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High levels of circulating glucocorticoid hormone may be important mediators for elevating resting metabolic rate upon severe injury or stress. We therefore investigated the effect of dexamethasone on mitochondrial biogenesis in rats (6 mg/kg daily) as well as in cells in culture (1 microM) over a period of 3 d. A marked stimulation of mitochondrial DNA transcription and increased levels of cytochrome c oxidase activity were found in skeletal muscle of rats and differentiated mouse C2C12 muscle cells, but not in other tissues, myoblasts, or other cell lines. The effect was inhibited by RU486. Therefore, increased occupancy of glucocorticoid receptors is necessary, but not sufficient to increase mitochondrial biogenesis and other, skeletal muscle specific factors are postulated. Expression of the mitochondrial transcription factor A was unchanged, suggesting a possible involvement of the recently described mitochondrial glucocorticoid receptor. Expression of uncoupling protein-3 was also unchanged. In conclusion, our results show that high levels of glucocorticoid hormone are sufficient to stimulate mitochondrial biogenesis; however, only in skeletal muscle. Increased mitochondrial mass in this tissue, without changes of the coupling state of the respiratory chain, might be the molecular basis for the elevated resting metabolic rate observed under high cortisol levels in humans.

The light strand promoter of mammalian mitochondrial DNA gives rise to a primary transcript, but also to the RNA primer necessary for initiation of replication and 7S DNA synthesis as well as 7S RNA. Here we have studied the turnover of 7S DNA in isolated rat liver mitochondria and whether import of mitochondrial transcription factor A (mtTFA), which is necessary for transcription initiation, increases its rate of synthesis. 7S DNA was present as two species, probably due to two different sites of RNA–DNA transition. Time course and pulse–chase experiments showed that the half-life of this DNA is ∼45 min. Import of mtTFA, produced in vitro, into the mitochondrial matrix in stoichiometric amounts significantly increased the rate of 7S DNA formation. We conclude that isolated rat liver mitochondria faithfully synthesize and degrade 7S DNA and that increased matrix levels of mtTFA are sufficient to increase its rate of synthesis, strongly supporting the hypothesis that this process is transcription primed.

Parathyroid hormone-related peptide (PTHrP) is expressed throughout the cardiovascular system including coronary endothelial cells. Factors involved in the regulation of cardiac PTHrP expression have not been examined before. This study investigates the influence of transforming growth factor (TGF)- β 1 on ventricular PTHrP expression. Coronary endothelial cells were isolated from ventricles of adult rats and PTHrP protein expression in these cultures was analysed by immunoblotting. TGF- β 1 caused a concentration-dependent reduction in PTHrP protein within 24 h. In transgenic mice over-expressing TGF- β 1 ventricular PTHrP protein expression and release was reduced compared to non-transgenic littermates. Similar concerns hold for PTHrP mRNA content (RT-PCR). Since ventricular TGF- β 1 expression increases under pathophysiological conditions like arterial hypertension, ventricular PTHrP expression was further determined in aging spontaneously hypertensive (SHR-SP) and normotensive rats. TGF- β 1 expression was increased in SHR-SP and ventricular PTHrP mRNA expression was downregulated at the age of 10 months. PTHrP expression did not recover in elder SHR-SP in which TGF- β 1 expression was normalized again. Finally, we investigated ventricular PTHrP expression in rats after banding of the ascending aorta which generates a pressure induced hypertrophy without an induction of TGF- β 1 expression. In ventricles from these animals, PTHrP expression was transiently increased and normalized at day 3. In conclusion, PTHrP expression was reduced under all conditions in which coronary endothelial cells were exposed to TGF- β 1 . PTHrP expression does not correlate with cardiac hypertrophy. Since coronary endothelial cells represent the majority of PTHrP producing cells in the ventricle its downregulation by TGF- β 1 seems to be relevant for the paracrine effects of PTHrP.

Fluid reabsorption from alveolar space is driven by active Na reabsorption via epithelial Na channels (ENaCs) and Na-K-ATPase. Both are inhibited by hypoxia. Here we tested whether hypoxia decreases Na transport by decreasing the number of copies of transporters in alveolar epithelial cells and in lungs of hypoxic rats. Membrane fractions were prepared from A549 cells exposed to hypoxia (3% O2) as well as from whole lung tissue and alveolar type II cells from rats exposed to hypoxia. Transport proteins were measured by Western blot analysis. In A549 cells, α1- and β1-Na-K-ATPase, Na/K/2Cl cotransport, and ENaC proteins decreased during hypoxia. In whole lung tissue, α1-Na-K-ATPase and Na/K/2Cl cotransport decreased. α- and β-ENaC mRNAs also decreased in hypoxic lungs. Similar results were seen in alveolar type II cells from hypoxic rats. These results indicate a slow decrease in the amount of Na-transporting proteins in alveolar epithelial cells during exposure to hypoxia that also occurs in vivo in lungs from hypoxic animals. The reduced number of transporters might account for the decreased transport activity and impaired edema clearance in hypoxic lungs.

The biogenesis of giant mitochondria in flight muscle of Locusta migratoria (L.) was analyzed at the molecular level. During the 2 weeks between the beginning of the last larval stage and the imago capable of sustained flight, individual mitochondria have been shown to enlarge 30-fold and the fractional mitochondrial volume of muscle cells increases fourfold [Brosemer, R.W., Vogell, W. and Bücher, Th. (1963) Biochem. Z. 338, 854-910]. Within the same period, the activity of cytochrome c oxidase, containing subunits encoded on mitochondrial DNA, increased twofold. However, no significant change in mitochondrial DNA copy number, and even a threefold decrease in mitochondrial transcripts, was observed. Mitochondrial translation rate, measured in isolated organelles, was twofold higher in larval muscle, which can be explained only partly by the higher content of mitochondrial RNAs. Thus, rather unusually, in this system of mitochondrial differentiation, the mitochondrial biosynthetic capacity correlates with the rate of organelle biogenesis rather than the steady-state concentration of a marker enzyme. The copy number of mitochondrial DNA does not seem to play a major role in determining either mitochondrial transcript levels or functional mass.

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.

