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Characterization and utilization of the flexor digitorum brevis for assessing skeletal muscle function

Characterization and utilization of the flexor digitorum brevis for assessing skeletal muscle... Background: The ability to assess skeletal muscle function and delineate regulatory mechanisms is essential to uncovering therapeutic approaches that preserve functional independence in a disease state. Skeletal muscle provides distinct experimental challenges due to inherent differences across muscle groups, including fiber type and size that may limit experimental approaches. The flexor digitorum brevis (FDB) possesses numerous properties that offer the investigator a high degree of experimental flexibility to address specific hypotheses. To date, surprisingly few studies have taken advantage of the FDB to investigate mechanisms regulating skeletal muscle function. The purpose of this study was to characterize and experimentally demonstrate the value of the FDB muscle for scientific investigations. Methods: First, we characterized the FDB phenotype and provide reference comparisons to skeletal muscles commonly used in the field. We developed approaches allowing for experimental assessment of force production, in vitro and in vivo microscopy, and mitochondrial respiration to demonstrate the versatility of the FDB. As proof-of principle, we performed experiments to alter force production or mitochondrial respiration to validate the flexibility the FDB affords the investigator. Results: The FDB is made up of small predominantly type IIa and IIx fibers that collectively produce less peak isometric force than the extensor digitorum longus (EDL) or soleus muscles, but demonstrates a greater fatigue resistance than the EDL. Unlike the other muscles, inherent properties of the FDB muscle make it amenable to multiple in vitro- and in vivo-based microscopy methods. Due to its anatomical location, the FDB can be used in cardiotoxin-induced muscle injury protocols and is amenable to electroporation of cDNA with a high degree of efficiency allowing for an effective means of genetic manipulation. Using a novel approach, we also demonstrate methods for assessing mitochondrial respiration in the FDB, which are comparable to the commonly used gastrocnemius muscle. As proof of principle, short-term overexpression of Pgc1α in the FDB increased mitochondrial respiration rates. Conclusion: The results highlight the experimental flexibility afforded the investigator by using the FDB muscle to assess mechanisms that regulate skeletal muscle function. Keywords: cDNA electroporation, Mitochondrial respiration, Muscle stimulation, Skeletal muscle function * Correspondence: Spangenburge14@ecu.edu Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27834, USA © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tarpey et al. Skeletal Muscle (2018) 8:14 Page 2 of 15 Background investigators have generally limited use of the FDB to Skeletal muscle is susceptible to a number of genetic, envir- isolated fiber imaging-based approaches or satellite cell onmental, and age-related pathologies that impair the tis- function in culture [10–13]. In this study, we demon- sue’s normal mechanical and metabolic function. This often strate the utility of the FDB as a model for assessing leads to the development of comorbidities and sometimes skeletal muscle function across a range of methodologies death. Defining the mechanisms that regulate the develop- commonly used within the skeletal muscle research ment of skeletal muscle dysfunction is critical for designing community, including isometric force production, in therapeutic interventions. Investigators currently employ a vitro and in vivo imaging, and mitochondrial respiration. variety of established methods for answering such ques- The full utility of the FDB is best demonstrated in stud- tions, but are often experimentally hampered by unique in- ies combining these physiological tests with genetic ma- herent heterogeneity between muscle groups and cells nipulation (cDNA electroporation), thus allowing the within the same muscle tissue. Muscles commonly used