Mitochondrial Energy Pathways Research Articles

Residual OXPHOS is required to drive primary and metastatic lung tumours in an orthotopic breast cancer model.

Fast adaptation of glycolytic and mitochondrial energy pathways to changes in the tumour microenvironment is a hallmark of cancer. Purely glycolytic ρ0 tumour cells do not form primary tumours unless they acquire healthy mitochondria from their micro-environment. Here we explored the effects of severely compromised respiration on the metastatic capability of 4T1 mouse breast cancer cells. 4T1 cell lines with different levels of respiratory capacity were generated; the Seahorse extracellular flux analyser was used to evaluate oxygen consumption rates, fluorescent confocal microscopy to assess the number of SYBR gold-stained mitochondrial DNA nucleoids, and the presence of the ATP5B protein in the cytoplasm and fluorescent in situ nuclear hybridization was used to establish ploidy. MinION nanopore RNA sequence analysis was used to compare mitochondrial DNA transcription between cell lines. Orthotopic injection was used to determine the ability of cells to metastasize to the lungs of female Balb/c mice. OXPHOS-deficient ATP5B-KO3.1 cells did not generate primary tumours. Severely OXPHOS compromised ρ0D5 cells generated both primary tumours and lung metastases. Cells generated from lung metastasis of both OXPHOS-competent and OXPHOS-compromised cells formed primary tumours but no metastases when re-injected into mice. OXPHOS-compromised cells significantly increased their mtDNA content, but this did not result in increased OXPHOS capacity, which was not due to decreased mtDNA transcription. Gene set enrichment analysis suggests that certain cells derived from lung metastases downregulate their epithelial-to-mesenchymal related pathways. In summary, OXPHOS is required for tumorigenesis in this orthotopic mouse breast cancer model but even very low levels of OXPHOS are sufficient to generate both primary tumours and lung metastases.

Early Chronic Fluoxetine Treatment of Ts65Dn Mice Rescues Synaptic Vesicular Deficits and Prevents Aberrant Proteomic Alterations.

Down syndrome (DS) is the most common form of inherited intellectual disability caused by trisomy of chromosome 21, presenting with intellectual impairment, craniofacial abnormalities, cardiac defects, and gastrointestinal disorders. The Ts65Dn mouse model replicates many abnormalities of DS. We hypothesized that investigation of the cerebral cortex of fluoxetine-treated trisomic mice may provide proteomic signatures that identify therapeutic targets for DS. Subcellular fractionation of synaptosomes from cerebral cortices of age- and brain-area-matched samples from fluoxetine-treated vs. water-treated trisomic and euploid male mice were subjected to HPLC-tandem mass spectrometry. Analysis of the data revealed enrichment of trisomic risk genes that participate in regulation of synaptic vesicular traffic, pre-synaptic and post-synaptic development, and mitochondrial energy pathways during early brain development. Proteomic analysis of trisomic synaptic fractions revealed significant downregulation of proteins involved in synaptic vesicular traffic, including vesicular endocytosis (CLTA, CLTB, CLTC), synaptic assembly and maturation (EXOC1, EXOC3, EXOC8), anterograde axonal transport (EXOC1), neurotransmitter transport to PSD (SACM1L), endosomal-lysosomal acidification (ROGDI, DMXL2), and synaptic signaling (NRXN1, HIP1, ITSN1, YWHAG). Additionally, trisomic proteomes revealed upregulation of several trafficking proteins, involved in vesicular exocytosis (Rab5B), synapse elimination (UBE3A), scission of endocytosis (DBN1), transport of ER in dendritic spines (MYO5A), presynaptic activity-dependent bulk endocytosis (FMR1), and NMDA receptor activity (GRIN2A). Chronic fluoxetine treatment of Ts65Dn mice rescued synaptic vesicular abnormalities and prevented abnormal proteomic changes in adult Ts65Dn mice, pointing to therapeutic targets for potential treatment of DS.

