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Aims:\ud \ud Heart failure with reduced ejection fraction (HFrEF) induces skeletal muscle mitochondrial abnormalities that contribute to exercise limitation; however, specific mitochondrial therapeutic targets remain poorly established. This study quantified the relationship and contribution of distinct mitochondrial respiratory states to prognostic whole‐body measures of exercise limitation in HFrEF.\ud \ud \ud \ud Methods and results:\ud \ud Male patients with HFrEF (n = 22) were prospectively enrolled and underwent ramp‐incremental cycle ergometry cardiopulmonary exercise testing to determine exercise variables including peak pulmonary oxygen uptake (V̇O2peak), lactate threshold (V̇O2LT), the ventilatory equivalent for carbon dioxide (V̇E/V̇CO2LT), peak circulatory power (CircPpeak), and peak oxygen pulse. Pectoralis major was biopsied for assessment of in situ mitochondrial respiration. All mitochondrial states including complexes I, II, and IV and electron transport system (ETS) capacity correlated with V̇O2peak (r = 0.40–0.64; P < 0.05), V̇O2LT (r = 0.52–0.72; P < 0.05), and CircPpeak (r = 0.42–0.60; P < 0.05). Multiple regression analysis revealed that combining age, haemoglobin, and left ventricular ejection fraction with ETS capacity could explain 52% of the variability in V̇O2peak and 80% of the variability in V̇O2LT, respectively, with ETS capacity (P = 0.04) and complex I (P = 0.01) the only significant contributors in the model.\ud \ud \ud \ud Conclusions:\ud \ud Mitochondrial respiratory states from skeletal muscle biopsies of patients with HFrEF were independently correlated to established non‐invasive prognostic cycle ergometry cardiopulmonary exercise testing indices including V̇O2peak, V̇O2LT, and CircPpeak. When combined with baseline patient characteristics, over 50% of the variability in V̇O2peak could be explained by the mitochondrial ETS capacity. These data provide optimized mitochondrial targets that may attenuate exercise limitations in HFrEF.

Accumulating evidence suggests that most elite cyclists have lower bone mineral density (BMD) values when compared with their nonelite counterparts (1) or sedentary young males (2, 3). This raises the question whether these ostensibly healthy athletes have a higher acute bone fracture risk and a higher risk of osteoporosis and associated comorbidities later in life. Although treatment of low BMD seems warranted in elite cyclists, the benefits of treatment for health and performance in this population remain to be established. In this viewpoint, we describe the etiology and consequences of impaired bone health in elite cyclists and discuss the need for interventions to optimize bone health in this unique population.

Background: Endurance training induces numerous cardio vascular and skeletal muscle adaptations, thereby increasing maximal oxygen uptake capacity (VO2max). Whether protein supplementation enhances these adaptations remains unclear.Objective: The present study was designed to determine the impact of protein supplementation on changes in VO2max during prolonged endurance training.Methods: We used a double-blind randomized controlled trial with repeated measures among 44 recreationally active, young males. Subjects performed 3 endurance training sessions per week for 10 wk. Supplements were provided immediately after each exercise session and daily before sleep, providing either protein (PRO group; n = 19; 21.5 +/- 0.4 y) or an isocaloric amount of carbohydrate as control (CON group; n = 21; 22.5 +/- 0.5 y). The VO2max, simulated 10-km time trial performance, and body composition (dual-energy X-ray absorptiometry) were measured before and after 5 and 10 wk of endurance training. Fasting skeletal muscle tissue samples were taken before and after 5 and 10 wk to measure skeletal muscle oxidative capacity, and fasting blood samples were taken every 2 wk to measure hematological factors.Results: VO2max increased to a greater extent in the PRO group than in the CON group after 5 wk (from 49.9 +/- 0.8 to 54.9 +/- 1.1 vs 50.8 +/- 0.9 to 53.0 +/- 1.1 mL . kg(-1) . min(-1); P 0.05) and 10 wk (-1.2 +/- 0.4 vs -0.2 +/- 0.2 kg; P Conclusions: Protein supplementation elicited greater gains in VO2max and stimulated lean mass accretion but did not improve skeletal muscle oxidative capacity and endurance performance during 10 wk of endurance training in healthy, young males.

