Cannabinoid receptor 1 expression is higher in muscle of old vs. young males, and increases upon resistance exercise in older adults

Aged skeletal muscle undergoes metabolic and structural alterations eventually resulting in a loss of muscle strength and mass, i.e. age-related sarcopenia. Therefore, novel targets for muscle growth purposes in elderly are needed. Here, we explored the role of the cannabinoid system in muscle plasticity through the expression of muscle cannabinoid receptors (CBs) in young and old humans. The CB1 expression was higher (+ 25%; p = 0.04) in muscle of old (≥ 65 years) vs. young adults (20–27 years), whereas CB2 was not differently expressed. Furthermore, resistance exercise tended to increase the CB1 (+ 11%; p = 0.055) and CB2 (+ 37%; p = 0.066) expression in muscle of older adults. Interestingly, increases in the expression of CB2 following resistance exercise positively correlated with changes in key mechanisms of muscle homeostasis, such as catabolism (FOXO3a) and regenerative capacity (Pax7, MyoD). This study for the first time shows that CB1 is differentially expressed with aging and that changes in CB2 expression upon resistance exercise training correlate with changes in mediators that play a central role in muscle plasticity. These data confirm earlier work in cells and mice showing that the cannabinoid system might orchestrate muscle growth, which is an incentive to further explore CB-based strategies that might counteract sarcopenia.

www.nature.com/scientificreports/ which regulates many cellular processes including proliferation, differentiation, apoptosis and energy metabolism. Whereas the signaling responses upon CB binding are well described in neural tissues, it is not understood how CB 1/2 affect (MAPK-mediated) downstream signaling in skeletal muscle tissue. Nevertheless, recent developments in mice indicate that CB signaling interferes with muscle metabolism 19 , muscle maintenance 15 and regenerative processes 13 . CB 1 antagonism in mice reversed age-related metabolic dysregulations 19,20 and improved muscle regeneration in a murine model of Duchenne muscular dystrophy 14 . Moreover, CB 1 knock-out in skeletal muscle protected old mice from insulin resistance and upregulated intermediates of the mTORC1 pathway, responsible for muscle protein synthesis (MPS) 15 . In vitro data confirmed that CB 1 inhibition stimulated mTORC1 and MPS in human myotubes 21 . Besides anabolic signaling, also muscle catabolism plays a crucial role in muscle maintenance. Unfortunately, there are no studies that investigated whether modifications in the cannabinoid tone affect catabolic mechanisms, such as the ubiquitin-proteasome system (e.g. FOXO and MuRF1) or autophagy (e.g. LC3b). Unpublished data from our lab showed that CB 2 was downregulated during muscle atrophying conditions such as unloading and CTX-induced injury 22 in mice (Supplementary Figure 1). Others demonstrated that CB 2 agonism improved, whereas CB 2 antagonism impaired, muscle regeneration [23][24][25] .
Despite these promising data indicating that CBs are interesting candidates to target (age-related) muscle wasting, little is known about the expression and modulation of CBs in human skeletal muscle tissue. In anticipation of CB (ant)agonists approval for human application, the present study explores for the first time muscle CB 1 and CB 2 expression in different age groups and in response to RE. We hypothesize that CB 1 is more expressed in muscle of old vs. young males, indicative for increased endocannabinoid activity that might be associated with age-related metabolic dysregulations 19,20 . No females were included since hormonal fluctuations in young females might interfere with the cannabinoid signaling. Furthermore, we hypothesize that RE downregulates CB 1 and upregulates CB 2 expression in muscle tissue of old adults (males and females), indicating an exercise-induced improvement in metabolic health and an improved myogenicity, respectively. Finally, we investigate whether responses in CB expression are related to anabolic, catabolic and regenerative muscle markers, and whether they occur in a fiber-specific way.
The change in expression (Post minus Pre RE) of the CB 2 was associated with the change in expression in these central markers of muscle maintenance (Fig. 3). Pearson correlations revealed that CB 2 correlated with total FOXO3a (r = 0.610; p = 0.006) and with both myogenic markers MyoD (r = 0.604; p = 0.006) and Pax7 (r = 0.491; p = 0.033), whereas CB 1 was not related to any of the markers of muscle maintenance.
