Effects of fasting on isolated murine skeletal muscle contractile function during acute hypoxia

Cameron A Schmidt; Emma J Goldberg; Tom D Green; Reema R Karnekar; Jeffrey J Brault; Spencer G Miller; Adam J Amorese; Dean J Yamaguchi; Espen E Spangenburg; Joseph M McClung

doi:10.1371/journal.pone.0225922

. 2020 Apr 23;15(4):e0225922. doi: 10.1371/journal.pone.0225922

Effects of fasting on isolated murine skeletal muscle contractile function during acute hypoxia

Cameron A Schmidt ^1,², Emma J Goldberg ^1,², Tom D Green ^1,², Reema R Karnekar ^1,², Jeffrey J Brault ^1,³, Spencer G Miller ³, Adam J Amorese ^1,², Dean J Yamaguchi ^4,⁵, Espen E Spangenburg ^1,², Joseph M McClung ^1,^2,^4,^*

Editor: Cameron J Mitchell⁶

PMCID: PMC7179920 PMID: 32324778

Abstract

Stored muscle carbohydrate supply and energetic efficiency constrain muscle functional capacity during exercise and are influenced by common physiological variables (e.g. age, diet, and physical activity level). Whether these constraints affect overall functional capacity or the timing of muscle energetic failure during acute hypoxia is not known. We interrogated skeletal muscle contractile properties in two anatomically distinct rodent hindlimb muscles that have well characterized differences in energetic efficiency (locomotory- extensor digitorum longus (EDL) and postural- soleus muscles) following a 24 hour fasting period that resulted in substantially reduced muscle carbohydrate supply. 180 mins of acute hypoxia resulted in complete energetic failure in all muscles tested, indicated by: loss of force production, substantial reductions in total adenosine nucleotide pool intermediates, and increased adenosine nucleotide degradation product—inosine monophosphate (IMP). These changes occurred in the absence of apparent myofiber structural damage assessed histologically by both transverse section and whole mount. Fasting and the associated reduction of the available intracellular carbohydrate pool (~50% decrease in skeletal muscle) did not significantly alter the timing to muscle functional impairment or affect the overall force/work capacities of either muscle type. Fasting resulted in greater passive tension development in both muscle types, which may have implications for the design of pre-clinical studies involving optimal timing of reperfusion or administration of precision therapeutics.

Introduction

Ischemic skeletal muscle necrosis occurs concurrently with several common clinical conditions (e.g. peripheral arterial disease, compartment syndrome, or diabetic necrosis) and is a complicating factor of successful muscle graft transplantation [1–3]. The severity of necrosis during an ischemic episode has long been considered a sole function of time, temperature, and magnitude of the hypoxic insult [4,5]. However, the timing of the events that precede irreversible functional impairment and necrosis during ischemia may also depend on other key variables including: metabolic rate; contractile efficiency; and the size of the stored carbohydrate pool [4]. Carbohydrate metabolism is key, as muscle energy supply becomes dependent on anaerobic fermentation of stored carbohydrate sources during ischemia [6–8]. Glycogen is the primary storage form of carbohydrate in skeletal muscle, and its storage/utilization can be influenced by acute environmental factors as well as chronic diseases [9–14].

Previous studies have examined the time dependent changes of metabolites and contractile function in rodent skeletal muscle following ischemia with reperfusion (I/R) [15–18]. Several important observations can be gleaned from these studies: First, locomotory (fast glycolytic) muscles experienced more damage compared to postural (slow oxidative) muscles [15,17]. Second, The degree of initial injury can have large effects on post ischemic recovery time [16]. Lastly, optimal reperfusion timing is related to changes in muscle metabolite levels during ischemia [18]. A major limitation of I/R studies is that it is difficult to distinguish between the functional impairment and/or damage that is attributable to the ischemia itself versus that caused by the reperfusion injury.

In a previous study, using an in vivo mouse hindlimb ischemia model (without reperfusion), we found that myonecrosis develops between three and six hours after the onset of ischemia and is accompanied by a complete loss of contractile function [19]. This led us to examine the <3-hour time domain in this study to better define the timing of muscle functional impairments and associated terminal metabolite changes that occur during acute hypoxia. We hypothesized that fasting, and associated reductions in stored muscle glycogen, would significantly shorten the amount of time that the muscles could remain functional during acute hypoxia.

To test this hypothesis, we utilized fasting to induce an approximate 50% decrease in resting muscle glycogen and employed a carefully controlled experimental system to assess the effects of carbohydrate reduction on isolated mouse hindlimb muscle function during acute hypoxia. Our data provide a novel characterization of hypoxic muscle mechanical/energetic failure and paint a detailed picture of the timing of these impairments. This information can be used in conjunction with existing in vivo rodent hindlimb ischemia/reperfusion studies [5,16,17,19] to generate new hypotheses regarding optimal timing of reperfusion or administration of precision therapeutics.

Materials and methods

Animals

Adult male BALB/c mice (N = 32), aged 16–24 weeks old, were obtained from Jackson Laboratories (Bar Harbor, ME). All work was approved by the Institutional Animal Care and Use Committee of East Carolina University. Animal care followed the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. Washington: National Academy Press, 1996. Animals had free access to water and food except during fasting protocols, during which animals had free access to water only.

Laser scanning confocal microscopy

Sarcomeric actin staining was performed in PFA fixed whole mount muscles, following permabilization with 30μg/ml saponin, using 200nM Alexa Fluor 488 conjugated phalloidin (Thermo Fisher, Waltham MA). Muscles were imaged in a glass bottom (#1.5) dish in Krebs Ringer solution. All imaging was performed using an Olympus FV1000 laser scanning confocal microscope (LSCM). Acquisition software was Olympus FluoView FSW (V4.2). The objective used was 60X oil immersion (NA = 1.35, Olympus Plan Apochromat UPLSAPO60X(F)) or 30X (NA = 1.05, Olympus Plan Apochromat UPLSAPO30XS). Images were 800x800 pixel with 2μs/pixel dwell time. Detector noise was reduced by application of a 3X line scanning kalman filter. Images were acquired in sequential scan mode. 2μM DAPI was used for nuclear counterstaining (Sigma Aldrich, St. Louis, MO) and was excited using the 405nm line of a multiline argon laser; emission was filtered using a 490nm dichroic mirror and 430-470nm barrier filter. AF488-phalloidin was excited using the 488nm line of a multiline argon laser; emission was filtered using a 560nm dichroic mirror and 505-540nm barrier filter. Zero detector offset was used for all images. The pinhole aperture diameter was set to 105um (1 Airy disc).

Dystrophin/laminin immunofluorescence in transverse muscle sections

EDL and soleus muscles were embedded in optimal cutting temperature medium (OCT), and frozen in liquid nitrogen cooled isopentane for cryosectioning. 10μm sections were cut using a CM-3050S cryostat (Leica, Wetzlar Germany) and collected on charged glass slides. Sections were then fixed in 1:1 acetone/methanol for 10 minutes at -20°C, rehydrated in 1X phosphate buffered saline (PBS), and blocked in 5% goat serum + 1X PBS for one hour at room temperature. Sections were then incubated with mouse anti-human monoclonal dystrophin antibody (Thermo-Fisher, MA5-13526), and rabbit anti-rat primary laminin antibody (Thermo-Fisher, A5-16287) at 4°C overnight. Sections were washed 3X for 10 minutes with cold 1X PBS and incubated for 1 hour with Alexa-fluor 594 conjugated goat anti-rabbit IgG or Alexa-fluor 488 conjugated goat anti-mouse (highly cross adsorbed) IgG2b secondary antibody (1:250, Invitrogen). Sections were mounted using Vectashield hard mount medium without Dapi (Vector Labs). Images were taken with an Evos FL auto microscope (Thermo Fisher, Waltham, MA) with a plan fluorite 20X cover slip corrected objective lens (NA = 0.5, air). The following excitation/emission filter cubes were used: GFP (470/22 nm Excitation; 510/42 nm Emission) and Texas Red (585/29 nm Excitation; 624/40 nm Emission). 4X and 20X magnification images were taken for each condition. Image processing was performed using ImageJ (NIH, v1.51f) [20].

Fasting

Mice were housed in a temperature-controlled facility on a 12-hour light-dark cycle with free access to food and water prior to fasting (dark cycle: beginning at 1900 hours, ending at 0700 hours). Mice were fasted for 24 hours to achieve a reduction of skeletal muscle glycogen of ~50%, compared to the fed state. The 24-hour fasting period began at the beginning of a light cycle (0700 hours) and was terminated at the end of the subsequent dark cycle (0700 hours). Mice had free access to water during fasting. All muscles (including control and fasted groups) were isolated for experiments immediately following the end of the dark cycle, between 0700 and 0800 hours. All experiments were performed in the summer season, between the months of May and August.