Work in the nematode Caenorhabditis elegans has led to fundamental insights into key biological mechanisms of ageing, which may be instrumental to develop strategies to increase life span and, more important, health span in humans. Dietary restriction (DR) and its surrogate, mild impairment of nutrient sensing pathways (insulin/IGF and/or mTOR axes) by genetic manipulation in the presence of normal food intake are among the most thoroughly investigated interventions, leading to life span extension in yeast, worms, flies and mice [1]. Surprisingly, it was shown that the effect of dietary restriction depends on the presence of two neurons (ASI) in C. elegans, whose ablation completely abrogates the effect [2]. Energy deprivation following dietary restriction leads to increased mitochondrial respiration, and the resulting, moderate increase in reactive oxygen species formation seems to be instrumental for life span extension as well [3,4] due to a process called “mitohormesis” [5]. Accordingly, moderate disturbance of the mitochondrial respiratory chain by mild mutations in several subunits was shown to be another fountain of youth [6–8]. In this issue of Molecular Metabolism, the Ristow group is closing the circle: They show that low amounts of three bona fide inhibitors of complex I of the respiratory chain extend life and health span in the worm [9]. They also show that this mimics dietary restriction, since it does not further extend survival of dietary restricted animals, and that again the effect of these inhibitors is gone if the two ASI neurons had been ablated [9]. They conclude that redox stress sensing in these two neurons may be the signal for the rest of the body to activate the “survive program”. Interestingly, in the same line the Dillin group had shown before that genetic interference with the respiratory chain specifically in neurons activates a beneficial, mitochondrial unfolded protein response in distant tissues, postulating a diffusible, however still unidentified, “mitokine” [10]. There are numerous other examples showing that neurons are necessary for the life span extending effect of various genetic and dietary interventions, and finally even a small molecule, the antidepressant mianserin, inhibiting neuronal serotonin transmission, let live worms longer [11]. This and the recent study of the Ristow team raises hope that maybe also in humans, pharmacologic interventions – specifically aiming at neuro-endocrine signaling instead of targeting peripheral organs – may be available in the near future to treat ageing-related diseases.

During development of the heart, mitochondria proliferate within cardiomyocytes. It is unclear whether this is a response to the increasing energy demand or whether it is part of the developmental program. To investigate the role of the electron transport chain (ETC) in this process, we used transgenic murine embryonic stem (ES) cells in which the green fluorescent protein gene is under control of the alpha-myosin heavy chain promoter (alpha-MHC), allowing easy monitoring of cardiomyocyte differentiation. Spontaneous contraction of these cells within embryoid bodies (EBs) was not affected by inhibition of the ETC, suggesting that early heart cell function is sufficiently supported by anaerobic ATP production. However, heart cell development was completely blocked when adding antimycin A, an inhibitor of ETC complex III, before initiation of differentiation, whereas KCN did not block differentiation, strongly suggesting that specifically complex III function rather than mitochondrial ATP production is necessary for early heart cell development. When the underlying mechanism was examined, we noticed that antimycin A but not KCN lead to inhibition of spontaneous intracellular Ca++ oscillations, whereas both substances decreased mitochondrial membrane potential, as expected. We postulate that mitochondrial complex III activity is necessary for these Ca++ oscillations, which in turn are a prerequisite for cardiomyocyte differentiation.

Keratin intermediate filaments (KIFs) protect the epidermis against mechanical force, support strong adhesion, help barrier formation, and regulate growth. The mechanisms by which type I and II keratins contribute to these functions remain incompletely understood. Here, we report that mice lacking all type I or type II keratins display severe barrier defects and fragile skin, leading to perinatal mortality with full penetrance. Comparative proteomics of cornified envelopes (CEs) from prenatal KtyI(-/-) and KtyII(-/-)(K8) mice demonstrates that absence of KIF causes dysregulation of many CE constituents, including downregulation of desmoglein 1. Despite persistence of loricrin expression and upregulation of many Nrf2 targets, including CE components Sprr2d and Sprr2h, extensive barrier defects persist, identifying keratins as essential CE scaffolds. Furthermore, we show that KIFs control mitochondrial lipid composition and activity in a cell-intrinsic manner. Therefore, our study explains the complexity of keratinopathies accompanied by barrier disorders by linking keratin scaffolds to mitochondria, adhesion, and CE formation.

The role of mitochondrial dysfunction in the development of insulin resistance and type 2 diabetes remains controversial. In order to specifically define the relationship between insulin receptor (InsR) signaling, insulin resistance, hyperglycemia, hyperlipidemia and mitochondrial function, we analyzed mitochondrial performance of insulin-sensitive, slow-oxidative muscle in four different mouse models. In obese but normoglycemic ob/ob mice as well as in obese but diabetic mice under high-fat diet, mitochondrial performance remained unchanged even though intramyocellular diacylglycerols (DAGs), triacylglycerols (TAGs), and ceramides accumulated. In contrast, in muscle-specific InsR knockout (MIRKO) and streptozotocin (STZ)-treated hypoinsulinemic, hyperglycemic mice, levels of mitochondrial respiratory chain complexes and mitochondrial function were markedly reduced. In STZ, but not in MIRKO mice, this was caused by reduced transcription of mitochondrial genes mediated via decreased PGC-1α expression. We conclude that mitochondrial dysfunction is not causally involved in the pathogenesis of obesity-associated insulin resistance under normoglycemic conditions. However, obesity-associated type 2 diabetes and accumulation of DAGs or TAGs is not associated with impaired mitochondrial function. In contrast, chronic hypoinsulinemia and hyperglycemia as seen in STZ-treated mice as well as InsR deficiency in muscle of MIRKO mice lead to mitochondrial dysfunction. We postulate that decreased mitochondrial mass and/or performance in skeletal muscle of non-diabetic, obese or type 2 diabetic, obese patients observed in clinical studies must be explained by genetic predisposition, physical inactivity, or other still unknown factors.

