Mitochondrial Telomerase Reverse Transcriptase Protects From Myocardial Ischemia/Reperfusion Injury by Improving Complex I Composition and Function.

BACKGROUND: The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), has protective functions in the cardiovascular system. TERT is not only present in the nucleus but also in mitochondria. However, it is unclear whether nuclear or mitochondrial TERT is responsible for the observed protection, and the appropriate tools are missing to dissect this. METHODS: We generated new mouse models containing TERT exclusively in the mitochondria (mitoTERT mice) or the nucleus (nucTERT mice) to finally distinguish between the functions of nuclear and mitochondrial TERT. Outcome after ischemia/reperfusion, mitochondrial respiration in the heart, and cellular functions of cardiomyocytes, fibroblasts, and endothelial cells, as well, were determined. RESULTS: All mice were phenotypically normal. Although respiration was reduced in cardiac mitochondria from TERT-deficient and nucTERT mice, it was increased in mitoTERT animals. The latter also had smaller infarcts than wild-type mice, whereas nucTERT animals had larger infarcts. The decrease in ejection fraction after 1, 2, and 4 weeks of reperfusion was attenuated in mitoTERT mice. Scar size was also reduced and vascularization increased. Mitochondrial TERT protected a cardiomyocyte cell line from apoptosis. Myofibroblast differentiation, which depends on complex I activity, was abrogated in TERT-deficient and nucTERT cardiac fibroblasts and completely restored in mitoTERT cells. In endothelial cells, mitochondrial TERT enhanced migratory capacity and activation of endothelial nitric oxide synthase. Mechanistically, mitochondrial TERT improved the ratio between complex I matrix arm and membrane subunits, explaining the enhanced complex I activity. In human right atrial appendages, TERT was localized in mitochondria and there increased by remote ischemic preconditioning. The telomerase activator TA-65 evoked a similar effect in endothelial cells, thereby increasing their migratory capacity, and enhanced myofibroblast differentiation. CONCLUSIONS: Mitochondrial, but not nuclear TERT, is critical for mitochondrial respiration and during ischemia/reperfusion injury. Mitochondrial TERT improves complex I subunit composition. TERT is present in human heart mitochondria, and remote ischemic preconditioning increases its level in those organelles. TA-65 has comparable effects ex vivo and improves the migratory capacity of endothelial cells and myofibroblast differentiation. We conclude that mitochondrial TERT is responsible for cardioprotection, and its increase could serve as a therapeutic strategy.

Telomerase as a Therapeutic Target in Cardiovascular Disease.

Shortened telomeres have been linked to numerous chronic diseases, most importantly coronary artery disease, but the underlying mechanisms remain ill defined. Loss-of-function mutations and deletions in telomerase both accelerate telomere shortening but do not necessarily lead to a clinical phenotype associated with atherosclerosis, questioning the causal role of telomere length in cardiac pathology. The differential extranuclear functions of the 2 main components of telomerase, telomerase reverse transcriptase and telomerase RNA component, offer important clues about the complex relationship between telomere length and cardiovascular pathology. In this review, we critically discuss relevant preclinical models, genetic disorders, and clinical studies to elucidate the impact of telomerase in cardiovascular disease and its potential role as a therapeutic target. We suggest that the antioxidative function of mitochondrial telomerase reverse transcriptase might be atheroprotective, making it a potential target for clinical trials. Graphic Abstract: A graphic abstract is available for this article.

Local Peroxynitrite Impairs Endothelial Transient Receptor Potential Vanilloid 4 Channels and Elevates Blood Pressure in Obesity.

