Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

Universidad Pablo de Olavide

Instituto de Salud Carlos III

Coenzyme Q (Q) is an obligatory component of both respiratory chain and uncoupling proteins. Also, Q acts as an antioxidant in cellular membranes. Several neurodegenerative diseases are associated with modifications of Q10 levels. For these reasons, therapies based on Q supplementation in the diet are currently studied in order to mitigate the symptoms of these diseases. However, the incorporation of exogenous Q also affects aging process in nematodes probably affecting reactive oxygen species (ROS) production. The aim of the present work is to clarify if supplementation with both Q10 and Q6 isoforms affects mitochondrial Q10 content, respiratory chain activity and ROS levels in human cells. Cells incorporated exogenously added Q10 and Q6 isoforms into mitochondria that produced changes in mitochondrial activity depending on the side chain length. Supplementation with Q10, but not with Q6, increased mitochondrial Q-dependent activities. However, Q6 affected the mitochondrial membrane potential, ROS production, and increased the protein levels of both catalase and Mn-superoxide dismutase (Mn-SOD). Also, Q6 induced a transient decrease in endogenous mitochondrial Q10 levels by increasing its catabolism. These results show that human cells supplemented with Q6 undergo a mitochondrial impairment, which is not observed with Q10 supplementation.

Specificity of coenzyme Q10 for a balanced function of respiratory chain and endogenous ubiquinone biosynthesis in human cells.

Coenzyme Q (Q) is an essential factor in the mitochondrial electron chain but also exerts important antioxidant functions in the rest of cell membranes of aerobic organisms. However, the mechanisms of distribution of Q among cell membranes are largely unclear. The aim of the present work is to study the mechanisms of distribution of endogenous Q(10) and exogenous Q(9) among cell membranes in human HL-60 cells. Endogenous Q(10) synthesized using the radiolabelled precursor [(14)C]-pHB was first detected in mitochondria, and it was later incorporated into mitochondria-associated membranes and endoplasmic reticulum (ER). Plasma membrane was the last location to incorporate [(14)C]-Q(10). Brefeldin A prevented Q(10) incorporation in plasma membrane. Exogenous Q(9) was preferably accumulated into the endo-lysosomal fraction but a significant amount was distributed among other cell membranes also depending on the brefeldin-A-sensitive endomembrane system. Our results indicate that mitochondria are the first location for new synthesized Q. Exogenous Q is mainly incorporated into an endo-lysosomal fraction, which is then rapidly incorporated to cell membranes mainly to MAM and mitochondria. We also demonstrate that both endogenous and dietary Q is distributed among endomembranes and plasma membrane by the brefeldin A-sensitive endo-exocytic pathway.

Coenzyme Q distribution in HL-60 human cells depends on the endomembrane system.

Free radicals have been implicated in the action of many chemotherapeutic drugs. Here we tested the hypothesis that camptothecin and other chemotherapeutic drugs, such as etoposide, doxorubicin, and methotrexate, induce an increase in coenzyme Q(10) levels as part of the antioxidant defense against free radical production under these anticancer treatments in cancer cell lines. Chemotherapy treatment induced both free radical production and an increase in coenzyme Q(10) levels in all the cancer cell lines tested. Reduced coenzyme Q(10) form levels were particularly enhanced. Coenzyme Q(10)-increased levels were associated with up-regulation of COQ genes expression, involved in coenzyme Q(10) biosynthesis. At the translational level, COQ7 protein expression levels were also increased. Furthermore, coenzyme Q(10) biosynthesis inhibition blocked camptothecin-induced coenzyme Q(10) increase, and enhanced camptothecin cytotoxicity. Our findings suggest that coenzyme Q(10) increase is implicated in the cellular defense under chemotherapy treatment and may contribute to cell survival.

Chemotherapy induces an increase in coenzyme Q10 levels in cancer cell lines.

It has recently been shown that the microtubule cytoskeleton is reformed during the execution phase of apoptosis. We demonstrate that this microtubule reformation occurs in many cell types and under different apoptotic stimuli. We confirm that the apoptotic microtubule network possesses a novel organization, whose nucleation appears independent of conventional gamma-tubulin ring complex containing structures. Our analysis suggests that microtubules are closely associated with the plasma membrane, forming a cortical ring or cellular "cocoon". Concomitantly other components of the cytoskeleton, such as actin and cytokeratins disassemble. We found that colchicine-mediated disruption of apoptotic microtubule network results in enhanced plasma membrane permeability and secondary necrosis, suggesting that the reformation of a microtubule cytoskeleton plays an important role in preserving plasma membrane integrity during apoptosis. Significantly, cells induced to enter apoptosis in the presence of the pan-caspase inhibitor z-VAD, nevertheless form microtubule-like structures suggesting that microtubule formation is not dependent on caspase activation. In contrast we found that treatment with EGTA-AM, an intracellular calcium chelator, prevents apoptotic microtubule network formation, suggesting that intracellular calcium may play an essential role in the microtubule reformation. We propose that apoptotic microtubule network is required to maintain plasma membrane integrity during the execution phase of apoptosis.

The apoptotic microtubule network preserves plasma membrane integrity during the execution phase of apoptosis.

