morphological defects in pink1 mutants, these manipulations fail to significantly rescue the reduced enzymatic activity of Complex I, indicating that functional defects observed at the level of Complex I enzymatic activity in pink1 mutant mitochondria do not arise from morphological defects. Our data indicate a central role for Complex I dysfunction in pink1-associated defects, and our genetic analyses with heterologous ETC enzymes suggest that Ndi1p-dependent NADH dehydrogenase activity largely acts downstream of, or in parallel to, Pink1 but upstream of Parkin and mitochondrial remodeling.     

AKIP1 Expression Modulates Mitochondrial Function in Rat Neonatal Cardiomyocytes

A kinase interacting protein 1 (AKIP1) is a molecular regulator of protein kinase A and nuclear factor kappa B signalling. Recent evidence suggests AKIP1 is increased in response to cardiac stress, modulates acute ischemic stress response, and is localized to mitochondria in cardiomyocytes. The mitochondrial function of AKIP1 is, however, still elusive. Here, we investigated the mitochondrial function of AKIP1 in a neonatal cardiomyocyte model of phenylephrine (PE)-induced hypertrophy. Using a seahorse flux analyzer we show that PE stimulated the mitochondrial oxygen consumption rate (OCR) in cardiomyocytes. This was partially dependent on PE mediated AKIP1 induction, since silencing of AKIP1 attenuated the increase in OCR. Interestingly, AKIP1 overexpression alone was sufficient to stimulate mitochondrial OCR and in particular ATP-linked OCR. This was also true when pyruvate was used as a substrate, indicating that it was independent of glycolytic flux. The increase in OCR was independent of mitochondrial biogenesis, changes in ETC density or altered mitochondrial membrane potential. In fact, the respiratory flux was elevated per amount of ETC, possibly through enhanced ETC coupling. Furthermore, overexpression of AKIP1 reduced and silencing of AKIP1 increased mitochondrial superoxide production, suggesting that AKIP1 modulates the efficiency of electron flux through the ETC. Together, this suggests that AKIP1 overexpression improves mitochondrial function to enhance respiration without excess superoxide generation, thereby implicating a role for AKIP1 in mitochondrial stress adaptation. Upregulation of AKIP1 during different forms of cardiac stress may therefore be an adaptive mechanism to protect the heart.     

Age-related alterations in oxidatively damaged proteins of mouse heart mitochondrial electron transport chain complexes

Mitochondrially generated ROS increase with age and are a major factor that damages proteins by oxidative modification. Accumulation of oxidatively damaged proteins has been implicated as a causal factor in the age-associated decline in tissue function. Mitochondrial electron transport chain (ETC) complexes I and III are the principle sites of ROS production, and oxidative modifications to their complex subunits inhibit their in vitro activity. We hypothesize that mitochondrial complex subunits may be primary targets for modification by ROS, which may impair normal complex activity. This study of heart mitochondria from young, middle-aged, and old mice reveals that there is an age-related decline in complex I and V activity that correlates with increased oxidative modification to their subunits. The data also show a specificity for modifications of the ETC complex subunits, i.e., several proteins have more than one type of adduct. We postulate that the electron leakage from ETC complexes causes specific damage to their subunits and increased ROS generation as oxidative damage accumulates, leading to further mitochondrial dysfunction, a cyclical process that underlies the progressive decline in physiologic function of the aged mouse heart.     

Aging selectively decreases oxidative capacity in rat heart interfibrillar mitochondria

Mitochondrial-derived oxidative injury contributes to cellular aging as well as to reperfusion-induced tissue damage. While the aging-heart suffers greater tissue damage following ischemia and reperfusion than the adult heart, the occurrence of aging-related alterations in mitochondrial oxidative metabolism in the elderly heart has remained uncertain. We determined if aging altered oxidative metabolism in either of the two populations of cardiac mitochondria, subsarcolemmal mitochondria (SSM) that reside beneath the plasma membrane or interfibrillar mitochondria (IFM) located between the myofibrils. SSM and IFM were isolated from 6-month adult and 24- and 28-month elderly Fischer 344 rat hearts. Aging-related alterations were limited to IFM, while SSM remained unaffected. Aging decreased the rate of oxidative phosphorylation in IFM, including when stimulated by electron donors specific for cytochrome oxidase. Cytochrome oxidase enzyme activity was decreased in IFM from aging hearts, while activity in SSM remained similar to adult controls. These findings allow future studies of aging-related decrements in oxidative function to focus upon IFM, while SSM provide an inherent control group of mitochondria that are free of aging-related alterations in oxidative function. The selective alteration of IFM during aging raises the possibility that the consequences of aging-induced mitochondrial dysfunction will be enhanced in specific subcellular regions of the senescent myocyte.     

Cardiac mitochondrial bioenergetics, oxidative stress, and aging

Mitochondria have been a central focus of several theories of aging as a result of their critical role in bioenergetics, oxidant production, and regulation of cell death. A decline in cardiac mitochondrial function coupled with the accumulation of oxidative damage to macromolecules may be causal to the decline in cardiac performance with age. In contrast, regular physical activity and lifelong caloric restriction can prevent oxidative stress, delay the onset of morbidity, increase life span, and reduce the risk of developing several pathological conditions. The health benefits of life long exercise and caloric restriction may be, at least partially, due to a reduction in the chronic amount of mitochondrial oxidant production. In addition, the available data suggest that chronic exercise may serve to enhance antioxidant enzyme activities, and augment certain repair/removal pathways, thereby reducing the amount of oxidative tissue damage. However, the characterization of age-related changes to cardiac mitochondria has been complicated by the fact that two distinct populations of mitochondria exist in the myocardium: subsarcolemmal mitochondria and interfibrillar mitochondria. Several studies now suggest the importance of studying both mitochondrial populations when attempting to elucidate the contribution of mitochondrial dysfunction to myocardial aging. The role that mitochondrial dysfunction and oxidative stress play in contributing to cardiac aging will be discussed along with the use of lifelong exercise and calorie restriction as countermeasures to aging. superoxide anion; longevity; postmitotic; calorie restriction; subsarcolemmal, interfibrillar, exercise     