BACKGROUND: Impaired endothelium-dependent vasodilation is a hallmark of obesity-induced hypertension. The recognition that Ca2+ signaling in endothelial cells promotes vasodilation has led to the hypothesis that endothelial Ca2+ signaling is compromised during obesity, but the underlying abnormality is unknown. In this regard, transient receptor potential vanilloid 4 (TRPV4) ion channels are a major Ca2+ influx pathway in endothelial cells, and regulatory protein AKAP150 (A-kinase anchoring protein 150) enhances the activity of TRPV4 channels. METHODS: We used endothelium-specific knockout mice and high-fat diet-fed mice to assess the role of endothelial AKAP150-TRPV4 signaling in blood pressure regulation under normal and obese conditions. We further determined the role of peroxynitrite, an oxidant molecule generated from the reaction between nitric oxide and superoxide radicals, in impairing endothelial AKAP150-TRPV4 signaling in obesity and assessed the effectiveness of peroxynitrite inhibition in rescuing endothelial AKAP150-TRPV4 signaling in obesity. The clinical relevance of our findings was evaluated in arteries from nonobese and obese individuals. RESULTS: We show that Ca2+ influx through TRPV4 channels at myoendothelial projections to smooth muscle cells decreases resting blood pressure in nonobese mice, a response that is diminished in obese mice. Counterintuitively, release of the vasodilator molecule nitric oxide attenuated endothelial TRPV4 channel activity and vasodilation in obese animals. Increased activities of inducible nitric oxide synthase and NADPH oxidase 1 enzymes at myoendothelial projections in obese mice generated higher levels of nitric oxide and superoxide radicals, resulting in increased local peroxynitrite formation and subsequent oxidation of the regulatory protein AKAP150 at cysteine 36, to impair AKAP150-TRPV4 channel signaling at myoendothelial projections. Strategies that lowered peroxynitrite levels prevented cysteine 36 oxidation of AKAP150 and rescued endothelial AKAP150-TRPV4 signaling, vasodilation, and blood pressure in obesity. Peroxynitrite-dependent impairment of endothelial TRPV4 channel activity and vasodilation was also observed in the arteries from obese patients. CONCLUSIONS: These data suggest that a spatially restricted impairment of endothelial TRPV4 channels contributes to obesity-induced hypertension and imply that inhibiting peroxynitrite might represent a strategy for normalizing endothelial TRPV4 channel activity, vasodilation, and blood pressure in obesity.

CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system-New mode of action for caffeine.

We show that the cyclin-dependent kinase inhibitor 1B (CDKN1B)/p27, previously known as a cell cycle inhibitor, is also localized within mitochondria. The migratory capacity of endothelial cells, which need intact mitochondria, is completely dependent on mitochondrial p27. Mitochondrial p27 improves mitochondrial membrane potential, increases adenosine triphosphate (ATP) content, and is required for the promigratory effect of caffeine. Domain mapping of p27 revealed that the N-terminus and C-terminus are required for those improvements. Further analysis of those regions revealed that the translocation of p27 into the mitochondria and its promigratory activity depend on serine 10 and threonine 187. In addition, mitochondrial p27 protects cardiomyocytes against apoptosis. Moreover, mitochondrial p27 is necessary and sufficient for cardiac myofibroblast differentiation. In addition, p27 deficiency and aging decrease respiration in heart mitochondria. Caffeine does not increase respiration in p27-deficient animals, whereas aged mice display improvement after 10 days of caffeine in drinking water. Moreover, caffeine induces transcriptome changes in a p27-dependent manner, affecting mostly genes relevant for mitochondrial processes. Caffeine also reduces infarct size after myocardial infarction in prediabetic mice and increases mitochondrial p27. Our data characterize mitochondrial p27 as a common denominator that improves mitochondria-dependent processes and define an increase in mitochondrial p27 as a new mode of action of caffeine.

Measurement of endothelium-dependent vasodilation in mice--brief report.

OBJECTIVE: Endothelium-dependent, flow-mediated vasodilation after an increase in shear stress at the endothelial lining of conduit arteries during reactive hyperemia after ischemia is a fundamental principle of vascular physiology adapting blood flow to demand of supplied tissue. Flow-mediated vasodilation measurements have been performed in human studies and are of diagnostic and prognostic importance, but have been impossible because of technical limitations in transgenic mice to date, although these represent the most frequently used animal model in cardiovascular research. APPROACH AND RESULTS: Using high-frequency ultrasound, we visualized, quantified, and characterized for the first time endothelium-dependent dilation of the femoral artery after temporal ischemia of the lower part of the hindlimb and demonstrated that the signaling was almost exclusively dependent on stimulation of endothelial nitric oxide synthase, similar to acetylcholine, completely abolished after pharmacological or genetic inhibition of endothelial nitric oxide synthase and endothelial denudation, substantially impaired in mice of increasing age and cholesterol-fed ApoE knock outs and increased by the dietary polyphenol (-)-epicatechin. Intra- and interindividual variability were similar to the human methodology. CONCLUSIONS: The physiology of flow-mediated vasodilation in mice resembles that in humans underscoring the significance of this novel technology to noninvasively, serially, and reliably quantify flow-mediated vasodilation in transgenic mice.

