Hydrogen peroxide: a metabolic by-product or a common mediator of ageing signals?

The reactive oxygen species that are generated by mitochondrial respiration, including hydrogen peroxide (H2O2), are potent inducers of oxidative damage and mediators of ageing. It is not clear, however, whether oxidative stress is the result of a genetic programme or the by-product of physiological processes. Recent findings demonstrate that a fraction of mitochondrial H2O2, produced by a specialized enzyme as a signalling molecule in the pathway of apoptosis, induces intracellular oxidative stress and accelerates ageing. We propose that genes that control H2O2 production are selected determinants of lifespan.

Diagnostic and Prognostic Utility of Circulating Cytochrome c in Acute Myocardial Infarction.

RATIONALE: In contrast to cardiomyocyte necrosis, which can be quantified by cardiac troponin, functional cardiomyocyte impairment, including mitochondrial dysfunction, has escaped clinical recognition in acute myocardial infarction (AMI) patients. OBJECTIVE: To investigate the diagnostic accuracy for AMI and prognostic prediction of in-hospital mortality of cytochrome c. METHODS AND RESULTS: We prospectively assessed cytochrome c serum levels at hospital presentation in 2 cohorts: a diagnostic cohort of patients presenting with suspected AMI and a prognostic cohort of definite AMI patients. Diagnostic accuracy for AMI was the primary diagnostic end point, and prognostic prediction of in-hospital mortality was the primary prognostic end point. Serum cytochrome c had no diagnostic utility for AMI (area under the receiver-operating characteristics curve 0.51; 95% confidence intervals 0.44-0.58; P=0.76). Among 753 AMI patients in the prognostic cohort, cytochrome c was detectable in 280 (37%) patients. These patients had higher in-hospital mortality than patients with nondetectable cytochrome c (6% versus 1%; P<0.001). This result was mainly driven by the high mortality rate observed in ST-segment-elevation AMI patients with detectable cytochrome c, as compared with those with nondetectable cytochrome c (11% versus 1%; P<0.001). At multivariable analysis, cytochrome c remained a significant independent predictor of in-hospital mortality (odds ratio 3.0; 95% confidence interval 1.9-5.7; P<0.001), even after adjustment for major clinical confounders (odds ratio 4.01; 95% confidence interval 1.20-13.38; P=0.02). CONCLUSIONS: Cytochrome c serum concentrations do not have diagnostic but substantial prognostic utility in AMI.

p66Shc deletion or deficiency protects from obesity but not metabolic dysfunction in mice and humans.

AIMS/HYPOTHESIS: Oxygen radicals generated by p66Shc drive adipogenesis, but contradictory data exist on the role of p66Shc in the development of obesity and the metabolic syndrome. We herein explored the relationships among p66Shc, adipose tissue remodelling and glucose metabolism using mouse models and human adipose tissue samples. METHODS: In wild-type (WT), leptin-deficient (ob/ob), p66Shc(-/-) and p66Shc(-/-) ob/ob mice up to 30 weeks of age, we analysed body weight, subcutaneous and visceral adipose tissue histopathology, glucose tolerance and insulin sensitivity, and liver and muscle fat accumulation. A group of mice on a high fat diet (HFD) was also analysed. A parallel study was conducted on adipose tissue collected from patients undergoing elective surgery. RESULTS: We found that p66Shc(-/-) mice were slightly leaner than WT mice, and p66Shc(-/-) ob/ob mice became less obese than ob/ob mice. Despite their lower body weight, p66Shc(-/-) mice accumulated ectopic fat in the liver and muscles, and were glucose intolerant and insulin resistant. Features of adverse adipose tissue remodelling induced by obesity, including adipocyte enlargement, apoptosis, inflammation and perfusion were modestly and transiently improved by p66Shc (also known as Shc1) deletion. After 12 weeks of the HFD, p66Shc(-/-) mice were leaner than but equally glucose intolerant and insulin resistant compared with WT mice. In 77 patients, we found a direct correlation between BMI and p66Shc protein levels. Patients with low p66Shc levels were less obese, but were not protected from other metabolic syndrome features (diabetes, dyslipidaemia and hypertension). CONCLUSIONS/INTERPRETATION: In mice and humans, reduced p66Shc levels protect from obesity, but not from ectopic fat accumulation, glucose intolerance and insulin resistance.

Dietary intake of cyanidin-3-glucoside induces a long-lasting cardioprotection from ischemia/reperfusion injury by altering the microbiota.

