Heavy-load and low-load blood flow restricted resistance training increases Na/K-ATPase subunit but not ClC-1 expression in untrained human skeletal muscle

Heavy-load and low-load blood flow restricted resistance training increases Na/K-ATPase subunit but not ClC-1 expression in untrained human skeletal muscle | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Heavy-load and low-load blood flow restricted resistance training increases Na/K-ATPase subunit but not ClC-1 expression in untrained human skeletal muscle Jakob Wang, Emil Rindom, Thomas Groennebaek, Peter Sieljacks, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1799620/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Apr, 2023 Read the published version in Journal of Muscle Research and Cell Motility → Version 1 posted 8 You are reading this latest preprint version Abstract Contractile function of skeletal muscle relies on the ability of muscle fibers to trigger and propagate action potentials (APs). These electrical signals are created by transmembrane ion transport through ion channels and membrane transporter systems. In this regard, the Cl- ion channel 1 (ClC-1) and the Na+/K--ATPase (NKA) are central for maintaining ion homeostasis across the sarcolemma during repetitive AP firing inherent of intense contractile activity. Therefore, this randomized controlled trial (RCT) aimed to investigate the changes in ClC-1 and specific NKA subunit isoform expression in response to 6 weeks (18 training sessions) of heavy-load resistance exercise (HLRE) and low-load blood flow restricted resistance exercise (BFRRE), respectively. Also, the potential associations between protein expression and contractile performance were investigated. We show that muscle ClC-1 abundance was not affected by either exercise modality, whereas NKA subunit isoforms 𝛼 2 and 𝛽 1 increased appx. 80-90% with BFRRE (p<0.05) and 70-80% with HLRE (p<0.05). No differential impact between exercise modalities was observed. At baseline, ClC-1 protein expression correlated inversely with dynamic knee extensor strength (r=-0.365, p=0.04). No correlation was observed between NKA subunit content and contractile performance at baseline. However, training-induced changes in NKA 𝛼 2 subunit (r=0.603, p<0.01) and 𝛽 1 subunit (r=0.453, p<0.05) correlated with changes in maximal voluntary contraction. These results suggest that the initial adaptation to resistance exercise does not involve changes in ClC-1 abundance in untrained skeletal muscle. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The ability to activate and control skeletal muscle fibers is dependent on excitation and propagation of action potentials (APs) in muscle fibres from the neuromuscular junction along the sarcolemma and into the T-tubular system (Sejersted and Sjogaard 2000). During exercise, preservation of muscle fiber excitability is dependent on the transmembrane ion gradients of Na + , K + and Cl - (Baekgaard Nielsen et al. 2017; Clausen and Nielsen 1994). This implicates that the abundance and function of membrane proteins involved in the regulation of these ions is essential for contractile performance. It is well documented that muscle activity results in accumulation of extracellular K + (Fitts 1994), which potentially could impact muscle fiber excitability. This is due to the associated depolarization of the membrane, which might compromise the function of voltage-gated Na + channels that are responsible for AP formation (Filatov, Pinter, and Rich 2005). During repeated AP firing, the Na + /K + -ATPase (NKA) plays a central role in counterbalancing the extracellular K + buildup (Clausen 2003). However, the rate of interstitial K + accumulation clearly exceeds the rate of re-uptake in human skeletal muscle during exercise (Nordsborg et al. 2003; Juel et al. 2000) and the ensuing loss of muscle excitability has been widely considered the primary driver of fatigue development during strenuous and high-intensity exercise in humans (Bangsbo et al. 1996; Nielsen et al. 2004). Equally important for preserving muscle fiber excitability during exercise is regulation of Cl - membrane conductance (G Cl ) in the active muscle fibers (de Paoli et al. 2013). The Cl - channel protein 1 (ClC-1) accounts for ~80% of the resting membrane conductance specifically in skeletal muscle (Pedersen et al. 2016). In contracting muscles, the onset of repeated AP firing triggers a substantial reduction of the G Cl in both rodent (Pedersen, Macdonald, et al. 2009; Pedersen, de Paoli, et al. 2009) and human muscle (Riisager et al. 2016; Leermakers et al. 2020). This inhibition of ClC-1 has been shown to augment high-intensity contractile endurance even in context of elevated extracellular K + (de Paoli et al. 2010). Thus, decreasing the ClC-1 abundance may constitute a beneficial adaptation to strenuous training with regards to preservation of excitability and consequently, muscle contractile performance. In recent years, low-load blood flow restricted resistance exercise (BFRRE) has emerged as a low-load alternative to high-load resistance exercise (HLRE) in promoting skeletal muscle adaptations (Wernbom, Augustsson, and Raastad 2008). During both HLRE and BFRRE, substantial increases in sEMG have been reported (Farup et al. 2015; Buckner et al. 2018), suggesting that both modes of exercise compromise muscle fiber excitability. In addition, the partial restriction of blood flow inherent of BFRRE exacerbates both the accumulation of metabolites (Fujita et al. 2007) and the ionic perturbations during exercise (Christiansen 2018), which may potentiate training-induced adaptations of transmembrane proteins. Interestingly, we demonstrated that 6 weeks of both BFRRE and HLRE markedly increased muscle strength (Sieljacks et al. 2019), whereas BFRRE selectively increased muscle strength-endurance capacity (Groennebaek et al. 2018). To our knowledge, no prior studies have investigated the impact of HLRE and BFRRE on ClC-1 and NKA subunit isoform expression in humans. Therefore, the aim of the present study was to investigate the impact of BFRRE and HLRE on ClC-1 protein and NKA subunit isoform abundance and to examine if these were related to skeletal muscle contractile properties in young, untrained individuals. We hypothesized; (1) that 6 weeks of BFRRE and HLRE would decrease ClC-1 expression and increase NKA subunit isoform abundance, and; (2) that BFRRE would accentuate this response compared to HLRE. Materials & Methods Subjects Thirty-four young, healthy, untrained men were enrolled in this randomized controlled trial. Inclusion and exclusion criteria as well as baseline characteristics of the subjects are available in detail elsewhere (Groennebaek et al. 2018). All participants provided both written and informed consent prior to enrollment. The study was approved by the Central Denmark Region Committee on Health Research Ethics (1-10-72-218-16) and registered in the database clinicaltrials.gov (NCT03380663). The study was conducted in conformity with the standards for human experimental trials outlined in the Declaration of Helsinki. Study Design The study was conducted as a randomized controlled trial. Each subject was randomly allocated to one of three intervention groups; BFRRE (n = 12), HLRE (n = 12) or non-exercise control (CON; n = 10). The total duration of the study was 9 weeks (Fig. 1). In week 1, subjects reported for baseline measures of muscle strength and strength-endurance capacity following collection of pre-intervention muscle biopsies. Furthermore, individual arterial occlusion pressure (AOP) was determined in the subjects prescribed to BFRRE. In week 2, a single-trial exercise experiment was conducted, intended for other investigatory points of interest reported elsewhere (Groennebaek et al. 2018). The subsequent 6 weeks comprised of the exercise intervention. In week 9, subjects reported for post-intervention assessment of muscle strength and strength-endurance capacity following collection of biopsies. The CON subjects were exposed to all experimental procedures of the study with exception of exercise. Exercise interventions Training comprised a total of 18 exercise sessions dispersed as three supervised sessions 3 week -1 (Monday Wednesday, Friday) throughout the intervention period. Exercise interventions are described in detail previously (Groennebaek et al. 2018). Briefly, training sessions comprised a standardized