Search

Epidermis is one of the most rapidly proliferating tissues in the body with high demands for adenosine triphosphate and cellular building blocks. In this study, we show that to meet these requirements, keratinocytes constitutively express HIF-1α, even in the presence of oxygen levels sufficient for HIF-1α hydroxylation. We previously reported that mice with severe epidermal mitochondrial dysfunction actually showed a hyperproliferative epidermis but rapidly died of systemic lactic acidosis and hypoglycemia, indicating excessive glycolysis. In this work, we interrogated HIF-1α function in glycolysis by its epidermal ablation combined with mitochondrial dysfunction, which resulted in decreased proliferation but even earlier lethality due to a severe barrier defect. Our data demonstrate that HIF-1α is indispensable for maintaining a high aerobic glycolytic flux necessary for supplying energy but also for synthetizing cellular building blocks such as lipids, which are both essential for proliferation as well as barrier formation. HIF-1α is stabilized in keratinocytes in the presence of oxygen by high levels of HIF-1α transcripts, low levels of prolyl-4-hydroxylases (PHD2 and PHD3), and a low cellular a-ketoglutarate/lactate ratio, likely inhibiting prolyl-4-hydroxylase activity. Our data suggest a key role for constitutive HIF-1α expression allowing a Warburg-like metabolism in healthy, highly proliferative keratinocytes, similar to that in tumor cells., (Copyright © 2024. Published by Elsevier Inc.)

Post-mitotic, non-proliferative dermal fibroblasts have crucial functions in maintenance and restoration of tissue homeostasis. They are involved in essential processes such as wound healing, pigmentation and hair growth, but also tumor development and aging-associated diseases. These processes are energetically highly demanding and error prone when mitochondrial damage occurs. However, mitochondrial function in fibroblasts and the influence of mitochondrial dysfunction on fibroblast-specific demands are still unclear. To address these questions, we created a mouse model in which accelerated cell-specific mitochondrial DNA (mtDNA) damage accumulates. We crossed mice carrying a dominant-negative mutant of the mitochondrial replicative helicase Twinkle (RosaSTOP system) with mice that express fibroblast-specific Cre Recombinase (Collagen1A2 Cre ERT ) which can be activated by Tamoxifen (Twinkle FIBRO ). Thus, we are able to induce mtDNA deletions and duplications in specific cells, a process which resembles the physiological aging process in humans, where this damage accumulates in all tissues. Upon proliferation in vitro, Tamoxifen induced Twinkle fibroblasts deplete most of their mitochondrial DNA which, although not disturbing the stoichiometry of the respiratory chain complexes, leads to reduced ROS production and mitochondrial membrane potential as well as an anti-inflammatory and anti-fibrotic profile of the cells. In Sodium Azide treated wildtype fibroblasts, without a functioning respiratory chain, we observe the opposite, a rather pro-inflammatory and pro-fibrotic signature. Upon accumulation of mitochondrial DNA mutations in vivo the Twinkle FIBRO mice are protected from fibrosis development induced by intradermal Bleomycin injections. This is due to dampened differentiation of the dermal fibroblasts into α-smooth-muscle-actin positive myofibroblasts in Twinkle FIBRO mice. We thus provide evidence for striking differences of the impact that mtDNA mutations have in contrast to blunted mitochondrial function in dermal fibroblasts and skin homeostasis. These data contribute to improved understanding of mitochondrial function and dysfunction in skin and provide mechanistic insight into potential targets to treat skin fibrosis in the future., (Copyright © 2024. Published by Elsevier B.V.)