for investigator to manipulate cellular protein levels effi- functional and mechanistic experiments include the exten- ciently in a setting that allows for accurate functional sor digitorum longus (EDL), soleus, plantaris, gastrocne- outcome analyses. mius, tibialis anterior (TA), and/or the quadriceps. These muscles each offer unique advantages across a host of Methods methodologies including measuring isometric force pro- Animals duction, susceptibility to muscle injury, mitochondrial res- All mice were 3–11-month-old C57BL/6 males at the time piration, protein content, and histology. For example, the of testing. Mice were purchased from Jackson laboratories, EDL is frequently used for measures of isometric force pro- housed in a temperature (22 °C) and light-controlled facil- duction or susceptibility to stretch-induced injury in order ity, and given free access to food and water. Mice were eu- to better understand and assess the efficacy of interventions thanized via isoflurane overdose. All animal procedures targeting Duchenne muscular dystrophy [1–3] and/or aging and usage were approved by the Institutional Review [4–6]. Meanwhile, studies investigating metabolic diseases Committee at East Carolina University. Animal care com- such as obesity and type II diabetes commonly measure plied with the Guide for the Care and Use of Laboratory skeletal muscle mitochondrial respiration using the red por- Animals, Institute of Laboratory Animal Resources, tion of the gastrocnemius muscle [7–9]. However, due to Commission on Life Sciences, National Research Council inherent differences across muscles, investigators are often (Washington: National Academy Press, 1996). forced to apply specific experimental approaches for each muscle. This specificity can limit the broad interpretation FDB dissection of the results. Critical to any procedure utilizing the FDB is careful dissec- Mechanistically driven research often utilizes DNA tion that prevents damage to the muscle (the muscle is sur- manipulation to alter protein expression in skeletal roundedbyanareaof dense connective tissue)(seeFig. 1). muscle. The electroporation of cDNA or shRNA into muscles often delivers inconsistent results due to both the size of the muscle and anatomical location, which each impede uniform distribution of cDNA. These fac- tors can lead to low transduction efficiencies, making physiological and biochemical assays unreliable. To over- come this technical limitation, investigators have tagged their gene of interest and used immunohistochemical procedures to determine changes in tagged fibers that are known to express their gene of interest. Unfortu- nately, this approach limits the assays that can be employed and rules out most functional assays. While transgenic models have proven a valuable resource, they remain a costly, time-consuming, and uncertain en- deavor. In contrast, the flexor digitorum brevis's (FDB) unique anatomical location coupled with its size makes the muscle amenable to cDNA electroporation allowing for high transduction efficiencies. Fig. 1 FDB dissection. A FDB prepared for dissection, displaying the The FDB is a skeletal muscle located in the base of the proximal tendon and tendons of the toes. B Increased magnification foot that has previously been used to isolate and culture of A with muscle outlined in black to define borders of muscle single muscle fibers. For reasons that are unclear, Tarpey et al. Skeletal Muscle (2018) 8:14 Page 3 of 15 To facilitate dissection, the toes were pinned to a cork- was used to normalize all measured myofiber lengths board, securing the foot in place with the sole of the foot fa- to optimal sarcomere length. cing upward. Next, the skin at the proximal end of the foot (above the calcaneus) was pinched allowing micro-scissors Individual fiber type and fiber size to make incisions along the lateral edges of the foot down Muscle fiber type analysis of the FDB was conducted on to the toes. Still pinching the skin above the calcaneus, samples from C57BL/6 mice, as previously described micro-scissors were used to separate the skin from the [16]. Sections were probed with primary antibodies underlying