warmup on stationary bicycle ergometer (Monark Ergomedic 818E, Monark, Varberg, Sweden) and submaximal knee extension repetitions in the same apparatus later used for exercise (TechnoGym selection-line, TechnoGym, Italy). Following warmup, subjects performed 4 sets of isolated knee extensions in accordance with the recommended principles inherent of the respective exercise modalities (Scott et al. 2015; American College of Sports 2009). BFRRE was conducted at 30% of 1RM with all sets performed to volitional fatigue. An individualized relative occlusion pressure corresponding to 50% of AOP was applied by use of 14 cm wide pneumatic cuffs (Delfi Medical, Vancouver, Canada). The cuffs were wrapped around the inguinal fold region of the thighs and were continuously inflated both during exercise and the 30 s inter-set recovery by means of a digital tourniquet (A.T.S 2200TS, Zimmer Surgical Inc. Ohio, USA). Relative exercise intensity for HLRE was 70% of 1RM. Participants were instructed to target 12 repetitions in each set, with sets being interspaced by 3 minutes of recovery. Appropriate exercise intensity was assured throughout the training period by re-evaluating 3 RM every other week (fig. 1) as well as progressively increasing training load in case of HLRE, as previously described (Groennebaek et al. 2018). Muscle strength and strength-endurance capacity Testing of muscle contractile performance comprised maximal isometric (maximal voluntary contraction; MVC) and dynamic strength (3RM) as well as local muscle strength-endurance capacity of the knee extensors. These were evaluated at baseline (pre) and 4 days after cessation of exercise (post). Testing of unilateral isometric MVC was conducted in an isokinetic dynamometer (Humac Norm, CSMI, Stoughton, Massachusetts, USA), as previously described (Sieljacks et al. 2019). Dynamic knee extensor strength was evaluated using a 3RM-test conducted in knee extension apparatus. Following the 3RM-test, the load was readjusted to 30% of the corresponding 1RM and subjects performed bilateral knee extensions to volitional failure. The total number of repetitions performed provided the measure of muscle strength-endurance capacity. Procedures for testing of maximal dynamic strength and strength-endurance capacity are described in detail elsewhere (Groennebaek et al. 2018). Muscle biopsies Muscle biopsies were harvested from the mid-section of vastus lateralis before and after the exercise intervention. Biopsies were obtained under local anesthesia (1% Lidocaine, Mylon Hospital, Norway) utilizing the Bergström needle technique. Prior to days of testing, subjects were instructed to refrain from strenuous physical activity. Furthermore, all biopsies were collected in the early morning following an overnight fast, to circumvent potential influence of nutritional and circadian confounders. After removal of visible fat and connective tissue an aliquot of the muscle sample (appx. 30 mg wet weight) was immediately snap-frozen in liquid nitrogen and subsequently stored at -80°C until further preparation and analysis. Immunoblotting Preparation of frozen muscle biopsies and determination of protein concentration was performed in accordance with Groennebaek et al (2018). Proteins were separated by SDS-PAGE on precast polyacrylamide gels (Bio-Rad, CA, USA) and subsequently electroblotted onto PVDF membranes (Bio-Rad, CA, USA). Equal loading of protein was controlled by visual examination using stain-free technology. Membranes were then blocked for 2 hours in 0.3% I-Block in solution with TBST at room temperature and incubated overnight at 4°C in appropriate primary anti-bodies. The primary antibodies utilized were as follows; ClC1 c-terminal (cat # ab189857, ABCAM, Cambridge, UK), NKA 𝛼 2 (cat # 07-674, Merck, Darmstadt, DE) and NKA 𝛽 1 (cat # 44759, Cell Signaling Technology, MA, US). All primary anti-bodies were used in a 1:1000 dilution of TBST and 5% BSA, with exception of NKA 𝛼 2 , which was diluted in 1:1000 TBST with 5% skim milk. After incubation in primary anti-bodies, membranes were incubated in secondary anti-bodies for 1 hour at room temperature with horseradish peroxidase-conjugated goat anti-rabbit (cat # 6721, ABCAM, Cambridge, UK) in 1:5000 TBST solution with 1% BSA, with exception of NKA 𝛼 2 which was incubated using skim milk. All proteins were visualized by chemilumescence (Thermo Fisher Scientific, MA, US). Arbitrary protein intensity was normalized and quantified by utilizing an UVP imaging system (UVP, CA, US). Statistical analysis Alterations in protein abundance were analyzed by a mixed effect linear model with group, time and group x time interaction as factors of interest. When significant group x time interactions were observed, pairwise comparisons were made between groups as well as separate timepoints (pre vs. post). The model was validated by test for equal standard deviations and visual examination of QQ-plots. Associations between cellular outcomes and muscle contractile performance were evaluated using linear regressions and Pearson’s correlation. All statistical analyses were conducted using STATA 15.0 (StataCorp, College Station, TX, USA) with an alfa level of 0.05 delineating statistically significant outcomes. Data are graphically presented as individual values and means ± SDs, whereas in-text data presented as means with corresponding 95%-CIs. All graphical presentation of data are performed using GraphPad Prism v.7 (GraphPad Software, La Jolla, CA, USA). Results Impact of Resistance Training on Protein Abundance Neither six weeks of HLRE or BFRRE influence ClC-1 protein abundance in untrained males (Fig. 2). As for the NKA, significant overall time x group interactions were observed for both 𝛼 2 subunits (p < 0.05) and 𝛽 1 subunits (p < 0.05). Specifically, 𝛼 2 -subunit expression increased 1.90 fold (0.80 ; 3.00 fold) and 1.70 fold (1.21 ; 2.19 fold), with 6 weeks of BFRRE (p = 0.04) and HLRE (p = 0.02), respectively. There was no difference in the change in NKA 𝛼 2 subunit isoform expression between BFRRE and HLRE at any timepoint. From baseline, NKA 𝛽 1 subunit isoform abundance increased 1.80 fold (1.20 ; 2.42 fold) and 1.78 fold (0.86 ; 2.70 fold) with BFRRE (p < 0.01) and HLRE (p < 0.05), whereas no change was observed following CON (Fig. 4A). At the post timepoint both BFRRE and HLRE was significantly different from the corresponding timepoint following CON (p < 0.05). No significant differences were observed between BFRRE and HLRE at any timepoint. Relationship Between Protein Expression and Contractile Performance To assess the potential relationships between muscle contractile performance and the protein expression of the membrane proteins of interest, we pooled all subjects (n = 33 due to missing biopsy material from one BFRRE subject) and investigated if baseline abundance of proteins correlated with measures of contractile performance at baseline (Fig. 5). At baseline a modest inverse correlation was observed between ClC-1 protein expression and dynamic strength (p < 0.5, r = -0.365) (Fig. 5C). No other significant associations between the protein expression of neither ClC-1 nor NKA subunit isoforms and measures of contractile performance were observed at baseline. As no distinct differences emerged on the impact of BFRRE and HLRE on the protein expression of any of the targets of interest, the two training groups were pooled (n = 22 due to missing post biopsy from one HLRE subject) to evaluate the relationship between alterations of protein content and changes in muscle contractile performance (Fig. 6). Positive linear correlations emerged between alteration of NKA 𝛼2 subunit isoform abundance and changes in MVC (p < 0.01, r = 0.603) (Fig. 6E). Also, a positive linear relationship was observed between differences NKA 𝛽 1 subunit isoform expression and changes in MVC (p < 0.05, r = 0.453) (Fig. 6H). Discussion The main finding of the present study was that 6 weeks of BFRRE and HLRE did not affect ClC-1 protein abundance in healthy, untrained individuals. Oppositely, both BFRRE and HLRE effectively increased content of NKA subunit isoforms expression in human skeletal muscle. Contrary to our hypothesis, BFRRE and HLRE impacted the NKA similarly. Finally, these increases in NKA subunit isoform expressions correlated with training-induced changes in MVC. Our results are in agreement with previous observations of unaltered ClC-1 content in