As expected, CB 1 and CB 2 were highly expressed in the sarcolemma. The expression of CB 1 in the m. Vastus Lateralis of older adults was more pronounced in type I vs. type II fibers (+ 288%; p < 0.001), whereas CB 2 was more expressed in type II vs. type I fibers (+ 268%; p = 0.001) (Fig. 4).

Discussion
Recently, it was shown in mice that CBs are key players in the regulation of myogenicity and muscle metabolism, both features that are affected with advancing age, and that contribute to sarcopenia. Consequently, it has been suggested that CB (ant)agonism is a promising strategy to counteract muscle degeneration, and more specifically age-related sarcopenia 15 . Unfortunately, CB modulation is not yet feasible in humans. Hence, the present explorative study for the first time investigates whether CB expression is different in old vs. young skeletal muscle tissue, and whether CB expression is responsive to anabolic stimuli such as RE. www.nature.com/scientificreports/ www.nature.com/scientificreports/ The findings of the present study demonstrate that CB 1 is differently expressed in skeletal muscle of young (20-27 years) vs. old adults (65-84 years), and that both CB 1 and CB 2 expression tend to increase upon chronic RE in old adults. Interestingly, the RE-induced change in CB 2 expression was associated with changes in the catabolic marker FOXO3a and with changes in markers of myogenicity, i.e. MyoD and Pax7. More interventional work in animals and humans is needed to unravel the mechanisms via which CBs might affect muscle plasticity in response to age and exercise.
It was reported that the cannabinoid tone (e.g. circulating AEA and 2-AG levels) was typically higher in metabolic conditions, such as obesity 17,26 , and that increased CB 1 expression related to a decreased metabolic health, as some 27,28 , but not all 17,29 , researchers observed higher CB 1 expression levels in the adipose tissue of overweight/obese adults when compared to lean. Furthermore, the cannabinoid tone positively correlated with (markers of) adiposity and insulin resistance 26,29 and CB 1 expression in visceral adipose tissue positively correlated with BMI 27 . Therefore, in the present study, it is conceivable that the increased CB 1 expression in the muscle of older vs. young adults reflects an increased cannabinoid tone and decreased metabolic health in the older adults. Although the older participants did not suffer from explicit metabolic conditions, it is very likely that their metabolic health is generally lower when compared to physically active, young adults (which might be reflected by their higher BMI, i.e. 27.2 in the older participants vs. 23.8 kg·m −2 in the young participants). Also, half of the older adults used statins to lower increased cholesterol levels.
The effect of age on CB 1 expression has previously been studied in mice, be it on the gene and not on the protein expression level. Evidence in mice was equivocal. CB 1 expression was lower in the m. Gastrocnemius of 1 year vs. 2 weeks old mice 30 , whereas others reported a higher CB 1 expression in the m. Gastrocnemius of 16-month old vs. 4-months old mice 19 . This latter observation is in accordance with our observation that CB 1 expression was higher in old vs. young adults. Besides metabolic health, it remains unclear which behavioral (e.g. physical activity) and/or biological (e.g. hormone levels) changes contribute to the effect of ageing on muscle CB 1 expression.
Whereas acute exercise upregulates circulating endocannabinoid levels (particularly AEA), the effect of chronic exercise on the endocannabinoid tone and on muscle CB expression remains poorly understood. We hypothesized that muscle CB 1 expression would decrease in response to a 12-weeks RE program, reflecting an exercise-induced improvement in metabolic health. Surprisingly, CB 1 expression increased following RE. This observation is very unlikely to reflect a deterioration in metabolic health, which is why alternative mechanisms might be at work. One hypothesis is that the increased CB expression can be explained by a change in the muscle fiber type composition since CB 1 and CB 2 expression was more pronounced in type I and type II muscle fibers, respectively. However, muscle MyHC type I and II expression did not alter due to RE, and if any changes were to be expected following RE, it would rather be an increase in type II fibers, which would have resulted in a relative decrease in CB 1 expression rather than an increase. Alternatively, it is possible that an RE-induced increase in mitochondrial density 31 explains the increases in CB 1 , as the receptor is highly expressed in mitochondria of human muscle tissue 32 . However, in contrast to endurance training, it remains debatable whether high-load RE increases the mitochondrial content 33 . Finally, RE-induced changes in the relative contribution and/or signaling of muscle-resident non-myogenic cell types 34,35 such as immune cells, vascular cells and neural cells might also explain part of the increase in CB 1 expression.