The anthocyanin class of flavonoids, including cyanidin-3-glucoside (C3G) present in berries, blood oranges and pigmented cereal crops, are food bioactives with antioxidant and anti-inflammatory action, capable to reduce myocardial ischemia/reperfusion (I/R) injury by unclear mechanism. Assessing the value of sporadic beneficial diet is critical for practical application. We aimed to determine whether and how the cardioptotective effect of dietary intake of anthocyanins persists. Gene expression, histology and resistance to I/R were investigated ex vivo in hearts from mice after a month beyond the cease of the C3G-enriched diet. Cardiac injury, oxidative stress and mitochondrial damage following I/R was effectively reduced in mice fed C3G-enriched diet, even after a month of wash out with standard diet. Cardioprotection was observed also in immune-deficient mice lacking mature B and T cells indicating the anti-inflammatory activity of C3G was not involved. Moreover, the transcription reprogramming induced by the C3G-enriched diets was rescued by the wash out treatment. Instead, we found C3G-enriched diet changed the microbiome and the transplantation of the fecal microbiota transferred the cardioprotection from mice fed C3G-enriched diet to mice fed standard diet. These findings established the effect of C3G dietary intake on gut microbiota determines long lasting cardioprotection.

Iron deficiency in patients with ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention.

BACKGROUND: Iron deficiency (ID) is a known co-morbidity and a potential therapeutic target in heart failure. Whether ID is frequent also in ST-segment elevation acute myocardial infarction (STEMI) patients and is associated with worse in-hospital outcomes has never been evaluated. METHODS: We defined ID as a serum ferritin < 100 µg/L or transferrin saturation < 20% at hospital admission. We assessed the association between ID and the primary endpoint (a composite of in-hospital mortality and Killip class ≥ 3). We explored the potential association between ID, circulating cell-free mitochondrial DNA (mtDNA), and cardiac magnetic resonance (CMR) parameters. RESULTS: Four-hundred-twenty STEMI patients undergoing primary percutaneous coronary intervention (pPCI) were included. Of them, 237 (56%) had ID. They had significantly higher admission high-sensitivity troponin and mtDNA levels as compared to non-ID patients (145 ±â€¯35 vs. 231 ±â€¯66 ng/L, P < 0.001; 917 [404-1748] vs. 1368 [908-4260] copies/µL; P < 0.003, respectively). A lower incidence of the primary endpoint (10% vs. 18%, P = 0.01) was observed in ID patients (adjusted OR 0.50 [95% CI 0.27-0.93]; P = 0.02). At CMR (n = 192), ID patients had a similar infarct size (21 ±â€¯18 vs. 21 ±â€¯19 g; P = 0.95), but a higher myocardial salvage index (0.56 ±â€¯0.30 vs. 0.43 ±â€¯0.27; P = 0.002), and a smaller microvascular obstruction extent (3.6 ±â€¯2.2 vs. 6.9 ±â€¯3.9 g; P < 0.001). CONCLUSIONS: Iron deficiency is frequent in STEMI patients, it is coupled with mitochondrial injury, and, paradoxically, with a better in-hospital outcome. This unexpected clinical result seems to be associated with a smaller myocardial reperfusion injury. The mechanisms underlying our findings and their potential clinical implications warrant further investigation.

Mitochondrial DNA copy number is regulated by cellular proliferation: a role for Ras and p66(Shc).

The abundance of mitochondria is regulated by biogenesis and division. These processes are controlled by cellular factors, given that, for example, mitochondria have to replicate their DNA prior to cell division. However, the mechanisms that allow a synchronization of cell proliferation with mitochondrial genome replication are still obscure. We report here our investigations on the role of proliferation and the contribution of Ras and p66Shc in the regulation of mitochondrial DNA copy number. Ras proteins mediate a variety of receptor-transduced mitogenic signals and appear to play an essential role in the cellular response to growth factors. P66Shc is a genetic determinant of life span in mammals and has been implicated in the regulation of receptor signaling and various mitochondrial functions. First, we confirmed previous reports showing that mitochondrial DNA is replicated during a specific phase of the cell cycle (the pre-S phase) and provided novel evidences that this process is regulated by mitogenic growth factors. Second, we showed that mitochondrial DNA replication is activated following Ras-induced cellular hyper-proliferation. Finally, we showed that p66Shc expression induces mitochondrial DNA replication, both in vitro and in vivo. We suggest that mitochondria are target of intracellular signaling pathways leading to proliferation, involving Ras and p66Shc, which might function to integrate cellular bio-energetic requirements and the inheritance of mitochondrial DNA in a cell cycle-dependent manner.

