High-intensity training induces non-stoichiometric changes in the mitochondrial proteome of human skeletal muscle without reorganisation of respiratory chain content

Mitochondrial defects are implicated in multiple diseases and aging. Exercise training is an accessible, inexpensive therapeutic intervention that can improve mitochondrial bioenergetics and quality of life. By combining multiple omics techniques with biochemical and in silico normalisation, we removed the bias arising from the training-induced increase in mitochondrial content to unearth an intricate and previously undemonstrated network of differentially prioritised mitochondrial adaptations. We show that changes in hundreds of transcripts, proteins, and lipids are not stoichiometrically linked to the overall increase in mitochondrial content. Our findings suggest enhancing electron flow to oxidative phosphorylation (OXPHOS) is more important to improve ATP generation than increasing the abundance of the OXPHOS machinery, and do not support the hypothesis that training-induced supercomplex formation enhances mitochondrial bioenergetics. Our study provides an analytical approach allowing unbiased and in-depth investigations of training-induced mitochondrial adaptations, challenging our current understanding, and calling for careful reinterpretation of previous findings.

Exercise and Mitochondrial Function: Importance and InferenceA Mini Review

: Skeletal muscles must generate and distribute energy properly in order to function perfectly. Mitochondria in skeletal muscle cells form vast networks to meet this need, and their functions may improve as a result of exercise. In the present review, we discussed exercise-induced mitochondrial adaptations, age-related mitochondrial decline, and a biomarker as a mitochondrial function indicator and exercise interference.

Placental mitochondrial function as a driver of angiogenesis and placental dysfunction

The placenta is a highly vascularized and complex foetal organ that performs various tasks, crucial to a healthy pregnancy. Its dysfunction leads to complications such as stillbirth, preeclampsia, and intrauterine growth restriction. The specific cause of placental dysfunction remains unknown. Recently, the role of mitochondrial function and mitochondrial adaptations in the context of angiogenesis and placental dysfunction is getting more attention. The required energy for placental remodelling, nutrient transport, hormone synthesis, and the reactive oxygen species leads to oxidative stress, stemming from mitochondria. Mitochondria adapt to environmental changes and have been shown to adjust their oxygen and nutrient use to best support placental angiogenesis and foetal development. Angiogenesis is the process by which blood vessels form and is essential for the delivery of nutrients to the body. This process is regulated by different factors, pro-angiogenic factors and anti-angiogenic factors, such as sFlt-1. Increased circulating sFlt-1 levels have been linked to different preeclamptic phenotypes. One of many effects of increased sFlt-1 levels, is the dysregulation of mitochondrial function. This review covers mitochondrial adaptations during placentation, the importance of the anti-angiogenic factor sFlt-1in placental dysfunction and its role in the dysregulation of mitochondrial function.

Skeletal muscle mitochondrial adaptations induced by long-term cigarette smoke exposure

Because patients with Chronic Obstructive Pulmonary Disease (COPD) are often physically inactive, it is still unclear whether the lower respiratory capacity in the locomotor muscles of these patients is due to cigarette smoking per se or is secondary to physical deconditioning. Accordingly, the purpose of this study was to examine mitochondrial alterations in the quadriceps muscle of 10 mice exposed to 8-months of cigarette smoke, a sedentary mouse model of emphysema, and 9 control mice, using immunoblotting, spectrophotometry, and high-resolution respirometry in permeabilized muscle fibers. Mice exposed to smoke displayed a two-fold increase in the oxidative stress marker, 4-HNE, (p < 0.05) compared with control mice. This was accompanied by significant decreases in protein expression of UCP3 (65%), ANT (58%), and mitochondrial complexes II-V (~60%-75%). In contrast, maximal ADP-stimulated respiration with complex I and II substrates (CON: 23.6 ± 6.6 and SMO: 19.2 ± 8.2 ρM·mg-1·s-1) or octanoylcarnitine (CON: 21.8 ± 9.0 and SMO: 16.5 ± 6.6 ρM·mg-1·s-1) measured in permeabilized muscle fibers, as well as citrate synthase activity, were not significantly different between groups. Collectively, our findings revealed that mice exposed to cigarette smoke for 8 months, which is typically associated with pulmonary inflammation and emphysema, exhibited a preserved mitochondrial respiratory capacity for various substrates, including free-fatty acid, in the skeletal muscle. However, the mitochondrial adaptations induced by cigarette smoke favored the development of chronic oxidative stress, which can indirectly contribute to augment the susceptibility to muscle fatigue and exercise intolerance.

Metabolic design in a model of extreme mammalian metabolism, the North American least shrew (Cryptotis parva)

Mitochondrial adaptations are fundamental to differentiated function and energetic homeostasis in mammalian cells. But the mechanisms that underlie these relationships remain poorly understood. Here, we investigated organ-specific mitochondrial morphology, connectivity and protein composition in a model of extreme mammalian metabolism, the Least shrew (Cryptotis parva). This was achieved through a combination of high-resolution 3D focused-ion-beam EM imaging and tandem-mass-tag MS proteomics. We demonstrate that liver and kidney mitochondrial content are equivalent to the heart permitting assessment of mitochondrial adaptations in different organs with similar metabolic demand. Muscle mitochondrial networks (cardiac and skeletal) are extensive, with a high incidence of nanotunnels - which collectively support the metabolism of large muscle cells. Mitochondrial networks were not detected in the liver and kidney as individual mitochondria are localized with sites of ATP consumption. This configuration is not observed in striated muscle, likely due to a homogenous ATPase distribution and the structural requirements of contraction. These results demonstrate distinct, fundamental mitochondrial structural adaptations for similar metabolic demand that are dependent on the topology of energy utilization process in a mammalian model of extreme metabolism.