musculature. The remaining skin flap was against myosin heavy chain type I (BA-F8), IIa (SC-71), peeled back and removed to expose the FDB and tendons IIb (BF-F3) (Development Studies Hybridoma Bank, of the toes. The proximal tendon was then cut, and while Univ of Iowa), and anti-dystrophin (Rb-9024, Thermo holding the tendon with forceps, the FDB was cut away Fisher, Waltham, MA), then imaged using an EVOS FL from the underlying fascia. The toe tendons were then cut auto microscope and accompanying software (Life to free the FDB from the foot. Technologies, Bothell, WA). Fiber type and fiber cross- sectional area (CSA) were assessed using ImageJ, as Myofiber isolation previously described [16]. After dissection of the FDB, the muscles were placed in fresh culture media (DMEM with glutamine, 2% sterile- Isometric force production filtered FBS, 0.1% gentamycin) supplemented with 4 mg/ Isometric force production and fatigue was assessed in ml collagenase A (Roche – 11088793001) for 90– EDL (n = 5), soleus (n = 4), and FDB (n = 6) muscles of 120 min at 37 °C in 5% CO as previously described C57BL/6 mice, as previously described [17] with slight [14]. The FDB muscle was placed in 2 mL of culture modifications. The FDB was exposed and the proximal media without collagenase and gently triturated against tendon was secured using silk suture. Fine-tip forceps the wall of the dish to release the fibers from the bundle were placed under the three medial toe tendons and using the cut end of a P1000 pipette tip. Isolated myofi- pulled gently down toward the toes before the three ten- bers were adhered to glass bottom dishes that were dons (Fig. 1) were secured with silk suture. We tied the coated with entactin-collagen-laminin (ECL Cell attach- three medial tendons because it is not possible to tie one ment matrix, #08110 Millipore). Fibers were returned to of the toes due to anatomical location, and tying the 37 °C in 5% CO for several hours and subsequently fourth tendon does not, in our experience, result in a dif- imaged as described below. ferent absolute force (data not shown). Each tendon was then cut just above the knot, and the FDB was gently lifted FDB muscle fiber length away from the foot. Micro-scissors were used to remove The FDB muscles of male and female C57BL/6 mice were any remaining connective tissue, releasing it from the foot. tied at the proximal tendon and three medial toe tendons The muscle was then tied to a force transducer and sus- with silk suture and fastened to a metal clip to maintain pended in oxygenated Krebs Ringer Buffer (KRB—[mM] resting tension. Myofibers were isolated as described 115 NaCl, 2.5 KCl, 1.8 CaCl ,2.2 Na HPO ,0.85 2 2 4 above, but were not adhered to glass bottom plates. NaH PO ) at room temperature. The muscle length was 2 4 Myofibers were imaged using a ×4 objective and an EVOS then adjusted until the FDB produced a peak twitch force, XL core microscope and accompanying software (Life at which point the optimal resting tension (Lo) was set Technologies, Bothell, WA). The lengths of approximately and the muscle was allowed to equilibrate for 10 min. So- 1000 myofibers were measured in ImageJ (version 1.6.0, leus muscles were prepared by tying a double square knot NIH, Bethesda, MD). When isolated, the myofibers were at the distal soleus tendon. The tendon was cut above the no longer at tension and therefore did not represent opti- knot and the posterior muscles were gently pulled away mal length. To account for this change in myofiber length, from the leg revealing the proximal soleus tendon, which a conversion factor was utilized using sarcomere length, was tied with a double square knot. The EDL was dis- as previously described by Dr. Richard Lieber [15]. When sected and tied as previously described [17]. EDL and so- at optimal length, the assumed mouse muscle optimal leus muscles were equilibrated in oxygenated room sarcomere length of a myofiber is 2.5 μmacross [15]. To temperature KRB at resting tension for 10 min. Following normalize myofiber lengths to sarcomere length, we equilibration, muscle tension was optimized by perform- measured sarcomere length in a subset of 