Small activating RNA induces myogenic differentiation of rat adipose-derived stem cells by upregulating MyoD

ABSTRACTPurpose:RNA activation (RNAa) is a mechanism of gene activation triggered by promoter-targeted small double stranded RNAs (dsRNAs), also known as small activating RNAs (saRNAs). Myogenic regulatory factor MyoD is regarded as the master activator of myogenic differentiation cascade by binding to enhancer of muscle specific genes. Stress urinary incontinence (SUI) is a condition primarily resulted from urethral sphincter deficiency. It is thus expected that by promoting differentiation of adipose-derived stem cells (ADSCs) into myoblasts by activating MyoD gene through RNAa may offer benefits to SUI.Materials and Methods:Rats ADSCs were isolated, proliferated in vitro, and identified by flow cytometry. Purified ADSCs were then transfected with a MyoD saRNA or control transfected. Real-time polymerase chain reaction (RT-PCR) and western blotting were used to detect MyoD mRNA and protein expression, respectively. Immunocytochemical staining was applied to determine the expression of desmin protein in transfected cells. Cell viability was measured by using CellTiter 96® AQueous One Solution Cell Proliferation Assay kit.Results:Transfection of a MyoD saRNA (dsMyoD) into ADSCs significantly induced the expression of MyoD at both the mRNA and protein levels, and inhibited cell proliferation. Desmin protein expression was detected in dsMyoD treated ADSCs 2 weeks later.Conclusion:Our findings show that RNAa mediated overexpression of MyoD can promote transdifferentiation of ADSCs into myoblasts and may help treat stress urinary incontinence (SUI)–a condition primarily resulted from urethral sphincter deficiency.

Low-level laser therapy modulates musculoskeletal loss in a skin burn model in rats

PURPOSE: To investigate the effectiveness of low-level laser therapy (LLLT) on gastrocnemius muscle morphology and Myod imunoexpression in a model of dorsal burn in rats. METHODS: Sixteen male Wistar rats were distributed into two groups: control group (CG): rats submitted to scald burn injury without treatment and laser treated group (LG): rats submitted to scald burn injury and treated with laser therapy. Fourteen days post-surgery, gastrocnemius muscle was evaluated being the specimens stained with HE and morphometric data was evaluated. MyoD expression was assessed by immunohistochemistry. RESULTS: The results showed that laser treated animals presented more organized tissue morphology compared to the non-treated animals, with a higher number of nucleus in the fibers. Also, the cross sectional area of the fibers and the MyoD immunoexpression in the laser treated groups was higher. CONCLUSION: Low-level laser therapy had positive effects on gastrocnemius muscle, improving tissue muscle morphology, increasing cross sectional area and MyoD immunoexpression. .

Os efeitos do laser de baixa potência no processo de reparo muscular após criolesão em ratos / The effects of low-level laser therapy on muscle healing process after cryolesion

O objetivo deste estudo foi verificar os efeitos da laserterapia de baixa potência no comprimento de onda ?=780nm entre diferentes períodos de tratamento 7, 14 e 21 dias e verificar a dose (10J/cm2 ou 50J/cm2) que promove melhor reparo muscular através das análises histopatológicas e imunohistoquímicas. Foram utilizados 54 ratos machos divididos em 3 grupos: GC: grupo controle (criolesão, sem tratamento); G10: criolesão do músculo tibial anterior (TA) e tratados com laser dose 10J/cm² e G50: criolesão do músculo TA e tratados com laser dose 50J/cm² que foram subdivididos em 3 subgrupos (n=6): 7, 14 e 21 dias de tratamento. Os achados histopatológicos revelaram maior organização das fibras musculares dos grupos tratados com laser 10J/cm² e 50J/cm² durante os períodos 7 e 14 dias em relação ao grupo controle; no período 21 dias os grupos apresentaram semelhanças na reparação tecidual. Em relação à área da lesão os grupos tratados com laser 10J/cm² e 50J/cm² durante 7 dias obtiveram diminuição significativa (p ? 0.05) da área da lesão em relação ao grupo controle, sendo que os grupos 14 e 21 dias não apresentaram diferenças significativas entre eles. Na contagem dos vasos o grupo tratado com laser 10J/cm² no 14° dia apresentou aumento dos vasos em relação ao grupo tratado com dose 50J/cm², mas não em relação ao grupo controle. Nos tempos de 7 e 21 dias os grupos não apresentaram diferença significativa entre si. Com relação às análises imunohistoquímicas da myoD no período de 7 dias os grupos tratados com laser 10J/cm² e 50J/cm² apresentaram maior imunomarcação comparada com o grupo controle, no período 14 e 21 dias a imunomarcação estava ausente.

The objective of this study was to assess the effects of 780nm low-level laser therapy at different periods of 7, 14 and 21 days after cryolesion, including the dose (10 or 50J/cm2) to promote a better muscle repair evidenced by histopathological and immumohistochemical analyses. Fifty-four male rats were divided into three groups: injured control group (CG) - injured animals without any treatment; injured 780nm laser treated group, at 10 J/cm² (G10) and injured 780nm laser treated group, at 50 J/cm² (G50). Each group was divided into 3 subgroups (n=6): 7, 14 and 21 days post-injury. Histopathological findings revealed better-organized muscle fibers in the G10 and G50 during the periods of 7 and 14 days compared to CG. The G10 and G50 during 7 days showed a significant reduction (p? 0.05) of lesion area compared to CG, without differences between groups treated for 14 and 21 days. The G10 showed an increase of the amount of vessels after 14 days compared to the G50, but not in relation to controls. With regard to the immumohistochemical analyses of the MyoD factor, The G10 and G50 during 7 days showed higher concentrations of immunomarkers than controls.

Acetyl-CoA carboxylase beta expression mediated by MyoD and muscle regulatory factor 4 is differentially affected by retinoic acid receptor and retinoid X receptor

Mammals have two major isoforms of acetyl-CoA carboxyase (ACC). The 275 kDa beta-form (ACC beta) is predominantly in heart and skeletal muscle while the 265 kDa alpha-form (ACC alpha) is the major isoform in lipogenic tissues such as liver and adipose tissue. ACC alpha is thought to control fatty acid oxidation by means of the ability of malonyl-CoA to inhibit carnitine palmitoyl-CoA transferase-1 (CPT-1), which is a rate-limiting enzyme of fatty acid oxidation in mitochondria. Previously, it was reported that MyoD and other muscle regulating factors (MRFs) up-regulate the expression of ACC beta by interactions between these factors and several cis-elements of ACC beta promoter. We described here that ACC beta expression mediated by MRFs is regulated by retinoic acids. Endogenous expression of ACCb in differentiated H9C2 myotube was significantly increased by retinoic acid treatment. However, on transient transfection assay in H9C2 myoblast, ACC beta promoter activity was suppressed by RXRa and more severely by RAR alpha. These effects on ACCb expression in myoblasts and myotubes by RXR alpha and RAR alpha seem to be mediated by their interactions with MRFs because no consensus sequence for RXR alpha and RAR alpha has been found in ACC beta promoter and retinoic acid receptors did not affect this promoter activities by itself. In transient transfection in NIH3T3 fibroblast, the activation of ACC beta promoter by MyoD, main MRF in myoblast, was significantly suppressed by RAR alpha and to a less extent by RXR alpha while the RXR alpha drastically augmented the activation by MRF4, major MRF in myotube. These results explained that retinoic acids differentially affected the action of MRFs according to their types and RXR alpha specially elevates the expression of muscle specific genes by stimulating the action of MRF4.