A p53-p66Shc signalling pathway controls intracellular redox status, levels of oxidation-damaged DNA and oxidative stress-induced apoptosis.

Correlative evidence links stress, accumulation of oxidative cellular damage and ageing in lower organisms and in mammals. We investigated their mechanistic connections in p66Shc knockout mice, which are characterized by increased resistance to oxidative stress and extended life span. We report that p66Shc acts as a downstream target of the tumour suppressor p53 and is indispensable for the ability of stress-activated p53 to induce elevation of intracellular oxidants, cytochrome c release and apoptosis. Other functions of p53 are not influenced by p66Shc expression. In basal conditions, p66Shc-/- and p53-/- cells have reduced amounts of intracellular oxidants and oxidation-damaged DNA. We propose that steady-state levels of intracellular oxidants and oxidative damage are genetically determined and regulated by a stress-induced signal transduction pathway involving p53 and p66Shc.

p66Shc, mitochondria, and the generation of reactive oxygen species.

Reactive oxygen species (ROS), mainly originated from mitochondrial respiration, are critical inducers of oxidative damage and involved in tissue dysfunction. It is not clear, however, whether oxidative stress is the result of an active gene program or it is the by-product of physiological processes. Recent findings demonstrate that ROS are produced by mitochondria in a controlled way through specialized enzymes, including p66Shc, and take part in cellular process aimed to ensure adaptation and fitness. Therefore, genes generating specifically ROS are selected determinants of life span in response to different environmental conditions.

Electrochemical study of hydrogen peroxide formation in isolated mitochondria.

Mitochondrial respiration generates reactive oxygen species that are involved in physiological and pathological processes. The majority of methods, with exception of electron paramagnetic resonance, used to evaluate the identity, the rate and the conditions of the reactive oxygen species produced by mitochondria, are mainly based on oxidation sensitive markers. Following latest electrochemical methodology, we implemented a novel electrochemical assay for the investigation of aerobic metabolism in preparations of isolated mitochondria through simultaneous measurement of O2 consumption and reactive species production. This electrochemical assay reveals active H2O2 production by respiring mouse liver mitochondria, and shows that ATP synthase activation and moderate depolarization increase the rate of H2O2 formation, suggesting that ATP synthesizing (state 3) mitochondria might contribute to oxidative stress or signaling.

The p66Shc knocked out mice are short lived under natural condition.

Deletion of the p66(Shc) gene results in lean and healthy mice, retards aging, and protects from aging-associated diseases, raising the question of why p66(Shc) has been selected, and what is its physiological role. We have investigated survival and reproduction of p66(Shc)-/- mice in a population living in a large outdoor enclosure for a year, subjected to food competition and exposed to winter temperatures. Under these conditions, deletion of p66(Shc) was strongly counterselected. Laboratory studies revealed that p66(Shc)-/- mice have defects in fat accumulation, thermoregulation, and reproduction, suggesting that p66(Shc) has been evolutionarily selected because of its role in energy metabolism. These findings imply that the health impact of targeting aging genes might depend on the specific energetic niche and caution should be exercised against premature conclusions regarding gene functions that have only been observed in protected laboratory conditions.

Circulating cytochrome c as potential biomarker of impaired reperfusion in ST-segment elevation acute myocardial infarction.

In patients with ST-segment elevation acute myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (pPCI), abrupt reperfusion can induce myocardial injury and apoptotic cell death. Reperfusion-induced myocardial damage, however, cannot be easily evaluated in clinical practice because of the lack of specific biomarkers. Cytochrome c, a mitochondrial protein, is released on reperfusion into the cytosol, where it triggers the apoptotic process. It can reach the external fluid and circulating blood when cell rupture occurs. We measured the cytochrome c circulating levels in patients with STEMI undergoing pPCI, and correlated them with the clinical signs of myocardial necrosis and reperfusion. The plasma creatine kinase-MB mass and serum cytochrome c (enzyme-linked immunosorbent assay method) were serially measured in 55 patients with STEMI undergoing pPCI. The angiographic and electrocardiographic signs of myocardial reperfusion were also assessed. Cytochrome c transiently increased in all patients with STEMI, with a curve that paralleled that of creatine kinase-MB. A significant relation was found between the peak values of the 2 biomarkers (R = 0.35, p = 0.01) and between the areas under the 2 curves (R = 0.33, p = 0.02). The creatine kinase-MB peak value correlated significantly with the clinical features of infarct extension. In contrast, the cytochrome c peak value correlated inversely with the myocardial blush grade. Patients with clinical signs of myocardial reperfusion injury had a significantly greater cytochrome c peak value than patients without reperfusion injury (median 1.65 ng/ml, interquartile range 1.20 to 2.20, vs 1.1 ng/ml, interquartile range 0.65 to 1.55; p = 0.04). In conclusion, serum cytochrome c is detectable in the early phase of STEMI treated with pPCI and is associated with clinical signs of impaired myocardial reperfusion.