Tissue stem cells and germ line or embryonic stem cells were shown to have reduced oxidative metabolism, which was proposed to be an adaptive mechanism to reduce damage accumulation caused by reactive oxygen species. However, an alternate explanation is that stem cells are less dependent on specialized cytoplasmic functions compared with differentiated cells, therefore, having a high nuclear-to-cytoplasmic volume ratio and consequently a low mitochondrial content. To determine whether stem cells rely or not on mitochondrial respiration, we selectively ablated the electron transport chain in the basal layer of the epidermis, which includes the epidermal progenitor/stem cells (EPSCs). This was achieved using a loxP-flanked mitochondrial transcription factor A (Tfam) allele in conjunction with a keratin 14 Cre transgene. The epidermis of these animals (TfamEKO) showed a profound depletion of mitochondrial DNA and complete absence of respiratory chain complexes. However, despite a short lifespan due to malnutrition, epidermal development and skin barrier function were not impaired. Differentiation of epidermal layers was normal and no proliferation defect or major increase of apoptosis could be observed. In contrast, mice with an epidermal ablation of prohibitin-2, a scaffold protein in the inner mitochondrial membrane, displayed a dramatic phenotype observable already in utero, with severely impaired skin architecture and barrier function, ultimately causing death from dehydration shortly after birth. In conclusion, we here provide unequivocal evidence that EPSCs, and probably tissue stem cells in general, are independent of the mitochondrial respiratory chain, but still require a functional dynamic mitochondrial compartment.

Here we demonstrate that the neuropeptide hormone thyrotropin (TSH), which controls thyroid hormone production, exerts a major nonclassical function in mitochondrial biology. Based on transcriptional, ultrastructural, immunohistochemical, and biochemical evidence, TSH up-regulates mitochondrial biogenesis and consequently activity in organ-cultured normal human epidermis in situ. Mitochondrial activity was assessed by measuring 2 key components of the respiratory chain. The abundance of mitochondria was assessed employing 2 independent morphological techniques: counting their numbers in human epidermis by high-magnification light microscopy of skin sections immunostained for mitochondria-selective cytochrome-c-oxidase subunit 1 (MTCO1) and transmission electron microscopy (TEM). Treatment with 10 mU/ml of TSH for 6 d strongly up-regulates the number of light-microscopically visualized, MTCO1-demarcated mitochondria. On the ultrastructural level, TEM confirms that TSH indeed stimulates mitochondrial proliferation and biogenesis in the perinuclear region of human skin epidermal keratinocytes. On the transcriptional level, TSH up-regulates MTCO1 mRNA (quantitative RT-PCR) and significantly enhances complex I and IV (cytochrome-c-oxidase) activity. This study pioneers the concept that mitochondrial energy metabolism and biogenesis in a normal, prototypic human epithelial tissue underlies potent neuroendocrine controls and introduces human skin organ culture as a clinically relevant tool for further exploring this novel research frontier in the control of mitochondrial biology.

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.

Noxious peripheral stimulation induces the expression of various proto-oncogenes in rat spinal neurons. However, proto-oncogene expression seems to differ depending on the mode of the stimulus. Here, we report that noxious cutaneous ultraviolet (UV) irradiation results in a nearly 8-fold increase in junD mRNA levels in the rat lumbar spinal cord. RNA slot-blotting and hybridization techniques revealed a transcriptional activation of the junD proto-oncogene after 6 h, but not 1 h following UV exposure. These results suggest that low-frequency ongoing afferent impulse discharge is reflected by an accumulation in junD transcripts.

Cytochrome c oxidase (COX), like other multi-subunit components of the respiratory chain, is controlled by both the nuclear and the mitochondrial genome. In order to find wether there is a close relationship between mRNAs encoded by the nucleus and by the mitochondrion, and between these mRNAs and enzyme activity, we compared six rat tissues (ventricle, liver, m. soleus, m. plantaris, and the white and red portions of m. gastrocnemius). We found a tenfold range for COX activity, a tenfold range for the contents of mRNA III (mitochondrial) and mRNA VIc (nuclear), a threefold range for total [poly(A)+] mRNA content and a sevenfold range for total RNA content in these tissues. The ratio of mRNA III to mRNA VIc was equal in each tissue, indicating the presence of a mechanism that coordinates the two genomes. There was a good correlation between mRNA content and COX activity (r = 0.78 for VIc, r = 0.77 for III; p less than 0.0001), demonstrating that the expression of this enzyme is mainly under pretranslational control.

A method is presented for the isolation of highly purified mitochondrial (mt)DNA from a crude DNA extract, making use of the different mobilities of covalently closed circular mtDNA vs. endonuclease-digested nuclear DNA in agarose gels. The preparation is virtually free of any contaminating linear DNA, as judged from its electron microscopic appearance, and can be used for further procedures such as polymerase chain reaction (PCR). Since isolation of mitochondria is not a prerequisite for this method, it can be applied to tissue samples in the mg range. In principle, the method can be applied to every eukaryotic species, provided a molecular hybridization probe is available which permits the position of mtDNA to be located in an agarose gel. This probe can be a cDNA, a DNA fragment generated by PCR, or mtDNA itself, if only the approximate size of the genome is known.

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.