Desmin gene mutations cause myopathies and cardiomyopathies. Our previously characterised R349P desminopathy mice, which carry the ortholog of the common human desmin mutation R350P, showed marked alterations in mitochondrial morphology and function in muscle tissue. By isolating skeletal muscle myoblasts from offspring of R349P desminopathy and p53 knock-out mice, we established an immortalised cellular disease model. Heterozygous and homozygous R349P desmin knock-in and wild-type myoblasts could be well differentiated into multinucleated spontaneously contracting myotubes. The desminopathy myoblasts showed the characteristic disruption of the desmin cytoskeleton and desmin protein aggregation, and the desminopathy myotubes showed the characteristic myofibrillar irregularities. Long-term electrical pulse stimulation promoted myotube differentiation and markedly increased their spontaneous contraction rate. In both heterozygous and homozygous R349P desminopathy myotubes, this treatment restored a regular myofibrillar cross-striation pattern as seen in wild-type myotubes. High-resolution respirometry of mitochondria purified from myotubes by density gradient ultracentrifugation revealed normal oxidative phosphorylation capacity, but a significantly reduced proton leak in mitochondria from the homozygous R349P desmin knock-in cells. Consistent with a reduced proton flux across the inner mitochondrial membrane, our quantitative proteomic analysis of the purified mitochondria revealed significantly reduced levels of ADP/ATP translocases in the homozygous R349P desmin knock-in genotype. As this alteration was also detected in the soleus muscle of R349P desminopathy mice, which, in contrast to the mitochondria purified from cultured cells, showed a variety of other dysregulated mitochondrial proteins, we consider this finding to be an early step in the pathogenesis of secondary mitochondriopathy in desminopathy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Accumulation of large-scale mitochondrial DNA (mtDNA) deletions and chronic, subclinical inflammation are concomitant during skin aging, thus raising the question of a causal link. To approach this, we generated mice expressing a mutant mitochondrial helicase (K320E-TWINKLE) in the epidermis to accelerate the accumulation of mtDNA deletions in this skin compartment. Mice displayed low amounts of large-scale deletions and a dramatic depletion of mtDNA in the epidermis and showed macroscopic signs of severe skin inflammation. The mtDNA alterations led to an imbalanced stoichiometry of mitochondrial respiratory chain complexes, inducing a unique combination of cytokine expression, causing a severe inflammatory phenotype, with massive immune cell infiltrates already before birth. Altogether, these data unraveled a previously unknown link between an imbalanced stoichiometry of the mitochondrial respiratory chain complexes and skin inflammation and suggest that severe respiratory chain dysfunction, as observed in few cells leading to a mosaic in aged tissues, might be involved in the development of chronic subclinical inflammation., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: During aging a mosaic of normal cells and cells with mitochondrial deficiency develops in various tissues including the heart. Whether this contributes to higher susceptibility for arrhythmia following myocardial infarction (MI) is unknown., Methods and Results: Myocardial cryoinfarction was performed in 12-month-old transgenic mice with accelerated accumulation of deletions in mitochondrial DNA. Occurrence and pathogenesis of arrhythmia was investigated after two weeks. Holter-ECG recordings revealed higher rates of premature ventricular complexes (incidence > 10/24 h: 100% vs. 20%; p = 0.048) and more severe spontaneous arrhythmia during stress test in mutant mice with MI as compared to control mice with MI. Mice with mitochondrial dysfunction exhibited longer spontaneous AV-blocks (467 ± 26 ms vs. 377 ± 24 ms; p = 0.013), an increased probability for induction of ventricular tachycardia during in vivo electrophysiological investigation (22% vs. 9%; p = 0.044), and a reduced conduction velocity in the infarct borderzone (38.5 ± 0.5 cm/s vs. 55.3 ± 0.9 cm/s; p = 0.001). Furthermore, mutant mice exhibited a significant reduction of the phospho-Cx43/Cx43 ratio in right (0.59 ± 0.04 vs. 0.85 ± 0.01; p = 0.027) and left ventricular myocardium (0.72 ± 0.01 vs. 0.86 ± 0.02; p = 0.023)., Conclusions: Aging-related cardiac mosaic respiratory chain dysfunction facilitates the occurrence of spontaneous and inducible cardiac arrhythmia after myocardial infarction and is associated with slowing of electrical impulse propagation in the infarct borderzone., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Here, we studied how epithelial energy metabolism impacts overall skin development by selectively deleting intraepithelial mtDNA in mice by ablating a key maintenance factor (Tfam(EKO)), which induces loss of function of the electron transport chain (ETC). Quantitative (immuno)histomorphometry demonstrated that Tfam(EKO) mice showed significantly reduced hair follicle (HF) density and morphogenesis, fewer intrafollicular keratin15+ epithelial progenitor cells, increased apoptosis, and reduced proliferation. Tfam(EKO) mice also displayed premature entry into (aborted) HF cycling by apoptosis-driven HF regression (catagen). Ultrastructurally, Tfam(EKO) mice exhibited severe HF dystrophy, pigmentary abnormalities, and telogen-like condensed dermal papillae. Epithelial HF progenitor cell differentiation (Plet1, Lrig1 Lef1, and β-catenin), sebaceous gland development (adipophilin, Scd1, and oil red), and key mediators/markers of epithelial-mesenchymal interactions during skin morphogenesis (NCAM, versican, and alkaline phosphatase) were all severely altered in Tfam(EKO) mice. Moreover, the number of mast cells, major histocompatibility complex class II+, or CD11b+ immunocytes in the skin mesenchyme was increased, and essentially no subcutis developed. Therefore, in contrast to their epidermal counterparts, pilosebaceous unit stem cells depend on a functional ETC. Most importantly, our findings point toward a frontier in skin biology: the coupling of HF keratinocyte mitochondrial function with the epithelial-mesenchymal interactions that drive overall development of the skin and its appendages.

