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Keratin intermediate filaments form dynamic filamentous networks, which provide mechanical stability, scaffolding, and protection against stress to epithelial cells. Keratins and other intermediate filaments have been increasingly linked to the regulation of mitochondrial function and homeostasis in different tissues and cell types. While deletion of keratin 8 (K8–/–) in mouse colon elicits a colitis-like phenotype, epithelial hyperproliferation, and blunted mitochondrial ketogenesis, the role of K8 in colonocyte mitochondrial function and energy metabolism is unknown. We used two K8 knockout mouse models and CRISPR/Cas9 K8–/– colorectal adenocarcinoma Caco-2 cells to answer this question. The results show that K8–/– colonocyte mitochondria in vivo are smaller and rounder and that mitochondrial motility is increased in K8–/– Caco-2 cells. Furthermore, K8−/− Caco-2 cells displayed diminished mitochondrial respiration and decreased mitochondrial membrane potential compared with controls, whereas glycolysis was not affected. The levels of mitochondrial respiratory chain complex proteins and mitochondrial regulatory proteins mitofusin-2 and prohibitin were decreased both in vitro in K8–/– Caco-2 cells and in vivo in K8–/– mouse colonocytes, and reexpression of K8 into K8–/– Caco-2 cells normalizes the mitofusin-2 levels. Mitochondrial Ca2+ is an important regulator of mitochondrial energy metabolism and homeostasis, and Caco-2 cells lacking K8 displayed decreased levels and altered dynamics of mitochondrial matrix and cytoplasmic Ca2+. In summary, these novel findings attribute an important role for colonocyte K8 in stabilizing mitochondrial shape and movement and maintaining mitochondrial respiration and Ca2+ signaling. Further, how these metabolically compromised colonocytes are capable of hyperproliferating presents an intriguing question for future studies. NEW & NOTEWORTHY: In this study, we show that colonocyte intermediate filament protein keratin 8 is important for stabilizing mitochondria and maintaining mitochondrial energy metabolism, as keratin 8-deficient colonocytes display smaller, rounder, and more motile mitochondria, diminished mitochondrial respiration, and altered Ca2+ dynamics. Changes in fusion-regulating proteins are rescued with reexpression of keratin 8. These alterations in colonocyte mitochondrial homeostasis contribute to keratin 8-associated colitis pathophysiology. [ABSTRACT FROM AUTHOR]

Critical power (CP) represents an important threshold for exercise performance and fatiguability. We sought to determine the extent to which sex, hemoglobin mass (Hbmass), and skeletal muscle characteristics influence CP. Before CP determination (i.e., 3–5 constant work rate trials to task failure), Hbmass and skeletal muscle oxidative capacity (τ) were measured and vastus lateralis (VL) muscle biopsy samples were collected from 12 females and 12 males matched for aerobic fitness relative to fat-free mass (FFM) [means (SD); V̇ o 2max: 59.2 (7.7) vs. 59.5 (7.1) mL·kg·FFM−1·min−1, respectively]. Males had a significantly greater CP than females in absolute units [225 (28) vs. 170 (43) W; P = 0.001] but not relative to body mass [3.0 (0.6) vs. 2.7 (0.6) W·kg·BM−1; P = 0.267] or FFM [3.6 (0.7) vs. 3.7 (0.8) W·kg·FFM−1; P = 0.622]. Males had significantly greater W′ (P ≤ 0.030) and greater Hbmass (P ≤ 0.016) than females, regardless of the normalization approach; however, there were no differences in mitochondrial protein content (P = 0.375), τ (P = 0.603), or MHC I proportionality (P = 0.574) between males and females. Whether it was expressed in absolute or relative units, CP was positively correlated with Hbmass (0.444 ≤ r ≤ 0.695; P < 0.05), mitochondrial protein content (0.413 ≤ r ≤ 0.708; P < 0.05), and MHC I proportionality (0.506 ≤ r ≤ 0.585; P < 0.05), and negatively correlated with τ when expressed in relative units only (−0.588 ≤ r ≤ −0.527; P < 0.05). Overall, CP was independent of sex, but variability in CP was related to Hbmass and skeletal muscle characteristics. The extent to which manipulations in these physiological parameters influence CP warrants further investigation to better understand the factors underpinning CP. NEW & NOTEWORTHY: In males and females matched for aerobic fitness [maximal oxygen uptake normalized to fat-free mass (FFM)], absolute critical power (CP) was greater in males, but relative CP (per kilogram body mass or FFM) was similar between sexes. CP correlated with hemoglobin mass, mitochondrial protein content, myosin heavy chain type I proportion, and skeletal muscle oxidative capacity. These findings demonstrate the importance of matching sexes for aerobic fitness, but further experiments are needed to determine causality. [ABSTRACT FROM AUTHOR]

Hypothalamic proopiomelanocortin (POMC) neurons are sensors of signals that reflect the energy stored in the body. Inducing mild stress in proopiomelanocortin neurons protects them from the damage promoted by the consumption of a high-fat diet, mitigating the development of obesity; however, the cellular mechanisms behind these effects are unknown. Here, we induced mild stress in a proopiomelanocortin neuron cell line by inhibiting Crif1. In proopiomelanocortin neurons exposed to high levels of palmitate, the partial inhibition of Crif1 reverted the defects in mitochondrial respiration and ATP production; this was accompanied by improved mitochondrial fusion/fission cycling. Furthermore, the partial inhibition of Crif1 resulted in increased reactive oxygen species production, increased fatty acid oxidation, and reduced dependency on glucose for mitochondrial respiration. These changes were dependent on the activity of CPT-1. Thus, we identified a CPT-1-dependent metabolic shift toward greater utilization of fatty acids as substrates for respiration as the mechanism behind the protective effect of mild stress against palmitate-induced damage of proopiomelanocortin neurons. NEW & NOTEWORTHY: Saturated fats can damage hypothalamic neurons resulting in positive energy balance, and this is mitigated by mild cellular stress; however, the mechanisms behind this protective effect are unknown. Using a proopiomelanocortin cell line, we show that under exposure to a high concentration of palmitate, the partial inhibition of the mitochondrial protein Crif1 results in protection due to a metabolic shift warranted by the increased expression and activity of the mitochondrial fatty acid transporter CPT-1. [ABSTRACT FROM AUTHOR]

The pathophysiology of muscle damage in peripheral artery disease (PAD) includes increased oxidant production and impaired antioxidant defenses. Epicatechin (EPI), a naturally occurring flavanol, has antioxidant properties that may mediate the beneficial effects of natural products such as cocoa. In a phase II randomized trial, a cocoa-flavanol-rich beverage significantly improved walking performance compared with a placebo in people with PAD. In the present work, the molecular mechanisms underlying the therapeutic effect of cocoa flavanols were investigated by analyzing baseline and follow-up muscle biopsies from participants. Increases in nuclear factor erythroid 2-related factor 2 (Nrf2) target antioxidants heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase [quinone] 1 (NQO1) in the cocoa group were significantly associated with reduced accumulation of central nuclei, a myopathy indicator, in type II muscle fibers (P ¼ 0.017 and P ¼ 0.023, respectively). Protein levels of the mitochondrial respiratory complex III subunit, cytochrome b-c1 complex subunit 2 (UQCRC2), were significantly higher in the cocoa group than in the placebo group (P ¼ 0.032), and increases in UQCRC2 were significantly associated with increased levels of Nrf2 target antioxidants HO-1 and NQO1 (P ¼ 0.001 and P ¼ 0.035, respectively). Exposure of non-PAD human myotubes to ex vivo serum from patients with PAD reduced Nrf2 phosphorylation, an indicator of activation, increased hydrogen peroxide production and oxidative stress, and reduced mitochondrial respiration. Treatment of myotubes with EPI in the presence of serum from patients with PAD increased Nrf2 phosphorylation and protected against PAD serum-induced oxidative stress and mitochondrial dysfunction. Overall, these findings suggest that cocoa flavanols may enhance antioxidant capacity in PAD via Nrf2 activation. NEW & NOTEWORTHY The current study supports the hypothesis that in people with PAD, cocoa flavanols activate Nrf2, thereby increasing antioxidant protein levels, protecting against skeletal muscle damage, and increasing mitochondrial protein abundance. These results suggest that Nrf2 activation may be an important therapeutic target for improving walking performance in people with PAD. [ABSTRACT FROM AUTHOR]