To further understand pathways coordinating the expression of nuclear genes encoding mitochondrial proteins, we studied mitochondrial biogenesis during differentiation of myoblasts to myotubes. This energy-demanding process was accompanied by a fivefold increase of ATP turnover, covered by an eightfold increase of mitochondrial activity. While no change in mitochondrial DNA copy number was observed, mRNAs as well as proteins for nucleus-encoded cytochrome c, cytochrome c oxidase subunit IV, and mitochondrial transcription factor A (TFAM) increased, together with total cellular RNA and protein levels. Detailed analysis of the cytochrome c promoter by luciferase reporter, binding affinity, and electrophoretic mobility shift assays as well as mutagenesis studies revealed a critical role for cyclic AMP responsive element binding protein 1 (CREB-I) for promoter activation. Expression of two CREB-1 isoforms was observed by using specific antibodies and quantitative reverse transcription-PCR, and a shift from phosphorylated CREB-1Δ in myoblasts to phosphorylated CREB-1α protein in myotubes was shown, while mRNA ratios remained unchanged. Chromatin immunoprecipitation assays confirmed preferential binding of CREB-1α in situ to the cytochrome c promoter in myotubes. Overexpression of constitutively active and dominant-negative forms supported the key role of CREB-1 in regulating the expression of genes encoding mitochondrial proteins during myogenesis and probably also in other situations of enhanced mitochondrial biogenesis. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Although numerous pathogenic changes within the mitochondrial respiratory chain (RC) have been associated with an elevated occurrence of apoptosis within the affected tissues, the mechanistic insight into how mitochondrial dysfunction initiates apoptotic cell death is still unknown. In this study, we show that the specific alteration of the cytochrome c oxidase (COX), representing a common defect found in mitochondrial diseases, facilitates mitochondrial apoptosis in response to oxidative stress. Our data identified an increased ceramide synthase 6 (CerS6) activity as an important pro-apoptotic response to COX dysfunction induced either by chemical or genetic approaches. The elevated CerS6 activity resulted in accumulation of the pro-apoptotic C16 : 0 ceramide, which facilitates the mitochondrial apoptosis in response to oxidative stress. Accordingly, inhibition of CerS6 or its specific knockdown diminished the increased susceptibility of COX-deficient cells to oxidative stress. Our results provide new insights into how mitochondrial RC dysfunction mechanistically interferes with the apoptotic machinery. On the basis of its pivotal role in regulating cell death upon COX dysfunction, CerS6 might potentially represent a novel target for therapeutic intervention in mitochondrial diseases caused by COX dysfunction.

This study investigated the influence of chronic beta(3)-adrenoceptor deficiency on myocardial function. Therefore, we investigated Ca(2+)-regulatory proteins, SERCA 2a activity, and myofibrillar and mitochondrial function in hearts of wild-type (WT, n=7) and beta(3)-adrenoceptor knockout mice (beta(3)-KNO, n=7). Morphometric heart analysis showed no difference between WT and beta(3)-KNO. No alterations were observed for the protein expression of the ryanodine receptor or phospholamban. However, in beta(3)-KNO mice, protein expression of SERCA 2a and phospholamban phosphorylation were significantly increased. These changes were accompanied by an increased SERCA 2a activity in beta(3)-KNO. Alterations in phospholamban phosphorylation were independent of alterations in beta(1)/beta(2)-adrenoceptor distribution and protein expression of G proteins in beta(3)-KNO. Measurement of myofibrillar Ca(2+) sensitivity showed no difference in the Ca(2+)/force relation for WT and beta(3)-KNO. The same seems to hold true for mitochondrial function since the protein expressions of cytochrome c, uncoupling protein 3 and cytochrome c oxidase subunit IV were similar in WT and beta(3)-KNO. The conclusion is that depression of beta(3)-adrenergic stimulation may modulate the protein expression of SERCA 2a and phospholamban phosphorylation, thereby improving sarcoplasmic reticulum Ca(2+) uptake. Thus, beta(3)-adrenergic depression may be a therapeutic aim in situations of impaired SERCA 2a activity, e.g. for the treatment of heart failure.

Mitochondrial transcription factor A (TFAM) stimulates transcription from mitochondrial DNA (mtDNA) promoters in vitro and in organello. To investigate whether changes of TFAM levels also modulate transcription and replication in situ, the protein was transiently overexpressed in cultured cells. Mitochondrial mRNAs were significantly elevated at early time points, when no expansion of the TFAM pool was yet observed, but were decreased when TFAM levels had doubled, resemb-ling in vitro results. HEK cells contain about 35 molecules of TFAM per mtDNA. High levels of TFAM were not associated with increases of full-length mtDNA, but nucleic acid species sensitive to RNAse H increased. Stimulation of transcription was more evident when TFAM was transiently overexpressed in cells pre-treated with ethidium bromide (EBr) having lowered mtDNA, TFAM and mitochondrial transcript levels. EBr rapidly inhibited mtDNA transcription, while decay of mtDNA was delayed and preferentially slowly migrating, relaxed mtDNA species were depleted. In conclusion, we show that transcription of mtDNA is submaximal in cultured cells and that a subtle increase of TFAM within the matrix is sufficient to stimulate mitochondrial transcription. Thus, this protein meets all criteria for being a key factor regulating mitochondrial transcription in vivo, but other factors are necessary for increasing mtDNA copy number, at least in cultured cells.

Spermatogenesis in the seminiferous tubuli of the testis occurs under a high proliferation rate, suggesting considerable oxygen consumption. Because of the lack of blood vessels, the oxygen partial pressure in the lumen of these tubuli is very low. We previously identified a testis isoform of the hypoxia-inducible factor (HIF)-1alpha in the mouse, termed mHIF-1alphaI.1. Here, we demonstrate that expression of mHIF-1alphaI.1 increases during puberty, further demonstrating its gene induction in postmeiotic germ cells. Using 5'-rapid amplification of cDNA ends, we identified a novel HIF-1alpha isoform in the human testis, called hHIF-1alphaTe. Like mHIF-1alphaI.1, hHIF-1alphaTe mRNA is derived from an alternative promoter-first exon combination, but with a different genomic organization and a different nucleotide sequence. Reverse transcription-polymerase chain reaction analysis confirmed that hHIF-1alphaTe is exclusively expressed in the testis. As determined by immunofluorescence of ejaculated sperm cells, HIF-1alpha protein is mainly localized in the postacrosomal head and in the midpiece of spermatozoa. Though overlapping with mitochondrial localization in human and mouse spermatozoa, neither hHIF-1alphaTe nor hHIF-1alpha associated with mitochondria. In contrast with the ubiquitously expressed HIF-1alpha protein and the mouse testis-specific mHIF-1alphaI.1 isoform, the hHIF-1alphaTe mRNA sequence predicts a protein with an N-terminal truncation of the DNA-binding domain. As shown by yeast two-hybrid assays, hHIF-1alphaTe still formed heterodimeric complexes with HIF-1beta. However, hHIF-1alphaTe was incapable of forming a DNA-binding HIF-1 complex. Overexpression of exogenous hHIF-1alphaTe resulted in the inhibition of the endogenous HIF-1 transcriptional activity, demonstrating that the testis-specific hHIF-1alphaTe isoform is a dominant-negative regulator of normal HIF-1 activity.

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.