Measurement of muscle mechanical function

Mice were sacrificed by cervical dislocation under isoflurane anesthesia (confirmed by lack of pedal withdrawal reflex). Extensor digitorum longus (EDL) or soleus muscles were carefully dissected and tied at both tendon ends with 5–0 silk sutures (Thermo Fisher, Waltham, MA). Muscles were tied to an anchor at the proximal end and a dual mode force transducer (Aurora 300B-LR, Aurora, ON, Canada) at the distal end in a vertical bath at 22°C. All protocols were performed in the absence of additional carbon fuel sources (i.e. amino acids, glucose, etc.) to restrict muscles to stored fuel supplies. The bath solution was a modified Krebs Ringer solution described previously [21]. All muscles were dissected and mounted within 15 mins of sacrifice. Muscles were equilibrated in the bath for 10 mins, and optimal length (L₀) was determined by stimulating twitch contractions (0.2ms pulse width, 1 pulse/train) at 10 second intervals and adjusting the length incrementally until maximal force was achieved. Supramaximal stimulation voltage for both muscle types was determined to be 20V. L_O (mm) was measured using a digital microcaliper (Thermo Fisher, Waltham, MA). A force frequency curve was developed for each muscle using stepwise increasing stimulation frequencies of 10, 20, 40, 60, 80, 100, and 120 Hz (.2ms pulse width, pulses/train = half of the stim. Freq.). Baths were aerated with 95%O₂/5%CO₂ (oxygenated; O₂ condition) during L_O determination and the initial force frequency curve. The aeration source was then either left the same or changed to 95%N₂/5%CO₂ (hypoxic; N₂ condition) to simulate an ischemia-like condition. The muscles were then equilibrated for 10 mins, and an initial isokinetic contraction protocol was elicited in the O₂ condition (100Hz isometric contraction for 0.8 seconds followed by a 3mm shortening phase over .3 seconds, then a return to L_o over 30s for the EDL; for the soleus 80Hz isometric contraction was elicited for 0.8 seconds followed by a 4mm shortening phase over .4 seconds, then a return to L_o over 30s). The aeration source was then either left the same or changed to 95%N₂/5%CO₂ (hypoxic condition), with experimental conditions alternated each time to reduce bias. The muscles were then equilibrated for 10 mins, followed by stimulated isokinetic contractions every 10 mins for 180 mins (18 total contractions). We chose this timing based on our previous observation that excitation contraction coupling is impaired in muscles isolated from BALB/c mice 180 minutes after in vivo induction of acute hindlimb ischemia (in the absence of histological signs of tissue necrosis) [19]. A second force frequency curve was measured following the 180-min. isokinetic protocol without changing the aeration source. Muscles were removed from the apparatus, blot dried on paper, weighed, and flash frozen in liquid nitrogen for biochemical analyses. Isometric time-tension integrals (TTI) were calculated by integrating over the isometric (phase I) portion of the curve and are expressed in units of Newton*second/square centimeter (N*s/cm²). Isokinetic work (W) was obtained by integrating the force over the length change during the shortening (phase II) portion of the protocol and is expressed in units of Joules/square centimeter (J/cm²).

Absolute isometric force measurements were normalized to mathematically approximated cross-sectional areas of the muscles. The cross-sectional area for each muscle was determined by dividing the mass of the muscle in grams (g) by the product of its optimal fiber length (L_f, cm) and estimated muscle density (1.06 g cm^-3). Muscle force production was expressed as specific force (N/cm²) determined by dividing the tension in Newtons (N) by the calculated muscle cross-sectional area. L_f was obtained by multiplying L_O by the standard muscle length to fiber length ratio (0.45 for adult mouse EDL; 0.71 for soleus) [22]. A gas calibrated Clark electrode (Innovative instruments, Lake Park, NC) was used to assess the oxygen saturation of the isolated bath medium under both aeration conditions prior to carrying out the experiments. O₂ conditions were approximately 90% saturation measured at the center of the bath (after 10 mins of aeration). N₂ conditions were <2% saturation.

Measurement of glycogen content in whole tissue

Skeletal muscle and liver tissues were flash frozen in liquid nitrogen and stored at -80°C. Glycogen assays were performed using acid hydrolysis and an enzyme coupled assay [23]. Briefly, tissue samples were digested/hydrolyzed under acidic conditions using 2N hydrochloric acid (Sigma Aldrich, St. Louis, MO) on a heating block at 95°C for 2 hours with additional vortexing. Samples were neutralized with equal volume 2N sodium hydroxide (Sigma). A small amount of tris HCl pH 7.0 (~1% of final volume) was added to buffer the solution. Samples were added to a clear 96 well plate in duplicate and were incubated with a solution containing: >2000U/L hexokinase (S. cerevisiae), >4000 U/L NAD⁺ dependent glucose-6-phosphate dehydrogenase (L. mesenteroides), 4mM ATP, 2mM Mg²⁺, and 2mM NAD⁺ (Hexokinase reagent solution; Thermo Fisher). Water was used in place of the reagent for background correction. A standard curve of D-glucose (Sigma Aldrich) was used to calculate the concentrations of hydrolyzed glucosyl units in each sample. Colorimetric measurement of NAD(P)H absorbance was made at 340nm using a Cytation 5 microtiter plate reader (Biotek, Winooski, VT). Liver samples were diluted 1:50 in water prior to enzyme coupled assays to obtain absorbance values within the range of the standard curve. Data were normalized to tissue mass and represented as nmoles glycogen/mg tissue wet weight. The response coefficient (R_Glyc) is defined as the fractional change in experimental group mean relative to the basal group (i.e. Mean Basal–Mean Experimental/Mean Basal*100).

Ultra-Performance Liquid Chromatography (UPLC) measurements of adenosine nucleotides in whole tissue

UPLC measurements of adenosine nucleotides in whole muscle tissue have been described in detail previously [24]. Briefly, isolated muscles were flash frozen in liquid nitrogen, homogenized in ice-cold perchloric acid using a glass on glass homogenizer, and centrifuged to remove precipitated proteins. Samples were neutralized using potassium hydroxide and centrifuged a second time, to remove perchlorate salt. Adenosine nucleotides and degradation products were assayed using an Acquity UPLC H class system (Waters, Milford, MA). Metabolites were identified by comparison of peak retention times of pure, commercially available standards (Sigma–Aldrich). These UPLC measures can provide an index of intracellular energetic state. The amount of IMP reflects longer periods of metabolic demand exceeding supply as the available adenylate pool is decreased via irreversible deamination of AMP to IMP. Over the timeframe of these stimulation protocols, IMP accumulation is a reliable measure of sustained mismatch between ATP supply and demand. (Adenosine triphosphate-ATP, adenosine diphosphate-ADP, adenosine monophosphate-AMP, and inosine monophosphate-IMP).

Statistical analysis

Data are represented by mean ± sample standard deviation (SD). Analyses and plotting were carried out using Graphpad prism (V8.01; Windows 10). Two-way analysis of variance (ANOVA) was used for comparison of group means. Assumption of equal variance was tested using a Brown-Forsythe test. Multiple comparisons were tested using Sidak’s method. Time series data were compared by fitting the curves for each group with an appropriate regression model (simple linear model for O₂ conditions; 3-parameter logistic growth model for N₂ conditions). Standard deviation of the residuals was used to determine goodness of fit for all curve fitting. Slopes (linear models) and P₅₀ values (i.e. the number of contractions to 50% of the initial force/work; non-linear models) were compared between fed/fasted groups using a sum of squares F-test. P values of < 0.05 were considered statistically significant for all analyses.

Results

Extensor digitorum longus (EDL) and soleus muscles were chosen for their known differences in thermodynamic efficiency [25]. The muscles also characteristically rely on different modes of energy metabolism for sustained contractions (glycolytic and oxidative energy metabolism respectively) [26]. We used a two-factor two-level experimental design (Fig 1) to test the hypothesis that fasting would significantly reduce the amount of time during hypoxia that the muscles could remain functional.

Fig 1 — Diagram showing the two-factor two-level experimental design implemented in this study. Groupwise comparisons were analyzed using two-way ANOVA. Time series data were analyzed by comparing regression parameters with a sum of squares F-test. P values ≤.05 were considered statistically significant. L_o is the optimal resting length of the muscle in mm. O₂ and N₂ aeration conditions were 95%O₂/5%CO₂ and 95%N₂/5%CO₂ respectively in Krebs Ringer solution at ~22°C.

Fasting is a well characterized and effective method of whole body carbohydrate reduction in mice, due to their high thermal conductivity and large surface area to body volume ratio [27]. This method was chosen for this study because it is independent of the confounding effects of exercise or contraction induced fatigue [28]. The mean change in bodyweight over the fasted period (24 hours) was 3.9 ± 0.12 grams, approximately 13% of the mean initial weight. We observed a large difference in stored glycogen levels between fed and fasted groups in both liver (~90% lower) (Table 1) and skeletal muscle (~50% lower) (Table 1). The baseline glycogen concentration was higher in the soleus than the EDL under both fed and fasted conditions. Additionally, soleus muscles had a lower mean glycogen concentration in the fasted group relative to the fed state (mean percent difference of 41.6% compared to 56.1% in the EDL groups; Table 1).

Table 1. Basal tissue glycogen concentrations in the liver and skeletal muscle of fed Vs. fasted groups.

