Nasser Al-Shanti

Powerful signals for weak muscles.

The aim of the present review is to summarise, evaluate and critique the different mechanisms involved in anabolic growth of skeletal muscle and the catabolic processes involved in cancer cachexia and sarcopenia of ageing. This is highly relevant, since they represent targets for future promising clinical investigations. Sarcopenia is an inevitable process associated with a gradual reduction in muscle mass and strength, associated with a reduction in motor unit number and atrophy of muscle fibres, especially the fast type IIa fibres. The loss of muscle mass with ageing is clinically important because it leads to diminished functional ability and associated complications. Cachexia is widely recognised as severe and rapid wasting accompanying disease states such as cancer or immunodeficiency disease. One of the main characteristics of cancer cachexia is asthenia or lack of strength, which is directly related to the muscle loss. Indeed, apart from the speed of loss, muscle wasting during cancer and ageing share many common metabolic pathways and mediators. In healthy young individuals, muscles maintain their mass and function because of a balance between protein synthesis and protein degradation associated with rates of anabolic and catabolic processes, respectively. Muscles grow (hypertrophy) when protein synthesis exceeds protein degradation. Conversely, muscles shrink (atrophy) when protein degradation dominates. These processes are not occurring independently of each other, but are finely coordinated by a web of intricate signalling networks. Such signalling networks are in charge of executing environmental and cellular cues that ultimately determine whether muscle proteins are synthesised or degraded. Increasing our understanding for the pathways involved in hypertrophy and atrophy and particularly the interaction of these pathways is essential in designing therapeutic strategies for both prevention and treatment of muscle wasting conditions with age and with disease.

C2 and C2C12 murine skeletal myoblast models of atrophic and hypertrophic potential: relevance to disease and ageing?

Reduced muscle mass and increased susceptibility to TNF‐induced degradation accompany inflamed ageing and chronic diseases. Furthermore, C2 myoblasts display diminished differentiation and increased susceptibility to TNF‐α‐induced cell death versus subcloned C2C12 cells, providing relevant models to assess: differentiation (creatine kinase), growth (protein), death (trypan‐blue) and anabolic/catabolic parameters (RT‐PCR) over 72 h ± TNF‐α (20 ng ml−1). At 48 and 72 h, respectively, larger myotubes and significantly higher CK activity (320.26 ± 6.82 vs. 30.71 ± 2.5, P < 0.05; 544.94 ± 27.7 vs. 39.4 ± 3.37 mU mg ml−1, P < 0.05), fold increases in myoD (21.45 ± 3.12 vs. 3.97 ± 1.76, P < 0.05; 31.07 ± 3.1 vs. 6.82 ± 1.93, P < 0.05) and myogenin mRNA (241.8 ± 40 vs. 36.80 ± 19.3, P < 0.05; 440 ± 100.5 vs. 201.1 ± 86, P < 0.05) were detected in C2C12 versus C2. C2C12 showed significant increases in IGF‐I mRNA (243.05 ± 3.87 vs. 105.75 ± 21.95, P < 0.05), reduced proliferation and significantly lower protein expression (1.21 ± 0.28 vs. 1.79 ± 0.29 mg ml−1, P < 0.05) at 72 h versus C2 cells. Significant temporal reductions in C2C12 IGFBP2 mRNA (28.02 ± 15.44, 13.82 ± 8.07, 6.92 ± 4.37, P < 0.05) contrasted increases in C2s (4.31 ± 3.31, 13.02 ± 9.92, 82.9 ± 58.9, P < 0.05) at 0, 48 and 72 h, respectively. TNF‐α increased cell death in C2s (2.67 ± 1.54%, 34.42 ± 5.39%, 29.71 ± 5.79% (0, 48, 72 h), P < 0.05), yet was without effect in C2C12s at 48 h but caused a small significant increase at 72 h (9.88 ± 4.02% (TNF‐α) vs. 6.17 ± 0.749% (DM), 72 h). TNF‐α and TNFRI mRNA were unchanged; however, larger reductions in IGF‐I (8.2‐ and 7.5‐fold vs. 4.5‐ and 4.1‐fold (48, 72 h)), IGF‐IR (2‐fold vs. no‐significant reduction (72 h)) and IGFBP5 (3.24 vs. 1.38 (48 h) and 2.21 vs. 1.71 (72 h), P < 0.05) mRNA were observed in C2 versus C2C12 with TNF‐α. This investigation provides insight into regulators of altered basal hypertrophy and TNF‐induced atrophy, providing a model for future investigation into therapeutic initiatives for ageing/wasting disorders. J. Cell. Physiol. 225: 240–250, 2010.

