Cell death-resistance of differentiated myotubes is associated with enhanced anti-apoptotic mechanisms compared to myoblasts
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- Xiao, R., Ferry, A.L. & Dupont-Versteegden, E.E. Apoptosis (2011) 16: 221. doi:10.1007/s10495-010-0566-9
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Skeletal muscle atrophy is associated with elevated apoptosis while muscle differentiation results in apoptosis resistance, indicating that the role of apoptosis in skeletal muscle is multifaceted. The objective of this study was to investigate mechanisms underlying apoptosis susceptibility in proliferating myoblasts compared to differentiated myotubes and we hypothesized that cell death-resistance in differentiated myotubes is mediated by enhanced anti-apoptotic pathways. C2C12 myoblasts and myotubes were treated with H2O2 or staurosporine (Stsp) to induce cell death. H2O2 and Stsp induced DNA fragmentation in more than 50% of myoblasts, but in myotubes less than 10% of nuclei showed apoptotic changes. Mitochondrial membrane potential dissipation was detected with H2O2 and Stsp in myoblasts, while this response was greatly diminished in myotubes. Caspase-3 activity was 10-fold higher in myotubes compared to myoblasts, and Stsp caused a significant caspase-3 induction in both. However, exposure to H2O2 did not lead to caspase-3 activation in myoblasts, and only to a modest induction in myotubes. A similar response was observed for caspase-2, -8 and -9. Abundance of caspase-inhibitors (apoptosis repressor with caspase recruitment domain (ARC), and heat shock protein (HSP) 70 and -25 was significantly higher in myotubes compared to myoblasts, and in addition ARC was suppressed in response to Stsp in myotubes. Moreover, increased expression of HSPs in myoblasts attenuated cell death in response to H2O2 and Stsp. Protein abundance of the pro-apoptotic protein endonuclease G (EndoG) and apoptosis-inducing factor (AIF) was higher in myotubes compared to myoblasts. These results show that resistance to apoptosis in myotubes is increased despite high levels of pro-apoptotic signaling mechanisms, and we suggest that this protective effect is mediated by enhanced anti-caspase mechanisms.
Apoptosis is a critical mechanism that allows multi-cellular organisms to maintain tissue integrity and function, and to eliminate damaged or superfluous cells. Dysregulation of apoptosis has been implicated in a number of diseases including cancer, which is often characterized by insufficient apoptosis [1, 2]. In contrast, other conditions, such as neurodegenerative (Parkinson and Alzheimer), and auto-immune diseases have been linked to excessive apoptosis [3, 4]. Apoptosis in skeletal muscle occurs under a number of different pathological and physiological circumstances, such as chronic heart failure , neuromuscular diseases [6–9], aging [10–12], and disuse-induced atrophy [13–17]. The specific role of apoptosis in skeletal muscle atrophy is currently unclear, but it is thought to contribute to the loss of nuclei in different models of muscle atrophy such as spinal cord injury , hind limb suspension [17, 18], immobilization , denervation , or chronic heart failure , although recently this role has been challenged . Gaining insight into the relevance and underlying mechanisms in the control of apoptosis might aid in identifying more efficient treatments for the loss of muscle mass [21, 22].
With regard to apoptosis, skeletal muscle is a unique tissue because of the multinucleated nature of muscle cells (or myofibers) and the flexibility in myonuclear number with hypertrophy and atrophy . In muscles undergoing atrophy induced by a variety of conditions, the individual myonuclei, instead of entire cells, may be eliminated by the process of ‘apoptotic nuclear death’ . Skeletal muscle also possesses remarkable recovery or regenerative capacity, and satellite cells have been shown to play an important role in this process [24, 25]. Enhanced satellite cell apoptosis, however, is thought to be related to compromised recovery potential in aged animals [26, 27] and therefore it is important to investigate mechanisms for apoptosis in both proliferating myoblasts (satellite cells) and in differentiated myotubes (skeletal muscle cells). The process by which multi- and mononucleated cells undergo apoptosis is likely quite different and we have recently shown that adult differentiated muscle cells utilize a caspase-independent mechanism for nuclear apoptosis . In addition, Siu et al.  recently showed that multinucleated myotubes exhibit both caspase-dependent and caspase-independent pathways of apoptosis in response to oxidative stress.