Two isoforms of Sister-Of-Mammalian Grainyhead have opposing functions in endothelial cells and in vivo.

OBJECTIVE: Sister-of-Mammalian Grainyhead (SOM) is a member of the Grainyhead family of transcription factors. In humans, 3 isoforms are derived from differential first exon usage and alternative splicing and differ only in their N terminal domain. SOM2, the only variant also present in mouse, induces endothelial cell migration and protects against apoptosis. The functions of the human specific isoforms SOM1 and SOM3 have not yet been investigated. Therefore we wanted to elucidate their functions in endothelial cells. APPROACH AND RESULTS: Overexpression of SOM1 in primary human endothelial cells induced migration, phosphorylation of Akt1 and endothelial nitric oxide synthase, and protected against apoptosis, whereas SOM3 had opposite effects; isoform-specific knockdowns confirmed the disparate effects on apoptosis. After reporter assays demonstrated that both are active transcription factors, microarray analyses revealed that they induce different target genes, which could explain the different cellular effects. Overexpression of SOM3 in zebrafish embryos resulted in increased lethality and severe deformations, whereas SOM1 had no deleterious effect. CONCLUSIONS: Our data demonstrate that the splice variant-derived isoforms SOM1 and SOM3 induce opposing effects in primary human endothelial cells and in a whole animal model, most likely through the induction of different target genes.

Dose-Dependent Effects of Lipopolysaccharide on the Endothelium-Sepsis versus Metabolic Endotoxemia-Induced Cellular Senescence.

The endothelium, the innermost cell layer of blood vessels, is not only a physical barrier between the bloodstream and the surrounding tissues but has also essential functions in vascular homeostasis. Therefore, it is not surprising that endothelial dysfunction is associated with most cardiovascular diseases. The functionality of the endothelium is compromised by endotoxemia, the presence of bacterial endotoxins in the bloodstream with the main endotoxin lipopolysaccharide (LPS). Therefore, this review will focus on the effects of LPS on the endothelium. Depending on the LPS concentration, the outcomes are either sepsis or, at lower concentrations, so-called low-dose or metabolic endotoxemia. Sepsis, a life-threatening condition evoked by hyperactivation of the immune response, includes breakdown of the endothelial barrier resulting in failure of multiple organs. A deeper understanding of the underlying mechanisms in the endothelium might help pave the way to new therapeutic options in sepsis treatment to prevent endothelial leakage and fatal septic shock. Low-dose endotoxemia or metabolic endotoxemia results in chronic inflammation leading to endothelial cell senescence, which entails endothelial dysfunction and thus plays a critical role in cardiovascular diseases. The identification of compounds counteracting senescence induction in endothelial cells might therefore help in delaying the onset or progression of age-related pathologies. Interestingly, two natural plant-derived substances, caffeine and curcumin, have shown potential in preventing endothelial cell senescence.

Dysfunction of the adhesion G protein-coupled receptor latrophilin 1 (ADGRL1/LPHN1) increases the risk of obesity.