To further refine the near-infrared spectroscopy (NIRS)-derived measure of skeletal muscle oxidative capacity in humans, we sought to determine whether the exercise stimulus intensity affected the τ value and/or influenced the magnitude of correlations with in vitro measures of mitochondrial content and in vivo indices of exercise performance. Males (n = 12) and females (n = 12), matched for maximal aerobic fitness per fat-free mass, completed NIRS-derived skeletal muscle oxidative capacity tests for the vastus lateralis following repeated contractions at 40% (`40) and 100% (τ100) of maximum voluntary contraction, underwent a skeletal muscle biopsy of the same muscle, and performed multiple intermittent isometric knee extension tests to task failure to establish critical torque (CT). The value of τ100 (34.4 ± 7.0 s) was greater than τ40 (24.2 ± 6.9 s, P < 0.001), but the values were correlated (r = 0.688; P < 0.001). The values of τ40 (r = -0.692, P < 0.001) and τ100 (r = -0.488, P = 0.016) correlated with myosin heavy chain I percentage and several markers of mitochondrial content, including COX II protein content in whole muscle (s40: r = -0.547, P = 0.006; τ100: r = -0.466, P = 0.022), type I pooled fibers (s40: r = -0.547, P = 0.006; τ100: r = -0.547, P = 0.006), and type II pooled fibers (s40: r = -0.516, P = 0.009; τ100: r = -0.635, P = 0.001). The value of τ40 (r = -0.702, P < 0.001), but not τ100 (r = -0.378, P = 0.083) correlated with critical torque (CT); however, neither value correlated with W0 (τ40: r = 0.071, P = 0.753; τ100: r = 0.054, P = 0.812). Overall, the NIRS method of assessing skeletal muscle oxidative capacity is sensitive to the intensity of skeletal muscle contraction but maintains relationships to whole body fitness, isolated limb critical intensity, and mitochondrial content regardless of intensity. [ABSTRACT FROM AUTHOR]

Induction of alternative, non-apoptotic cell death programs such as cell-lethal autophagy and mitophagy represent possible strategies to combat glioblastoma (GBM). Here we report that VLX600, a novel iron chelator and oxidative phosphorylation (OXPHOS) inhibitor, induces a caspase-independent type of cell death that is partially rescued in adherent U251 ATG5/7 (autophagy related 5/7) knockout (KO) GBM cells and NCH644 ATG5/7 knockdown (KD) glioma stem-like cells (GSCs), suggesting that VLX600 induces an autophagy-dependent cell death (ADCD) in GBM. This ADCD is accompanied by decreased oxygen consumption, increased expression/mitochondrial localization of BNIP3 (BCL2 interacting protein 3) and BNIP3L (BCL2 interacting protein 3 like), the induction of mitophagy as demonstrated by diminished levels of mitochondrial marker proteins [e.g., COX4I1 (cytochrome c oxidase subunit 4I1)] and the mitoKeima assay as well as increased histone H3 and H4 lysine tri-methylation. Furthermore, the extracellular addition of iron is able to significantly rescue VLX600-induced cell death and mitophagy, pointing out an important role of iron metabolism for GBM cell homeostasis. Interestingly, VLX600 is also able to completely eliminate NCH644 GSC tumors in an organotypic brain slice transplantation model. Our data support the therapeutic concept of ADCD induction in GBM and suggest that VLX600 may be an interesting novel drug candidate for the treatment of this tumor.NEW & NOTEWORTHY Induction of cell-lethal autophagy represents a possible strategy to combat glioblastoma (GBM). Here, we demonstrate that the novel iron chelator and OXPHOS inhibitor VLX600 exerts pronounced tumor cell-killing effects in adherently cultured GBM cells and glioblastoma stem-like cell (GSC) spheroid cultures that depend on the iron-chelating function of VLX600 and on autophagy activation, underscoring the context-dependent role of autophagy in therapy responses. VLX600 represents an interesting novel drug candidate for the treatment of this tumor. [ABSTRACT FROM AUTHOR]

Mitochondria rely upon the coordination of protein import, protein translation, and proper functioning of oxidative phosphorylation (OXPHOS) complexes I-V to sustain the activities of life for an organism. Each process is dependent upon the function of profoundly large protein complexes found in the mitochondria [translocase of the outer mitochondrial membrane (TOMM) complex, translocase of the inner mitochondrial membrane (TIMM) complex, OXPHOS complexes, mitoribosomes]. These massive protein complexes, in some instances more than one megadalton, are built up from numerous protein subunits of varying sizes, including many proteins that are ≤100-150 amino acids. However, these small proteins, termed microproteins, not only act as cogs in large molecular machines but also have important steps in inhibiting or promoting the intrinsic pathway of apoptosis, coordinate responses to cellular stress, and even act as hormones. This review focuses on microproteins that occupy the mitochondria and are critical for its function. Although the microprotein field is relatively new, researchers have long recognized the existence of these mitochondrial proteins as critical components of virtually all aspects of mitochondrial biology. Thus, recent studies estimating that hundreds of new microproteins of unknown function exist and are missing from current genome annotations suggests that the mitochondrial "microproteome" is a rich area for future biological investigation. [ABSTRACT FROM AUTHOR]

This article discusses the role of reactive oxygen species (ROS) in cardiac electrophysiology and the impact of Scn1b loss on susceptibility to oxidative stress and the development of arrhythmias. The dysregulation of voltage-gated ion channels, particularly sodium channels, can lead to electrophysiological abnormalities and potentially deadly arrhythmias. Mouse models of Scn1b deletion exhibit cardiac arrhythmia, and the loss of Scn1b also affects mitochondrial function, redox systems, and ROS scavenging. The findings suggest that ROS scavenging has a protective impact on arrhythmia development and may be a potential target for treatment. The article also highlights the need to examine comorbidities associated with increased ROS generation as potential risk factors for sudden unexpected death in epilepsy (SUDEP). [Extracted from the article]

Decorin, a small leucine-rich proteoglycan with multiple biological functions, is known to evoke autophagy and mitophagy in both endothelial and cancer cells. Here, we investigated the effects of soluble decorin on mitochondrial homeostasis using live cell imaging and ex vivo angiogenic assays. We discovered that decorin triggers mitochondrial depolarization in triplenegative breast carcinoma, HeLa, and endothelial cells. This bioactivity was mediated by the protein core in a time- and dosedependent manner and was specific for decorin insofar as biglycan, the closest homolog, failed to trigger depolarization. Mechanistically, we found that the bioactivity of decorin to promote depolarization required the MET receptor and its tyrosine kinase. Moreover, two mitochondrial interacting proteins, mitostatin and mitofusin 2, were essential for downstream decorin effects. Finally, we found that decorin relied on the canonical mitochondrial permeability transition pore to trigger tumor cell mitochondrial depolarization. Collectively, our study implicates decorin as a soluble outside-in regulator of mitochondrial dynamics. [ABSTRACT FROM AUTHOR]

As a central hub for cellular metabolism and intracellular signaling, the mitochondrion is a pivotal organelle, dysfunction of which has been linked to several human diseases including neurodegenerative disorders and in particular Parkinson's disease. An inherent challenge that mitochondria face is the continuous exposure to diverse stresses that increase their likelihood of dysregulation. In response, eukaryotic cells have evolved sophisticated quality control mechanisms to monitor, identify, repair, and/or eliminate abnormal or misfolded proteins within the mitochondrion and/or the dysfunctional mitochondrion itself. Chaperones identify unstable or otherwise abnormal conformations in mitochondrial proteins and can promote their refolding to recover their correct conformation and stability. However, if repair is not possible, the abnormal protein is selectively degraded to prevent potentially damaging interactions with other proteins or its oligomerization into toxic multimeric complexes. The autophagic-lysosomal system and the ubiquitin-proteasome system mediate the selective and targeted degradation of such abnormal or misfolded protein species. Mitophagy (a specific kind of autophagy) mediates the selective elimination of dysfunctional mitochondria, to prevent the deleterious effects of the dysfunctional organelles within the cell. Despite our increasing understanding of the molecular responses toward dysfunctional mitochondria, many key aspects remain relatively poorly understood. Here, we review the emerging mechanisms of mitochondrial quality control including quality control strategies coupled to mitochondrial import mechanisms. In addition, we review the molecular mechanisms regulating mitophagy, with an emphasis on the regulation of PINK1/Parkin-mediated mitophagy in cellular physiology and in the context of Parkinson's disease cell biology. [ABSTRACT FROM AUTHOR]