In general, cell differentiation results in increased apoptosis resistance which is particularly true for cancer cells and is a major problem in cancer therapy [2, 30]. Indeed, in muscle, increased apoptosis-resistance has also been observed in differentiated myotubes compared to proliferating myoblasts [31–35]. Apoptosis and cellular differentiation are two biologically distinct processes that nonetheless share biochemical and morphological features that imply that they are closely related . For example, as nuclear disruption is viewed as an irreversible step in the apoptotic process, terminal differentiation of many cell types including erythrocytes, keratinocytes, and lens fiber epithelial cells are characterized by complete removal of the nucleus . Also, caspases have been extensively used as a surrogate to monitor apoptosis, because of their central role in this process, but caspases have also been found to play non-apoptotic roles in pathways such as the inflammatory response, immune cell proliferation, and differentiation [38, 39]. Importantly, prodifferentiation functions of caspase-3 have been implicated for osteoclasts , bone marrow stromal cells , neurons , neural and glial progenitor cells [43, 44], and muscle cells , and these observations have led to the proposition that cell differentiation may be a modified or curtailed form of cell death [36, 46]. Larsen et al.  recently showed that caspase-3 promotes cell differentiation in muscle cells through the induction of DNA strand breaks, but it remains unclear how muscle cells regulate caspase-mediated differentiation and cell death signaling simultaneously and restrict apoptosis while undergoing differentiation. Therefore, the goal of current study was to investigate underlying mechanisms of the altered apoptosis susceptibility in myogenic C2C12 cells in response to differentiation. It seems contradictive that caspase activation is elevated with both differentiation and apoptosis induction, but differentiated myotubes are more resistant to apoptosis. We suggest that pathways are in existence in differentiated myotubes that inhibit apoptosis even under conditions where apoptosis would be favorable, such as high caspase activities, to prevent the death of postmitotic muscle tissue. Therefore, we hypothesize that differentiated muscle cells (myotubes) are more resistant to apoptosis than undifferentiated cells (myoblasts), because of enhanced anti-apoptosis pathways.
Cell culture and differentiation
The C2C12 cell line was grown as described previously with a few revisions . Briefly, C2C12 cells were maintained as myoblasts in subconfluent conditions in growth medium (GM: Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine serum [FBS]) at 37°C in a humidified, 10% CO2 incubator. To induce differentiation from myoblasts to myotubes, cells at 95–100% confluency were switched to differentiation medium (DM: DMEM plus 2% horse serum) and maintained in DM for 3 days. Myogenin gene expression was used to show the highly differentiated cells after 3 days in DM.
Induction of apoptosis
Two apoptosis inducers, H2O2 and staurosporine (Stsp), were used to investigate the mechanisms of cell death in undifferentiated myoblasts and in differentiated myotubes, because previous studies have indicated that they induced programmed cell death in muscle cells [29, 33, 49]. Cells were treated for 3–6 h with different concentrations of either H2O2 (10 μM – 10 mM) or staurosporine (Stsp: 0.1–0.5 μM) to determine the optimal concentration and time point for further experiments (see Fig. 2). The concentration of H2O2 or staurosporine that induced about a 50–60% cell death in myoblasts was chosen for further experiments (1000 μM H2O2 and 0.5 μM Stsp).
Terminal transferase dUTP nick end labeling (TUNEL) assay
The susceptibility to apoptosis was measured in C2C12 myoblasts and myotubes using TUNEL assay according to the manufacturer’s recommendations with minor revision (Roche Molecular Biochemicals, Pleasanton, CA) after a 3–6 h treatment with different concentrations of either H2O2 (10 μM–10 mM) or staurosporine (Stsp: 0.1–0.5 μM). Briefly, after incubation with apoptosis inducers, myoblasts and differentiated myotubes were rinsed with PBS and fixed in 2% paraformaldehyde at room temperature for 20 minutes (min), blocked in 3% H2O2 for 10 min, and permeabilized with 0.1% Triton X and 0.1% sodium citrate. Cells were incubated with TUNEL reaction mix diluted 1:3 with dilution buffer (Roche Molecular Biochemicals, Pleasanton, CA) in a humidified chamber at 37°C for 1 h, followed by incubation with streptavidin-POD enzyme conjugate at 37°C for 30 min. Signal amplification was performed using the tyramide signal amplification system (TSA, Perkin Elmer, Boston, MA) at a dilution of 1:200 (TSA fluorescein : amplification buffer) for 30 min as described previously . TUNEL positive nuclei were counted in five separate fields (×200 magnification) using a Zeiss Observer.D1 (Zeiss, Thornwood, NY) microscope with GFP filter. Total nuclear number was visualized by Hoechst staining (4 μg/ml) under UV filter. Apoptosis was presented as the percentage of TUNEL positive nuclei compared to total nuclei. Since there are always some undifferentiated cells after differentiation, bright field pictures were used to determine whether nuclei staining positive for TUNEL and Hoechst were inside the myotubes.