P66Shc signals to age.

Oxygen metabolism is thought to impact on aging through the formation of reactive oxygen species (ROS) that are supposed to damage biological molecules. The study of p66(Shc), a crucial regulator of ROS level involved in aging dysfunction, suggests that the incidence of degenerative disease and longevity are determined by a specific signaling function of ROS other than their unspecific damaging property.

p66Shc-generated oxidative signal promotes fat accumulation.

Reactive oxygen species (ROS) and insulin signaling in the adipose tissue are critical determinants of aging and age-associated diseases. It is not clear, however, if they represent independent factors or they are mechanistically linked. We investigated the effects of ROS on insulin signaling using as model system the p66(Shc)-null mice. p66(Shc) is a redox enzyme that generates mitochondrial ROS and promotes aging in mammals. We report that insulin activates the redox enzyme activity of p66(Shc) specifically in adipocytes and that p66(Shc)-generated ROS regulate insulin signaling through multiple mechanisms, including AKT phosphorylation, Foxo localization, and regulation of selected insulin target genes. Deletion of p66(Shc) resulted in increased mitochondrial uncoupling and reduced triglyceride accumulation in adipocytes and in vivo increased metabolic rate and decreased fat mass and resistance to diet-induced obesity. In addition, p66(Shc-/-) mice showed impaired thermo-insulation. These findings demonstrate that p66(Shc)-generated ROS regulate the effect of insulin on the energetic metabolism in mice and suggest that intracellular oxidative stress might accelerate aging by favoring fat deposition and fat-related disorders.

A hyperstructure approach to mitochondria.

Several questions in our understanding of mitochondria are unanswered. These include how the ratio of mitochondrial (mt)DNA to mitochondria is maintained, how the accumulation of defective, rapidly replicating mitochondrial DNA is avoided, how the ratio of mitochondria to cells is adjusted to fit cellular needs, and why any proteins are synthesized in mitochondria rather than simply imported. In bacteria, large hyperstructures or assemblies of proteins, mRNA, lipids and ions have been proposed to constitute a level of organization intermediate between macromolecules and whole cells. Here, we suggest how the concept of hyperstructures together with other concepts developed for bacteria such as transcriptional sensing and spontaneous segregation may provide answers to mitochondrial problems. In doing this, we show how the problem of the very existence of mtDNA brings its own solution.

The life span determinant p66Shc localizes to mitochondria where it associates with mitochondrial heat shock protein 70 and regulates trans-membrane potential.

P66Shc regulates life span in mammals and is a critical component of the apoptotic response to oxidative stress. It functions as a downstream target of the tumor suppressor p53 and is indispensable for the ability of oxidative stress-activated p53 to induce apoptosis. The molecular mechanisms underlying the apoptogenic effect of p66Shc are unknown. Here we report the following three findings. (i) The apoptosome can be properly activated in vitro in the absence of p66Shc only if purified cytochrome c is supplied. (ii) Cytochrome c release after oxidative signals is impaired in the absence of p66Shc. (iii) p66Shc induces the collapse of the mitochondrial trans-membrane potential after oxidative stress. Furthermore, we showed that a fraction of cytosolic p66Shc localizes within mitochondria where it forms a complex with mitochondrial Hsp70. Treatment of cells with ultraviolet radiation induced the dissociation of this complex and the release of monomeric p66Shc. We propose that p66Shc regulates the mitochondrial pathway of apoptosis by inducing mitochondrial damage after dissociation from an inhibitory protein complex. Genetic and biochemical evidence suggests that mitochondria regulate life span through their effects on the energetic metabolism (mitochondrial theory of aging). Our data suggest that mitochondrial regulation of apoptosis might also contribute to life span determination.