Human skeletal muscle mitochondrial dynamics in relation to oxidative capacity and insulin sensitivity.

AIMS/HYPOTHESIS: Mitochondria operate in networks, adapting to external stresses and changes in cellular metabolic demand and are subject to various quality control mechanisms. On the basis of these traits, we here hypothesise that the regulation of mitochondrial networks in skeletal muscle is hampered in humans with compromised oxidative capacity and insulin sensitivity. METHODS: In a cross-sectional design, we compared four groups of participants (selected from previous studies) ranging in aerobic capacity and insulin sensitivity, i.e. participants with type 2 diabetes (n = 11), obese participants without diabetes (n = 12), lean individuals (n = 10) and endurance-trained athletes (n = 12); basal, overnight fasted muscle biopsies were newly analysed for the current study and we compared the levels of essential mitochondrial dynamics and quality control regulatory proteins in skeletal muscle tissue. RESULTS: Type 2 diabetes patients and obese participants were older than lean participants and athletes (58.6 ± 4.0 and 56.7 ± 7.2 vs 21.8 ± 2.5 and 25.1 ± 4.3 years, p < 0.001, respectively) and displayed a higher BMI (32.4 ± 3.7 and 31.0 ± 3.7 vs 22.1 ± 1.8 and 21.0 ± 1.5 kg/m2, p < 0.001, respectively) than lean individuals and endurance-trained athletes. Fission protein 1 (FIS1) and optic atrophy protein 1 (OPA1) protein content was highest in muscle from athletes and lowest in participants with type 2 diabetes and obesity, respectively (FIS1: 1.86 ± 0.79 vs 0.79 ± 0.51 AU, p = 0.002; and OPA1: 1.55 ± 0.64 vs 0.76 ± 0.52 AU, p = 0.014), which coincided with mitochondrial network fragmentation in individuals with type 2 diabetes, as assessed by confocal microscopy in a subset of type 2 diabetes patients vs endurance-trained athletes (n = 6). Furthermore, lean individuals and athletes displayed a mitonuclear protein balance that was different from obese participants and those with type 2 diabetes. Mitonuclear protein balance also associated with heat shock protein 60 (HSP60) protein levels, which were higher in athletes when compared with participants with obesity (p = 0.048) and type 2 diabetes (p = 0.002), indicative for activation of the mitochondrial unfolded protein response. Finally, OPA1, FIS1 and HSP60 correlated positively with aerobic capacity (r = 0.48, p = 0.0001; r = 0.55, p < 0.001 and r = 0.61, p < 0.0001, respectively) and insulin sensitivity (r = 0.40, p = 0.008; r = 0.44, p = 0.003 and r = 0.48, p = 0.001, respectively). CONCLUSIONS/INTERPRETATION: Collectively, our data suggest that mitochondrial dynamics and quality control in skeletal muscle are linked to oxidative capacity in humans, which may play a role in the maintenance of muscle insulin sensitivity. CLINICAL TRIAL REGISTRY: numbers NCT00943059, NCT01298375 and NL1888 Graphical abstract.

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

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.

MicroRNA-382 silencing induces a mitonuclear protein imbalance and activates the mitochondrial unfolded protein response in muscle cells.

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.

An unbiased silencing screen in muscle cells identifies miR-320a, miR-150, miR-196b, and miR-34c as regulators of skeletal muscle mitochondrial metabolism.

OBJECTIVE: Strategies improving skeletal muscle mitochondrial capacity are commonly paralleled by improvements in (metabolic) health. We and others previously identified microRNAs regulating mitochondrial oxidative capacity, but data in skeletal muscle are limited. Therefore, the present study aimed to identify novel microRNAs regulating skeletal muscle mitochondrial metabolism. METHODS AND RESULTS: We conducted an unbiased, hypothesis-free microRNA silencing screen in C2C12 myoblasts, using >700 specific microRNA inhibitors, and investigated a broad panel of mitochondrial markers. After subsequent validation in differentiated C2C12 myotubes, and exclusion of microRNAs without a human homologue or with an adverse effect on mitochondrial metabolism, 19 candidate microRNAs remained. Human clinical relevance of these microRNAs was investigated by measuring their expression in human skeletal muscle of subject groups displaying large variation in skeletal muscle mitochondrial capacity. CONCLUSION: The results show that that microRNA-320a, microRNA-196b-3p, microRNA-150-5p, and microRNA-34c-3p are tightly related to in vivo skeletal muscle mitochondrial function in humans and identify these microRNAs as targets for improving mitochondrial metabolism.