Mitochondrial morphology and dynamics in <Emphasis Type="Italic">Triticum aestivum</Emphasis> roots in response to rotenone and antimycin A

Mitochondria are dynamic organelles, capable of fusion and fission as a part of cellular responses to various signals, such as the shifts in the redox status of a cell. The mitochondrial electron transport chain (ETC.) is involved in the generation of reactive oxygen species (ROS), with complexes I and III contributing the most to this process. Disruptions of ETC. can lead to increased ROS generation. Here, we demonstrate the appearance of giant mitochondria in wheat roots in response to simultaneous application of the respiratory inhibitors rotenone (complex I of mitochondrial ETC.) and antimycin A (complex III of mitochondrial ETC.). The existence of such megamitochondria was temporary, and following longer treatment with inhibitors mitochondria resumed their conventional size and oval shape. Changes in mitochondrial morphology were accompanied with a decrease in mitochondrial potential and an unexpected increase in oxygen consumption. Changes in mitochondrial morphology and activity may result from the fusion and fission of mitochondria induced by the disruption of mitochondrial ETC. Results from experiments with the inhibitor of mitochondrial fission Mdivi-1 suggest that the retarded fission may facilitate plant mitochondria to appear in a fused shape. The processes of mitochondrial fusion and fission are involved in the regulation of the efficacy of the functions of the respiratory chain complexes and ROS metabolism during stresses. The changes in morphology of mitochondria, along with the changes in their functional activity, can be a part of the strategy of the plant adaptation to stresses.     

Age-related increases in oxidatively damaged proteins of mouse kidney mitochondrial electron transport chain complexes

Mitochondrial dysfunction generates reactive oxygen species (ROS) which damage essential macromolecules. Oxidative modification of proteins, DNA, and lipids has been implicated as a major causal factor in the age-associated decline in tissue function. Mitochondrial electron transport chain complexes I and III are the principal sites of ROS production, and oxidative modifications to the complex subunits inhibit their in vitro activity. Therefore, we hypothesize that mitochondrial complex subunits may be primary targets for oxidative damage by ROS which may impair normal complex activity by altering their structure/function leading to mitochondrial dysfunction associated with aging. This study of kidney mitochondria from young, middle-aged, and old mice reveals that there are functional decreases in complexes I, II, IV, and V between aged compared to young kidney mitochondria and these functional declines directly correlate with increased oxidative modification to particular complex subunits. We postulate that the electron leakage from complexes causes specific damage to their subunits and increased ROS generation as oxidative damage accumulates, leading to further mitochondrial dysfunction, a cyclical process that underlies the progressive decline in physiologic function seen in aged mouse kidney. In conclusion, increasing mitochondrial dysfunction may play a key role in the age-associated decline in tissue function.     

Biphasic modulation of the mitochondrial electron transport chain in myocardial ischemia and reperfusion

Mitochondrial electron transport chain (ETC) is the major source of reactive oxygen species during myocardial ischemia-reperfusion (I/R) injury. Ischemic defect and reperfusion-induced injury to ETC are critical in the disease pathogenesis of postischemic heart. The properties of ETC were investigated in an isolated heart model of global I/R. Rat hearts were subjected to ischemia for 30 min followed by reperfusion for 1 h. Studies of mitochondrial function indicated a biphasic modulation of electron transfer activity (ETA) and ETC protein expression during I/R. Analysis of ETAs in the isolated mitochondria indicated that complexes I, II, III, and IV activities were diminished after 30 min of ischemia but increased upon restoration of flow. Immunoblotting analysis and ultrastructural analysis with transmission electron microscopy further revealed marked downregulation of ETC in the ischemic heart and then upregulation of ETC upon reperfusion. No significant difference in the mRNA expression level of ETC was detected between ischemic and postischemic hearts. However, reperfusion-induced ETC biosynthesis in myocardium can be inhibited by cycloheximide, indicating the involvement of translational control. Immunoblotting analysis of tissue homogenates revealed a similar profile in peroxisome proliferator-activated receptor-γ coactivator-1α expression, suggesting its essential role as an upstream regulator in controlling ETC biosynthesis during I/R. Significant impairment caused by ischemic and postischemic injury was observed in the complexes I- III. Analysis of NADH ferricyanide reductase activity indicated that injury of flavoprotein subcomplex accounts for 50% decline of intact complex I activity from ischemic heart. Taken together, our findings provide a new insight into the molecular mechanism of I/R-induced mitochondrial dysfunction.     

Microtubule-targeted agents: when mitochondria become essential to chemotherapy

Microtubule-Targeting Agents (MTAs) constitute a class of drugs largely used for cancer treatment in adults and children. In cancer cells, they suppress microtubule dynamics, and induce cell death via the mitochondrial intrinsic pathway. To date, links between mitochondria and microtubule network disturbance in MTAs mechanism of action are not obvious. The aim of the present contribution is to provide elements that could answer to the question: how far are mitochondria essential to anticancer chemotherapy that targets the microtubule cytoskeleton? We review the main molecular candidates to link microtubule alteration with the apoptotic mitochondrial pathway control. Involvement of direct targeting of mitochondria in MTA efficacy is also discussed. Furthermore, we line up current evidence and emerging concepts on the participation of both mitochondria and microtubule in MTA neurotoxic side effects. To decipher the interconnections between the mitochondrial and the microtubule networks may help to improve cancer cell response to chemotherapy.