Muscle mitochondrial health : ageing, physical activity and molecular mechanisms

Muscle mass and strength are reported to decline with age. Due to the vital role the muscle in daily life activities, increased loss of muscle mass and strength is associated with functional decline, decreased quality of life and increased hospitalisation rates. Therefore, maintaining skeletal muscle mass and strength with age is a key component in healthy ageing. One of the postulated causes of the decline in mass and strength is the decline in the amount and the quality of the muscle mitochondria, collectively referred to as mitochondrial capacity. Nevertheless, the exact contribution of the mitochondria is still under debate. Therefore, this thesis aims to obtain a better understanding of the role of skeletal muscle mitochondria during ageing. Understanding the role of the contributors to skeletal muscle ageing on the physiologic and molecular level could focus intervention strategies to ultimately to sustain muscle mass and strength with age. Current methods to assess mitochondrial capacity in humans are either invasive, such as the sampling of muscle tissue via a muscle biopsy, or less accessible, due to expensive and specialised equipment, such as 31P-MRS. In recent years, the assessment of mitochondrial capacity using near-infrared spectroscopy (NIRS) has offered relief to these limitations. Yet, additional effort is needed to extend the use of NIRS to also study the effect of age on mitochondrial capacity. For example, previous studies compared groups of subjects with large expected differences in mitochondrial capacity, and therefore it was unknown if NIRS is able to detect relatively smaller differences in mitochondrial capacity, such as might be expected between a young and older population. In chapter 2 we demonstrated that NIRS was able to detect differences in mitochondrial capacity in the gastrocnemius muscle in a homogenous population of high- and low-fitness males, with a smaller expected difference in mitochondrial capacity than was previously assessed. In chapter 3 we additionally showed that NIRS is able to detect differences in mitochondrial capacity the gastrocnemius muscle in a population of a high- and low-fitness females. Furthermore, we show that NIRS correlates with other measures of oxidative capacity, underlining the physiological relevance of NIRS assessment of mitochondrial capacity. This demonstrates that NIRS could be a valuable tool to study muscle mitochondrial capacity in an ageing population In chapter 4 we used NIRS to assess the effect of age on mitochondrial capacity in a population of older (65-71 years) and young (19-25 years) males. Due to the interaction between physical activity, mitochondrial capacity and age, the two age groups were selected based on self-reported, similar physical activity, which was verified using a 5-day accelerometry measurement. We showed that NIRS was able to detect differences in mitochondrial capacity between the two age groups in the gastrocnemius and vastus lateralis, but not tibialis anterior. This showed that not all muscle groups display similar mitochondrial ageing and, because we observed these effects despite similar physical activity, the lower mitochondrial capacity is likely a direct effect of ageing and cannot be completely prevented by physical activity. Nevertheless, a higher mitochondrial capacity was correlated with spending more time in moderate-to-vigorous physical activity, suggesting that physical activity might ameliorate part of the age-related decline in mitochondrial capacity. In chapter 5 we used transcriptome sequencing to identify molecular mechanisms of ageing in vastus lateralis muscle biopsies in the aforementioned population. The significant regulated processes in older compared to young muscle included: cell-adhesion, the matrisome, innervation and inflammation, which were largely upregulated, and oxidative metabolism, which was downregulated. In accordance with the transcriptome results, the protein expression of some mitochondrial respiratory complexes was lower in older compared to young muscle. Moreover, the expression of these complexes in the older group was correlated with in vivo mitochondrial capacity in the vastus lateralis. This showed that the observed lower mitochondrial capacity could be explained by a lower expression of mitochondrial complex proteins and further substantiated the use of NIRS to measure mitochondrial capacity in vivo. In chapter 6 and 7 we explored the role of protein propionylation as regulatory factor of mitochondrial and muscle function in aging. Post-translational protein modifications are an important regulatory mechanism for protein functionality and offers the cell a rapid and reversible mechanism to respond to changes in the environment. Protein propionylation might be an important post-translational modification in muscle physiology and ageing. In chapter 6 we turned to a pathophysiological human model in which levels of propionyl-CoA were elevated and protein propionylation was increased. We showed that fibroblasts from patients in this pathological state have impaired mitochondrial function compared to healthy donor cells, possibly due to aberrant propionylation of proteins involved in mitochondrial respiration. Furthermore, increasing propionylation by exposure to pathophysiological concentrations of propionate induced impaired mitochondrial function in cultured fibroblasts and liver cells. Yet, this effect was not observed in cultured muscle cells, possibly due to differences in metabolic handling of propionyl-CoA. Despite an absence of the effect of propionate exposure and increased propionylation on mitochondrial function in muscle cells, in chapter 7 we showed that exposure to propionate impairs skeletal muscle differentiation. Concomitant with this observation, we observed an increase in histone protein propionylation and acetylation. The increase in propionylation and acetylation occurred on regions of the genome that regulate muscle differentiation, possibly revealing an additional mechanism by which propionyl-CoA and propionylation can influence muscle cellular fates. In chapter 8 the conclusions are presented and the main findings of this thesis are further discussed. For example, we further discuss the way forward for NIRS assessment of mitochondrial capacity. Additionally, we discuss the limitations of the study design used in chapter 4 and 5. Lastly, the plausible role of protein propionylation in skeletal muscle ageing is discussed. In conclusion, this thesis aimed to obtain a better understanding of the role of mitochondria in skeletal muscle ageing. We obtained better understanding on the non-invasive assessment of skeletal muscle mitochondrial capacity using NIRS and newly applied this method to study the effect of age on mitochondrial capacity in locomotor muscles. Furthermore, we obtained better understanding on the molecular mechanisms of ageing and identified that the age-related decline in mitochondrial capacity in skeletal muscle occurs despite similar physical activity, although we demonstrated this effect is muscle dependent. Lastly, we explored the effect of protein propionylation on skeletal muscle cells. Although we did not find direct effects of increased propionylation on mitochondrial function in muscle, we observed that increased propionylation was associated with impaired muscle differentiation and propionylation could therefore play a role in muscle physiology and ageing

