MicroRNA-204-5p modulates mitochondrial biogenesis in C2C12 myotubes and associates with oxidative capacity in humans

Abstract

Using an unbiased high-throughput microRNA (miRNA)-silencing screen combined with functional readouts for mitochondrial oxidative capacity in C2C12 myocytes, we previously identified 19 miRNAs as putative regulators of skeletal muscle mitochondrial metabolism. In the current study, we highlight miRNA-204-5p, identified from this screen, and further studied its role in the regulation of skeletal muscle mitochondrial function. Following silencing of miRNA-204-5p in C2C12 myotubes, gene and protein expression were assessed using quantitative polymerase chain reaction, microarray analysis, and western blot analysis, while morphological changes were studied by confocal microscopy. In addition, miRNA-204-5p expression was quantified in human skeletal muscle biopsies and associated with in vivo mitochondrial oxidative capacity. Transcript levels of PGC-1α (3.71-fold; p < .01), predicted as an miR-204-5p target, as well as mitochondrial DNA copy number (p < .05) and citrate synthase activity (p = .06) were increased upon miRNA-204-5p silencing in C2C12 myotubes. Silencing of miRNA-204-5p further resulted in morphological changes, induced gene expression of autophagy marker light chain 3 protein b (LC3B; q = .05), and reduced expression of the mitophagy marker FUNDC1 (q = .01). Confocal imaging revealed colocalization between the autophagosome marker LC3B and the mitochondrial marker OxPhos upon miRNA-204-5p silencing. Finally, miRNA-204-5p was differentially expressed in human subjects displaying large variation in oxidative capacity and its expression levels associated with in vivo measures of skeletal muscle mitochondrial function. In summary, silencing of miRNA-204-5p in C2C12 myotubes stimulated mitochondrial biogenesis, impacted on cellular morphology, and altered expression of markers related to autophagy and mitophagy. The association between miRNA-204-5p and in vivo mitochondrial function in human skeletal muscle further identifies miRNA-204-5p as an interesting modulator of skeletal muscle mitochondrial metabolism.

Keywords: C2C12; microRNA; mitochondria; mitophagy; skeletal muscle.

Figure 1

miRNA‐204‐5p silencing induces mitochondrial biogenesis through PGC‐1α induction. (a) Representation of the binding between the 3′‐UTR of PGC‐1α mRNA and miRNA‐204‐5p in different species. (b) Activity assay of a luciferase reporter constructs harboring the PGC‐1α 3′‐UTR after transfection with a synthetic precursor for miRNA‐204‐5p. Data shown are the result of three independently executed duplicate experiments. (c) Relative PGC‐1α gene expression upon miRNA‐204‐5p silencing compared to Negative Control A (NegA), 24 and 48 hr posttransfection in C2C12 myotubes. (d) Citrate synthase activity following 24 and 48 hr transfection of anti‐miRNA‐204‐5p versus NegA in C2C12 myotubes. (d) Mitochondrial DNA copy number and nuclear DNA upon miRNA‐204‐5p silencing versus NegA. *p < .05 and **p < .01 (n = 3). 3′‐UTR, 3′‐untranslated region; miRNA, microRNA; mRNA, messenger RNA; PGC‐1α, peroxisome proliferator‐activated receptor‐gamma coactivator‐1α

Figure 2

Silencing miRNA‐204‐5p induces the development of an aberrant morphology in C2C12 myotubes. C2C12 myoblasts were differentiated for 5 days prior transfection with either Negative Control A or an LNA targeting miRNA‐204‐5p, and were subsequently observed for 72 hr. The development of the aberrant phenotype was consistently observed 48 hr posttransfection. (a–e) C2C12 myotubes transfected with of Negative Control A. (f–j) C2C12 myotubes transfected with an LNA directed against miRNA‐204‐5p. Panels are bright‐field micrographs. Red arrows indicate C2C12 myotubes with atypical morphology. Scale bar = 400 μm (n = 3). LNA, locked nucleic acid; miRNA, microRNA

Figure 3

miRNA‐204‐5p silencing only affects fully differentiated C2C12 myotubes. Undifferentiated C2C12 myoblasts were transfected with either Negative Control A or an LNA targeting miRNA‐204‐5p, and were subsequently observed for 7 days. (a–d) Negative Control A. (e–h) Transfection of an LNA against miRNA‐204‐5p. Red arrows indicate C2C12 myotubes with atypical morphology. Panels are bright‐field micrographs (n = 2). Scale bar =  400 μm. LNA, locked nucleic acid; miRNA, microRNA

Figure 4

miRNA‐204‐5p silencing does not increase LC3B‐I or LC3B‐II protein abundance. (a) Representation of the binding between the 3′‐UTR of LC3B (MAP1LC3B) mRNA and miRNA‐204‐5p across different species. (b) Gene expression analysis (microarray analysis) following anti‐miRNA‐204‐5p (24 hr) in differentiated C2C12 myotubes. *q < .05 (FDR adjusted q‐value intensity‐based moderated T‐statistic, n = 3). (c) LC3B‐I and LC3B‐II protein levels were quantified using western blot analysis 12, 24, 36, 48, and 72 hr posttransfection with either Negative Control A or anti‐miRNA‐204‐5p. In addition, transfected cells were compared to chloroquine treated C2C12 myotubes as a positive control (n = 3). 3′‐UTR, 3′‐untranslated region; FDR, false discovery rate; LC3B, light chain 3 protein b; miRNA, microRNA; mRNA, messenger RNA; NC, negative control

Figure 5

miRNA‐204‐5p silencing leads to the activation of LC3B and the accumulation of autophagosomes in C2C12 myotubes. C2C12 myoblasts were differentiated for 5 days and transfection with either Negative Control A (a) or an LNA targeting miRNA‐204‐5p (b) for 48 hr. Immunocytochemistry targeting the autophagosome marker LC3B (green) or nuclei (blue) was performed. (c) Twenty‐four hours chloroquine incubation (1 μg/ml) was used as a positive control for autophagosome accumulation (n = 4). LC3B, light chain 3 protein b; LNA, locked nucleic acid; miRNA, microRNA

Figure 6

miRNA‐204‐5p silencing targets mitochondria for autophagy. C2C12 myotubes silenced with an anti‐miRNA‐204‐5p displayed colocalization of the autophagosome marker LC3B (green) and the mitochondrial marker OxPhos (red), indicating that mitochondria are targeted for autophagy (mitophagy). Scale bar = 25 μm (n = 3). DAPI, 4′,6‐diamidino‐2‐phenylindole; LC3B, light chain 3 protein b; miRNA, microRNA

Figure 7

miRNA‐204‐5p levels in human biopsies correlates with oxidative capacity in vivo. (a) miRNA‐204‐5p expression was assessed in skeletal muscle (vastus lateralis) biopsies from Type 2 diabetic (T2DM), obese (O), lean sedentary (LS), and athletic (A) subjects. *p < .05 one‐way ANOVA with Tukey's post hoc testing for multiple comparisons. Pearson's correlation between miRNA‐204‐5p expression and (b) VO₂ max (normalized for lean body mass) and between miRNA‐204‐5p expression and (c) PCr recovery rate, a measure for in vivo mitochondrial capacity. ANOVA, analysis of variance; miRNA, microRNA; PCr, phosphocreatine; T2DM, Type 2 diabetes mellitu; VO₂ max, maximal aerobic capacity