The development of skeletal muscle (myogenesis) is a well-orchestrated process where myoblasts withdraw from the cell cycle and differentiate into myotubes. Signaling by fluxes in intracellular calcium (Ca2+) is known to contribute to myogenesis, and increased mitochondrial biogenesis is required to meet the metabolic demand of mature myotubes. However, gaps remain in the understanding of how intracellular Ca2+ signals can govern myogenesis. Polycystin-2 (PC2 or TRPP1) is a nonselective cation channel permeable to Ca2+. It can interact with intracellular calcium channels to control Ca2+ release and concurrently modulates mitochondrial function and remodeling. Due to these features, we hypothesized that PC2 is a central protein in mediating both the intracellular Ca2+ responses and mitochondrial changes seen in myogenesis. To test this hypothesis, we created CRISPR/Cas9 knockout (KO) C2C12 murine myoblast cell lines. PC2 KO cells were unable to differentiate into myotubes, had impaired spontaneous Ca2+ oscillations, and did not develop depolarization-evoked Ca2+ transients. The autophagic-associated pathway beclin-1 was downregulated in PC2 KO cells, and direct activation of the autophagic pathway resulted in decreased mitochondrial remodeling. Re-expression of full-length PC2, but not a calcium channel dead pathologic mutant, restored the differentiation phenotype and increased the expression of mitochondrial proteins. Our results establish that PC2 is a novel regulator of in vitro myogenesis by integrating PC2-dependent Ca2+ signals and metabolic pathways. [ABSTRACT FROM AUTHOR]

Pulmonary hypertension (PH) is a debilitating condition characterized by increased pulmonary arterial pressures and remodeling of pulmonary arteries, leading to right heart failure. Women have a higher prevalence of PH, whereas men have more severe disease and poorer outcomes. Animal models also show female-predominant disease. Despite the known sex differences in PH, little is known about how pathogenesis differs between the sexes. There is growing evidence of mitochondrial dysfunction, as well as altered mitophagy in PH. We hypothesized that sexual dimorphism contributes to mitochondrial dysfunction and altered mitophagy in PH. Using mouse lung endothelial cells, we exposed both wild-type and Parkin(/( cells to hypoxia and measured the effects on mitochondrial function and mitophagy-associated proteins. Our results show that females have more Parkin expression at baseline as well as increased mitochondrial respiratory capacity when exposed to oxidative stress. Inhibition of Parkin increased metabolic activity but reduced cell proliferation but to different degrees depending on sex, with results differing by sex. Our findings demonstrate sexual dimorphism in mitophagy-associated proteins and in mitochondrial respiration, which may help shed light on how the pathogenesis of PH may differ between the sexes. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is an inherited muscle wasting disease. Metabolic impairments and oxidative stress are major secondary mechanisms that severely worsen muscle function in DMD. Here, we sought to determine whether germline reduction or ablation of sarcolipin (SLN), an inhibitor of sarco/endoplasmic reticulum (SR) Ca2+ ATPase (SERCA), improves muscle metabolism and ameliorates muscle pathology in the mdx mouse model of DMD. Glucose and insulin tolerance tests show that glucose clearance rate and insulin sensitivity were improved in the SLN haploinsufficient mdx (mdx:sln+/-) and SLN-deficient mdx (mdx:sln-/-) mice. The histopathological analysis shows that fibrosis and necrosis were significantly reduced in muscles of mdx:sln+/- and mdx:sln-/- mice. SR Ca2+ uptake, mitochondrial complex protein levels, complex activities, mitochondrial Ca2+ uptake and release, and mitochondrial metabolism were significantly improved, and lipid peroxidation and protein carbonylation were reduced in the muscles of mdx:sln+/- and mdx:sln-/- mice. These data demonstrate that reduction or ablation of SLN expression can improve muscle metabolism, reduce oxidative stress, decrease muscle pathology, and protects the mdx mice from glucose intolerance. [ABSTRACT FROM AUTHOR]

Endurance exercise triggers skeletal muscle adaptations, including enhanced insulin signaling, glucose metabolism, and mitochondrial biogenesis. However, exercise-induced skeletal muscle adaptations may not occur in some cases, a condition known as exercise resistance. Methylglyoxal (MG) is a highly reactive dicarbonyl metabolite and has detrimental effects on the body such as causing diabetic complications, mitochondrial dysfunction, and inflammation. This study aimed to clarify the effect of methylglyoxal on skeletal muscle molecular adaptations following endurance exercise. Mice were randomly divided into four groups (n = 12/group): sedentary control group, voluntary exercise group, MG-treated group, and MG-treated with voluntary exercise group. Mice in the voluntary exercise group were housed in a cage with a running wheel, whereas mice in the MG-treated groups received drinking water containing 1% MG. Four weeks of voluntary exercise induced several molecular adaptations in the plantaris muscle, including increased expression of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α), mitochondria complex proteins, Toll-like receptor 4 (TLR4), 72-kDa heat shock protein (HSP72), hexokinase II, and glyoxalase 1; this also enhanced insulin-stimulated Akt Ser473 phosphorylation and citrate synthase activity. However, these adaptations were suppressed with MG treatment. In the soleus muscle, the exercise-induced increases in the expression of TLR4, HSP72, and advanced glycation end products receptor 1 were inhibited with MG treatment. These findings suggest that MG is a factor that inhibits endurance exercise-induced molecular responses including mitochondrial adaptations, insulin signaling activation, and the upregulation of several proteins related to mitochondrial biogenesis, glucose handling, and glycation in primarily fast-twitch skeletal muscle. [ABSTRACT FROM AUTHOR]

We hypothesize that intrauterine hypoxia (HPX) alters the mitochondrial phenotype in fetal hearts contributing to developmental programming. Pregnant guinea pigs were exposed to normoxia (NMX) or hypoxia (HPX, 10.5% O2), starting at early [25 days (25d), 39d duration] or late gestation (50d, 14d duration). Near-term (64d) male and female fetuses were delivered by hysterotomy from anesthetized sows, and body/organ weights were measured. Left ventricles of fetal hearts were excised and frozen for measurement of expression of complex (I-V) subunits, fusion (Mfn2/OPA1) and fission (DRP1/Fis1) proteins, and enzymatic rates of I and IV from isolated mitochondrial proteins. Chronic HPX decreased fetal body weight and increased relative placenta weight regardless of timing. Early-onset HPX increased I, III, and V subunit levels, increased complex I but decreased IV activities in males but not females (all P < 0.05). Late-onset HPX decreased (P < 0.05) I, III, and V levels in both sexes but increased I and decreased IV activities in males only. Both HPX conditions decreased cardiac mitochondrial DNA content in males only. Neither early- nor late-onset HPX had any effect on Mfn2 levels but increased OPA1 in both sexes. Both HPX treatments increased DRP1/Fis1 levels in males. In females, early-onset HPX increased DRP1 with no effect on Fis1, whereas late-onset HPX increased Fis1 with no effect on DRP1. We conclude that both early- and late-onset HPX disrupts the expression/activities of select complexes that could reduce respiratory efficiency and shifts dynamics toward fission in fetal hearts. Thus, intrauterine HPX disrupts the mitochondrial phenotype predominantly in male fetal hearts, potentially altering cardiac metabolism and predisposing the offspring to heart dysfunction. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is the leading cause of dementia and presents a considerable disease burden. Its pathology involves substantial neuronal loss, primarily attributed to neuronal apoptosis. Although sirtuin 4 (SIRT4) has been implicated in regulating apoptosis in various diseases, the role of SIRT4 in AD pathology remains unclear. The study used APP/PS1 mice as an animal model of AD and amyloid-β (Aβ)1-42-treated HT-22 cells as an AD cell model. SIRT4 expression was determined by quantitative real-time polymerase chain reaction, Western blot, and immunofluorescence. A Sirt4 knockdown model was established by intracranial injection of lentivirus-packaged sh-SIRT4 and cellular lentivirus transfection. Immunohistochemistry and flow cytometry were used to examine Aβ deposition in mice and apoptosis, respectively. Protein expression was assessed by Western blot analysis. The UCSC and JASPAR databases were used to predict upstream transcription factors of Sirt4. Subsequently, the binding of transcription factors to Sirt4 was analyzed using a dual-luciferase assay and chromatin immunoprecipitation. SIRT4 expression was upregulated in both APP/PS1 mice and Aβ-treated HT-22 cells compared with their respective control groups. Sirt4 knockdown in animal and cellular models of AD resulted in reduced apoptosis, decreased Aβ deposition, and amelioration of learning and memory impairments in mice. Mechanistically, SIRT4 modulates apoptosis via the mTOR pathway and is negatively regulated by the transcription factor signal transducer and activator of transcription 2 (STAT2). Our study findings suggest that targeting the STAT2-SIRT4-mTOR axis may offer a new treatment approach for AD. NEW & NOTEWORTHY The study reveals that in Alzheimer's disease models, SIRT4 expression increases, contributing to neuronal apoptosis and amyloid-β deposition. Reducing SIRT4 lessens apoptosis and amyloid-β accumulation, improving memory in mice. This process involves the mTOR pathway, regulated by STAT2 transcription factor. These findings suggest targeting the STAT2-SIRT4-mTOR axis as a potential Alzheimer's treatment strategy.