trained cyclists following <7 weeks of high-intensity endurance training (Thomassen et al. 2018). Accordingly, these short intervention periods may be insufficient to changes in ClC-1 expression. Alternatively, the observation of unaltered protein expression level could also suggest that the ClC-1 is sufficiently intrinsic regulated during in vivo muscle contractile activity. Of cause, from the present study, we cannot disclose if longer periods of training may stimulate reductions of ClC-1 content. In fact, lower overall ClC-1 abundance has been reported in well-trained cyclists compared to recreationally active individuals (Thomassen et al. 2018). Also, muscles from physically active rats exhibit a reduced resting membrane conductance compared with those of sedentary rats, despite displaying similar resting membrane potentials (Broch-Lips et al. 2011). Hence, it could be speculated that habitual physical activity may modulate ClC-1 expression in the long term. In the current study, when pooling all subjects at baseline, a modest inverse relationship was observed between ClC-1 abundance and dynamic strength. The fact that ClC-1 abundance at baseline did not correlate with neither MVC nor strength-endurance capacity, confines our ability to firmly conclude that ClC-1 content poses a limitation of muscle contractile performance per se. Opposed to our hypothesis, the selective increase in muscle strength-endurance capacity observed with BFRRE training was not mirrored by a pronounced decrease in ClC-1 abundance prompted by increased metabolite accumulation or exacerbated ionic perturbations inherent of BFRRE. Potentially, the augmented strength-endurance capacity may more so adhere to task-specificity (i.e. high number of reps) and/or distinct differences in neural innervation patterns of BFRRE and HLRE as previously discussed (Groennebaek et al. 2018). It has previously been questioned if reduced excitability is a hallmark of fatigue development in humans during normal exercise. In example, 3 minutes of sustained isometric contraction of the quadriceps at 30% of MVC markedly reduced the twitch response by >50% while substantially elevating venous plasma K+, however, no change in compound surface AP (M-wave) was observed (West et al. 1996). This could suggest, that the posttranslational regulation of ClC-1 in working skeletal muscles may sufficiently preserve excitability during in vivo contractile activity in humans. At the onset of muscle contractions, several cellular signals partake in the tightly regulated intrinsic inhibition of ClC-1, including lactate accumulation (de Paoli et al. 2010), intracellular acidification (Palade and Barchi 1977; Pedersen et al. 2004), rise in adenosine nucleotides (Leermakers et al. 2020) and most predominantly activation of Protein Kinase C (PKC) mediated by Ca2+-release from the sarcoplasmatic reticulum (de Paoli et al. 2013; Pedersen, Macdonald, et al. 2009). Notably, de Paoli et al. (2013) showed that abolishing PKC-dependent ClC-1 inhibition caused a marked loss of contractile endurance. Furthermore, a biphasic relationship between GCl and excitability was reported with ~70% inhibition of the ClC-1 channels optimizing the contractile endurance of the working muscles. Intriguingly, similar degrees of acute ClC-1 inhibition has been reported in both isolated human (Riisager et al. 2016) and rodent muscles (Pedersen, Macdonald, et al. 2009; Pedersen, de Paoli, et al. 2009; Riisager et al. 2014) upon repeated activation, emphasizing the importance of intrinsic ClC-1 regulation in the maintenance of muscle excitability. This notion is further supported by findings that rodent hindlimb disuse models exhibit increases in resting GCl entirely attributable to dysregulated PKC-mediated inhibition of ClC-1 (Pierno et al. 2007). Future studies investigating the impact of long-term exercise (e.g. >12 weeks) on ClC-1 abundance and the acute effects of exercise on upstream regulators of GCl are strongly warranted. We investigated the content of specific NKA subunit isoforms 𝛼 2 and 𝛽 1 . These isoforms were chosen as they are highly expressed in skeletal muscle (Pirkmajer and Chibalin 2016). Previously, a 15% upregulation of 𝛼 2 isoforms, with no apparent change in 𝛽 1 isoforms, has been reported following 7 weeks of cycle ergometer exercise (Nielsen et al. 2004). Oppositely, we observed that subunit content increased uniformly both in response to BFRRE (88% and 81% for 𝛼 2 and 𝛽 1 , respectively) and HLRE (70% and 78% for 𝛼 2 and 𝛽 1 , respectively), supporting the previous findings of no differential regulation of these specific NKA subunit isoforms in response to HLRE (Dela, Holten, and Juel 2004). Also, our similar findings between training groups suggest that no distinguished factors inherent of BFRRE affect the NKA more profoundly compared to traditional HLRE. A human cross-sectional study previously reported a positive correlation between NKA expression, assessed by 3H-ouabain binding and MVC (Klitgaard and Clausen 1989). Intriguingly, we found similar correlations between training-induced changes in NKA subunit content and MVC (n = 22, Fig. 6E and 6H), suggesting that increases in NKA subunit abundance might contribute to the increases in maximal force generation capacity. Intuitively, we expected that NKA subunit abundance would be more closely related to changes in strength-endurance capacity rather than MVC, as the need for clearance of increased extracellular K+ would be more pronounced during repeated contractions. Significant correlations between HLRE-induced changes in NKA concentration and muscle strength-endurance capacity at 70% of 1RM has previously been reported (Medbo et al. 2001). Potentially, the contraction intensity of 30% of 1RM employed when evaluating strength-endurance capacity in the present study, may have been insufficient to evoke increases in extracellular K+ limiting excitability (Sjogaard, Adams, and Saltin 1985). Conclusion The present study is the first to investigate the impact of BFRRE and HLRE on ClC-1 and NKA abundance in human skeletal muscle. We report that 6 weeks of neither resistance training modality affects the expression of ClC-1 in untrained individuals. Furthermore, at baseline ClC-1 abundance correlated inversely with dynamic muscle strength, however no such correlations were observed between ClC-1 expression and MVC and local muscle strength-endurance capacity. These findings suggest that lowering of overall the ClC-1 abundance is not a part of the short-term adaptation to resistance exercise. Accordingly, this points to intrinsic ClC-1 regulation as the key mechanism responsible for maintaining GCl during in vivo muscle activation. Finally, BFRRE and HLRE elicited similar increases in NKA subunit isoforms 𝛼 2 and 𝛽 1 , which correlated with training-induced increases in MVC. Declarations Grants This study was supported by grants from Novo Nordisk Foundation (NNF15OC0016674) and Aarhus University Research Foundation (AUFF-E-2O15-FLS-7-32). Disclosures The authors declare no conflicts of interests, financial or otherwise . Acknowledgements We would like to thank Gitte Kaiser Hartvigsen for technical assistance. We also acknowledge Jon Hagen Herskind and Anders Gravholdt for tremendous efforts in conducting the randomized controlled trial. Author contributions THP, KV and FdP contributed to the conception and study design. JW wrote the first manuscript draft. JW, ER, TG, JEJ, JF, PS and FdP contributed with data acquisition. All authors interpreted data, critically revised the manuscript, provided intellectual and scientific contributions, and approved the final version of the manuscript submitted for publication. References American College of Sports, Medicine. 2009. 'American College of Sports Medicine position stand. 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'Water and ion shifts in skeletal muscle of humans with intense dynamic knee extension', Am J Physiol , 248: R190-6. Thomassen, M., M. Hostrup, R. M. Murphy, B. A. Cromer, C. Skovgaard, T. P. Gunnarsson, P. M. Christensen, and J. Bangsbo. 2018. 'Abundance of ClC-1 chloride channel in human skeletal muscle: fiber type specific differences and effect of training', J Appl Physiol (1985) , 125: 470-78. Wernbom, M., J. Augustsson, and T. Raastad. 2008. 'Ischemic strength training: a low-load alternative to heavy resistance exercise?', Scand J Med Sci Sports , 18: 401-16. West, W., A. Hicks, R. McKelvie, and J. O'Brien. 