Tissue	Condition	Glycogen (nmol/mg)	StDev (nmol/mg)	% Fed Group
Liver	Fed	387.9^*	88.43
	Fasted	42.0^*	20.7	10.8
EDL	Fed	34.4^*^#	8.0
	Fasted	19.3^*^#	3.2	56.1
Soleus	Fed	61.9^*^#	17.5
	Fasted	25.8^*^#	3.6	41.6

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Units are nanomoles hydrolyzed glucosyl units/milligram tissue wet weight (nmol/mg). Data analyzed using two-way ANOVA.

*p <.05 Fed V. Fasted Groups.

^#p <.05 EDL V. Sol. N = 4. Sample standard deviation (StDev).

Fasting had no effect on the isometric force-frequency relationship at baseline or under any of the tested conditions in the EDL (Fig 2A) or soleus (Fig 2B), indicating reduced carbohydrate pool size did not alter excitation-contraction coupling. Specific force values for both muscles were consistent with those obtained previously [21]. Additionally, we observed characteristic reductions in maximal specific force following the O₂ protocols (and completely impaired force production following the N₂ protocols) in both muscles (Fig 2A and 2B). Notably, the isometric force capacity during each protocol did not differ between the fed and fasted groups in either the EDL (Fig 2C) or the soleus (Fig 2D). Similarly, the work capacity over the course of the protocols did not differ for either muscle between the fed and fasted states (Fig 2E and 2F). As expected, the force and work capacities were greatly reduced under the N₂ conditions compared to O₂ conditions.

Given that no substantial differences in force or work capacities were observed, we next examined whether the timing of muscle functional impairments would differ between the fed and fasted states. The time-tension integral (TTI) of the isometric portion of each contraction was plotted as a function of the number of contractions (or time) during each protocol for the EDL (Fig 3A) and soleus (Fig 3B). This measurement represents the ability of the muscle to perform sustained non-shortening contractions. Additionally, the length-time integral of the isokinetic portion of each contraction was also plotted against the number of contractions for the EDL (Fig 3C) and soleus (Fig 3D). This measurement represents the ability of the muscle to perform shortening work. Both sets of curves were characterized by an inverse linear relationship under O₂ conditions and a distinctly non-linear inverse relationship under N₂ conditions during the time and frequency domains of the experiments. The muscles from the fasted groups experienced more rapid reduction in both TTI and work (Fig 3A–3D). Passive tension was measured at the start of each contraction, and the mean maximal values observed during the protocols are reported for the EDL (Fig 3E) and soleus (Fig 3F). This measurement represents stiffening of the muscle, which may be due to several possible factors, including impaired calcium reuptake or cellular swelling due to uncontrolled fluid uptake [29]. None of the muscles experienced substantial changes in passive tension during the O₂ protocol. Greater increases in maximal passive tension occurred in the fasted groups (compared to fed groups) in both muscle types under N₂ conditions. To account for the possibility that the muscles were accumulating excessive water, the wet weights of the EDL (Fig 3G) and soleus (Fig 3H) were plotted. No differences in wet weight between the fed and fasted states were observed in either muscle and all the tested muscles accumulated additional weight following the N₂ protocol.

Fig 3 — Isometric time-tension integrals (TTI) of each contraction over the course of 18 contractions (or 180 minutes) under each condition for the EDL (A) and soleus (B). Isokinetic length-tension integrals (isokinetic work) of each contraction for the EDL (C) and soleus (D). Maximal passive tension developed during the protocol (measured at the start of each contraction) for the EDL (E) and soleus (F). Muscle wet weights obtained at the end of each protocol for the EDL (G) and soleus (H). N = 8/treatment/group (EDL), N = 7/treatment/group soleus. Data are presented as mean ± SD. For the O₂ condition data, the slope of each line was determined using a simple linear regression model. For N₂ data, the number of contractions to 50% initial force/work (P₅₀) was estimated using non-linear regression. Parameter values (A-D) were compared using a sum of squares F-test. Solid black bars = Fed Group. Crosshatched bars = Fasted Group. Group means (E-H) were compared using two-way ANOVA. ****p <.0001 statistically significant effect of bath aeration condition. ns = no significant effect of feeding condition. # p <.05 statistically significant effect of feeding condition; #### p <.0001 statistically significant effect of feeding condition (Sidak’s multiple comparison test).

We next measured the muscle glycogen levels following the O₂ and N₂ protocols. The N₂ protocol reduced glycogen concentrations in all the muscles tested, relative to the O₂ condition (Table 2). Additionally, glycogen concentrations were lower in the fasted soleus groups compared to the fed groups under both O₂ and N₂ conditions (Table 2). However, glycogen concentrations did not differ between fed and fasted groups in the EDL muscles. Using the response coefficient (R_Glyc), allowed for comparison of each group mean to the basal values that are presented in Table 1. The patterns among both muscle types were similar when represented this way. The largest differences observed were between O₂ and N₂ conditions and were not substantially different between fed and fasted groups. Interestingly, the smallest difference in glycogen concentration observed was in the fasted O₂ condition for each muscle. This observation likely indicates the use of alternative (oxygen dependent) fuel sources.

Table 2. Tissue glycogen concentrations in EDL and soleus muscles of fed V. fasted mice following O₂ or N₂ protocols.

Tissue	Group	Condition	Glycogen(nmol/mg)	StDev(nmol/mg)	R_Glyc (%)
EDL	Fed	O₂	22.4^†	4.4	-34.8
		N₂	7.9^†	5.1	-77.0
	Fasted	O₂	18.8^†	7.3	-2.5
		N₂	7.6^†	1.3	-60.6
Soleus	Fed	O₂	42.1^†^*	6.6	-31.9
		N₂	29.0^†^*	1.6	-53.1
	Fasted	O₂	23.5^†^*	8.8	-8.9
		N₂	9.8^†^*	4.2	-62.0

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Units are nanomoles hydrolyzed glucosyl units/milligram tissue wet weight (nmol/mg). The Response Coefficient (R_Glyc) indicates the percent change relative to the baseline group means (Presented in Table 1). Sample standard deviation (StDev). Group means were compared using two-way ANOVA.

*p <.05 Fed V. Fasted Groups.

^†p <.05 O₂ V. N₂ Groups. N = 4/group.

Total adenosine nucleotide (TAN) concentrations were examined as a measure of the aggregate tissue energetic state at baseline and at the end of each protocol. Reductions in the concentrations of the total adenosine nucleotide pool, and accumulation of IMP, are measures of the muscles’ inability to resynthesize ATP [24]. Hypoxia resulted in statistically significant reduction in the all the adenosine nucleotides measured, except for adenosine monophosphate (AMP) in the soleus (Fig 4A–4F). A significant increase in the AMP degradation product inosine monophosphate (IMP) following hypoxia was observed in all groups tested (Fig 4G and 4H). This observation supports the notion that the muscles were in a state of energetic failure at the completion of the protocols. No differences were observed between the fed and fasted groups in either muscle type for any of the measured nucleotides (Fig 4A–4H).

Fig 4 — Whole muscle tissue adenosine triphosphate (ATP) concentrations at baseline and after the O₂ and N₂ protocols for the EDL (A) and soleus (B). Adenosine diphosphate (ADP) concentrations for the EDL (C) and soleus (D). Adenosine monophosphate (AMP) concentrations for the EDL (E) and soleus (F). Inosine monophosphate (IMP) concentrations for the EDL (G) and soleus (H). Solid black bars = Fed Group. Crosshatched bars = Fasted Group. N = 3/treatment/group. Data are presented as mean ± SD. Group means were compared using a two-way ANOVA. ****p <.0001, **p <.005, *p <.05 statistically significant effect of bath aeration condition. ns = no significant effect of feeding condition.

Previous reports have indicated that dystrophin IF staining is rapidly reduced in skeletal and cardiac muscle during early myonecrosis [19,30]. Immunofluorescent staining for the sarcolemmal protein dystrophin and the extracellular matrix protein laminin was performed on a subset of transverse sectioned muscles to assess the possibility that muscles were incurring damage during the contraction protocols. No apparent changes were observed in the EDL (Fig 5A) or soleus (Fig 5C) under O₂ or N₂ conditions, indicating that the muscle tissue remained intact during the experiments. Degradation of myofibrillar structures are another well characterized indicator of myonecrosis development [31]. Parallel assessments were made to accompany the dystrophin/laminin stain. Fibrous actin was stained in fixed whole mount muscle specimens utilizing optical sectioning to assess the intramyofibrillar-IMF and perinuclear-PN regions of the myofibers at baseline and following the N₂ protocol in the EDL (Fig 5B) and soleus (Fig 5D). Together these qualitative assessments did not reveal any indication of damage.

Fig 5 — To control for the possibility that the muscles were structurally damaged during the contraction protocols, we performed immunofluorescence against sarcolemmal and extracellular matrix proteins. Image panels of dystrophin (green), and laminin (red) stained transverse EDL (A) and soleus (C) muscle sections under each of the conditions tested. Sarcomeric actin was stained using phalloidin (Cyan) in fixed/permeabilized whole mount muscles at baseline or following 180 mins of severe hypoxia (95% N₂); EDL (B) and soleus (D). Optical sectioning facilitated imaging in the intra-myofibrillar (IMF) and perinuclear (PN) regions of the muscle fibers. Scale bars are 1000μm (A, B Left Panel), 200 μm (A,B right panels), and 25 μm (B,D). N = 1/timepoint.

