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

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 ischemia is not known. 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) under conditions of reduced carbohydrate supply. 180 mins of acute ischemia 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. Restriction of the available intracellular carbohydrate pool by fasting (~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 did cause rapid development of passive tension in both muscle types, which may have implications for optimal timing of reperfusion or administration of precision therapeutics.

3 49 Introduction 50 Ischemic skeletal muscle necrosis occurs concurrently with several common clinical 51 conditions (e.g. peripheral arterial disease, compartment syndrome, or diabetic necrosis) 52 and is a complicating factor of successful muscle graft transplantation (1)(2)(3). The severity 53 of necrosis during an ischemic episode has long been considered a sole function of time, 54 temperature, and magnitude of the hypoxic insult(4,5). However, the timing of the events 55 that precede irreversible functional impairment and necrosis during ischemia may also 56 depend on other key variables including: metabolic rate; contractile efficiency; and the size 57 of the stored carbohydrate pool(4). Carbohydrate metabolism is key, as muscle energy 58 supply becomes dependent on anaerobic fermentation of stored carbohydrate sources 59 during ischemia(6-8). Glycogen is the primary storage form of carbohydrate in skeletal 60 muscle, and its storage/utilization can be influenced by acute environmental factors as 61 well as chronic diseases(9-14).  73 In a previous study, using an in vivo mouse hindlimb ischemia model (without reperfusion), 74 we found that myonecrosis develops between three and six hours after the onset of 4 75 ischemia and is accompanied by a complete loss of contractile function (19). This led us 76 to examine the <3-hour time domain in this study to better characterize the exact temporal 77 nature of muscle functional impairments and metabolite changes that occur under 78 ischemic conditions. We hypothesized that reductions in stored muscle glycogen would 79 significantly shorten the amount of time that the muscles could remain functional during 80 ischemia. To test this hypothesis, we utilized fasting to induce an approximate 50% 81 decrease in resting muscle glycogen and employed a carefully controlled experimental 82 system to assess the effects of carbohydrate depletion on isolated mouse hindlimb muscle 83 function during severe hypoxia and nutrient deprivation. Our data provide a novel 84 characterization of ischemic muscle mechanical/energetic failure and paint a detailed 85 picture of the timing of these impairments. This information will provide a valuable 86 resource to be used in conjunction with studies of ischemia/reperfusion in mouse hindlimb 87 ischemia models.

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St. Louis, MO) and was excited using the 405nm line of a multiline argon laser; emission 117 was filtered using a 490nm dichroic mirror and 430-470nm barrier filter. BODIPY and 118 AF488-phalloidin were excited using the 488nm line of a multiline argon laser; emission 119 was filtered using a 560nm dichroic mirror and 505-540nm barrier filter. Dylight 594 (GS-120 IB 4 ) was excited using a 559nm laser diode; emission was filtered using a 575-675nm 121 barrier filter. Zero detector offset was used for all images. The pinhole aperture diameter 122 was set to 105um (1 Airy disc). NAD(P)H autofluorescence has been shown to be highly 123 localized to skeletal muscle mitochondria(21). NAD(P)H autofluorescence was excited 124 using a mode locked pulsed laser (Mai Tai, Spectra Physics, Santa Clara, CA) tuned to 125 720nm. Emission was collected using separate non-descanned detectors.

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Unpaired two-tailed t-tests were used for between group comparisons. For comparison of 246 means, p values of < 0.05 were considered statistically significant.

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Extensor digitorum longus (EDL) and soleus muscles were chosen for their known 250 differences in thermodynamic efficiency(28). The muscles also characteristically rely on 251 different modes of energy metabolism (glycolytic and oxidative metabolism 252 respectively)(29). The specialized nature of each muscle is highlighted for illustrative 253 purposes by whole mount imaging (Fig 1), contrasting the dramatically different

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Fasting had no effect on the isometric force-frequency relationship at baseline or under 287 any of the tested conditions in the EDL (Fig 2A) or soleus (Fig 2B), indicating reduced 288 carbohydrate pool size did not alter excitation-contraction coupling. Specific force values 289 for both muscles were consistent with those obtained previously(24). Additionally, we 290 observed characteristic reductions in maximal specific force following the O 2 protocols 291 (and completely impaired force production following the N 2 protocols) in both muscles ( Fig   292   2A,B). Notably, the isometric force capacity during each protocol did not differ between 293 the fed and fasted groups in either the EDL (Fig 2C) or the soleus (Fig 2D). Similarly, the 294 work capacity over the course of the protocols did not differ for either muscle between the 295 fed and fasted states (Fig 2E,F). As expected, the force and work capacities were greatly

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Given that no substantial differences in force or work capacities were observed, we next protocol for the EDL (Fig 3A) and soleus (Fig 3B). This measurement represents the 313 ability of the muscle to perform sustained non-shortening contractions. Additionally, the 314 length-time integral of the isokinetic portion of each contraction was also plotted against 315 the number of contractions for the EDL (Fig 3C) and soleus (Fig 3D). This measurement

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( Fig 3E) and soleus (Fig 3F). This measurement represents stiffening of the muscle, which 14 324 may be due to several possible factors, including impaired calcium reuptake or cellular 325 swelling due to uncontrolled fluid uptake(31). None of the muscles experienced substantial 326 changes in passive tension during the O 2 protocol. Large increases in passive tension 327 occurred in both muscles under N 2 conditions. Interestingly, passive tension development 328 occurred earlier in the fasted groups (Fig 3E,F). To account for the possibility that the 329 muscles were accumulating excessive water, the wet weights of the EDL (Fig 3G) and 330 soleus (Fig 3H) were plotted. No differences in wet weight between the fed and fasted 331 states were observed in either muscle, and all the tested muscles accumulated additional 332 weight following the N 2 protocol.

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TAN pool decreased slightly in both EDL (Fig 4C) and soleus (Fig 4D) muscles compared 368 to our reported baseline values (Fig 4 A,B), but did not differ between fed and fasted 369 groups. Following N 2 protocols, there were large decreases in the TAN pool in both the 370 EDL (Fig 4E) and soleus (Fig 4F), with accompanying increases in the tissue IMP 371 concentrations (Fig 4G,H). However, no substantial differences were observed between 372 the fed and fasted groups for either muscle type.

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Previous reports have indicated that dystrophin IF staining is rapidly reduced in skeletal 384 and cardiac muscle during early myonecrosis(19,32). Immunofluorescent staining for the 385 sarcolemmal protein dystrophin and the extracellular matrix protein laminin was performed 386 on a subset of transverse sectioned muscles to assess the possibility that muscles were 387 incurring damage during the contraction protocols. No apparent changes were observed 388 in the EDL (Fig 5A) or soleus (Fig 5C)  the N 2 protocol in the EDL (Fig 5B) and soleus (Fig 5D). Together these assessments did

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Investigating the key factors that affect the timing of muscle energetic failure during 479 ischemia will aid in identifying optimal windows for therapeutic intervention. We predicted 480 that the amount of stored carbohydrate is one such factor, as it is a major contributor to 481 anaerobic energy metabolism and is influenced by several physiologically relevant 482 conditions. We conclude that mouse hindlimb muscles maintain a large pool of stored