Mitochondria have been recognized as key organelles in cardiac physiology and are potential targets for clinical interventions to improve cardiac function. Mitochondrial dysfunction has been accepted as a major contributor to the development of heart failure. The main function of mitochondria is to meet the high energy demands of the heart by oxidative metabolism. Ionic homeostasis in mitochondria directly regulates oxidative metabolism, and any disruption in ionic homeostasis causes mitochondrial dysfunction and eventually contractile failure. The mitochondrial ionic homeostasis is closely coupled with inner mitochondrial membrane potential. To regulate and maintain ionic homeostasis, mitochondrial membranes are equipped with ion transporting proteins. Ion transport mechanisms involving several different ion channels and transporters are highly efficient and dynamic, thus helping to maintain the ionic homeostasis of ions as well as their salts present in the mitochondrial matrix. In recent years, several novel proteins have been identified on the mitochondrial membranes and these proteins are actively being pursued in research for roles in the organ as well as organelle physiology. In this article, the role of mitochondrial ion channels in cardiac function is reviewed. In recent times, the major focus of the mitochondrial ion channel field is to establish molecular identities as well as assigning specific functions to them. Given the diversity of mitochondrial ion channels and their unique roles in cardiac function, they present novel and viable therapeutic targets for cardiac diseases. [ABSTRACT FROM AUTHOR]

Mitochondria are recognized as signaling organelles, because under stress, mitochondria can trigger various signaling pathways to coordinate the cell's response. The specific pathway(s) engaged by mitochondria in response to mitochondrial energy defects in vivo and in high-energy tissues like the heart are not fully understood. Here, we investigated cardiac pathways activated in response to mitochondrial energy dysfunction by studying mice with cardiomyocyte-specific loss of the mitochondrial phosphate carrier (SLC25A3), an established model that develops cardiomyopathy as a result of defective mitochondrial ATP synthesis. Mitochondrial energy dysfunction induced a striking pattern of acylome remodeling, with significantly increased posttranslational acetylation and malonylation. Mass spectrometry-based proteomics further revealed that energy dysfunction-induced remodeling of the acetylome and malonylome preferentially impacts mitochondrial proteins. Acetylation and malonylation modified a highly interconnected interactome of mitochondrial proteins, and both modifications were present on the enzyme isocitrate dehydrogenase 2 (IDH2). Intriguingly, IDH2 activity was enhanced in SLC25A3- deleted mitochondria, and further study of IDH2 sites targeted by both acetylation and malonylation revealed that these modifications can have site-specific and distinct functional effects. Finally, we uncovered a novel cross talk between the two modifications, whereby mitochondrial energy dysfunction-induced acetylation of sirtuin 5 (SIRT5), inhibited its function. Because SIRT5 is a mitochondrial deacylase with demalonylase activity, this finding suggests that acetylation can modulate the malonylome. Together, our results position acylations as an arm of the mitochondrial response to energy dysfunction and suggest a mechanism by which focal disruption to the energy production machinery can have an expanded impact on global mitochondrial function. [ABSTRACT FROM AUTHOR]

Peroxynitrite (PN), generated from the reaction of nitric oxide (NO) and superoxide, is implicated in the pathogenesis of ischemic and neurodegenerative brain injuries. Mitochondria produce NO from mitochondrial NO synthases and superoxide by the electron transport chain. Our objective was to detect the generation of PN of mitochondrial origin and characterize its effects on mitochondrial respiratory function. Freshly isolated brain nonsynaptosomal mitochondria from C57Bl/6 (wild type, WT) and endothelial NO synthase knockout (eNOS-KO) mice were treated with exogenous PN (0.1, 1, 5 mmol/L) or a PN donor (SIN-1; 50 mmol/L) or a PN scavenger (FeTMPyP; 2.5 mmol/L). Oxygen consumption rate (OCR) was measured using Agilent Seahorse XFe24 analyzer and mitochondrial respiratory parameters were calculated. Mitochondrial membrane potential, superoxide, and PN were determined from rhodamine 123, dihydroethidium, and DAX-J2 PON green fluorescence measurements, respectively. Mitochondrial protein nitrotyrosination was determined by Western blots. Both exogenous PN and SIN-1 decreased respiratory function in WT isolated brain mitochondria. FeTMPyP enhanced state III and state IVo mitochondrial respiration in both WT and eNOS-KO mitochondria. FeTMPyP also elevated state IIIu respiration in eNOS-KO mitochondria. Unlike PN, neither SIN-1 nor FeTMPyP depolarized the mitochondria. Although mitochondrial protein nitrotyrosination was unaffected by SIN-1 or FeTMPyP, FeTMPyP reduced mitochondrial PN levels. Mitochondrial superoxide levels were increased by FeTMPyP but were unaffected by PN or SIN-1. Thus, we present the evidence of functionally significant PN generation in isolated brain mitochondria. Mitochondrial PN activity was physiologically relevant in WT mice and pathologically significant under conditions with eNOS deficiency. [ABSTRACT FROM AUTHOR]

Myoblast differentiation is a crucial process for myogenesis. Mitochondria function as an energyproviding machine that is critical to this process, and mitochondrial dysfunction can prevent myoblasts from fusing into myotubes. However, the molecular mechanisms underlying the dynamic regulation of mitochondrial networks remain poorly understood. In the present study, we found that the PTEN induced kinase 1 (PINK1)/Parkin (an E3 ubiq TIN- protein ligase) pathway is activated at the early stage of myoblast differentiation. Moreover, downregulation of mitofusin 2 (Mfn2) and increased dynamin-related protein 1 (Drp1) resulted in loosely formed mitochondria during this period. Furthermore, selective knockdown of the mitochondrial matrix protein Lon peptidase-1 (LonP1) at the early stage of myoblast differentiation induced mitochondrial depolarization and suppressed the PINK1/Parkin pathway and reduced Mfn2 and Drp1 levels, which blocked mitochondrial remodeling and myoblast differentiation. Overall, these data demonstrate that LonP1 promotes myoblast differentiation by regulating PINK1/Parkin-mediated mitochondrial remodeling. [ABSTRACT FROM AUTHOR]