Summary Background Trials that assessed the impact of protein supplementation on endurance training adaptations have reported conflicting findings. Objective To determine the impact of protein supplementation during chronic endurance training on aerobic capacity, body composition and exercise performance in healthy and clinical populations. Design A systematic database search was conducted for randomised controlled trials addressing the effects of protein supplementation during endurance training on aerobic capacity, body composition and exercise performance in PubMed, Embase, Web of Science, and CINAHL. Meta-analyses were performed to outline the overall effects of protein supplementation with all studies containing endurance training components. The effects of endurance training and add-on effects of protein supplementation were evaluated by the meta-analyses with endurance training-focused studies. Results Nineteen studies and 1162 participants contributed to the analyses. Compared with the control group, the protein supplementation group demonstrated greater improvements in aerobic capacity measured by mixed peak oxygen uptake (VO2peak) and peak workload power (Wpeak) (standardised mean difference [SMD] = 0.36, 95% confidence interval [CI]: 0.05 to 0.67), and VO2peak (mean difference [MD] = 0.89 mL‧kg−1‧min−1, 95% CI: 0.07 to 1.70); had a greater lean mass gain (MD = 0.32 kg, 95% CI: 0.07 to 0.58); and had a greater improvement in time trial performance (MD = −29.1s, 95% CI:-55.3 to −3.0). Secondary analyses showed that, in addition to the substantial improvement in VO2peak (MD = 3.67 mL‧kg−1‧min−1, 95% CI: 2.32 to 5.03) attributed to endurance training, protein supplementation provided an additional 26.4% gain in VO2peak (MD = 0.97 mL‧kg−1‧min−1, 95% CI: −0.03 to 1.97). Conclusion Protein supplementation further increased aerobic capacity, stimulated lean mass gain, and improved time trial performance during chronic endurance training in healthy and clinical populations. Prospero registration number (CRD42020155239).

Skeletal muscle responds to exercise by a diversity of processes that collectively contribute to short-term and structural adaptations to the demanded performance capacities. There is common consensus that, in general, adequate nutrient availability during and after exercise is important to maximise skeletal muscle adaptation and ultimately performance. At the same time, several knowledge gaps remain regarding the precise mechanisms underlying these effects on adaptation, the most optimal nutrient composition in relation to type of exercise, optimal timing etc. This dissertation addresses some of these unsolved issues by studying the role of carbohydrates and proteins during adaptation following different forms of exercise. The first part (chapters 2 – 4) focusses on carbohydrate availability with resistance exercise, whereas the second part (chapters 5 - 7) specifically addresses the effects and potential of protein supplementation with endurance training. In chapter 2 we reviewed the existing literature regarding the role of skeletal muscle glycogen with endurance and resistance exercise. Based on this review we concluded that the role of muscle glycogen levels and/or carbohydrate availability on the skeletal muscle adaptive response to resistance exercise requires further scientific attention. To experimentally explore this, we assessed the impact of a pre-exercise meal that differed in macronutrient content on skeletal muscle glycogen levels and acute transcriptional level analysing specific mRNAs in the post-resistance exercise period in chapter 3. Specifically, after a glycogen depleting endurance exercise session in the morning, subjects received an isocaloric mixed meal containing different amounts of carbohydrates and fat 2 hours before a resistance exercise session in the afternoon, while ample protein was provided throughout the day. We hypothesized that some of the selected mRNAs associated with substrate metabolism and mitochondrial biogenesis would differ between the nutritional conditions, without any changes in proteolytic genes. The findings described in chapter 3 demonstrated that muscle mRNA responses related to exercise adaptation were minimally affected by the pre-exercise meals that differed in macronutrient composition. In chapter 4, derived from the same study, we describe the analysis of a number of plasma cytokine patterns during the day to investigate whether these mediators were affected by carbohydrate availability. We hypothesized that some selected cytokines would differ between nutritional conditions, whereas other circulating cytokines suggested to be involved in skeletal muscle adaptation would not respond differently. Our main finding was that a pre-exercise meal in general did not influence plasma cytokine responses in the post-resistance exercise period. Findings of chapter 3 and 4 contribute to the view that carbohydrate availability during resistance exercise is of minor importance when aiming for an acute positive skeletal muscle adaptive response. In addition, our data question the importance of carbohydrates as both substrate for resistance exercise and as modulator of the skeletal muscle response that underlies adaptation. Yet, at present it might be premature to change carbohydrate recommendations for individuals performing resistance exercise. Shifting our focus to proteins, we first reviewed the effects and possible underlying physiological mechanisms of protein supplementation on the adaptive response to endurance training in Chapter 5. To further explore these insights, we performed a double-blind randomised controlled trial with repeated measures to determine whether protein supplementation impacts the adaptive response to endurance training. In chapter 6 we provide proof-of-concept that protein supplementation elicited greater increases in VO2max and stimulated lean mass gain in response to prolonged endurance training. To our knowledge, this was the first double-blind randomised controlled trial with repeated measures showing that protein supplementation enhances the adaptive response to endurance training. These remarkable effects of protein on VO2max that were observed give rise to questions regarding their underlying mechanisms. To this end, we analysed the muscle transcriptome to gain insight into changes in the steady-state gene expression. In chapter 7, we demonstrated that prolonged endurance training changed expression of genes involved in extracellular matrix organisation, energy metabolism and oxidative phosphorylation. Changes in extracellular matrix organisation tended to be greater in the protein group than in the control group and these greater transcriptional changes may reflect the enhanced physiological adaptation as a result of protein supplementation.

