Optimization of permeabilized fibers preparation for mitochondrial respiration measurements using Design of Experiments methodology.

International audienceTo optimize the permeabilized fibers (pf) preparation from mouse Tibialis anterior in our lab, we used the Design of Experiments (DoE) methodology thatevaluates the impact of 6 experimental conditions or factors, on the pf respiration parameters (Pyruvate Malate Succinate respiration (PMS leak) andrespiratory control ratio (RCRPMS)), to provide a maximum of information using a limited number of experiments and animals

Characterization of mitochondrial respiratory complexes involved in the regulation of myoblast differentiation

International audienceDuring myoblast differentiation, mitochondria undergo numerous changes that are necessary for the progression of the myogenic program. Notably, we previously showed that alteration in mitochondrial activity was able to control the expression of keys regulator of cell cycle withdrawal and terminal differentiation. Here, we assessed whether inhibition of one of the respiratory complexes was a key factor in the regulation of myogenic differentiation in C2C12 cells, and was associated with alteration in reactive oxygen species (ROS) production. C2C12 cells were treated from proliferation to differentiation with specific inhibitors of mitochondrial complexes at a concentration that were inhibiting respiration but not altering cell morphology. Proliferation was significantly repressed with inhibition of complexes I, II, and III, or mitochondrial protein synthesis (using Chloramphenicol treatment), while complex IV inhibition did not alter myoblast proliferation compared to control cells. Moreover, inhibition of complexes I and II altered cell cycle regulators, with p21 protein expression upregulated since proliferation and p27 protein expression reduced at differentiation. Myotubes formation and myogenin expression were blunted with complexes I and II inhibitors while MyoD protein expression was maintained, suggesting an alteration in its transcriptional activity. Finally, a decrease in overall ROS production was observed with continuous inhibition of mitochondrial complexes I-IV. In summary, our data provide evidence that complexes I and II may be the primary regulators of C2C12 myogenic differentiation. This occurs through specific regulation of myogenic rather than cell cycle regulators expression and ROS production at mitochondrial rather than cell level

Involvement of mitochondrial NAD+-dependent deacetylase Sirt3 in the regulation of myoblast differentiation

National audienceSirt3, one of the seven mammalian sirtuins, is a mitochondrial nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, and has been shown to control multiple key metabolic pathways. Sirt3 deacetylases and activates a large number of mitochondrial enzymes implicated in the activity of respiratory chain and ATP production, TCA and Urea cycles. We have previously shown that mitochondrial activity is importantly involved in the regulation of myoblast differentiation. Since sirt3 modulates mitochondrial activity, we have investigated its influence on myoblast differentiation. In this purpose, we have first evaluated endogen sirt3 expression during C2C12 myoblast differentiation and then we examined the effect of its overexpression or inhibition on the differentiation processes and on mitochondrial activity. We have shown that sirt3 protein expression increased after the induction of myoblast differentiation. A transient sirt3 overexpression in C2C12 myoblasts induced the expression of Myogenin and p21 even in proliferative conditions (10% of Fetal Bovine Serum). On the other hand, stable inhibition of sirt3 expression, using short hairpin sirt3 (sh sirt3) in C2C12 myoblasts resulted in: 1) abrogation of terminal differentiation reflected by a sharp decrease of the fusion index and a significant decrease of Myogenin, MyoD and Troponin T protein expression; 2) a decrease in mitochondrial density and activity reflected by alterations in PGC1-alpha, VDAC and citrate synthase expression, and a decrease in respiratory chain complexes activity and myoblast maximal respiration. These data suggest that sirt3 plays an important role in the regulation of myoblast differentiation through its influence on mitochondrial activity

Role of M3 short isoform of NAD+ dependent deacetylase SIRT3 in skeletal muscle homeostasis

Role of M3 short isoform of NAD+ dependent deacetylase SIRT3 in skeletal muscle homeostasis. 11. Journée de l’École Doctorale en Sciences du Mouvement Humain « SANTE ET PERFORMANCE

SIRT3, a mitochondrial NAD(+)-dependent deacetylase, is snvolved in the regulation of myoblast differentiation