Obesity is one of the diseases with severe health consequences and rapidly increasing worldwide prevalence. Understanding the complex network of food intake and energy balance regulation is an essential prerequisite for pharmacological intervention with obesity. G protein-coupled receptors (GPCRs) are among the main modulators of metabolism and energy balance. They, for instance, regulate appetite and satiety in certain hypothalamic neurons, as well as glucose and lipid metabolism and hormone secretion from adipocytes. Mutations in some GPCRs, such as the melanocortin receptor type 4 (MC4R), have been associated with early-onset obesity. Here, we identified the adhesion GPCR latrophilin 1 (ADGRL1/LPHN1) as a member of the regulating network governing food intake and the maintenance of energy balance. Deficiency of the highly conserved receptor in mice results in increased food consumption and severe obesity, accompanied by dysregulation of glucose homeostasis. Consistently, we identified a partially inactivating mutation in human ADGRL1/LPHN1 in a patient suffering from obesity. Therefore, we propose that LPHN1 dysfunction is a risk factor for obesity development.

Caffeine Inhibits Oxidative Stress- and Low Dose Endotoxemia-Induced Senescence-Role of Thioredoxin-1.

The maintenance of Thioredoxin-1 (Trx-1) levels, and thus of cellular redox homeostasis, is vital for endothelial cells (ECs) to prevent senescence induction. One hallmark of EC functionality, their migratory capacity, which depends on intact mitochondria, is reduced in senescence. Caffeine improves the migratory capacity and mitochondrial functionality of ECs. However, the impact of caffeine on EC senescence has never been investigated. Moreover, a high-fat diet, which can induce EC senescence, results in approximately 1 ng/mL lipopolysaccharide (LPS) in the blood. Therefore, we investigated if low dose endotoxemia induces EC senescence and concomitantly reduces Trx-1 levels, and if caffeine prevents or even reverses senescence. We show that caffeine precludes H2O2-triggered senescence induction by maintaining endothelial NO synthase (eNOS) levels and preventing the elevation of p21. Notably, 1 ng/mL LPS also increases p21 levels and reduces eNOS and Trx-1 amounts. These effects are completely blocked by co-treatment with caffeine. This prevention of senescence induction is similarly accomplished by the permanent expression of mitochondrial p27, a downstream effector of caffeine. Most importantly, after senescence induction by LPS, a single bolus of caffeine inhibits the increase in p21. This treatment also blocks Trx-1 degradation, suggesting that the reversion of senescence is intimately associated with a normalized redox balance.

Oxidative stress-induced degradation of thioredoxin-1 and apoptosis is inhibited by thioredoxin-1-actin interaction in endothelial cells.

OBJECTIVE: Thioredoxin-1 (Trx-1), one important antioxidative enzyme in endothelial cells, is required for apoptosis inhibition. Apoptosis induction is dependent on cytoskeletal changes, which depend on actin rearrangements. Therefore, we wanted to elucidate whether a physical interaction exists between Trx-1 and actin and what the functional consequences are. METHODS AND RESULTS: Combined immunoprecipitation/mass spectrometry identified actin as a new binding partner for Trx-1. A separate pool of Trx-1 forms a complex with apoptosis signaling kinase 1. Actin is required for stress fiber formation; thus, the interaction of actin with Trx-1 might interfere with this process. Stress fiber formation, which is directly linked to the phosphorylation of focal adhesion kinase (FAK), occurs as early as 1 hour after H(2)O(2) treatment. It is inhibited by Trx-1 overexpression, treatment with exogenous Trx-1, or inhibition of FAK. Prolonged incubation with H(2)O(2) induced stress fiber formation, reduced Trx-1 protein levels, and increased apoptosis. All these processes were inhibited by preincubation with the FAK inhibitor PF573228. On the contrary, incubation with PF573228 1 hour after H(2)O(2) treatment did not block stress fiber formation, degradation of Trx-1, or apoptosis. CONCLUSIONS: These data demonstrate that the actin-Trx-1 complex protects Trx-1 from degradation and, thus, endothelial cells from apoptosis. Reciprocally, Trx-1 prevents stress fiber formation.

Protective Effects of Curcumin in Cardiovascular Diseases-Impact on Oxidative Stress and Mitochondria.