Regulation of mitochondrial dynamics and energetics in the diabetic renal proximal tubule by the b2-adrenergic receptor agonist formoterol. Am J Physiol Renal Physiol 319: F773-F779, 2020. First published September 21, 2020; doi:10.1152/ajprenal.00427.2020.--Diabetes is a prevalent metabolic disease that contributes to 50% of all endstage renal disease and has limited treatment options. We previously demonstrated that the b2-adrenergic receptor agonist formoterol induced mitochondrial biogenesis and promoted recovery from acute kidney injury. Here, we assessed the effects of formoterol on mitochondrial dysfunction and dynamics in renal proximal tubule cells (RPTCs) treated with high glucose and in a mouse model of type 2 diabetes. RPTCs exposed to 17 mM glucose exhibited increased electron transport chain (ETC) complex I, II, III, and V protein levels and reduced ATP levels and uncoupled oxygen consumption rate compared with RPTCs cultured in the absence of glucose or osmotic controls after 96 h. ETC proteins, ATP, and oxygen consumption rate were restored in RPTCs treated with formoterol. RPTCs exposed to high glucose had increased phosphodynamin- related protein 1 (Drp1), a mitochondrial fission protein, and decreased mitofusin 1 (Mfn1), a mitochondrial fusion protein. Formoterol treatment restored phospho-Drp1 and Mfn1 to control levels. Db/db and nondiabetic (db/m) mice (10 wk old) were treated with formoterol or vehicle for 3 wk and euthanized. Db/db mice showed increased renal cortical ETC protein levels in complexes I, III, and V and decreased ATP; these changes were prevented by formoterol. Phospho-Drp1 was increased and Mfn1 was decreased in db/db mice, and formoterol restored both to control levels. Together, these findings demonstrate that hyperglycemic conditions in vivo and exposure of RPTCs to high glucose similarly alter mitochondrial bioenergetic and dynamics profiles and that treatment with formoterol can reverse these effects. Formoterol may be a promising strategy for treating early stages of diabetic kidney disease. [ABSTRACT FROM AUTHOR]

Members of the mitochondrial carrier family (SLC25) transport a variety of compounds across the inner membrane of mitochondria. These transport steps provide building blocks for the cell and link the pathways of the mitochondrial matrix and cytosol. An increasing number of diseases and pathologies has been associated with their dysfunction. In this review, the molecular basis of these diseases is explained based on our current understanding of their transport mechanism. [ABSTRACT FROM AUTHOR]

Chronic imaging of mitochondria in the murine cerebral vasculature using in vivo twophoton microscopy. Am J Physiol Heart Circ Physiol 318: H1379- H1386, 2020. First published April 24, 2020; doi:10.1152/ajpheart. 00751.2019.--Mitochondria are important regulators of cerebral vascular function in health and disease, but progress in understanding their roles has been hindered by methodological limitations. We report the first in vivo imaging of mitochondria specific to the cerebral endothelium in real time in the same mouse for extended periods. Mice expressing Dendra2 fluorescent protein in mitochondria (mito- Dendra2) in the cerebral vascular endothelium were generated by breeding PhAM-floxed and Tie2-Cre mice. We used mito-Dendra2 expression, cranial window implantation, and two-photon microscopy to visualize mitochondria in the cerebral vascular endothelium of mice. Immunohistochemistry and mitochondrial staining were used to confirm the localization of the mitochondrial signal to endothelial cells and the specificity of mito-Dendra2 to mitochondria. Mito- Dendra2 and Rhodamine B-conjugated dextran allowed simultaneous determinations of mitochondrial density, vessel diameters, area, and mitochondria-to-vessel ratio in vivo, repeatedly, in the same mouse. Endothelial expression of mito-Dendra2 was confirmed in vitro on brain slices and aorta. In addition, we observed an overlapping mito-Dendra2 and Chromeo mitochondrial staining of cultured brain microvascular endothelial cells. Repeated imaging of the same location in the cerebral microcirculation in the same mouse demonstrated stability of mito-Dendra2. While the overall mitochondrial signal was stable over time, mitochondria within the same endothelial cell were mobile. In conclusion, our results indicate that the mito-Dendra2 signal and vascular parameters are suitable for real-time and longitudinal examination of mitochondria in vivo in the cerebral vasculature of mice. NEW & NOTEWORTHY We introduce an innovative in vivo approach to study mitochondria in the cerebral circulation in their physiological environment by demonstrating the feasibility of longterm imaging and three-dimensional reconstruction. We postulate that the appropriate combination of Cre/Lox system and two-photon microscopy will contribute to a better understanding of the role of mitochondria in not only endothelium but also the different cell types of the cerebral circulation. [ABSTRACT FROM AUTHOR]

Acetaldehyde dehydrogenase 2 (ALDH2) is an enzyme involved in redox homeostasis as well as the detoxification process in alcohol metabolism. Nearly 8% of the world's population have an inactivating mutation in the ALDH2 gene. However, the expression patterns and specific functions of ALDH2 in skeletal muscles are still unclear. Herein, we report that ALDH2 is expressed in skeletal muscle and is localized to the mitochondrial fraction. Oxidative muscles had a higher amount of ALDH2 protein than glycolytic muscles. We next comprehensively investigated whether ALDH2 knockout in mice induces mitochondrial adaptations in gastrocnemius muscle (for example, content, enzymatic activity, respiratory function, supercomplex formation, and functional networking). We found that ALDH2 deficiency resulted in partial mitochondrial dysfunction in gastrocnemius muscle because it increased mitochondrial reactive oxygen species (ROS) emission (2=,7=-dichlorofluorescein and MitoSOX oxidation rate during respiration) and the frequency of regional mitochondrial depolarization. Moreover, we determined whether ALDH2 deficiency and the related mitochondrial dysfunction trigger mitochondrial stress and quality control responses in gastrocnemius muscle (for example, mitophagy markers, dynamics, and the unfolded protein response). We found that ALDH2 deficiency upregulated the mitochondrial serine protease Omi/HtrA2 (a marker of the activation of a branch of the mitochondrial unfolded protein response). In summary, ALDH2 deficiency leads to greater mitochondrial ROS production, but homeostasis can be maintained via an appropriate stress response. [ABSTRACT FROM AUTHOR]

Disruption of mitochondrial dynamics is an important pathogenic event in both acute and chronic kidney diseases, but the underlying mechanism remains poorly understood. Here, we report the regulation of mitofusin-2 (Mfn2; a key mitochondrial fusion protein) by microRNA-214 (miR-214) in renal ischemia-reperfusion that contributes to mitochondrial fragmentation, renal tubular cell death, and ischemic acute kidney injury (AKI). miR-214 was induced, whereas Mfn2 expression was decreased, in mouse ischemic AKI and cultured rat kidney proximal tubular cells (RPTCs) following ATP depletion treatment. Overexpression of miR-214 decreased Mfn2. Conversely, inhibition of miR-214 with anti-miR-214 prevented Mfn2 downregulation in RPTCs following ATP depletion. Anti-miR-214 further ameliorated mitochondrial fragmentation and apoptosis, whereas overexpression of miR-214 increased apoptosis, in ATPdepleted RPTCs. To test regulation in vivo, we established a mouse model with miR-214 specifically deleted from kidney proximal tubular cells (PT-miR-214+/+). Compared with wild-type mice, PT-miR- 214+/+ mice had less severe tissue damage, fewer apoptotic cells, and better renal function after ischemic AKI. miR-214 induction in ischemic AKI was suppressed in PT-miR-214-/- mice, accompanied by partial preservation of Mfn2 in kidneys. These results unveil the miR-214/Mfn2 axis that contributes to the disruption of mitochondrial dynamics and tubular cell death in ischemic AKI, offering new therapeutic targets. [ABSTRACT FROM AUTHOR]