Discussion

Skeletal muscle is among the most metabolically dynamic tissues in the body, and is capable of sustaining a 100-fold change in ATP utilization rate during contraction [32]. The total cost of ATP during contraction is proportional to the duration, intensity, and type (i.e. shortening vs. non-shortening) [33]. Glycogen is the primary storage form of glucose in skeletal muscle, and is a major source of fuel during most forms of muscle activity [34].

Importantly, glycogen is also the primary source of stored fuel utilized to regenerate ATP via substrate level phosphorylation in anaerobic glycolysis during severe hypoxia [33]. Depletion of stored muscle glycogen by fasting or exhaustive exercise results in impaired fatigue resistance and recovery in isolated rodent muscles under normoxic conditions [34–36]. Under hypoxic conditions, this effect would be expected to lead to cumulative reductions in energetic capacity due to the inability to resynthesize ATP and phospho-creatine (PCr) that is used in support of contraction or resting metabolic processes.

Overnight fasting in rodents results in more dramatic metabolic effects than human overnight fasting, but induces experimentally reproduceable reductions in systemic carbohydrate stores that are similar to more extreme physiological conditions such as hyperinsulinemia, hypoglycemia, or post exercise recovery [11,14,27,37]. Fasting was used in this study because it is independent of the confounding effects of exercise or contraction induced fatigue [28]. Notably, the glycogen values observed in this study differ from several previous reports in that fed state control values for both muscles are relatively high, and that the soleus glycogen levels are substantially higher than the EDL (values between muscles did not differ in the previous reports) [38–40].

Muscle glycogen concentration is a physiologically dynamic parameter that is influenced by experimental conditions such as assay method and normalization factor, as well as biological conditions such as parental genetic background and metabolic state [41–43]. Though we cannot directly account for specific confounders that explain the discrepancy in this study, there are two likely candidates that should be considered for further investigation: 1.) Muscle glycogen levels have been shown to vary with season and diurnal cycle in mice [44] and rats [45,46], with peaks in the dark-light cycle transition period (the time at which animals were sacrificed in this study). 2.) Soleus muscles have been shown to be more sensitive to insulin stimulated glucose uptake and glycogen synthesis in both mice [38] and rats [47], and insulin stimulated glucoregulatory responses have been shown to differ among inbred mouse strains [48]. Taken together, the described findings support the possibility that stored muscle glycogen values may have been influenced by seasonal, circadian, or hormonal variation intrinsic to the genetic background of the mice used in this study.

We were somewhat surprised to find that fasting associated reductions in muscle glycogen levels had no substantial effect on the timing or magnitude of isolated muscle functional impairment under hypoxic conditions. Our findings indicate that both muscle types retain a large pool of stored glycogen that is non-essential for reserve mechanical force production during hypoxia. It is not clear what the reserve glycogen pool contributes to in vivo during fasting. Future studies could be directed to investigate its’ potential involvement in the maintenance of systemic glucose homeostasis through the production of free amino acids (i.e. alanine and glutamine) or lactate which can be converted to glucose in the liver [49,50].

In mouse EDL and soleus muscles, as much as 50% of the resting metabolic rate has been attributed to maintenance of intracellular calcium homeostasis [51]. We observed greater maximal passive tension development in the fasted group relative to the fed group under N₂ conditions in both muscle types. This phenomenon is most likely indicative of progressive impairment of calcium handling as the capacity for ATP re-synthesis was gradually depleted [24]. This effect may have implications for reperfusion timing, as it has been noted that calcium handling impairment prior to reperfusion is associated with poor salvage outcomes [4,52].

Skeletal muscle fiber types are categorized by a range of intrinsic metabolic and mechanical properties [53]. Human muscles are generally of mixed fiber type, but mouse muscles consist of more homogeneous fiber type distributions, making them a practical model for studying fiber type specific effects (soleus: 1:1 slow type I/fast type IIa; EDL: 9:1 fast type IIb/fast type IIa) [26,54]. At face value, it may seem intuitive that fast glycolytic fiber types would be better suited to performance during hypoxia due to their preference for stored carbohydrate dependent energy metabolism [34,35,55]. However, several studies have indicated a high degree of sensitivity of fast glycolytic muscles to ischemia/reperfusion injury [16,17,56]. One important contributing factor to this effect is an energetic inefficiency of contraction due to interactions at the level of the acto-myosin crossbridges [25,57].

In this study, we observed that Soleus muscles stored more glycogen at baseline, had greater specific force/work capacities, and produced absolute force for a longer period during hypoxia compared to EDL muscles. The observations regarding greater glycogen content in the soleus muscle compared to EDL muscles are not consistent with previous reports [39,40,58], but the observations of improved mechanical function during hypoxia in soleus compared to EDL muscles have been previously reported using small muscles isolated from rats [56]. Though the absolute differences in glycogen concentrations between groups were larger in the soleus compared to the EDL, the response coefficient (R_Glyc) which facilitates interpretation of group differences relative to their baseline concentration, indicated that the patterns of utilization were not different between the two types of muscles. We interpret these findings to mean that the greater basal glycogen concentration observed in the soleus muscles was likely not the primary factor underlying it’s enhanced ischemic mechanical performance.

There are several important limitations to this study. First, the carbohydrate reduction associated with fasting is not complete, leaving approximately half of the fed state muscle glycogen available during experimental hypoxia. Though this is independent of confounding effects associated with other methods of glycogen depletion [28], there are other effects of fasting that may confound observed outcomes [27]. Second, though we were able to assess muscle contractile function in time series, we were only able to assess changes in muscle metabolite levels (e.g. adenine nucleotides, glycogen, etc.) at baseline and after the 180-minute protocols. Additional experimentation is necessary to fully characterize the time-dependent changes in key metabolites during hypoxia. Finally, our experimental system utilizing isolated muscle is highly controlled for the effects of hypoxia but lacks the biological variability and complexity of ischemia. We hope that the observations in this study can be used to inform development of hypotheses that can be further tested using in vivo preclinical models of ischemia.

Conclusion

Identification of key factors that affect the timing of muscle energetic failure during hypoxia will aid in identifying optimal windows for therapeutic intervention in ischemic disease. We predicted that the amount of stored carbohydrate is one such factor, as it is a major contributor to anaerobic energy metabolism and is influenced by several physiologically relevant conditions. We conclude that mouse hindlimb muscles maintain a large pool of stored carbohydrate that is utilized during fasting but does not contribute substantially to the timing of functional decline during acute hypoxia. The carbohydrate lowering effects associated with fasting did not substantially affect the total capacity or timing of contractile function impairment in either muscle type. However, fasting did result in substantial increases in passive tension development during hypoxia, which may have implications for the design of follow up studies in vivo. We also found that soleus muscles maintained a greater total force capacity and became impaired more slowly than EDL muscles, independent of glycogen utilization during the experimental period. This finding supports several previous observations and bolsters the notion that susceptibility to hypoxia-induced impairment is not uniform across muscle types.

Data Availability

All relevant data are available at DOI 10.17605/OSF.IO/PZT5G.

Funding Statement

This work was made possible by grants awarded by the National Institutes of Health (NIH.gov) to JMM (R01HL125695), EES (R01AR066660) and JJB (R01AR070200).

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PLoS One. doi: 10.1371/journal.pone.0225922.r001

Decision Letter 0

Cameron J Mitchell

18 Dec 2019

PONE-D-19-31637

Effects of fasting induced carbohydrate depletion on murine ischemic skeletal muscle function.

PLOS ONE

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The reviews have found merit in your study however, they have both expressed concerns about the presentation of the data which would need to be addressed to make the manuscript suitable for publication. In particular the applicability of your model to ischemia needs to be addressed. It is also important to reconcile the reported muscle glycogen concentrations with those commonly reported in the literature.

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The bar graphs used would be much more informative if individual data points were overlaid to show the true variability in the data.

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Reviewer #1: The main idea of this manuscript is investigating the role of muscle glycogen amount for ischemic muscle function. The authors hypothesized that reduced muscle glycogen would shorten the functional time of ischemic muscle. To test this hypothesis, the authors used fasting to reduce glycogen storage and measured muscle functions with hypoxia condition in vitro. Although the idea was interesting, reduced muscle glycogen did not likely change the muscle functions with/without ischemia (rejected hypothesis). I recommend several changes as below to improve the manuscript.

Fig 1. Need quantification for each image. Vessel density should be normalized by fiber area. Without quantification, this figure can be a supplemental figure. Analyzed fiber number and total animal number should be added to the legend. Make more connections between figure 1 and rest of figures. Otherwise, this figure is little bit out of place.

Fig 2. To help readers better understand this and later figures, Please add scheme (cartoon) of experiment, which will help to understand this and later figures. Also add statistical analysis method for this figure to the legend. Since data have more than 2 factors (O2 vs N2 and Fed vs. Fasting), 2-way ANOVA should be used. (Please note, the Methods section only mentioned a 2 tail student test for group comparison) (Minor comments for Fig 2C-F, adding crosshatch as a fill for the fasting graph (but keeping the color) would make the graph easier to read.)