Cardiovascular diseases (CVDs) contribute to a large part of worldwide mortality. Similarly, two of the major risk factors for these diseases, aging and obesity, are also global problems. Aging, the gradual decline of body functions, is non-modifiable. Obesity, a modifiable risk factor for CVDs, also predisposes to type 2 diabetes mellitus (T2DM). Moreover, it affects not only the vasculature and the heart but also specific fat depots, which themselves have a major impact on the development and progression of CVDs. Common denominators of aging, obesity, and T2DM include oxidative stress, mitochondrial dysfunction, metabolic abnormalities such as altered lipid profiles and glucose metabolism, and inflammation. Several plant substances such as curcumin, the major active compound in turmeric root, have been used for a long time in traditional medicine and for the treatment of CVDs. Newer mechanistic, animal, and human studies provide evidence that curcumin has pleiotropic effects and attenuates numerous parameters which contribute to an increased risk for CVDs in aging as well as in obesity. Thus, curcumin as a nutraceutical could hold promise in the prevention of CVDs, but more standardized clinical trials are required to fully unravel its potential.

Triiodothyronine improves contractile recovery of human atrial trabeculae after hypoxia/reoxygenation.

BACKGROUND: In patients undergoing interventional or surgical coronary revascularization, subclinical hypothyroidism is common and associated with worse outcome, including the need for postoperative inotropic support. In isolated rat hearts with global ischemia/reperfusion, exogenous triiodothyronine (T3) reduces infarct size. Aim of this study was, to investigate whether or not exogenous T3 protects human myocardium from ischemia/reperfusion injury. METHODS: Right atrial trabeculae from patients undergoing routine coronary artery bypass grafting were isolated and transferred to Tyrode's buffer. Electrically initiated (1 Hz) contractile stress (mN/mm2) was recorded for 10 min at baseline (95% O2/ 5% CO2, glucose). Sixty min hypoxia were induced by changing buffer gas and increasing stimulation rate (95% N2/ 5% CO2, choline chloride, 3 Hz) before return to reoxygenation for 30 min. T3 (500 µg/l) vs. NaOH (solvent control) was administered A) throughout (n = 11 vs. n = 9) or B) only 15 min before and during reoxygenation (n = 12 vs. n = 13). Western blot analyses of established cardioprotective signaling proteins were performed. RESULTS: At baseline, contractile stress was comparable. T3 improved the cumulative recovery of contractile stress during reoxygenation from 41 ± 16 with NaOH to 55 ± 11% of baseline with T3, when given continuously in A or from 52 ± 13 with NaOH to 63 ± 11% of baseline with T3 when given just before and during reoxygenation in B. The ratio of mitochondrial complex I matrix arm to membrane NADH:ubiquinone oxidoreductase subunits (NDUF)V2 to NDUFA9 was reduced, reflecting increased complex I activity. CONCLUSION: T3 increases contractile recovery of human right atrial trabeculae from hypoxia/reoxygenation.

Flavanol Consumption in Healthy Men Preserves Integrity of Immunological-Endothelial Barrier Cell Functions: Nutri(epi)genomic Analysis.

SCOPE: While cocoa flavanol (CF) consumption improves cardiovascular risk biomarkers, molecular mechanisms underlying their protective effects are not understood. OBJECTIVE: To investigate nutri(epi)genomic effects of CF and identify regulatory networks potential mediating vascular health benefits. METHODS AND RESULTS: Twenty healthy middle-aged men consume CF (bi-daily 450 mg) or control drinks for 1 month. Microarray analysis identifies 2235 differentially expressed genes (DEG) involved in processes regulating immune response, cell adhesion, or cytoskeleton organization. Distinct patterns of DEG correlate with CF-related changes in endothelial function, arterial stiffness, and blood pressure. DEG profile negatively correlates with expression profiles of cardiovascular disease patients. CF modulated DNA methylation profile of genes implicates in cell adhesion, actin cytoskeleton organization, or cell signaling. In silico docking analyses indicate that CF metabolites have the potential of binding to cell signaling proteins and transcription factors. Incubation of plasma obtained after CF consumption decrease monocyte to endothelial adhesion and dose-dependently increase nitric oxide-dependent chemotaxis of circulating angiogenic cells further validating the biological functions of CF metabolites. CONCLUSION: In healthy humans, CF consumption may mediate vascular protective effects by modulating gene expression and DNA methylation towards a cardiovascular protective effect, in agreement with clinical results, by preserving integrity of immunological-endothelial barrier functions.

