non-invasive-assessment-of-dorsiflexor-muscle-function-in-mice

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Deakin University

Skeletal muscle tissue undergoes adaptive changes in response to stress and the genes that control these processes are incompletely characterised. NDRG2 (N-myc downstream-regulated gene 2), a stress- and growth-related gene, was investigated in skeletal muscle growth and adaption. While NDRG2 expression levels were found to be up-regulated in both differentiated human and mouse myotubes compared with undifferentiated myoblasts, the suppression of NDRG2 in C2C12 myoblasts resulted in slowed myoblast proliferation. The increased expression levels of the cell cycle inhibitors, p21 Waf1/Cip1 and p27 Kip1, and of various muscle differentiation markers in NDRG2-deficient myoblasts indicate that a lack of NDRG2 promoted cell cycle exiting and the onset of myogenesis. Furthermore, the analysis of NDRG2 regulation in C2C12 myotubes treated with catabolic and anabolic agents and in skeletal muscle from human subjects following resistance exercise training revealed NDRG2 gene expression to be down-regulated during hypertrophic conditions, and conversely, up-regulated during muscle atrophy. Together, these data demonstrate that NDRG2 expression is highly responsive to different stress conditions in skeletal muscle and suggest that the level of NDRG2 expression may be critical to myoblast growth and differentiation.

NDRG2, a novel regulator of myoblast proliferation, is regulated by anabolic and catabolic factors.

The stress-responsive, tumor suppressor N-myc downstream-regulated gene 2 (Ndrg2) is highly expressed in striated muscle. In response to anabolic and catabolic signals, Ndrg2 is suppressed and induced, respectively, in mouse C2C12 myotubes. However, little is known about the mechanisms regulating Ndrg2 expression in muscle, as well as the biological role for Ndrg2 in differentiated myotubes. Here, we show that Ndrg2 is a target of a peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) and estrogen-related receptor alpha (ERRα) transcriptional program and is induced in response to endurance exercise, a physiological stress known also to increase PGC-1α/ERRα activity. Analyses of global gene and protein expression profiles in C2C12 myotubes with reduced levels of NDRG2, suggest that NDRG2 affects muscle growth, contractile properties, MAPK signaling, ion and vesicle transport and oxidative phosphorylation. Indeed, suppression of NDRG2 in myotubes increased protein synthesis and the expression of fast glycolytic myosin heavy chain isoforms, while reducing the expression of embryonic myosin Myh3, other contractile-associated genes and the MAPK p90 RSK1. Conversely, enhanced expression of NDRG2 reduced protein synthesis, and furthermore, partially blocked the increased protein synthesis rates elicited by a constitutively active form of ERRα. In contrast, suppressing or increasing levels of NDRG2 did not affect mRNA expression of genes involved in mitochondrial biogenesis that are regulated by PGC-1α or ERRα. This study shows that in C2C12 myotubes Ndrg2 is a novel PGC-1α/ERRα transcriptional target, which influences protein turnover and the regulation of genes involved in muscle contraction and function. 

Ndrg2 is a PGC-1α/ERRα target gene that controls protein synthesis and expression of contractile-type genes in C2C12 myotubes.

Deuterated water (²H₂O), a stable isotopic tracer, provides a convenient and reliable way to label multiple cellular biomass components (macromolecules), thus permitting the calculation of their synthesis rates. Here, we have combined ²H₂O labelling, GC-MS analysis and a novel cell fractionation method to extract multiple biomass components (DNA, protein and lipids) from the one biological sample, thus permitting the simultaneous measurement of DNA (cell proliferation), protein and lipid synthesis rates. We have used this approach to characterize the turnover rates and metabolism of a panel of mammalian cells in vitro (muscle C2C12 and colon cancer cell lines). Our data show that in actively-proliferating cells, biomass synthesis rates are strongly linked to the rate of cell division. Furthermore, in both proliferating and non-proliferating cells, it is the lipid pool that undergoes the most rapid turnover when compared to DNA and protein. Finally, our data in human colon cancer cell lines reveal a marked heterogeneity in the reliance on the de novo lipogenic pathway, with the cells being dependent on both 'self-made' and exogenously-derived fatty acid.

Article	Year
NDRG2, a novel regulator of myoblast proliferation, is regulated by anabolic and catabolic factors. The Journal of physiology\| PubMed ID: 19204049	2009
Ndrg2 is a PGC-1α/ERRα target gene that controls protein synthesis and expression of contractile-type genes in C2C12 myotubes. Biochimica et biophysica acta\| PubMed ID: 24008097	2013
Analysis of Mammalian Cell Proliferation and Macromolecule Synthesis Using Deuterated Water and Gas Chromatography-Mass Spectrometry. Metabolites\| PubMed ID: 27754354	2016

Victoria C. Foletta

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