AIM: The transcriptional factor HIF-1α is recognized for its contribution to cardioprotection against acute ischemia/reperfusion injury. Adaptation to chronic hypoxia (CH) is known to stabilize HIF-1α and increase myocardial ischemic tolerance. However, the precise role of HIF-1α in mediating the protective effect remains incompletely understood. METHODS: Male wild-type (WT) mice and mice with partial Hif1a deficiency (hif1a +/-) were exposed to CH for 4 weeks, while their respective controls were kept under normoxic conditions. Subsequently, their isolated perfused hearts were subjected to ischemia/reperfusion to determine infarct size, while RNA-sequencing of isolated cardiomyocytes was performed. Mitochondrial respiration was measured to evaluate mitochondrial function, and western blots were performed to assess mitophagy. RESULTS: We demonstrated enhanced ischemic tolerance in WT mice induced by adaptation to CH compared with their normoxic controls and chronically hypoxic hif1a +/- mice. Through cardiomyocyte bulk mRNA sequencing analysis, we unveiled significant reprogramming of cardiomyocytes induced by CH emphasizing mitochondrial processes. CH reduced mitochondrial content and respiration and altered mitochondrial ultrastructure. Notably, the reduced mitochondrial content correlated with enhanced autophagosome formation exclusively in chronically hypoxic WT mice, supported by an increase in the LC3-II/LC3-I ratio, expression of PINK1, and degradation of SQSTM1/p62. Furthermore, pretreatment with the mitochondrial division inhibitor (mdivi-1) abolished the infarct size-limiting effect of CH in WT mice, highlighting the key role of mitophagy in CH-induced cardioprotection. CONCLUSION: These findings provide new insights into the contribution of HIF-1α to cardiomyocyte survival during acute ischemia/reperfusion injury by activating the selective autophagy pathway.

• MeSH
• faktor 1 indukovatelný hypoxií - podjednotka alfa * metabolismus   genetika   MeSH
• fyziologická adaptace fyziologie   MeSH
• hypoxie * metabolismus   MeSH
• infarkt myokardu * metabolismus   patologie   genetika   MeSH
• kardiomyocyty metabolismus   patologie   MeSH
• mitofagie * fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Mitochondrial Ca2+-independent phospholipase A2γ (iPLA2γ/PNPLA8) was previously shown to be directly activated by H2O2 and release free fatty acids (FAs) for FA-dependent H+ transport mediated by the adenine nucleotide translocase (ANT) or uncoupling protein 2 (UCP2). The resulting mild mitochondrial uncoupling and consequent partial attenuation of mitochondrial superoxide production lead to an antioxidant effect. However, the antioxidant role of iPLA2γ in the brain is not completely understood. Here, using wild-type and iPLA2γ-KO mice, we demonstrate the ability of tert-butylhydroperoxide (TBHP) to activate iPLA2γ in isolated brain mitochondria, with consequent liberation of FAs and lysophospholipids. The liberated FA caused an increase in respiratory rate, which was fully inhibited by carboxyatractyloside (CATR), a specific inhibitor of ANT. Employing detailed lipidomic analysis, we also demonstrate a typical cleavage pattern for TBHP-activated iPLA2γ, reflecting cleavage of glycerophospholipids from both sn-1 and sn-2 positions releasing saturated FAs, monoenoic FAs, and predominant polyunsaturated FAs. The acute antioxidant role of iPLA2γ-released FAs is supported by monitoring both intramitochondrial superoxide and extramitochondrial H2O2 release. We also show that iPLA2γ-KO mice were more sensitive to stimulation by pro-inflammatory lipopolysaccharide, as reflected by the concomitant increase in protein carbonyls in the brain and pro-inflammatory IL-6 release in the serum. These data support the antioxidant and anti-inflammatory role of iPLA2γ in vivo. Our data also reveal a substantial decrease of several high molecular weight cardiolipin (CL) species and accumulation of low molecular weight CL species in brain mitochondria of iPLA2γ-KO mice. Collectively, our results support a key role of iPLA2γ in the remodeling of lower molecular weight immature cardiolipins with predominantly saturated acyl chains to high molecular weight mature cardiolipins with highly unsaturated PUFA acyl chains, typical for the brain.