ObjectivesChronic systemic low grade inflammation is associated with the age-related loss of muscle mass. Resistance exercise has been suggested to reduce or lower chronic systemic low grade inflammation. However, systemic chronic low-grade inflammation may adversely affect the adaptive response to exercise training. We investigated the effect of resistance exercise training on systemic chronic low-grade inflammation in older adults. In addition, we studied the association between systemic chronic low-grade inflammation and the adaptive response to exercise training.Design/setting/participantsFrail and pre-frail older adults (61 subjects) performed 24 weeks of progressive resistance exercise training. Frailty was assessed using the Fried frailty criteria.MeasurementsLean body mass (DXA), strength (1RM), circulating levels of IL-1β, IL-6, IL-8 and TNF-α were measured prior to exercise training, after 12 weeks of training, and after 24 weeks of training.ResultsProlonged progressive resistance exercise training did not affect circulating levels of IL-6, IL-8 and TNF-α. However, exercise training led to a small but significant increase of 0.052 pg/mL in IL-1β. Higher circulating levels of TNF-α, IL-8 and IL-6 during the training period were negatively associated with strength gains for the leg press. A doubling of plasma TNF-α, IL-8 or IL-6 resulted in reduced strength gains for leg press with coefficients of −3.52, −3.42 and −1.54 respectively. High levels of circulating TNF-α were also associated with decreased strength gains for the leg extension (coefficient −1.50). Inflammatory cytokines did not appear to have an effect on gains in lean mass.ConclusionOur findings suggest that increased levels of plasma cytokines (TNF-α, IL-6 and IL-8) are associated with lower strength gains during resistance exercise training.

The significance of carbohydrates for endurance training has been well established, whereas the role of protein and the adaptive response with endurance training is unclear. Therefore, the aim of this perspective is to discuss the current evidence on the role of dietary protein and the adaptive response with endurance training. On a metabolic level, a single bout of endurance training stimulates the oxidation of several amino acids. Although the amount of amino acids as part of total energy expenditure during exercise is relatively low compared to other substrates (e.g., carbohydrates and fat), it may depress the rates of skeletal muscle protein synthesis, and thereby have a negative effect on training adaptation. A low supply of amino acids relative to that of carbohydrates may also have negative effects on the synthesis of capillaries, synthesis and turn-over of mitochondrial proteins and proteins involved in oxygen transport including hamoglobin and myoglobin. Thus far, the scientific evidence demonstrating the significance of dietary protein is mainly derived from research with resistance exercise training regimes. This is not surprising since the general paradigm states that endurance training has insignificant effects on skeletal muscle growth. This could have resulted in an underappreciation of the role of dietary protein for the endurance athlete. To conclude, evidence of the role of protein on endurance training adaptations and performance remains scarce and is mainly derived from acute exercise studies. Therefore, future human intervention studies must unravel whether dietary protein is truly capable of augmenting endurance training adaptations and ultimately performance.