RNAseq of old and young males with equal physical activity

18 older (65-71 years) and 20 young (19-25 years) males were recruited based on similar, self-reported physical activity, which was verified using accelerometry measurements. Gene expression profiles of vastus lateralis biopsies obtained by RNA sequencing. A clear ageing signature was observed despite similar physcical activity

RNAseq of old and young males with equal physical activity

18 older (65-71 years) and 20 young (19-25 years) males were recruited based on similar, self-reported physical activity, which was verified using accelerometry measurements. Gene expression profiles of vastus lateralis biopsies obtained by RNA sequencing. A clear ageing signature was observed despite similar physcical activity

In vivo assessment of muscle mitochondrial function in healthy, young males in relation to parameters of aerobic fitness

Purpose: The recovery of muscle oxygen consumption (mV˙ O2) after exercise provides a measure of skeletal muscle mitochondrial capacity, as more and better-functioning mitochondria will be able to restore mV˙ O2 faster to the pre-exercise state. The aim was to measure muscle mitochondrial capacity using near-infrared spectroscopy (NIRS) within a healthy, normally active population and relate this to parameters of aerobic fitness, investigating the applicability and relevance of using NIRS to assess muscle mitochondrial capacity non-invasively. Methods: Mitochondrial capacity was analysed in the gastrocnemius and flexor digitorum superficialis (FDS) muscles of eight relatively high-aerobic fitness (V˙ O2peak ≥ 57 mL/kg/min) and eight relatively low-aerobic fitness male subjects (V˙ O2peak ≤ 47 mL/kg/min). Recovery of whole body V˙ O2, i.e. excess post-exercise oxygen consumption (EPOC) was analysed after a cycling protocol. Results: Mitochondrial capacity, as analysed using NIRS, was significantly higher in high-fitness individuals compared to low-fitness individuals in the gastrocnemius, but not in the FDS (p = 0.0036 and p = 0.20, respectively). Mitochondrial capacity in the gastrocnemius was significantly correlated with V˙ O2peak (R2 = 0.57, p = 0.0019). Whole body V˙ O2 recovery was significantly faster in the high-fitness individuals (p = 0.0048), and correlated significantly with mitochondrial capacity in the gastrocnemius (R2 = 0.34, p = 0.028). Conclusion: NIRS measurements can be used to assess differences in mitochondrial muscle oxygen consumption within a relatively normal, healthy population. Furthermore, mitochondrial capacity correlated with parameters of aerobic fitness (V˙ O2peak and EPOC), emphasising the physiological relevance of the NIRS measurements

In vivo assessment of muscle mitochondrial function in healthy, young males in relation to parameters of aerobic fitness

