The expansion of myogenic progenitors (MPs) in the adult muscle stem cell niche is critical for the regeneration of skeletal muscle. Activation of quiescent MPs depends on the dismantling of the basement membrane and increased access to growth factors such as fibroblast growth factor-2 (FGF2). Here, we demonstrate using microRNA (miRNA) profiling in mouse and human myoblasts that the capacity of FGF2 to stimulate myoblast proliferation is mediated by miR-29a. FGF2 induces miR-29a expression and inhibition of miR-29a using pharmacological or genetic deletion decreases myoblast proliferation. Next generation RNA sequencing from miR-29a knockout myoblasts (Pax7 CE/1 ; miR-29a flox/flox ) identified members of the basement membrane as the most abundant miR-29a targets. Using gain-and loss-of-function experiments, we confirm that miR-29a coordinately regulates Fbn1, Lamc1, Nid2, Col4a1, Hspg2 and Sparc in myoblasts in vitro and in MPs in vivo. Induction of FGF2 and miR-29a and downregulation of its target genes precedes muscle regeneration during cardiotoxin (CTX)-induced muscle injury. Importantly, MP-specific tamoxifen-induced deletion of miR-29a in adult skeletal muscle decreased the proliferation and formation of newly formed myofibers during both CTX-induced muscle injury and after a single bout of eccentric exercise. Our results identify a novel miRNAbased checkpoint of the basement membrane in the adult muscle stem cell niche. Strategies targeting miR-29a might provide useful clinical approaches to maintain muscle mass in disease states such as ageing that involve aberrant FGF2 signaling. STEM CELLS 2016;34:768-780 SIGNIFICANCE STATEMENTSkeletal muscle mass and function is critical for the maintenance of health, and the decline of muscle mass during aging inversely correlates with mortality. Adult muscle stem cells provide an important target for strategies to maintain muscle mass, but the molecular mechanisms that control the activation of these stem cells in vivo are still incompletely understood. We demonstrate that microRNA-29a is a novel downstream target of FGF2, and that miR-29a mediates the dismantling of the basement membrane in the adult muscle stem cell niche that is required for the proliferation of myogenic progenitors during muscle regeneration. We propose the FGF2/miR-29a pathway as a novel target to prevent a decrease in muscle mass in disease states such as ageing where FGF2 signaling is overly activated.
BackgroundPseudoalteromonas haloplanktis is a cold-adapted γ-proteobacterium isolated from Antarctic sea ice. It is characterized by remarkably high growth rates at low temperatures. P. haloplanktis is one of the model organisms of cold-adapted bacteria and has been suggested as an alternative host for the soluble overproduction of heterologous proteins which tend to form inclusion bodies in established expression hosts. Despite the progress in establishing P. haloplanktis as an alternative expression host the cell densities obtained with this organism, which is unable to use glucose as a carbon source, are still low. Here we present the first fed-batch cultivation strategy for this auspicious alternative expression host.ResultsThe key for the fed-batch cultivation of P. haloplanktis was the replacement of peptone by casamino acids, which have a much higher solubility and allow a better growth control. In contrast to the peptone medium, on which P. haloplanktis showed different growth phases, on a casamino acids-containing, phosphate-buffered medium P. haloplanktis grew exponentially with a constant growth rate until the stationary phase. A fed-batch process was established by feeding of casamino acids with a constant rate resulting in a cell dry weight of about 11 g l-1 (OD540 = 28) which is a twofold increase of the highest densities which have been obtained with P. haloplanktis so far and an eightfold increase of the density obtained in standard shake flask cultures.The cell density was limited in the fed-batch cultivation by the relatively low solubility of casamino acids (about 100 g l-1), which was proven by pulse addition of casamino acid powder which increased the cell density to about 20 g l-1 (OD540 = 55).ConclusionThe growth of P. haloplanktis to higher cell densities on complex medium is possible. A first fed-batch fermentation strategy could be established which is feasible to be used in lab-scale or for industrial purposes. The substrate concentration of the feeding solution was found to influence the maximal biomass yield considerably. The bottleneck for growing P. haloplanktis to high cell densities still remains the availability of a highly concentrated substrate and the reduction of the substrate complexity. However, our results indicate glutamic acid as a major carbon source, which provides a good basis for further improvement of the fed-batch process.
Akt kinases are important mediators of the insulin signal, and some Akt substrates are directly involved in glucose homeostasis. Recently, Girdin has been described as an Akt substrate that is expressed ubiquitously in mammals. Cells overexpressing Girdin show an enhanced Akt activity. However, not much is known about Girdin's role in insulin signaling. We therefore analyzed the role of Girdin in primary human myotubes and found a correlation between Girdin expression and insulin sensitivity of the muscle biopsy donors, as measured by a hyperinsulinemic-euglycemic clamp. To understand this finding on a cellular level, we then investigated the function of Girdin in C2C12 mouse myoblasts. Girdin knock-down reduced Akt and insulin receptor substrate-1 phosphorylation. In contrast, stable overexpression of Girdin in C2C12 cells strikingly increased insulin sensitivity through a massive upregulation of the insulin receptor and enhanced tyrosine phosphorylation of insulin receptor substrate-1. Furthermore, Akt and c-Abl kinases were constitutively activated. To investigate medium-term insulin responses we measured glucose incorporation into glycogen. The Girdin overexpressing cells showed a high basal glycogen synthesis that peaked already at 1nM insulin. Taken together, we characterized Girdin as a new and major regulator of the insulin signal in myoblasts and skeletal muscle.
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