Evaluation of Muscle microRNA Expression in Relation to Human Peripheral Insulin Sensitivity: A Cross-Sectional Study in Metabolically Distinct Subject Groups.

In recent years, several microRNAs (miRNAs)-post-transcriptional regulators of gene expression-have been linked to the regulation of peripheral insulin sensitivity. Many of these studies, however, have been conducted in cell or animal models and the few human studies available lack adequate measurements of peripheral insulin sensitivity. In the present study, we examined the expression of 25 miRNAs, putatively involved in (peripheral) insulin sensitivity, in skeletal muscle biopsies from extensively phenotyped human individuals, widely ranging in insulin sensitivity. To identify miRNAs expressed in skeletal muscle and associated with insulin sensitivity and type 2 diabetes, a comprehensive PubMed-based literature search was performed. Subsequently, the expression of selected miRNAs was determined by RT-qPCR using predesigned 384-well Pick-&-Mix miRNA PCR Panel plates in muscle biopsies from type 2 diabetes patients, non-diabetic obese/overweight individuals, lean sedentary individuals and endurance-trained athletes. In all subjects, peripheral insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp. The literature search resulted in 25 candidate miRNAs, 6 of which were differentially expressed in human type 2 diabetes compared to non-diabetic obese/overweight individuals. In turn, four of these miRNAs, i.e., miRNA27a-3p (r = -0.45, p = 0.0012), miRNA-29a-3p (r = -0.40, p = 0.0052), miRNA-29b-3p (r = -0.70, p < 0.0001) and miRNA-29c-3p (r = -0.50, p = 0.0004) demonstrated strong negative correlations with peripheral insulin sensitivity across all four subject groups. We identified miR-27a-3p and all members of the miRNA-29 family as potential regulatory players in insulin sensitivity in humans. These miRNA's may represent interesting novel targets for maintaining or improving insulin sensitivity.

Mitochondrial dynamics, quality control and miRNA regulation in skeletal muscle: implications for obesity and related metabolic disease.

The western dietary habits and sedentary lifestyle largely contributes to the growing epidemic of obesity. Mitochondria are at the front line of cellular energy homoeostasis and are implicated in the pathophysiology of obesity and obesity-related metabolic disease. In recent years, novel aspects in the regulation of mitochondrial metabolism, such as mitochondrial dynamics, mitochondrial protein quality control and post-transcriptional regulation of genes coding for mitochondrial proteins, have emerged. In this review, we discuss the recent findings concerning the dysregulation of these processes in skeletal muscle in obesogenic conditions.

Quantitative Analysis of MicroRNAs in Vaccinia virus Infection Reveals Diversity in Their Susceptibility to Modification and Suppression.

Vaccinia virus (VACV) is a large cytoplasmic DNA virus that causes dramatic alterations to many cellular pathways including microRNA biogenesis. The virus encodes a poly(A) polymerase which was previously shown to add poly(A) tails to the 3' end of cellular miRNAs, resulting in their degradation by 24 hours post infection (hpi). Here we used small RNA sequencing to quantify the impact of VACV infection on cellular miRNAs in human cells at both early (6 h) and late (24 h) times post infection. A detailed quantitative analysis of individual miRNAs revealed marked diversity in the extent of their modification and relative change in abundance during infection. Some miRNAs became highly modified (e.g. miR-29a-3p, miR-27b-3p) whereas others appeared resistant (e.g. miR-16-5p). Furthermore, miRNAs that were highly tailed at 6 hpi were not necessarily among the most reduced at 24 hpi. These results suggest that intrinsic features of human cellular miRNAs cause them to be differentially polyadenylated and altered in abundance during VACV infection. We also demonstrate that intermediate and late VACV gene expression are required for optimal repression of some miRNAs including miR-27-3p. Overall this work reveals complex and varied consequences of VACV infection on host miRNAs and identifies miRNAs which are largely resistant to VACV-induced polyadenylation and are therefore present at functional levels during the initial stages of infection and replication.