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Morphofunctional and Biochemical Approaches for Studying Mitochondrial Changes during Myoblasts Differentiation

Morphofunctional and Biochemical Approaches for Studying Mitochondrial Changes during Myoblasts... SAGE-Hindawi Access to Research Journal of Aging Research Volume 2011, Article ID 845379, 16 pages doi:10.4061/2011/845379 Research Article Morphofunctional and Biochemical Approaches for Studying Mitochondrial Changes during Myoblasts Differentiation 1 2 1 1 Elena Barbieri, Michela Battistelli, Lucia Casadei, Luciana Vallorani, 1 1 1 1 Giovanni Piccoli, Michele Guescini, Anna Maria Gioacchini, Emanuela Polidori, Sabrina 1 1 3 1 2, 4 Zeppa, Paola Ceccaroli, Laura Stocchi, Vilberto Stocchi, and Elisabetta Falcieri Department of Biomolecular Sciences, University of Urbino Carlo Bo, Via I Maggetti, 26, 61029 Urbino (PU), Italy DISUAN, University of Urbino Carlo Bo, 61029 Urbino, Italy Department of Biopathology, Tor Vergata University of Rome, 00133 Rome, Italy IGM, CNR, Orthopedic Rizzoli Institute, 40136 Bologna, Italy Correspondence should be addressed to Elena Barbieri, elena.barbieri@uniurb.it Received 8 November 2010; Revised 15 February 2011; Accepted 4 March 2011 Academic Editor: Alberto Sanz Copyright © 2011 Elena Barbieri et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This study describes mitochondrial behaviour during the C2C12 myoblast differentiation program and proposes a proteomic approach to mitochondria integrated with classical morphofunctional and biochemical analyses. Mitochondrial ultrastructure variations were determined by transmission electron microscopy; mitochondrial mass and membrane potential were analysed by Mitotracker Green and JC-1 stains and by epifluorescence microscope. Expression of PGC1α, NRF1α,and Tfam genes controlling mitochondrial biogenesis was studied by real-time PCR. The mitochondrial functionality was tested by cytochrome c oxidase activity and COXII expression. Mitochondrial proteomic profile was also performed. These assays showed that mitochondrial biogenesis and activity significantly increase in differentiating myotubes. The proteomic profile identifies 32 differentially expressed proteins, mostly involved in oxidative metabolism, typical of myotubes formation. Other notable proteins, such as superoxide dismutase (MnSOD), a cell protection molecule, and voltage-dependent anion-selective channel protein (VDAC1) involved in the mitochondria-mediated apoptosis, were found to be regulated by the myogenic process. The integration of these approaches represents a helpful tool for studying mitochondrial dynamics, biogenesis, and functionality in comparative surveys on mitochondrial pathogenic or senescent satellite cells. 1. Introduction death, and cell differentiation [6–13]. In particular, mito- chondrial activity is involved in the regulation of myoblast Skeletal muscle represents an important model for studying differentiation through myogenin expression, the activity mitochondrial behaviour during cell growth and differentia- of myogenic factors, and by control of c-Myc expression tion. Myoblasts cultured in vitro, if induced by cell confluence [8, 14, 15]. Furthermore, differentiation appears to be and serum deprivation, follow a myogenic program, which a program which is dependent on both mitochondrial includes an active proliferation, withdrawal from the cell function and mitochondrial biogenesis, as indicated by the cycle, synthesis of muscle-specific proteins, and fusion into rapid increase in mitochondrial mass/volume, mtDNA copy multinucleated myotubes [1, 2]. This event is accomplished number, mitochondrial enzyme activities, and mRNA levels by the activation of specific myogenic regulatory factors within the first 48 hrs of myoblast differentiation [6, 7]. (MRFs) [3–5]. Mitochondrial DNA transcription and replication are key Recent studies suggest that mitochondria are involved events in cellular differentiation, which requires interaction in the regulation of the skeletal muscle physiology and play a critical role in cell growth, cell proliferation, cell between the nucleus and the mitochondrion [16]. 2 Journal of Aging Research Several aging theories are associated with mitochon- microscope (RM) with a digital Nikon DN100 acquisition drial damage or with a decline in mitochondrial energy system. Twenty optical fields were randomly chosen. Data production in which links between mitochondria genome were expressed as means ± S.E.M. expression and senescence symptoms are not always rec- ognized [17–19]. Our interest is particularly focused on 2.3. Mitochondrial Ultrastructure. Undifferentiated and dif- the role that mitochondria may play in the proliferative ferentiated cell monolayers were washed and fixed with 2.5% and differentiation capacity of satellite stem cells. It is well glutaraldehyde in 0.1 M phosphate buffer for 15 min, gently documented that with aging, satellite stem cells lose both scraped, and centrifuged at 1200 rpm. mitogenic and myogenesis abilities and may decrease in Cell pellets, as well as purified mitochondria, were numbers in both mice and humans [20–23]. The C2C12 further fixed by glutaraldehyde for 1 h. All specimens were cell line satellite myoblasts could offer a suitable model for OsO postfixed, alcohol dehydrated, and embedded in studying mitochondrial behaviour during the differentiation araldite, as previously described [24]. Thin sections were program. stainedwith uranylacetate andleadcitrate andanalysed In this study, we combined a morphological and bio- with a Philips CM10 electron microscope. Mitochondrial molecular approach to analyze changes in mitochondrial density was calculated in 20 different areas of 10 × 15 cm at phenotype, ultrastructure, biogenesis, and functional activity 28000 magnification. Mitochondrial sizes were evaluated at during C2C12 myoblast differentiation. Although the contri- 28000 magnification using the Philips CM10 microscope and bution of the proteomic profile of mitochondria during the Megaview software system. myogenesis program is significant, it has not been described in the literature. In this paper we aim to better define the 2.4. Mitochondrial Mass and Membrane Potential. The flu- involvement of mitochondria in the regulation of muscle orescent dye Mito Tracker Green FM (Molecular Probes), cell differentiation and discover new proteins potentially which covalently binds to mitochondrial proteins by reacting involved in the crosstalk between nuclei and mitochondria. with free thiol groups of cysteine residues regardless of membrane potential (DWm) and JC-1 (Molecular Probes), a mitochondrial membrane potential sensor, were used 2. Material and Methods to monitor mitochondrial mass and membrane potential 2.1. Cell Line. Mouse C2C12 myoblasts were grown in flasks respectively [25, 26]. The medium was removed from the in the presence of Dulbecco’s modified Eagle’s medium culture dish and replaced with prewarmed growth medium (DMEM) supplemented with 10% heat-inactivated fetal containing 100 nM Mito Tracker Green or 2 μg/mL JC-1. bovine serum (FBS), 2 mM glutamine at 37 C, and 5% After incubation for 20 min at 37 C, cells were immediately CO . To induce myogenic differentiation, when 80%–90% washed twice in cold PBS and analyzed using a Zeiss LSM 510 confluence was obtained, the medium was changed to metaconfocal microscope. The variation of JC-1 signals was DMEM supplemented with 1% FBS. Cells were analyzed at also analyzed by Zeiss LSM Image Examiner software. the undifferentiated stage and at the early-, middle-, and late-differentiation stage. In order to eliminate divergences in 2.5. Nucleic Acid Extraction and cDNA Synthesis. At each the differentiation time points analyzed, we assessed several differentiation step, plates (n = 3) were washed with PBS, differentiation markers. The cells, grown in the presence and nucleic acids were isolated. Total DNA and total RNA of 10% fetal calf serum until 80% cell confluence, were were extracted using QIAmp DNA kit (Qiagen, Chatsworth, considered undifferentiated cells, corresponding to day 0 of Calif, USA) and RNeasy Mini Kit (Qiagen, Chatsworth, Calif, the differentiation process (T0). To induce differentiation, USA), respectively, following the manufacturer’s instruc- cells at T0 were switched to differentiation medium. They tions. Nucleic acid concentrations were estimated spec- were analyzed in the early-differentiation stage, 24 h after trophotometrically (DU-640; Beckman Instruments, Milan, serum removal (T1), in mid-differentiation, 3–5 days after Italy) at 260 nm. One microgram of DNase-treated total serum removal, when myotubes containing one of two nuclei RNA was reverse transcribed using Omniscript RT (Qia- appeared (T3–5), and in the late-differentiation stage, that gen, Chatsworth, Calif, USA) and random hexamers in a is, 7–10 days after serum removal, in the presence of long final volume of 20 μL as suggested in the manufacturer’s multinucleated myotubes (T7-10). protocol. 