Heat shock was induced by placing myoblasts at 60% confluency at 42°C for 6 h followed by 16 h at 37°C. After this period either H2O2 or Stsp were administered for 3 h and cells were assayed for cell viability as described below.
Cell viability was measured using the Trypan Blue exclusion assay. This assay is based on the fact that dead cells take up Trypan blue dye while viable cells exclude the dye. C2C12 cells were plated with 40,000 cells per well on 6 well plates; cells were then treated with H2O2 or StSp for 3 h as described above or treated as control. After incubation, cells were trypsinized, spun down, and resuspended in serum-free DMEM after which 0.4% Trypan Blue (Cellgro, Manassas, VA) was added in a 1:1 dilution for 2 min and the number of viable cells was counted with a hemocytometer. The number of viable cells was expressed as a percentage of controls.
Assessment of mitochondrial membrane potential (∆ψm)
∆ψm was measured as an early event in the initiation of apoptosis, since a decrease of ∆ψm preceeds leakage of pro-apoptotic proteins involved in caspase-dependent and independent apoptosis from mitochondria into the cytosol. After treatment with H2O2 and StSp, ∆ψm was assessed using the cationic dye, JC-1 (5,5′, 6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide), according to the manufacturer’s protocol (Biotium, Hayward, CA). In healthy cells the dye stains the cells bright red due to accumulation of the dye within the mitochondria. In apoptotic cells the dye is unable to accumulate within the mitochondria because of the collapse of the mitochondrial membrane potential and the dye stains the cells green. The ratio of red divided by green fluorescence is calculated and a decrease in this ratio indicates a decrease in mitochondrial membrane potential. C2C12 cells were plated at approximately 60% confluency on 6-well plates and were exposed to 1000 μM H2O2, 0.5 μM StSp or control conditions for 3 h, rinsed with PBS, and followed by incubation with JC-1 reagent 1:100 at 37°C for 30 min. After incubation, cells were again rinsed twice with PBS and images were obtained using TRITC (red, 590 nm) and GFP (green, 530 nm) filters on a fluorescent microscope (Zeiss Axio Observer). Care was taken to obtain pictures with identical exposure times and pictures were analyzed using Axio Vision software (Zeiss). The automated measurement program with identical user defined parameters for densitometric and geometric variables was used to determine fluorescent intensity for both filters. The ratio of the sum of intensities of red divided by green fluorescence was determined and expressed as the ∆ψm index.
Caspase activity determination
Enzymatic activities of caspase-2, -3, -8 and -9 were measured by using caspase specific fluorogenic substrates as described previously . Briefly, after apoptosis treatments, floating and adherent myoblasts or myotubes were collected and lysed in whole lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with leupeptin (1 mg/ml) and PMSF (1 mM) and protein concentrations were determined by a BCA assay (Pierce Biotechnology, Rockford, IL). For each reaction, 1 μM of specific caspase substrate including Ac-VDVAD-AMC (caspase-2), Ac-IETD-AMC (caspase-8), Ac-LEHD-AMC (caspase-9) or Ac-DEVD-AMC (caspase-3) was mixed with assay buffer (100 mM HEPES (pH 7.4), 10 mM DTT, 0.1% CHAPS and 10% sucrose) and equal amounts of protein followed by incubation at 37°C for 1 h (all substrates from Peptides International, Louisville, KY). Liberation of free AMC from the fluorogenic substrates (representing caspase activity) was detected by using a Spectra Max fluorescent microplate reader (Molecular Devices), with an excitation wavelength of 380 nm and an emission wavelength of 460 nm. AMC liberated from the substrates was compared to a standard curve prepared with known amounts of free AMC and the concentration of AMC was calculated.