30 myofibers. ing maximal twitch stimulations and adjusting the muscle The distance between 10 sarcomeres of each myofiber was length until peak force was achieved. Twitch stimulations measured, and an average sarcomere length was calculated were performed 30 s apart to avoid fatiguing the muscle. across all 30 myofibers. The optimal sarcomere length Muscles were then stimulated 60 s apart at 10, 20, 40, 60, (2.5 μm) divided by the average measured sarcomere 80, 100, and 120 Hz to generate a force frequency curve. length produced a conversion factor of 1.14, which Muscles were rested for an additional 1 min before Tarpey et al. Skeletal Muscle (2018) 8:14 Page 4 of 15 completing a 10-min stimulation protocol to determine for sectioning and H&E staining, as previously described fatigue resistance. The fatigue protocol stimulated muscles [16]. at 30 Hz every 2 s for a period of 600 s for a total of 300 contractions. Optimal muscle length was recorded and Skeletal muscle high-resolution mitochondrial muscles were blotted to remove excess KRB before being respirometry weighed. An optimum voltage of 20 V was established Preparation of permeabilized gastrocnemius and FDB prior to the experiments to ensure maximal stimulation of muscle fiber bundles the FDB, EDL, and soleus (data not shown). Absolute Respirometry was conducted on isolated permeabilized muscle force data were converted to specific force (N/ gastrocnemius and FDB muscle bundles excised from cm ) using previously described equations for the the same limb of C57BL/6 mice (n = 4). A portion of the mathematical estimation of muscle CSA [18]and red gastrocnemius was dissected and used for the prep- physiological cross-sectional area (PCSA) [19]. The pri- aration of permeabilized fiber bundles, as previously de- mary difference between CSA and PCSA is the inclusion scribed [21]. Red gastrocnemius muscle was used as the of the muscle fiber length to muscle length ratio in the comparative tissue as it is commonly used to assess PCSA equation. We used both corrected methods to pro- murine skeletal muscle mitochondrial respiration [8, 21]. vide a wider compatibility with the literature. The protocol for preparing permeabilized FDB muscle fiber bundles was adapted from previously described Passive contractile properties methods on the permeabilization of red gastrocnemius Passive contractile properties were assessed in EDL and muscle bundles [21] and is outlined below. The FDBs FDB muscles from C57BL/6 male mice (n = 4), as previ- were dissected and immediately added to ice-cold buffer ously described [20], with slight modifications. The EDL X ([mM]—7.23 K EGTA, 2.77 CaK EGTA, 20 imidazole, 2 2 and FDB muscles dissected and tied to a force trans- 20 taurine, 5.7 ATP, 14.3 phosphocreatine, 6.56 ducer as described above. Muscles were equilibrated for MgCl ·6H O, 50 MES, pH 7.1, 295 mosmol/kgH O). 2 2 2 10 min in oxygenated KRB at room temperature. Fol- Using a dissecting microscope, connective tissue, fat, lowing equilibration, muscle tension was optimized by and blood vessels were removed carefully to avoid performing maximal twitch stimulations and adjusting muscle loss. FDBs were cut into bundles and divided the muscle length until peak force was achieved. Twitch into groups of three to four bundles weighing 1.5–2. stimulations were performed 30 s apart to avoid fa- 0 mg wet weight. Bundle groups were then perme- tiguing the muscle. The muscle reference length was abilized in buffer X containing 22.5 μg/ml saponin with measured as the Lo before undergoing a passive stretch continuous rotation at 4 °C for 5 min. Muscle bundles of 105, 110, 115, 120, 125, and 130% of Lo. Muscles were were promptly transferred to ice-cold buffer Z ([mM]— blotted to remove excess KRB and then weighed. Data 110 K-MES, 35 KCl, 1 EGTA, 5 K HPO ,3 2 4 were corrected to CSA and PCSA. MgCl ·6H O, 5 mg/ml BSA, pH 7.4, 295 mosmol/ 2 2 kgH O) and washed with continuous rotation at 4 °C for Cardiotoxin (CTX) injury 15 min. Comparisons were made in C57BL/6 mice between a CTX-treated FDB (Naja nigricollis, #02152238, MP Bio- Mitochondrial respiration medicals, Santa Ana, CA) and PBS-treated contralateral