2.2. Estimation of Myoblast Fusion. Myoblasts and myotubes 2.6. Construction of the Reference Plasmid pDGC. To con- were methanol fixed and air dried under different experi- struct the reference plasmid pDGC, a 98 bp amplicon of mental conditions. They were then stained with water 1 : 10 the mouse GAPDH, Acc. no. NM 008084, and a 100 bp May Grunwald-Giemsa solution, washed, and mounted to sequence of mouse mtDNA located within the COXII,Acc. evaluate cell fusion. Cells were considered fused if they no. NP 904331.1, were inserted into the TA cloning and contained two nuclei within one cytoplasmic continuity as HindIII restriction sites, respectively, of the polylinker region reported by Ferri et al. [5]. The fusion percentage was of pDrive (Qiagen, Chatsworth, Calif, USA). The resultant evaluated as the number of nuclei in myotubes divided by the dual-insert plasmid of 4048 bp, renamed pDGC, was purified total number of nuclei in myoblasts and myotubes magnified by using DNA plasmid purification Kit (Qiagen, Chatsworth, by 100 (×40 objective) using a TE 2000-S Nikon reverted Calif, USA) and was verified as having only one copy Journal of Aging Research 3 of each insert by restriction enzyme digestion as well as 2.9. Enzymatic Activity of Cytochrome c Oxidase. Cyto- DNA sequencing. Plasmid concentration was estimated spec- chrome c oxidase activity was determined spectrophotomet- trophotometrically (DU-640; Beckman Instruments, Milan, rically using the Cytochrome c Oxidase Assay Kit (Sigma, Italy) at 260 nm and was adjusted to give a stock solution of MO, USA). Reactions were started by the addition of fer- 1×10 molecule/μL. Further 10-fold serial dilutions down to rocytochrome c. The difference in extinction coefficients 1 mM a concentration of 1 × 10 molecule/μL were prepared. (Δε ) between ferrocytochrome c and ferricytochrome c is 21.84 at 550 nm. One unit of enzyme will oxidize 1.0 μmole of ferrocytochrome c per minute at pH 7.0 at 25 C. The 2.7. Determination of mtDNA Content and mRNA Expression proteins were determined according to the method of Lowry Levels by Quantitative Real-Time PCR. All quantitative real- et al. [29] using bovine serum albumin as the standard. time PCR reaction were carried out in a Bio-Rad iCycler iQ Multi-Color Real-Time PCR Detection System using 2x Quantitect SYBR Green PCR kit (Qiagen). The PCR 2.10. Proteomic Analysis. Mitochondria were resuspended in conditions were set up as follows: hot start at 95 Cfor urea lysis buffer (8 M urea, 4% CHAPS, 65 mM DTE, and 10 min then 40 cycles of the two steps at 95 Cfor 30sec 40 mM Tris base) and sonicated for 5 s on ice. Following and at 60 C for 30 sec. Reaction mix (25 μL final volume) centrifugation at 21000 g, protein concentration was deter- consisted of 12.5 μL Mix Hot-Start (Qiagen), total DNA mined by Bradford assay [30]. Aliquots were then stored (50 ng) or cDNA (1 μL) template, 2 μL SYBR Green, and at −80 C until use. Two dimensional electrophoresis (2- 0.3 μMofeachprimer (Table 1). Threshold cycle (Ct) was DE) was carried out as previously described [31]. Briefly, determined on the linear phase of PCRs using the iCycler isoelectric focusing was made on Immobiline strips pro- iQ Optical System software version 3 (BioRad, Milan, viding a nonlinear pH 3–10 gradient (GE Healthcare Italy, Italy). The specificity of the amplification products obtained Milan, Italy) using an IPGphore system (GE Healthcare) was confirmed by examining thermal denaturation plots, and applying an increasing voltage from 200 V to 3500 V by sample separation in a 3% DNA agarose gel and by during the first 3 h, then stabilized at 5000 V for 20 h. After sequencing. A precise determination of mitochondrial DNA IPG strip equilibration, the second dimension was carried (mtDNA) copy number was determined amplifying both out in a Laemmli system on 9%–16% polyacrylamide linear COXII and GAPDH as mtDNA and nDNA targets, respec- gradient gels (18 cm×20 cm×1.5 mm) at 40 mA/gel constant tively. Quantification of mtDNA was performed by reference current, until the dye front reached gel bottom. Forty-five μg to a single recombinant plasmid (pDGC) containing a copy (analytical runs) or 500 μg (semipreparative runs) of proteins of each target DNA sequence (mitochondrial and nuclear). were used for each electrophoretic run. COXII and GAPDH gene copy number were determined by Analytical gels were stained with silver nitrate [32], while interpolating the threshold cycle (Ct) from standard curves semipreparative gels for mass spectrometry analysis were that were obtained using serial dilution of the recombinant stained with Brilliant Blue G-Colloidal (Sigma- Aldrich, plasmid pDGC. The mtDNA/nDNA ratio was obtained, Saint Louis, USA) according to the manufacturer’s proce- relating the mitochondrial and nuclear DNA quantities. The dure. Gel images were acquired by Fluor-S MAX multi- relative expression of Tfam, PGC1-α transcription factors, imaging system (BioRad Laboratories Italy, Segrate, Italy), and COXII were quantified using 1 μLof cDNA template and the data were analysed with ImageMaster 2D Platinum and the PCR condition already described above. The amount software. To test the significant differences in the relative of each target transcript was related to that of the reference protein levels for each spot, a paired Student’s t-test statistic gene (the ribosomal protein S16) using the method described was applied at a significant level of P< .05. by Pfall [28]. In fact, previous experiments have shown The gel digestion procedure was adapted from that S16 mRNA is stable during the differentiation process Shevchenko et al. [33] as previously described [34]. [5]. All oligonucleotide primers were designed using Primer LC-ESI-MS/MS analysis was performed using a Q-TOF Express version 1.0 (Perkin-Elmer Applied Biosystem) from microTM mass spectrometer (Micromass, Manchester, UK) the GenBank database and are listed in Table 1. equipped with a Z-spray nanoflow electrospray ion source and a CapLC system. The sample was analyzed using a Symmetry C18 nano column (Waters, Milford, Mass, 2.8. Preparation of Mitochondria for Enzymatic and Proteomic USA) as an analytical column. For protein identification, Analyses. About 3 × 10 cells were harvested and washed MS/MS spectra were searched by MASCOT (Matrix sci- with 1 × PBS buffer. The pellet was resuspended in 5 mL ence,www.matrixscience.com, UK) using the database of of an ice-cold solution containing 5 mM K -Hepes, pH NCBI nr. For unmatched peptides, however, good quality 7.4, 210 mM mannitol, 1 mM EGTA, 70 mM sucrose, and MS/MS spectra were manually sequenced using de novo 55 μg/mL digitonin and homogenized by 10 strokes in an sequencing process (carried out by PepSeq of the Masslynx ice-cold glass homogenizer. Nonlysed cells and nuclei were 4.0 software, Micromass), and the obtained sequence was pelleted by centrifugation at 750 g for 20 min at 4 C, and subsequently used in Expasy TagIdent. the supernatant was centrifuged again at 8000 g for 15 min at 4 C. The resulting mitochondrial pellet was resuspended in 1 mL of 5 mM K -Hepes, pH 7.4, 210 mM mannitol, and 2.11. Statistical Analysis. Unless noted otherwise, the results 70 mM sucrose at 37 C and treated for cytochrome oxidase were expressed as mean values ± S.E.M. for the indicated activity and proteomic analysis as described below. number of measurements. Results from PCR real-time 4 Journal of Aging Research Table 1: List of primer pairs. Genes Primers (forward) Primers (reverse) References Mouse COXII 5 -CATCTGAAGACGTCCTCCACTCAT-3 5 -TCGGTTTGATGTTACTGTTGCTTGAT-3 this study Mouse TfamA 5 -GGGAGCTACCAGAAGCAGAA-3 5 -CTTTGTATGCTTTCCACTCAGC-3 this study Mouse PGC1-α 5 -CGGAAATCATATCCAACCAG-3 5 -TGAGGACCGCTAGCAAGTTTG-3 [27] Mouse S16 5 -TGAAGGGTGGTGGACATGTG-3 5 -AATAAGCTACCAGGGCCTTTGA-3 [5] Mouse GAPDH 5 -TGACGTGCCGCCTGGAGAAA-3 5 -AGTGTAGCCCAAGATGCCCTTCAG-3 [27] Table 2: Mitochondrial area and number variability during differentiation by means of ultrastructural observations of resin-embedded sections. Δ cell mitochondria Δ isolated mitochondria Number of Differentiation day area/10 × 15 cm total area/10 × 15 cm total mitochondria/10×15 cm surface surface total area T = 0 3.30E−02 ± 0.005 6.90E−02 ± 0.009 6 ± 0.89 T = 1 9.30E−02 ± 0.008 9.80E−02 ± 0.008 10 ± 1.14 T = 4 8.20E−02 ± 0.004 8.10E−02 ± 0.004 13 ± 0.91 T = 7 3.40E−02 ± 0.008 5.40E−02 ± 0.005 15 ± 0.86 analysis were compared with the ANOVA test, followed by (f). It then steadily decreases (f, i), showing minimal values a post hoc test using Tukey’s multiple comparison test. The in the late differentiation stage (l). TEM of isolated mito- threshold of significance for the ANOVA and the Tukey’s test chondria further highlights mitochondrial changes. Table 2 was fixed at P ≤ .05. represents mitochondrial number and area variability during differentiation. They undergo a progressive rounding from 0 (Figure 1,inset c) to 7day (Figure 1, inset l) after differen- 3. Results tiation induction. Analysis of mitochondria suggests a numerical increase 3.1. Cell Differentiation. The monolayer organization, as of mitochondrial cristae from the undifferentiated to differ- directly analysed at RM and by means of Giemsa stain- entiated condition (Figure 1, insets: c, f, i, and l) probably ing, deeply changes from undifferentiated myoblasts to correlated with the reported increase in enzymatic activities myotubes. In the undifferentiated condition (Figures 1(a), [6]. 1(b),and 1(c)), myoblasts appear as fusiform or star-shaped Figure 2 describes mitochondrial characteristics during cells, mostly flattened and closely adherent to the substrate. differentiation, analysed by confocal microscopy, after Mito At the initial differentiation stage (Figures 1(d), 1(e),and Tracker green (a–d) and JC-1 (e–h) staining, both specific 1(f)), intercellular spaces disappear, cells progressively align, mitochondrial dyes. The first covalently binds to mito- and, occasionally, elongate. Four days after differentiation chondrial proteins and is generally considered an available induction (Figures 1(g), 1(h),and 1(i)), early myotubes, with indicator of mitochondrial mass. The second undergoes 2 or more centrally located nuclei, appear (T = 4, fusion characteristic fluorescence changes according to the mito- index = 38 ± 3.4%). The late differentiation condition (7 chondrial membrane ΔΨ, thus revealing functional mito- days) is characterized by the presence of highly structured chondrial alterations. In myoblasts (a, b,c,and d),both myotubes (Figures 1(j), 1(k),and 1(l)). These are 100– fluorescent probes show a perinuclear mitochondrial dis- 600 μm syncytia and contain even more than 20 nuclei, tribution. Indeed, at initial differentiation stages, numerous mainly centrally located or, occasionally, aligned in parallel mitochondria can be identified as clearly distinguishable rows (T = 7, fusion index 84.6 ± 6%). single organelles. Moreover, after differentiation induc- tion, mitochondrial mass increased appearing uniform in 3.2. Morphofunctional Changes in Mitochondrial