1996. 'The relationship between plasma potassium, muscle membrane excitability and force following quadriceps fatigue', Pflugers Arch , 432: 43-9. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 04 Apr, 2023 Read the published version in Journal of Muscle Research and Cell Motility → Version 1 posted Editorial decision: Major revision 08 Aug, 2022 Reviewers agreed at journal 07 Aug, 2022 Reviews received at journal 13 Jul, 2022 Reviewers agreed at journal 05 Jul, 2022 Reviewers invited by journal 04 Jul, 2022 Editor assigned by journal 04 Jul, 2022 Submission checks completed at journal 04 Jul, 2022 First submitted to journal 27 Jun, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1799620","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":118315659,"identity":"e2e98dd5-58c8-437d-9885-61c01ef7da35","order_by":0,"name":"Jakob Wang","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jakob","middleName":"","lastName":"Wang","suffix":""},{"id":118315660,"identity":"64932a0a-c004-431a-8536-62869bf084a1","order_by":1,"name":"Emil Rindom","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Emil","middleName":"","lastName":"Rindom","suffix":""},{"id":118315661,"identity":"b777563a-2884-4057-a291-2ccc28e7bbf3","order_by":2,"name":"Thomas Groennebaek","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Groennebaek","suffix":""},{"id":118315662,"identity":"9ba25c94-e107-4696-9dbf-a1c29bc1b10c","order_by":3,"name":"Peter Sieljacks","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Sieljacks","suffix":""},{"id":118315663,"identity":"c754d6cd-e4d5-42c7-b274-a6b330a0ff25","order_by":4,"name":"Jesper Emil Jakobsgaard","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jesper","middleName":"Emil","lastName":"Jakobsgaard","suffix":""},{"id":118315664,"identity":"c784ae53-f2a1-432a-a066-939d2a116600","order_by":5,"name":"Jean Farup","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jean","middleName":"","lastName":"Farup","suffix":""},{"id":118315665,"identity":"4a119e04-bdcc-470d-be35-ce13b449eb59","order_by":6,"name":"Kristian Vissing","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kristian","middleName":"","lastName":"Vissing","suffix":""},{"id":118315666,"identity":"7490d750-3575-43c1-b940-d140499e34be","order_by":7,"name":"Thomas Holm Pedersen","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"Holm","lastName":"Pedersen","suffix":""},{"id":118315667,"identity":"bb2aa789-9c21-49b7-a4cf-37d9d7a70ec4","order_by":8,"name":"Frank Vincenzo Paoli","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYBAC9oYExscMBiBmAohgNiCohedAArMxyVrYpCFMorWw5x6rLiiwieZnT2D8zMNgbUxYC8+7tNszDNJyZ/Y8YJbmYUg3I6jFXiLH7DaPweHcDTcSGIBaDtsQtgWopRikZf+NBObfRGthBtsiAQwHoBbCDgP6JVka5JcZZx62Wc4xSCfC++y5Bz8X/LHJ7W9PPnzjTYW1YQNBPQw8MAYjUDHhWEHRMgpGwSgYBaMABwAArKs10/CLuowAAAAASUVORK5CYII=","orcid":"","institution":"Aarhus University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Frank","middleName":"Vincenzo","lastName":"Paoli","suffix":""}],"badges":[],"createdAt":"2022-06-27 11:44:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1799620/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1799620/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10974-023-09644-6","type":"published","date":"2023-04-04T20:24:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":23714583,"identity":"ccfea1c8-3e5a-47b9-96c5-bf897b0a00c0","added_by":"auto","created_at":"2022-07-11 15:03:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":38184,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStudy overview.\u003c/strong\u003e The total study length was 9 weeks and comprised a single-trial study investigating endpoints intended for publication elsewhere and a 6-week intervention study. For the intervention study, BFRRE and HLRE groups conducted training 3 times per week while CON served as non-exercise control. During the intervention period 3RM was re-evaluated every other week to adjust exercise load accordingly. Biopsies and testing of contractile performance were performed before and after the 6-week intervention period. BFRRE, blood flow restricted exercise; CON, non-exercise control; HLRE, heavy load resistance exercise; MVC, maximal voluntary contraction; RM, repetition maximum; SEC, strength-endurance capacity. \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-1799620/v1/be5a476069d476f8ee87e8f8.png"},{"id":23714582,"identity":"0503d01e-8a98-49df-acaf-266ceb5e3f0c","added_by":"auto","created_at":"2022-07-11 15:03:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":55025,"visible":true,"origin":"","legend":"\u003cp\u003eClC-1 protein abundance of CON, BFRRE, and HLRE groups. Representable immunoblots are shown in upper panel. \u003cstrong\u003e(A)\u003c/strong\u003e raw-data expressed arbitrary units (AU) at pre (filled circles) and post (open circles) timepoints. Data are presented as individual values (circles) and corresponding group means ± SD (red whiskers). \u003cstrong\u003e(B)\u003c/strong\u003e changes in protein abundance expressed as fold changes from pre. Data are presented as mean fold changes ± SD (bars) at pre (filled) and post (open) timepoints. Overall effects are designated in the upper left corner of A.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-1799620/v1/dfd934a1da15d212cbf3e99a.png"},{"id":23715131,"identity":"7d20ee9e-0dff-4d3c-a79c-be0c2ee8f769","added_by":"auto","created_at":"2022-07-11 15:08:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":59984,"visible":true,"origin":"","legend":"\u003cp\u003eNKA 𝛼\u003csup\u003e2\u003c/sup\u003e subunit protein abundance of CON, BFRRE, and HLRE groups. Representable immunoblots are shown in upper panel. (A) raw-data expressed arbitrary units (AU) at pre (filled circles) and post (open circles) timepoints. Data are presented as individual values (circles) and corresponding group means ± SD (red whiskers). (B) changes in protein abundance expressed as fold changes from pre. Data are presented as mean fold changes ± SD (bars) at pre (filled) and post (open) timepoints. * denotes difference from pre-values within groups (P\u0026lt;0.05). Overall effects are designated in the upper left corner of A.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-1799620/v1/114bb6c023124b476466d197.png"},{"id":23714586,"identity":"333eaed5-2c17-45e3-bc06-216ab1de94e7","added_by":"auto","created_at":"2022-07-11 15:03:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":56365,"visible":true,"origin":"","legend":"\u003cp\u003eNKA 𝛽\u003csup\u003e1\u003c/sup\u003e subunit protein abundance of CON, BFRRE, and HLRE groups. Representable immunoblots are shown in upper panel. (A) raw-data expressed arbitrary units (AU) at pre (filled circles) and post (open circles) timepoints. Data are presented as individual values (circles) and corresponding group means ± SD (red whiskers). (B) changes in protein abundance expressed as fold changes from pre. Data are presented as mean fold changes ± SD (bars) at pre (filled) and post (open) timepoints. * denotes difference from pre-values within groups (P\u0026lt;0.05), ** denotes difference from pre-values within groups (P\u0026lt;0.01), C denotes difference from corresponding timepoint in CON. Overall effects are designated in the upper right corner of A.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-1799620/v1/083c94f9b37bd0d36e55e6eb.png"},{"id":23714584,"identity":"7c52ef1a-3d6a-4a14-b06d-2cf4fd7eaaab","added_by":"auto","created_at":"2022-07-11 15:03:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":193941,"visible":true,"origin":"","legend":"\u003cp\u003eCross-sectional baseline correlation of protein expression and contractile performance (n = 33 due to one missing value in BFRRE group). (A) ClC-1 and SEC, (B) ClC-1 and MVC, (C) ClC-1 and dynamic strength, (D) NKA 𝛼2 subunit and SEC, (E) NKA 𝛼\u003csup\u003e2 \u003c/sup\u003esubunit and MVC, (F) NKA 𝛼\u003csup\u003e2\u003c/sup\u003e subunit and dynamic strength, (G) NKA 𝛽\u003csup\u003e1\u003c/sup\u003e subunit and SEC, (H) NKA 𝛽\u003csup\u003e1\u003c/sup\u003e subunit and MVC, (I) NKA 𝛽1 subunit and dynamic strength. SEC = strength-endurance capacity, MVC = maximal voluntary contraction, RM = repetition maximum. Strength and level of significance are designated in the upper right corner of each graph.