RNA isolation and assessment of mRNA abundance
Total RNA from C2C12 myoblasts and myotubes was isolated with the RNAqueous Kit (Ambion, Austin, TX) according to the manufacturer’s instructions. Total RNA was treated with DNase (Ambion) and its integrity was checked using Agilent Bioanalyzer (Santa Clara, CA) before measurement of mRNA abundance by real time RT-PCR, as previous described . 1 μg of RNA was reverse transcribed to cDNA with the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). Real time PCR primers were designed using Beacon Designer software (Bio-Rad Laboratories) and obtained from Integrated DNA Technologies (Coralville, IA). The primers and corresponding mouse mRNAs (gene, forward primer, and reverse primer) investigated in this study were apoptosis repressor with caspase recruitment domain (ARC, 5′-CAAGAAGAGGATGAATCTGAAG-3′, 5′-TTGGCAGTAGGTGTCTCG-3′), the 70 kD heat shock protein (HSP70, 5′-GAACTCTTGATGTGTCGTTACTG-3′, 5′-CCGCCTGTCTTAATCTGTGG-3′), 25 kD heat shock protein (HSP25, 5′-AAGGAAGGCGTGGTGGAG-3′, 5′-ACTGCGTGACTGCTTTGG-3′) and myogenin (Myogenin, 5′-ACAATCTGCACTCCCTTACG-3′, 5′-CGTCTGGGAAGGCAACAG-3′). PCR reactions were conducted on iQ5 real time PCR detection system (Bio-Rad Laboratories) and a SYBRgreen-based protocol was applied, RNA abundance for each gene of interest is normalized to the geometric mean, a factor calculated by GeNorm software (PrimerDesign, Southampton, Hants) based on the expression of 18S ribosomal RNA (18S, 5′-AATGAGCCATTCGCAGTTTC-3′, 5′-CTCTGTTCCGCCTAGTCCTG-3′) and ubiquitin C (UBC, 5′-AGGTCAAACAGGAAGACAGACGTA-3′, 5′-TGTGCTTGTTCTTGGGTGTGA-3′) in the same sample as described previously .
Cytosolic and nuclear fractions from myoblasts and myotubes were obtained using the method described by Siu et al. . Briefly, myoblasts and myotubes were homogenized in lysis buffer (10 mM NaCl, 1.5 mM MgCl2, 20 mM HEPES, pH 7.4, 20% glycerol, 0.1% Triton X-100 and 1 mM dithiothreitol) and centrifuged for 5 min at 4°C. Supernatants were collected as the cytosolic fractions. The nuclear pellet was resuspended in lysis buffer and 5 M NaCl was added to lyse the nuclei. The mixture was rotated for 1 h at 4°C and centrifuged at 14,000 rpm for 15 min at 4°C. The supernatant containing the nuclear protein was collected. Purity of the fractions was confirmed with histone and CuZnSOD antibodies for nuclear and cytosolic fractions, respectively.
Western blot analysis of proteins was performed as described previously with minor modifications . Briefly, C2C12 myoblasts and myotubes treated with H2O2 or Stsp were lysed in lysis buffer (see above), which was supplemented with 1 mM PMSF, 1 μg/ml leupeptin, 1 μg/ml aprotinin and 1 μg/ml pepstatin. Protein concentration of the supernatants was determined using the Bradford assay. For determination of apoptosis repressor with CARD domain (ARC), endonuclease G (EndoG), apoptosis inducing factor (AIF), HSP25 and HSP70 protein content, 10–100 μg total protein was loaded and separated on 4–15% acrylamide gradient gel (Bio-Rad), followed by transfer proteins to PVDF membranes with 0.22 μm pore size. Membranes were incubated in Odyssey Blocking Buffer (Li-Cor, Lincoln, NE) for 1-h at room temperature followed by incubation with ARC (1:200; ProSci Incorporated, Poway, CA), HSP25 (1:2000; Enzo Life Sciences, Plymouth, PA), HSP70 (1:500; Cell Signaling, Danvers, MA), EndoG (1:1000; Cell Signaling), or AIF (1:200; Santa Cruz Biotechnologies, Santa Cruz, CA) antibodies overnight at 4°C, washed, and further incubated with highly cross-absorbed infra red-labeled secondary antibodies (1:20,000; Li-Cor) for 20 min at room temperature. Membranes were scanned using Odyssey infrared imaging system (Li-Cor) to detect specific antibody binding and quantification. Normalization of bands was performed using β-actin antibody (1:2,000; Abcam, Cambridge, MA).