Fig 3. Add statistical analysis (such as * marks) for each figure and add statistical analysis method (2 way ANOVA is recommended) to the legend. (Same minor comments for Fig 3G-H, adding crosshatch to the fasting graph would improve clarity of the image)

Fig 4. Consider change the way the figure is presented. It would be nice combine ATP, ADP, AMP values of all conditions in each single graph like Fig 4G-H. Also add statistical analysis in graph and methods to the legend.

Fig 5. Add quantification graph for image analysis, including number of sections and number of mice with statistical analysis method.

Result. Line 319-320. The author claims that “the muscles from the fasted group experienced more rapid reduction in both TTI and work”. However, Fig 3c and 3d did not support this claim due to lack of statistical analysis. Error bars of fed and fasted group graph seem to overlap each other at almost every time point. Please provide detailed support/explanation for this claim.

Reviewer #2: This manuscript studies the effects of fasting and hypoxia in ex vivo soleus and EDL mouse muscles that are stimulated to contract. The measurements include contractile function, passive tension and metabolites such as glycogen, IMP, and TAN. The premise is that the hypoxia, which is produced by incubated muscles in solution gassed with N2, is a model for ischemia. This premise if flawed. Fasting is used as an intervention to reduce tissue carbohydrate supply. Fasting does lower carbohydrate content of muscles, but that is not the only thing that fasting does. The text needs to be revised to use more direct and accurate language to describe the experimental approach. The major metabolic measurement is glycogen, and some of the values disagree with literature values, which undermines confidence in the data.

MAJOR

The title is misleading and should be revised to something more accurate, such as “Effects of fasting and ex vivo hypoxia on murine skeletal muscle contractile function.” The authors don’t have to use this exact wording, but “ischemia” should not be used, “hypoxia” should be used, and the function should be specified.

Ischemia refers to low blood flow. The muscles are studied ex vivo without any flow, whether they are oxygenated or not. Low oxygen is not the only consequence of ischemia. There is no convincing evidence that this is a good model for ischemia. The repeated use of the word “ischemia” or “ischemic” to describe the experiment should be eliminated. The experiment is studying hypoxia, not ischemia, and the text in the entire manuscript should be revised accordingly. Eliminating or at least deemphasizing the assertion that the experimental approach is an ischemia model would be helpful. If the authors are determined to comment on how this model has relevance to ischemia, they need to provide specific and direct evidence to support this assertion, and to also directly acknowledge the limitations of this experimental approach as an ischemia model.

The abstract refers to “conditions of reduced carbohydrate supply” before using a more informative description of “fasting.” The abstract should identify the duration of fasting. The text throughout should also not suggest that all fasting does is reduce carbohydrate supply or glycogen levels. It is OK to indicate that this might be an important consequence of fasting for the effects on contraction function, but there should be a more accurate description of what fasting represents and recognition that reducing glycogen is not everything it does.

A major point made by the authors is that glycogen concentration is much higher in the soleus than the EDL. This result has not been observed in earlier research. Glycogen of soleus was not much greater for mouse soleus compared to EDL (Jorgensen J Biol Chem. 2004. 279(2):1070-9; Bonen J Appl Physiol 1994. 76(4):1753-8). The authors should address what might account for the discrepant results and provide evidence that their results are consistent with results of a number of earlier studies.

The muscle glycogen concentrations are higher than usually reported for mouse EDL and soleus. The value in Table 1 for fed soleus (61.9 nmol/mg) is very high compared to the literature. There should be citations of literature values for glycogen and an explanation for the high values in this study compared to the literature.

The light/dark cycle times should be stated, and the times when fasting began and when muscles were sampled should be stated.

The Methods section (lines 148-150) on fasting refers to a pilot study and cites a study (ref 23) that is not from this group of authors. It is confusing to know if the authors performed a pilot study or not, and why they cited this study.

In the statistics section (lines 242-243), it stated that both SEM and SD are used with the data. Either one or the other should be used. SD is more informative.

The Discussion should acknowledge important limitations of the study. One would be that only one timepoint was studied for metabolite concentrations. Measurements at several timepoints would make the study more informative.

The final sentence of the Introduction is that the “This information …ischemia models.” The Conclusion states that the results are valuable for therapeutic intervention (lines 479 and 488). It is unclear why this information will be valuable for either these ischemia models or for therapeutic interventions. It should be directly stated why this information will be valuable.

Figure 4 should include text on the figure itself to indicate which A-F panels are from the O2 treatment and which are from N2 treatment.

In the Introduction (line 76), it stated that the experiment was intended to determine the “exact temporal nature…”, but only one time point (3 hours) was studied, so this study doesn’t determine the “exact temporal nature” of the results.

**********

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Reviewer #1: No

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PLoS One. 2020 Apr 23;15(4):e0225922. doi: 10.1371/journal.pone.0225922.r002

Author response to Decision Letter 0

28 Jan 2020

Additional Editor Comments:

The bar graphs used would be much more informative if individual data points were overlaid to show the true variability in the data.

-We thank the editor for this suggestion and agree that the recommended change would improve the manuscript. We have modified all bar graphs accordingly. Figures: 2C-F; 3G-H; and 4A-H.

Review Comments to the Author

-We thank the reviewer for their time and for helping us improve the quality of this manuscript with constructive feedback. We have addressed each comment below and summarized the changes made below each comment.

-We agree with this assessment and thank the reviewer for this suggestion. In order to keep the manuscript message focused, we have removed the original Figure 1 and replaced it with a schematic diagram that summarizes the experiments (Also addressed in the second comment). We have also removed the paragraph that referred to the original figure 1 from the discussion:

“The dynamic requirements of ATP during muscle contraction require a similarly dynamic supply of carbon fuel sources that are derived from both blood and intracellular stores. Physiological and anatomical adaptations (i.e. capillary density, size of stored energy substrate pools, and mitochondrial density/function) are known to facilitate large differences in capacity for spontaneous vs. sustained exercise in different species(44). Similar adaptive differences are highlighted in the distinct anatomical, mechanical, and thermodynamic properties of mouse locomotory EDL and postural soleus muscles(23,27,45,46). These adaptive differences, combined with their similar size and relatively homogeneous fiber type compositions(28), make these muscles excellent candidates for comparative studies of muscle energy metabolism.”

-We apologize for this omission. We have updated the statistical analysis methods section to include details of the 2-way ANOVA. We have updated the methods section:

Lines: 238-248 “Data are represented by mean ± sample standard deviation (SD). Analyses and plotting were carried out using Graphpad prism (V8.01; Windows 10). Two-way analysis of variance (ANOVA) was used for comparison of group means. Assumption of equal variance was tested using a Brown-Forsythe test. Multiple comparisons were tested using Sidak’s method. Time series data were compared by fitting the curves for each group with an appropriate regression model (simple linear model for O2 conditions; 3-parameter logistic growth model for N2 conditions). Standard deviation of the residuals was used to determine goodness of fit for all curve fitting. Slopes (linear models) and P50 values (i.e. the number of contractions to 50% of the initial force/work; non-linear models) were compared between fed/fasted groups using a sum of squares F-test. P values of < 0.05 were considered statistically significant for all analyses”.

Additionally, we have added crosshatch patterns to the fasted group in the bar graphs as requested. Additionally, we have included the individual data points on the bar graphs to more clearly represent the sample variability.

-We have updated the legends in all figure panels to include details of the relevant statistical analysis. For example: Figure 3 legend:

Lines 340-349 “N=8/treatment/group (EDL), N=7/treatment/group soleus. Data are presented as mean ± SD. For the O2 condition data, the slope of each line was determined using a simple linear regression model. For N2 data, the number of contractions to 50% initial force/work (P50) was estimated using non-linear regression. Parameter values (A-D) were compared using a sum of squares F-test. Solid black bars = Fed Group. Crosshatched bars = Fasted Group. Group means (E-H) were compared using two-way ANOVA. ****p<.0001 statistically significant effect of bath aeration condition. ns = no significant effect of feeding condition. # p<.05 statistically significant effect of feeding condition; #### p<.0001 statistically significant effect of feeding condition (Sidak’s multiple comparison test)”.

-We agree that the recommended changes would improve the interpretation of the figure. We have reorganized the graphs in Figure 4 accordingly. Additionally, we have added individual data points to the bar graphs in the manuscript to better represent sample variability. Finally, we have updated the figure legend to include description of the statistical analysis and relevant symbols indicating statistical significance to the graphs.

Fig 5. Add quantification graph for image analysis, including number of sections and number of mice with statistical analysis method.

-We understand the reviewer’s concern that the images are not quantified. The images in Figure 5 are intended to demonstrate that muscle is not becoming significantly damaged during the experiment. The images were intended to be qualitative, but thorough in that they demonstrate a lack of damage using multiple histological methods. To address this concern, we have updated the figure legend to highlight the qualitative nature of the images as well as the number of muscles used:

Lines: 409-410, 418 “Fig 5: Qualitative assessment of structural integrity of the muscles following experimental protocols.” “N=1/timepoint”).