• Publikační typ
• časopisecké články MeSH

Prostate cancer is one of the most prominent cancers diagnosed in males. Contrasting with other cancer types, glucose utilization is not increased in prostate carcinoma cells as they employ different metabolic adaptations involving mitochondria as a source of energy and intermediates required for rapid cell growth. In this regard, prostate cancer cells were associated with higher activity of mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH), the key rate limiting component of the glycerophosphate shuttle, which connects mitochondrial and cytosolic processes and plays significant role in cellular bioenergetics. Our research focused on the role of mGPDH biogenesis and regulation in prostate cancer compared to healthy cells. We show that the 42 amino acid presequence is cleaved from N-terminus during mGPDH biogenesis. Only the processed form is part of the mGPDH dimer that is the prominent functional enzyme entity. We demonstrate that mGPDH overexpression enhances the wound healing ability in prostate cancer cells. As mGPDH is at the crossroad of glycolysis, lipogenesis and oxidative metabolism, regulation of its activity by intramitochondrial processing might represent rapid means of cellular metabolic adaptations.

• MeSH
• glycerolfosfátdehydrogenasa metabolismus   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• mitochondrie genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádory prostaty genetika   metabolismus   MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Biogenesis of F1Fo ATP synthase, the key enzyme of mitochondrial energy provision, depends on transmembrane protein 70 (TMEM70), localized in the inner mitochondrial membrane of higher eukaryotes. TMEM70 absence causes severe ATP-synthase deficiency and leads to a neonatal mitochondrial encephalocardiomyopathy in humans. However, the exact biochemical function of TMEM70 remains unknown. Using TMEM70 conditional knockout in mice, we show that absence of TMEM70 impairs the early stage of enzyme biogenesis by preventing incorporation of hydrophobic subunit c into rotor structure of the enzyme. This results in the formation of an incomplete, pathologic enzyme complex consisting of F1 domain and peripheral stalk but lacking Fo proton channel composed of subunits c and a. We demonstrated direct interaction between TMEM70 and subunit c and showed that overexpression of subunit c in TMEM70-/- cells partially rescued TMEM70 defect. Accordingly, TMEM70 knockdown prevented subunit c accumulation otherwise observed in F1-deficient cells. Altogether, we identified TMEM70 as specific ancillary factor for subunit c. The biologic role of TMEM70 is to increase the low efficacy of spontaneous assembly of subunit c oligomer, the key and rate-limiting step of ATP-synthase biogenesis, and thus to reach an adequately high physiologic level of ATP synthase in mammalian tissues.-Kovalčíková, J., Vrbacký, M., Pecina, P., Tauchmannová, K., Nůsková, H., Kaplanová, V., Brázdová, A., Alán, L., Eliáš, J., Čunátová, K., Kořínek, V., Sedlacek, R., Mráček, T., Houštěk, J. TMEM70 facilitates biogenesis of mammalian ATP synthase by promoting subunit c incorporation into the rotor structure of the enzyme.