A minimum of three plates was used for each individual condition. Statistical computations were performed using SigmaStat (SSI, Richmond, CA). For comparisons of means between treatments, a one-way or two-way ANOVA was applied where appropriate. In case of significant differences, Tukey multiple comparisons test was used. Statistical significance was set at P < 0.05.
C2C12 muscle cell differentiation and apoptosis
Differentiated C2C12 are resistant to apoptosis
∆ψm dissipation is attenuated in differentiated myotubes
Differential activation of caspases in myoblasts and myotubes
Enhanced anti-apoptotic mechanisms in differentiated myotubes
Elevated EndoG expression in myotubes
Differentiated skeletal muscle cells are more resistant to apoptosis than undifferentiated cells [31, 32, 60, 61] and this phenomenon may reflect a mechanism by which the organism can maintain postmitotic muscle cells . How this resistance is conferred is not completely understood. In this study, we found that the apoptosis resistance in myotubes is associated with the elevation of anti-apoptotic proteins, such as ARC and HSPs and that elevation of HSPs reduced myoblast cell death, indicating that these proteins are indeed protective in a muscle environment. We further determined that ∆ψm dissipation was inhibited in myotubes indicating a protection of the mitochondrial pathway of apoptosis in myotubes.
Potential pathways for apoptosis in muscle cells
The mitochondrial pathway of apoptosis begins with the permeabilization of the mitochondrial outer membrane and can be either mitochondrial permeability transition pore (mPTP) dependent or independent . Increased permeabilization causes dissipation of the ∆ψm followed by the release of apoptotic proteins, including cytochrome c, AIF and EndoG [63, 64]. Cytochrome c release induces activation of caspase-9, and subsequent cleavage of caspase-3, which in turn induces apoptosis in most mononucleated cell types. Caspases are thought to be the central proteases responsible for apoptosis and therefore, enhanced resistance to apoptosis such as occurs in myotubes, was expected to be associated with lower caspase activities. Surprisingly, activities of caspase-2, -3, -8 and -9 were all dramatically higher in myotubes, compared to myoblasts, even though apoptosis in response to cell death inducers H2O2 and Stsp was markedly lower in myotubes compared to myoblasts. This indicates that most likely caspases have different functions in differentiated muscle cells compared to undifferentiated muscle cells and other mononucleated cells. Indeed, a number of cellular processes that do not involve apoptosis has been attributed to caspases, such as T-cell activation , maintenance of embryonic endothelial cells , monocyte, osteogenic and myogenic differentiation [41, 45, 66–68], and fusion of trophoblasts . More importantly, caspase-3 cleavage of muscle specific proteins has been implicated as the initial step in triggering muscle proteolysis during atrophy . Caspases can therefore be viewed as regular signal-transducing molecules which is a phenomenon also suggested by a recent study in which the proteolytic events of caspases were investigated . Even though caspases likely play alternative roles in differentiated myotubes, induction of apoptosis through Stsp in our study still caused a significant increase in caspase-2, -3, -8 and -9 activities even though ∆ψm dissipation was attenuated compared to myoblasts. Since apoptosis was not increased in the face of these elevated caspase activities, we hypothesized that the anti-apoptotic system in myogenic cells may be strengthened after differentiation. Of interest is the fact that in response to H2O2 caspases did not increase in myoblasts even though cell death is high in these cells. It is possible that these cells undergo a different or additional form of cell death, such as necrosis, and still show DNA fragmentation as shown by the TUNEL assay. Since H2O2 is an inducer of oxidative stress the underlying pathways leading to cell death are likely different than those in response to Stsp and may not involve caspases. Indeed, morphologically the myoblasts treated with H2O2 showed different characteristics than those treated with Stsp. It is also possible that the attenuated response to H2O2 in myotubes is regulated by differential expression of anti-oxidant systems in differentiated cells, but this possibility was not investigated here.