Additionally, we have clarified this point in the results to avoid any confusion:

Lines: 406-407 “Together these qualitative assessments did not reveal any indication of damage”).

-Excellent point, we apologize for any apparent over-interpretation of the data. We have added additional analysis to include a more direct measure of the rate of muscle mechanical failure for statistical comparison. In brief, we performed linear regression for O2 data sets and non-linear regression for N2 data sets. We selected a three-parameter logistic growth model for the N2 data sets, because it gave a similar fit (determined by standard deviation of the residuals) compared to a four-parameter model but required fewer model assumptions. We then compared curve parameters between groups using a sum of squares F-test. The parameters were: slope (for O2 groups) and P50 (for N2 groups; P50= number of contractions or time to reach 50% of the initial force/work value predicted by the model).

Please note that these additional analyses did not result in any major changes to our previous interpretation of the data. In summary, our conclusion is that fasted groups did reach 50% of initial force/work faster than fed groups. However, the effect of feeding condition was negligible from a practical perspective because it only separated the groups by a matter of minutes.

We have highlighted our interpretation of our observations in the results:

Lines 316-320 “Both sets of curves were characterized by an inverse linear relationship under O2 conditions and a distinctly non-linear inverse relationship under N2 conditions during the time and frequency domains of the experiments. The muscles from the fasted group experienced more rapid reduction in both TTI and work (Fig A-D)”.

We have updated the methods section to include description of the additional analysis:

Lines 242-248 “Time series data were compared by fitting the curves for each group with an appropriate regression model (simple linear model for O2 conditions; 3-parameter logistic growth model for N2 conditions). Standard deviation of the residuals was used to determine goodness of fit for all curve fitting. Slopes (linear models) and P50 (i.e. the number of contractions to 50% of the initial force/work; non-linear models) were compared using a sum of squares F-test.”. We have updated Figure 3, the legend, and the methods section to reflect these changes.

-We thank the reviewer for their time and careful consideration of our manuscript. The provided comments have been very helpful in improving the quality of the work. We have provided individual responses to the comments below, and included brief summaries of the changes made as well as line information that links with the manuscript.

MAJOR

-We agree with this concern and have revised the title and main body text to reflect these changes:

New title: Lines 1-2 “Effects of fasting on isolated murine skeletal muscle contractile function during acute hypoxia”.

Lines: 26-27 Example of use in revised text: “Whether these constraints affect overall functional capacity or the timing of muscle energetic failure during acute hypoxia is not known”.

-We have revised the text to avoid any misinterpretation of the data by changing the word ‘ischemia’ to ‘hypoxia’ where it refers directly to the model or its interpretation. Example of use in the text:

Example Lines78-80 “We hypothesized that fasting, and associated reductions in stored muscle glycogen, would significantly shorten the amount of time that the muscles could remain functional during acute hypoxia”.

-Excellent point. We have revised the abstract to reflect these changes.

Lines: 27-32 “We interrogated skeletal muscle contractile properties in two anatomically distinct hindlimb muscles that have well characterized differences in energetic efficiency (locomotory- extensor digitorum longus (EDL) and postural- soleus muscles) following a 24-hour fasting period that resulted in substantially reduced muscle carbohydrate supply”.

Additional lines: 79-81 “We hypothesized that fasting, and associated reductions in stored muscle glycogen, would significantly shorten the amount of time that the muscles could remain functional during acute hypoxia”.

-We thank the reviewer for this suggestion, and agree that additional support from the literature will strengthen the interpretation of our data. We have added several additional references to the manuscript:

• Ryder JW, Kawano Y, Galuska D, Fahlman R, Wallberg-Henriksson H, Charron MJ, et al. Postexercise glucose uptake and glycogen synthesis in skeletal muscle from GLUT4-deficient mice. FASEB J. 1999;13(15):2246–56.

• Sandström ME, Abbate F, Andersson DC, Zhang SJ, Westerblad H, Katz A. Insulin-independent glycogen supercompensation in isolated mouse skeletal muscle: Role of phosphorylase inactivation. Pflugers Arch Eur J Physiol. 2004;448(5):533–8.

• Jørgensen SB, Viollet B, Andreelli F, Frøsig C, Birk JB, Schjerling P, et al. Knockout of the α2 but Not α1, 5′-AMP-activated Protein Kinase Isoform Abolishes 5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside- but Not Contraction-induced Glucose Uptake in Skeletal Muscle. J Biol Chem. 2004;279(2):1070–9.

• Hunter RW, Treebak JT, Wojtaszewski JFP, Sakamoto K. Molecular mechanism by which AMP-activated protein kinase activation promotes glycogen accumulation in muscle. Diabetes. 2011;60(3):766–74.

• Azpiazu I, Manchester J, Skurat A V., Roach PJ, Lawrence JC. Control of glycogen synthesis is shared between glucose transport and glycogen synthase in skeletal muscle fibers. Am J Physiol - Endocrinol Metab. 2000;278(2 41-2):234–43.

• Helander I, Westerblad H, Katz A. Effects of glucose on contractile function, [Ca 2+] i, and glycogen in isolated mouse skeletal muscle. Am J Physiol - Cell Physiol. 2002;282(6 51-6):1306–12.

• Bonen A, Mcdermott J, Tan M. Glycogenesis and Glyconeogenesis in Skeletal: Effects of pH and Hormones. Am J Physiol - Endocrinol Metab. 1990;258(4):693–700.

• Bonen A, Homonko DA. Effects of exercise and glycogen depletion on glyconeogenesis in muscle. J Appl Physiol. 1994;76(4):1753–8.

We have also modified the discussion to include the following paragraph:

Lines: 436-457 “Overnight fasting in rodents results in more dramatic metabolic effects than human overnight fasting, but induces experimentally reproduceable reductions in systemic carbohydrate stores that are similar to more extreme physiological conditions such as hyperinsulinemia, hypoglycemia, or post exercise recovery(11,14,29,39). Fasting was used in this study because it is independent of the confounding effects of exercise or contraction induced fatigue(30). There is a range of reported glycogen values available in the literature for mouse skeletal muscle and liver tissues which are likely influenced by assay method, normalization factor, as well as genetic background and physiological state of the animals(40–45). The fed state values observed for mouse soleus and EDL muscles in this study fall well within the range of normal variability observed in the literature(40–47). Additionally, the magnitude of tissue glycogen reduction observed with 24-hour fasting were concomitant with other available data(29)”.

-Addressed in response to the previous comment above. We agree that the values may be interpreted as high compared to some studies, but there are other studies available that report even higher values. Ultimately the absolute values reported in the literature are likely very sensitive to physiological (e.g. insulin/glucagon stimulation, stress responses, diurnal cycle, etc.) and technical variables (e.g. assay method, normalization factor, etc.). To address this concern, we have added the references indicated above to highlight the range of values observed in other studies, and modified the language in our discussion to reflect the variability of reported absolute values:

Lines: 441-447 “There is a range of reported glycogen values available in the literature for mouse skeletal muscle and liver tissues which are likely influenced by assay method, normalization factor, as well as genetic background and physiological state of the animals(40–45). The fed state values observed for mouse soleus and EDL muscles in this study fall well within the range of normal variability observed in the literature(40–47). Additionally, the magnitude of tissue glycogen reduction observed with 24-hour fasting were concomitant with other available data(29)”.

The light/dark cycle times should be stated, and the times when fasting began and when muscles were sampled should be stated.

-Excellent suggestion. We apologize for this oversight. We have revised the methods section to reflect these changes:

Lines: 141-146 “Mice were housed in a temperature-controlled facility on a 12-hour light dark cycle with free access to food and water prior to fasting. Mice were fasted for 24 hours to achieve a reduction of skeletal muscle glycogen of ~50% (compared to the fed state). The 24-hour fasting period began at the beginning of a light cycle and was terminated at the end of the subsequent dark cycle. Mice had free access to water during fasting. Muscles were isolated for experiments immediately following the end of the fasting period.”.

- We apologize for the confusing way that this was presented. We modified the methods section to clarify the procedure:

In the statistics section (lines 242-243), it stated that both SEM and SD are used with the data. Either one or the other should be used. SD is more informative.

-We apologize for the confusing way that this was presented in the methods. We have modified the methods section to reflect only use only sample standard deviation:

Line: 238 “Data are represented by mean ± sample standard deviation (SD)”.

-This is an excellent point. We have added additional discussion of the limitations of our study design:

Lines: 490-502 “There are several important limitations to this study. First, the carbohydrate reduction associated with fasting is not complete, leaving approximately half of the fed state muscle glycogen available during experimental hypoxia. Though this is independent of confounding effects associated with other methods of glycogen depletion(30), there are other effects of fasting that may confound observed outcomes(29). Second, though we were able to assess muscle contractile function in time series, we were only able to assess changes in muscle metabolite levels (e.g. adenine nucleotides, glycogen, etc.) at baseline and after the 180-minute protocols. Additional experimentation is necessary to fully characterize the time-dependent changes in key metabolites during hypoxia. Finally, our experimental system utilizing isolated muscle is highly controlled for the effects of hypoxia but lacks the biological variability and complexity of ischemia. We hope that the observations in this study can be used to inform development of hypotheses that can be further tested using in vivo preclinical models of ischemia”.