Aryl Hydrocarbon Receptor-Dependent and -Independent Pathways Mediate Curcumin Anti-Aging Effects.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor whose activity can be modulated by polyphenols, such as curcumin. AhR and curcumin have evolutionarily conserved effects on aging. Here, we investigated whether and how the AhR mediates the anti-aging effects of curcumin across species. Using a combination of in vivo, in vitro, and in silico analyses, we demonstrated that curcumin has AhR-dependent or -independent effects in a context-specific manner. We found that in Caenorhabditis elegans, AhR mediates curcumin-induced lifespan extension, most likely through a ligand-independent inhibitory mechanism related to its antioxidant activity. Curcumin also showed AhR-independent anti-aging activities, such as protection against aggregation-prone proteins and oxidative stress in C. elegans and promotion of the migratory capacity of human primary endothelial cells. These AhR-independent effects are largely mediated by the Nrf2/SKN-1 pathway.

The transcription factor Grainyhead like 3 (GRHL3) affects endothelial cell apoptosis and migration in a NO-dependent manner.

Migratory capacity and resistance to apoptosis are crucial for proper endothelial function. In a screen for anti-apoptotic genes in a breast cancer cell line, we identified Grainyhead like 3 (GRHL3). Therefore, the aim of our study was to investigate whether GRHL3 is expressed in endothelial cells and moreover, to determine its role in migration, apoptosis and senescence. GRHL3 is expressed in human endothelial cells. GRHL3 is required for endothelial cell migration. The underlying mechanism is independent of vascular endothelial growth factor. GRHL3 induces Akt and endothelial nitric oxide synthase phosphorylation and its expression is increased by physiological concentrations of nitric oxide. Nitric oxide dependent migration is completely dependent on GRHL3 expression. Moreover, GRHL3 inhibits apoptosis of endothelial cells in an eNOS-dependent manner. Thus, loss of GRHL3 may result in endothelial dysfunction in vivo. One may consider new therapeutic strategies with the aim to conserve GRHL3 expression in the vasculature.

Extra-Nuclear Functions of the Transcription Factor Grainyhead-Like 3 in the Endothelium-Interaction with Endothelial Nitric Oxide Synthase.

We previously demonstrated that the transcription factor Grainyhead-like 3 (GRHL3) has essential functions in endothelial cells by inhibiting apoptosis and promoting migration as well as activation of endothelial nitric oxide synthase (eNOS). We now show that a large portion of the protein is localized to myo-endothelial projections of murine arteries suggesting extra-nuclear functions. Therefore, we generated various deletion mutants to identify the nuclear localization signal (NLS) of GRHL3 and assessed potential extra-nuclear functions. Several large-scale deletion mutants were incapable of activating a GRHL3-dependent reporter construct, which could either be due to deficiencies in transcriptional activation or to impaired nuclear import. One of these mutants encompassed a predicted bipartite NLS whose deletion led to the retention of GRHL3 outside the nucleus. Interestingly, this mutant retained functions of the full-length protein as it could still inhibit pathways inducing endothelial cell apoptosis. As apoptosis protection by GRHL3 depends on NO-production, we examined whether GRHL3 could interact with eNOS and showed a direct interaction, which was enhanced with the extra-nuclear GRHL3 variant. The observation that endogenous GRHL3 also interacts with eNOS in intact murine arteries corroborated these findings and substantiated the notion that GRHL3 has important extra-nuclear functions in the endothelium.

Selenoprotein T Protects Endothelial Cells against Lipopolysaccharide-Induced Activation and Apoptosis.