The mitochondrial electron transport chain (ETC) is the most important source of reactive oxygen species (ROS) in mammalian cells. To assess its relevance to the endogenous generation of oxidative DNA damage in the nucleus, we have compared the background (steady-state) levels of oxidative DNA base modifications sensitive to the repair glycosylase Fpg (mostly 7,8-dihydro-8-oxoguanine) in wild-type HeLa cells and HeLa rho0 cells. The latter are depleted of mitochondrial DNA and therefore are unable to produce ROS in the ETC. Although the levels of ROS measured by flow cytometry and redox-sensitive probes in rho0 cells were only 10-15% those of wild-type cells, steady-state levels of oxidative DNA base modifications were the same as in wild-type cells. Mitochondrial generation of ROS was then stimulated in HeLa wild-type cells using inhibitors interfering with the ETC. Although mitochondrial ROS production was raised up to 6-fold, none of the substances nor their combinations induced additional oxidative base modifications in the nuclear DNA. This was also true for glutathione-depleted cells. The results indicate that the contribution of mitochondria to the endogenously generated background levels of oxidative damage in the nuclear DNA is negligible.

Mitochondrial transcription factor A (TFAM) has been shown to stimulate transcription from mitochondrial DNA promoters in vitro. In order to determine whether changes in TFAM levels also regulate RNA synthesis in situ, recombinant human precursor proteins were imported into the matrix of rat liver mitochondria. After uptake of wt-TFAM, incorporation of [alpha-32P]UTP into mitochondrial mRNAs as well as rRNAs was increased 2-fold (P0.05), whereas import of truncated TFAM lacking 25 amino acids at the C-terminus had no effect. Import of wt-TFAM into liver mitochondria from hypothyroid rats stimulated RNA synthesis up to 4-fold. We conclude that the rate of transcription is submaximal in freshly isolated rat liver mitochondria and that increasing intra-mitochondrial TFAM levels is sufficient for stimulation. The low transcription rate associated with the hypothyroid state observed in vivo as well as in organello seems to be a result of low TFAM levels, which can be recovered by treating animals with T3 in vivo or by importing TFAM in organello. Thus, this protein meets the criteria for being a key factor in regulating mitochondrial gene expression in vivo.

Biogenesis of mitochondria is happening constantly due to the physiological and developmental situation of a cell. As mitochondrial biogenesis is a complex process producing about 20 % of cellular protein, the expression of the 1000 genes involved is expected to be coordinated and regulated tightly. The variety of physiological stimuli and differentiation states lead to the idea of a complex network connecting many different regulatory pathways. By analysing nuclear encoded mitochondrial genes some of the factors involved in the regulation and coordination of mitochondrial gene expression were identified. These factors include general transcription factors such as Sp1 or YY1, as well as transcription factors specific for mitochondrial genes like the nuclear respiratory factors NRF1 and 2. An important control function linked to the physiological situation of a cell is triggered by hormones such as steroid and thyroid hormones. Even cell type-specific regulatory proteins like the myogenin transcription factor family have a strong influence on some mitochondrial genes in the specific cellular background. The regulatory function of most of these proteins can be modulated and enhanced by the coactivators PGC-1a and b and PRC. Although regulatory pathways have been characterized in more detail in recent years, no regulation mechanism has been shown to work on all analysed mitochondrial genes, and the general concept of mitochondrial regulation still remains unclear.

Abstract —In isolated cardiac myocytes, the direct effects of angiotensin II on cellular growth and gene expression were shown to be mediated by endothelin via the endothelin subtype A (ET A ) receptor. To determine whether this pathway is also involved in the cardiovascular adaptations to a chronic activation of the renin-angiotensin system in vivo, the effects of a selective ET A receptor antagonist (LU 127043) were investigated in adult rats with renal artery stenosis. Four groups of rats (n=107) were studied over a period of 10 days after surgery: (1) sham-operated animals with saline administration, (2) rats subjected to left renal artery clipping with saline administration, (3) sham-operated rats with LU 127043 administration, and (4) rats subjected to left renal artery clipping with LU 127043 administration. LU 127043 (50 mg/kg) or saline was given by gavage twice daily starting 1 day before the operation. In clipped rats with saline administration, plasma renin activity, the ratio of left ventricular weight to body weight, and mRNAs for β-myosin heavy chain and atrial natriuretic peptide were significantly elevated as early as 2 days after surgery. Blood pressure started to rise on the third postoperative day and attained a steady state hypertensive level by day 6. Blockade of ET A receptors had no effects on plasma renin activity or the time course of hypertension in clipped animals but completely prevented left ventricular hypertrophy and the re-expression of the β-myosin heavy chain and atrial natriuretic peptide genes on day 2. While the expressions of the β-myosin heavy chain and atrial natriuretic peptide genes were not different from saline-treated, clipped animals after day 4, the development of left ventricular hypertrophy remained markedly blunted (−50%) during ET A receptor blockade until day 10. These results show that a continuous blockade of ET A receptors significantly attenuates the development of left ventricular hypertrophy and, more transiently, fetal gene expression in the early phase of renovascular hypertension. Since neither blood pressure nor the increase in plasma renin activity was significantly altered by ET A receptor blockade, the inhibitory influences of the ET A receptor antagonist on left ventricular hypertrophy and gene expression were mediated most likely through a direct blockade of myocardial ET A receptors.