• MeSH
• genotyp MeSH
• genový knockout metody   MeSH
• HEK293 buňky MeSH
• kultivované buňky MeSH
• lidé MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• mitochondriální protonové ATPasy genetika   metabolismus   MeSH
• myši knockoutované MeSH
• myši MeSH
• proteolipidy metabolismus   MeSH
• regulace genové exprese MeSH
• tamoxifen farmakologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Hypertrophic pancreatic islets (PI) of Goto Kakizaki (GK) diabetic rats contain a lower number of β-cells vs. non-diabetic Wistar rat PI. Remaining β-cells contain reduced mitochondrial (mt) DNA per nucleus (copy number), probably due to declining mtDNA replication machinery, decreased mt biogenesis or enhanced mitophagy. We confirmed mtDNA copy number decrease down to <30% in PI of one-year-old GK rats. Studying relations to mt nucleoids sizes, we employed 3D superresolution fluorescent photoactivable localization microscopy (FPALM) with lentivirally transduced Eos conjugate of mt single-stranded-DNA-binding protein (mtSSB) or transcription factor TFAM; or by 3D immunocytochemistry. mtSSB (binding transcription or replication nucleoids) contoured "nucleoids" which were smaller by 25% (less diameters >150 nm) in GK β-cells. Eos-TFAM-visualized nucleoids, composed of 72% localized TFAM, were smaller by 10% (immunochemically by 3%). A theoretical ~70% decrease in cell nucleoid number (spatial density) was not observed, rejecting model of single mtDNA per nucleoid. The β-cell maintenance factor Nkx6.1 mRNA and protein were declining with age (>12-fold, 10 months) and decreasing with fasting hyperglycemia in GK rats, probably predetermining the impaired mtDNA replication (copy number decrease), while spatial expansion of mtDNA kept nucleoids with only smaller sizes than those containing much higher mtDNA in non-diabetic β-cells.

• MeSH
• beta-buňky metabolismus   patologie   MeSH
• DNA vazebné proteiny genetika   MeSH
• experimentální diabetes mellitus genetika   metabolismus   patologie   MeSH
• homeodoménové proteiny genetika   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• mitochondriální DNA genetika   MeSH
• mitochondrie genetika   patologie   MeSH
• mitofagie genetika   MeSH
• pankreas exokrinní metabolismus   MeSH
• potkani Wistar MeSH
• replikace DNA genetika   MeSH
• transkripční faktory genetika   MeSH
• variabilita počtu kopií segmentů DNA genetika   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Metformin is widely prescribed as a first-choice antihyperglycemic drug for treatment of type 2 diabetes mellitus, and recent epidemiological studies showed its utility also in cancer therapy. Although it is in use since the 1970s, its molecular target, either for antihyperglycemic or antineoplastic action, remains elusive. However, the body of the research on metformin effect oscillates around mitochondrial metabolism, including the function of oxidative phosphorylation (OXPHOS) apparatus. In this study, we focused on direct inhibitory mechanism of biguanides (metformin and phenformin) on OXPHOS complexes and its functional impact, using the model of isolated brown adipose tissue mitochondria. We demonstrate that biguanides nonspecifically target the activities of all respiratory chain dehydrogenases (mitochondrial NADH, succinate, and glycerophosphate dehydrogenases), but only at very high concentrations (10-2-10-1 M) that highly exceed cellular concentrations observed during the treatment. In addition, these concentrations of biguanides also trigger burst of reactive oxygen species production which, in combination with pleiotropic OXPHOS inhibition, can be toxic for the organism. We conclude that the beneficial effect of biguanides should probably be associated with subtler mechanism, different from the generalized inhibition of the respiratory chain.

• MeSH
• biguanidy farmakologie   MeSH
• fenformin farmakologie   MeSH
• glycerolfosfátdehydrogenasa metabolismus   MeSH
• hnědá tuková tkáň cytologie   MeSH
• hypoglykemika farmakologie   MeSH
• krysa rodu rattus MeSH
• kyselina jantarová metabolismus   MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• metformin farmakologie   MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• oxidace-redukce účinky léků   MeSH
• peroxid vodíku farmakologie   MeSH
• potkani Wistar MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Mild constitutive hyperbilirubinemia is associated with a reduced risk of cardiovascular diseases, diabetes, and cancer. Since these pathologies are associated with aging, inflammation, and oxidative stress, we investigated whether hyperbilirubinemia interferes with ROS homeostasis in cell cultures and with inflammation, senescence, and mitochondrial dysfunction in aged rats. Human embryonic kidney cells and rat primary fibroblasts showed a dose-dependent decrease in the ratio of oxidized/reduced glutathione, intracellular H2O2 levels, and mitochondrial ROS production, with increasing bilirubin concentrations in the culture media. Compared to their normobilirubinemic siblings, aged hyperbilirubinemic Gunn rats showed significantly smaller amounts of visceral fat, better glucose tolerance, and decreased serum levels of proinflammatory cytokines TNFα, IL-1β, and IL-18. Simultaneously, livers from Gunn rats showed decreased expression of senescence markers and cell cycle inhibitors p21 and p16. Mitochondria from aged Gunn rats showed higher respiration and lower H2O2 production compared to controls. In conclusion, we demonstrated that mildly elevated serum bilirubin is generally associated with attenuation of oxidative stress and with better anthropometric parameters, decreased inflammatory status, increased glucose tolerance, fewer signs of cellular senescence, and enhanced mitochondrial function in aged rats.