We investigated sex differences in mitochondrial Ca2+ handling properties in mouse fast-twitch skeletal muscle. Changes in cytoplasmic Ca2+ concentration ([Ca2+]cyto) were measured in vivo using tibialis anterior muscles from male and female mice. The muscles were exposed to increasing concentrations of cyclopiazonic acid [CPA; sarcoplasmic reticulum (SR) Ca2+- ATPase inhibitor] (from 10 to 30 to 50 μMat 10 min intervals). Thirty minutes after treatment, [Ca2+]cyto was increased by 31.6 ± 2.0% and 13.5 ± 4.5% of initial [Ca2+]cyto in male and female muscles, respectively, and there was a significant difference between sexes. However, muscle preincubation for 5 min with 10 M carbonyl cyanide-4- (trifluoromethoxy) phenylhydrazone (an inhibitor of mitochondria Ca2+ uptake) eradicated this difference between sexes with respect to the CPA-induced [Ca2+]cyto increase. Both intermyofibrillar mitochondrial number and volume, assessed in longitudinal fiber sections, were higher in females compared with males (mitochondria number: 13.1 ± 1.0 in males vs. 19.9 ± 2.3 in females; mitochondrial volume: 0.034 ± 0.004 μm3/μm3 fiber volume in males vs. 0.066 ± 0.008 μm3/μm3 fiber volume in females, both P < 0.05). There were no sex differences in the content of SR Ca2+-ATPase, mitochondrial Ca2+ uniporter, mitofusin (Mfn) 1, or Mfn2. These results suggest that 1) mitochondrial Ca2+ uptake ability is greater in female than male myocytes, and 2) this superior Ca2+ uptake ability of female myocytes is due, partly, to the higher intermyofibrillar mitochondrial content but not to the expression of mitochondrial proteins related to mitochondrial Ca2+ uptake. [ABSTRACT FROM AUTHOR]

One of the major characteristics of hyperglycemic states such as type 2 diabetes is increased reactive oxygen species (ROS) generation. Since mitochondria are a major source of ROS, it is vital to understand the involvement of these organelles in the pathogenesis of ROSmediated conditions. Therefore, we investigated mitochondrial function and ROS production in cerebral blood vessels of 21-wk-old Zucker diabetic fatty obese rats and their lean controls. We have previously shown that in the early stages of insulin resistance, and short periods of type 2 diabetes mellitus, only mild differences exist in mitochondrial function. In the present study, we examined mitochondrial respiration, mitochondrial protein expression, and ROS production in large-surface cerebral arteries. We used 21-wk-old animals exposed to peak glucose levels for 7 wk and compared them with our previous studies on younger diabetic animals. We found that the same segments of mitochondrial respiration (basal respiration and proton leak) were diminished in diabetic groups as they were in younger diabetic animals. Levels of rattin, a rat humanin analog, tended to decrease in the diabetic group but did not reach statistical significance (P = 0.08). Other mitochondrial proteins were unaffected, which might indicate the existence of compensatory mechanisms with extension of this relatively mild form of diabetes. Superoxide levels were significantly higher in large cerebral vessels of diabetic animals compared with the control group. In conclusion, prolonged dietary diabetes leads to stabilization, rather than deterioration, of metabolic status in the cerebral circulation, despite continued overproduction of ROS. [ABSTRACT FROM AUTHOR]

Endocannabinoids (ECs) mediate effects via cannabinoid receptor types 1 and 2 (CB1 and 2) and transient receptor potential channel-vanilloid subfamily member 1 (TRPV1) channels. In high-fat diet (HFD)-induced obese mice overactivity of the EC system and inhibition of CB1 increase skeletal muscle glucose uptake. We explored the role of TRPV1. Male TRPV1-/-(WT) and TRPV1-/-(KO)-mice were fed (20 wk) a standard laboratory diet (SLD) or HFD. An intraperitoneal glucose tolerance test was performed. RT-PCR was performed to measure mRNA of genes involved in glucose/lipid metabolism and the EC system in soleus (SOL) and extensor digitorum longus (EDL) muscles. Cultured L6 cells were used to measure glucose uptake in skeletal muscle. HFD mice weighed more and had higher insulin levels than SLD mice, with no genotype differences. Basal and peak glucose were higher in HFD mice irrespective of genotype, but glucose cleared faster in HFD WT vs. HFD KO-mice. 2-Arachidonoylglycerol augmented insulin-induced glucose uptake in skeletal L6-cells, an effect blocked by the TRPV1 antagonist SB-366791. In EDL, fatty acid amide hydrolase (FAAH) mRNA was increased in KO vs. WT mice, irrespective of diet. Pyruvate dehydrogenase kinase isozyme 4 (PDK4) and mitochondrial uncoupling protein 3 (UCP3) were elevated and FA desaturase 2 (FADS2) mRNA lower in HFD mice, irrespective of genotype. CB1 and stearoyl-CoA desaturase 1 (SCD1) were lower in HFD WT mice only. In SOL, PDK4, UCP3, hormone-sensitive lipase (LIPE), fatty acid translocase (CD36), and carnitine palmitoyl transferase 2 (CPT2) were elevated and SCD1, FAAH, FADS2, and Troponin 1 (TNNC1) mRNA lower in HFD mice, irrespective of genotype. In conclusion, TRPV1 regulates glucose disposal in HFD mice. We propose that TRPV1 plays a role in coordinating glucose metabolism in EDL under conditions of metabolic stress. [ABSTRACT FROM AUTHOR]

The heart requires high-energy production, but metabolic ability declines in the failing heart. Nicotinamide phosphoribosyl-transferase (Nampt) is a rate-limiting enzyme in the salvage pathway of nicotinamide adenine dinucleotide (NAD) synthesis. NAD is directly involved in various metabolic processes and may indirectly regulate metabolic gene expression through sirtuin 1 (Sirt1), an NAD-dependent protein deacetylase. However, how Nampt regulates cardiac function and metabolism in the failing heart is poorly understood. Here we show that pressure-overload (PO)- induced heart failure is exacerbated in both systemic Nampt heterozygous knockout (Nampt-/-) mice and mice with cardiac-specific Nampt overexpression (Tg-Nampt). The NAD level declined in Nampt-/- mice under PO (wild: 377 pmol/mg tissue; Nampt-/-: 119 pmol/mg tissue; P = 0.028). In cultured cardiomyocytes, Nampt knockdown diminished mitochondrial NAD content and ATP production (relative ATP production: wild: 1; Nampt knockdown: 0.56; P = 0.0068), suggesting that downregulation of Nampt induces mitochondrial dysfunction. On the other hand, the NAD level was increased in Tg-Nampt mice at baseline but not during PO, possibly due to increased consumption of NAD by Sirt1. The expression of Sirt1 was increased in Tg-Nampt mice, in association with reduced overall protein acetylation. PO-induced downregulation of metabolic genes was exacerbated in Tg-Nampt mice. In cultured cardiomyocytes, Nampt and Sirt1 cooperatively suppressed mitochondrial proteins and ATP production, thereby promoting mitochondrial dysfunction. In addition, Nampt overexpression upregulated inflammatory cytokines, including TNF- and monocyte chemoattractant protein-1. Thus endogenous Nampt maintains cardiac function and metabolism in the failing heart, whereas Nampt overexpression is detrimental during PO, possibly due to excessive activation of Sirt1, suppression of mitochondrial function, and upregulation of proinflammatory mechanisms. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S) attenuates N-methyl-D-aspartate receptor-R1 (NMDA-R1) and mitigates diabetic renal damage; however, the molecular mechanism is not well known. Whereas NMDA-R1 facilitates Ca2+ permeability, H2S is known to inhibit L-type Ca2+ channel. High Ca2+ activates cyclophilin D (CypD), a gatekeeper protein of mitochondrial permeability transition pore (MPTP), thus facilitating molecular exchange between matrix and cytoplasm causing oxidative outburst and cell death. We tested the hypothesis of whether NMDA-R1 mediates Ca2+ influx causing CypD activation and MPTP opening leading to oxidative stress and renal injury in diabetes. We also tested whether H2S treatment blocks Ca2+ channel and thus inhibits CypD and MPTP opening to prevent renal damage. C57BL/6J and Akita (C57BL/6J-Ins2Akita) mice were treated without or with H2S donor GYY4137 (0.25 mg·kg-1·day-1 ip) for 8 wk. In vitro studies were performed using mouse glomerular endothelial cells. Results indicated that low levels of H2S and increased expression of NMDA-R1 in diabetes induced Ca2+ permeability, which was ameliorated by H2S treatment. We observed cytosolic Ca2+ influx in hyperglycemic (HG) condition along with mitochondrial-CypD activation, increased MPTP opening, and oxidative outburst, which were mitigated with H2S treatment. Renal injury biomarker KIM-1 was upregulated in HG conditions and normalized following H2S treatment. Inhibition of NMDA-R1 by pharmacological blocker MK-801 revealed similar results. We conclude that NMDA-R1-mediated Ca2+ influx in diabetes induces MPTP opening via CypD activation leading to increased oxidative stress and renal injury, and H2S protects diabetic kidney from injury by blocking mitochondrial Ca2+ permeability through NMDA-R1 pathway. [ABSTRACT FROM AUTHOR]

