Figure 1.
Differentiated myotubes have higher mitochondrial volume and elevated total glutathione level.
(A) Morphological differences between undifferentiated myoblasts and terminally differentiated, fused myotubes shown by phase-contrast microscopy. (B) The effect of myoblast differentiation on expression levels of Pax7, myogenin and PCNA (differentiation markers) monitored on days 0-4 by Western analysis of total cell extracts. The level of GAPDH was used as a loading control. (C) The level of total glutathione (GSSG+GSH) in total cell extracts of control myoblasts and myotubes expressed as µM/mg protein. (D) Changes in expression level of MnSOD, Cu/ZnSOD and catalase as a function of myoblasts differentiation. The level of GAPDH was used as a loading control. (E) The effect of myoblasts differentiation on mitochondrial biogenesis, as calculated by specific activity of citrate synthase localized exclusively in the mitochondria. (F) Quantification of normalized levels of MnSOD (to the citrate synthase activity), and Cu/ZnSOD and catalase (to the expression level of GAPDH) in myoblasts and myotubes based on analysis of three independent Western blots. * indicates p ≤ 0.05 relative to myoblasts.
Figure 2.
Mitochondrial genome of myoblasts is highly sensitive to oxidant-induced damage.
(A) The amount of ROS generated by 0.005, 0.05 and 0.5 U/ml of GOx detected by fluorescence of DCF at 0, 5, 15, 30 and 60 min in cultured myoblasts and myotubes. Integrity of the nuclear (B) and mitochondrial (C) genomes of myoblasts and myotubes cultured for 1 h in various concentrations of GOx analyzed by amplification of 9kb and 10kb of nuclear- and mitochondrial-specific DNA fragments, respectively, by long-amplicon (LA)-PCR technique. (D) Mitochondrial genome copy number was analyzed by amplification of the 117bp mitochondrial genome-specific DNA fragment. The mitochondrial genome copy number in UT myoblasts and myotubes were set as 1. The graphs are based on PCR amplification with three independently isolated DNA for each experimental point and shown as mean ± standard error (s.e.m.). The integrity in untreated (UT) control myoblasts was set as 1. The integrity in UT myotubes is shown relative to UT myoblasts or set as 1. Myotubes at day 4 of differentiation were used. * indicates p ≤ 0.05 relative to UT controls.
Figure 3.
The nuclear extracts of myotubes are deficient in DNA polymerase and DNA ligase activity.
(A) Purity of the myoblast and myotube nuclear and mitochondrial fractions monitored by Western analysis with nucleolin and the 56kDa subunit of ATP synthase antibodies. (B) Total repair synthesis of uracil (U), 5OH-uracil (5OHU) and tetrahydrofuran (THF); (C) APE1 endonuclease activity in nuclear extracts of myoblasts and myotubes. (D) The effect of myoblast differentiation on expression levels of Lig3 and APE1 (key base excision repair enzymes) monitored on days 0-4 by Western analysis of total cell extracts. The level of GAPDH was used as a loading control. Activities of (E) DNA polymerase and (F) DNA ligase in nuclear extracts of myoblasts and myotubes. Schematic representation of each repair reaction is shown above the representative radiogram. Relative repair efficiency for each reaction is based on analysis of at least three independently isolated nuclear extracts. Activities in the nuclear extracts of myoblasts were set as 1. Myotubes at day 4 of differentiation were used. * indicate p ≤ 0.05. P, final repair product; INT, repair intermediates; S, substrate; M, marker; Nc, negative control.
Figure 4.
Mitochondrial extracts of myoblasts accumulate DNA repair intermediates.
(A) Total repair synthesis activity of tetrahydrofuran (THF) containing oligo3 duplex in the mitochondrial extract of myoblasts and myotubes. Schematic representation of repair reaction is shown above the radiogram. Repair efficiency is based on analysis of at least three independently isolated mitochondrial extracts for each cell type. Activity in the mitochondrial extracts of myoblasts was set as 1. * indicates p ≤ 0.05. (B) Repair efficiency of the mitochondrial genome of myoblasts and myotubes after GOx treatment was monitored by amplification of a 10kb mitochondrial-specific DNA fragment by LA-PCR. The level of integrity in UT myoblasts and myotubes was set as 1. (C) The relative number of mitochondrial genomes in myoblasts and myotubes was based on PCR amplification of 117bp mitochondrialDNA-specific fragment. The graphs represent PCR amplification of three independently isolated DNA for each experimental point and shown as mean ± standard error (s.e.m.). Myotubes at day 4 of differentiation were used. P, final repair product; INT, repair intermediates; UT, untreated control.
Figure 5.
Ectopic expression of EXOG increases resistance to oxidant-induced DNA damage in myoblasts.
(A) The expression level of EXOG-FLAG-tagged was monitored by Western analysis with FLAG-HRP conjugated antibody. (B) The integrity of the mitochondrial genome in myoblasts and myotubes transfected with vector or EXOG expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity in UT control (empty vector transfected) myoblasts and myotubes was set as 1. (C) The integrity of the mitochondrial genome of the myoblasts transfected with vector or mitochondrial specific OGG1 expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity of the genome was monitored by amplification of the 10kb mitochondrial genome-specific DNA fragment and normalized by mitochondrial genome copy number. The graphs are based on PCR reaction of three independently isolated DNA for each experimental point and shown as mean ± standard error (s.e.m.). Myotubes at day 4 of differentiation were used. * indicates p ≤ 0.05. UT, untreated control.
Figure 6.
Oxidative stress induces apoptosis in myoblasts.
(A) Viability of the myoblasts and myotubes upon GOx treatment determined by MTT assay. Viability of untreated, control myoblasts and myotubes was set as 1. (B) Flow-cytometry staining using Anexin V/PI of myoblasts 4 h after oxidative challenge. Camptothecin (1 µM) was used as a positive control to induce apoptosis. (C) Western analysis of myoblasts treated with 0.01 U GOx/ml at various times. Activation of caspase-9 indicates activation of the intrinsic apoptotic pathway. (D) The viability of oxidatively stressed myoblasts was improved by pre-treatment with the pan-specific caspase inhibitor z-VAD-fmk, suggesting inhibition of apoptosis induced by GOx treatment. The graphs are based on three independently experiments and shown as mean ± standard error (s.e.m.). Myotubes at day 4 of differentiation were used. * indicates p ≤ 0.05. UT, untreated control.