Background: Substantial research has been done on the impact of carbohydrate and fat availability on endurance exercise adaptation, though its role in the acute adaptive response to resistance exercise has yet to be fully characterized. Purpose: We aimed to assess the effects of a pre-resistance exercise isocaloric mixed meal containing different amounts of carbohydrates and fat, on post-resistance exercise gene expression associated with muscle adaptation. Methods: Thirteen young (age 21.2 ± 1.6 year), recreationally trained (VO2max 51.3 ± 4.8 ml/kg/min) men undertook an aerobic exercise session of 90-min continuous cycling (70% VO2max) in the morning with pre- and post-exercise protein ingestion (10 and 15 g casein in a 500 ml beverage pre- and post-exercise, respectively). Subjects then rested for 2 h and were provided with a meal consisting of either 3207 kJ; 52 g protein; 51 g fat; and 23 g carbohydrate (FAT) or 3124 kJ; 53 g protein; 9 g fat; and 109 g carbohydrate (CHO). Two hours after the meal, subjects completed 5 × 8 repetitions (80% 1-RM) for both bilateral leg press and leg extension directly followed by 25 g of whey protein (500 ml beverage). Muscle biopsies were obtained from the vastus lateralis at baseline (morning) and 1 and 3 h post-resistance exercise (afternoon) to determine intramuscular mRNA response. Results: Muscle glycogen levels were significantly decreased post-resistance exercise, without any differences between conditions. Plasma free fatty acids increased significantly after the mixed meal in the FAT condition, while glucose and insulin were higher in the CHO condition. However, PDK4 mRNA quantity was significantly higher in the FAT condition at 3 h post-resistance exercise compared to CHO. HBEGF, INSIG1, MAFbx, MURF1, SIRT1, and myostatin responded solely as a result of exercise without any differences between the CHO and FAT group. FOXO3A, IGF-1, PGC-1a, and VCP expression levels remained unchanged over the course of the day. Conclusion: We conclude that mRNA quantity associated with muscle adaptation after resistance exercise is not affected by a difference in pre-exercise nutrient availability. PDK4 was differentially expressed between CHO and FAT groups, suggesting a potential shift toward fat oxidation and reduced glucose oxidation in the FAT group.

Carbohydrate availability is proposed as a potential regulator of cytokine responses. We aimed to evaluate the effect of a preresistance exercise carbohydrate meal versus fat meal on plasma cytokine responses to resistance exercise after an endurance exercise earlier that day. Thirteen young, healthy, recreationally active males performed two experimental days with endurance exercise in the morning and resistance exercise in the afternoon. Either a carbohydrate (110 g carbohydrate, 52 g protein, 9 g fat; ~750 kcal) or an isocaloric fat meal (20 gr carbohydrate, 52 g protein, 51 g fat) was provided 2 h before resistance exercise. Blood was taken at baseline and at regular time intervals to measure circulating plasma cytokine levels (e.g. IL-6, IL-8, IL-10, IL-15, TNFα, ANGPTL4, decorin and MCP-1). Plasma glucose and insulin were higher in the postprandial period before the start of the resistance exercise on the carbohydrate condition, while free fatty acids were reduced. At 2 h postresistance exercise, IL-6 concentrations were higher in the fat condition compared to the carbohydrate condition (P < 0.05). In addition, in both conditions IL-6 levels were higher at all time points compared with baseline (P < 0.05). The pattern of increase in plasma IL-8 and IL-10 did not differ significantly between conditions (P > 0.05). There were no differences between conditions on TNFα levels and levels remain constant when compared with baseline (P > 0.05). ANGPTL4, IL-15, Decorin and MCP-1 showed no differences between the fat and carbohydrate condition (P > 0.05). The composition of the pre-exercise meal did in general not influence cytokine responses in the postresistance exercise period, except postresistance exercise circulating plasma IL-6 levels being higher in the fat condition compared with carbohydrate. Our findings support the view that pre-exercise carbohydrate availability does not have a major impact on acute responses of circulating plasma cytokines in humans.