Purpose: The recovery of muscle oxygen consumption (mV˙ O2) after exercise provides a measure of skeletal muscle mitochondrial capacity, as more and better-functioning mitochondria will be able to restore mV˙ O2 faster to the pre-exercise state. The aim was to measure muscle mitochondrial capacity using near-infrared spectroscopy (NIRS) within a healthy, normally active population and relate this to parameters of aerobic fitness, investigating the applicability and relevance of using NIRS to assess muscle mitochondrial capacity non-invasively. Methods: Mitochondrial capacity was analysed in the gastrocnemius and flexor digitorum superficialis (FDS) muscles of eight relatively high-aerobic fitness (V˙ O2peak ≥ 57 mL/kg/min) and eight relatively low-aerobic fitness male subjects (V˙ O2peak ≤ 47 mL/kg/min). Recovery of whole body V˙ O2, i.e. excess post-exercise oxygen consumption (EPOC) was analysed after a cycling protocol. Results: Mitochondrial capacity, as analysed using NIRS, was significantly higher in high-fitness individuals compared to low-fitness individuals in the gastrocnemius, but not in the FDS (p = 0.0036 and p = 0.20, respectively). Mitochondrial capacity in the gastrocnemius was significantly correlated with V˙ O2peak (R2 = 0.57, p = 0.0019). Whole body V˙ O2 recovery was significantly faster in the high-fitness individuals (p = 0.0048), and correlated significantly with mitochondrial capacity in the gastrocnemius (R2 = 0.34, p = 0.028). Conclusion: NIRS measurements can be used to assess differences in mitochondrial muscle oxygen consumption within a relatively normal, healthy population. Furthermore, mitochondrial capacity correlated with parameters of aerobic fitness (V˙ O2peak and EPOC), emphasising the physiological relevance of the NIRS measurements.</p

The effect of a single bout of exercise on vitamin B2 status is not different between high-and low-fit females

High-fitness individuals have been suggested to be at risk of a poor vitamin B2 (riboflavin) status due to a potentially higher vitamin B2 demand, as measured by the erythrocyte glutathione reductase (EGR) activation coefficient (EGRAC). Longer-term exercise interventions have been shown to result in a lower vitamin B2 status, but studies are contradictory. Short-term exercise effects potentially contribute to discrepancies between studies but have only been tested in limited study populations. This study investigated if vitamin B2 status, measured by EGRAC, is affected by a single exercise bout in females who differ in fitness levels, and that represents long-term physical activity. At baseline and overnight after a 60-min cycling bout at 70% ˙VO2 peak, EGR activity and EGRAC were measured in 31 young female adults, divided into a high-fit (VO2 peak ≥ 47 mL/kg/min, N = 15) and low-fit (VO2 peak ≤ 37 mL/kg/min, N = 16) group. A single exercise bout significantly increased EGR activity in high-fit and low-fit females (Ptime = 0.006). This response was not affected by fitness level (Ptime*group = 0.256). The effect of exercise on EGRAC was not significant (Ptime = 0.079) and not influenced by EGR activity. The exercise response of EGRAC was not significantly different between high-fit and low-fit females (Ptime*group = 0.141). Thus, a single exercise bout increased EGR activity, but did not affect EGRAC, indicating that vitamin B2 status was not affected. The exercise response on EGRAC and EGR did not differ between high-fit and low-fit females

Novel standardized method for extracellular flux analysis of oxidative and glycolytic metabolism in peripheral blood mononuclear cells

Analyzing metabolism of peripheral blood mononuclear cells (PBMCs) provides key opportunities to study the pathophysiology of several diseases, such as type 2 diabetes, obesity and cancer. Extracellular flux (XF) assays provide dynamic metabolic analysis of living cells that can capture ex vivo cellular metabolic responses to biological stressors. To obtain reliable data from PBMCs from individuals, novel methods are needed that allow for standardization and take into account the non-adherent and highly dynamic nature of PBMCs. We developed a novel method for extracellular flux analysis of PBMCs, where we combined brightfield imaging with metabolic flux analysis and data integration in R. Multiple buffy coat donors were used to demonstrate assay linearity with low levels of variation. Our method allowed for accurate and precise estimation of XF assay parameters by reducing the standard score and standard score interquartile range of PBMC basal oxygen consumption rate and glycolytic rate. We applied our method to freshly isolated PBMCs from sixteen healthy subjects and demonstrated that our method reduced the coefficient of variation in group mean basal oxygen consumption rate and basal glycolytic rate, thereby decreasing the variation between PBMC donors. Our novel brightfield image procedure is a robust, sensitive and practical normalization method to reliably measure, compare and extrapolate XF assay data using PBMCs, thereby increasing the relevance for PBMCs as marker tissue in future clinical and biological studies, and enabling the use of primary blood cells instead of immortalized cell lines for immunometabolic experiments.</p