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-1799620/v1/fd47bc2546fa5dd540efee3c.png"},{"id":23714587,"identity":"e07938a9-a690-4956-8842-491f0bc4488e","added_by":"auto","created_at":"2022-07-11 15:03:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":115367,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelations between training-induced changes protein expression and contractile performance (n = 22 due to one missing value in the BFRRE and HLRE group, respectively). (A) ClC-1 and SEC, (B) ClC-1 and MVC, (C) ClC-1 and dynamic strength, (D) NKA 𝛼\u003csup\u003e2\u003c/sup\u003e subunit and SEC, (E) NKA 𝛼\u003csup\u003e2\u003c/sup\u003e subunit and MVC, (F) NKA 𝛼2 subunit and dynamic strength, (G) NKA 𝛽\u003csup\u003e1\u003c/sup\u003e subunit and SEC, (H) NKA 𝛽\u003csup\u003e1\u003c/sup\u003e subunit and MVC, (I) NKA 𝛽\u003csup\u003e1\u003c/sup\u003e subunit and dynamic strength. BFRRE subjects (circles) and HLRE subjects (triangles). SEC = strength-endurance capacity, MVC = maximal voluntary contraction, RM = repetition maximum. Strength and level of significance are designated in the upper or lower right corner of each graph.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-1799620/v1/e53482274de6a787f5d3ae77.png"},{"id":44724721,"identity":"6f81ce30-f9df-4a93-9fe2-46c73547abd2","added_by":"auto","created_at":"2023-10-16 20:34:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":827310,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1799620/v1/62845ac1-7f32-4815-835b-32daccb86aaf.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Heavy-load and low-load blood flow restricted resistance training increases Na/K-ATPase subunit but not ClC-1 expression in untrained human skeletal muscle","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe ability to activate and control skeletal muscle fibers is dependent on excitation and propagation of action potentials (APs) in muscle fibres from the neuromuscular junction along the sarcolemma and into the T-tubular system\u0026nbsp;(Sejersted and Sjogaard 2000). During exercise, preservation of muscle fiber excitability is dependent on the transmembrane ion gradients of Na\u003csup\u003e+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e-\u003c/sup\u003e (Baekgaard Nielsen et al. 2017; Clausen and Nielsen 1994). This implicates that the abundance and function of membrane proteins involved in the regulation of these ions is essential for contractile performance.\u003c/p\u003e\n\u003cp\u003eIt is well documented that muscle activity results in accumulation of extracellular K\u003csup\u003e+\u003c/sup\u003e (Fitts 1994), which potentially could impact muscle fiber excitability. This is due to the associated depolarization of the membrane, which might compromise the function of voltage-gated Na\u003csup\u003e+\u003c/sup\u003e channels that are responsible for AP formation\u0026nbsp;(Filatov, Pinter, and Rich 2005). During repeated AP firing, the Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e-ATPase (NKA) plays a central role in counterbalancing the extracellular K\u003csup\u003e+\u003c/sup\u003e buildup\u0026nbsp;(Clausen 2003). However, the rate of interstitial K\u003csup\u003e+\u003c/sup\u003e accumulation clearly exceeds the rate of re-uptake in human skeletal muscle during exercise\u0026nbsp;(Nordsborg et al. 2003; Juel et al. 2000)\u0026nbsp;and the ensuing loss of muscle excitability has been widely considered the primary driver of fatigue development during strenuous and high-intensity exercise in humans\u0026nbsp;(Bangsbo et al. 1996; Nielsen et al. 2004).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEqually important for preserving muscle fiber excitability during exercise is regulation of Cl\u003csup\u003e-\u003c/sup\u003e membrane conductance (G\u003csub\u003eCl\u003c/sub\u003e) in the active muscle fibers\u0026nbsp;(de Paoli et al. 2013). The Cl\u003csup\u003e-\u003c/sup\u003e channel protein 1 (ClC-1) accounts for ~80% of the resting membrane conductance specifically in skeletal muscle\u0026nbsp;(Pedersen et al. 2016). In contracting muscles, the onset of repeated AP firing triggers a substantial reduction of the G\u003csub\u003eCl\u003c/sub\u003e in both rodent\u0026nbsp;(Pedersen, Macdonald, et al. 2009; Pedersen, de Paoli, et al. 2009)\u0026nbsp;and human muscle\u0026nbsp;(Riisager et al. 2016; Leermakers et al. 2020). This inhibition of ClC-1 has been shown to augment high-intensity contractile endurance even in context of elevated extracellular K\u003csup\u003e+\u003c/sup\u003e (de Paoli et al. 2010). Thus, decreasing the ClC-1 abundance may constitute a beneficial adaptation to strenuous training with regards to preservation of excitability and consequently, muscle contractile performance.\u003c/p\u003e\n\u003cp\u003eIn recent years, low-load blood flow restricted resistance exercise (BFRRE) has emerged as a low-load alternative to high-load resistance exercise (HLRE) in promoting skeletal muscle adaptations\u0026nbsp;(Wernbom, Augustsson, and Raastad 2008). During both HLRE and BFRRE, substantial increases in sEMG have been reported\u0026nbsp;(Farup et al. 2015; Buckner et al. 2018), suggesting that both modes of exercise compromise muscle fiber excitability. In addition, the partial restriction of blood flow inherent of BFRRE exacerbates both the accumulation of metabolites\u0026nbsp;(Fujita et al. 2007)\u0026nbsp;and the ionic perturbations during exercise\u0026nbsp;(Christiansen 2018), which may potentiate training-induced adaptations of transmembrane proteins. Interestingly, we demonstrated that 6 weeks of both BFRRE and HLRE markedly increased muscle strength\u0026nbsp;(Sieljacks et al. 2019), whereas BFRRE selectively increased muscle strength-endurance capacity\u0026nbsp;(Groennebaek et al. 2018). To our knowledge, no prior studies have investigated the impact\u0026nbsp;of HLRE and BFRRE on ClC-1 and NKA subunit isoform expression in humans.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTherefore, the aim of the present study was to investigate the impact of BFRRE and HLRE on ClC-1 protein and NKA subunit isoform abundance and to examine if these were related to skeletal muscle contractile properties in young, untrained individuals. We hypothesized; (1) that 6 weeks of BFRRE and HLRE would decrease ClC-1 expression and increase NKA subunit isoform abundance, and; (2) that BFRRE would accentuate this response compared to HLRE.\u003c/p\u003e"},{"header":"Materials \u0026 Methods ","content":"\u003ch2\u003eSubjects\u003c/h2\u003e\n\u003cp\u003eThirty-four young, healthy, untrained men were enrolled in this randomized controlled trial. Inclusion and exclusion criteria as well as baseline characteristics of the subjects are available in detail elsewhere (Groennebaek et al. 2018).\u003c/p\u003e\n\u003cp\u003eAll participants provided both written and informed consent prior to enrollment. The study was approved by the Central Denmark Region Committee on Health Research Ethics (1-10-72-218-16) and registered in the database clinicaltrials.gov (NCT03380663). The study was conducted in conformity with the standards for human experimental trials outlined in the Declaration of Helsinki.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eStudy Design\u003c/h2\u003e\n\u003cp\u003eThe study was conducted as a randomized controlled trial. Each subject was randomly allocated to one of three intervention groups; BFRRE (n = 12), HLRE (n = 12) or non-exercise control (CON; n = 10). The total duration of the study was 9 weeks (Fig. 1). In week 1, subjects reported for baseline measures of muscle strength and strength-endurance capacity following collection of pre-intervention muscle biopsies. Furthermore, individual arterial occlusion pressure (AOP) was determined in the subjects prescribed to BFRRE. In week 2, a single-trial exercise experiment was conducted, intended for other investigatory points of interest reported elsewhere\u0026nbsp;(Groennebaek et al. 2018). The subsequent 6 weeks comprised of the exercise intervention. In week 9, subjects reported for post-intervention assessment of muscle strength and strength-endurance capacity following collection of biopsies. The CON subjects were exposed to all experimental procedures of the study with exception of exercise.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eExercise interventions\u003c/h2\u003e\n\u003cp\u003eTraining comprised a total of 18 exercise sessions dispersed as three supervised sessions 3 week\u003csup\u003e-1\u003c/sup\u003e (Monday Wednesday, Friday) throughout the intervention period.