Ca2+/calmodulin-dependent transcriptional pathways: potential mediators of skeletal muscle growth and development

The loss of muscle mass with age and disuse has a significant impact on the physiological and social well‐being of the aged; this is an increasingly important problem as the population becomes skewed towards older age. Exercise has psychological benefits but it also impacts on muscle protein synthesis and degradation, increasing muscle tissue volume in both young and older individuals. Skeletal muscle hypertrophy involves an increase in muscle mass and cross‐sectional area and associated increased myofibrillar protein content. Attempts to understand the molecular mechanisms that underlie muscle growth, development and maintenance, have focused on characterising the molecular pathways that initiate, maintain and regenerate skeletal muscle. Such understanding may aid in improving targeted interventional therapies for age‐related muscle loss and muscle wasting associated with diseases. Two major routes through which skeletal muscle development and growth are regulated are insulin‐like growth factor I (IGF‐I) and Ca2+/calmodulin‐dependent transcriptional pathways. Many reviews have focused on understanding the signalling pathways of IGF‐I and its receptor, which govern skeletal muscle hypertrophy. However, alternative molecular signalling pathways such as the Ca2+/calmodulin‐dependent transcriptional pathways should also be considered as potential mediators of muscle growth. These latter pathways have received relatively little attention and the purpose herein is to highlight the progress being made in the understanding of these pathways and associated molecules: calmodulin, calmodulin kinases (CaMKs), calcineurin and nuclear factor of activated T‐cell (NFAT), which are involved in skeletal muscle regulation. We describe: (1) how conformational changes in the Ca2+ sensor calmodulin result in the exposure of binding pockets for the target proteins (CaMKs and calcineurin). (2) How Calmodulin consequently activates either the Ca2+/calmodulin‐dependent kinases pathways (via CaMKs) or calmodulin‐dependent serine/threonine phosphatases (via calcineurin). (3) How calmodulin kinases alter transcription in the nucleus through the phosphorylation, deactivation and translocation of histone deacetylase 4 (HDAC4) from the nucleus to the cytoplasm. (4) How calcineurin transmits signals to the nucleus through the dephosphorylation and translocation of NFAT from the cytoplasm to the nucleus.

Beneficial synergistic interactions of TNF-α and IL-6 in C2 skeletal myoblasts—Potential cross-talk with IGF system

The interaction effects of tumour necrosis factor-alpha (TNF-α) and interlukin-6 (IL-6) on skeletal muscle proliferation and differentiation remains controversial. We therefore investigated the potential interactive effects of TNF-α and IL-6 on murine C2 skeletal myoblast survival, differentiation and proliferation. A novel and unexpected positive temporal interaction between TNF-α and IL-6 on cell growth was identified (90%), with maximal beneficial effects obtained in myoblasts treated with TNF-α (10 ng/ml) for 24 h prior to being dosed with IL-6 (2.5 ng/ml) for a further 24 h. This combined treatment significantly (p < 0.05) increased the level of total cellular protein (330%), extracellular signal-regulated kinase (ERK) phosphorylation (55%), and S-phase of cell cycle (2.5-fold), confirming cell growth. The expression of mRNAs of key regulators of muscle mass: insulin-like growth factor binding protein-5, insulin-like growth factor-II (IGF-II), IGF-I receptor (IGF-IR) and IGF-II receptor (IGF-IIR) were also significantly (p < 0.05) increased by 1600-, 1.6-, 27- and 6-fold, respectively, giving an indication of the regulatory mechanisms of this interaction. Moreover, in response to this treatment, the expression level of signal-transducing glycoprotein 130 (gp130) was induced up to 3.5-fold but not after either treatments alone. This may not only explain the beneficial effects of this treatment on skeletal myoblast numbers but also define a functional role of gp130 in skeletal muscle cells. Our data suggest that in the presence of TNF-α/IL-6 functions positively and potentially also cooperatively with the IGF system to achieve the maximal beneficial effect on skeletal myoblast numbers.

Pro- and anti-apoptotic roles for IGF-I in TNF-α-induced apoptosis: A MAP kinase mediated mechanism

Objective. The concept of skeletal muscle homeostasis—often viewed as the net balance between two separate processes, namely protein degradation and protein synthesis—are not occurring independently of each other, but are finely co-ordinated by a web of intricate signalling networks. Materials and methods. Using rodent muscle cell lines we have investigated TNF-α/IGF-I interactions, in an attempt to mimic and understand mechanisms underlying the wasting process. Results and conclusion. When myoblast cells are incubated with TNF-α (10 ng ml− 1) maximal damage (∼21% ± 0.7 myoblast death, p < 0.05) was induced. Co-incubation of TNF-α (10 ng ml− 1) with IGF-I resulted in cell survival (∼50% reduction in myoblast death, p < 0.05), however, myotube formation was not evident. In contrast, a novel role of IGF-I has been identified whereby co-incubation of muscle cells with IGF-I (1.5 ng ml− 1) and a non-apoptotic dose of TNF-α (1.25 ng ml− 1; sufficient to block differentiation) unexpectedly were shown not to rescue a block on differentiation but to facilitate significant myoblast death (p < 0.05). Interestingly, pre-administration of PD98059, a MAPK signal-blocking agent followed by co-incubation of 1.25 ng ml− 1 TNF-α and 1.5 ng ml− 1 IGF-I, reduced death to baseline levels (p < 0.05). We show for the first time that IGF-I can be apoptotic in the absence of TNF-α-induced cell death.

Reduction of myoblast differentiation following multiple population doublings in mouse C2C12 cells: A model to investigate ageing?

Ageing skeletal muscle displays declines in size, strength, and functional capacity. Given the acknowledged role that the systemic environment plays in reduced regeneration (Conboy et al. [2005] Nature 433: 760–764), the role of resident satellite cells (termed myoblasts upon activation) is relatively dismissed, where, multiple cellular divisions in‐vivo throughout the lifespan could also impact on muscular deterioration. Using a model of multiple population doublings (MPD) in‐vitro thus provided a system in which to investigate the direct impact of extensive cell duplications on muscle cell behavior. C2C12 mouse skeletal myoblasts (CON) were used fresh or following 58 population doublings (MPD). As a result of multiple divisions, reduced morphological and biochemical (creatine kinase, CK) differentiation were observed. Furthermore, MPD cells had significantly increased cells in the S and decreased cells in the G1 phases of the cell cycle versus CON, following serum withdrawal. These results suggest continued cycling rather than G1 exit and thus reduced differentiation (myotube atrophy) occurs in MPD muscle cells. These changes were underpinned by significant reductions in transcript expression of: IGF‐I and myogenic regulatory factors (myoD and myogenin) together with elevated IGFBP5. Signaling studies showed that decreased differentiation in MPD was associated with decreased phosphorylation of Akt, and with later increased phosphorylation of JNK1/2. Chemical inhibition of JNK1/2 (SP600125) in MPD cells increased IGF‐I expression (non‐significantly), however, did not enhance differentiation. This study provides a potential model and molecular mechanisms for deterioration in differentiation capacity in skeletal muscle cells as a consequence of multiple population doublings that would potentially contribute to the ageing process. J. Cell. Biochem. 112: 3773–3785, 2011.

The role of insulin-like-growth factor binding protein 2 (IGFBP2) and phosphatase and tensin homologue (PTEN) in the regulation of myoblast differentiation and hypertrophy.

The complex actions of the insulin-like-growth factor binding proteins (IGFBPs) in skeletal muscle are becoming apparent, with IGFBP2 being implicated in skeletal muscle cell proliferation and differentiation (Ernst et al., 1992; Sharples et al., 2010). Furthermore, PTEN signalling has been linked to IGFBP2 action in other cell types by co-ordinating downstream Akt signalling, a known modulator of myoblast differentiation. The present study therefore aimed to determine the interaction between IGFBP2 and PTEN on myoblast differentiation. It has previously been established that C2C12 cells have high IGFBP2 gene expression upon transfer to low serum media, and that expression reduces rapidly as cells differentiate over 72 h [1]. Wishing to establish a potential role for IGFBP2 in this model, a neutralising IGFBP2 antibody was administered to C2C12 myoblasts upon initiation of differentiation. Myoblasts subsequently displayed reduced morphological differentiation (myotube number), biochemical differentiation (creatine kinase) and myotube hypertrophy (myotube area) with an early reduction in Akt phosphorylation. Knock-down of phosphatase and tensin homologue (PTEN) using siRNA in the absence of the neutralising antibody did not improve differentiation or hypertrophy vs. control conditions, however, in the presence of the neutralising IGFBP2 antibody, differentiation was restored and importantly hypertrophy exceeded that of control levels. Overall, these data suggest that; 1) reduced early availability of IGFBP2 can inhibit myoblast differentiation at later time points, 2) knock-down of PTEN levels can restore myoblast differentiation in the presence of neutralising IGFBP2 antibody, and 3) PTEN inhibition acts as a potent inducer of myotube hypertrophy when the availability of IGFBP2 is reduced in C2C12 myoblasts.

Sirtuin 1 regulates skeletal myoblast survival and enhances differentiation in the presence of resveratrol

Sirtuin 1 also known as NAD‐dependent deacetylase sirtuin 1, is a protein that in humans is encoded by the Sirt1 gene. Sirt1 is an enzyme that deacetylates proteins that contribute to cellular regulation and is a key regulator of cell defenses and survival in response to stress. Deletion of Sirt1 abolishes the increase in lifespan induced by calorie restriction or sublethal cytokine stress, indicating that Sirt1 promotes longevity and survival. We have demonstrated that administration of a sublethal dose of tumour necrosis factor‐α (TNF‐α; 1.25 ng ml−1) inhibits myotube formation, and co‐incubation with insulin‐like growth factor I (IGF‐I; 1.5 ng ml−1) facilitates C2 myoblast death rather than rescuing differentiation. A higher dose of TNF‐α (10 ng ml−1) resulted in significant apoptosis, which was rescued by IGF‐I (1.5 ng ml−1; 50% rescue; P < 0.05). We aimed to investigate the role of Sirt1 in the conflicting roles of IGF‐I. Quantitative real‐time PCR revealed that Sirt1 expression was elevated in myoblasts following incubation of 10 ng ml−1 TNF‐α or 1.25 ng ml−1 TNF‐α plus IGF‐I (fivefold and 7.2‐fold increases versus control, respectively; P < 0.05). A dose of 10 ng ml−1 TNF‐α induced ∼21 ± 0.7% apoptosis, which was reduced (∼50%; P < 0.05) when administered with IGF‐I. Likewise, Sirt1 expression was elevated following 10 ng ml−1 TNF‐α administration, but was reduced (∼30%; P < 0.05) in the presence of IGF‐I. C2C12 myoblasts, a subclone of the C2 cell line produced for their differentiation potential and used to examine intrinsic ageing, unlike C2 cells, do not die in the presence of TNF‐α and do not upregulate Sirt1. As conditions that induced the greatest myoblast stress/damage resulted in elevated Sirt1 expression, we investigated the effects of Sirt1 gene silencing. Treatment with 10 ng ml−1 TNF‐α or co‐incubation with 1.25 ng ml−1 TNF‐α and 1.5 ng ml−1 IGF‐I resulted in apoptosis (20.33 ± 2.08 and 19 ± 2.65%, respectively), which was increased when myoblasts were pretreated with Sirt1 small interfering RNA (31 ± 2.65 and 27.33 ± 2.52%, respectively; P < 0.05) and was reduced (14.33 ± 3.05%, P < 0.05 and 12.78 ± 4.52%, P= 0.054) by resveratrol, which also significantly rescued the block on differentiation. In conclusion, Sirt1 expression increases in conditons of stress, potentially serving to reduce or dampen myoblast death.

Inhibitory effects of IL‐6 on IGF‐1 activity in skeletal myoblasts could be mediated by the activation of SOCS‐3

In elderly people, low and high levels of insulin‐like growth factor 1 (IGF‐1) and interleukin‐6 (IL‐6), respectively, are well documented and may contribute to reduced muscle mass and poor muscle function of ageing and suggesting a biological interactions between IGF‐1 and IL‐6. However, the dual effect of IGF‐1/IL‐6 on skeletal muscle differentiation and proliferation has not been fully investigated. We therefore hypothesised that IL‐6 impairs the biological activity of IGF‐1 in skeletal muscle through inhibiting its signalling pathways, ERK1/2 and Akt. Our aim was to examine the combined effects of these factors on models of muscle wasting, with objectives to examine skeletal muscle differentiation and proliferation using the murine C2 skeletal muscle cell line. Cells were cultured with DM, IGF‐1 and IL‐6 alone (control treatments), or co‐cultured with IGF‐1/IL‐6. Co‐incubation of C2 cells in IGF‐1 plus IL‐6 resulted in maximal cell death (22 ± 4%; P < 0.005) compared with control treatments (14 ± 2.9%). This was also confirmed by cyclin D1 expression levels in co‐incubation treatments (7 ± 3.5%; P < 0.05) compared with control treatments (∼23%). The expression levels of myogenic‐specific transcriptional factor mRNAs (myoD and myogenin) were also significantly (P < 0.005) reduced by 70% and 90%, respectively, under the co‐incubation regimes, compared with control treatments. Signalling investigations showed significant phosphorylation reduction by 20%, (P < 0.05) of ERK1/2 and Akt in co‐incubation treatments relative to either treatment alone. Expression studies for SOCS‐3 (1.6‐fold ± 0.08, P < 0.05) and IRS‐1 (0.65‐fold ± 0.13 P < 0.005) mRNAs showed significant elevation and reduction for both genes, respectively, in co‐treatments relative to control treatments. These data may suggest that IL‐6 exerts its inhibitory effects on IGF‐1 signalling pathways (ERK1/2 and Akt) through blocking its receptor substrate IRS‐1 by SOCS‐3. J. Cell. Biochem. 113: 923–933, 2012.

PD98059 enhances C2 myoblast differentiation through p38 MAPK activation: a novel role for PD98059.

Cell differentiation is usually accompanied by irreversible cell cycle exit, which is a critical step for skeletal muscle differentiation. We therefore hypothesise that PD98059 that blocks the MAP kinase kinase (MEK) pathway (proliferation pathway) when administrated to murine C2 skeletal myoblasts will arrest cell cycle and, consequently, enhances differentiation relative to untreated controls. In this study, we aimed to examine this hypothesis using phenotypic differentiation, biochemical assays, flow cytometry and real-time PCR in C2 cells cultured for 48 h in differentiation media only (untreated) or supplemented with either a single dose of 10 ng/ml IGF-I or 20 muM PD98059 for 48 h. Creatine kinase (CK) activity was increased by 7.5-fold (P<0.05) in the presence of PD98059, whereas untreated and IGF-I-treated cells induced 4.5- and 4-fold increase respectively when compared with baseline controls. Increased CK values in the presence of PD98059 were not only associated with myotube formation but also associated with cell cycle arrest in G1 phase (86+/-3.2%; P<0.05). Moreover, the expression of myogenic-specific transcriptional factor mRNAs (MyoD and myogenin) was significantly higher in PD-treated cells (4.7+/-0.15 and 314+/-10.2 ng/reaction respectively; P<0.05) than untreated (2.0+/-0.2 and 233+/-11 ng/reaction respectively) or IGF-treated cells (3.2+/-0.24 and 296+/-16.2 ng/reaction respectively). Unexpectedly, Id3 mRNA, the potent negative regulator of muscle differentiation, was also expressed at significantly higher levels in PD-treated cells (77+/-0.346 ng/reaction; P<0.05) than untreated (49+/-7.7 ng/reaction) or IGF-I-treated cells (47+/-0.7 ng/reaction). Furthermore, expression of the muscle differentiation-specific genes (IGF-binding protein-5, IGF-II receptor and IGF-II) was also increased significantly in PD-treated cells when compared with untreated cells. Phosflow analysis showed a significant increase in the levels of phosphorylation of p38 mitogen-activated protein kinase (49.0+/-6.7%, P<0.05) in PD-treated cells when compared with DM-treated cells (31.7+/-5.7%). These findings uncover a previously unconsidered positive effect of PD98059 on C2 myoblast differentiation and identify the pathway(s) underlying PD-induced C2 myoblast differentiation.