Mitofusin 2 Deficiency Affects Energy Metabolism and Mitochondrial Biogenesis in MEF Cells.

Mitofusin 2 (Mfn2), mitochondrial outer membrane protein which is involved in rearrangement of these organelles, was first described in pathology of hypertension and diabetes, and more recently much attention is paid to its functions in Charcot-Marie-Tooth type 2A neuropathy (CMT2A). Here, cellular energy metabolism was investigated in mouse embryonic fibroblasts (MEF) differing in the presence of the Mfn2 gene; control (MEFwt) and with Mfn2 gene depleted MEFMfn2-/-. These two cell lines were compared in terms of various parameters characterizing mitochondrial bioenergetics. Here, we have shown that relative rate of proliferation of MEFMfn2-/- cells versus control fibroblasts depend on serum supplementation of the growth media. Moreover, MEFMfn2-/- cells exhibited significantly increased respiration rate in comparison to MEFwt, regardless of serum supplementation of the medium. This effect was correlated with increased level of mitochondrial markers (TOM20 and NAO) as well as mitochondrial transcription factor A (TFAM) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) protein levels and unchanged total ATP content. Interestingly, mitochondrial DNA content in MEFMfn2-/- cells was not reduced. Fundamentally, these results are in contrast to a commonly accepted belief that mitofusin 2 deficiency inevitably results in debilitation of mitochondrial energy metabolism. However, we suggest a balance between negative metabolic consequences of mitofusin 2 deficiency and adaptive processes exemplified by increased level of PGC-1α and TFAM transcription factor which prevent an excessive depletion of mtDNA and severe impairment of cell metabolism

Oxygen consumption by MEFwt and MEF^Mfn2-/- cells.

<p>The cells were grown in media supplemented with either FBS or BCS. Oxygen consumption was measured polarographically at 37°C in a substrate free buffered saline solution (PBS with Mg²⁺ and Ca²⁺), and after sequential administration of 1 mM pyruvate, 5 mM glucose, 0.1 mg/ml oligomycin and 1 μM CCCP. Data are expressed as pmoles O₂ x s^-1/mg protein and show mean values ± S.D. n = 4; *p<0.01, **p<0.001, ***p<0.0001.</p

Mitochondrial membrane potential in MEFwt and MEF^Mfn2-/- cells.

<p>The cells were grown in the medium supplemented with FBS. Mitochondrial membrane potential was determined fluorimetrically with JC-1 probe using flow cytometry. JC-1 fluorescence was measured in TO-PRO-3 negative cells (more than 95% of total population), which were assumed as 100%. Ordinate shows a proportion of cells with energized mitochondria. Data show mean values ± S.D. n = 6.</p

Relative effects of oligomycin and iodoacetate on ATP content in MEF cells incubated with or without glucose in the reaction buffer.

<p>The cells were grown in the medium supplemented with FBS. Data shown in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134162#pone.0134162.g008" target="_blank">Fig 8</a> are presented as ratios between amount of ATP in cells incubated with oligomycin (olig) or iodoacetate (IA) and untreated cells of the same type (MEFwt_, or MEF^Mfn2-/-, respectively). Amount of ATP in cells untreated with oligomycin or CCCP was assumed to be 1. Results represent mean values of the ratio ± S.D. for n = 3–5. p values were calculated using paired <i>t</i>-Student test in a relation to ATP formation rate in cells not treated with inhibitors.</p><p>Relative effects of oligomycin and iodoacetate on ATP content in MEF cells incubated with or without glucose in the reaction buffer.</p

Respiratory capacity of MEFwt and MEF^Mfn2-/- cells.

<p>The cells were grown in media supplemented with either FBS or BCS, as indicated. Respiratory capacity was expressed as a ratio between maximal (upon addition of CCCP) and minimal (in the presence of oligomycin prior to addition CCCP) rates of oxygen consumption. Results represent mean values of the ratio ± S.D. n = 4.</p><p>Respiratory capacity of MEFwt and MEF^Mfn2-/- cells.</p

Effect of mitofusin deficiency on the amount of TOM20 protein and mitochondrial mass in MEF cells.

<p>The cells were grown in the medium supplemented with FBS. (A). Relative amount of mitochondrial marker TOM20 in MEFwt and MEF^Mfn2-/- cells were tested immunochemically by Western blotting and normalized to immunoreactivity of PCNA. Representative blot as well as mean values of immunoreactivity ± S.D. for TOM20 are shown. Immunoreactivity of this protein in wild type cells is assumed as 100%, n = 4, **p<0.001. (B) Comparison of three loading markers (PCNA, β-actin and GAPDH) level in MEFwt and MEF^Mfn2-/- cells. There are no differences in the immunoreactivity between these proteins in both cell lines tested. (C) Mitochondrial mass was estimated by NAO staining of cardiolipin. Fluorescence was measured with flow cytometry. n = 6, *** p < 0.0005.</p

Effect of mitofusin deficiency on mtDNA content in MEF cell.

<p>Amount of mtDNA was determined in MEFwt and MEF^Mfn2-/- cells grown in the medium supplemented with FBS by real time PCR. Quantification was based on mtDNA to nuclear thymidylate kinase (ntDNA) gene encoding ratio. Mean values and standard deviations (indicated by error bars) were calculated from at least 6 independent experiments.</p

Effect of mitofusin deficiency on ATP-ase (complex V) activity.

<p>The cells were grown in the medium supplemented with FBS. In-gel activity of entire F1Fo complex and separated F1 subunit in MEFwt and MEF^Mfn2-/- cells were measured after blue-native polyacrylamide gel electrophoresis. One representative image out of three independent experiments is shown.</p