Skeletal muscle mass is maintained by the balance between muscle protein synthesis and breakdown rates such that overall muscle net protein balance (NPB) remains essentially unchanged over the course of the day. A prolonged negative NPB will result in a loss of skeletal muscle proteins. Ageing or prolonged periods of disuse (due to illness or a sedentary lifestyle) exacerbate the loss of skeletal muscle mass. It has been speculated that the capacity of skeletal muscle tissue to recover from prolonged periods of muscle unloading is reduced with advancing age. Therefore, a greater understanding of the proposed mechanisms underpinning the unloading induced atrophy, and the eventual incomplete recovery that generally follows, will certainly contribute to the development of more effective nutritional or lifestyle strategies to attenuate muscle mass loss and support healthy ageing. The lack of skeletal muscle recovery following unloading in the elderly may be explained by an observed phenomenon commonly coined ‘anabolic resistance’ of muscle protein synthesis rates. Specifically, frequent periods of disuse, simplified as ‘inactive muscle’, will desensitize skeletal muscle to hyperaminoacidaemia and ultimately lead to a reduced muscle protein synthetic response when compared with the response of active skeletal muscle to the same stimuli. There is no general consensus on whether anabolic resistance of muscle protein synthesis rates is truly an inherent intrinsic characteristic of ageing muscle or a self-induced product of a sedentary lifestyle. Hitherto, the role of muscle protein breakdown in unloading-induced disturbances of protein metabolism has not been studied profoundly. However, it is assumed that any change in muscle protein breakdown rates during unloading is an adaptive response to changes in muscle protein synthesis rates. Of course, more in vivo human research is necessary to understand the quantitative role of muscle protein breakdown during periods of unloading. Thus, due to the sparse knowledge regarding muscle protein breakdown rates in unloading-mediated muscle atrophy, contemporary interventions generally focus on nutritional strategies known to stimulate muscle protein synthesis rates. Recent work from our lab has revealed that the type of protein consumed can have a fundamental impact on postprandial muscle protein synthesis rates (Pennings et al. 2011). Specifically, we found that ingestion of a meal-like amount of whey protein (20 g) is more effective at stimulating postprandial muscle protein synthesis rates than ingestion of an isonitrogenous amount of casein hydrolysate or micellar casein (Pennings et al. 2011). We speculated that the superiority of whey protein for the stimulation of postprandial muscle protein synthesis rates is probably attributable to both its speed of digestion/absorption and its higher leucine content. Thus, meal-induced leucinaemia appears to be a primary driver of the anabolic response. Interestingly, Glover et al. (2008) investigated whether increasing the blood amino acid availability can overcome the anabolic resistance of muscle protein synthesis rates to amino acid provision induced by immobilization. It was observed across a wide range of amino acid concentrations that the immobilized limb revealed a blunted muscle protein synthetic response when compared to the non-immobilized limb. Notably, even a high-dose amino acid infusion could not overcome the anabolic resistance of muscle protein synthesis rates imposed by unloading. These data also suggest that eating large amounts of protein may be a fruitless nutritional strategy to offset muscle mass loss during unloading. Despite these results in young adults (Glover et al. 2008), it provides perspective for more research into the phenomenon of immobilization-induced anabolic resistance among the elderly. As a matter of fact, the effects of different anabolic stimuli, such as intact proteins or free dietary amino acid supplementation, after prolonged periods of unloading during ageing remain largely unknown. Recently, an article published in The Journal of Physiology by Magne et al. (2012) provided new insights into nutritional strategies that may help recover muscle mass after a prolonged period of unloading. The authors used a rodent (male Wister rats aged 22–24 months) unilateral hind-limb cast immobilization model. After 10, 20, 30 and 40 days of recovery from an 8 day immobilisation period, muscle protein synthesis rates, markers of proteolysis and intramuscular anabolic signalling protein phosphorylation were measured. The authors observed that ∼20% of the gastrocnemius muscle mass was lost following immobilisation. Muscle protein synthesis rates were depressed in the postprandial state after prolonged mechanical unloading in the elderly rats and did not normalize during reloading. In addition, the authors report that leucine supplementation was not as effective as whey or high-protein diets in maximizing skeletal muscle mass after 40 days of recovery. This is an interesting finding since leucine is presumed to be highly anabolic toward the stimulation of muscle protein synthesis rates and often used as a pharmaconutrient to improve muscle recovery after prolonged periods of unloading. However, it has recently been demonstrated that simply supplementating the diet with free dietary leucine (7.5 g