Background & Aims: To determine if diabetic and insulin-resistant states cause mitochondrial dysfunction in liver or if there is long term adaptation of mitochondrial function to these states, mice were (i) fed with a high-fat diet to induce obesity and T2D (HFD), (ii) had a genetic defect in insulin signaling causing whole body insulin resistance, but not full blown T2D (IR/IRS-1(+/-) mice), or (iii) were analyzed after treatment with streptozocin (STZ) to induce a T1D-like state., Methods: Hepatic lipid levels were measured by thin layer chromatography. Mitochondrial respiratory chain (RC) levels and function were determined by Western blot, spectrophotometric, oxygen consumption and proton motive force analysis. Gene expression was analyzed by real-time PCR and microarray., Results: HFD caused insulin resistance and hepatic lipid accumulation, but RC was largely unchanged. Livers from insulin resistant IR/IRS-1(+/-) mice had normal lipid contents and a normal RC, but mitochondria were less well coupled. Livers from severely hyperglycemic and hypoinsulinemic STZ mice had massively depleted lipid levels, but RC abundance was unchanged. However, liver mitochondria isolated from these animals showed increased abundance and activity of the RC, which was better coupled., Conclusions: Insulin resistance, induced either by obesity or genetic manipulation and steatosis do not cause mitochondrial dysfunction in mouse liver. Also, mitochondrial dysfunction is not a prerequisite for liver steatosis. However, severe insulin deficiency and high blood glucose levels lead to an enhanced performance and better coupling of the RC. This may represent an adaptation to fuel overload and the high energy-requirement of an unsuppressed gluconeogenesis., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Ras-related GTPases of the Miro family have been implicated in mitochondrial homeostasis and microtubule-dependent transport. They consist of two GTP-binding domains separated by calcium-binding motifs and of a C-terminal transmembrane domain that targets the protein to the outer mitochondrial membrane. We disrupted the single Miro-encoding gene in Dictyostelium discoideum and observed a substantial growth defect that we attribute to a decreased mitochondrial mass and cellular ATP content. However, mutant cells even showed an increased rate of oxygen consumption, while glucose consumption, mitochondrial transmembrane potential and production of reactive oxygen species were unaltered. Processes characteristic of the multicellular stage of the D. discoideum life cycle were also unaltered. Although mitochondria occasionally use microtubules for transport in D. discoideum, their size and distribution were not visibly affected. We found Miro in all branches of the eukaryotic tree with the exception of a few protist lineages (mainly those lacking typical mitochondria). Trypanosomatids and ciliates possess structurally unique homologs lacking the N-terminal or the C-terminal GTPase domain, respectively. We propose that in D. discoideum, as in yeasts and plants, Miro plays roles in mitochondrial homeostasis, but the ability to build a complex that regulates its association to kinesin for microtubule-dependent transport probably arose in metazoans., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Isolation of mitochondria by current methods relies mainly on their physicochemical properties. Here we describe an alternative approach to obtain functional mitochondria from human cells in a fast, reproducible, and standardized procedure. The new approach is based on superparamagnetic microbeads conjugated to anti-TOM22 antibody. The bead conjugates label the cytoplasmic part of the human mitochondrial membrane protein TOM22 and, thus, allow for a gentle isolation of mitochondria in a high gradient magnetic field. By comparing the MACS (magnetic cell separation) approach with mitochondria isolation methods using differential centrifugation and ultracentrifugation we demonstrate that the MACS approach provides the highest yield of isolated mitochondria. The quality, enrichment, and purity of mitochondria isolated with this protocol are comparable to mitochondria obtained using the ultracentrifuge method, and a typical separation procedure takes only approximately 1 to 2h from initial cell homogenization. Mitochondria isolated with the new approach are sufficient for protein import, blue native gel electrophoresis, and other mitochondrial assays.