During hibernation, small mammals, including the 13-lined ground squirrel (Ictidomys tridecemlineatus), cycle between two distinct metabolic states: torpor, where metabolic rate is suppressed by β95% and body temperature falls to ~5°C, and interbout euthermia (IBE), where both metabolic rate and body temperature rapidly increase to euthermic levels. Suppression of whole animal metabolism during torpor is paralleled by rapid, reversible suppression of mitochondrial respiration. We hypothesized that these changes in mitochondrial metabolism are regulated by posttranslational modifications to mitochondrial proteins. Differential two-dimensional gel electrophoresis and two-dimensional blue-native PAGE revealed differences in the isoelectric point of several liver mitochondrial proteins between torpor and IBE. Quadrupole time-of-flight LC/MS and matrix- assisted laser desorption/ionization MS identified these as proteins involved in -oxidation, the tricarboxylic acid cycle, reactive oxygen species detoxification, and the electron transport system (ETS). Immunoblots revealed that subunit 1 of ETS complex IV was acetylated during torpor but not IBE. Phosphoprotein staining revealed significantly greater phosphorylation of succinyl-CoA ligase and the flavoprotein subunit of ETS complex II in IBE than torpor. In addition, the 75-kDa subunit of ETS complex I was 1.5-fold more phosphorylated in torpor. In vitro treatment with alkaline phosphatase increased the maximal activity of complex I from liver mitochondria isolated from torpid, but not IBE, animals. By contrast, phosphatase treatment decreased complex II activity in IBE but not torpor. These findings suggest that the rapid changes in mitochondrial metabolism in hibernators are mediated by posttranslational modifications of key metabolic enzymes, perhaps by intramitochondrial kinases and deacetylases. [ABSTRACT FROM AUTHOR]

Reduced physical performance reduces quality of life in patients with chronic obstructive pulmonary disease (COPD). Impaired physical performance is, in part, a consequence of reduced muscle mass and function, which is accompanied by mitochondrial dysfunction. We recently showed that miR-542-3p and miR-542-5p were elevated in a small cohort of COPD patients and more markedly in critical care patients. In mice, these microRNAs (miRNAs) promoted mitochondrial dysfunction suggesting that they would affect physical performance in patients with COPD, but we did not explore the association of these miRNAs with disease severity or physical performance further. We therefore quantified miR-542-3p/5p and mitochondrial rRNA expression in RNA extracted from quadriceps muscle of patients with COPD and determined their association with physical performance. As miR-542-3p inhibits ribosomal protein synthesis its ability to inhibit protein synthesis was also determined in vitro. Both miR-542-3p expression and -5p expression were elevated in patients with COPD (5-fold P < 0.001) and the degree of elevation associated with impaired lung function (transfer capacity of the lung for CO in % and forced expiratory volume in 1 s in %) and physical performance (6-min walk distance in %). In COPD patients, the ratio of 12S rRNA to 16S rRNA was suppressed suggesting mitochondrial ribosomal stress and mitochondrial dysfunction and miR-542-3p/5p expression was inversely associated with mitochondrial gene expression and positively associated with p53 activity. miR-542-3p suppressed RPS23 expression and maximal protein synthesis in vitro. Our data show that miR-542-3p and -5p expression is elevated in COPD patients and may suppress physical performance at least in part by inhibiting mitochondrial and cytoplasmic ribosome synthesis and suppressing protein synthesis. NEW & NOTEWORTHY miR-542-3p and -5p are elevated in the quadriceps muscle of patients with chronic obstructive pulmonary disease (COPD) in proportion to the severity of their lung disease. These microRNAs inhibit mitochondrial and cytoplasmic protein synthesis suggesting that they contribute to impaired exercise performance in COPD. [ABSTRACT FROM AUTHOR]

Excess fat intake can increase lipid oxidation and expression of mitochondrial proteins, indicating remodeling of the mitochondrial proteome. Yet intermediates of lipid oxidation also accumulate, indicating a relative insufficiency to completely oxidize lipids. We investigated remodeling of the mitochondrial proteome to determine mechanisms of changes in lipid oxidation following high-fat feeding. C57BL/6J mice consumed a high-fat diet (HFD, 60% fat from lard) or a low-fat diet (LFD, 10% fat) for 12 wk. Mice were fasted for 4 h and then anesthetized by pentobarbital sodium overdose for tissue collection. A mitochondrial-enriched fraction was prepared from gastrocnemius muscles and underwent proteomic analysis by high-resolution mass spectrometry. Mitochondrial respiratory efficiency was measured as the ratio of ATP production to O2 consumption. Intramuscular acylcarnitines were measured by liquid chromatography-mass spectrometry. A total of 658 mitochondrial proteins were identified: 40 had higher abundance and 14 had lower abundance in mice consuming the HFD than in mice consuming the LFD. Individual proteins that changed with the HFD were primarily related to β-oxidation; there were fewer changes to the electron transfer system. Gene set enrichment analysis indicated that the HFD increased pathways of lipid metabolism and β-oxidation. Intramuscular concentrations of select acylcarnitines (C18:0) were greater in the HFD mice and reflected dietary lipid composition. Mitochondrial respiratory ATP production-to-O2 consumption ratio for lipids was not different between LFD and HFD mice. After the 60% fat diet, remodeling of the mitochondrial proteome revealed upregulation of proteins regulating lipid oxidation that was not evident for all mitochondrial pathways. The accumulation of lipid metabolites with obesity may occur without intrinsic dysfunction to mitochondrial lipid oxidation. [ABSTRACT FROM AUTHOR]

The 2016 Colorado Trail Race (CTR) was an ultra-endurance mountain bike race in which competitors cycled for up to 24 h/day between altitudes of 1,675 and 4,025 m to complete 800 km and 21,000 m of elevation gain. In one athlete, we had the unique opportunity to characterize skeletal muscle protein synthesis and mitochondrial respiration in response to a normal activity control period (CON) and the CTR. We hypothesized that mitochondrial protein synthesis would be elevated and mitochondrial respiration would be maintained during the extreme stresses of the CTR. Titrated and bolus doses of ADP were provided to determine substrate-specific oxidative phosphorylation (OXPHOS) and electron transport system (ETS) capacities in permeabilized muscle fibers via high-resolution respirometry. Protein synthetic rates were determined by daily oral consumption of deuterium oxide (2H2O). The endurance athlete had OXPHOS (226 pmol·s-1·mg tissue-1) and ETS (231 pmol·s-1·mg tissue-1) capacities that rank among the highest published to date in humans. Mitochondrial (3.2-fold), cytoplasmic (2.3-fold), and myofibrillar (1.5-fold) protein synthesis rates were greater during CTR compared with CON. With titrated ADP doses, the apparent Km of ADP, OXPHOS, and ETS increased after the CTR. With provision of ADP boluses after the CTR, the addition of fatty acids (-12 and -14%) mitigated the decline in OXPHOS and ETS capacity during carbohydrate-supported respiration (-26 and -31%). In the face of extreme stresses during the CTR, elevated rates of mitochondrial protein synthesis may contribute to rapid adaptations in mitochondrial bioenergetics. NEW & NOTEWORTHY The mechanisms that maintain skeletal muscle function during extreme stresses remain incompletely understood. In the current study, greater rates of mitochondrial protein synthesis during the energetic demands of ultra-endurance exercise may contribute to rapid adaptations in mitochondrial bioenergetics. The endurance athlete herein achieved mitochondrial respiratory capacities among the highest published for humans. Greater mitochondrial protein synthesis during ultra-endurance exercise may contribute to improved mitochondrial respiration and serve as a mechanism to resist cellular energetic stresses. [ABSTRACT FROM AUTHOR]