Content. myotubes (e and f). Mitochondrial membrane potential Changes in mitochondrial ultrastructure were determined also increased, highlighted by JC-1 main red staining (g), by transmission electron microscopy (TEM). Figure 1 shows still more evident in late differentiation condition shown the progression of C2C12 cell differentiation and the related in (h). Graphs of lower panel show the increasing level of mitochondrial behaviour. Their number per area signifi- red fluorescence JC-1 intensity from myoblasts (i) to late cantly increases from the undifferentiated condition (c), myotubes (j). through the initial (f) and the intermediate (i) differentiation stages, to the final phase, characterized by myotubes, which show the maximal mitochondrial content (l). Conversely, the 3.3. mtDNA Content. To ensure accurate quantification of size of single mitochondria, appears to change throughout mtDNA, we applied a PCR-based assay using a dual-insert differentiation. It increases in the undifferentiated stage (c) reference plasmid, containing both mtDNA and nuclear reaching maximal values at initial differentiation condition DNA targets [35]. In this work, pDrive plasmid was used to Journal of Aging Research 5 Table 3: Identification of mitochondrial protein differentially expressed during myogenesis. No. Protein Score NCBI nr Peptides MW PI Localization IFGVTTLDIVR, Malate dehydrogenase, 1 162 DEMSMM 35589 8.93 Mitochondrial matrix VDFPQDQLATLTGR, mitochondrial (MDH2) IQEAGTEVVK VAVLGASGGIGQPLSLLLK, Malate dehydrogenase IFGVTTLDIVRANTFVAELK, 2 345 DEMSMM 35589 8.93 Mitochondrial matrix precursor (MDH2) VDFPQDQLATLTGRIQEAGTEVVK, MIAEAIPELK TIPIDGDFFSYTR, Aldehyde dehydrogenase 2, 3 mitochondrial (Aldh2); puta- 143 Q3TVM2 MOUSE 56560 7.03 Mitochondrial matrix VAEQTPLTALYVANLIK, tive uncharacterized protein EAGFPPGVVNIVPGFGPTAGAAIASHEGVDK TIPIDGDFFSYTR, LGPALATGNVVVMK, TFVQENVYDEFVER, Aldehyde dehydrogenase 4 411 I48966 56502 7.53 Mitochondrial matrix TEQGPQVDETQFK, precursor, mitochondrial GYFIQPTVFGDVK, TIEEVVGR, YGLAAAVFTK LTFDSSFSPNTGK, Voltage-dependent anion Mitochondrial outer 5 161 VDAC1 MOUSE 32331 8.55 VTQSNFAVGYK, channel 1 (VDAC1) membrane LTLSALLDGK GYGFGLIK, WTEYGLTFTEK, Voltage-dependent anion Mitochondrial outer LTFDSSFSPNTGK, 6 344 VDAC1 MOUSE 32331 8.55 channel 1 (VDAC1) membrane VTQSNFAVGYK, VNNSSLIGLGYTQTLKPGIK, LTLSALLDGK TYYMSAGLQPVPIVFR, Pyruvate dehydrogenase DFLIPIGK, 7 (lipoamide) beta. (Pdhb 288 Q99LW9 MOUSE 34814 5.63 Mitochondrial matrix IMEGPAFNFLDAPAVR, protein) VTGADVPMPYAK, VLEDNSVPQVK 6 Journal of Aging Research Table 3: Continued. No. Protein Score NCBI nr Peptides MW PI Localization DLQNVNITLR, ILFRPVASQLPR, IYTSIGEDYDER, Mitochondrial VLPSITTEILK, 8 Prohibitin 396 A39682 29802 5.57 intermembrane space FDAGELITQR, AAIISAEGDSK, AAELIANSLATAGDGLIELR, NITYLPAGQSVLLQLPQ ATP synthase D chain, ANVAKPGLVDDFEK, Mitochondrial inner 9 131 ATP5H MOUSE 18607 5.52 mitochondrial (ATP5H) membrane YTALVDQEEKEDVK Ubiquinol-cytochrome c TDLTDYLNR, Mitochondrial inner 10 135 Q3THM1 MOUSE 52806 5.89 reductase core protein 1 membrane IQEVDAQMLR AAAEVNQEYGLDPK, Fumarate hydratase precursor, 11 170 UFRT 54429 9.06 Mitochondrial AIEMLGGELGSK, mitochondrial (FH) VAALTGLPFVTAPNK Superoxide dismutase 12 72 DSRTN GDVTTQVALQPALK 24659 8.96 Mitochondrial matrix precursor DINQEVYNFLATAGAK, SQFTITPGSEQIR, Aconitase 2, mitochondrial NTIVTSYNR, 13 340 Q3UDK9 MOUSE 85376 8.08 Mitochondrial matrix (ACO2) FNPETDFLTGK, NAVTQEFGPVPDTAR, WVVIGDENYGEGSSR Dihydrolipoamide ADGSTQVIDTK, 14 139 Q99LD3 MOUSE 54238 7.99 Mitochondrial matrix dehydrogenase (DLDH) EANLAAAFGHPINF Journal of Aging Research 7 Table 3: Continued. No. Protein Score NCBI nr Peptides MW PI Localization LVLEVAQHLGESTVR, TIAMDGTEGLVR, VLDSGAPIK, IPVGPETLGR, IMNVIGEPIDER, VVDLLAPYAK, ATP synthase, H+ IGLFGGAGVGK, transporting mitochondrial Mitochondrial inner 15 998 Q3TFD7 MOUSE 56207 5.25 TVLIMELINNVAK, F1 complex, beta subunit membrane (ATP5B) EGNDLYHEMIESGVINLK, VALVYGQMNEPPGAR, VALTGLTVAEYFR, FTQAGSEVSALLGR, AIAELGIYPAVDPLDSTSR, IMDPNIVGNEHYDVAR, ILQDYK, FLSQPFQVAEVFTGHMGK DASVVGFFR, Endoplasmic reticulum, also Protein disulfide isomerase A3 16 94 PDIA3 MOUSE 56472 5.88 present in mitochondria (see GFPTIYFSPANK, (Pdia3) discussion). ELNDFISYLQR 8 Journal of Aging Research (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l) Figure 1: Undifferentiated (a, b, c), early differentiation (d, e, f), intermediate differentiation (g, h, i) and late differentiation stages (j, k, l), are indicated by RM (a, d, g, j), Giemsa staining (b, e, h, k), and TEM (c, f, i, l). Mitochondrial morphology is further detailed by the correspondent insets, showing TEM analysis of isolated mitochondria. C2C12 cell differentiation morphological progression is evident, as well as mitochondrial behaviour in the various stages. (a, b, d, e, g, h, j, k): Bar = 20 μm; (c,f,i,l): Bar = 0.5 μm; insets, Bar = 0.1 μm. Journal of Aging Research 9 (a) (b) (c) (d) (e) (f) (g) (h) 0 0 50 50 150 150 200 200 250 250 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250 (i) (j) Figure 2: Confocal microscopy of C2C12 myoblasts (a–d) and late myotubes (e–h), after Mito Tracker (a, b, e, f) and JC-1 (c, d, g, h) staining. Graphs of lower panel show the different fluorescence JC-1 intensity in myoblasts (i) and late myotubes (j). (a–h): Bar = 20 μm. construct the reference plasmid pDGC, containing a single coactivator 1 alpha (PGC1α), and the mitochondrial tran- copy of COXII and GAPDH segments, the mitochondrial scription factor A (Tfam) were quantified using RT real-time and nuclear target genes, respectively. PCR during differentiation. PGC1α induces mitochondrial As shown in Figure 3(a), twenty-four hours after dif- biogenesis by interacting with several nuclear transcription ferentiation induction, the relative amount of mtDNA factors [36–39], and Tfam is involved in the mitochondrial undergoes a 2-fold increment at the intermediate period of genome transcription [40, 41], replication [42], and it is also differentiation (T = 3) reaching a plateau level at the final crucial for maintaining mitochondrial DNA [43]. stage of maturation (T = 7). As shown in Figure 3(b), PGC-1α expression does not change during the first 24 h from the induction of 3.4. mRNA Expression Level of Mitochondrial Biogenesis differentiation while progressively increasing up to 9.2-fold “Master” Genes. An increase in mitochondrial biogenesis in differentiated myotubes on the 7th day compared to the reflects an enhanced expression of nuclear and mitochon- myoblasts at time T0. drial genes [36–38]. Two master genes involved in the mitochondrial biogenesis, the nuclear transcriptional coac- The Tfam expression level during the myoblasts dif- tivator peroxisome proliferative activated receptor, gamma, ferentiation is slightly shifted compared to the PGC-1α 10 Journal of Aging Research PGC1α expression mtDNA content ∗∗ 2.5 14 ∗∗ ∗∗ 1.5 0.5 T = 1 T = 3 T = 5 T = 7 T = 1 T = 3 T = 5 T = 7 Differentiation days Differentiation days (a) (b) T -fam expression ∗∗ 1.5 0.5 T = 1 T = 3 T = 5 T = 7 Differentiation days (c) Figure 3: Evaluation of mitochondrial biogenesis during myoblast differentiation. In (a), determination by real-time PCR of mtDNA content expressed as mtDNA/nDNA ratio (COXII/GAPDH), as described in Section 2. In (b), quantitative analyses of PGC-1α and T-fam by real- time PCR. The amount of each target transcript was related to that of the reference gene (the ribosomal protein S16). Data are expressed as the mean ± SEM of three experiments; all samples were analyzed in triplicate. Results from PCR real-time analysis were compared with the ANOVA test, followed by a post hoc test using Tukey’s multiple comparison test. The threshold of significance for the ANOVA and the ∗ ∗∗ Tukey’s test was fixed at P ≤ .05; P ≤ .01. expression; in fact, it increased significantly between days 3–7 differentiation, we performed a 2D page on mitochondria (Figure 3(c)). isolated from C2C12 myoblasts over a 7-day time span differentiation. A total of 994 spots (mean) could be resolved on a silver-stained large 2DE gel, where we loaded 45 μg 3.5. Cytochrome c Oxidase Activity and COXII Expression of total protein. A larger amount of protein per spot was Level. The mitochondrial enzymatic activities of cytochrome oxidase reflecting the respiratory chain activities were signif- necessary for protein identification, thus we used preparative icantly higher in myoblasts able to differentiate (Figure 4(a)). gels stained with Brilliant Blue G-Colloidal. To evaluate the possible presence of cellular contaminants, we compared the In addition, we evaluated the expression level of the cor- responding gene coding for the subunit II of mitochondrial mitochondrial map with that of the whole cellular lysate in which we had previously identified several cytosolic and cytochrome c oxidase (COXII), which represents a target membrane proteins [46]. The comparison of 2D maps of gene for mitochondrial transcriptional activity [27, 44, 45]. On days 3–7, the mitochondrial COXII transcript levels mitochondria and whole cell lysate allowed us to state that the preparation of mitochondria contained little or no were significantly higher than in proliferating myoblasts (Figure 4(b)). cellular contaminants. The study of quantitative changes of individual pro- teins in a purified mitochondrial fraction showed that 32 3.6. Changes in Mitochondrial Proteomic Profile. To high- light significant changes in mitochondrial proteome during mitochondrial proteins increased significantly in abundance mtDNA/nDNA (a.u.) Relative mRNA level (a.u.) Relative mRNA level (a.u.) Journal of Aging Research 11 COX enzimatic activity COXII expression 1.8 2.5 ∗∗ 1.6 1.4 1.2 1.5 0.8 0.6 0.4 0.5 0.2 0 0 T = 1 T = 3 T = 5 T = 7 T = 1 T = 3 T = 5 T = 7 Differentiation days Differentiation days (a) (b) Figure 4: Time course change of cytochrome oxidase (COX) enzymatic activity and transcription level of cytochrome oxidase subunit II (COXII) gene at progressive differentiation stages. (a) Quantitative analysis enzymatic activity. (b) The expression level of COXII is related to S16 mRNA gene level. Results from PCR real-time analysis were compared with the ANOVA test, followed by a post hoc test using Tukey’s ∗ ∗∗ multiple comparison test. The threshold of significance for the ANOVA and the Tukey’s test was fixed at P ≤ .05; P ≤ .01. 