Ranolazine promotes muscle differentiation and reduces oxidative stress in C2C12 skeletal muscle cells

Figures

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  Fig. 1. RAN effect on cell growth. a Experimental scheme for growth curve and viability determination. b C2C12 cells were seeded in 60 × 15 mm culture dishes at 20 % confluence and grown in GM medium with or without RAN, and DM medium. Medium was changed every 24 h and the experiment lasted until control cells achieved 40 % of confluence (3 days). As shown in panel b, RAN increases C2C12 proliferative potential without cytotoxic effects. c To confirm the results obtained by growth curve, we analyzed p53 protein content: Western blot analysis (panel c) shows the p53 significant increment in DM condition, confirming an inhibition of the cell cycle only in this condition, as reported in literature. In GM and GM-RAN cells p53 levels remain comparable with basal condition. d Experimental scheme for RAN treatment during proliferative, differentiation and post differentiation phases. Representative immunoblots of analyzed proteins are shown. Anova test: >p ≤ 0.05, t test: * p ≤ 0.05 vs. GM

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  Fig. 2. RAN action on proliferative myoblasts: 24 h. To investigate whether RAN could enhance myogenic phenotype acquisition, we analyzed myoblasts morphology and Myf5, p21 and MyoD protein expression. Immunofluorescence images and quantification (20X): RAN increased the signal of Myf5, p21 and MyoD proteins in respect of GM and DM at 24 h. These images showed the important morphological changes and the different localization of the signal in myoblasts treated with RAN with respect to GM and DM control. Objective: 20X. t test: *p ≤ 0.05 or **0.01 vs. GM

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  Fig. 3. RAN action on proliferative myoblasts: 48–72 h. Intracellular calcium signaling and p70S6 kinase play an important role in the early stages of myogenesis. We studied whether RAN could act on CaMKII protein expression and p70S6 activation during proliferation phase. a Immunofluorescence analysis and quantification (20X): RAN increased the signal of CaMKII protein expression compared with GM at 48 h. b Phalloidin staining and phase contrast images corroborated the morphological changes observed in proliferative myoblasts (Fig. 2). c Western blot analysis: RAN significantly increased MyoD and p21 protein content at 72 h of proliferation. d Western blot analysis: RAN did not induce myoblast proliferation activating p70S6 kinase at 72 h. Representative immunoblots are shown. Objective: 20X. Anova test: >p ≤ 0.05, t test: *p ≤ 0.05 or **0.01 vs. GM

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  Fig. 4. RAN action on mitochondria in C2C12 myoblasts. We analyzed RAN action on mitochondria and ROS levels during the proliferation phase utilizing a specific staining procedure: a b, c MITO CytoPainter staining (40X): in all proliferative phases (a 24 h, b 48 h, c 72 h), RAN did not modify the number of mitochondria compared with control (GM and DM) as shown in quantification (e). d Cell Rox® staining (20X): RAN appears to be able to decrease ROS synthesis in the basal state (f) and after H2O2-induced oxidative stress

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  Fig. 5. RAN action during differentiation process. To study RAN effects during differentiation, 70 % confluent C2C12 cells were placed in DM with RAN. RAN was not added in the medium (DM) in the control cells. In our in vitro differentiation model, early myotubes appeared after 24–48 h and neo myotubes formation was completed in 72 h. To analyze whether RAN could promote hypertrophy process in neo-formed myotubes, we also treated cells with RAN for 24 h. RAN was not added in the control cells. a Western blot quantification data during the differentiation process indicated that RAN significantly improved MyHC and Myogenin protein levels, but did not activate p70S6 and ERK kinases. b Phase contrast images at the end of differentiation showed how RAN increased myotubes dimension, as reported in the graph of myotubes area quantification. Objective: 20X. c In neo-formed myotubes, RAN raises MyHC protein amount. RAN decreases p70S6 and ERKs kinases activation. Representative immunoblots are shown. Anova test: >p ≤ 0.05, t test: *p ≤ 0.05 or **0.01 vs. DM or vs. DMt = 72 h, or °p ≤ 0.05 vs. DM96 h (c)

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  Fig. 6. RAN action during the differentiation process: morphological studies. a An important marker of muscle differentiation progression is the increase of MyHC positive cells. MyHC immunofluorescence (20X) indicated that in RAN-treated cells the number of MyHC-positive myotubes was significantly higher compared with control (24 h), as reported in the quantification graph. b During the proliferation phase we observed that RAN could influence CaMKII signaling. In the same manner, after 48 h of differentiation, RAN could improve pCaMKII protein expression compared with DM control (20X), as reported in the quantification. t test: *p ≤ 0.05 vs. DM

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  Fig. 7. RAN action on mitochondria during differentiation and on OPN protein. We evaluated RAN action on mitochondria during the differentiation phase by specific staining procedure: a, b, c Mito CytoPainter assay and quantification: RAN treatment did not change the number of activated mitochondria [33,34] compared with DM control, in all differentiation phases (a 24 h, b 48 h, c 72 h). Objective: 40X. d OPN has been described as a component of the inflammation of skeletal muscle. After 72 h of differentiation RAN treatment significant decreased OPN protein expression compared with DM control (20X), as reported in the quantification graph. t test: *p ≤ 0.05 or **0.01 vs. DM