The regions of troponin I (TnI) responsible for Ca2+-dependent activation and Ca2+ sensitivity of the actin-myosin subfragment 1-tropomyosin ATPase (acto-S1-TM) activity have been determined. A colorimetric ATPase assay at pH 7.8 has been applied to reconstituted skeletal muscle thin filaments at actin:S1:TM ratios of 6:1:2. Several TnI fragments (TnI-(104-115), TnI-(1-116), and TnI-(96-148)) and TnI mutants with single amino acid substitutions within the inhibitory region (residues 104-115) were assayed to determine their roles on the regulatory function of TnI. TnI-(104-115) is sufficient for achieving maximum inhibition of the acto-S1-TM ATPase activity and its importance was clearly shown by the reduced potency of TnI mutants with single amino acid substitutions within this region. However, the function of the inhibitory region is modulated by other regions of TnI as observed by the poor inhibitory activity of TnI-(1-116) and the increased potency of the inhibitory region by TnI-(96-148). The regulatory complex composed of TnI-(96-148) plus troponin T-troponin C complex (TnT.C) displays the same Ca2+ sensitivity (pCa50) as intact troponin (Tn) or TnI plus TnT.C while those regulatory complexes composed of TnT.C plus either TnI-(104-115) or TnI-(1-116) had an increase in their pCa50 values. This indicates that the Ca2+ sensitivity or responsiveness of the thin filament is controlled by TnI residues 96-148. The ability of Tn to activate the acto-S1-TM ATPase activity in the presence of calcium to the level of the acto-S1 rate was mimicked by the regulatory complex composed of TnI-(1-116) plus TnT.C and was not seen with complexes composed with either TnI-(104-115) or TnI-(96-148). This indicates that the N terminus of TnI in conjunction with TnT controls the degree of activation of the ATPase activity. Although the TnI inhibitory region (104-115) is the Ca2+-sensitive switch which changes binding sites from actin-TM to TnC in the presence of calcium, its function is modulated by both the C-terminal and N-terminal regions of TnI. Thus, distinct regions of TnI control different aspects of Tn's biological function.

In order to test the hypothesis that mitochondrial transcription factor A (mtTFA) regulates mitochondrial transcription in vivo, mtTFA was overexpressed in HeLa cells and imported into isolated rat liver mitochondria. Five hours after transfection with an eukaryotic expression vector, mitochondrial transcripts for cytochrome-c-oxidase subunit I and 12 S rRNA were increased over controls. In the presence of rat liver mitochondria, the 29 kDa mtTFA, generated by in vitro translation, was processed to a 24 kDa protein which was protected from protease digestion. This demonstrates that mtTFA was imported into the matrix. Incorporation of 32P-UTP into mitochondrial transcripts was stimulated following import of mtTFA. We conclude that the intracellular and intramitochondrial concentration of mtTFA, respectively, indeed regulates mitochondrial transcription. (Mol Cell Biochem 174: 227–230, 1997)

Using treatment with vanadate solutions, we extracted native cardiac troponin I and troponin C (cTnI and cTnC) from skinned fibers of porcine right ventricles. These proteins were replaced by exogenously supplied TnI and TnC isoforms, thereby restoring Ca2+-dependent regulation. Force then depended on the negative logarithm of Ca2+ concentration (pCa) in a sigmoidal manner, the pCa for 50% force development, pCa50, being about 5.5. For reconstitution we used fast-twitch rabbit skeletal muscle TnI and TnC (sTnI and sTnC), bovine cTnI and cTnC or recombinant sTnIs that were altered by site-directed mutagenesis. Incubation with TnI inhibited isometric tension in TnI-extracted fibers in the absence of Ca2+, but restoration of Ca2+ dependence required incubation with both TnI and TnC. Relaxation at low Ca2+ levels and the steepness of the force/pCa relation depended on the concentration of exogenously supplied TnI in the reconstitution solution (range 20-150 "mu"M), while Ca2+ sensitivity, i.e. the pCa50, was dependent on the isoform, and also on the concentration of TnC in the reconstitution solution. At pH 6.7, skinned fibers reconstituted with optimal concentrations of sTnC and sTnI (120 "mu"M and 150 "mu"M, respectively) were more sensitive to Ca2+ than those reconstituted with cTnC and cTnI (difference in pCa50 approx. 0.2 units). Rabbit sTnI was cloned and expressed in Escherichia coli using a high yield expression plasmid. We introduced point mutations into the TnI inhibitory region comprising the sequence of the minimal common TnC/actin binding site (-G104-K-F-K-R-P-P-L-R-R-V-R115-). The four mutants produced by substitution of T for P110, G for P110, G for L111, and G for K105 were chosen, based on previous work with synthetic peptides showing that single amino acid substitution in this region diminished the capacity of these peptides to inhibit acto-S1 ATPase or contraction of skinned fibers. Therefore, all amino acid residues of the inhibitory region are thought to contribute to biological activity of TnI. However, each of the recombinant TnIs could substitute for endogenous TnI. In combination with exogenous TnC, Ca2+ dependence could be restored when gly110sTnI, thr110sTnI or gly111sTnI was used for reconstitution. The mutant gly105sTnI, on the other hand, reduced the ability of skinned fibers to relax at low Ca2+ concentrations and it caused an increase in Ca2+ sensitivity.

Skinned fibres from porcine ventricles exhibited a higher Ca2+ sensitivity (pCa50, i.e. -log10 Ca2+ concentration required for half-maximal activation, for force generation) than atrial fibres. The thiadiazinone derivative EMD 53998 increased Ca2+ sensitivity and Ca2+ efficacy in both preparations. The drug effect depended on the isoform of troponin (Tn). Using the vanadate method TnI and TnC could be partly extracted and replaced by foreign tropin or by the TnI subunit of added foreign troponins. We investigated the relationship between pCa and force development before and after replacement of TnI with foreign troponin (bovine ventricular troponin, cTn, or rabbit skeletal muscle troponin, sTn) in the presence and absence of EMD 53998. Substitution with bovine cTn increased Ca2+ sensitivity to a value characteristic of bovine ventricular skinned fibres (pCa50=5.4) and was further increased by EMD 53998. Substitution with sTn also increased Ca2+ sensitivity, but subsequent addition of EMD 53998 caused little further increase in Ca2+ sensitivity. Following extraction of TnI with vanadate, skinned fibres contracted in a Ca2+-independent manner and failed to relax at a pCa of 8. Relaxation could be induced, however, by bovine ventricular TnI and rabbit skeletal muscle recombinant TnI. This relaxation could be reversed by EMD 53998 (100 μM). The Ca2+-independent force of contracted fibres could also be depressed by a TnI inhibitory peptide, (cTnI 137–148) and, in addition, this effect was antagonized by EMD 53998. These results suggest that EMD 53998 antagonizes the inhibitory action of TnI, possibly by interfering with the interaction of the TnI inhibitory region with actin.

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.