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExercise interventions are described in detail previously (Groennebaek et al. 2018). Briefly, training sessions comprised a standardized warmup on stationary bicycle ergometer (Monark Ergomedic 818E, Monark, Varberg, Sweden) and submaximal knee extension repetitions in the same apparatus later used for exercise (TechnoGym selection-line, TechnoGym, Italy). Following warmup, subjects performed 4 sets of isolated knee extensions in accordance with the recommended principles inherent of the respective exercise modalities (Scott et al. 2015; American College of Sports 2009). BFRRE was conducted at 30% of 1RM with all sets performed to volitional fatigue. An individualized relative occlusion pressure corresponding to 50% of AOP was applied by use of 14 cm wide pneumatic cuffs (Delfi Medical, Vancouver, Canada). The cuffs were wrapped around the inguinal fold region of the thighs and were continuously inflated both during exercise and the 30 s inter-set recovery by means of a digital tourniquet (A.T.S 2200TS, Zimmer Surgical Inc. Ohio, USA). Relative exercise intensity for HLRE was 70% of 1RM. Participants were instructed to target 12 repetitions in each set, with sets being interspaced by 3 minutes of recovery. Appropriate exercise intensity was assured throughout the training period by re-evaluating 3 RM every other week (fig. 1) as well as progressively increasing training load in case of HLRE, as previously described (Groennebaek et al. 2018). \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eMuscle strength and strength-endurance capacity\u003c/h2\u003e\n\u003cp\u003eTesting of muscle contractile performance comprised maximal isometric (maximal voluntary contraction; MVC) and dynamic strength (3RM) as well as local muscle strength-endurance capacity of the knee extensors. These were evaluated at baseline (pre) and 4 days after cessation of exercise (post).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTesting of unilateral isometric MVC was conducted in an isokinetic dynamometer (Humac Norm, CSMI, Stoughton, Massachusetts, USA), as previously described\u0026nbsp;(Sieljacks et al. 2019).\u003c/p\u003e\n\u003cp\u003eDynamic knee extensor strength was evaluated using a 3RM-test conducted in knee extension apparatus. Following the 3RM-test, the load was readjusted to 30% of the corresponding 1RM and subjects performed bilateral knee extensions to volitional failure. The total number of repetitions performed provided the measure of muscle strength-endurance capacity. Procedures for testing of maximal dynamic strength and strength-endurance capacity are described in detail elsewhere (Groennebaek et al. 2018). \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eMuscle biopsies\u003c/h2\u003e\n\u003cp\u003eMuscle biopsies were harvested from the mid-section of vastus lateralis before and after the exercise intervention. Biopsies were obtained under local anesthesia (1% Lidocaine, Mylon Hospital, Norway) utilizing the Bergstr\u0026ouml;m needle technique. Prior to days of testing, subjects were instructed to refrain from strenuous physical activity. Furthermore, all biopsies were collected in the early morning following an overnight fast, to circumvent potential influence of nutritional and circadian confounders.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter removal of visible fat and connective tissue an aliquot of the muscle sample (appx. 30 mg wet weight) was immediately snap-frozen in liquid nitrogen and subsequently stored at -80\u0026deg;C until further preparation and analysis.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eImmunoblotting\u003c/h2\u003e\n\u003cp\u003ePreparation of frozen muscle biopsies and determination of protein concentration was performed in accordance with Groennebaek et al (2018). Proteins were separated by SDS-PAGE on precast polyacrylamide gels (Bio-Rad, CA, USA) and subsequently electroblotted onto PVDF membranes (Bio-Rad, CA, USA). Equal loading of protein was controlled by visual examination using stain-free technology. Membranes were then blocked for 2 hours in 0.3% I-Block in solution with TBST at room temperature and incubated overnight at 4\u0026deg;C in appropriate primary anti-bodies. The primary antibodies utilized were as follows; ClC1 c-terminal (cat # ab189857, ABCAM, Cambridge, UK), NKA 𝛼\u003csup\u003e2\u003c/sup\u003e (cat # 07-674, Merck, Darmstadt, DE) and NKA 𝛽\u003csup\u003e1\u003c/sup\u003e (cat # 44759, Cell Signaling Technology, MA, US). All primary anti-bodies were used in a 1:1000 dilution of TBST and 5% BSA, with exception of NKA 𝛼\u003csup\u003e2\u003c/sup\u003e, which was diluted in 1:1000 TBST with 5% skim milk. After incubation in primary anti-bodies, membranes were incubated in secondary anti-bodies for 1 hour at room temperature with horseradish peroxidase-conjugated goat anti-rabbit (cat # 6721, ABCAM, Cambridge, UK) in 1:5000 TBST solution with 1% BSA, with exception of NKA 𝛼\u003csup\u003e2\u003c/sup\u003e which was incubated using skim milk. All proteins were visualized by chemilumescence (Thermo Fisher Scientific, MA, US). Arbitrary protein intensity was normalized and quantified by utilizing an UVP imaging system (UVP, CA, US).\u003c/p\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eAlterations in protein abundance were analyzed by a mixed effect linear model with group, time and group x time interaction as factors of interest. When significant group x time interactions were observed, pairwise comparisons were made between groups as well as separate timepoints (pre vs. post). The model was validated by test for equal standard deviations and visual examination of QQ-plots. Associations between cellular outcomes and muscle contractile performance were evaluated using linear regressions and Pearson\u0026rsquo;s correlation. All statistical analyses were conducted using STATA 15.0 (StataCorp, College Station, TX, USA) with an alfa level of 0.05 delineating statistically significant outcomes. Data are graphically presented as individual values and means \u0026plusmn; SDs, whereas in-text data presented as means with corresponding 95%-CIs. All graphical presentation of data are performed using GraphPad Prism v.7 (GraphPad Software, La Jolla, CA, USA). \u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eImpact of Resistance Training on Protein Abundance\u003c/h2\u003e\n\u003cp\u003eNeither six weeks of HLRE or BFRRE influence ClC-1 protein abundance in untrained males (Fig. 2). As for the NKA, significant overall time x group interactions were observed for both 𝛼\u003csup\u003e2\u003c/sup\u003e subunits (p \u0026lt; 0.05) and 𝛽\u003csup\u003e1\u003c/sup\u003e subunits (p \u0026lt; 0.05). Specifically, 𝛼\u003csup\u003e2\u003c/sup\u003e-subunit expression increased 1.90 fold (0.80 ; 3.00 fold) and 1.70 fold (1.21 ; 2.19 fold), with 6 weeks of BFRRE (p = 0.04) and HLRE (p = 0.02), respectively. There was no difference in the change in NKA 𝛼\u003csup\u003e2\u003c/sup\u003e subunit isoform expression between BFRRE and HLRE at any timepoint.\u003c/p\u003e\n\u003cp\u003eFrom baseline, NKA 𝛽\u003csup\u003e1\u0026nbsp;\u003c/sup\u003esubunit isoform abundance increased 1.80 fold (1.20 ; 2.42 fold) and 1.78 fold (0.86 ; 2.70 fold) with BFRRE (p \u0026lt; 0.01) and HLRE (p \u0026lt; 0.05), whereas no change was observed following CON (Fig. 4A). At the post timepoint both BFRRE and HLRE was significantly different from the corresponding timepoint following CON (p \u0026lt; 0.05). No significant differences were observed between BFRRE and HLRE at any timepoint.\u003c/p\u003e\n\u003ch2\u003eRelationship Between Protein Expression and Contractile Performance\u003c/h2\u003e\n\u003cp\u003eTo assess the potential relationships between muscle contractile performance and the protein expression of the membrane proteins of interest, we pooled all subjects (n = 33 due to missing biopsy material from one BFRRE subject) and investigated if baseline abundance of proteins correlated with measures of contractile performance at baseline (Fig. 5). At baseline a modest inverse correlation was observed between ClC-1 protein expression and dynamic strength (p \u0026lt; 0.5, r = -0.365) (Fig. 5C). No other significant associations between the protein expression of neither ClC-1 nor NKA subunit isoforms and measures of contractile performance were observed at baseline.