Abstract B100: Perfluoroalkyl substances (PFAS) induce platinum resistance in ovarian cancer through altering mitochondrial function

Abstract Platinum resistance is a major barrier to the effective treatment of advanced-stage ovarian cancer; however, factors leading to the development of resistance are not well understood. In fact, the role of environmental endocrine disrupting chemicals, such as perfluoroalkyl substances (PFAS), have seldom been explored. This is important because select PFAS have been linked to adverse reproductive outcomes in females including endometriosis, oocyte apoptosis, infertility, and increased even ovarian cancer risk at very high exposure levels. The relationship between PFAS and adverse female reproductive outcomes, as well as reports of other endocrine disruptors leading to therapy resistance, led, in the current study, to the evaluation of the effect of PFAS exposure on response to carboplatin in ovarian cancer cells. We recently showed that select PFAS, at human-relevant concentrations, induced carboplatin resistance in OVCAR-3 and Caov-3 cells; however, the mechanism underlying the onset of resistance remains largely unknown. Since platinum-resistant ovarian cancer is characterized, in part, by increased mitochondrial networks and enhanced bioenergetic capacities, the central hypothesis of this study is that PFAS induce alterations to mitochondrial biology. Mitochondrial endpoints of interest included redox ratio and superoxide production. To measure redox ratio, nicotinamide dinucleotide (NADH) and flavin adenine dinucleotide (FAD), which are inherently fluorescent, were measured using a fluorescence plate reader. Since NADH and FAD are metabolites produced via oxidative phosphorylation and glycolysis, evaluating redox ratio can provide an understanding of which pathway is preferred in the presence of environmental stimuli like PFAS. In OVCAR-3 and Caov-3 cells, baseline NADH:FAD ratios were 3.3 and 3.5, respectively. After 48 hours of PFAS exposure, NADH:FAD ratios of OVCAR-3 and Caov-3 cells ranged from 8-10 or 6-10, respectively. This increase after PFAS exposure could suggest enhanced energy production capabilities that warrant further investigation. As an additional measure, superoxide production was measured using flow cytometry. OVCAR-3 and Caov-3 cells were exposed to PFAS for 6 days prior to isolating cell pellets and incubating them with MitoSOX (mitochondrial) and SYTOX Blue (nuclear) dyes. After 15–30-minute incubations, samples were run on the flow cytometer. Compared to controls, superoxide production in PFAS-exposed ovarian cancer cells increased by up to 200%. Often, cancer cells have elevated levels of reactive oxygen species due to enhanced energy production and signaling pathways, thus this finding further suggests that PFAS affect mitochondrial energy pathways. Altogether, these data show that PFAS exposure, which can induce carboplatin resistance in OVCAR-3 and Caov-3 cells, increases redox ratios and superoxide production in ovarian cancer cells. Therefore, targeting alterations in mitochondrial biology arising from PFAS exposure using precision medicine could be effective in overcoming platinum resistance in ovarian cancer. Citation Format: Brittany P. Rickard, Marta Overchuk, Vesna A. Chappell, Carl D. Bortner, Victoria L. Bae-Jump, Suzanne E. Fenton, Imran Rizvi. Perfluoroalkyl substances (PFAS) induce platinum resistance in ovarian cancer through altering mitochondrial function [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B100.

Dysregulation of Human Juvenile Huntington's Disease Brain Proteomes in Cortex and Putamen Involves Mitochondrial and Neuropeptide Systems.

Huntington's disease (HD) is a genetic neurodegenerative disease caused by trinucleotide repeat CAG expansions in the human HTT gene. Early onset juvenile HD (JHD) in children is the most severe form of the disease caused by high CAG repeat numbers of the HTT gene. To gain understanding of human HD mechanisms hypothesized to involve dysregulated proteomes of brain regions that regulate motor and cognitive functions, this study analyzed the proteomes of human JHD cortex and putamen brain regions compared to age-matched controls. JHD and age-matched control brain tissues were assessed for CAG repeat numbers of HTT by PCR. Human brain JHD brain cortex regions of BA4 and BA6 with the putamen region (n = 5) were analyzed by global proteomics, compared to age-matched controls (n = 7). Protein interaction pathways were assessed by gene ontology (GO), STRING-db, and KEGG bioinformatics. JHD brain tissues were heterozygous for one mutant HTT allele containing 60 to 120 CAG repeats, and one normal HTT allele with 10 to 19 CAG repeats. Proteomics data for JHD brain regions showed dysregulated mitochondrial energy pathways and changes in synaptic systems including peptide neurotransmitters. JHD compared to control proteomes of cortex and putamen displayed (a) proteins present only in JHD, (b) proteins absent in JHD, and (c) proteins that were downregulated or upregulated. Human JHD brain cortex and putamen regions display significant dysregulation of proteomes representing deficits in mitochondrial and synaptic neurotransmission functions. These findings advance understanding of JHD brain molecular mechanisms associated with HD disabilities.