Sepsis is an exaggerated immune response upon infection with lipopolysaccharide (LPS) as the main causative agent. LPS-induced activation and apoptosis of endothelial cells (EC) can lead to organ dysfunction and finally organ failure. We previously demonstrated that the first twenty amino acids of the Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) are sufficient to inhibit EC apoptosis. To identify genes whose regulation by LPS is affected by this N-terminal APEX1 peptide, EC were transduced with an expression vector for the APEX1 peptide or an empty control vector and treated with LPS. Following RNA deep sequencing, genes upregulated in LPS-treated EC expressing the APEX1 peptide were identified bioinformatically. Selected candidates were validated by semi-quantitative real time PCR, a promising one was Selenoprotein T (SELENOT). For functional analyses, an expression vector for SELENOT was generated. To study the effect of SELENOT expression on LPS-induced EC activation and apoptosis, the SELENOT vector was transfected in EC. Immunostaining showed that SELENOT was expressed and localized in the ER. EC transfected with the SELENOT plasmid showed no activation and reduced apoptosis induced by LPS. SELENOT as well as APEX1(1-20) can protect EC against activation and apoptosis and could provide new therapeutic approaches in the treatment of sepsis.

Role of Mitochondrial Protein Import in Age-Related Neurodegenerative and Cardiovascular Diseases.

Mitochondria play a critical role in providing energy, maintaining cellular metabolism, and regulating cell survival and death. To carry out these crucial functions, mitochondria employ more than 1500 proteins, distributed between two membranes and two aqueous compartments. An extensive network of dedicated proteins is engaged in importing and sorting these nuclear-encoded proteins into their designated mitochondrial compartments. Defects in this fundamental system are related to a variety of pathologies, particularly engaging the most energy-demanding tissues. In this review, we summarize the state-of-the-art knowledge about the mitochondrial protein import machinery and describe the known interrelation of its failure with age-related neurodegenerative and cardiovascular diseases.

Accessing Mitochondrial Protein Import in Living Cells by Protein Microinjection.

Mitochondrial protein biogenesis relies almost exclusively on the expression of nuclear-encoded polypeptides. The current model postulates that most of these proteins have to be delivered to their final mitochondrial destination after their synthesis in the cytoplasm. However, the knowledge of this process remains limited due to the absence of proper experimental real-time approaches to study mitochondria in their native cellular environment. We developed a gentle microinjection procedure for fluorescent reporter proteins allowing a direct non-invasive study of protein transport in living cells. As a proof of principle, we visualized potential-dependent protein import into mitochondria inside intact cells in real-time. We validated that our approach does not distort mitochondrial morphology and preserves the endogenous expression system as well as mitochondrial protein translocation machinery. We observed that a release of nascent polypeptides chains from actively translating cellular ribosomes by puromycin strongly increased the import rate of the microinjected pre-protein. This suggests that a substantial amount of mitochondrial translocase complexes was involved in co-translational protein import of endogenously expressed pre-proteins. Our protein microinjection method opens new possibilities to study the role of mitochondrial protein import in cell models of various pathological conditions as well as aging processes.

Endothelial hyaluronan synthase 3 aggravates acute colitis in an experimental model of inflammatory bowel disease.

The association between hyaluronan (HA) accumulation and increased inflammation in the colon suggests that HA is a potential therapeutic target in inflammatory bowel disease (IBD). However, whether patients with IBD would benefit from interference with HA synthesis is unknown. Here, we used pharmacological and genetic approaches to investigate the impact of systemic and partial blockade of HA synthesis in the Dextran Sodium Sulfate (DSS)-induced colitis model. To systemically inhibit HA production, we used 4-Methylumbelliferone (4-MU), whereas genetic approaches included the generation of mice with global or inducible cell-type specific deficiency in the Hyaluronan synthase 3 (Has3). We found that 4-MU treatment did not ameliorate but exacerbated disease severity characterized by increased body weight loss and enhanced colon tissue destruction compared to control mice without colitis. In contrast, global Has3 deficiency had a profound protective effect as reflected by a low colitis score and reduced infiltration of immune cells into the colon. To get further mechanistic insight into the proinflammatory role of HAS3, we deleted Has3 in a cell-type specific manner. Interestingly, while lack of Has3 expression in intestinal epithelial and smooth muscle cells had no effect or was rather proinflammatory, mice with Has3 deficiency in the endothelium were strongly protected against acute colitis. We conclude that endothelium-derived HAS3 plays a critical role in driving experimental colitis, warranting future studies on cell type-specific therapeutic interference with HA production in human IBD.