Hydrolytic Activity of Mitochondrial F1FO-ATP Synthase as a Target for Myocardial Ischemia-Reperfusion Injury: Discovery and In Vitro and In Vivo Evaluation of Novel Inhibitors.

F1FO-ATP synthase is the mitochondrial complex responsible for ATP production. During myocardial ischemia, it reverses its activity, hydrolyzing ATP and leading to energetic deficit and cardiac injury. We aimed to discover novel inhibitors of ATP hydrolysis, accessing the druggability of the target within ischemia(I)/reperfusion(R) injury. New molecular scaffolds were revealed using ligand-based virtual screening methods. Fifty-five compounds were tested on isolated murine heart mitochondria and H9c2 cells for their inhibitory activity. A pyrazolo[3,4-c]pyridine hit structure was identified and optimized in a hit-to-lead process synthesizing nine novel derivatives. Three derivatives significantly inhibited ATP hydrolysis in vitro, while in vivo, they reduced myocardial infarct size (IS). The novel compound 31 was the most effective in reducing IS, validating that inhibition of F1FO-ATP hydrolytic activity can serve as a target for cardioprotection during ischemia. Further examination of signaling pathways revealed that the cardioprotection mechanism is related to the increased ATP content in the ischemic myocardium and increased phosphorylation of PKA and phospholamban, leading to the reduction of apoptosis.

Targeting Oxidative Stress with Polyphenols to Fight Liver Diseases.

Reactive oxygen species (ROS) are important second messengers in many metabolic processes and signaling pathways. Disruption of the balance between ROS generation and antioxidant defenses results in the overproduction of ROS and subsequent oxidative damage to biomolecules and cellular components that disturb cellular function. Oxidative stress contributes to the initiation and progression of many liver pathologies such as ischemia-reperfusion injury (LIRI), non-alcoholic fatty liver disease (NAFLD), and hepatocellular carcinoma (HCC). Therefore, controlling ROS production is an attractive therapeutic strategy in relation to their treatment. In recent years, increasing evidence has supported the therapeutic effects of polyphenols on liver injury via the regulation of ROS levels. In the current review, we summarize the effects of polyphenols, such as quercetin, resveratrol, and curcumin, on oxidative damage during conditions that induce liver injury, such as LIRI, NAFLD, and HCC.

Downregulation of Sirtuin 1 Does Not Account for the Impaired Long-Term Potentiation in the Prefrontal Cortex of Female APPswe/PS1dE9 Mice Modelling Alzheimer's Disease.

Alzheimer's disease (AD), which predominantly affects women, involves at its onset a metabolic deregulation associated with a synaptic failure. Here, we performed a behavioral, neurophysiological and neurochemical characterization of 9-month-old female APPswe/PS1dE9 (APP/PS1) mice as a model of early AD. These animals showed learning and memory deficits in the Morris water maze, increased thigmotaxis and anxiety-like behavior and showed signs of fear generalization. Long-term potentiation (LTP) was decreased in the prefrontal cortex (PFC), but not in the CA1 hippocampus or amygdala. This was associated with a decreased density of sirtuin-1 in cerebrocortical synaptosomes and a decreased density of sirtuin-1 and sestrin-2 in total cerebrocortical extracts, without alterations of sirtuin-3 levels or of synaptic markers (syntaxin, synaptophysin, SNAP25, PSD95). However, activation of sirtuin-1 did not affect or recover PFC-LTP deficit in APP/PS1 female mice; instead, inhibition of sirtuin-1 increased PFC-LTP magnitude. It is concluded that mood and memory dysfunction in 9-month-old female APP/PS1 mice is associated with a parallel decrease in synaptic plasticity and in synaptic sirtuin-1 levels in the prefrontal cortex, although sirtiun1 activation failed to restore abnormal cortical plasticity.

Preservation of Mitochondrial Health in Liver Ischemia/Reperfusion Injury.