Metabolomic changes in lactating multiparous naturally MAP-infected Holstein-Friesian dairy cows suggest changes in mitochondrial energy pathways

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative organism of Johne's Disease, a chronic intestinal infection of ruminants. Infected cows begin shedding MAP within the asymptomatic, subclinical stage of infection before clinical signs, such as weight loss, diarrhoea and reduced milk yields develop within the clinical stages of disease. Herein, we examine the milk metabolomic profiles of naturally MAP-infected Holstein-Friesian cows. The study used biobanked milk samples which were collected 73.4 ± 3.79 (early lactation) and 143 ± 3.79 (mean ± SE) (mid-lactation) days post-calving from 5 MAP-infected and 5 control multiparous cows. The milk metabolome was assessed using flow infusion electrospray high-resolution mass spectrometry (FIE-HRMS) for sensitive, non-targeted metabolite fingerprinting. Metabolite fingerprinting assessments using partial least squares discriminate analyses (PLS-DA) indicated that lactation stage was a larger source of variation than MAP status. Examining each lactation stage separately for changes associated to MAP-infection status identified 45 metabolites, 33 in early lactation and 12 in mid-lactation, but only 6 metabolites were targeted in both stages of lactation. Pathway enrichment analysis suggested that MAP affected the malate-aspartate shuffle during early lactation. Pearson's correlation analysis indicated relationships between milk lactose concentrations in mid-lactation and 6 metabolites that were tentatively linked to MAP-infection status. The targeted metabolites were suggestive of wider changes in the bioenergetic metabolism that appear to be an acceleration of the effects of progressing lactation in healthy cows. Additionally, milk lactose concentrations suggest that MAP reduces the availability of lactose derivatives.

Engineering Supramolecular Nanomedicine for Targeted Near Infrared-triggered Mitochondrial Dysfunction to Potentiate Cisplatin for Efficient Chemophototherapy.

Combinatorial cancer therapies based on nanomedicine have emerged as a promising strategy to achieve potentiated treatment efficiency. Herein, cisplatin (CDDP) prodrug (Pt-CD) and a mitochondria-targeted near-infrared (NIR) photosensitizer IR780 were combined to construct a multifunctional nanomedicine IR780@Pt NPs through a supramolecular self-assembly strategy. Targeted mitochondrial dysfunction of cancer cells was sufficiently induced under NIR laser irradiation through both photothermal and photodynamic effects, inhibiting the overactive mitochondrial energy pathways of cancer cells. The mitochondrial dysfunction significantly attenuated the crosstalk between mitochondria and nucleus via the cellular ATP energy chain, leading to obvious down-regulation of the key proteins of the nucleotide excision repair (NER) pathway. Thereby, the chemotherapeutic effect of CDDP could be significantly potentiated because of reduced DNA lesion repair capacity by ERCC1-XPF nuclease system. Moreover, IR780@Pt NPs exhibited excellent NIR fluorescence and photoacoustic (PA) imaging capacity for in vivo imaging-guided NIR laser treatment. Ultimately, the IR780@Pt NPs mediated combinatorial chemophototherapy achieved potentiated anticancer efficacy against cancer cells in vitro and tumor inhibition performance in vivo. Overall, this study highlighted the significance of nanomedicine mediated targeted induction of mitochondrial dysfunction to potentiate chemotherapy for efficient combinatorial cancer therapy.