The expression of the alpha fast (alpha f) and beta tropomyosin (TM) genes has been analysed with muscle-specific and common cDNA probes after unilateral nerve section of the pectoralis major muscle (PM) in 4-week-old chickens. The following were observed in denervated muscles. (1) The beta TM mRNA, which was repressed during development, reaccumulates in a biphasic curve with the increase in the beta TM protein lagging behind the changes in its mRNA. Accordingly, no beta TM is seen in products translated in vitro from total and polyA+ RNA obtained 1 week after denervation. No such translation block is seen with RNA obtained from control or muscles denervated for 6 weeks. (2) No changes in the alpha fTM mRNA and corresponding protein are observed. (3) RNA processing of the two genes is not changed. (4) In the contralateral muscles, transitory increases in alpha f and beta TM mRNAs are observed while the corresponding proteins remain unchanged. Our data suggest that muscle fibres display early and long-term responses to the loss of neural input which might result from a combination of changes produced by regenerative processes and reprogramming of existing fibres. Moreover, in contrast to normal development, no reciprocal changes of alpha f and beta TM expression are seen in denervated muscles.

We determined the steady-state level of mRNA for myosin heavy chain (MHC) from airway smooth muscle during maturation in domestic swine. Tissues were excised, and airway smooth muscle was dissected from three neonatal (NEO), three 2-wk-old swine (2ws), three 10-wk-old swine (10ws), and three adult swine. Total RNA was isolated, fractionated, and transferred to a nitrocellulose membrane (Northern blot). A single-stranded oligonucleotide of 63-nt was synthesized corresponding to the 3' coding region of the chicken gizzard MHC cDNA. This region appeared to be highly conserved (92% nucleotide sequence homology with the corresponding portion of rabbit uterine smooth muscle MHC cDNA). Northern blots, which were loaded with equivalent quantities of total RNA, were probed with gamma 32P-labeled synthetic oligonucleotide, and, under stringent washing conditions, the 5' end-labeled DNA was hybridized to a single band of the expected molecular weight. The mRNA for total myosin was quantified using autoradiograms of blots, and signal intensity was measured as integrated areas expressed as arbitrary densitometric units x mm (AU). The content of mRNA for MHC was substantially greater in NEO than in more mature animals; maximal area was 1.33 +/- 0.15 AU for NEO, 0.33 +/- 0.05 AU for 2ws, 0.30 +/- 0.04 AU for 10ws, and 0.34 +/- 0.08 AU for adult swine (P < 0.05, NEO versus 2ws, 10ws, and adult). Rehybridization of each blot with a 28S ribosomal RNA probe confirmed comparable total RNA loadings for all tissue samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Uptake of L-glutamate was studied in isolated adult rat heart myocytes in the presence and absence of oxygen. Normoxic incubation of cardiomyocytes with glutamate concentrations varying from 0.0625 to 0.33 mM led to an apparent Km-value for glutamate of 0.18 mM. However, incubation of cardiomyocytes with glutamate concentrations up to 10 mM did not saturate glutamate uptake. We therefore assume that the high affinity glutamate carrier is saturated and further nonspecific transport mechanisms are responsible for the non-saturation kinetics. The initial glutamate uptake rate from the incubation medium, under normoxia, amounted to 24.3 ±1.6 pmol min-1 mg-1 protein and increased under anoxia to 38.1 ± 3.8 pmol min-1 mg-1 protein. Even this increased glutamate uptake could not prevent a depletion of intracellular glutamate in anaerobic cardiomyocytes. Under reoxygenation glutamate increased again to control values with a rate of 930 pmol min-1 mg-1 protein within 15 min. Glutamate uptake from the incubation medium after reoxygenation amounted to 31.9 ± 2.5 pmol min-1 mg-1 protein. Therefore the glutamate uptake from extracellular space cannot be responsible for the rapid increase in intracellular glutamate concentration after reoxygenation. Cardiomyocytes must replenish glutamate by other mechanisms, e.g. intracellular proteolysis.

International audience; We have disrupted expression of the mitochondrial Friedreich ataxia protein frataxin specifically in murine hepatocytes to generate mice with impaired mitochondrial function and decreased oxidative phosphorylation. These animals have a reduced life span and develop multiple hepatic tumors. Livers also show increased oxidative stress, impaired respiration and reduced ATP levels paralleled by reduced activity of iron-sulfur cluster (Fe/S) containing proteins (ISP), which all leads to increased hepatocyte turnover by promoting both apoptosis and proliferation. Accordingly, phosphorylation of the stress-inducible p38 MAP kinase was found to be specifically impaired following disruption of frataxin. Taken together, these findings indicate that frataxin may act as a mitochondrial tumor suppressor protein in mammals.

In yeast strains (S. cerevisiae) carrying a point mutation of the ATP2 gene, which destabilizes the beta subunit of F1 ATP synthase in vitro, the growth rate was reduced significantly, demonstrating that the mutation is also deleterious in vivo. Immunoblots showed that levels of the mutated beta, but also of the wild-type alpha subunit were increased in the mutated strains, together with levels of the corresponding mRNAs (approximately 1.6-fold). Northern analysis showed that this was due to both the appearance of new transcript species as well as upregulation of the cognate transcripts, strongly indicating that the increase was probably due to activation of transcription. Levels of other mitochondrial proteins, e.g. cytochrome c oxidase, were unaffected. We conclude that a specific signal communicates the actual performance of the ATP synthase inside the mitochondria to the nuclear genes encoding its subunits.

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.

SummaryAging is a progressive decline of body function, during which many tissues accumulate few cells with high levels of deleted mitochondrial DNA (mtDNA), leading to a defect of mitochondrial functions. Whether this mosaic mitochondrial deficiency contributes to organ dysfunction is unknown. To investigate this, we generated mice with an accelerated accumulation of mtDNA deletions in the myocardium, by expressing a dominant-negative mutant mitochondrial helicase. These animals accumulated few randomly distributed cardiomyocytes with compromised mitochondrial function, which led to spontaneous ventricular premature contractions and AV blocks at 18 months. These symptoms were not caused by a general mitochondrial dysfunction in the entire myocardium, and were not observed in mice at 12 months with significantly lower numbers of dysfunctional cells. Therefore, our results suggest that the disposition to arrhythmia typically found in the aged human heart might be due to the random accumulation of mtDNA deletions and the subsequent mosaic respiratory chain deficiency.