Multi-omics reveal intricate network of mitochondrial adaptations to training in human skeletal muscle

Defects in mitochondria have been implicated in multiple diseases and aging; therefore, interventions able to counteract these changes can improve quality of life. Exercise training is a readily accessible and inexpensive therapeutic intervention; however, the complexity of training-induced mitochondrial adaptations in skeletal muscle remains poorly understood. Here, we describe an intricate and previously undemonstrated network of differentially prioritised training-induced adaptations in human skeletal muscle mitochondria. We show that changes in hundreds of transcripts, proteins, and lipids are not stoichiometrically linked to the increase in mitochondrial content. Moreover, we demonstrate a prioritisation of specific mitochondrial functional protein networks at different stages of the training intervention, including an initial deprioritisation of oxidative phosphorylation (OXPHOS) and a prioritisation of TCA cycle and fatty acid β-oxidation linked mitochondrial respiration. This indicates that enhancing electron flow to OXPHOS may be more important to improve ATP generation in skeletal muscle than increasing the abundance of the OXPHOS machinery. Our research unearths the elaborate and multi-layered nature of the adaptive response to exercise and provides a valuable resource that can be mined to maximise the therapeutic benefits of exercise.

β2-Adrenergic Signaling Modulates Mitochondrial Function and Morphology in Skeletal Muscle in Response to Aerobic Exercise

The molecular mechanisms underlying skeletal muscle mitochondrial adaptations induced by aerobic exercise (AE) are not fully understood. We have previously shown that AE induces mitochondrial adaptations in cardiac muscle, mediated by sympathetic stimulation. Since direct sympathetic innervation of neuromuscular junctions influences skeletal muscle homeostasis, we tested the hypothesis that β2-adrenergic receptor (β2-AR)-mediated sympathetic activation induces mitochondrial adaptations to AE in skeletal muscle. Male FVB mice were subjected to a single bout of AE on a treadmill (80% Vmax, 60 min) under β2-AR blockade with ICI 118,551 (ICI) or vehicle, and parameters of mitochondrial function and morphology/dynamics were evaluated. An acute bout of AE significantly increased maximal mitochondrial respiration in tibialis anterior (TA) isolated fiber bundles, which was prevented by β2-AR blockade. This increased mitochondrial function after AE was accompanied by a change in mitochondrial morphology towards fusion, associated with increased Mfn1 protein expression and activity. β2-AR blockade fully prevented the increase in Mfn1 activity and reduced mitochondrial elongation. To determine the mechanisms involved in mitochondrial modulation by β2-AR activation in skeletal muscle during AE, we used C2C12 myotubes, treated with the non-selective β-AR agonist isoproterenol (ISO) in the presence of the specific β2-AR antagonist ICI or during protein kinase A (PKA) and Gαi protein blockade. Our in vitro data show that β-AR activation significantly increases mitochondrial respiration in myotubes, and this response was dependent on β2-AR activation through a Gαs-PKA signaling cascade. In conclusion, we provide evidence for AE-induced β2-AR activation as a major mechanism leading to alterations in mitochondria function and morphology/dynamics. β2-AR signaling is thus a key-signaling pathway that contributes to skeletal muscle plasticity in response to exercise.

The role of hepatocellular eNOS in NAFLD development and hepatic mitochondrial adaptations to exercise

Whole body loss of endothelial nitric oxide synthase (eNOS) worsens hepatic mitochondrial function and exacerbates nonalcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) development and progression. However, the precise role of eNOS in hepatocytes in the contribution to NAFLD has not been established. Here, we use gain- and loss- of-function approaches including a hepatocyte-specific eNOS knockout mouse model (eNOShep-/-), and lifestyle interventions (diet and exercise), to investigate the role of hepatocellular eNOS in NAFLD/NASH development and hepatic mitochondrial adaptations to exercise. Ablation of hepatocellular eNOS via genetic and viral knockout exacerbated hepatic steatosis and inflammation, decreased hepatic mitochondrial fatty acid oxidation and respiration, and impaired mitophagy. Conversely, overexpressing hepatocellular eNOS via viral approaches increased hepatocyte respiration, markers of mitophagy, while attenuating NASH progression. Interestingly, these detriments were not rescued by BNIP3 overexpression or nitric oxide (NO) donors in eNOS deficient hepatocytes. In addition, elevated H2O2 emission and hepatic steatosis in eNOShep-/- mice was completely ablated with 10 weeks of voluntary wheel running exercise. Interestingly, eNOShep-/- male mice had a blunted exercise-induced increase in hepatic fatty acid oxidation. eNOShep-/- mice also had impaired markers of energy sensing ability of the cell and attenuated activation of the autophagy initiating factor ULK1. While mitochondrial respiration and markers of mitochondrial content were not increased with exercise, female mice showed markers of mitochondrial biogenesis. Collectively, these data uncover the important and novel role of hepatocellular eNOS in exercise-induced hepatic mitochondrial adaptations, and help to further the understanding behind the mechanistic role of eNOS in NAFLD development.