Abstract P5-06-01: Distinctive coding and non-coding RNA profiles of pre-menopausal and post-menopausal benign breast

Abstract Background: Despite a paucity of data on the benign breast transcriptome, reduction mammoplasty tissue is commonly used as a control for RNA biomarker discovery in breast cancer (BC). We evaluated the transcriptome of benign breast to characterize pre-menopausal and post-menopausal coding and non-coding RNA profiles. These profiles will provide insight into the metabolic landscape of benign breast and inform transcriptomic studies of BC and peri-tumoral microenvironments. Methods: With institutional approval, cryobanked fresh breast tissues from reduction mammoplasties (age 15-62y; follow-up: 4-10 y with no cancer events), with histologically-confirmed benign tissues with similar epithelial: stromal tissues were grouped into 4 age sets (< 30y, N=10; 30-39y, N=11; 40-49y, N=10; ≥50y (clinically post-menopausal), N=10). Following RNA extraction, library preparation (Illumina TruSeq Stranded mRNA kit), and sequencing (Illumina HiSeq 4000), reads were processed (MAP-RSeq v3.0.0) and aligned (STAR aligner; hg38). Differential expression (DE) analysis (edgeR 2.6.2) identified DE genes from normalized RPKM reads (absolute log2 fold change (FC) > 1 and false discovery rate (FDR) < 0.10), corrected for intra-group biases using medians (absolute FC cut-off of >1.5). Over-representation analysis [Ingenuity pathway analysis (IPA), Ingenuity® Systems] and gene set enrichment analysis [(GSEA), GeneTrail 2.0] identified significantly-enriched pathways. Results: Across the 4 age sets, 561 DE genes were identified. Compared to the post-menopausal (PM) set, the number of DE genes was highest in <30 y set (N= 372) and decreased with increasing age (N= 170, age 30-39 set and N=20, age 40-49 set), generating up-regulated (PMup) and down-regulated (PMdown) PM transcriptomic profiles. The top PMdown DE genes included RANKL, WNT4, MKI67, extracellular matrix, and lactation-related genes (lactoferrin ,MUC4, MUC16, p < 0.01). Top PMdown canonical pathways were cell cycle-related (CDK1, CCNA2, CCNB2, ESPL1, TOP2A)(p< 0.001). Top PMup genes included those involved in adipogenesis, NPY1R, NPY2R, unsaturated fatty acid synthesis and eicosanoid signaling (P< 0.001). Top PMup canonical pathways included acyl co-A hydrolysis, stearate biosynthesis, acute phase response and RXR signaling (P < 0.001). GSEA of the entire gene set (N=15,466; ranked in order of 561 DE genes) identified 16 significant pathways, functionally grouped as mitochondrial energy metabolism, proteasome, and unsaturated fatty acid biosynthesis and signaling (PPAR pathway). While coding RNAs comprised 85% of DE transcripts, lncRNA comprised ˜5%. Top DE lncRNAs and precursor miRNAs included MEG3, a tumor suppressor, LIN00092, miR22 and miR1182 (p < 0.0001). Conclusions: Pre and post-menopausal benign breast tissues have distinctive transcriptomic profiles. PM benign breast has down-regulated proliferation/cell-cycle-related pathways, and up-regulated genes in mitochondrial energy pathways, lipogenesis and inflammatory pathways. Notably, many of the top DE coding and non-coding RNAs in PM benign breast have been implicated in BC progression, highlighting their value in better understanding breast carcinogenesis and the need to characterize their functional roles in normal aging and menopause. Citation Format: Carter JM, Nair AA, Davila JI, Heinzen EP, Hoskin TL, Winham SJ, Radisky DC, Degnim AC, Visscher DW. Distinctive coding and non-coding RNA profiles of pre-menopausal and post-menopausal benign breast [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-06-01.

Elucidating the Metabolic Plasticity of Cancer: Mitochondrial Reprogramming and Hybrid Metabolic States.