Measurements of high-resolution O consumption were FDB 4 days (n = 4) and 10 days post-treatment (n = 2). made using the OROBOROS Oxygraph-2K (Oroboros Sterile 8-mm-long 31G syringes were prepared with Instruments, Innsbruck, Austria) at 37 °C with a starting 10 μLof 10 μM CTX or 10 μL of sterile 1× PBS, as pre- oxygen concentration of ~ 300–350 μM as previously viously described [16]. Following isoflurane-induced described [22]. Experiments were conducted in buffer Z anesthesia, the base of the feet of four mice was cleaned containing 20 mM creatine monohydrate and 25 μM with alcohol wipes. CTX was injected at the proximal blebbistatin. Mitochondrial respiration was assessed by portion of the foot with the needle positioned under the the sequential addition of substrates at a final concentra- skin and toward the toes. PBS was injected into the tion of pyruvate 4 mM, malate 0.5 mM, glutamate contralateral foot. Mice were sacrificed at the appropri- 5 mM, ADP 2.5 mM, succinate 5 mM, cytochrome c ate time, and FDBs were dissected for the measurement 5 μM, rotenone 10 μM, antimycin A 5 μM, ascorbic acid of force production, as described above. Data were cor- 2 mM, and TMPD 0.5 mM (N,N,N′,N′-tetramethyl-p- rected to PCSA. phenylenediamine dihydrochloride). Integrity of the mitochondrial membrane was confirmed by excluding Hematoxylin and eosin staining gastrocnemius muscle bundles and FDB muscle bundles FDB muscles subjected to 10 days CTX treatment were that produced a > 10 or > 20% increase in respiration, re- flash frozen in optimal cutting temperature (OCT) solution spectively, following exogenous cytochrome c addition. Tarpey et al. Skeletal Muscle (2018) 8:14 Page 5 of 15 A different exclusion criteria was used for the gastrocne- NA 1.35) and an Olympus FV1000 LSM operating mius and FDB muscle bundles as preliminary testing in- FV10-ASW 4.2 acquisition software. dicated a greater percent increase in respiration was common in FDB fiber bundles compared to gastrocne- cDNA electroporation and high-resolution respirometry of mius fiber bundles, following the addition of cytochrome skeletal muscle overexpressing Pgc1α c. Upon completion of the protocol, muscle bundles Electroporation of cDNA into FDBs was performed as were rinsed in distilled H O, freeze-dried (Labconco, previously described [25]. Briefly, the feet of seven Kansas City, MO), and weighed (Orion Cahn C-35, C57BL/6 male and female anesthetized mice were Thermo Electron, Beverly, MA). Respiration rates for in- cleaned with an alcohol wipe and the footpads were tact gastrocnemius muscle bundles are commonly cor- injected with 10 μl of 2 mg/mL hyaluronidase suspended rected to dry weight; however, due to differences in in sterile-filtered KRB using an 8-mm-long 31 gauge connective tissue content of the two muscle groups, JO sterile needle. Approximately 1 h, later mice underwent values were also corrected to total protein and citrate anesthesia for a second time. Feet were again cleaned synthase (CS) activity. CS activity was measured using with an alcohol wipe and one foot received 30 μgof kit CS0720. Chemicals and reagents were purchased green fluorescent protein (GFP)-tagged PGC1α plasmid from Sigma Aldrich. and the contralateral foot was injected with YFP cDNA. Following a full recovery from anesthesia, mice were Microscopy anesthetized for a third time ~ 10 min later. Platinum In vitro electrodes were inserted under the skin and positioned FDB muscle fibers were isolated from C57BL/6 mice perpendicular to the FDB and parallel to one another at and attached to 35-mm glass bottom dishes coated with the heel and footpad beneath the toes. The FDB was ECL, as previously discussed. Isolated myofibers were stimulated with 20 pulses of 20 ms duration at 1 Hz and stained with mitotracker deep red (M2246, Thermo 100 V [25]. Mice were sacrificed 14 days later, and FDBs Fisher) and NucBlue (R37605, Thermo Fisher) in DMEM were dissected and imaged under a fluorescent micro- for 30 min. Fibers were washed three times with 2 mL scope to confirm plasmid expression. Intact FDB muscle KRB. Fibers were imaged using a single photon confocal samples were then prepared and