Lysine acetylation is a reversible posttranslational modification and is particularly important in the regulation of mitochondrial metabolic enzymes. Acetylation uses acetyl-CoA derived from fuel metabolism as a cofactor, thereby linking nutrition to metabolic activity. In the present study, we investigated how mitochondrial acetylation status in the heart is controlled by food intake and how these changes affect mitochondrial metabolism. We found that there was a significant increase in cardiac mitochondrial protein acetylation in mice fed a long-term high-fat diet and that this change correlated with an increase in the abundance of the mitochondrial acetyltransferase-related protein GCN5L1. We showed that the acetylation status of several mitochondrial fatty acid oxidation enzymes (long-chain acyl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase, and hydroxyacyl-CoA dehydrogenase) and a pyruvate oxidation enzyme (pyruvate dehydrogenase) was significantly upregulated in high-fat diet-fed mice and that the increase in long-chain and short-chain acyl-CoA dehydrogenase acetylation correlated with increased enzymatic activity. Finally, we demonstrated that the acetylation of mitochondrial fatty acid oxidation proteins was decreased after GCN5L1 knockdown and that the reduced acetylation led to diminished fatty acid oxidation in cultured H9C2 cells. These data indicate that lysine acetylation promotes fatty acid oxidation in the heart and that this modification is regulated in part by the activity of GCN5L1. [ABSTRACT FROM AUTHOR]

Obstructive kidney disease is a common complication in the clinic. Downregulation of aquaporins (AQPs) in obstructed kidneys has been thought as a key factor leading to the polyuria and impairment of urine-concentrating capability after the release of kidney obstruction. The present study was to investigate the role of mitochondrial complex-1 in modulating AQPs in obstructive nephropathy. Following 7-day unilateral ureteral obstruction (UUO), AQP1, AQP2, AQP3, and vasopressin 2 (V2) receptor were remarkably reduced as determined by qRT-PCR and/or Western blotting. Notably, inhibition of mitochondrial complex-1 by rotenone markedly reversed the downregulation of AQP1, AQP2, AQP3, and V2. In contrast, AQP4 was not affected by kidney obstruction or rotenone treatment. In a separate study, rotenone also attenuated AQPs' downregulation after 48-h UUO. To study the potential mechanisms in mediating the rotenone effects on AQPs, we examined the regulation of the COX-2/microsomal prostaglandin E synthase (mPGES)-1/PGE2/EP pathway and found that COX-2, mPGES-1, and renal PGE2 content were all significantly elevated in obstructive kidneys, which was not affected by rotenone treatment. For EP receptors, EP2 and EP4 but not EP1 and EP3 were upregulated in obstructive kidneys. Importantly, rotenone strikingly suppressed EP1 and EP4 but not EP2 and EP3 receptors. However, treatment of EP1 antagonist SC-51322 could not affect AQPs' reduction in obstructed kidneys. Collectively, these findings suggested an important role of mitochondrial dysfunction in modulating AQPs and V2 receptor in obstructive nephropathy possibly via prostaglandin-independent mechanisms. [ABSTRACT FROM AUTHOR]

The histidine nucleotide-binding protein, Hint2, is a mitochondrial phosphoramidase expressed in liver, brown fat, pancreas, and muscle. The livers of Hint2 knockout (Hint2-/-) mice accumulate triglycerides and show a pattern of mitochondrial protein lysine hyperacetylation. The extent and nature of the lysine acetylation changes and the response of Hint2-/- mice to nutritional challenges that elicit a modification of protein acetylation have not been investigated. To compare the adaptation of Hint2-/- and control (Hint2+/+) mice with episodes of fasting and high-fat diet (HFD), we subjected animals to either feeding ad libitum or fasting for 24 h, and to either a HFD or control diet for 8 wk. Triglyceride content was higher in Hint2-/- than in Hint2+/+ livers, whereas plasma triglycerides were fourfold lower. Malonyl-CoA levels were increased twofold in Hint2-/- livers. After 24 h fasting, Hint2-/- displayed a decrease in body temperature, commensurate with a decrease in mass of brown fat and downregulation of uncoupling protein 1. HFD-treated Hint2-/- livers showed more steatosis, and plasma insulin and cholesterol were higher than in Hint+/+ mice. Several proteins identified as substrates of sirtuin 3 and 5 and active in intermediary and ketone metabolism were hyperacetylated in liver and brown fat mitochondria after both HFD and fasting regimens. Glutamate dehydrogenase activity was downregulated in fed and fasted livers, and this was attributed to an increase in acetylation and ADP-ribosylation. The absence of Hint2 deregulates the posttranslational modification of several mitochondrial proteins, which impedes the adaptation to episodes of nutritional stress. [ABSTRACT FROM AUTHOR]

It has been proposed that diet-induced obesity at thermoneutrality (TN; 29°C) is reduced by a UCP1-dependent thermogenesis; however, it has not been shown how UCP1-dependent thermogenesis can be activated in the absence of sympathetic activity. A recent study provides such a mechanism by showing that dietary bile acids (BAs) suppress obesity in mice fed a high-fat diet (HFD) by a mechanism dependent on type 2 deiodinase (DIO2); however, neither a role for UCP1 nor the influence of sympathetic activity was properly assessed. To test whether the effects of BAs on adiposity are independent of Ucp1 and cold-activated thermogenesis, obesity phenotypes were determined in C57BL6/J.+/+ (WT) and C57BL6/J.Ucp1.-/- mice (Ucp1-KO) housed at TN and fed a HFD with or without 0.5% (wt/wt) cholic acid (CA) for 9 wk. CA in a HFD reduced adiposity and hepatic lipogenesis and improved glucose tolerance in WT but not in Ucp1-KO mice and was accompanied by increases in food intake and energy expenditure (EE). In iBAT, CA increased Ucp1 mRNA and protein levels 1.5- and twofold, respectively, and increased DIO2 and TGR5 protein levels in WT mice. Despite enhanced Dio2 expression in Ucp1-KO and Ucp1-KO-CA treated mice, this did not enhance the ability of BAs to reduce obesity. By comparing the effects of BAs on WT and Ucp1-KO mice at TN, our study showed that BAs suppress diet-induced obesity by increasing EE through a mechanism dependent on Ucp1 expression, which is likely independent of adrenergic signaling. [ABSTRACT FROM AUTHOR]

Calpain 1 (CPN1) is a ubiquitous cysteine protease that exists in both cytosol and cardiac mitochondria. Mitochondrial CPN1 (mit-CPN1) is located in the intermembrane space and matrix. Activation of mit-CPN1 within the intermembrane space increases cardiac injury by releasing apoptosis-inducing factor from mitochondria during ischemia-reperfusion (IR). We asked if activation of mit-CPN1 is involved in mitochondrial injury during IR. MDL-28170 (MDL) was used to inhibit CPN1 in buffer-perfused hearts following 25-min ischemia and 30-min reperfusion. MDL treatment decreased the release of lactate dehydrogenase into coronary effluent compared with untreated hearts, indicating that inhibition of CPN1 decreases cardiac injury. MDL also prevented the cleavage of spectrin (a substrate of CPN1) in cytosol during IR, supporting that MDL treatment decreased cytosolic calpain activation. In addition, MDL markedly improved calcium retention capacity compared with untreated heart, suggesting that MDL treatment decreases mitochondrial permeability transition pore opening. In addition, we found that IR led to decreased complex I activity, whereas inhibition of mit-CPN1 using MDL protected complex I. Pyruvate dehydrogenase content was decreased following IR. However, pyruvate dehydrogenase content was preserved in MDL-treated mitochondria. Taken together, MDL treatment decreased cardiac injury during IR by inhibiting both cytosolic and mit-CPN1. Activation of mit-CPN1 increases cardiac injury during IR by sensitizing mitochondrial permeability transition pore opening and impairing mitochondrial metabolism through damage of complex I. [ABSTRACT FROM AUTHOR]