-We apologize for any apparent over-interpretation of the data. We have modified the introduction and discussion to clarify our intent and interpretation:

Lines: 86-90 “Our data provide a novel characterization of hypoxic muscle mechanical/energetic failure and paint a detailed picture of the timing of these impairments. This information can be used in conjunction with existing in vivo rodent hindlimb ischemia/reperfusion studies(5,16,17,19) to generate new hypotheses regarding optimal timing of reperfusion or administration of precision therapeutics”.

Additional lines: 40-42 “Fasting resulted in greater passive tension development in both muscle types, which may have implications for the design of pre-clinical studies involving optimal timing of reperfusion or administration of precision therapeutics”.

Figure 4 should include text on the figure itself to indicate which A-F panels are from the O2 treatment and which are from N2 treatment.

-We apologize for this oversight. We have reorganized the panels in this figure to include the basal, O2, and N2 conditions within each graph for each nucleotide. The feeding conditions and bath conditions are both indicated within each graph. Figure 4A-H. Additionally, we have added cross-hatch patterns to the fasted group bars to help differentiate between the two levels. Finally, we have included individual data points in order to enhance visualization of the data variability.

-We thank the reviewer for this insight and appreciate the concern. Though we were only able to measure metabolite levels at the terminal endpoint of the experiments, we interpret the force production and work measurements to be informative of the overall energetic state of the muscle during the time course of the experiments. We have modified the introduction to clarify our intent and interpretation of the limitations of the study:

Lines: 76-79 “This led us to examine the <3-hour time domain in this study to better define the timing of muscle functional impairments and associated terminal metabolite changes that occur during acute hypoxia”.

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PLoS One. doi: 10.1371/journal.pone.0225922.r003

Decision Letter 1

Cameron J Mitchell

10 Feb 2020

PONE-D-19-31637R1

Effects of fasting on isolated murine skeletal muscle contractile function during acute hypoxia.

PLOS ONE

Dear Dr. McClung,

==============================

Although some improvements have been made to the manuscript the measurement of glycogen is still a major concern. In order for this manuscript to be suitable for publication all of the reviewer’s comments must be specifically addressed.

==============================

We would appreciate receiving your revised manuscript by Mar 26 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
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We look forward to receiving your revised manuscript.

Kind regards,

Cameron J. Mitchell, PhD

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: (No Response)

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: (No Response)

Reviewer #2: No

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: The authors made a number of revisions that have improved the manuscript. However, they have not adequately addressed issues related to the questions about the glycogen results. The current text is not forthcoming in acknowledging or explaining the discrepant glycogen values in this manuscript compared to the literature. The specific concerns are described below.

The authors’ response did not address the fact that their observation of much higher glycogen in the soleus than the EDL, and this result has not been observed in earlier research. Glycogen of soleus was not much greater for mouse soleus compared to EDL (Jorgensen J Biol Chem. 2004. 279(2):1070-9; Bonen J Appl Physiol 1994. 76(4):1753-8). The authors should address what might account for the discrepant results and provide evidence that their results are consistent with results of a number of earlier studies. The authors cited the 2 publications above (which found soleus values were slightly, but not significantly lower for soleus versus EDL) and 6 additional publications, none of which reported glycogen for both soleus and EDL in mice. The authors did not acknowledge that their results of 80% greater glycogen in soleus compared to EDL are at odds with the published literature, and they offered no data that supported their discrepant results. There needs to be a direct explanation in the manuscript related to the unusual findings in this study.

The value for soleus glycogen in Table 1 (61.9 nmol/mg) is much greater than previously published values. The authors state that other studies have reported even higher values, but they don’t point out which studies reported higher values. In the revised manuscript, they state the observed values “in this study fall well within the range of normal availability observed in the literature”, and they cite 8 publications (#40-47). However, several of these publications do not report mouse soleus or EDL glycogen values, and the studies that do report glycogen in these muscle do not report values higher than 61.9 nmol/mg. Some cited studies only reported gastrocnemius glycogen, or single fiber glycogen, or not glycogen concentrations (only glycogenesis rates). Citations that do not include mouse soleus and or EDL glycogen concentrations should not be cited to support the statement in the manuscript. The text needs to be revised to be accurate. The text refers to values being influenced by “assay method, normalization factor, as well as genetic and physiological state of the animals.” This statement is true, but it doesn’t explain the much greater value in this study. If there is any specific evidence that the genetic or physiological state of the mice, or the particular assay used in this study led to much increased soleus glycogen values, it should be specifically identified rather than this general statement without any direct support of its validity.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

PLoS One. 2020 Apr 23;15(4):e0225922. doi: 10.1371/journal.pone.0225922.r004

Author response to Decision Letter 1

20 Mar 2020

We sincerely apologize for failing to appropriately address the concerns regarding the reported muscle glycogen values. The references included in the last resubmission were chosen to highlight a range of potential factors that reflect variation in reported values for mouse skeletal muscle glycogen, but clearly, we missed the mark. We appreciate the reviewer’s concerns and thank them for their ongoing efforts in aiding our improvement of the quality of this manuscript. Below, we attempt to address all the key concerns that we interpret from the provided comments.

1. “The authors should address what might account for the discrepant results”

We have added additional text to the discussion. Lines 448-458. “Though we cannot directly account for specific confounders that explain the discrepancy in this study, there are two likely candidates that should be considered for further investigation: 1.) Muscle glycogen levels have been shown to vary with season and diurnal cycle in mice(44) and rats(45,46), with peaks in the dark-light cycle transition period (the time at which animals were sacrificed in this study). 2.) Soleus muscles have been shown to be more sensitive to insulin stimulated glucose uptake and glycogen synthesis in both mice(38) and rats(47), and insulin stimulated glucoregulatory responses have been shown to differ among inbred mouse strains(48). Taken together, the described findings support the possibility that stored muscle glycogen values may have been influenced by seasonal, circadian, or hormonal variation intrinsic to the genetic background of the mice used in this study”.

2. “provide evidence that their results are consistent with results of a number of earlier studies”

Our observed glycogen values differed from three prior studies that assessed mouse EDL and soleus muscle glycogen content using similar assay methods (cited in the paper and listed below; these were the best matched studies we could find in terms of methodology and including both EDL and soleus muscles). We have altered the manuscript to clearly acknowledge that the results differ from the cited studies and added discussion of potential confounding variables (see response described above). We have found it difficult to directly account for the discrepancies. However, there are a few methodological and environmental differences between this study, and the cited references (particularly contribution of diurnal variation, seasonal variation, and parental genetic background variation as discussed). We have been fully transparent regarding our methods/data analysis and feel that the methodological description in this manuscript is thorough and comparable to all of the published studies that we have reviewed. Additionally, all supporting raw data will be available to the scientific community via open science framework if the manuscript is accepted for publication (in accordance with Plos One’s publishing requirements). We value the importance of ensuring that the data are of high quality and reproducibility. However, we also appreciate that biological data is variable and subject to substantial influence from a multitude of confounding factors. If there are any additional specific methodological concerns, we will enthusiastically address them. We don’t want to risk sharing inaccurate data, but we also want to avoid denying the scientific community access to data solely on the basis that it differs from previous findings.

Some additional technical considerations: For brevity, we reported overall sample size for the groups by using the smallest group size (as this has the largest effect on the ANOVA power). The actual sample sizes for the fed state basal EDL and soleus is N=17 and N=10 respectively. This occurred because of additional myography (twitch protocols) and biochemical assays (UPLC amino acid profiles) that ultimately were not included in the manuscript in order to keep the supporting evidence concise. The soleus glycogen data does have two outliers (87.2 and 96.2 nmol/mg) that skew the mean toward a higher value, however, we did not have any reasonable evidence that the values should be excluded from the data set (61.9±17.5nmol/mg with outliers; 54.4±8.6 nmol/mg without outliers; Mean±SD). Even if the outliers were excluded, the soleus glycogen would have been higher than the EDL values (34.4±8.0 nmol/mg), and both were higher than the values reported in the cited references.

We do not have the original lysates used to determine the glycogen values from the original study and re-performing all of the assays would necessitate re-performing the entire study. However, in the interest of ensuring that our assay methods were sound, we did attempt to assess the reproducibility of the assay. For this quality control (QC) assay, we performed additional glycogen measurements in muscles isolated from BALB/c mice. However, the mice that we had available were younger and of the opposite sex than those used in the study, therefore it is difficult to rule out contributions of biological variability from the reproduced assays. We were able to match the circadian timing for sacrifice, but not seasonal timing (Summer for study vs. winter for QC). However, we provide the data for comparison below. Though the higher glycogen values in the soleus observed in the study were not reproduced, we interpret the consistent values for the tibialis anterior muscles to indicate that the assay is sound and reproducible, and that the previously observed discrepancy was likely derived from biological variation rather than technical variation. Some of the difference in values between study and QC values may also be attributable to large differences in muscle size between the two groups. In both cases the samples were run on the same plate, in triplicate, with R2 of the standard curve ≥.99.

