Numerous studies suggest that Creatine (Cr) supplementation may help to treat various neuromuscular diseases in which mitochondrial impairment plays a causative role. Our purpose is to focus on the effect of Cr on mitochondrial responses in oxidatively-stressed, differentiating C2C12 myoblasts. Mitochondrial morphology, network and function were monitored using cytofluorimetric, ultrastructural, and molecular techniques. Oxidative insult with H2O2 (0,3 mM-1h) induced severe ultrastructural damages to the majority of mitochondria, showing typical swelling of the matrix and loss of cristae. Cr-supplementation, 3 and 10 mM Cr, 24 h prior to H2O2 treatment, was found to significantly prevent these effects in both prealoading conditions. Confocal microscopy allowed imaging of mitochondrial network morphology that in oxidatively injured myoblasts appeared fragmented. Cr supplementation seems to partially preserve the network organization in both Cr prealoading concentrations. Flow cytometric analysis of NAO/cardiolipin interaction showed that H2O2 treatment caused a significant peroxidation of cardiolipin within 24 hours post-oxidative injury; 3 and 10 mM Cr pre-loading prevented the oxidation with a protection rate of 32% and of 18%, respectively. Cytofluorimetric data also revealed either a modulation in mitochondria membrane potential per mitochondria unit using a tetramethyl rhodamine methyl ester (TMRM) as potentiometric fluorescent dye and mitotracker green (MTG) for mitochondrial mass (TMRM/MTG ratio), or mitochondrial dysfunctionmasked by a ATP synthase reverse mode operating after addition of oligomycin (5uM), an ATP synthase inhibitor. In particular, only the Cr-treated myoblasts showed an increase in TMRM /MTG ratio compared to oxidatively-stressed myoblasts, after 1 and 24h. Furthermore a significant higher ratio of oligomycin- insensitive fraction was found in 3 mM Cr-pretreated cells (1h), as compared to trolox (a reference antioxidant), strengthening the TMRM /MTG ratio data observed in Cr-preloading conditions. Thus, Cr not only reduces H2O2-induced mito dysfunction, but also acts in the absence of a relevant contribution of ATP-synthase, differently from trolox condition with slighlty differences between the two Cr concentration conditions used. Expression of nuclear encoded genes controlling mitochondrial biogenesis (PGC-1α, NRF- 1α and Tfam) were also studied by RT realtime PCR as well as mitochondrial proteins profile and western blot of specific metabolic target such us AMPK and its downstream signaling substrate acetyl-CoA carboxylase (ACC). Analysis of C2C12 cells treated with H2O2 and H2O2+Cr, showed that PGC-1α expression levels were markedly increased immediately after oxidative stress in both conditions as compared to controls, and positively correlated with the severity of mitochondrial cell damage, probably due to a compensatory cell response. Interestingly, Cr preloading increased the phosphorylation of AMPK and especially ACC. On the other hand, Cr stimulates AMPK a key energy-sensitive target that controls numerous metabolic and cellular processes. On the whole, our data indicate that under oxidative stress conditions, where the expression of PGC-1α and mitochondrial biogenesis are likely to be stimulated, Cr preloading may favour an enhanced adaptive mechanism which allows myoblasts to increase the regulation of cellular energy metabolism and minimize oxidative damage in the course of myogenesis.

Creatine
 supplementation enhances the mitochondrial function in oxidatively injured myoblasts

BARBIERI, ELENA;CALCABRINI, CINZIA;GUESCINI, MICHELE;VALLORANI, LUCIANA;LUCHETTI, FRANCESCA;CANONICO, BARBARA;CIACCI, CATERINA;GIOACCHINI, ANNA MARIA;BATTISTELLI, MICHELA;FALCIERI, ELISABETTA;STOCCHI, VILBERTO;SESTILI, PIERO
2013-01-01

Abstract

Numerous studies suggest that Creatine (Cr) supplementation may help to treat various neuromuscular diseases in which mitochondrial impairment plays a causative role. Our purpose is to focus on the effect of Cr on mitochondrial responses in oxidatively-stressed, differentiating C2C12 myoblasts. Mitochondrial morphology, network and function were monitored using cytofluorimetric, ultrastructural, and molecular techniques. Oxidative insult with H2O2 (0,3 mM-1h) induced severe ultrastructural damages to the majority of mitochondria, showing typical swelling of the matrix and loss of cristae. Cr-supplementation, 3 and 10 mM Cr, 24 h prior to H2O2 treatment, was found to significantly prevent these effects in both prealoading conditions. Confocal microscopy allowed imaging of mitochondrial network morphology that in oxidatively injured myoblasts appeared fragmented. Cr supplementation seems to partially preserve the network organization in both Cr prealoading concentrations. Flow cytometric analysis of NAO/cardiolipin interaction showed that H2O2 treatment caused a significant peroxidation of cardiolipin within 24 hours post-oxidative injury; 3 and 10 mM Cr pre-loading prevented the oxidation with a protection rate of 32% and of 18%, respectively. Cytofluorimetric data also revealed either a modulation in mitochondria membrane potential per mitochondria unit using a tetramethyl rhodamine methyl ester (TMRM) as potentiometric fluorescent dye and mitotracker green (MTG) for mitochondrial mass (TMRM/MTG ratio), or mitochondrial dysfunctionmasked by a ATP synthase reverse mode operating after addition of oligomycin (5uM), an ATP synthase inhibitor. In particular, only the Cr-treated myoblasts showed an increase in TMRM /MTG ratio compared to oxidatively-stressed myoblasts, after 1 and 24h. Furthermore a significant higher ratio of oligomycin- insensitive fraction was found in 3 mM Cr-pretreated cells (1h), as compared to trolox (a reference antioxidant), strengthening the TMRM /MTG ratio data observed in Cr-preloading conditions. Thus, Cr not only reduces H2O2-induced mito dysfunction, but also acts in the absence of a relevant contribution of ATP-synthase, differently from trolox condition with slighlty differences between the two Cr concentration conditions used. Expression of nuclear encoded genes controlling mitochondrial biogenesis (PGC-1α, NRF- 1α and Tfam) were also studied by RT realtime PCR as well as mitochondrial proteins profile and western blot of specific metabolic target such us AMPK and its downstream signaling substrate acetyl-CoA carboxylase (ACC). Analysis of C2C12 cells treated with H2O2 and H2O2+Cr, showed that PGC-1α expression levels were markedly increased immediately after oxidative stress in both conditions as compared to controls, and positively correlated with the severity of mitochondrial cell damage, probably due to a compensatory cell response. Interestingly, Cr preloading increased the phosphorylation of AMPK and especially ACC. On the other hand, Cr stimulates AMPK a key energy-sensitive target that controls numerous metabolic and cellular processes. On the whole, our data indicate that under oxidative stress conditions, where the expression of PGC-1α and mitochondrial biogenesis are likely to be stimulated, Cr preloading may favour an enhanced adaptive mechanism which allows myoblasts to increase the regulation of cellular energy metabolism and minimize oxidative damage in the course of myogenesis.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11576/2610791
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