In search of tumor-specific mitochondrial DNA (mtDNA) mutations in head and neck squamous cell cancer, we found heteroplasmy in the blood of two individuals, i.e., these individuals carried two alleles of mtDNA. In both cases, the tumor was found to be homoplasmic, i.e., it contained only one of the two mtDNA alleles present in blood. More interestingly, in one case the tumor had acquired the wild-type allele, while in the other case it contained the mutant allele only. Sequencing of the whole 16.5 kb mtDNA showed that the observed heteroplasmic positions in the D-loop region, nucleotides 152 and 16187, respectively, were the only differences between tumor and blood mtDNA genotypes in these individuals. Our findings thus strongly support the hypothesis that accumulation of mtDNA mutations in solid tumors occurs by clonal and random expansion of pre-existing alleles and is not necessary for the metabolic changes generally associated with tumor formation, the Warburg effect.

The energy metabolism of the epidermis has been the subject of controversy; thus we characterized the mitochondrial phenotype of human primary keratinocytes and fibroblasts, in cell culture and in human skin sections. We found that keratinocytes respire as much as fibroblasts, however, maximal activities of the respiratory chain (RC) complexes were 2- to 5-fold lower, whereas expression levels of RC proteins were similar. Maximal activities of aconitase and isocitrate dehydrogenase, two mitochondrial enzymes especially vulnerable to superoxide, were lower than in fibroblasts. Indeed, superoxide anion levels were much higher in keratinocytes, and keratinocytes displayed higher lipid peroxidation levels and a lower reduced glutathione/oxidized glutathione ratio, indicating enhanced oxidative stress. Although superoxide dismutase activity and especially expression of the mitochondrial superoxide dismutase, Mn-SOD, were drastically lower in keratinocytes, explaining the high superoxide levels, glutathione peroxidase activity and protein were almost undetectable in fibroblasts. Catalase activity and hydrogen peroxide levels were similar. In summary, we could show that keratinocytes actively use the mitochondrial RC not only for adenosine 5' triphosphate synthesis but also for the accumulation of superoxide anions, even at the expense of mitochondrial functional capacity, indicating that superoxide-driven mitochondrial impairment might be a prerequisite for keratinocyte differentiation.