measured for mito- laser scanning microscope with a ×60 oil immersion ob- chondrial respiration as outline above. jective (Olympus, Plan Apochromat, NA 1.35) and exci- tation was achieved using the 405- and 488-nm lines of Statistics a multiline argon laser. Data distributions were assessed and data that did not conform to a normal distribution were log base 10 trans- In vivo formed. Force frequency contractile data were analyzed Second harmonic generation describes the optical effect via two-way ANOVA with Tukey multiple comparisons. produced from the passage of laser pulses through Muscle mass, fiber type, time to max and half- highly polarized, non-centro-symmetrical materials such relaxation, and fatigue data were analyzed via one-way as myosin. When polarized at the appropriate wave- ANOVA with Tukey multiple comparisons. In cases length, these materials emit light at half the wavelength where data could not be transformed to a normal distri- of that entering, producing high-resolution images with- bution, a Kruskal-Wallis test with Dunn multiple com- out the need for fluorescent probes that are subject to parisons was performed. ANOVAs were completed photobleaching and phototoxicity. Furthermore, the near using GraphPad Prism 7.03. Respiratory data and CS ac- infrared wavelengths used allow for deep tissue penetra- tivity were analyzed via paired two-tailed t-tests with an tion without the need for invasive procedures [23]. alpha level of 0.05 using Microsoft Excel. All data are C57BL/6 mice were anesthetized before having the skin presented as mean ± SEM. covering the FDB removed, exposing the FDB muscle. Once exposed, the FDB was hydrated with sterile KRB Results and the mouse was laid prone on a glass cover slip (#1.5, FDB fiber type and comparative characteristics Leica), as previously described [24] (Fig. 1C). Myosin Phenotypic characterization of the FDB demonstrates it and nicotinamide-containing molecules were excited at is a smaller muscle than the EDL and soleus and dis- 900 and 720 nm using a mode locked Ti:Sapphire pulsed plays a smaller fiber length to muscle length ratio rela- laser (Mai Tai Deep See HP series, Spectra-Physics, tive to the EDL and soleus, as measured by Brooks and Santa Clasa, CA), and emission was recorded using non- Faulkner [19] (Table 1). The FDB myofiber length is descanned detection with a FV10 MRV/G filter set at highly variable (Fig. 2A, B), although an analysis of the 450 and 420 nm, respectively. All images were taken myofiber length frequency indicates a majority of the using a ×60 oil immersion objective (Plan Apochromat, myofibers are approximately 500–700 μm (Fig. 2C). The Tarpey et al. Skeletal Muscle (2018) 8:14 Page 6 of 15 Table 1 Characteristics of the FDB compared to the EDL and soleus muscles FDB EDL Soleus Muscle mass (mg) 6.7 ± 0.4* 11.2 ± 0.7 10.0 ± 0.4 Optimal muscle length (mm) 9.5 ± 0.2* 14.2 ± 0.3 13.4 ± 0.3 Fiber length (mm) 0.744 ± 0.01 5.44 ± 0.12 [19] 7.84 ± 0.22 [19] Fiber to muscle length ratio 0.079 ± 0.002 0.45 ± 0.004 [19] 0.69 ± 0.006 [19] Fiber type % - Type I 4.4 ± 2.9 0 35 - Type IIa 43.9 ± 1.9 3 53 - Type IIx 51.6 ± 4.8 25 11 - Type IIb 0 72 1 [26][26] CSA (μm ± SD) - Type I 488 ± 67 – 943 ± 198 - Type IIa 927 ± 314 634 ± 142‡ 790 ± 149‡ - Type IIx 1267 ± 462 –– - Type IIb – 772 ± 120 – [35][35] Numbers in [brackets] represent the data citation *FDB v.s. EDL; p < 0.05 ‡Figure represents a combination of type IIa and type IIx muscle fibers. Data are mean ± SEM unless otherwise stated FDB exhibits a mixed muscle fiber type comprising of Isometric contractions and force production predominantly type IIa fibers (43.9 ± 1.9%) and type IIx The respective whole muscle length and contractile kin- fibers (51.6 ± 4.8%) with a small population of type I etics of the FDB, EDL, and soleus are demonstrated in fibers (4.4 ± 2.9%) (Fig. 2D). The cross-sectional area Fig. 3A–F. The FDB produces less peak force than either (CSA) of the individual muscle fiber types were all sig- the EDL or soleus muscle (Fig. 4A–C). The comparative nificantly different, with the largest CSA found in type relationship between the FDB, EDL, and soleus specific IIx fibers, followed