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Pharmacological inhibition of myostatin/TGF-? receptor/pSmad3 signaling rescues muscle regenerative responses in mouse model of type 1 diabetes.

ABSTRACT: To study the influence of acute experimental diabetes on the regenerative potential of muscle stem (satellite) cells in mice.Male C57BL/6 young mice were injected with a single dose of streptozotocin (STZ, 180 mg/kg, ip) to induce diabetes. The diabetic mice were treated with insulin (0.75 U/kg, ip), follistatin (12 ?g/kg, im) or Alk5 inhibitor (5 ?mol/L per kg, sc) once a day. On the first day when high glucose levels were found, cardiotoxin (CTX) was focally injected into tibialis anterior and gastronemius muscles of the mice. The muscles were harvested 3 d and 5 d after CTX injection, and myofibers and satellite cells were isolated. Quantitative ex-vivo and in-vivo assays of myogenic potential were used to evaluate the muscle regenerative responses.The satellite cells from the diabetic mice 3 d after CTX injection fail to activate, and the repair of muscle deteriorates, resembling that observed in old control mice. Furthermore, the satellite cells have excessive levels of myostatin, TGF-? receptor 1, pSmad3 and the cell cycle inhibitor p15, while the level of TGF-?1 remain unchanged. Treatment of the diabetic mice with insulin rescued muscle regenerative responses, and restored the expression levels of myostatin, TGF-? receptor 1, pSmad3, and p15 to those similar of healthy controls. Treatment of the diabetic mice with the myostatin antagonist follistatin, or with the Alk5 inhibitor of TGF-? receptor 1 (which did not diminish the blood glucose levels) rescued muscle regenerative responses and attenuated the myostatin/TGF? receptor/pSmad3 signaling.The muscle regenerative responses are incapacitated and repair of the tissue fails within hours after the initiation of hyperglycemia in a mouse model of type 1 diabetes, but stem cell function is rescued by insulin, as well as follistatin or an Alk5 inhibitor that blocks TGF-? receptor signaling.

SUBMITTER: Jeong J

PROVIDER: S-EPMC4003029 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Pharmacological inhibition of myostatin/TGF-β receptor/pSmad3 signaling rescues muscle regenerative responses in mouse model of type 1 diabetes.

Jeong Jaemin J Conboy Michael J MJ Conboy Irina M IM

Acta pharmacologica Sinica 20130617 8

<h4>Aim</h4>To study the influence of acute experimental diabetes on the regenerative potential of muscle stem (satellite) cells in mice.<h4>Methods</h4>Male C57BL/6 young mice were injected with a single dose of streptozotocin (STZ, 180 mg/kg, ip) to induce diabetes. The diabetic mice were treated with insulin (0.75 U/kg, ip), follistatin (12 μg/kg, im) or Alk5 inhibitor (5 μmol/L per kg, sc) once a day. On the first day when high glucose levels were found, cardiotoxin (CTX) was focally injecte ...[more]

PMID: 23770987

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Project description:BackgroundshRNA lentiviral vectors are extensively used for gene knockdowns in mammalian cells, and non-target shRNAs typically are considered the proper experimental control for general changes caused by RNAi. However, the effects of non-target lentivirus controls on the modulation of cell signaling pathways remain largely unknown. In this study, we evaluated the effect of control lentiviral transduction on oxytocin receptor (OXTR) expression through the ERK/MAPK pathway in mouse and human skeletal muscle cells, on myogenic activity, and in vivo on mouse muscle regeneration. Furthermore, we mined published data for the influence of viral infections on OXTR levels in human populations and found that unrelated viral pathologies have a common consequence: diminished levels of OXTR.MethodsWe examined the change in OXTR mRNA expression upon transduction with control and Smad3-targeting viral vectors through real time RT-PCR and Western blotting, and confirmed with immunofluorescence. Changes in Smad3 and OXTR expression were examined both in vitro with mouse and human myoblasts and in vivo in mouse satellite cells. The general effects of viral infections on OXTR downregulation in humans were also examined by analyzing published Gene Expression Omnibus (GEO) datasets. The change in myoblast myogenic activity caused by the viral transduction (the percent of Pax7 + Ki67+ cells) was examined by immunofluorescence.ResultsResults shown in this work establish that lentiviral control vectors significantly downregulate OXTR expression at mRNA and protein levels and diminish key downstream effectors of OXTR, ERK signaling, reducing the myogenic proliferation of infected cells. This effect is evolutionarily conserved between mouse and human myogenic cells, and it manifests in satellite cells after control lentiviral transduction of mice in vivo. Furthermore, an examination of published datasets uncovered similar OXTR downregulation in humans that are afflicted with different viral infections. Additionally, cells transduced with Smad3-targeting shRNA downregulate OXTR even more than cells transduced with control viruses.ConclusionsOur work suggests that experimental cohorts transduced with control viruses may not behave the same as un-transduced cells and animals, specifically that control viral vectors significantly change the intensity of key cell-signaling pathways, such as OXTR/ERK. Our results further demonstrate that lentiviral transduction significantly decreases myogenic proliferation and suggest that viral infections in general may play a role in decreasing muscle health and regeneration, a decline in metabolic health, and a lower sense of well-being, as these rely on effective OXTR signaling. Additionally, our data suggest pathway crosstalk between TGF-β/pSmad3 and OXTR, implying that sustained attenuation of the TGF-β/pSmad3 pathway will reduce pro-regenerative OXTR/pERK signaling.

Dataset Information

Pharmacological inhibition of myostatin/TGF-? receptor/pSmad3 signaling rescues muscle regenerative responses in mouse model of type 1 diabetes.

Publications

Pharmacological inhibition of myostatin/TGF-β receptor/pSmad3 signaling rescues muscle regenerative responses in mouse model of type 1 diabetes.

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