\u003c/p\u003e\n\u003cp\u003eAs no distinct differences emerged on the impact of BFRRE and HLRE on the protein expression of any of the targets of interest, the two training groups were pooled (n = 22 due to missing post biopsy from one HLRE subject) to evaluate the relationship between alterations of protein content and changes in muscle contractile performance (Fig. 6). Positive linear correlations emerged between alteration of NKA 𝛼2 subunit isoform abundance and changes in MVC (p \u0026lt; 0.01, r = 0.603) (Fig. 6E). Also, a positive linear relationship was observed between differences NKA 𝛽\u003csup\u003e1\u003c/sup\u003e subunit isoform expression and changes in MVC (p \u0026lt; 0.05, r = 0.453) (Fig. 6H).\u003c/p\u003e\n\u003ch2\u003e\u0026nbsp;\u003c/h2\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe main finding of the present study was that 6 weeks of BFRRE and HLRE did not affect ClC-1 protein abundance in healthy, untrained individuals. Oppositely, both BFRRE and HLRE effectively increased content of NKA subunit isoforms expression in human skeletal muscle. Contrary to our hypothesis, BFRRE and HLRE impacted the NKA similarly. Finally, these increases in NKA subunit isoform expressions correlated with training-induced changes in MVC.\u003c/p\u003e\n\u003cp\u003eOur results are in agreement with previous observations of unaltered ClC-1 content in trained cyclists following \u0026lt;7 weeks of high-intensity endurance training (Thomassen et al. 2018). Accordingly, these short intervention periods may be insufficient to changes in ClC-1 expression. Alternatively, the observation of unaltered protein expression level could also suggest that the ClC-1 is sufficiently intrinsic regulated during in vivo muscle contractile activity. Of cause, from the present study, we cannot disclose if longer periods of training may stimulate reductions of ClC-1 content. In fact, lower overall ClC-1 abundance has been reported in well-trained cyclists compared to recreationally active individuals (Thomassen et al. 2018). Also, muscles from physically active rats exhibit a reduced resting membrane conductance compared with those of sedentary rats, despite displaying similar\u003c/p\u003e\n\u003cp\u003eresting membrane potentials (Broch-Lips et al. 2011). Hence, it could be speculated that habitual physical activity may modulate ClC-1 expression in the long term.\u003c/p\u003e\n\u003cp\u003eIn the current study, when pooling all subjects at baseline, a modest inverse relationship was observed between ClC-1 abundance and dynamic strength. The fact that ClC-1 abundance at baseline did not correlate with neither MVC nor strength-endurance capacity, confines our ability to firmly conclude that ClC-1 content poses a limitation of muscle contractile performance per se.\u003c/p\u003e\n\u003cp\u003eOpposed to our hypothesis, the selective increase in muscle strength-endurance capacity observed with BFRRE training was not mirrored by a pronounced decrease in ClC-1 abundance prompted by increased metabolite accumulation or exacerbated ionic perturbations inherent of BFRRE. Potentially, the augmented strength-endurance capacity may more so adhere to task-specificity (i.e. high number of reps) and/or distinct differences in neural innervation patterns of BFRRE and HLRE as previously discussed (Groennebaek et al. 2018).\u003c/p\u003e\n\u003cp\u003eIt has previously been questioned if reduced excitability is a hallmark of fatigue development in humans during normal exercise. In example, 3 minutes of sustained isometric contraction of the quadriceps at 30% of MVC markedly reduced the twitch response by \u0026gt;50% while substantially elevating venous plasma K+, however, no change in compound surface AP (M-wave) was observed (West et al. 1996). This could suggest, that the posttranslational regulation of ClC-1 in working skeletal muscles may sufficiently preserve excitability during in vivo contractile activity in humans. At the onset of muscle contractions, several cellular signals partake in the tightly regulated intrinsic inhibition of ClC-1, including lactate accumulation (de Paoli et al. 2010), intracellular acidification (Palade and Barchi 1977; Pedersen et al. 2004), rise in adenosine nucleotides (Leermakers et al. 2020) and most predominantly activation of Protein Kinase C (PKC) mediated by Ca2+-release from the sarcoplasmatic reticulum (de Paoli et al. 2013; Pedersen, Macdonald, et al. 2009). Notably, de Paoli et al. (2013) showed that abolishing PKC-dependent ClC-1 inhibition caused a marked loss of\u003c/p\u003e\n\u003cp\u003econtractile endurance. Furthermore, a biphasic relationship between GCl and excitability was reported with ~70% inhibition of the ClC-1 channels optimizing the contractile endurance of the working muscles. Intriguingly, similar degrees of acute ClC-1 inhibition has been reported in both isolated human (Riisager et al. 2016) and rodent muscles (Pedersen, Macdonald, et al. 2009; Pedersen, de Paoli, et al. 2009; Riisager et al. 2014) upon repeated activation, emphasizing the importance of intrinsic ClC-1 regulation in the maintenance of muscle excitability. This notion is further supported by findings that rodent hindlimb disuse models exhibit increases in resting GCl entirely attributable to dysregulated PKC-mediated inhibition of ClC-1 (Pierno et al. 2007). Future studies investigating the impact of long-term exercise (e.g. \u0026gt;12 weeks) on ClC-1 abundance and the acute effects of exercise on upstream regulators of GCl are strongly warranted.\u003c/p\u003e\n\u003cp\u003eWe investigated the content of specific NKA subunit isoforms 𝛼\u003csup\u003e2\u003c/sup\u003e and 𝛽\u003csup\u003e1\u003c/sup\u003e. These isoforms were chosen as they are highly expressed in skeletal muscle (Pirkmajer and Chibalin 2016). Previously, a 15% upregulation of 𝛼\u003csup\u003e2\u003c/sup\u003e isoforms, with no apparent change in 𝛽\u003csup\u003e1\u003c/sup\u003e isoforms, has been reported following 7 weeks of cycle ergometer exercise (Nielsen et al. 2004). Oppositely, we observed that subunit content increased uniformly both in response to BFRRE (88% and 81% for 𝛼\u003csup\u003e2\u003c/sup\u003e and 𝛽\u003csup\u003e1\u003c/sup\u003e, respectively) and HLRE (70% and 78% for 𝛼\u003csup\u003e2\u003c/sup\u003e and 𝛽\u003csup\u003e1\u003c/sup\u003e, respectively), supporting the previous findings of no differential regulation of these specific NKA subunit isoforms in response to HLRE (Dela, Holten, and Juel 2004). Also, our similar findings between training groups suggest that no distinguished factors inherent of BFRRE affect the NKA more profoundly compared to traditional HLRE.\u003c/p\u003e\n\u003cp\u003eA human cross-sectional study previously reported a positive correlation between NKA expression, assessed by 3H-ouabain binding and MVC (Klitgaard and Clausen 1989). Intriguingly, we found similar correlations between training-induced changes in NKA subunit content and MVC (n = 22, Fig. 6E and 6H), suggesting that increases in NKA subunit abundance might contribute to the increases in maximal force generation capacity. Intuitively, we expected that NKA subunit abundance\u003c/p\u003e\n\u003cp\u003ewould be more closely related to changes in strength-endurance capacity rather than MVC, as the need for clearance of increased extracellular K+ would be more pronounced during repeated contractions. Significant correlations between HLRE-induced changes in NKA concentration and muscle strength-endurance capacity at 70% of 1RM has previously been reported (Medbo et al. 2001). Potentially, the contraction intensity of 30% of 1RM employed when evaluating strength-endurance capacity in the present study, may have been insufficient to evoke increases in extracellular K+ limiting excitability (Sjogaard, Adams, and Saltin 1985).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study is the first to investigate the impact of BFRRE and HLRE on ClC-1 and NKA abundance in human skeletal muscle. We report that 6 weeks of neither resistance training modality affects the expression of ClC-1 in untrained individuals. Furthermore, at baseline ClC-1 abundance correlated inversely with dynamic muscle strength, however no such correlations were observed between ClC-1 expression and MVC and local muscle strength-endurance capacity. These findings suggest that lowering of overall the ClC-1 abundance is not a part of the short-term adaptation to resistance exercise. Accordingly, this points to intrinsic ClC-1 regulation as the key mechanism responsible for maintaining GCl during in vivo muscle activation. Finally, BFRRE and HLRE elicited similar increases in NKA subunit isoforms 𝛼\u003csup\u003e2\u003c/sup\u003e and 𝛽\u003csup\u003e1\u003c/sup\u003e, which correlated with training-induced increases in MVC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eGrants\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from Novo Nordisk Foundation (NNF15OC0016674) and Aarhus University Research Foundation (AUFF-E-2O15-FLS-7-32).