17 14 27 15 3 1 2 30 31 5 6 (a) (b) Figure 5: Image of a silver-stained 2-DE gel of 45 μg purified mitochondrial proteins from C2C12 myoblasts at 0 (a) and 7 (b) days of differentiation time. Differentially expressed spots are indicated by arrows and numbered according to Table 3. (Figure 5). The proteins showing the greatest expression This was also interesting for the superoxide dismutase changes were also characterized by electrospray ionisation (MnSOD), a voltage-dependent anion-selective channel pro- (ESI) tandem mass spectrometry. In particular, the major tein 1 (VDAC1), and the protein disulfide-isomerase A3 changes occurred between T1and T4timeofdifferentiation, (Pdia3) that were differentially expressed during differenti- while fewer differences were shown between T0-T1and T4– ation. T7(Table 3). The main mitochondrial proteins which could be 4. Discussion detected in fully differentiated syncytia were involved in the citric acid cycle (malate dehydrogenase: MDH2, fumarate In this study, we described temporal mitochondrial changes hydratase: FH, and aconitase 2: ACO2) or belong to the during the myogenic program of C2C12 myoblasts by pyruvate dehydrogenase complex (pyruvate dehydrogenase, analyzing complementary key parameters for mitochon- lipoamide beta: PDHB, dihydrolipoamide dehydrogenase), drial dynamics, biogenesis, and functionality. Of particular complex III (ubiquinol-cytochrome c reductase core protein interest is the contribution of the proteomic approach to 1: UQCRC1) and complex V (ATP synthase, H+ transporting better define the pattern of mitochondrial protein expression mitochondrial F1 complex, beta subunit: ATP5B, and ATP accompanying differentiation in myotubes and potentially synthase d chain: ATP5H) of the respiratory chain. involved in the crosstalk between nuclei and mitochondria. Protein (U/mg) Relative mRNA level (a.u.) 12 Journal of Aging Research Morphological analysis performed by fluorescence mitochondrial biogenesis. This shift could be explained by microscopy with markers of mitochondrial mass/volume and the biological cycle of mitochondria [56]. Mitochondrial ΔΨ, as well as ultrastructural analysis, allowed us to acquire fission is preceded by an extension of the organelles and the more information regarding the mitochondrial organization mtDNA replication phase. Although there is a slight shifting, and dynamics in C2C12 myoblast differentiation. the correlation between the number of copies of mtDNA and Mitochondrial organization in myoblasts was perinu- mitochondrial biogenesis is positive (r = 0.85, data not clear, and it was possible to discriminate individual mito- shown). chondrion by both MitoTracker Green and JC1 staining. In several studies, the measure of mtDNA copy number This type of mitochondrial distribution is described in the has been considered proportional to the number of mito- literature for other cell types including fibroblasts [47], chondria, a golden star for mitochondrial density [57–60]. pancreatic acinar cells [48, 49], astrocytes, and neurons [50]. However, changes in mitochondrial abundance regardless of On the contrary, in myotubes, morphological observa- the mtDNA copy number may occur, especially in peculiar tion by epifluorescence did not allow us to discriminate conditions such as during alterations in the rates of intracel- individual mitochondrion, showing homogeneous staining, lular ROS generation [61]. Franko et al., investigating C2F3 representative of the mitochondrial network, well described mouse myoblasts, showed that an increment in mtDNA does in skeletal muscle tissue [51, 52]. TEM analysis showed a not always correlate with the proliferation of mitochondria mitochondrial remodeling during differentiation and align- or with their activity [62]. In this investigation, the mtDNA ment of organelles along the myotubes. copy number of C2C12 myoblasts significantly increased At this level, we cannot show the formation of a network during the early-intermediate differentiation phases (T = equal to that which is found in skeletal muscle fibers, 1and T3) up to 2-fold remaining constant during the where mitochondria are arranged in crystal structures closely myotube maturation. Likewise, over the course of myoblast related to the sarcoplasm [51, 52]. Indeed, the sarcomeres of differentiation in rat cell line L6, a small but significant myotubes are only sketched [53], but they may support the increase in mitochondrial DNA copy number was observed development of a mitochondrial network during myotube by [27]. Furthermore, in a recent study on the regulation of maturation. mitochondrial biogenesis during myogenesis, mtDNA copy The mitochondrial counting per area of cell surface, number was determined as a marker for mitochondrial obtained by TEM, showed that the number of mitochondria density using QPCR, and the mtDNA copy number was 4- increased from undifferentiated to differentiated conditions. fold higher in fully differentiated myotubes than it was in Mature myotubes contained approximately 2-fold more myoblasts [60]. mitochondria than myoblasts. However, in the first 24 Interestingly, during differentiation, an increased hours after induction of differentiation, the mitochondria mtDNA transcriptional activity and oxidative metabolism increased in size up to 3-fold gradually decreasing in size only correspond to an enhanced mitochondrial biogenesis, as after the intermediate phases to reach the same size observed highlighted by the upregulation of COXII mRNA levels and in myoblasts at T = 0, in mature myotubes (T = 7). This 2 2 cytochrome c oxidase activity (r = 0.83 and r = 0.97, observation suggests that mitochondria first undergo fusion resp., data not shown). and then fission, which allows their distribution during In our investigation, we integrated the mitochondrial syncytia formation as previously reported [54]. Nevertheless, changes observed by multiple key determinants with pro- the mitochondria in myotubes showed a greater extension teomic analysis. of mitochondrial cristae than mitochondria in myoblasts. In the literature, mitochondrial proteomic maps of Marked stimulation of the biosynthesis of the phospho- differentiating myoblasts are not available; hence, this work lipid cardiolipin during the differentiation phases has been presents the first proteomic profile of mitochondria during observed in previous studies on L6E9 myoblasts and other the myogenesis program. Previously, proteome-based inves- cells [54, 55]. It is probably necessary to supply the proper tigations have been carried out to provide a description amount of functional mitochondrial inner membrane for the of the myogenic differentiation program [46, 63, 64]. We respiratory chain proteins involved in oxidative metabolism employed a proteomic approach using two dimensional [7]. electrophoresis, particularly helpful for investigating the All the parameters observed through morphological subset of cellular proteins, such as organellar proteins, due analysis confirm a linear increase in mitochondrial bio- to the reduced complexity of the protein sample [65]. genesis during differentiation. The morphological analysis corroborates the progression of the myogenic process and In particular, analyzing the differentially expressed pro- teins in the mitochondrial proteome map during the myo- the increase in biochemical markers such as the transcription factors of mitochondrial biogenesis PGC-1 α and Tfam. genic process, we observed that also the enzymes involved in cellular respiration, such as pyruvate dehydrogenase, MDH2, Of particular interest was the timing of mtDNA repli- cation compared to mitochondrial biogenesis. Although FH, ACO2, and more markedly HB and 5B ATP syn- mitochondrial biogenesis increased linearly during differ- thase subunits, representative of oxidative phosphorylation, increase linearly with the mitochondrial biogenesis showing entiation, mtDNA content increased significantly from the early days of differentiation already reaching a plateau at a positive correlation (r = 0.915, data not shown). These the intermediate stage. Hence, our investigation shows a findings are consistent with the differentiating cells’ greater slight difference in timing between DNA replication and reliance on aerobic metabolism compared to the glycolytic Journal of Aging Research 13 metabolism that characterizes the undifferentiated myoblasts scenario of mitochondrial dynamics, biogenesis and func- [7]. tionality useful in comparative surveys of mitochondrial Moreover, our observations are in agreement with Moyes pathogenic or senescent satellite cells. and coworkers who demonstrated an mRNA increment for pyruvate dehydrogenase, citrate synthase, isocitrate dehydro- Acknowledgments genase, cytochrome c oxidase, and NADH dehydrogenase [6]. The increment of Krebs cycle and respiratory chain The authors thank Dr. Rosa Curci (Orthopedic Rizzoli proteins supports the augmented mitochondrial function- Institute, Bologna, Italy) for providing confocal microscope ality also confirmed by the COX enzymatic activity during images. They wish to thank Professor Timothy Bloom, myoblast differentiation [6, 7]. Centro Linguistico di Ateneo of the University of Urbino, for The data obtained using the proteomic approach are a critical reading of the paper. consistent with the increase in mitochondrial function and membrane depolarization highlighted by JC-1 and in agree- ment with the increase of mitochondrial cristae observed References by TEM. These data support the evidence described by [1] M. E. Pownall, M. K. Gustafsson, and C. P. Emerson Jr., Sauvanet (2010) assuming that mitochondrial bioenergetics “Myogenic regulatory factors and the specification of muscle and dynamics are linked and that mitochondrial morphology progenitors in vertebrate embryos,” Annual Review of Cell and reflects their functional status [66]. Developmental Biology, vol. 18, pp. 747–783, 2002. Proteomic analysis revealed other notable proteins [2] L. A. Sabourin and M. A. 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Morphofunctional and Biochemical Approaches for Studying Mitochondrial Changes during Myoblasts Differentiation