Adipogenesis of preadipocytes in culture has been frequently used to study the molecular basis and effect of drugs on fat cell conversion. However, after adipogenic induction, cells respond to the inducing agent with various speeds of conversion and fat accumulation, which complicates direct molecular and biochemical analyses. Here we present a simple and sensitive method to detect and quantify fat accumulation inside cells by flow cytometry. Using this method, we detected elevated levels of cytoplasmic granularity that correlated well with an increased level of fat accumulated inside cells after adipogenic conversion. We further demonstrated the ability of this method to monitor and quantify fat cell maturation within a complex population of cells and to identify and collect the fat cells with similar fat storage for further analysis. Flow cytometry offers distinct advantages over existing detection systems for cytoplasmic lipid staining and lipid extraction and could represent a powerful analytical tool to monitor the effect of chemicals and biological molecules on fat cell conversion and maturation. Moreover, in combination with a cell sorting facility, our method offers a simple and efficient means of collecting fat cells of specific status for further analysis.

Background: Previous studies have suggested a neuroendocrine defect underlying uremic hypogonadism, characterized by a reduced secretion of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH)., Methods: We studied the GnRH-producing GT1-7 cell line and the LH-producing LbetaT-2 pituitary cell line under uremic conditions to investigate whether substances circulating in uremic plasma directly affect hypothalamic or pituitary hormone secretion. The cells were incubated with serum from 5/6-nephrectomized or sham-nephrectomized castrated rats, respectively. Furthermore, GT1 cells were incubated with delipidated sera, serum subfractions separated by molecular weight, or several peptide hormones. Cellular viability, apoptosis rate and extracellular hormone degradation were assessed separately. GnRH and LH were measured by RIA in supernatants and cell lysates. GnRH gene expression was assessed by Northern blot., Results: Uremic serum caused a reduction of extracellular GnRH concentration by 31%, whereas intracellular GnRH increased by 12%. This effect was independent of serum lipids and enzymatic GnRH degradation but was abolished by trypsin digestion. Cellular viability, apoptosis rates and GnRH gene expression did not differ between the two groups. The inhibitory activity was recovered from the high-molecular weight fraction, whereas the fraction <5 kD had stimulatory activity. In contrast, uremic serum did not affect LH secretion from LbetaT-2 cells, indicating that the hypoactivity of the hypothalamo-pituitary gonadotrope unit results from an inhibition at the hypothalamic rather than the pituitary level., Conclusions: Our results suggest that uremic serum contains macromolecular and hydrophilic peptide(s) able to specifically suppress the neurosecretion of GnRH from GT1-7 cells.

The steady state concentration of cytochrome c oxidase subunit I mRNA and 12 S rRNA, respectively, measured by a quantitative reverse transcription/polymerase chain reaction method, was 4 and 15 molecules per molecule of mt DNA in rat liver and 2 and 9 molecules in rat muscle, respectively. These results imply that in the mitochondrial compartment, the molar concentration of all thirteen mRNAs by far exceeds the concentration of ribosomes, a situation fundamentally different from the cytosolic compartment. Following thyroid hormone treatment, both mitochondrial transcripts increased, in parallel with the mRNA encoding mitochondrial transcription factor A. We conclude that this transcription factor might be the rate limiting factor for mitochondrial transcription in vivo, at least under these conditions.

In this report, we show that the actual number of target molecules of the polymerase chain reaction can be determined by measuring the concentration of product accumulating in consecutive cycles. The equation describing product accumulation, log Nn = log eff x n + log N0, can be analyzed by linear regression and the molar concentration of target at cycle zero, N0, is obtained. Using this new approach, the actual content of mitochondrial DNA was determined in rat tissues and ranged from 116 x 10(9) molecules/g in fast-twitch skeletal muscle to 743 x 10(9) molecules/g in liver. Using morphometric data from the literature, mitochondria were found to contain 1 to 3 DNA molecules. There was no relation between the oxidative capacity of a tissue and its content of mitochondrial DNA, indicating that transcriptional and posttranscriptional mechanisms rather than gene dosage, as postulated by others, determine to what extent the mitochondrial genome is expressed.