Aerobic glycolysis, also referred to as the Warburg effect, has been regarded as the dominant metabolic phenotype in cancer cells for a long time. More recently, it has been shown that mitochondria in most tumors are not defective in their ability to carry out oxidative phosphorylation (OXPHOS). Instead, in highly aggressive cancer cells, mitochondrial energy pathways are reprogrammed to meet the challenges of high energy demand, better utilization of available fuels and macromolecular synthesis for rapid cell division and migration. Mitochondrial energy reprogramming is also involved in the regulation of oncogenic pathways via mitochondria-to-nucleus retrograde signaling and post-translational modification of oncoproteins. In addition, neoplastic mitochondria can engage in crosstalk with the tumor microenvironment. For example, signals from cancer-associated fibroblasts can drive tumor mitochondria to utilize OXPHOS, a process known as the reverse Warburg effect. Emerging evidence shows that cancer cells can acquire a hybrid glycolysis/OXPHOS phenotype in which both glycolysis and OXPHOS can be utilized for energy production and biomass synthesis. The hybrid glycolysis/OXPHOS phenotype facilitates metabolic plasticity of cancer cells and may be specifically associated with metastasis and therapy-resistance. Moreover, cancer cells can switch their metabolism phenotypes in response to external stimuli for better survival. Taking into account the metabolic heterogeneity and plasticity of cancer cells, therapies targeting cancer metabolic dependency in principle can be made more effective.

Abstract P6-01-07: Mitochondria-nuclear communication regulates epithelial-mesenchymal transition and metastasis in triple negative breast cancer

Abstract For triple negative breast cancer (TNBC), the driver pathways are still poorly understood. Advances in cancer metabolism research over the last decade have enhanced and modified our understanding on Warburg effect. It is now known that mitochondria in tumors are not always defective in their ability to carry out oxidative phosphorylation. Instead, in proliferating cells, mitochondrial energy pathways are reprogrammed to meet the challenges of macromolecular synthesis and to escape from apoptosis. Tumor initiating cells (TICs) maintain cancer stem cell properties and are known to play significant role in TNBC metastasis. Mitochondrial retrograde regulation (MRR) is a bidirectional communication between mitochondria and nucleus. MRR is triggered by mitochondrial functional demands and it responds in a continuous manner to change metabolic needs of the cell. Using transmitochondrial cybrid (cybrid) technology, we generated different cybrid models under common nuclear backgrounds of benign breast epithelium or TNBC. Mitochondria from cells with different cancer potential such as benign breast epithelium, moderately metastatic and highly metastatic breast cancer cell lines were studied under the common nuclear background to understand MRR-regulated TIC properties and cancer pathways. Using genomic, metabolomic, and proteomic approaches, we confirmed the significance of mitochondrial character in the regulation of epithelial mesenchymal transition (EMT), TIC and metastatic properties. Altogether, our results suggest that MRR is critical in TNBC TIC character and stemness. Citation Format: Park JH, Jung KH, Sirupangi T, Vithayathil S, Jin F, Putluri V, Piyarathna DWB, Yotnda P, Bhat VB, Sreekumar A, Lewis MT, Coarfa C, Putluri N, Creighton CJ, Wong L-JC, Kaipparettu BA. Mitochondria-nuclear communication regulates epithelial-mesenchymal transition and metastasis in triple negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-01-07.

An Impaired Respiratory Electron Chain Triggers Down-regulation of the Energy Metabolism and De-ubiquitination of Solute Carrier Amino Acid Transporters

Hundreds of genes have been associated with respiratory chain disease (RCD), the most common inborn error of metabolism so far. Elimination of the respiratory electron chain by depleting the entire mitochondrial DNA (mtDNA, ρ(0) cells) has therefore one of the most severe impacts on the energy metabolism in eukaryotic cells. In this study, proteomic data sets including the post-translational modifications (PTMs) phosphorylation and ubiquitination were integrated with metabolomic data sets and selected enzyme activities in the osteosarcoma cell line 143B.TK(-) A shotgun based SILAC LC-MS proteomics and a targeted metabolomics approach was applied to elucidate the consequences of the ρ(0) state. Pathway and protein-protein interaction (PPI) network analyses revealed a nonuniform down-regulation of the respiratory electron chain, the tricarboxylic acid (TCA) cycle, and the pyruvate metabolism in ρ(0) cells. Metabolites of the TCA cycle were dysregulated, such as a reduction of citric acid and cis-aconitic acid (six and 2.5-fold), and an increase of lactic acid, oxalacetic acid (both twofold), and succinic acid (fivefold) in ρ(0) cells. Signaling pathways such as GPCR, EGFR, G12/13 alpha, and Rho GTPases were up-regulated in ρ(0) cells, which could be indicative for the mitochondrial retrograde response, a pathway of communication from mitochondria to the nucleus. This was supported by our phosphoproteome data, which revealed two main processes, GTPase-related signal transduction and cytoskeleton organization. Furthermore, a general de-ubiquitination in ρ(0) cells was observed, for example, 80S ribosomal proteins were in average threefold and SLC amino acid transporters fivefold de-ubiquitinated. The latter might cause the observed significant increase of amino acid levels in ρ(0) cells. We conclude that an elimination of the respiratory electron chain, e.g. mtDNA depletion, not only leads to an uneven down-regulation of mitochondrial energy pathways, but also triggers the retrograde response.