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Hindawi Publishing Corporation
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Copyright © 2011 Elena Barbieri et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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2090-2204
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10.4061/2011/845379
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SAGE-Hindawi Access to Research Journal of Aging Research Volume 2011, Article ID 845379, 16 pages doi:10.4061/2011/845379 Research Article Morphofunctional and Biochemical Approaches for Studying Mitochondrial Changes during Myoblasts Differentiation 1 2 1 1 Elena Barbieri, Michela Battistelli, Lucia Casadei, Luciana Vallorani, 1 1 1 1 Giovanni Piccoli, Michele Guescini, Anna Maria Gioacchini, Emanuela Polidori, Sabrina 1 1 3 1 2, 4 Zeppa, Paola Ceccaroli, Laura Stocchi, Vilberto Stocchi, and Elisabetta Falcieri Department of Biomolecular Sciences, University of Urbino Carlo Bo, Via I Maggetti, 26, 61029 Urbino (PU), Italy DISUAN, University of Urbino Carlo Bo, 61029 Urbino, Italy Department of Biopathology, Tor Vergata University of Rome, 00133 Rome, Italy IGM, CNR, Orthopedic Rizzoli Institute, 40136 Bologna, Italy Correspondence should be addressed to Elena Barbieri, elena.barbieri@uniurb.it Received 8 November 2010; Revised 15 February 2011; Accepted 4 March 2011 Academic Editor: Alberto Sanz Copyright © 2011 Elena Barbieri et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This study describes mitochondrial behaviour during the C2C12 myoblast differentiation program and proposes a proteomic approach to mitochondria integrated with classical morphofunctional and biochemical analyses. Mitochondrial ultrastructure variations were determined by transmission electron microscopy; mitochondrial mass and membrane potential were analysed by Mitotracker Green and JC-1 stains and by epifluorescence microscope. Expression of PGC1α, NRF1α,and Tfam genes controlling mitochondrial biogenesis was studied by real-time PCR. The mitochondrial functionality was tested by cytochrome c oxidase activity and COXII expression. Mitochondrial proteomic profile was also performed. These assays showed that mitochondrial biogenesis and activity significantly increase in differentiating myotubes. The proteomic profile identifies 32 differentially expressed proteins, mostly involved in oxidative metabolism, typical of myotubes formation. Other notable proteins, such as superoxide dismutase (MnSOD), a cell protection molecule, and voltage-dependent anion-selective channel protein (VDAC1) involved in the mitochondria-mediated apoptosis, were found to be regulated by the myogenic process. The integration of these approaches represents a helpful tool for studying mitochondrial dynamics, biogenesis, and functionality in comparative surveys on mitochondrial pathogenic or senescent satellite cells. 1. Introduction death, and cell differentiation [6–13]. In particular, mito- chondrial activity is involved in the regulation of myoblast Skeletal muscle represents an important model for studying differentiation through myogenin expression, the activity mitochondrial behaviour during cell growth and differentia- of myogenic factors, and by control of c-Myc expression tion. Myoblasts cultured in vitro, if induced by cell confluence [8, 14, 15]. Furthermore, differentiation appears to be and serum deprivation, follow a myogenic program, which a program which is dependent on both mitochondrial includes an active proliferation, withdrawal from the cell function and mitochondrial biogenesis, as indicated by the cycle, synthesis of muscle-specific proteins, and fusion into rapid increase in mitochondrial mass/volume, mtDNA copy multinucleated myotubes [1, 2]. This event is accomplished number, mitochondrial enzyme activities, and mRNA levels by the activation of specific myogenic regulatory factors within the first 48 hrs of myoblast differentiation [6, 7]. (MRFs) [3–5]. Mitochondrial DNA transcription and replication are key Recent studies suggest that mitochondria are involved events in cellular differentiation, which requires interaction in the regulation of the skeletal muscle physiology and play a critical role in cell growth, cell proliferation, cell between the nucleus and the mitochondrion [16]. 2 Journal of Aging Research Several aging theories are associated with mitochon- microscope (RM) with a digital Nikon DN100 acquisition drial damage or with a decline in mitochondrial energy system. Twenty optical fields were randomly chosen. Data production in which links between mitochondria genome were expressed as means ± S.E.M. expression and senescence symptoms are not always rec- ognized [17–19]. Our interest is particularly focused on 2.3. Mitochondrial Ultrastructure. Undifferentiated and dif- the role that mitochondria may play in the proliferative ferentiated cell monolayers were washed and fixed with 2.5% and differentiation capacity of satellite stem cells. It is well glutaraldehyde in 0.1 M phosphate buffer for 15 min, gently documented that with aging, satellite stem cells lose both scraped, and centrifuged at 1200 rpm. mitogenic and myogenesis abilities and may decrease in Cell pellets, as well as purified mitochondria, were numbers in both mice and humans [20–23]. The C2C12 further fixed by glutaraldehyde for 1 h. All specimens were cell line satellite myoblasts could offer a suitable model for OsO postfixed, alcohol dehydrated, and embedded in studying mitochondrial behaviour during the differentiation araldite, as previously described [24]. Thin sections were program. stainedwith uranylacetate andleadcitrate andanalysed In this study, we combined a morphological and bio- with a Philips CM10 electron microscope. Mitochondrial molecular approach to analyze changes in mitochondrial density was calculated in 20 different areas of 10 × 15 cm at phenotype, ultrastructure, biogenesis, and functional activity 28000 magnification. Mitochondrial sizes were evaluated at during C2C12 myoblast differentiation. Although the contri- 28000 magnification using the Philips CM10 microscope and bution of the proteomic profile of mitochondria during the Megaview software system. myogenesis program is significant, it has not been described in the literature. In this paper we aim to better define the 2.4. Mitochondrial Mass and Membrane Potential. The flu- involvement of mitochondria in the regulation of muscle orescent dye Mito Tracker Green FM (Molecular Probes), cell differentiation and discover new proteins potentially which covalently binds to mitochondrial proteins by reacting involved in the crosstalk between nuclei and mitochondria. with free thiol groups of cysteine residues regardless of membrane potential (DWm) and JC-1 (Molecular Probes), a mitochondrial membrane potential sensor, were used 2. Material and Methods to monitor mitochondrial mass and membrane potential 2.1. Cell Line. Mouse C2C12 myoblasts were grown in flasks respectively [25, 26]. The medium was removed from the in the presence of Dulbecco’s modified Eagle’s medium culture dish and replaced with prewarmed growth medium (DMEM) supplemented with 10% heat-inactivated fetal containing 100 nM Mito Tracker Green or 2 μg/mL JC-1. bovine serum (FBS), 2 mM glutamine at 37 C, and 5% After incubation for 20 min at 37 C, cells were immediately CO . To induce myogenic differentiation, when 80%–90% washed twice in cold PBS and analyzed using a Zeiss LSM 510 confluence was obtained, the medium was changed to metaconfocal microscope. The variation of JC-1 signals was DMEM supplemented with 1% FBS. Cells were analyzed at also analyzed by Zeiss LSM Image Examiner software. the undifferentiated stage and at the early-, middle-, and late-differentiation stage. In order to eliminate divergences in 2.5. Nucleic Acid Extraction and cDNA Synthesis. At each the differentiation time points analyzed, we assessed several differentiation step, plates (n = 3) were washed with PBS, differentiation markers. The cells, grown in the presence and nucleic acids were isolated. Total DNA and total RNA of 10% fetal calf serum until 80% cell confluence, were were extracted using QIAmp DNA kit (Qiagen, Chatsworth, considered undifferentiated cells, corresponding to day 0 of Calif, USA) and RNeasy Mini Kit (Qiagen, Chatsworth, Calif, the differentiation process (T0). To induce differentiation, USA), respectively, following the manufacturer’s instruc- cells at T0 were switched to differentiation medium. They tions. Nucleic acid concentrations were estimated spec- were analyzed in the early-differentiation stage, 24 h after trophotometrically (DU-640; Beckman Instruments, Milan, serum removal (T1), in mid-differentiation, 3–5 days after Italy) at 260 nm. One microgram of DNase-treated total serum removal, when myotubes containing one of two nuclei RNA was reverse transcribed using Omniscript RT (Qia- appeared (T3–5), and in the late-differentiation stage, that gen, Chatsworth, Calif, USA) and random hexamers in a is, 7–10 days after serum removal, in the presence of long final volume of 20 μL as suggested in the manufacturer’s multinucleated myotubes (T7-10). protocol. 