Sirtuin 3 (SIRT3), one of the seven mammalian sirtuins, is a mitochondrial NAD(+)-dependent deacetylase known to control key metabolic pathways. SIRT3 deacetylases and activates a large number of mitochondrial enzymes involved in the respiratory chain, in ATP production, and in both the citric acid and urea cycles. We have previously shown that the regulation of myoblast differentiation is tightly linked to mitochondrial activity. Since SIRT3 modulates mitochondrial activity, we decide to address its role during myoblast differentiation. For this purpose, we first investigated the expression of endogenous SIRT3 during C2C12 myoblast differentiation. We further studied the impact of SIRT3 silencing on both the myogenic potential and the mitochondrial activity of C2C12 cells. We showed that SIRT3 protein expression peaked at the onset of myoblast differentiation. The inhibition of SIRT3 expression mediated by the stable integration of SIRT3 short inhibitory RNA (SIRT3shRNA) in C2C12 myoblasts, resulted in: 1) abrogation of terminal differentiation - as evidenced by a marked decrease in the myoblast fusion index and a significant reduction of Myogenin, MyoD, Sirtuin 1 and Troponin T protein expression - restored upon MyoD overexpression; 2) a decrease in peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1 alpha) and citrate synthase protein expression reflecting an alteration of mitochondrial density; and 3) an increased production of reactive oxygen species (ROS) mirrored by the decreased activity of manganese superoxide dismutase (MnSOD). Altogether our data demonstrate that SIRT3 mainly regulates myoblast differentiation via its influence on mitochondrial activity

Role of mitochondrial and cytosolic isoforms of NAD+ dependent deacetylase SIRT3 in skeletal muscle homeostasis

Role of mitochondrial and cytosolic isoforms of NAD+ dependent deacetylase SIRT3 in skeletal muscle homeostasis. EUROMIT 2014, 9. International Meeting on Mitochondrial Patholog

Nramp1 Is Not a Major Determinant in the Control of Brucella melitensis Infection in Mice

Brucella, the causative agent of brucellosis in animals and humans, can survive and proliferate within macrophages. Macrophages mediate mouse resistance to various pathogens through the expression of the Nramp1 gene. The role of this gene in the control of Brucella infection was investigated. When BALB/c mice (Nramp1(s)) and C.CB congenic mice (Nramp1(r)) were infected with Brucella melitensis, the number of Brucella organisms per spleen was significantly larger in the C.CB mice than in the BALB/c mice during the first week postinfection (p.i.). This Nramp1-linked susceptibility to Brucella was temporary, since similar numbers of Brucella were recovered from the two strains of mice 2 weeks p.i. The effect of Nramp1 expression occurred within splenocytes intracellularly infected by Brucella. However, there was no difference between in vitro replication rates of Brucella in macrophages isolated from the two strains of mice infected in vivo or in Nramp1 RAW264 transfectants. In mice, infection with Brucella induced an inflammatory response, resulting in splenomegaly and recruitment of phagocytes in the spleen, which was amplified in C.CB mice. Reverse transcription-PCR (RT-PCR), performed 5 days p.i., showed that inducible nitric oxide synthase, tumor necrosis factor alpha (TNF-α), interleukin-12 p40 (IL-12p40), gamma interferon (IFN-γ), and IL-10 mRNAs were similarly induced in spleens of the two strains. In contrast, the mRNA of KC, a C-X-C chemokine, was induced only in infected C.CB mice at this time. This pattern of mRNA expression was maintained at 14 days p.i., with IFN-γ and IL-12p40 mRNAs being more intensively induced in the infected C.CB mice, but TNF-α mRNA was no longer induced. The higher recruitment of neutrophils observed in the spleens of infected C.CB mice could explain the temporary susceptibility of C.CB mice to B. melitensis infection. In contrast to infections with Salmonella, Leishmania, and Mycobacterium, the expression of the Nramp1 gene appears to be of limited importance for the natural resistance of mice to Brucella

Role of mitochondrial and cytosolic isoforms of NAD+ dependent deacetylase SIRT3 in skeletal muscle homeostasis

Role of mitochondrial and cytosolic isoforms of NAD+ dependent deacetylase SIRT3 in skeletal muscle homeostasis. EUROMIT 2014, 9. International Meeting on Mitochondrial Patholog

Rôle physiologique des isoformes M1 et M3 de Sirt3 dans le tissu musculaire

Rôle physiologique des isoformes M1 et M3 de Sirt3 dans le tissu musculaire. Colloque Meetochondri