Similar to CB 1 , also CB 2 expression tended to be increased upon RT. Pharmaceutical CB 2 agonism increased the expression of satellite cell growth markers such as MyoD and myogenin 23 , while CB 2 knock-out decreased MyoD and myogenin expression during murine muscle regeneration 13 . Therefore, the increased CB 2 expression in the present study might reflect an improved myogenic capacity, e.g. satellite cell content, in response to RE. Indeed, MyoD and Pax7 expression significantly increased upon RE, and the increase of both markers positively correlated with the increase in CB 2 expression. Together, these data indicate that there might be regulatory networks that relate CB 2 biology to satellite cell myogenicity. However, there is need for more evidence to explore a potential association between muscle progenitor cells and CBs, especially in humans.
Whereas evidence in mice 30 and frogs 36 showed that CB 1 gene expression was higher in predominantly fast (e.g. Gastrocnemius, Tibialis Anterior) vs. slow (e.g. Soleus) muscles, our data show that CB 1 expression was more highly abundant in type I than in type II fibers of the m. Vastus Lateralis of older adults (based on colorimetric analysis of individual muscle fibers). From a metabolic perspective, it seems indeed more logical that CB 1 , which www.nature.com/scientificreports/ is an important regulatory player in peripheral oxidative capacity 32 , is more abundant in type I fibers, which exhibit a higher oxidative capacity than type II fibers.
In conclusion, cell culture and murine experiments suggested that CBs can be a promising target to treat cachexia and sarcopenia through modulation of the metabolism and muscle regenerative capacity. Yet, the role of CB modulation as a window of opportunity to treat muscle devastating conditions in (old) humans remains unstudied. The present explorative study for the first time shows that CB 1 is differentially expressed in muscle of old (i.e. higher expression) vs. young (i.e. lower expression) adults, and that both CB 1 and CB 2 expression increases upon RE, the most important anabolic intervention for muscle growth purposes. Interestingly, REinduced increases in CB 2 correlated with markers of myogenicity (MyoD and Pax7). These data imply that CB modulation might be a promising tool to combat muscle degeneration. As a next step, human intervention studies should be performed to confirm whether CBs are promising targets to improve the muscle phenotype in age-related sarcopenia and/or muscle devastating conditions, such as cancer cachexia.  (Red, panel a,b) or CB 2 (Red, panel c,d). CB 1 and CB 2 expression was quantified in a fiber type-specific way (type I vs. type II fibers), in 16  . All participants were recreationally active, but did not engage in a consistent training program or any sport at a competitive level. Muscle tissues were collected in previously executed studies at our laboratory (2014-2019; Exercise Physiology Research Group; KU Leuven; Belgium) and have been stored at − 80 °C immediately after completion of the study. The only inclusion criteria for selection were age (< 30 years or ≥ 65 years, respectively) and sufficiently muscle tissue available (≥ 15 mg). Exclusion criteria were BMI < 20 or > 35 kg·m −2 , unstable body weight (i.e. 2-kg change during the past 6 months), smoking, disease (cancer, liver, renal, musculoskeletal, neurodegenerative or unstable cardiovascular dysfunctions), smoking or supplementation of cannabis or cannabis-related products (e.g. CBD oil), regular protein supplementation and structural RE or endurance training. None of the young participants took any medication. The medicine intake of the older participants reflected the real-life situation, e.g. a large proportion of the older participants took blood thinners (n = 4; 29%; e.g. asaflow), statins (n = 7; 50%; e.g. simvastatin), beta blockers (n = 7; 50%; e.g. bisoprolol), antacids (n = 3; 21%; e.g. nexiam) and medication to treat osteoporosis (n = 1; 7%) and to treat benign prostatic hyperplasia (n = 2; 14%). All muscle biopsies were sampled by the same MD, and all samples have been prepared simultaneously (August 2020) for analyses by the same researcher. This study was approved by the Ethics Committee Research UZ/KU Leuven (S58361, S61809) and conformed to the Declaration of Helsinki. All participants gave their written informed consents.  ) with the same exclusion criteria as reported in experiment 1. The medicine intake of the older participants reflected the real-life situation, e.g. a large proportion of the older participants took blood thinners (n = 6; 32%; e.g. assaflow), statins (n = 9; 47%; e.g. simvastatin), beta blockers (n = 7; 37%; e.g. bisoprolol), antacids (n = 4; 21%; e.g. nexiam), antidepressants (n = 1; 5%), sleep medication (n = 1; 5%), and medication against migraine (n = 2; 11%), to treat osteoporosis (n = 2; 11%) and to treat benign prostatic hyperplasia (n = 1; 5%). Muscle biopsies were sampled before (Pre) and after (Post) 12-weeks RE program. All biopsies were taken at least 72 h after RE to exclude interference of acute exercise on muscle molecular signaling 37 . Three supervised training sessions per week started with a 10-min warm-up on a cycle ergometer, followed by lower extremity exercises (leg press, leg extension and calf raises). In the first 6 weeks, 2 sets of 12-15 repetitions at 70% of the 1-repetition maximum (1RM) were performed, and in the last 6 weeks, participants completed 3 sets of 10-12 repetitions at 80% of the 1RM. This study was approved by the Ethics Committee Research UZ/KU Leuven (S61809) and conformed to the Declaration of Helsinki. All participants gave their written informed consents.

Experiment 2 (resistance exercise in old adults
Muscle biopsy procedure. Prior to muscle biopsies, fasted participants reported at the lab and rested for 45 min. A needle biopsy (~ 150 mg) of the m. vastus lateralis was performed under local anaesthesia (2% xylocaine, 1 mL subcutaneously) with a 5-mm Bergström-type needle at the Pre and Post experimental session. The muscle sample was immediately frozen in liquid nitrogen and stored at − 80 °C for later biochemical analyses.
Protein extraction and western blot analysis. As previously described 3  Next, the blue (cell nuclei) and green (MyHC I) signal was removed, and the red signal (CB 1 and CB 2 expression) was quantified in a fiber-type specific way.
Muscle strength. The 1-RM was measured on the leg press device as previously described 39 . Shortly, participants started with a warm-up set of 8 repetitions at ~ 50% of the estimated 1-RM, followed by a set of 3 repetitions at ~ 70% of the estimated 1-RM. Subsequent lifts were single repetitions with progressively heavier resistances until failure. A 2-min recovery period was provided between each attempt. The heaviest successful lift was determined as 1-RM. Participants were familiarized with the 1-RM leg press procedure. The 1-RM was assessed at baseline (PRE), after 6 weeks of RE and after 12 weeks of RE (POST).
Muscle volume and density. As previously reported 3 , a computed tomography scan (Somatom Force ® , Siemens Medical Solutions, Erlangen, Germany) was used to measure the muscle volume of the left upper leg. Four thick axial images of 5 mm were obtained at the midpoint of the distance between the medial edge of the trochanter major and the intercondyloid fossa of the femur. Standard Hounsfield units (HU) ranges for muscle (0-100) were used to segment muscle area, and were corrected for bone marrow. The 4 slices were put together as one slice of 20 mm. Total muscle volume was determined by an expert radiologist, with a software program developed at the University Hospital.
Statistics. Data are presented as mean ± SEM or mean (range), and were tested for normality with a Kolmogorov-Smirnov test. Conditions were compared with a two-tailed Independent-Sample t Test or Mann-Whitney U Test (experiment 1), or a two-tailed Pair-Sampled t Test or Wilcoxon signed-rank test (experiment 2) (SPSS 20, IBM, Chicago, IL). Where relevant, Cohen's D (d) was calculated as an index of effect size (0.2 = small, 0.5 = medium, > 0.8 = large). Correlations were assessed by the Pearson (normal data) or Spearman (non-normal data) correlation coefficient analyses (experiment 2, SPSS 20). Significance (*) was accepted at p < 0.05 and trends ( † ) were set at p = 0.05-0.10.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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