d−1) is not an effective strategy to increase muscle mass or strength, in healthy elderly men consuming the recommended dietary allowance for protein (Verhoeven et al. 2009). With respect to the animal in vivo measurements of muscle protein synthesis by Magne et al. (2012), the positive leucine-induced effect on the stimulation of muscle protein synthesis rates failed to translate into muscle growth. Moreover, other factors involved in stimulating muscle protein synthesis rates were also altered in favour of muscle protein synthesis, despite the lack of long-term benefits. For example, free leucine supplementation increased the amount of phosphorylation of S6 and 4EBP1 proteins and at the same time generated a decrease in chymotrypsin- and trypsin-like activities of the 26S proteasome associated with a P-FOXO3a/FOXO3a ratio in favour of antiproteolytic processes. However, when the duration of muscle protein synthesis increment is inadequate to generate a significant muscle protein accretion, the effect on muscle mass is negligible, which is in accordance with the authors’ conclusion. Unlike free leucine supplementation, whey and high-protein diets resulted in a ∼60% regain of the amount of muscle lost with immobilization. This could be explained by the larger increases in muscle protein synthesis rates after the whey and high-protein diets. Despite the positive effects of free leucine supplementation on muscle protein synthesis rates, it appears that intact proteins are essential to gain muscle mass. Notwithstanding, proteins enriched with leucine might be applicable to accelerate muscle regain or prevent muscle loss. Magne et al. (2012) are to be praised for studying the role of leucine and even more for underpinning the importance of intact proteins such as whey or consumption of higher protein diets in the recovery of muscle mass during or after a period of muscle disuse. The next step is to translate the authors’ work to a human model. Therefore, we need immobilization trials with elderly people to investigate whether the animal findings are similar to that of in vivo human work. Notable is that Drummond et al. (2012) examined the effect of 7 days of bed rest and amino acid supplementation on muscle protein synthesis rates in humans. As in previous trials, Drummond et al. (2012) showed that 7 days of bed rest blunted the essential amino acid-induced increase in muscle protein synthesis rates. In addition, the workers studied different types of amino acid transporters, to decipher whether a reduced amino acid-transporter response was partly responsible for the desensitization to amino acidaemia of muscle during disuse. To stimulate mTORC1 signalling exogenous amino acids first have to enter the muscle cell via active transport. Interestingly, both amino acid transporters, LAT1 and SNAT2, are associated with mTORC1 activation and muscle protein synthesis rates. Indeed, in addition to the impaired response in muscle protein synthesis following an amino acid stimulation the authors found a decrease in amino acid-transporter content, thus providing some clue as to how anabolic resistance of muscle proteins synthesis rates to amino acids manifests during unloading. Taken together, the work from Magne et al. (2012) and other referenced authors contributes to the development of nutritional strategies to recover muscle mass after periods of muscle disuse (e.g. injury or illness) in the elderly. Importantly, the authors’ findings point to a thesis that high-quality proteins, such as whey, that are easily digested (and already high in leucine) are superior to create an anabolic environment, especially after periods of disuse. This message is one that those supplementing their diet with free dietary leucine with a wish of building bigger muscles might not want to hear! In conclusion, the addition of free leucine next to our habitual diet is not the answer to prevent muscle loss or accelerate muscle regain. More work is needed to explore the anabolic properties of our nutrition in general, with the functional properties of different proteins in particular.

It is well established that glycogen depletion affects endurance exercise performance negatively. Moreover, numerous studies have demonstrated that post-exercise carbohydrate ingestion improves exercise recovery by increasing glycogen resynthesis. However, recent research into the effects of glycogen availability sheds new light on the role of the widely accepted energy source for adenosine triphosphate (ATP) resynthesis during endurance exercise. Indeed, several studies showed that endurance training with low glycogen availability leads to similar and sometimes even better adaptations and performance compared to performing endurance training sessions with replenished glycogen stores. In the case of resistance exercise, a few studies have been performed on the role of glycogen availability on the early post-exercise anabolic response. However, the effects of low glycogen availability on phenotypic adaptations and performance following prolonged resistance exercise remains unclear to date. This review summarizes the current knowledge about the effects of glycogen availability on skeletal muscle adaptations for both endurance and resistance exercise. Furthermore, it describes the role of glycogen availability when both exercise modes are performed concurrently.