Muscle Sample size (N) Mean Glycogen (nmol/mg tissue wet weight) Standard deviation

Tibialis Anterior (study) 18 38.7 6.7

Tibialis Anterior (QC Assay) 6 32.3 2.6

EDL (study) 17 34.4 8.0

EDL (QC) 6 23.1 2.4

Soleus (Study) 10 61.9 17.5

Soleus (QC) 6 27.3 6.7

3. “The authors did not acknowledge that their results of 80% greater glycogen in soleus compared to EDL are at odds with the published literature, and they offered no data that supported their discrepant results”

We have added additional discussion to the manuscript to fully acknowledge the discrepancy and to try to explain potential sources of variability in the data. Lines 436-455 “Overnight fasting in rodents results in more dramatic metabolic effects than human overnight fasting, but induces experimentally reproduceable reductions in systemic carbohydrate stores that are similar to more extreme physiological conditions such as hyperinsulinemia, hypoglycemia, or post exercise recovery(1–4). Fasting was used in this study because it is independent of the confounding effects of exercise or contraction induced fatigue(5). Notably, the glycogen values observed in this study differ from several previous reports in that fed state control values for both muscles are relatively high, and that the soleus glycogen levels are substantially higher than the EDL (values between muscles did not differ in the previous reports)(6–8).

Muscle glycogen concentration is a physiologically dynamic parameter that is influenced by experimental conditions such as assay method and normalization factor, as well as biological conditions such as parental genetic background and metabolic state (9–11). Though we cannot directly account for specific confounders that explain the discrepancy in this study, there are two likely candidates that should be considered for further investigation: 1.) Muscle glycogen levels have been shown to vary with season and diurnal cycle in mice(12) and rats(13,14), with peaks in the dark-light cycle transition period (the time at which animals were sacrificed in this study). 2.) Soleus muscles have been shown to be more sensitive to insulin stimulated glucose uptake and glycogen synthesis in both mice(6) and rats(15), and insulin stimulated glucoregulatory responses have been shown to differ among inbred mouse strains(16). Taken together, the described findings support the possibility that stored muscle glycogen values may have been influenced by seasonal, circadian, or hormonal variation intrinsic the genetic background of the mice used in this study”.

Additional Lines 486-498 “In this study, we observed that Soleus muscles stored more glycogen at baseline, had greater specific force/work capacities, and produced absolute force for a longer period during hypoxia compared to EDL muscles. The observations regarding greater glycogen content in the soleus muscle compared to EDL muscles are not consistent with previous reports(7,8,17), but the observations of improved mechanical function during hypoxia in soleus compared to EDL muscles have been previously reported using small muscles isolated from rats(18). Though the absolute differences in glycogen concentrations between groups were larger in the soleus compared to the EDL, the response coefficient (RGlyc) which facilitates interpretation of group differences relative to their baseline concentration, indicated that the patterns of utilization were not different between the two types of muscles. We interpret these findings to mean that the greater basal glycogen concentration observed in the soleus muscles was likely not the primary factor underlying it’s enhanced ischemic mechanical performance“.

4. “The authors state that other studies have reported even higher values, but they don’t point out which studies reported higher values”

a. We apologize for any confusion regarding our interpretation of the additional references provided in the previous submission. Our intent was to show that the measured values fell within a reasonable range of values relative to previous reports (i.e. that the values were physically possible). We have been careful to ensure that any specific references to published data are clearly linked to relevant text (see above paragraphs and additional references). Additionally, we have removed any references that do not directly reference the EDL and/or Soleus muscles.

5. “Citations that do not include mouse soleus and or EDL glycogen concentrations should not be cited to support the statement in the manuscript.”

a. We have removed two of the cited references that did not specifically assess glycogen content in mouse EDL and/or soleus muscles.

6. “If there is any specific evidence that the genetic or physiological state of the mice, or the particular assay used in this study led to much increased soleus glycogen values, it should be specifically identified rather than this general statement without any direct support of its validity.”

a. As discussed, the discrepancies in the measured values are difficult to account for directly. However, there are a few candidate sources of variation that have been more thoroughly discussed in the manuscript in an attempt to account for the differences. These include: references describing seasonal and diurnal variation in muscle glycogen content in mice and rats. Diurnal variation in muscle glycogen typically peaks at the end of the dark cycle/beginning of the light cycle, when the animals in this study were sacrificed. Thus, we think it reasonable to suggest that this may be a confounding variable here. Additionally, soleus muscles in mice and rats have been shown to be more sensitive to insulin stimulated glucose uptake.

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PLoS One. doi: 10.1371/journal.pone.0225922.r005

Decision Letter 2

Cameron J Mitchell

2 Apr 2020

PONE-D-19-31637R2

Effects of fasting on isolated murine skeletal muscle contractile function during acute hypoxia.

PLOS ONE

Dear Dr. McClung,

We would appreciate receiving your revised manuscript by May 17 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Before the manuscript can be published please address the minor comments raised by reviewer 2.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #2: Because circadian and seasonal effects are described as important for glycogen values, please specify for each experiment (if is the same for every experiment, that can be noted and it can be stated only once):

1) The times in the day when lights were turned on and turned off.

2) The times in the day when samples were collected.

3) The month(s) when experiments were performed,

**********

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Reviewer #2: No

PLoS One. 2020 Apr 23;15(4):e0225922. doi: 10.1371/journal.pone.0225922.r006

Author response to Decision Letter 2

3 Apr 2020

1) The times in the day when lights were turned on and turned off.

2) The times in the day when samples were collected.

3) The month(s) when experiments were performed

We thank the reviewer for their continued time and effort. We appreciate the constructive nature of the feedback and feel that the manuscript has been substantially improved by our exchanges. To address the outlined concerns, we have added additional details to the methods section:

Lines 141-149 “Mice were housed in a temperature-controlled facility on a 12-hour light-dark cycle with free access to food and water prior to fasting (dark cycle: began at 1900 hours, ended at 0700 hours). Mice were fasted for 24 hours to achieve a reduction of skeletal muscle glycogen of ~50%, compared to the fed state. The 24-hour fasting period started at the beginning of a light cycle (0700 hours) and was terminated at the end of the subsequent dark cycle (0700 hours). Mice had free access to water during fasting. All muscles (including control and fasted groups) were isolated for experiments immediately following the end of the dark cycle, between 0700 and 0800 hours. All experiments were performed in the summer season, between the months of May and August”.

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PLoS One. doi: 10.1371/journal.pone.0225922.r007

Decision Letter 3

Cameron J Mitchell

6 Apr 2020

Effects of fasting on isolated murine skeletal muscle contractile function during acute hypoxia.

PONE-D-19-31637R3

Dear Dr. McClung,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Cameron J. Mitchell, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0225922.r008

Acceptance letter

Cameron J Mitchell

10 Apr 2020

PONE-D-19-31637R3

Effects of fasting on isolated murine skeletal muscle contractile function during acute hypoxia.

Dear Dr. McClung:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Cameron J. Mitchell

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

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Data Availability Statement

All relevant data are available at DOI 10.17605/OSF.IO/PZT5G.

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PERMALINK

Effects of fasting on isolated murine skeletal muscle contractile function during acute hypoxia

Cameron A Schmidt

Emma J Goldberg

Tom D Green

Reema R Karnekar

Jeffrey J Brault

Spencer G Miller

Adam J Amorese

Dean J Yamaguchi

Espen E Spangenburg

Joseph M McClung

Roles

Abstract

Introduction

Materials and methods

Animals

Laser scanning confocal microscopy

Dystrophin/laminin immunofluorescence in transverse muscle sections

Fasting

Measurement of muscle mechanical function

Measurement of glycogen content in whole tissue

Ultra-Performance Liquid Chromatography (UPLC) measurements of adenosine nucleotides in whole tissue

Statistical analysis

Results

Fig 1. Experimental design.

Table 1. Basal tissue glycogen concentrations in the liver and skeletal muscle of fed Vs. fasted groups.

Fig 2. Effects of carbohydrate depletion on excitation-contraction coupling and force/work capacities.

Fig 3. Effects of carbohydrate depletion on the timing of functional impairment during hypoxia and nutrient deprivation (HND).

Table 2. Tissue glycogen concentrations in EDL and soleus muscles of fed V. fasted mice following O2 or N2 protocols.

Fig 4. Adenosine nucleotide profiles of fed/fasted EDL and soleus muscles at baseline and following 180 min. O2 or N2 protocols.

Fig 5. Qualitative assessment of structural integrity of the muscles following experimental protocols.

Discussion

Conclusion

Data Availability

Funding Statement

References

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Cameron J Mitchell

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Author response to Decision Letter 0

Decision Letter 1

Cameron J Mitchell

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Author response to Decision Letter 1

Decision Letter 2

Cameron J Mitchell

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Author response to Decision Letter 2

Decision Letter 3

Cameron J Mitchell

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Acceptance letter

Cameron J Mitchell

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Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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Table 2. Tissue glycogen concentrations in EDL and soleus muscles of fed V. fasted mice following O₂ or N₂ protocols.

Fig 4. Adenosine nucleotide profiles of fed/fasted EDL and soleus muscles at baseline and following 180 min. O₂ or N₂ protocols.