Liver ischemia-reperfusion injury (LIRI) is a major cause of the development of complications in different clinical settings such as liver resection and liver transplantation. Damage arising from LIRI is a major risk factor for early graft rejection and is associated with higher morbidity and mortality after surgery. Although the mechanisms leading to the injury of parenchymal and non-parenchymal liver cells are not yet fully understood, mitochondrial dysfunction is recognized as a hallmark of LIRI that exacerbates cellular injury. Mitochondria play a major role in glucose metabolism, energy production, reactive oxygen species (ROS) signaling, calcium homeostasis and cell death. The diverse roles of mitochondria make it essential to preserve mitochondrial health in order to maintain cellular activity and liver integrity during liver ischemia/reperfusion (I/R). A growing body of studies suggest that protecting mitochondria by regulating mitochondrial biogenesis, fission/fusion and mitophagy during liver I/R ameliorates LIRI. Targeting mitochondria in conditions that exacerbate mitochondrial dysfunction, such as steatosis and aging, has been successful in decreasing their susceptibility to LIRI. Studying mitochondrial dysfunction will help understand the underlying mechanisms of cellular damage during LIRI which is important for the development of new therapeutic strategies aimed at improving patient outcomes. In this review, we highlight the progress made in recent years regarding the role of mitochondria in liver I/R and discuss the impact of liver conditions on LIRI.

Loss of epigenetic information as a cause of mammalian aging.

All living things experience an increase in entropy, manifested as a loss of genetic and epigenetic information. In yeast, epigenetic information is lost over time due to the relocalization of chromatin-modifying proteins to DNA breaks, causing cells to lose their identity, a hallmark of yeast aging. Using a system called "ICE" (inducible changes to the epigenome), we find that the act of faithful DNA repair advances aging at physiological, cognitive, and molecular levels, including erosion of the epigenetic landscape, cellular exdifferentiation, senescence, and advancement of the DNA methylation clock, which can be reversed by OSK-mediated rejuvenation. These data are consistent with the information theory of aging, which states that a loss of epigenetic information is a reversible cause of aging.

Potent and Specific Activators for Mitochondrial Sirtuins Sirt3 and Sirt5.

Sirtuins are NAD+-dependent protein deacylases involved in metabolic regulation and aging-related diseases. Specific activators for seven human Sirtuin isoforms would be important chemical tools and potential therapeutic drugs. Activators have been described for Sirt1 and act via a unique N-terminal domain of this isoform. For most other Sirtuin isoforms, including mitochondrial Sirt3-5, no potent and specific activators have yet been identified. We here describe the identification and characterization of 1,4-dihydropyridine-based compounds that either act as pan Sirtuin activators or specifically stimulate Sirt3 or Sirt5. The activators bind to the Sirtuin catalytic cores independent of NAD+ and acylated peptides and stimulate turnover of peptide and protein substrates. The compounds also activate Sirt3 or Sirt5 in cellular systems regulating, e.g., apoptosis and electron transport chain. Our results provide a scaffold for potent Sirtuin activation and derivatives specific for Sirt3 and Sirt5 as an excellent basis for further drug development.

Shaping of Hepatic Ischemia/Reperfusion Events: The Crucial Role of Mitochondria.

Hepatic ischemia reperfusion injury (HIRI) is a major hurdle in many clinical scenarios, including liver resection and transplantation. Various studies and countless surgical events have led to the observation of a strong correlation between HIRI induced by liver transplantation and early allograft-dysfunction development. The detrimental impact of HIRI has driven the pursuit of new ways to alleviate its adverse effects. At the core of HIRI lies mitochondrial dysfunction. Various studies, from both animal models and in clinical settings, have clearly shown that mitochondrial function is severely hampered by HIRI and that its preservation or restoration is a key indicator of successful organ recovery. Several strategies have been thus implemented throughout the years, targeting mitochondrial function. This work briefly discusses some the most utilized approaches, ranging from surgical practices to pharmacological interventions and highlights how novel strategies can be investigated and implemented by intricately discussing the way mitochondrial function is affected by HIRI.

Mitochondrial and metabolic dysfunction in ageing and age-related diseases.

Organismal ageing is accompanied by progressive loss of cellular function and systemic deterioration of multiple tissues, leading to impaired function and increased vulnerability to death. Mitochondria have become recognized not merely as being energy suppliers but also as having an essential role in the development of diseases associated with ageing, such as neurodegenerative and cardiovascular diseases. A growing body of evidence suggests that ageing and age-related diseases are tightly related to an energy supply and demand imbalance, which might be alleviated by a variety of interventions, including physical activity and calorie restriction, as well as naturally occurring molecules targeting conserved longevity pathways. Here, we review key historical advances and progress from the past few years in our understanding of the role of mitochondria in ageing and age-related metabolic diseases. We also highlight emerging scientific innovations using mitochondria-targeted therapeutic approaches.