2.2. Estimation of Myoblast Fusion. Myoblasts and myotubes 2.6. Construction of the Reference Plasmid pDGC. To con- were methanol fixed and air dried under different experi- struct the reference plasmid pDGC, a 98 bp amplicon of mental conditions. They were then stained with water 1 : 10 the mouse GAPDH, Acc. no. NM 008084, and a 100 bp May Grunwald-Giemsa solution, washed, and mounted to sequence of mouse mtDNA located within the COXII,Acc. evaluate cell fusion. Cells were considered fused if they no. NP 904331.1, were inserted into the TA cloning and contained two nuclei within one cytoplasmic continuity as HindIII restriction sites, respectively, of the polylinker region reported by Ferri et al. [5]. The fusion percentage was of pDrive (Qiagen, Chatsworth, Calif, USA). The resultant evaluated as the number of nuclei in myotubes divided by the dual-insert plasmid of 4048 bp, renamed pDGC, was purified total number of nuclei in myoblasts and myotubes magnified by using DNA plasmid purification Kit (Qiagen, Chatsworth, by 100 (×40 objective) using a TE 2000-S Nikon reverted Calif, USA) and was verified as having only one copy Journal of Aging Research 3 of each insert by restriction enzyme digestion as well as 2.9. Enzymatic Activity of Cytochrome c Oxidase. Cyto- DNA sequencing. Plasmid concentration was estimated spec- chrome c oxidase activity was determined spectrophotomet- trophotometrically (DU-640; Beckman Instruments, Milan, rically using the Cytochrome c Oxidase Assay Kit (Sigma, Italy) at 260 nm and was adjusted to give a stock solution of MO, USA). Reactions were started by the addition of fer- 1×10 molecule/μL. Further 10-fold serial dilutions down to rocytochrome c. The difference in extinction coefficients 1 mM a concentration of 1 × 10 molecule/μL were prepared. (Δε ) between ferrocytochrome c and ferricytochrome c is 21.84 at 550 nm. One unit of enzyme will oxidize 1.0 μmole of ferrocytochrome c per minute at pH 7.0 at 25 C. The 2.7. Determination of mtDNA Content and mRNA Expression proteins were determined according to the method of Lowry Levels by Quantitative Real-Time PCR. All quantitative real- et al. [29] using bovine serum albumin as the standard. time PCR reaction were carried out in a Bio-Rad iCycler iQ Multi-Color Real-Time PCR Detection System using 2x Quantitect SYBR Green PCR kit (Qiagen). The PCR 2.10. Proteomic Analysis. Mitochondria were resuspended in conditions were set up as follows: hot start at 95 Cfor urea lysis buffer (8 M urea, 4% CHAPS, 65 mM DTE, and 10 min then 40 cycles of the two steps at 95 Cfor 30sec 40 mM Tris base) and sonicated for 5 s on ice. Following and at 60 C for 30 sec. Reaction mix (25 μL final volume) centrifugation at 21000 g, protein concentration was deter- consisted of 12.5 μL Mix Hot-Start (Qiagen), total DNA mined by Bradford assay [30]. Aliquots were then stored (50 ng) or cDNA (1 μL) template, 2 μL SYBR Green, and at −80 C until use. Two dimensional electrophoresis (2- 0.3 μMofeachprimer (Table 1). Threshold cycle (Ct) was DE) was carried out as previously described [31]. Briefly, determined on the linear phase of PCRs using the iCycler isoelectric focusing was made on Immobiline strips pro- iQ Optical System software version 3 (BioRad, Milan, viding a nonlinear pH 3–10 gradient (GE Healthcare Italy, Italy). The specificity of the amplification products obtained Milan, Italy) using an IPGphore system (GE Healthcare) was confirmed by examining thermal denaturation plots, and applying an increasing voltage from 200 V to 3500 V by sample separation in a 3% DNA agarose gel and by during the first 3 h, then stabilized at 5000 V for 20 h. After sequencing. A precise determination of mitochondrial DNA IPG strip equilibration, the second dimension was carried (mtDNA) copy number was determined amplifying both out in a Laemmli system on 9%–16% polyacrylamide linear COXII and GAPDH as mtDNA and nDNA targets, respec- gradient gels (18 cm×20 cm×1.5 mm) at 40 mA/gel constant tively. Quantification of mtDNA was performed by reference current, until the dye front reached gel bottom. Forty-five μg to a single recombinant plasmid (pDGC) containing a copy (analytical runs) or 500 μg (semipreparative runs) of proteins of each target DNA sequence (mitochondrial and nuclear). were used for each electrophoretic run. COXII and GAPDH gene copy number were determined by Analytical gels were stained with silver nitrate [32], while interpolating the threshold cycle (Ct) from standard curves semipreparative gels for mass spectrometry analysis were that were obtained using serial dilution of the recombinant stained with Brilliant Blue G-Colloidal (Sigma- Aldrich, plasmid pDGC. The mtDNA/nDNA ratio was obtained, Saint Louis, USA) according to the manufacturer’s proce- relating the mitochondrial and nuclear DNA quantities. The dure. Gel images were acquired by Fluor-S MAX multi- relative expression of Tfam, PGC1-α transcription factors, imaging system (BioRad Laboratories Italy, Segrate, Italy), and COXII were quantified using 1 μLof cDNA template and the data were analysed with ImageMaster 2D Platinum and the PCR condition already described above. The amount software. To test the significant differences in the relative of each target transcript was related to that of the reference protein levels for each spot, a paired Student’s t-test statistic gene (the ribosomal protein S16) using the method described was applied at a significant level of P< .05. by Pfall [28]. In fact, previous experiments have shown The gel digestion procedure was adapted from that S16 mRNA is stable during the differentiation process Shevchenko et al. [33] as previously described [34]. [5]. All oligonucleotide primers were designed using Primer LC-ESI-MS/MS analysis was performed using a Q-TOF Express version 1.0 (Perkin-Elmer Applied Biosystem) from microTM mass spectrometer (Micromass, Manchester, UK) the GenBank database and are listed in Table 1. equipped with a Z-spray nanoflow electrospray ion source and a CapLC system. The sample was analyzed using a Symmetry C18 nano column (Waters, Milford, Mass, 2.8. Preparation of Mitochondria for Enzymatic and Proteomic USA) as an analytical column. For protein identification, Analyses. About 3 × 10 cells were harvested and washed MS/MS spectra were searched by MASCOT (Matrix sci- with 1 × PBS buffer. The pellet was resuspended in 5 mL ence,www.matrixscience.com, UK) using the database of of an ice-cold solution containing 5 mM K -Hepes, pH NCBI nr. For unmatched peptides, however, good quality 7.4, 210 mM mannitol, 1 mM EGTA, 70 mM sucrose, and MS/MS spectra were manually sequenced using de novo 55 μg/mL digitonin and homogenized by 10 strokes in an sequencing process (carried out by PepSeq of the Masslynx ice-cold glass homogenizer. Nonlysed cells and nuclei were 4.0 software, Micromass), and the obtained sequence was pelleted by centrifugation at 750 g for 20 min at 4 C, and subsequently used in Expasy TagIdent. the supernatant was centrifuged again at 8000 g for 15 min at 4 C. The resulting mitochondrial pellet was resuspended in 1 mL of 5 mM K -Hepes, pH 7.4, 210 mM mannitol, and 2.11. Statistical Analysis. Unless noted otherwise, the results 70 mM sucrose at 37 C and treated for cytochrome oxidase were expressed as mean values ± S.E.M. for the indicated activity and proteomic analysis as described below. number of measurements. Results from PCR real-time 4 Journal of Aging Research Table 1: List of primer pairs. Genes Primers (forward) Primers (reverse) References Mouse COXII 5 -CATCTGAAGACGTCCTCCACTCAT-3 5 -TCGGTTTGATGTTACTGTTGCTTGAT-3 this study Mouse TfamA 5 -GGGAGCTACCAGAAGCAGAA-3 5 -CTTTGTATGCTTTCCACTCAGC-3 this study Mouse PGC1-α 5 -CGGAAATCATATCCAACCAG-3 5 -TGAGGACCGCTAGCAAGTTTG-3 [27] Mouse S16 5 -TGAAGGGTGGTGGACATGTG-3 5 -AATAAGCTACCAGGGCCTTTGA-3 [5] Mouse GAPDH 5 -TGACGTGCCGCCTGGAGAAA-3 5 -AGTGTAGCCCAAGATGCCCTTCAG-3 [27] Table 2: Mitochondrial area and number variability during differentiation by means of ultrastructural observations of resin-embedded sections. Δ cell mitochondria Δ isolated mitochondria Number of Differentiation day area/10 × 15 cm total area/10 × 15 cm total mitochondria/10×15 cm surface surface total area T = 0 3.30E−02 ± 0.005 6.90E−02 ± 0.009 6 ± 0.89 T = 1 9.30E−02 ± 0.008 9.80E−02 ± 0.008 10 ± 1.14 T = 4 8.20E−02 ± 0.004 8.10E−02 ± 0.004 13 ± 0.91 T = 7 3.40E−02 ± 0.008 5.40E−02 ± 0.005 15 ± 0.86 analysis were compared with the ANOVA test, followed by (f). It then steadily decreases (f, i), showing minimal values a post hoc test using Tukey’s multiple comparison test. The in the late differentiation stage (l). TEM of isolated mito- threshold of significance for the ANOVA and the Tukey’s test chondria further highlights mitochondrial changes. Table 2 was fixed at P ≤ .05. represents mitochondrial number and area variability during differentiation. They undergo a progressive rounding from 0 (Figure 1,inset c) to 7day (Figure 1, inset l) after differen- 3. Results tiation induction. Analysis of mitochondria suggests a numerical increase 3.1. Cell Differentiation. The monolayer organization, as of mitochondrial cristae from the undifferentiated to differ- directly analysed at RM and by means of Giemsa stain- entiated condition (Figure 1, insets: c, f, i, and l) probably ing, deeply changes from undifferentiated myoblasts to correlated with the reported increase in enzymatic activities myotubes. In the undifferentiated condition (Figures 1(a), [6]. 