• MeSH
• bilirubin krev   MeSH
• fibroblasty metabolismus   patologie   MeSH
• hyperbilirubinemie krev   patologie   MeSH
• intracelulární prostor metabolismus   MeSH
• kultivované buňky MeSH
• metabolické nemoci komplikace   patologie   MeSH
• mitochondrie metabolismus   MeSH
• potkani Gunn MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• stárnutí patologie   MeSH
• zánět komplikace   patologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Mitochondrial DNA (mtDNA) is compacted in ribonucleoprotein complexes called nucleoids, which can divide or move within the mitochondrial network. Mitochondrial nucleoids are able to aggregate into clusters upon reaction with intercalators such as the mtDNA depletion agent Ethidium Bromide (EB) or anticancer drug Doxorobicin (DXR). However, the exact mechanism of nucleoid clusters formation remains unknown. Resolving these processes may help to elucidate the mechanisms of DXR-induced cardiotoxicity. Therefore, we addressed the role of two key nucleoid proteins; mitochondrial transcription factor A (TFAM) and mitochondrial single-stranded binding protein (mtSSB); in the formation of mitochondrial nucleoid clusters during the action of intercalators. We found that both intercalators cause numerous aberrations due to perturbing their native status. By blocking mtDNA replication, both agents also prevented mtDNA association with TFAM, consequently causing nucleoid aggregation into large nucleoid clusters enriched with TFAM, co-existing with the normal nucleoid population. In the later stages of intercalation (>48h), TFAM levels were reduced to 25%. In contrast, mtSSB was released from mtDNA and freely distributed within the mitochondrial network. Nucleoid clusters mostly contained nucleoids with newly replicated mtDNA, however the nucleoid population which was not in replication mode remained outside the clusters. Moreover, the nucleoid clusters were enriched with p53, an anti-oncogenic gatekeeper. We suggest that mitochondrial nucleoid clustering is a mechanism for protecting nucleoids with newly replicated DNA against intercalators mediating genotoxic stress. These results provide new insight into the common mitochondrial response to mtDNA stress and can be implied also on DXR-induced mitochondrial cytotoxicity.

• MeSH
• buňky Hep G2 MeSH
• DNA vazebné proteiny metabolismus   MeSH
• doxorubicin MeSH
• ethidium MeSH
• GTP-fosfohydrolasy metabolismus   MeSH
• jaterní mitochondrie metabolismus   MeSH
• lidé MeSH
• mitochondriální DNA metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• poškození DNA MeSH
• proteiny asociované s mikrotubuly metabolismus   MeSH
• transkripční faktory metabolismus   MeSH
• transportní proteiny mitochondriální membrány metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The relationship of the inner mitochondrial membrane (IMM) cristae structure and intracristal space (ICS) to oxidative phosphorylation (oxphos) is not well understood. Mitofilin (subunit Mic60) of the mitochondrial contact site and cristae organizing system (MICOS) IMM complex is attached to the outer membrane (OMM) via the sorting and assembly machinery/topogenesis of mitochondrial outer membrane β-barrel proteins (SAM/TOB) complex and controls the shape of the cristae. ATP synthase dimers determine sharp cristae edges, whereas trimeric OPA1 tightens ICS outlets. Metabolism is altered during hypoxia, and we therefore studied cristae morphology in HepG2 cells adapted to 5% oxygen for 72 h. Three dimensional (3D), super-resolution biplane fluorescence photoactivation localization microscopy with Eos-conjugated, ICS-located lactamase-β indicated hypoxic ICS expansion with an unchanged OMM (visualized by Eos-mitochondrial fission protein-1). 3D direct stochastic optical reconstruction microscopy immunocytochemistry revealed foci of clustered mitofilin (but not MICOS subunit Mic19) in contrast to its even normoxic distribution. Mitofilin mRNA and protein decreased by ∼20%. ATP synthase dimers vs monomers and state-3/state-4 respiration ratios were lower during hypoxia. Electron microscopy confirmed ICS expansion (maximum in glycolytic cells), which was absent in reduced or OMM-detached cristae of OPA1- and mitofilin-silenced cells, respectively. Hypoxic adaptation is reported as rounding sharp cristae edges and expanding cristae width (ICS) by partial mitofilin/Mic60 down-regulation. Mitofilin-depleted MICOS detaches from SAM while remaining MICOS with mitofilin redistributes toward higher interdistances. This phenomenon causes partial oxphos dormancy in glycolytic cells via disruption of ATP synthase dimers.-Plecitá-Hlavatá, L., Engstová, H., Alán, L., Špaček, T., Dlasková, A., Smolková, K., Špačková, J., Tauber, J., Strádalová, V., Malínský, J., Lessard, M., Bewersdorf, J., Ježek, P. Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down-regulation concomitant to MICOS clustering.