PEG35 as a Preconditioning Agent against Hypoxia/Reoxygenation Injury.

Pharmacological conditioning is a protective strategy against ischemia/reperfusion injury, which occurs during liver resection and transplantation. Polyethylene glycols have shown multiple benefits in cell and organ preservation, including antioxidant capacity, edema prevention and membrane stabilization. Recently, polyethylene glycol 35 kDa (PEG35) preconditioning resulted in decreased hepatic injury and protected the mitochondria in a rat model of cold ischemia. Thus, the study aimed to decipher the mechanisms underlying PEG35 preconditioning-induced protection against ischemia/reperfusion injury. A hypoxia/reoxygenation model using HepG2 cells was established to evaluate the effects of PEG35 preconditioning. Several parameters were assessed, including cell viability, mitochondrial membrane potential, ROS production, ATP levels, protein content and gene expression to investigate autophagy, mitochondrial biogenesis and dynamics. PEG35 preconditioning preserved the mitochondrial function by decreasing the excessive production of ROS and subsequent ATP depletion, as well as by recovering the membrane potential. Furthermore, PEG35 increased levels of autophagy-related proteins and the expression of genes involved in mitochondrial biogenesis and fusion. In conclusion, PEG35 preconditioning effectively ameliorates hepatic hypoxia/reoxygenation injury through the enhancement of autophagy and mitochondrial quality control. Therefore, PEG35 could be useful as a potential pharmacological tool for attenuating hepatic ischemia/reperfusion injury in clinical practice.

IGL-2 as a Unique Solution for Cold Static Preservation and Machine Perfusion in Liver and Mitochondrial Protection.

Hypothermic static cold storage and machine perfusion strategies remain the clinical standard of care for liver graft preservation. Recently, the protection of the mitochondrial function and the energetic levels derived from it has emerged as one of the key points for organ preservation. However, the complex interactions between liver mitochondrial protection and its relation with the use of solutions/perfusates has been poorly investigated. The use of an alternative IGL-2 solution to Belzer MPS one for hypothermic oxygenated perfusion (HOPE), as well as in static cold storage, introduce a new kind of perfusate to be used for liver grafts subjected to HOPE strategies, either alone or in combination with hypothermic static preservation strategies. IGL-2 not only protected mitochondrial integrity, but also avoided the mixture of different solutions/perfusates reducing. Thus, the operational logistics and times prior to transplantation, a critical factor when suboptimal organs such as donation after circulatory death or steatotic ones, are used for transplantation. The future challenges in graft preservation will go through (1) the improvement of the mitochondrial status and its energetic status during the ischemia and (2) the development of strategies to reduce ischemic times at low temperatures, which should translate in a better transplantation outcome.

Blueberry Counteracts Prediabetes in a Hypercaloric Diet-Induced Rat Model and Rescues Hepatic Mitochondrial Bioenergetics.

The paramount importance of a healthy diet in the prevention of type 2 diabetes is now well recognized. Blueberries (BBs) have been described as attractive functional fruits for this purpose. This study aimed to elucidate the cellular and molecular mechanisms pertaining to the protective impact of blueberry juice (BJ) on prediabetes. Using a hypercaloric diet-induced prediabetic rat model, we evaluated the effects of BJ on glucose, insulin, and lipid profiles; gut microbiota composition; intestinal barrier integrity; and metabolic endotoxemia, as well as on hepatic metabolic surrogates, including several related to mitochondria bioenergetics. BJ supplementation for 14 weeks counteracted diet-evoked metabolic deregulation, improving glucose tolerance, insulin sensitivity, and hypertriglyceridemia, along with systemic and hepatic antioxidant properties, without a significant impact on the gut microbiota composition and related mechanisms. In addition, BJ treatment effectively alleviated hepatic steatosis and mitochondrial dysfunction observed in the prediabetic animals, as suggested by the amelioration of bioenergetics parameters and key targets of inflammation, insulin signaling, ketogenesis, and fatty acids oxidation. In conclusion, the beneficial metabolic impact of BJ in prediabetes may be mainly explained by the rescue of hepatic mitochondrial bioenergetics. These findings pave the way to support the use of BJ in prediabetes to prevent diabetes and its complications.

Chenodeoxycholic Acid Has Non-Thermogenic, Mitodynamic Anti-Obesity Effects in an In Vitro CRISPR/Cas9 Model of Bile Acid Receptor TGR5 Knockdown.

Bile acids (BA) have shown promising effects in animal models of obesity. However, the said effects are thought to rely on a thermogenic effect, which is questionably present in humans. A previous work has shown that the BA chenodeoxycholic acid (CDCA) can revert obesity and accelerate metabolism in animal and cell culture models. Thus, the aim of this study was to understand if this obesity reduction is indeed thermogenically-dependent. A CRISPR/Cas9 model of TGR5 (BA receptor) knockdown in 3T3-L1 adipocytes was developed to diminish thermogenic effects. Various parameters were assessed, including mitochondrial bioenergetics by Seahorse flux analysis, oxidative stress and membrane potential by fluorometry, intermediary metabolism by NMR, protein content assessment by Western Blot, gene expression by qPCR, and confocal microscopy evaluation of mitophagy. CDCA was still capable, for the most part, of reversing the harmful effects of cellular obesity, elevating mitophagy and leading to the reduction of harmed mitochondria within the cells, boosting mitochondrial activity, and thus energy consumption. In summary, CDCA has a non-thermogenic, obesity reducing capacity that hinges on a healthy mitochondrial population, explaining at least some of these effects and opening avenues of human treatment for metabolic diseases.

Determination of Oxidative Phosphorylation Complexes Activities.

Mitochondria possess a genome that codes for proteins, in the same fashion as the nuclear genome. However, the small, circular mitochondrial DNA (mtDNA) molecule has a reduced base pair content, for it can only code for 2 rRNA, 22 tRNA molecules, and 13 proteins, all of them part of the mitochondrial respiratory chain. As such, all of the other mitochondrial components derive from nuclear genome. This separation leads to a requirement for a well-tuned coordination between both genomes, in order to produce fully functional mitochondria. A vast number of pathologies have been demonstrated to involve, to some extent, alterations in mitochondrial function that, no doubt, can be caused by alterations to the respiratory chain activity. As such, several methods and techniques have been developed to assess both content and function of mitochondrial proteins, in order to help understand mitochondrial involvement on the pathogenesis of disease. In this chapter, we will address some of these methods, with the main focus being on isolated mitochondria.

Sestrin2 and mitochondrial quality control: Potential impact in myogenic differentiation.

Mitochondria are highly dynamic organelles capable of adapting their network, morphology, and function, playing a role in oxidative phosphorylation and many cellular processes in most cell types. Skeletal muscle is a very plastic tissue, subjected to many morphological changes following diverse stimuli, such as during myogenic differentiation and regenerative myogenesis. For some time now, mitochondria have been reported to be involved in myogenesis by promoting a bioenergetic remodeling and assisting myoblasts in surviving the process. However, not much is known about the interplay between mitochondrial quality control and myogenic differentiation. Sestrin2 (SESN2) is a well described regulator of autophagy and antioxidant responses and has been gaining attention due to its role in aging-associated pathologies and redox signaling promoted by reactive oxygen species (ROS) in many tissues. Current evidence involving SESN2-associated pathways suggest that it can act as a potential regulator of mitochondrial quality control following induction by ROS under stress conditions, such as during myogenesis. Yet, there are no studies directly assessing SESN2 involvement in myogenic differentiation. This review provides novel insights pertaining the involvement of SESN2 in myogenic differentiation by analyzing the interactions between ROS and mitochondrial remodeling.

miR-378a-3p Participates in Metformin's Mechanism of Action on C2C12 Cells under Hyperglycemia.

Metformin is the most used biguanide drug for the treatment of type 2 diabetes mellitus. Despite being mostly known for its hepatic anti-gluconeogenic effect, it is also known to modulate microRNAs (miRNAs, miRs) associated with metabolic diseases. The latter mechanism could be relevant for better understanding metformin's mechanisms underlying its biological effects. In the current work, we found that metformin increases miR-378a-3p expression (p < 0.002) in C2C12 myoblasts previously exposed to hyperglycemic conditions. While the inhibition of miR-378a-3p was shown to impair metformin's effect in ATP production, PEPCK activity and the expression of Tfam. Finally, mitophagy, an autophagic process responsible for the selective degradation of mitochondria, was found to be induced by miR-378a-3p (p < 0.04). miR-378a-3p stimulated mitophagy through a process independent of sestrin-2 (SESN2), a stress-responsible protein that has been recently demonstrated to positively modulate mitophagy. Our findings provide novel insights into an alternative mechanism of action of metformin involving miR-378a-3, which can be used in the future for the development of improved therapeutic strategies against metabolic diseases.