1(b),and 1(c)), myoblasts appear as fusiform or star-shaped Figure 2 describes mitochondrial characteristics during cells, mostly flattened and closely adherent to the substrate. differentiation, analysed by confocal microscopy, after Mito At the initial differentiation stage (Figures 1(d), 1(e),and Tracker green (a–d) and JC-1 (e–h) staining, both specific 1(f)), intercellular spaces disappear, cells progressively align, mitochondrial dyes. The first covalently binds to mito- and, occasionally, elongate. Four days after differentiation chondrial proteins and is generally considered an available induction (Figures 1(g), 1(h),and 1(i)), early myotubes, with indicator of mitochondrial mass. The second undergoes 2 or more centrally located nuclei, appear (T = 4, fusion characteristic fluorescence changes according to the mito- index = 38 ± 3.4%). The late differentiation condition (7 chondrial membrane ΔΨ, thus revealing functional mito- days) is characterized by the presence of highly structured chondrial alterations. In myoblasts (a, b,c,and d),both myotubes (Figures 1(j), 1(k),and 1(l)). These are 100– fluorescent probes show a perinuclear mitochondrial dis- 600 μm syncytia and contain even more than 20 nuclei, tribution. Indeed, at initial differentiation stages, numerous mainly centrally located or, occasionally, aligned in parallel mitochondria can be identified as clearly distinguishable rows (T = 7, fusion index 84.6 ± 6%). single organelles. Moreover, after differentiation induc- tion, mitochondrial mass increased appearing uniform in 3.2. Morphofunctional Changes in Mitochondrial Content. myotubes (e and f). Mitochondrial membrane potential Changes in mitochondrial ultrastructure were determined also increased, highlighted by JC-1 main red staining (g), by transmission electron microscopy (TEM). Figure 1 shows still more evident in late differentiation condition shown the progression of C2C12 cell differentiation and the related in (h). Graphs of lower panel show the increasing level of mitochondrial behaviour. Their number per area signifi- red fluorescence JC-1 intensity from myoblasts (i) to late cantly increases from the undifferentiated condition (c), myotubes (j). through the initial (f) and the intermediate (i) differentiation stages, to the final phase, characterized by myotubes, which show the maximal mitochondrial content (l). Conversely, the 3.3. mtDNA Content. To ensure accurate quantification of size of single mitochondria, appears to change throughout mtDNA, we applied a PCR-based assay using a dual-insert differentiation. It increases in the undifferentiated stage (c) reference plasmid, containing both mtDNA and nuclear reaching maximal values at initial differentiation condition DNA targets [35]. In this work, pDrive plasmid was used to Journal of Aging Research 5 Table 3: Identification of mitochondrial protein differentially expressed during myogenesis. No. Protein Score NCBI nr Peptides MW PI Localization IFGVTTLDIVR, Malate dehydrogenase, 1 162 DEMSMM 35589 8.93 Mitochondrial matrix VDFPQDQLATLTGR, mitochondrial (MDH2) IQEAGTEVVK VAVLGASGGIGQPLSLLLK, Malate dehydrogenase IFGVTTLDIVRANTFVAELK, 2 345 DEMSMM 35589 8.93 Mitochondrial matrix precursor (MDH2) VDFPQDQLATLTGRIQEAGTEVVK, MIAEAIPELK TIPIDGDFFSYTR, Aldehyde dehydrogenase 2, 3 mitochondrial (Aldh2); puta- 143 Q3TVM2 MOUSE 56560 7.03 Mitochondrial matrix VAEQTPLTALYVANLIK, tive uncharacterized protein EAGFPPGVVNIVPGFGPTAGAAIASHEGVDK TIPIDGDFFSYTR, LGPALATGNVVVMK, TFVQENVYDEFVER, Aldehyde dehydrogenase 4 411 I48966 56502 7.53 Mitochondrial matrix TEQGPQVDETQFK, precursor, mitochondrial GYFIQPTVFGDVK, TIEEVVGR, YGLAAAVFTK LTFDSSFSPNTGK, Voltage-dependent anion Mitochondrial outer 5 161 VDAC1 MOUSE 32331 8.55 VTQSNFAVGYK, channel 1 (VDAC1) membrane LTLSALLDGK GYGFGLIK, WTEYGLTFTEK, Voltage-dependent anion Mitochondrial outer LTFDSSFSPNTGK, 6 344 VDAC1 MOUSE 32331 8.55 channel 1 (VDAC1) membrane VTQSNFAVGYK, VNNSSLIGLGYTQTLKPGIK, LTLSALLDGK TYYMSAGLQPVPIVFR, Pyruvate dehydrogenase DFLIPIGK, 7 (lipoamide) beta. (Pdhb 288 Q99LW9 MOUSE 34814 5.63 Mitochondrial matrix IMEGPAFNFLDAPAVR, protein) VTGADVPMPYAK, VLEDNSVPQVK 6 Journal of Aging Research Table 3: Continued. No. Protein Score NCBI nr Peptides MW PI Localization DLQNVNITLR, ILFRPVASQLPR, IYTSIGEDYDER, Mitochondrial VLPSITTEILK, 8 Prohibitin 396 A39682 29802 5.57 intermembrane space FDAGELITQR, AAIISAEGDSK, AAELIANSLATAGDGLIELR, NITYLPAGQSVLLQLPQ ATP synthase D chain, ANVAKPGLVDDFEK, Mitochondrial inner 9 131 ATP5H MOUSE 18607 5.52 mitochondrial (ATP5H) membrane YTALVDQEEKEDVK Ubiquinol-cytochrome c TDLTDYLNR, Mitochondrial inner 10 135 Q3THM1 MOUSE 52806 5.89 reductase core protein 1 membrane IQEVDAQMLR AAAEVNQEYGLDPK, Fumarate hydratase precursor, 11 170 UFRT 54429 9.06 Mitochondrial AIEMLGGELGSK, mitochondrial (FH) VAALTGLPFVTAPNK Superoxide dismutase 12 72 DSRTN GDVTTQVALQPALK 24659 8.96 Mitochondrial matrix precursor DINQEVYNFLATAGAK, SQFTITPGSEQIR, Aconitase 2, mitochondrial NTIVTSYNR, 13 340 Q3UDK9 MOUSE 85376 8.08 Mitochondrial matrix (ACO2) FNPETDFLTGK, NAVTQEFGPVPDTAR, WVVIGDENYGEGSSR Dihydrolipoamide ADGSTQVIDTK, 14 139 Q99LD3 MOUSE 54238 7.99 Mitochondrial matrix dehydrogenase (DLDH) EANLAAAFGHPINF Journal of Aging Research 7 Table 3: Continued. No. Protein Score NCBI nr Peptides MW PI Localization LVLEVAQHLGESTVR, TIAMDGTEGLVR, VLDSGAPIK, IPVGPETLGR, IMNVIGEPIDER, VVDLLAPYAK, ATP synthase, H+ IGLFGGAGVGK, transporting mitochondrial Mitochondrial inner 15 998 Q3TFD7 MOUSE 56207 5.25 TVLIMELINNVAK, F1 complex, beta subunit membrane (ATP5B) EGNDLYHEMIESGVINLK, VALVYGQMNEPPGAR, VALTGLTVAEYFR, FTQAGSEVSALLGR, AIAELGIYPAVDPLDSTSR, IMDPNIVGNEHYDVAR, ILQDYK, FLSQPFQVAEVFTGHMGK DASVVGFFR, Endoplasmic reticulum, also Protein disulfide isomerase A3 16 94 PDIA3 MOUSE 56472 5.88 present in mitochondria (see GFPTIYFSPANK, (Pdia3) discussion). ELNDFISYLQR 8 Journal of Aging Research (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l) Figure 1: Undifferentiated (a, b, c), early differentiation (d, e, f), intermediate differentiation (g, h, i) and late differentiation stages (j, k, l), are indicated by RM (a, d, g, j), Giemsa staining (b, e, h, k), and TEM (c, f, i, l). Mitochondrial morphology is further detailed by the correspondent insets, showing TEM analysis of isolated mitochondria. C2C12 cell differentiation morphological progression is evident, as well as mitochondrial behaviour in the various stages. (a, b, d, e, g, h, j, k): Bar = 20 μm; (c,f,i,l): Bar = 0.5 μm; insets, Bar = 0.1 μm. Journal of Aging Research 9 (a) (b) (c) (d) (e) (f) (g) (h) 0 0 50 50 150 150 200 200 250 250 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250 (i) (j) Figure 2: Confocal microscopy of C2C12 myoblasts (a–d) and late myotubes (e–h), after Mito Tracker (a, b, e, f) and JC-1 (c, d, g, h) staining. Graphs of lower panel show the different fluorescence JC-1 intensity in myoblasts (i) and late myotubes (j). (a–h): Bar = 20 μm. construct the reference plasmid pDGC, containing a single coactivator 1 alpha (PGC1α), and the mitochondrial tran- copy of COXII and GAPDH segments, the mitochondrial scription factor A (Tfam) were quantified using RT real-time and nuclear target genes, respectively. PCR during differentiation. PGC1α induces mitochondrial As shown in Figure 3(a), twenty-four hours after dif- biogenesis by interacting with several nuclear transcription ferentiation induction, the relative amount of mtDNA factors [36–39], and Tfam is involved in the mitochondrial undergoes a 2-fold increment at the intermediate period of genome transcription [40, 41], replication [42], and it is also differentiation (T = 3) reaching a plateau level at the final crucial for maintaining mitochondrial DNA [43]. stage of maturation (T = 7). As shown in Figure 3(b), PGC-1α expression does not change during the first 24 h from the induction of 3.4. mRNA Expression Level of Mitochondrial Biogenesis differentiation while progressively increasing up to 9.2-fold “Master” Genes. An increase in mitochondrial biogenesis in differentiated myotubes on the 7th day compared to the reflects an enhanced expression of nuclear and mitochon- myoblasts at time T0. drial genes [36–38]. Two master genes involved in the mitochondrial biogenesis, the nuclear transcriptional coac- The Tfam expression level during the myoblasts dif- tivator peroxisome proliferative activated receptor, gamma, ferentiation is slightly shifted compared to the PGC-1α 10 Journal of Aging Research PGC1α expression mtDNA content ∗∗ 2.5 14 ∗∗ ∗∗ 1.5 0.5 T = 1 T = 3 T = 5 T = 7 T = 1 T = 3 T = 5 T = 7 Differentiation days Differentiation days (a) (b) T -fam expression ∗∗ 1.5 0.5 T = 1 T = 3 T = 5 T = 7 Differentiation days (c) Figure 3: Evaluation of mitochondrial biogenesis during myoblast differentiation. In (a), determination by real-time PCR of mtDNA content expressed as mtDNA/nDNA ratio (COXII/GAPDH), as described in Section 2. In (b), quantitative analyses of PGC-1α and T-fam by real- time PCR. The amount of each target transcript was related to that of the reference gene (the ribosomal protein S16). Data are expressed as the mean ± SEM of three experiments; all samples were analyzed in triplicate. Results from PCR real-time analysis were compared with the ANOVA test, followed by a post hoc test using Tukey’s multiple comparison test. The threshold of significance for the ANOVA and the ∗ ∗∗ Tukey’s test was fixed at P ≤ .05; P ≤ .01. expression; in fact, it increased significantly between days 3–7 differentiation, we performed a 2D page on mitochondria (Figure 3(c)). isolated from C2C12 myoblasts over a 7-day time span differentiation. A total of 994 spots (mean) could be resolved on a silver-stained large 2DE gel, where we loaded 45 μg 3.5. Cytochrome c Oxidase Activity and COXII Expression of total protein. A larger amount of protein per spot was Level. The mitochondrial enzymatic activities of cytochrome oxidase reflecting the respiratory chain activities were signif- necessary for protein identification, thus we used preparative icantly higher in myoblasts able to differentiate (Figure 4(a)). gels stained with Brilliant Blue G-Colloidal. To evaluate the possible presence of cellular contaminants, we compared the In addition, we evaluated the expression level of the cor- responding gene coding for the subunit II of mitochondrial mitochondrial map with that of the whole cellular lysate in which we had previously identified several cytosolic and cytochrome c oxidase (COXII), which represents a target membrane proteins [46]. The comparison of 2D maps of gene for mitochondrial transcriptional activity [27, 44, 45]. On days 3–7, the mitochondrial COXII transcript levels mitochondria and whole cell lysate allowed us to state that the preparation of mitochondria contained little or no were significantly higher than in proliferating myoblasts (Figure 4(b)). cellular contaminants. The study of quantitative changes of individual pro- teins in a purified mitochondrial fraction showed that 32 3.6. Changes in Mitochondrial Proteomic Profile. To high- light significant changes in mitochondrial proteome during mitochondrial proteins increased significantly in abundance mtDNA/nDNA (a.u.) Relative mRNA level (a.u.) Relative mRNA level (a.u.) Journal of Aging Research 11 COX enzimatic activity COXII expression 1.8 2.5 ∗∗ 1.6 1.4 1.2 1.5 0.8 0.6 0.4 0.5 0.2 0 0 T = 1 T = 3 T = 5 T = 7 T = 1 T = 3 T = 5 T = 7 Differentiation days Differentiation days (a) (b) Figure 4: Time course change of cytochrome oxidase (COX) enzymatic activity and transcription level of cytochrome oxidase subunit II (COXII) gene at progressive differentiation stages. (a) Quantitative analysis enzymatic activity. (b) The expression level of COXII is related to S16 mRNA gene level. Results from PCR real-time analysis were compared with the ANOVA test, followed by a post hoc test using Tukey’s ∗ ∗∗ multiple comparison test. The threshold of significance for the ANOVA and the Tukey’s test was fixed at P ≤ .05; P ≤ .01. 