Abstract BS3-1: Dominance of mitochondria in determining the malignant phenotype

Abstract Mitochondria are semiautonomous organelles within cells that play an important role in cellular energy metabolism, free radical generation, and apoptosis. Mitochondria contain their own genome (mtDNA), which encodes a number of proteins critical for energy metabolism, particularly in oxidative phosphorylation. They are also the major source of generating reactive oxygen species (ROS) in the cell. Advances in cancer metabolism research over the last decade have enhanced and modified our understanding on Warburg effect. It is now known that mitochondria in tumors are not always defective in their ability to carry out oxidative phosphorylation. Instead, in proliferating cells, mitochondrial energy pathways are reprogrammed to meet the challenges of macromolecular synthesis and to escape from apoptosis. Mitochondrial retrograde regulation (MRR) is a bidirectional communication between mitochondria and nucleus. MRR is triggered by mitochondrial functional demands and it responds in a continuous manner to the changing metabolic needs of the cell. At present, it is not clear whether mitochondrial genomic status or metabolomic reprogramming affect nuclear genome stability. Also, limited evidence is available on the significance of proteins involved in the inter-genomic cross talk in the regulation of tumorigenesis. Transmitochondrial cybrids (cybrids) are a great utility for the study of the functional effects of mitochondria in a defined nuclear background. Cybrids are constructed by fusing enucleated cells harboring mitochondria of interest with ρ0 cells (mtDNA-depleted cells). Our approach of discovering mitochondria regulated pathways using cybrid models and their in vitro and in vivo validation in different breast cancer cell lines and patient derived xenograft models suggest that mitochondrial metabolic character and retrograde signaling are playing important roles in oncogenic transformation and metastasis. Importantly, mitochondria-nuclear crosstalk is critical in the regulation of some of the major cancer pathways. Compared to hormone regulated/responsive breast cancers, triple negative breast cancer patients suffer from worse overall survival, significantly shorter disease-free and post-recurrence survival. Due to the lack of known oncogenic drivers for triple negative breast cancer, clinical benefit from currently available targeted therapies is limited, and new therapeutic strategies are urgently needed. Thus, understanding MRR and mitochondria mediated oncogenic signature is critical in understanding the currently limited known etiology and treatment resistance of certain subgroups of cancers like the triple negative breast cancer. Citation Format: Kaipparettu BA. Dominance of mitochondria in determining the malignant phenotype. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr BS3-1.

04 The regulation of mitochondrial energy metabolism by L-carnitine lowering agents in ischaemia-reperfusion injury

Mitochondrial L-carnitine system plays an important role in energy metabolism of the cardiac cells. Administration of both mildronate and sodium pivalate decreases myocardial concentration of L- carnitine. However, mildronate induces...

05 The cardioprotective effect of mildronate is associated with decreased CPT I-dependent fatty acid metabolism in mitochondria

Mildronate is a cardioprotective agent whose mechanism of action is based on decrease in L-carnitine concentration in heart tissue and concomitant decrease in fatty acid metabolism. The present study was...