The potential role of sestrin 2 in liver regeneration.

Liver regeneration is a remarkably complex phenomenon conserved across all vertebrates, enabling the restoration of lost liver mass in a matter of days. Unfortunately, extensive damage to the liver may compromise this process, often leading to the death of affected individuals. Ischemia/reperfusion injury (IRI) is a common source of damage preceding regeneration, often present during liver transplantation, resection, trauma, or hemorrhagic shock. Increased oxidative stress and mitochondrial dysfunction are key hallmarks of IRI, which can jeopardize the liver's ability to regenerate. Therefore, a better understanding of both liver regeneration and IRI is of important clinical significance. In the current review, we discuss the potential role of sestrin 2 (SESN2), a novel anti-aging protein, in liver regeneration and ischemia/reperfusion preceding regeneration. We highlight its beneficial role in protecting cells from mitochondrial dysfunction and oxidative stress as key aspects of its involvement in liver regeneration. Additionally, we describe how its ability to promote the expression of Nrf2 bears significant importance in this context. Finally, we focus on a potential novel link between SESN2, mitohormesis and ischemic preconditioning, which could explain some of the protective effects of preconditioning.

Blueberry Consumption Challenges Hepatic Mitochondrial Bioenergetics and Elicits Transcriptomics Reprogramming in Healthy Wistar Rats.

An emergent trend of blueberries' (BB) "prophylactic" consumption, due to their phytochemicals' richness and well-known health-promoting claims, is widely scaled-up. However, the benefits arising from BB indiscriminate intake remains puzzling based on incongruent preclinical and human data. To provide a more in-depth elucidation and support towards a healthier and safer consumption, we conducted a translation-minded experimental study in healthy Wistar rats that consumed BB in a juice form (25 g/kg body weight (BW)/day; 14 weeks' protocol). Particular attention was paid to the physiological adaptations succeeding in the gut and liver tissues regarding the acknowledged BB-induced metabolic benefits. Systemically, BB boosted serum antioxidant activity and repressed the circulating levels of 3-hydroxybutyrate (3-HB) ketone bodies and 3-HB/acetoacetate ratio. Moreover, BB elicited increased fecal succinic acid levels without major changes on gut microbiota (GM) composition and gut ultra-structural organization. Remarkably, an accentuated hepatic mitochondrial bioenergetic challenge, ensuing metabolic transcriptomic reprogramming along with a concerted anti-inflammatory pre-conditioning, was clearly detected upon long-term consumption of BB phytochemicals. Altogether, the results disclosed herein portray a quiescent mitochondrial-related metabolomics and hint for a unified adaptive response to this nutritional challenge. The beneficial or noxious consequences arising from this dietary trend should be carefully interpreted and necessarily claims future research.

The Evaluation of Mitochondrial Membrane Potential Using Fluorescent Dyes or a Membrane-Permeable Cation (TPP+) Electrode in Isolated Mitochondria and Intact Cells.

The proton electrochemical gradient generated by respiratory chain activity accounts for over 90% of all available ATP and, as such, its evaluation and accurate measurements regarding its total values and fluctuations is an invaluable component in the understanding of mitochondrial functions. Consequently, alterations in electric potential across the inner mitochondrial membrane generated by differential protonic accumulations and transport are known as the mitochondrial membrane potential, or Δψ, and are reflective of the functional metabolic status of mitochondria. There are several experimental approaches to measure Δψ, ranging from fluorometric evaluations to electrochemical probes. Here we discuss the advantages and disadvantages of several of these methods, ranging from one that is dependent on the movement of a particular ion (tetraphenylphosphonium (TPP+) with a selective electrode) to the selection of a fluorescent dye from various types to achieve the same goal. The evaluation of the accumulation and movements of TPP+ across the inner mitochondrial membrane, or the fluorescence of accumulated dye particles, is a sensitive and accurate method of evaluating the Δψ in respiring mitochondria (either isolated or still inside the cell).