The subcellular distribution of adenine nucleotides, phosphocreatine and intermediates of the malate-aspartate cycle was investigated in adult rat heart myocytes under normoxia and anoxia. Cytosolic and mitochondrial concentrations of metabolites were determined by a fractionation method using digitonin. Under normoxia, cytosolic/mitochondrial gradients were found for ATP (c/m = 4), AMP (c/m less than 0.01), citrate (c/m = 0.5), aspartate (c/m = 3), glutamate (c/m = 2), while phosphocreatine and glutamine were confined to the cytosolic space. No gradients were found for malate and 2-oxoglutarate. The results show that the transport of electrons from the cytosol into the mitochondria is supported by the glutamate gradient and by a high glutamate/aspartate ratio inside the mitochondria (Glu/Asp = 15) which is maintained by the energy-dependent Glu-Asp exchange across the mitochondrial membrane. Under anoxia, cytosolic glutamate is transaminated with pyruvate, yielding alanine and 2-oxoglutarate, which is oxidized to succinate inside the mitochondria and leaves the cell. The data indicate that stimulation of transamination is caused by a mass action effect following a decrease in cytosolic 2-oxoglutarate which may be due to succinate-2-oxoglutarate exchange across the mitochondrial membrane. Inhibition of the energy-dependent inward transport of glutamate may support this process.

Mitochondrial disorders are clinically and genetically heterogeneous and are associated with a variety of disease mechanisms. Defects of mitochondrial protein synthesis account for the largest subgroup of disorders manifesting with impaired respiratory chain capacity; yet, only a few have been linked to dysfunction in the protein components of the mitochondrial ribosomes. Here, we report a subject presenting with dyskinetic cerebral palsy and partial agenesis of the corpus callosum, while histochemical and biochemical analyses of skeletal muscle revealed signs of mitochondrial myopathy. Using exome sequencing, we identified a homozygous variant c.215C>T in MRPS25, which encodes for a structural component of the 28S small subunit of the mitochondrial ribosome (mS25). The variant segregated with the disease and substitutes a highly conserved proline residue with leucine (p.P72L) that, based on the high-resolution structure of the 28S ribosome, is predicted to compromise inter-protein contacts and destabilize the small subunit. Concordant with the in silico analysis, patient’s fibroblasts showed decreased levels of MRPS25 and other components of the 28S subunit. Moreover, assembled 28S subunits were scarce in the fibroblasts with mutant mS25 leading to impaired mitochondrial translation and decreased levels of multiple respiratory chain subunits. Crucially, these abnormalities were rescued by transgenic expression of wild-type MRPS25 in the mutant fibroblasts. Collectively, our data demonstrate the pathogenicity of the p.P72L variant and identify MRPS25 mutations as a new cause of mitochondrial translation defect.

Most of the known disease associated point mutations in mitochondrial DNA (for overviews see Wallace1 and Shoubridge and Molnar2) affect mitochondrial tRNA genes leading to impaired translation of the mitochondrial encoded subunits of the oxidative phosphorylation complexes I, III, IV, and V. Among these, three different point mutations in the mitochondrial tRNALeu(CUN) gene have been identified so far. These rare mutations were reported only in single individuals and were found to be associated with skeletal muscle disorders, including chronic progressive external ophthalmoplegia (CPEO),3 isolated skeletal myopathy,4 and chronic fatigue syndrome.5 However, the detailed pathogenic mechanisms of these tRNA point mutations leading to mitochondrial dysfunction in skeletal muscle have remained elusive. From a diagnostic point of view it is noteworthy that the mitochondrial genome is highly variable, which in turn leads to difficulties in proving the pathogenic role of any particular heteroplasmic mtDNA mutation.6 Therefore, it has become common practice to provide correlation between the mutation load (degree of mtDNA heteroplasmy) and the biochemical pathology (for example, cytochrome c oxidase (COX) negativity) in individual muscle fibres or cybrids. Here, we report a novel heteroplasmic point mutation in the mitochondrial tRNALeu(CUN) gene associated with a mild form of CPEO. This mutation causes, due to its location in the highly conserved DHU-stem, a severe decrease in the steady state levels of this tRNA, explaining the impaired translation of mitochondrial encoded proteins. Interestingly, this mutation strictly co-segregates with one allele of a heteroplasmic D-loop polymorphism. This causes pseudo-correlation of the neutral polymorphism with a pathological biochemical phenotype and might be responsible for routing of the pathogenic mutation into skeletal muscle. ### Case description A 28 year old female patient presented with a history of mild exercise intolerance starting in early childhood. At the age of 13 years she developed …

In earlier research, we identified a 43-kDa c-ErbAalpha1 protein (p43) in the mitochondrial matrix of rat liver. In the present work, binding experiments indicate that p43 displays an affinity for triiodothyronine (T3) similar to that of the T3 nuclear receptor. Using in organello import experiments, we found that p43 is targeted to the organelle by an unusual process similar to that previously reported for MTF1, a yeast mitochondrial transcription factor. DNA-binding experiments demonstrated that p43 specifically binds to four mitochondrial DNA sequences with a high similarity to nuclear T3 response elements (mt-T3REs). Using in organello transcription experiments, we observed that p43 increases the levels of both precursor and mature mitochondrial transcripts and the ratio of mRNA to rRNA in a T3-dependent manner. These events lead to stimulation of mitochondrial protein synthesis. In transient-transfection assays with reporter genes driven by the mitochondrial D loop or two mt-T3REs located in the D loop, p43 stimulated reporter gene activity only in the presence of T3. All these effects were abolished by deletion of the DNA-binding domain of p43. Finally, p43 overexpression in QM7 cells increased the levels of mitochondrial mRNAs, thus indicating that the in organello influence of p43 was physiologically relevant. These data reveal a novel hormonal pathway functioning within the mitochondrion, involving a truncated form of a nuclear receptor acting as a potent mitochondrial T3-dependent transcription factor.