The Forkhead Transcription Factor Hcm1 Promotes Mitochondrial Biogenesis and Stress Resistance in Yeast

In Saccharomyces cerevisiae, the forkhead transcription factor Hcm1 is involved in chromosome segregation, spindle pole dynamics, and budding. We found that Hcm1 interacts with the histone deacetylase Sir2 and shifts from cytoplasm to the nucleus in the G(1)/S phase or in response to oxidative stress stimuli. The nuclear localization of Hcm1 depends on the activity of Sir2 as revealed by activators and inhibitors of the sirtuins and the Δsir2 mutant. Hcm1-overexpressing cells display more mitochondria that can be attributed to increased amounts of Abf2, a protein involved in mitochondrial biogenesis. These cells also show higher rates of oxygen consumption and improved resistance to oxidative stress that would be explained by increased catalase and Sod2 activities and molecular chaperones such as Hsp26, Hsp30, and members of Hsp70 family. Microarray analyses also reveal increased expression of genes involved in mitochondrial energy pathways and those allowing the transition from the exponential to the stationary phase. Taken together, these results describe a new and relevant role of Hcm1 for mitochondrial functions, suggesting that this transcription factor would participate in the adaptation of cells from fermentative to respiratory metabolism.

443: HAND1 Gene Expression Is Associated Mitochondrial Energy Pathways and Is Altered during Myocardial Recovery from Heart Failure Following LVAD Combination Therapy

HAND1 Gene Expression Is Associated Mitochondrial Energy Pathways and Is Altered during Myocardial Recovery from Heart Failure Following LVAD Combination Therapy E. Lara-Pezzi, L.E. Felkin, R. George, M.H. Yacoub, E.J. Birks, P.J. Barton, Harefield Heart Science Centre, Imperial College London, Harefield, Middlesex, United Kingdom; Harefield Hospital, Royal Brompton and Harefield NHS Trust, Harefield, Middlesex, United Kingdom

Post-transcriptional Silencing and Functional Characterization of theDrosophila melanogasterHomolog of HumanSurf1

Mutations in Surf1, a human gene involved in the assembly of cytochrome c oxidase (COX), cause Leigh syndrome, the most common infantile mitochondrial encephalopathy, characterized by a specific COX deficiency. We report the generation and characterization of functional knockdown (KD) lines for Surf1 in Drosophila. KD was produced by post-transcriptional silencing employing a transgene encoding a dsRNA fragment of the Drosophila homolog of human Surf1, activated by the UAS transcriptional activator. Two alternative drivers, Actin5C-GAL4 or elav-GAL4, were used to induce silencing ubiquitously or in the CNS, respectively. Actin5C-GAL4 KD produced 100% egg-to-adult lethality. Most individuals died as larvae, which were sluggish and small. The few larvae reaching the pupal stage died as early imagos. Electron microscopy of larval muscles showed severely altered mitochondria. elav-GAL4-driven KD individuals developed to adulthood, although cephalic sections revealed low COX-specific activity. Behavioral and electrophysiological abnormalities were detected, including reduced photoresponsiveness in KD larvae using either driver, reduced locomotor speed in Actin5C-GAL4 KD larvae, and impaired optomotor response as well as abnormal electroretinograms in elav-GAL4 KD flies. These results indicate important functions for SURF1 specifically related to COX activity and suggest a crucial role of mitochondrial energy pathways in organogenesis and CNS development and function.

Altered expression of mitochondrial genes in response to fracture in old rats

Background Old rats require more time for bone to bridge a fracture gap than young rats. To explore possible mitochondrial dysfunction in this delay, we measured levels of mRNA derived from mitochondrial genes in healing fractures of young, adult, and old rats.Methods Diaphyseal femoral fractures were induced in female rats at 6, 26, and 52 weeks of age (young, adult, and old rats, respectively). At baseline, at 3 days, and 1, 2, 4, and 6 weeks after fracture, the fracture site was harvested. Total RNA was extracted, and cRNA was prepared and hybridized to 54 Affymetrix U34A microarrays (2 rats/array and 3 arrays/age/time point).Results Radiographic union occurred progressively later with age. Of the 107 mitochondria-related genes reviewed, all 8 located within the mitochondrial DNA and some nuclear genes (of the electron transport chain and tricarboxylic acid cycle) showed a prolonged reduction in gene expression after fracture in the oldest rats, to about half the level of expression detected in young rats at 6 weeks after fracture. The transcript levels of apoptotic genes increased after fracture in rats of all three ages, while the transcript levels of 23 mitochondriarelated genes were largely unaltered after fracture.Interpretation Delayed fracture healing in old rats is associated with reduced mRNA expression of genes forming the mitochondrial energy pathways.