• MeSH
• adenosintrifosfát biosyntéza   MeSH
• ATP-synthetasa (komplexy) metabolismus   MeSH
• buňky Hep G2 MeSH
• down regulace MeSH
• fyziologická adaptace fyziologie   MeSH
• interakční proteinové domény a motivy MeSH
• kyslík * MeSH
• lidé MeSH
• mitochondriální dynamika fyziologie   MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• mitochondrie fyziologie   MeSH
• multiproteinové komplexy fyziologie   MeSH
• podjednotky proteinů MeSH
• regulace genové exprese fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Data segmentation and object rendering is required for localization super-resolution microscopy, fluorescent photoactivation localization microscopy (FPALM), and direct stochastic optical reconstruction microscopy (dSTORM). We developed and validated methods for segmenting objects based on Delaunay triangulation in 3D space, followed by facet culling. We applied them to visualize mitochondrial nucleoids, which confine DNA in complexes with mitochondrial (mt) transcription factor A (TFAM) and gene expression machinery proteins, such as mt single-stranded-DNA-binding protein (mtSSB). Eos2-conjugated TFAM visualized nucleoids in HepG2 cells, which was compared with dSTORM 3D-immunocytochemistry of TFAM, mtSSB, or DNA. The localized fluorophores of FPALM/dSTORM data were segmented using Delaunay triangulation into polyhedron models and by principal component analysis (PCA) into general PCA ellipsoids. The PCA ellipsoids were normalized to the smoothed volume of polyhedrons or by the net unsmoothed Delaunay volume and remodeled into rotational ellipsoids to obtain models, termed DVRE. The most frequent size of ellipsoid nucleoid model imaged via TFAM was 35 × 45 × 95 nm; or 35 × 45 × 75 nm for mtDNA cores; and 25 × 45 × 100 nm for nucleoids imaged via mtSSB. Nucleoids encompassed different point density and wide size ranges, speculatively due to different activity stemming from different TFAM/mtDNA stoichiometry/density. Considering twofold lower axial vs. lateral resolution, only bulky DVRE models with an aspect ratio >3 and tilted toward the xy-plane were considered as two proximal nucleoids, suspicious occurring after division following mtDNA replication. The existence of proximal nucleoids in mtDNA-dSTORM 3D images of mtDNA "doubling"-supported possible direct observations of mt nucleoid division after mtDNA replication.

• MeSH
• algoritmy * MeSH
• analýza hlavních komponent * MeSH
• buňky Hep G2 MeSH
• DNA vazebné proteiny metabolismus   MeSH
• fluorescenční mikroskopie * MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• mitochondriální DNA chemie   metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• molekulární modely MeSH
• zobrazování trojrozměrné * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH