Amarjit Saini

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.

Longevity and skeletal muscle mass: the role of IGF signalling, the sirtuins, dietary restriction and protein intake.

Advancing age is associated with a progressive loss of skeletal muscle (SkM) mass and function. Given the worldwide aging demographics, this is a major contributor to morbidity, escalating socio‐economic costs and ultimately mortality. Previously, it has been established that a decrease in regenerative capacity in addition to SkM loss with age coincides with suppression of insulin/insulin‐like growth factor signalling pathways. However, genetic or pharmacological modulations of these highly conserved pathways have been observed to significantly enhance life and healthspan in various species, including mammals. This therefore provides a controversial paradigm in which reduced regenerative capacity of skeletal muscle tissue with age potentially promotes longevity of the organism. This paradox will be assessed and considered in the light of the following: (i) the genetic knockout, overexpression and pharmacological models that induce lifespan extension (e.g. IRS‐1/s6K KO, mTOR inhibition) versus the important role of these signalling pathways in SkM growth and adaptation; (ii) the role of the sirtuins (SIRTs) in longevity versus their emerging role in SkM regeneration and survival under catabolic stress; (iii) the role of dietary restriction and its impact on longevity versus skeletal muscle mass regulation; (iv) the crosstalk between cellular energy metabolism (AMPK/TSC2/SIRT1) and survival (FOXO) versus growth and repair of SkM (e.g. AMPK vs. mTOR); and (v) the impact of protein feeding in combination with dietary restriction will be discussed as a potential intervention to maintain SkM mass while increasing longevity and enabling healthy aging.

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.

Evidence for Vitamin D Receptor Expression and Direct Effects of 1α,25(OH)2D3 in Human Skeletal Muscle Precursor Cells

Presence of the vitamin D receptor and direct effects of vitamin D on the proliferation and differentiation of muscle precursor cells have been demonstrated in animal models. However, the effects and mechanisms of vitamin D actions in human skeletal muscle, and the presence of the vitamin D receptor in human adult skeletal muscle, remain to be established. Here, we investigated the role of vitamin D in human muscle cells at various stages of differentiation. We demonstrate that the components of the vitamin D-endocrine system are readily detected in human muscle precursor cells but are low to nondetectable in adult skeletal muscle and that human muscle cells lack the ability to convert the inactive vitamin D-metabolite 25-hydroxy-vitamin D3 to the active 1α,25-dihydroxy-vitamin D3 (1α,25(OH)2D3). In addition, we show that 1α,25(OH)2D3 inhibits myoblast proliferation and differentiation by altering the expression of cell cycle regulators and myogenic regulatory factors, with associated changes in forkhead box O3 and Notch signaling pathways. The present data add novel information regarding the direct effects of vitamin D in human skeletal muscle and provide functional and mechanistic insight to the regulation of myoblast cell fate decisions by 1α,25(OH)2D3.

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.

Adult stem cells: the therapeutic potential of skeletal muscle

Embryonic stem cells have revolutionised our understanding of normal and deregulated growth and development. The potential to produce cells and tissues as needed offers enormous therapeutic potential. The use of these cells, however, is accompanied by ongoing ethical, religious and biomedical issues. The expansion potential and plasticity of adult stem cells have therefore received much interest. Adult skeletal muscle is highly adaptable, responding to both the hypertrophic and degenerative stresses placed upon it. This extreme plasticity is in part regulated by resident stem cells. In addition to regenerating muscle, if exposed to osteogenic or adipogenic inducers, these cells spontaneously form osteoblasts or adipocytes. The potential for and heterogeneity of muscle stem cells is underscored by the observation that CD45+ muscle side population cells are capable of reconstituting bone marrow in lethally irradiated mice and of contributing to neo-vascularisation of regenerating muscle. Finally, first attempts to replace infarcted myocardium relied on injection of skeletal myoblasts into the heart. Cells successfully engrafted and cardiac function was improved. Harnessing their differentiation/trans-differentiation capacity provides enormous potential for adult stem cells. In this review, current understanding of the different stem cells within muscle will be discussed as will their potential utility for regenerative medicine.

A Semi-automated Programme for Tracking Myoblast Migration Following Mechanical Damage: Manipulation by Chemical Inhibitors

Background: Potential roles for undifferentiated skeletal muscle stem cells or satellite cells in muscle hypertrophy and repair have been reported, however, the capacity, the mode and the mechanisms underpinning migration have not been investigated. We hypothesised that damaged skeletal myoblasts would elicit a mesenchymal-like migratory response, which could be precisely tracked and subsequently manipulated. Methods: We therefore established a model of mechanical damage and developed a MATLABTM tool to measure the migratory capacity of myoblasts in a non-subjective manner. Results: Basal migration following damage was highly directional, with total migration distances of 948µm ± 239µm being recorded (average 0-24 hour distances: 491µm ± 113µm and 24-48 hour distances: 460µm ± 218µm). Pharmacological inhibition of MEK or PI3-K using PD98059 (20µM) or LY294002 (5µm), resulted in significant reduction of overall cell migration distances of 38% (p<0.001) and 39.5% (p<0.0004), respectively. Using the semi-automated cell tracking using MATLABTM program we validated that not only was migration distance reduced as a consequence of reduced cell velocity, but critically also as a result of altered directionality of migration. Conclusion: These studies demonstrate that murine myoblasts in culture migrate and provide a good model for studying responsiveness to damage in vitro. They illustrate for the first time the powerful tool that MATLABTM provides in determining that both velocity and directional capacity influence the migratory potential of cellular movement with obvious implications for homing and for metastases.

C2 Skeletal Myoblast Survival, Death, Proliferation and Differentiation: Regulation by Adra1d

IGF-I positively impacts on muscle anabolism/regeneration. Using C2 skeletal myoblasts, we previously reported high dose TNF-α -induced (10 ng.ml-1) cell death is rescued by IGF-I. However, non-myotoxic low dose TNF-α (1.25 ng.ml-1) elicits a MAPK-mediated apoptotic response when co-incubated with IGF-I (1.5 ng.ml-1). Our aim was to investigate these conflicting roles of IGF-I in our model. Insulin array and qRT-PCR identified Adra1d as a potential regulatory gene that was up-regulated in survival and down-regulated under apoptotic conditions. TNF-α administration (1.25 or 10 ng.ml-1) induced significant decreases (∼50% both incubations) in Adra1d expression relative to DM. IGF-I addition to high dose TNF-α (10 ng.ml-1) induced myoblast survival and matched a significant (P < 0.05) increase in Adra1d expression. By contrast, IGF-I addition to low dose TNF-α (1.25 ng.ml-1) induced elevated death resulting in a significant (P < 0.05) decline (∼55%) in Adra1d expression. Pre-administration of PD98059 (20 uM), which rescues death induced by co-incubation of low dose TNF-α with IGF-I, Adra1d levels were again comparable to DM control. Since Adra1d was elevated following incubations that induced myoblast survival, we investigated effects of Adra1d siRNA gene silencing under these conditions. Adra1d knockdown resulted in significantly higher levels of cell death under all incubations suggesting Adra1d expression is essential for skeletal muscle cell survival.

Omega-3 fatty acid EPA improves regenerative capacity of mouse skeletal muscle cells exposed to saturated fat and inflammation

Sarcopenic obesity is characterised by high fat mass, low muscle mass and an elevated inflammatory environmental milieu. We therefore investigated the effects of elevated inflammatory cytokine TNF-α (aging/obesity) and saturated fatty acid, palmitate (obesity) on skeletal muscle cells in the presence/absence of EPA, a-3 polyunsaturated fatty acid with proposed anti-inflammatory, anti-obesity activities. In the present study we show that palmitate was lipotoxic, inducing high levels of cell death and blocking myotube formation. Cell death under these conditions was associated with increased caspase activity, suppression of differentiation, reductions in both creatine kinase activity and gene expression of myogenic factors; IGF-II, IGFBP-5, MyoD and myogenin. However, inhibition of caspase activity via administration of Z-VDVAD-FMK (caspase-2), Z-DEVD-FMK (caspase-3) and ZIETD-KMK (caspase 8) was without effect on cell death. By contrast, lipotoxicity associated with elevated palmitate was reduced with the MEK inhibitor PD98059, indicating palmitate induced cell death was MAPK mediated. These lipotoxic conditions were further exacerbated in the presence of inflammation via TNF-α co-administration. Addition of EPA under cytotoxic stress (TNF-α) was shown to partially rescue differentiation with enhanced myotube formation being associated with increased MyoD, myogenin, IGF-II and IGFBP-5 expression. EPA had little impact on the cell death phenotype observed in lipotoxic conditions but did show benefit in restoring differentiation under lipotoxic plus cytotoxic conditions. Under these conditions Id3 (inhibitor of differentiation) gene expression was inversely linked with survival rates, potentially indicating a novel role of EPA and Id3 in the regulation of apoptosis in lipotoxic/cytotoxic conditions. Additionally, signalling studies indicated the combination of lipo- and cyto-toxic effects on the muscle cells acted through ceramide, JNK and MAPK pathways and blocking these pathways using PD98059 (MEK inhibitor) and Fumonisin B1 (ceramide inhibitor) significantly reduced levels of cell death. These findings highlight novel pathways associated with in vitro models of lipotoxicity (palmitate-mediated) and cytotoxicity (inflammatory cytokine mediated) in the potential targeting of molecular modulators of sarcopenic obesity.