Abstract
Proper mitochondrial function plays a central role in cellular metabolism. Various diseases as well as aging are associated with diminished mitochondrial function. Previously, we identified 19 miRNAs putatively involved in the regulation of mitochondrial metabolism in skeletal muscle, a highly metabolically active tissue. In the current study, these 19 miRNAs were individually silenced in C2C12 myotubes using antisense oligonucleotides, followed by measurement of the expression of 27 genes known to play a major role in regulating mitochondrial metabolism. Based on the outcomes, we then focused on miR‐382‐5p and identified pathways affected by its silencing using microarrays, investigated protein expression, and studied cellular respiration. Silencing of miRNA‐382‐5p significantly increased the expression of several genes involved in mitochondrial dynamics and biogenesis. Conventional microarray analysis in C2C12 myotubes silenced for miRNA‐382‐5p revealed a collective downregulation of mitochondrial ribosomal proteins and respiratory chain proteins. This effect was accompanied by an imbalance between mitochondrial proteins encoded by the nuclear and mitochondrial DNA (1.35‐fold, p < 0.01) and an induction of HSP60 protein (1.31‐fold, p < 0.05), indicating activation of the mitochondrial unfolded protein response (mtUPR). Furthermore, silencing of miR‐382‐5p reduced basal oxygen consumption rate by 14% ( p < 0.05) without affecting mitochondrial content, pointing towards a more efficient mitochondrial function as a result of improved mitochondrial quality control. Taken together, silencing of miR‐382‐5p induces a mitonuclear protein imbalance and activates the mtUPR in skeletal muscle, a phenomenon that was previously associated with improved longevity.
Highlights
Mitochondria play a central role in the regulation of cellular metabolism
We previously conducted an unbiased high throughput miRNA silencing screen in C2C12 myoblasts and myotubes to identify novel miRNAs involved in the regulation of skeletal muscle mitochondrial metabolism
We extended these findings and determined the expression of 27 genes related to mitochondrial metabolism, after individual silencing of these 19 candidate miRNAs in C2C12
Summary
Mitochondria play a central role in the regulation of cellular metabolism. Various pathologies as well as aging have been associated with mitochondrial dysfunction (Dominic et al, 2014; Johannsen & Ravussin, 2009; Lin & Beal, 2006). During the last decade miRNAs have been reported to be present in mitochondria, and to regulate various aspects of mitochondrial function (Barrey et al, 2011; Bian et al, 2010; El Azzouzi et al, 2013; Li et al, 2014; Mohamed et al, 2014; Sripada, Tomar, & Singh, 2012) In this context, we recently performed an unbiased, hypothesis‐free, high throughput miRNA silencing screen in C2C12 muscle cells, and identified 19 miRNAs, which upon silencing, had a positive impact on mitochondrial metabolism (Dahlmans et al, 2017). We singled out miR‐382‐5p and show that silencing of miR‐382‐5p leads to a collective downregulation of the transcripts encoding for the mitochondrial ribosomal proteins, induces a mitonuclear protein imbalance and activates the mtUPR
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