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The identification of non-coding RNA species, previously thought of as 'junk' DNA, adds a new dimension of complexity to the regulation of DNA, RNA and protein. Micro RNAs are short non-coding RNA species that control gene expression, are dysregulated in settings of cardiac and skeletal muscle disease and have emerged as promising therapeutic targets. Micro RNAs specifically enriched in cardiac and skeletal muscle are called myomiRs and play an important role in cardiac pathology and skeletal muscle biology. Moreover, micro RNA profiles are altered in response to exercise and disease; thus, their potential as therapeutic drug targets is being widely explored. In the cardiovascular field, therapeutic inhibition of micro RNAs has been shown to be effective in improving cardiac outcome in preclinical cardiac disease models. Micro RNAs that promote skeletal muscle regeneration are attractive therapeutic targets in muscle wasting conditions where regenerative capacity is compromised. [ABSTRACT FROM AUTHOR]

In models of cancer cachexia, inhibiting type IIB activin receptors (ActRIIBs) reverse muscle wasting and prolongs survival, even with continued tumor growth. ActRIIB mediates signaling of numerous TGF-β proteins; of these, we demonstrate that activins are the most potent negative regulators of muscle mass. To determine whether activin signaling in the absence of tumor-derived factors induces cachexia, we used recombinant serotype 6 adeno-associated virus (rAAV6) vectors to increase circulating activin A levels in C57BL/6 mice. While mice injected with control vector gained ~10% of their starting body mass (3.8±0.4 g) over 10 wk, mice injected with increasing doses of rAAV6:activin A exhibited weight loss in a dose-dependent manner, to a maximum of --12.4% ( --4.2±1.1 g). These reductions in body mass in rAAV6:activin-injected mice correlated inversely with elevated serum activin A levels (7- to 24-fold). Mechanistically, we show that activin A reduces muscle mass and function by stimulating the ActRIIB pathway, leading to deleterious consequences, including increased transcription of atrophy-related ubiquitin ligases, decreased Akt/mTOR-mediated protein synthesis, and a profibrotic response. Critically, we demonstrate that the muscle wasting and fibrosis that ensues in response to excessive activin levels is fully reversible. These findings highlight the therapeutic potential of targeting activins in cachexia.--Chen, J. L., Walton, K. L., Winbanks, C. E., Murphy, K. T., Thomson, R. E., Makanji, Y., Qian, H., Lynch, G. S., Harrison, C. A., Gregorevic, P. Elevated expression of activins promotes muscle wasting and cachexia. [ABSTRACT FROM AUTHOR]

Recognised by their de novo expression of alpha-smooth muscle actin (SMA), recruitment of myofibroblasts is key to the pathogenesis of fibrosis in chronic kidney disease. Increasingly, we realise that epithelial-mesenchymal transition (EMT) may be an important source of these cells. In this study we describe a novel model of renal EMT. Rat kidney explants were finely diced on gelatin-coated Petri dishes and cultured in serum-supplemented media. Morphology and immunocytochemistry were used to identify mesenchymal (vimentin+, α-smooth muscle actin (SMA)+, desmin+), epithelial (cytokeratin+), and endothelial (RECA+) cells at various time points. Cell outgrowths were all epithelial in origin (cytokeratin+) at day 3. By day 10, 50±12% (mean±SE) of cytokeratin+ cells double-labelled for SMA, indicating EMT. Lectin staining established a proximal tubule origin. By day 17, cultures consisted only ofmyofibroblasts (SMA+/cytokeratin-). Explanting is a reproducible ex vivo model of EMT. The ability tomodify this change in phenotype provides a useful tool to study the regulation andmechanisms of renal tubulointerstitial fibrosis. [ABSTRACT FROM AUTHOR]

Although the canonical transforming growth factor β signaling pathway represses skeletal muscle growth and promotes muscle wasting, a role in muscle for the parallel bone morphogenetic protein (BMP) signaling pathway has not been defined. We report, for the first time, that the BMP pathway is a positive regulator of muscle mass. Increasing the expression of BMP7 or the activity of BMP receptors in muscles induced hypertrophy that was dependent on Smad1/5-mediated activation of mTOR signaling. In agreement, we observed that BMP signaling is augmented in models of muscle growth. Importantly, stimulation of BMP signaling is essential for conservation of muscle mass after disruption of the neuromuscular junction. Inhibiting the phosphorylation of Smad1/5 exacerbated denervation-induced muscle atrophy via an HDAC4-myogenin-dependent process, whereas increased BMP-Smad1/5 activity protected muscles from denervation-induced wasting. Our studies highlight a novel role for the BMP signaling pathway in promoting muscle growth and inhibiting muscle wasting, which may have significant implications for the development of therapeutics for neuromuscular disorders.

MicroRNAs (miRs) are emerging as prominent players in the regulation of many biological processes, including myogenic commitment and skeletal muscle formation. Members of the TGF-β family can influence the proliferation and myogenic differentiation of cells, although it is presently not clear what role miRNAs play in the TGF-β-mediated control of myogenic differentiation. Here, we demonstrate in the myogenic C2C12 cell line, and in primary muscle cells, that miR-206 and miR-29-two miRs that act on transcriptional events implicated in muscle differentiation are down-regulated by TGF-β. We further demonstrate that TGF-β treatment of myogenic cells is associated with increased expression of histone deacetylase 4 (HDAC4), a key inhibitor of muscle differentiation that has been identified as a target for regulation by miR-206 and miR-29. We confirmed that increased expression of miR-206 and miR-29 resulted in the translational repression of HDAC4 in the presence or absence of TGF-β via interaction with the HDAC4 3'-untranslated region. Importantly, we found that miR-206 and miR-29 can attenuate the inhibitory actions of TGF-β on myogenic differentiation. Furthermore, we present evidence that the mechanism by which miR-206 and miR-29 can inhibit the TGF-β-mediated up-regulation of HDAC4 is via the inhibition of Smad3 expression, a transducer of TGF-β signaling. These findings identify a novel mechanism of interaction between TGF-β and miR-206 and -29 in the regulation of myogenic differentiation through HDAC4.

Glycoconjugates are complex macromolecules present in all tissues throughout the body. Depending on the tissue region, glycoconjugates express different carbohydrate moieties, which can be used to both distinguish cell type and examine changes in cell phenotype.Although the periodic acid-schiff (PAS) method has long been used to study the distribution of glycoconjugates, the usefulness of the technique is severely limited by its lack of specificity. A more specific technique makes use of the affinity that plant-derived lectins have for different carbohydrate moieties in glycoconjugates. Binding of lectins is therefore a particularly useful adjunct to conventional histology when it is important to characterise cell type. These well-characterised binding patterns have proved particularly valuable in helping us understand the pathogenesis of kidney disease, changes in cell surface carbohydrates on normal and neoplastic cells in tumours, and blood group biology.When labeled with a reporter molecule such as biotin or gold, lectin binding can be easily identified using light and electron microscopy. In this chapter, we describe the appropriate experimental protocols for light and electron microscopic examination of lectin binding, emphasising their utility in characterising nephron segments in renal disease.