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interests, financial or otherwise\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Gitte Kaiser Hartvigsen for technical assistance. We also acknowledge Jon Hagen Herskind and Anders Gravholdt for tremendous efforts in conducting the randomized controlled trial. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTHP, KV and FdP contributed to the conception and study design. JW wrote the first manuscript draft. JW, ER, TG, JEJ, JF, PS and FdP contributed with data acquisition. All authors interpreted data, critically revised the manuscript, provided intellectual and scientific contributions, and approved the final version of the manuscript submitted for publication. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAmerican College of Sports, Medicine. 2009. \u0026apos;American College of Sports Medicine position stand. Progression models in resistance training for healthy adults\u0026apos;, \u003cem\u003eMed Sci Sports Exerc\u003c/em\u003e, 41: 687-708.\u003c/li\u003e\n \u003cli\u003eBaekgaard Nielsen, O., F. 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Dascombe. 2015. \u0026apos;Exercise with blood flow restriction: an updated evidence-based approach for enhanced muscular development\u0026apos;, \u003cem\u003eSports Med\u003c/em\u003e, 45: 313-25.\u003c/li\u003e\n \u003cli\u003eSejersted, O. M., and G. Sjogaard.\u0026nbsp;2000. \u0026apos;Dynamics and consequences of potassium shifts in skeletal muscle and heart during exercise\u0026apos;, \u003cem\u003ePhysiol Rev\u003c/em\u003e, 80: 1411-81.\u003c/li\u003e\n \u003cli\u003eSieljacks, P., J. Wang, T. Groennebaek, E. Rindom, J. E. Jakobsgaard, J. Herskind, A. Gravholt, A. B. Moller, R. V. Musci, F. V. de Paoli, K. L. Hamilton, B. F. Miller, and K. Vissing.\u0026nbsp;2019. \u0026apos;Six Weeks of Low-Load Blood Flow Restricted and High-Load Resistance Exercise Training Produce Similar Increases in Cumulative Myofibrillar Protein Synthesis and Ribosomal Biogenesis in Healthy Males\u0026apos;, \u003cem\u003eFront Physiol\u003c/em\u003e, 10: 649.\u003c/li\u003e\n \u003cli\u003eSjogaard, G., R. P. Adams, and B. Saltin. 1985. \u0026apos;Water and ion shifts in skeletal muscle of humans with intense dynamic knee extension\u0026apos;, \u003cem\u003eAm J Physiol\u003c/em\u003e, 248: R190-6.\u003c/li\u003e\n \u003cli\u003eThomassen, M., M. Hostrup, R. M. Murphy, B. A. Cromer, C. Skovgaard, T. P. Gunnarsson, P. M. Christensen, and J. Bangsbo. 2018. \u0026apos;Abundance of ClC-1 chloride channel in human skeletal muscle: fiber type specific differences and effect of training\u0026apos;, \u003cem\u003eJ Appl Physiol (1985)\u003c/em\u003e, 125: 470-78.\u003c/li\u003e\n \u003cli\u003eWernbom, M., J. Augustsson, and T. Raastad. 2008. \u0026apos;Ischemic strength training: a low-load alternative to heavy resistance exercise?\u0026apos;, \u003cem\u003eScand J Med Sci Sports\u003c/em\u003e, 18: 401-16.\u003c/li\u003e\n \u003cli\u003eWest, W., A. Hicks, R. McKelvie, and J. O\u0026apos;Brien. 1996. \u0026apos;The relationship between plasma potassium, muscle membrane excitability and force following quadriceps fatigue\u0026apos;, \u003cem\u003ePflugers Arch\u003c/em\u003e, 432: 43-9.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-muscle-research-and-cell-motility","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jure","sideBox":"Learn more about [Journal of Muscle Research and Cell Motility](http://link.springer.com/journal/10974)","snPcode":"10974","submissionUrl":"https://submission.nature.com/new-submission/10974/3","title":"Journal of Muscle Research and Cell Motility","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-1799620/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1799620/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eContractile function of skeletal muscle relies on the ability of muscle fibers to trigger and propagate action potentials (APs). These electrical signals are created by transmembrane ion transport through ion channels and membrane transporter systems. In this regard, the Cl- ion channel 1 (ClC-1) and the Na+/K--ATPase (NKA) are central for maintaining ion homeostasis across the sarcolemma during repetitive AP firing inherent of intense contractile activity. Therefore, this randomized controlled trial (RCT) aimed to investigate the changes in ClC-1 and specific NKA subunit isoform expression in response to 6 weeks (18 training sessions) of heavy-load resistance exercise (HLRE) and low-load blood flow restricted resistance exercise (BFRRE), respectively. Also, the potential associations between protein expression and contractile performance were investigated. We show that muscle ClC-1 abundance was not affected by either exercise modality, whereas NKA subunit isoforms 𝛼\u003csup\u003e2\u003c/sup\u003e and 𝛽\u003csup\u003e1 \u003c/sup\u003eincreased appx. 80-90% with BFRRE (p\u0026lt;0.05) and 70-80% with HLRE (p\u0026lt;0.05). No differential impact between exercise modalities was observed. At baseline, ClC-1 protein expression correlated inversely with dynamic knee extensor strength (r=-0.365, p=0.04). No correlation was observed between NKA subunit content and contractile performance at baseline. However, training-induced changes in NKA 𝛼\u003csup\u003e2\u003c/sup\u003e subunit (r=0.603, p\u0026lt;0.01) and 𝛽\u003csup\u003e1\u003c/sup\u003e subunit (r=0.453, p\u0026lt;0.05) correlated with changes in maximal voluntary contraction. These results suggest that the initial adaptation to resistance exercise does not involve changes in ClC-1 abundance in untrained skeletal muscle.\u003c/p\u003e","manuscriptTitle":"Heavy-load and low-load blood flow restricted resistance training increases Na/K-ATPase subunit but not ClC-1 expression in untrained human skeletal muscle","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-11 15:03:43","doi":"10.21203/rs.3.rs-1799620/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-08-08T08:49:14+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"16a23d36-a743-40e1-bd34-115e5f377ff8","date":"2022-08-07T07:45:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-07-13T05:40:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"b85eef67-3179-4f12-9a53-522f3463d976","date":"2022-07-05T22:21:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-07-04T09:14:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-07-04T07:21:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-07-04T07:21:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Muscle Research and Cell Motility","date":"2022-06-27T11:40:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-muscle-research-and-cell-motility","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jure","sideBox":"Learn more about [Journal of Muscle Research and Cell Motility](http://link.springer.com/journal/10974)","snPcode":"10974","submissionUrl":"https://submission.nature.com/new-submission/10974/3","title":"Journal of Muscle Research and Cell Motility","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"dc3fd402-7c99-4d01-8abf-b872d2439d13","owner":[],"postedDate":"July 11th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T20:30:07+00:00","versionOfRecord":{"articleIdentity":"rs-1799620","link":"https://doi.org/10.1007/s10974-023-09644-6","journal":{"identity":"journal-of-muscle-research-and-cell-motility","isVorOnly":false,"title":"Journal of Muscle Research and Cell Motility"},"publishedOn":"2023-04-04 20:24:51","publishedOnDateReadable":"April 4th, 2023"},"versionCreatedAt":"2022-07-11 15:03:43","video":"","vorDoi":"10.1007/s10974-023-09644-6","vorDoiUrl":"https://doi.org/10.1007/s10974-023-09644-6","workflowStages":[]},"version":"v1","identity":"rs-1799620","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1799620","identity":"rs-1799620","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}