When Is a Biopsy-Proven Diagnosis Necessary Before Stereotactic Ablative Radiotherapy for Lung Cancer? A Decision Analysis

BACKGROUND: The practice of treating a solitary pulmonary nodule (SPN) suspicious for stage I non-small cell lung cancer (NSCLC) with stereotactic ablative radiotherapy (SABR) in the absence of pathology is growing. In the absence of randomized evidence, the appropriate prior probability threshold of lung cancer of when such a strategy is warranted can be informed using decision analysis. METHODS: A decision tree and Markov model were constructed to evaluate the relative merits of surveillance, a PET scan-directed SABR strategy (without pathology), or a PET scan-biopsy-SABR strategy, when faced with an SPN at different prior probabilities for lung cancer. Diagnostic characteristics, as well as disease, treatment, and toxicity parameters, were extracted from the literature. Deterministic analysis and probabilistic sensitivity analyses were performed to inform the appropriate lung cancer prior probability threshold between treatment strategies. RESULTS: In the reference case analysis, the prior probability threshold between surveillance and PET scan-biopsy-SABR was 17.0%; between PET scan-directed SABR and PET scan-biopsy-SABR, the threshold was 85.0%. The latter finding was confirmed on probabilistic sensitivity analysis (85.2%; 95% CI, 80.0% to 87.2%). This predicted lung cancer prior probability threshold was most sensitive to the diagnostic sensitivity of transthoracic biopsy (range, 77.2% to 94.0%) and the detection rate of false negatives on CT scan surveillance (range, 82.4% to 92.3 %). CONCLUSIONS: This model suggests that if there are concerns about morbidity related to biopsy for an SPN, a PET scan-directed SABR strategy is warranted when the prior probability of lung cancer exceeds a point estimate of 85%

Propionate hampers differentiation and modifies histone propionylation and acetylation in skeletal muscle cells

Protein acylation via metabolic acyl-CoA intermediates provides a link between cellular metabolism and protein functionality. A process in which acetyl-CoA and acetylation are fine-tuned is during myogenic differentiation. However, the roles of other protein acylations remain unknown. Protein propionylation could be functionally relevant because propionyl-CoA can be derived from the catabolism of amino acids and fatty acids and was shown to decrease during muscle differentiation. We aimed to explore the potential role of protein propionylation in muscle differentiation, by mimicking a pathophysiological situation with high extracellular propionate which increases propionyl-CoA and protein propionylation, rendering it a model to study increased protein propionylation. Exposure to extracellular propionate, but not acetate, impaired myogenic differentiation in C2C12 cells and propionate exposure impaired myogenic differentiation in primary human muscle cells. Impaired differentiation was accompanied by an increase in histone propionylation as well as histone acetylation. Furthermore, chromatin immunoprecipitation showed increased histone propionylation at specific regulatory myogenic differentiation sites of the Myod gene. Intramuscular propionylcarnitine levels are higher in old compared to young males and females, possibly indicating increased propionyl-CoA levels with age. The findings suggest a role for propionylation and propionyl-CoA in regulation of muscle cell differentiation and ageing, possibly via alterations in histone acylation.</p

Single and Joined Behaviour of Circulating Biomarkers and Metabolic Parameters in High-Fit and Low-Fit Healthy Females

Biomarkers are important in the assessment of health and disease, but are poorly studied in still healthy individuals with a (potential) different risk for metabolic disease. This study investigated, first, how single biomarkers and metabolic parameters, functional biomarker and metabolic parameter categories, and total biomarker and metabolic parameter profiles behave in young healthy female adults of different aerobic fitness and, second, how these biomarkers and metabolic parameters are affected by recent exercise in these healthy individuals. A total of 102 biomarkers and metabolic parameters were analysed in serum or plasma samples from 30 young, healthy, female adults divided into a high-fit (V̇O2peak ≥ 47 mL/kg/min, N = 15) and a low-fit (V̇O2peak ≤ 37 mL/kg/min, N = 15) group, at baseline and overnight after a single bout of exercise (60 min, 70% V̇O2peak). Our results show that total biomarker and metabolic parameter profiles were similar between high-fit and low-fit females. Recent exercise significantly affected several single biomarkers and metabolic parameters, mostly related to inflammation and lipid metabolism. Furthermore, functional biomarker and metabolic parameter categories corresponded to biomarker and metabolic parameter clusters generated via hierarchical clustering models. In conclusion, this study provides insight into the single and joined behavior of circulating biomarkers and metabolic parameters in healthy females, and identified functional biomarker and metabolic parameter categories that may be used for the characterisation of human health physiology