Negative regulators of cell death are elevated in myotubes
Like other vital biological processes, tight regulation of apoptosis is required to ensure a delicate balance of cellular life and death. The resistance to particularly caspase-induced apoptosis in differentiated muscle cells has likely evolved to protect this tissue from accidental loss of cells and/or nuclei due to caspase activation for different processes such as proteolysis. Indeed, several gene families are involved in the negative regulation of apoptosis. In skeletal muscle, for example, cytochrome c release is regulated, in part, by the Bcl-2 family of proteins , while inhibitors of apoptosis (IAP) directly suppress activity of caspase-3 and -9 . More recently, a muscle specific anti-apoptotic protein ARC has caused interest because of its extensive anti-apoptotic activity and its links to myogenic differentiation . ARC is an endogenous inhibitor that is thought to target caspase-2 and -8 and plays a role in the mitochondria-mediated pathway by preventing cytochrome c release from mitochondria .We show here that ARC expression in differentiated myotubes is indeed ten times higher than in myoblasts, indicating that this inhibitor may be involved in the apoptosis resistance of myotubes. In addition, ARC may be responsible for the lack of increased caspase activities in response to H2O2 and could therefore be specific in protecting against oxidative stress-induced apoptosis, but this possibility needs further investigation. Upon exposure to apoptosis inducers, ARC mRNA abundance decreased in myotubes, as has been shown for protein abundance in response to H2O2 . Also, ARC protein abundance decreased in response to H2O2 in myotubes coinciding with the decrease in ∆ψm indicating that in myotubes ARC may be an important protective protein for ∆ψm dissipation.
Heat shock proteins (HSPs) are commonly believed to function as a protective mechanism in response to various stressful events including high temperature, oxidative stress, hypoxia, and other cellular damages [76, 77]. In addition, HSPs are known to inhibit apoptosis through caspase-dependent and -independent mechanisms [57, 78–84]. We observed that the HSP70 and HSP25 mRNA abundance was significantly elevated in myotubes compared to myoblasts (see also ), suggesting that HSPs may play a role in the protection of myotubes from apoptosis in situations where caspase activity is high for different processes, such as differentiation. It has previously been shown that elevated HSPs are protective against calcium- and mitochondrial-related damage in muscle myotubes in vitro . We showed that in myoblasts an elevation of HSPs in response to hyperthermia protected from cell death induced by H2O2 and Stsp, indicating that the enhanced abundance of these proteins in myotubes likely confers a similar cell protective effect.
AIF and EndoG as regulators of apoptosis in myotubes
Caspase-independent pathways for apoptosis have recently been shown to be important in apoptotic nuclear loss in skeletal muscle . EndoG and AIF are two apoptogenic factors released from mitochondria upon apoptotic stimulation, which have been thought to play an important role in regulating apoptosis, associated to disuse or age related muscle atrophy [11, 16, 28]. Since EndoG and AIF are highly elevated in myotubes, we suggest that these molecules may function as apoptosis inducers in differentiated muscle cells where the abundance of molecules capable of inhibiting caspases is very high. AIF, which is capable of DNA fragmentation and subsequent apoptosis , was elevated in both cytoplasmic and nuclear fractions of myotubes, but did not change abundance or localization in response to the induction of apoptosis by H2O2 and Stsp, indicating that it is likely not involved in the induction of apoptosis in myotubes. Interestingly, EndoG decreased in the cytosolic fraction of myotubes in response to H2O2 and Stsp indicting that it could be translocated to the nucleus, but our analysis was not sensitive enough to detect this translocation.
In conclusion, our study demonstrates that myogenic cells develop an apoptosis-resistant phenotype during differentiation that is coincident with the elevation of inhibitors of cell death, such as ARC and HSPs. We propose that these molecules inhibit cell death of muscle cells in the face of elevated apoptotic molecules such as caspases. In addition, the fact that the mitochondrial membrane potential of myotubes is resistant to the induction of apoptosis compared to myoblasts indicates that mechanisms upstream from the mitochondrial membrane dissipation may also play a role in the decreased susceptibility to cell death of myotubes. Moreover, this study suggests that caspase-independent cell death molecules, such as EndoG, may be important for the induction of apoptosis in differentiated muscle cells, but it remains to be determined what mechanisms are underlying the high cell death in myoblasts.
This research was supported by National Institute on Health grant # AG028925 and AR053967.
Conflict of interest
The authors declare that they have no conflict of interest.