Coenzyme Q (CoQ) is a key component of the mitochondrial respiratory chain carrying electrons from complexes I and II to complex III and it is an intrinsic component of the respirasome. CoQ concentration is highly regulated in cells in order to adapt the metabolism of the cell to challenges of nutrient availability and stress stimuli. At least 10 proteins have been shown to be required for CoQ biosynthesis in a multi-peptide complex and COQ7 is a central regulatory factor of this pathway. We found that the first 765 bp of the 3'-untranslated region (UTR) of COQ7 mRNA contains cis-acting elements of interaction with RNA-binding proteins (RBPs) HuR and hnRNP C1/C2. Binding of hnRNP C1/C2 to COQ7 mRNA was found to require the presence of HuR, and hnRNP C1/C2 silencing appeared to stabilize COQ7 mRNA modestly. By contrast, lowering HuR levels by silencing or depriving cells of serum destabilized and reduced the half-life of COQ7 mRNA, thereby reducing COQ7 protein and CoQ biosynthesis rate. Accordingly, HuR knockdown decreased oxygen consumption rate and mitochondrial production of ATP, and increased lactate levels. Taken together, our results indicate that a reduction in COQ7 mRNA levels by HuR depletion causes mitochondrial dysfunction and a switch toward an enhanced aerobic glycolysis, the characteristic phenotype exhibited by primary deficiency of CoQ10. Thus HuR contributes to efficient oxidative phosphorylation by regulating of CoQ10 biosynthesis.

RNA-binding proteins regulate cell respiration and coenzyme Q biosynthesis by post-transcriptional regulation of COQ7.

Coenzyme Q (CoQ) deficiency syndrome includes clinically heterogeneous mitochondrial diseases that show a variety of severe and debilitating symptoms. A multiprotein complex encoded by nuclear genes carries out CoQ biosynthesis. Mutations in any of these genes are responsible for the primary CoQ deficiency, but there are also different conditions that induce secondary CoQ deficiency including mitochondrial DNA (mtDNA) depletion and mutations in genes involved in the fatty acid β-oxidation pathway. The diagnosis of CoQ deficiencies is determined by the decrease of its content in skeletal muscle and/or dermal skin fibroblasts. Dietary CoQ supplementation is the only available treatment for these deficiencies that require a rapid and distinct diagnosis. Here we review methods for determining CoQ content by HPLC separation and identification using alternative approaches including electrochemical detection and mass spectrometry. Also, we review procedures to determine the CoQ biosynthesis rate using labeled precursors.

Biochemical Assessment of Coenzyme Q Deficiency.

Fatty acids and glucose are the main bioenergetic substrates in mammals. Impairment of mitochondrial fatty acid oxidation causes mitochondrial myopathy leading to decreased physical performance. Here, we report that haploinsufficiency of , a member of the aarF domain-containing mitochondrial protein kinase family, in human is associated with liver dysfunction and severe mitochondrial myopathy with lipid droplets in skeletal muscle. In order to better understand the etiology of this rare disorder, we generated a heterozygous  knockout mouse model to perform in vivo and cellular studies using integrated analysis of physiological and omics data (transcriptomics-metabolomics). The data showed that +/- mice exhibited impaired fatty acid oxidation, liver dysfunction, and mitochondrial myopathy in skeletal muscle resulting in lower physical performance. Significant decrease in Coenzyme Q (CoQ) biosynthesis was observed and supplementation with CoQ partially rescued the phenotype both in the human subject and mouse model. These results indicate that ADCK2 is involved in organismal fatty acid metabolism and in CoQ biosynthesis in skeletal muscle. We propose that patients with isolated myopathies and myopathies involving lipid accumulation be tested for possible  defect as they are likely to be responsive to CoQ supplementation.

 Haploinsufficiency Reduces Mitochondrial Lipid Oxidation and Causes Myopathy Associated with CoQ Deficiency.

Coenzyme Q (CoQ ) deficiency is a rare disease characterized by a decreased accumulation of CoQ in cell membranes. Considering that CoQ synthesis and most of its functions are carried out in mitochondria, CoQ deficiency cases are usually considered a mitochondrial disease. A relevant feature of CoQ deficiency is that it is the only mitochondrial disease with a successful therapy available, the CoQ supplementation. Defects in components of the synthesis machinery caused by mutations in COQ genes generate the primary deficiency of CoQ . Mutations in genes that are not directly related to the synthesis machinery cause secondary deficiency. Cases of CoQ deficiency without genetic origin are also considered a secondary deficiency. Both types of deficiency can lead to similar clinical manifestations, but the knowledge about primary deficiency is deeper than secondary. However, secondary deficiency cases may be underestimated since many of their clinical manifestations are shared with other pathologies. This review shows the current state of secondary CoQ deficiency, which could be even more relevant than primary deficiency for clinical activity. The analysis covers the fundamental features of CoQ deficiency, which are necessary to understand the biological and clinical differences between primary and secondary CoQ deficiencies. Further, a more in-depth analysis of CoQ secondary deficiency was undertaken to consider its origins, introduce a new way of classification, and include aging as a form of secondary deficiency.

Daniel J. M. Fernandez-Ayala

Centro Andaluz de Biología del Desarrollo,
CSIC-UPO-JA,
Centro Andaluz de Biología del Desarrollo, CSIC-UPO-JA

Universidad Pablo de Olavide

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Daniel J. M. Fernandez-Ayala

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Universidad Pablo de Olavide

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Centro Andaluz de Biología del Desarrollo,
CSIC-UPO-JA,
Centro Andaluz de Biología del Desarrollo, CSIC-UPO-JA