17 14 27 15 3 1 2 30 31 5 6 (a) (b) Figure 5: Image of a silver-stained 2-DE gel of 45 μg purified mitochondrial proteins from C2C12 myoblasts at 0 (a) and 7 (b) days of differentiation time. Differentially expressed spots are indicated by arrows and numbered according to Table 3. (Figure 5). The proteins showing the greatest expression This was also interesting for the superoxide dismutase changes were also characterized by electrospray ionisation (MnSOD), a voltage-dependent anion-selective channel pro- (ESI) tandem mass spectrometry. In particular, the major tein 1 (VDAC1), and the protein disulfide-isomerase A3 changes occurred between T1and T4timeofdifferentiation, (Pdia3) that were differentially expressed during differenti- while fewer differences were shown between T0-T1and T4– ation. T7(Table 3). The main mitochondrial proteins which could be 4. Discussion detected in fully differentiated syncytia were involved in the citric acid cycle (malate dehydrogenase: MDH2, fumarate In this study, we described temporal mitochondrial changes hydratase: FH, and aconitase 2: ACO2) or belong to the during the myogenic program of C2C12 myoblasts by pyruvate dehydrogenase complex (pyruvate dehydrogenase, analyzing complementary key parameters for mitochon- lipoamide beta: PDHB, dihydrolipoamide dehydrogenase), drial dynamics, biogenesis, and functionality. Of particular complex III (ubiquinol-cytochrome c reductase core protein interest is the contribution of the proteomic approach to 1: UQCRC1) and complex V (ATP synthase, H+ transporting better define the pattern of mitochondrial protein expression mitochondrial F1 complex, beta subunit: ATP5B, and ATP accompanying differentiation in myotubes and potentially synthase d chain: ATP5H) of the respiratory chain. involved in the crosstalk between nuclei and mitochondria. Protein (U/mg) Relative mRNA level (a.u.) 12 Journal of Aging Research Morphological analysis performed by fluorescence mitochondrial biogenesis. This shift could be explained by microscopy with markers of mitochondrial mass/volume and the biological cycle of mitochondria [56]. Mitochondrial ΔΨ, as well as ultrastructural analysis, allowed us to acquire fission is preceded by an extension of the organelles and the more information regarding the mitochondrial organization mtDNA replication phase. Although there is a slight shifting, and dynamics in C2C12 myoblast differentiation. the correlation between the number of copies of mtDNA and Mitochondrial organization in myoblasts was perinu- mitochondrial biogenesis is positive (r = 0.85, data not clear, and it was possible to discriminate individual mito- shown). chondrion by both MitoTracker Green and JC1 staining. In several studies, the measure of mtDNA copy number This type of mitochondrial distribution is described in the has been considered proportional to the number of mito- literature for other cell types including fibroblasts [47], chondria, a golden star for mitochondrial density [57–60]. pancreatic acinar cells [48, 49], astrocytes, and neurons [50]. However, changes in mitochondrial abundance regardless of On the contrary, in myotubes, morphological observa- the mtDNA copy number may occur, especially in peculiar tion by epifluorescence did not allow us to discriminate conditions such as during alterations in the rates of intracel- individual mitochondrion, showing homogeneous staining, lular ROS generation [61]. Franko et al., investigating C2F3 representative of the mitochondrial network, well described mouse myoblasts, showed that an increment in mtDNA does in skeletal muscle tissue [51, 52]. TEM analysis showed a not always correlate with the proliferation of mitochondria mitochondrial remodeling during differentiation and align- or with their activity [62]. In this investigation, the mtDNA ment of organelles along the myotubes. copy number of C2C12 myoblasts significantly increased At this level, we cannot show the formation of a network during the early-intermediate differentiation phases (T = equal to that which is found in skeletal muscle fibers, 1and T3) up to 2-fold remaining constant during the where mitochondria are arranged in crystal structures closely myotube maturation. Likewise, over the course of myoblast related to the sarcoplasm [51, 52]. Indeed, the sarcomeres of differentiation in rat cell line L6, a small but significant myotubes are only sketched [53], but they may support the increase in mitochondrial DNA copy number was observed development of a mitochondrial network during myotube by [27]. Furthermore, in a recent study on the regulation of maturation. mitochondrial biogenesis during myogenesis, mtDNA copy The mitochondrial counting per area of cell surface, number was determined as a marker for mitochondrial obtained by TEM, showed that the number of mitochondria density using QPCR, and the mtDNA copy number was 4- increased from undifferentiated to differentiated conditions. fold higher in fully differentiated myotubes than it was in Mature myotubes contained approximately 2-fold more myoblasts [60]. mitochondria than myoblasts. However, in the first 24 Interestingly, during differentiation, an increased hours after induction of differentiation, the mitochondria mtDNA transcriptional activity and oxidative metabolism increased in size up to 3-fold gradually decreasing in size only correspond to an enhanced mitochondrial biogenesis, as after the intermediate phases to reach the same size observed highlighted by the upregulation of COXII mRNA levels and in myoblasts at T = 0, in mature myotubes (T = 7). This 2 2 cytochrome c oxidase activity (r = 0.83 and r = 0.97, observation suggests that mitochondria first undergo fusion resp., data not shown). and then fission, which allows their distribution during In our investigation, we integrated the mitochondrial syncytia formation as previously reported [54]. Nevertheless, changes observed by multiple key determinants with pro- the mitochondria in myotubes showed a greater extension teomic analysis. of mitochondrial cristae than mitochondria in myoblasts. In the literature, mitochondrial proteomic maps of Marked stimulation of the biosynthesis of the phospho- differentiating myoblasts are not available; hence, this work lipid cardiolipin during the differentiation phases has been presents the first proteomic profile of mitochondria during observed in previous studies on L6E9 myoblasts and other the myogenesis program. Previously, proteome-based inves- cells [54, 55]. It is probably necessary to supply the proper tigations have been carried out to provide a description amount of functional mitochondrial inner membrane for the of the myogenic differentiation program [46, 63, 64]. We respiratory chain proteins involved in oxidative metabolism employed a proteomic approach using two dimensional [7]. electrophoresis, particularly helpful for investigating the All the parameters observed through morphological subset of cellular proteins, such as organellar proteins, due analysis confirm a linear increase in mitochondrial bio- to the reduced complexity of the protein sample [65]. genesis during differentiation. The morphological analysis corroborates the progression of the myogenic process and In particular, analyzing the differentially expressed pro- teins in the mitochondrial proteome map during the myo- the increase in biochemical markers such as the transcription factors of mitochondrial biogenesis PGC-1 α and Tfam. genic process, we observed that also the enzymes involved in cellular respiration, such as pyruvate dehydrogenase, MDH2, Of particular interest was the timing of mtDNA repli- cation compared to mitochondrial biogenesis. Although FH, ACO2, and more markedly HB and 5B ATP syn- mitochondrial biogenesis increased linearly during differ- thase subunits, representative of oxidative phosphorylation, increase linearly with the mitochondrial biogenesis showing entiation, mtDNA content increased significantly from the early days of differentiation already reaching a plateau at a positive correlation (r = 0.915, data not shown). These the intermediate stage. Hence, our investigation shows a findings are consistent with the differentiating cells’ greater slight difference in timing between DNA replication and reliance on aerobic metabolism compared to the glycolytic Journal of Aging Research 13 metabolism that characterizes the undifferentiated myoblasts scenario of mitochondrial dynamics, biogenesis and func- [7]. tionality useful in comparative surveys of mitochondrial Moreover, our observations are in agreement with Moyes pathogenic or senescent satellite cells. and coworkers who demonstrated an mRNA increment for pyruvate dehydrogenase, citrate synthase, isocitrate dehydro- Acknowledgments genase, cytochrome c oxidase, and NADH dehydrogenase [6]. The increment of Krebs cycle and respiratory chain The authors thank Dr. Rosa Curci (Orthopedic Rizzoli proteins supports the augmented mitochondrial function- Institute, Bologna, Italy) for providing confocal microscope ality also confirmed by the COX enzymatic activity during images. 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