by IIa and I, respectively (Fig. 2E, F). force is highly dependent on whether force is normalized Fig. 2 FDB fiber size, type, and cross-sectional area (CSA). A, B Example images of single myofibers isolated from FDB muscle. Blue arrows indicate viable myofibers. Red arrows indicate non-viable myofibers. Only viable myofiber lengths were measured. C Relative frequency of FDB muscle fiber lengths. D Fiber type percentage of whole FDB muscles (n =4). E Average individual CSA (μm ) of each muscle fiber type in the FDB. F Representative FDB cross section stained for type I (blue), type IIa (green), type IIx (no stain), type IIb (red), and dystrophin (purple). *, p < 0.05. Data are mean ± SEM Tarpey et al. Skeletal Muscle (2018) 8:14 Page 7 of 15 Fig. 3 Comparison of FDB, EDL, and soleus muscle length, and in vitro isometric contraction profile. Example images of A FDB, B EDL, and C soleus muscle after the tendons were secured with silk suture for subsequent isometric force assessment. Example tetanic (100 Hz) tracings for each muscle: D FDB, E EDL, F soleus to CSA (Fig. 4B) or PCSA (Fig. 4C). The FDB is more fa- CTX-induced muscle injury prevents force production tigue resistant than the EDL and the soleus (Fig. 4D). FDB muscles injected with CTX failed to produce meas- The FDB exhibits differing contractile kinetics compared urable force at 4 days post injection (Fig. 5A, B). to the EDL. Relative to the soleus, the FDB displays a However, force began to recover by 10 days post injec- statistically similar time to max force and half-relaxation tion, although force production still remained impaired time (Fig. 4E, F). relative to contralateral control limbs (Fig. 5C, D). H&E- stained muscle sections showed a greater number of nu- Passive contractile properties clei, which were predominantly centrally located, in FDB Using a previously described protocol [20] with slight muscle injected with CTX, compared to PBS-injected modifications, we assessed the passive mechanical prop- FDB muscle sections, following 10 days of recovery erties of the FDB and EDL muscles. In response to a (Fig. 5E, F). graded increase in muscle length (strain), both the FDB and EDL recorded increasing force (mN/mm )ateach Comparison of mitochondrial respiration between red length up to 125% of Lo. At 130% Lo, both the FDB and gastrocnemius and FDB muscle state 4 EDL did not demonstrate any further increases in force Mitochondrial respiration rates for complex I leak , state3ADP state3ADP output compared to 125% Lo. The relationship between complex I ,complex Iand II ,complex state3ADP the FDB and EDL in response to muscle lengthening II , and complex IV were consistently higher in was substantially altered by the method of force red gastrocnemius muscle compared to FDB muscle normalization. When normalized to CSA, the FDB (Fig. 6A). FDB mitochondrial respiratory data, consistently exhibited significantly more force than the normalized to muscle dry weight, is displayed alone in EDL (Fig. 4G). However, when normalized to PCSA Fig. 6B. Themagnitudeof thedifferencein respiration (accounting for the respective fiber length to muscle between red gastrocnemius and FDB muscle was length ratios), the EDL exhibited significantly more force dependent on the corrective method employed. than the FDB (Fig. 4H). Correcting to muscle dry weight demonstrated the Tarpey et al. Skeletal Muscle (2018) 8:14 Page 8 of 15 Fig. 4 Comparison of in vitro force production characteristics and fatigue resistance. Force frequency curves were generated from FDB (n = 6), 2 2 EDL (n = 5), and soleus muscles (n = 4). A Absolute force (mN). B Specific force (N/cm ) corrected to CSA. C Specific force (N/cm ) corrected to PCSA. D Percent decline in baseline force during a 10-min fatigue resistance protocol. E Time (s) to maximum force. F Half-relaxation time (s). Time to maximum force and half-relaxation times for each muscle were averaged from 100 Hz contractions. F–H Comparison of passive stress lengthening contractions between the FDB and EDL (mN/mm ) corrected to CSA and PCSA, respectively. *, FDB v.s. EDL p < 0.05; †, FDB v.s. soleus p < 0.05; #, EDL v.s. soleus p < 0.05. Data are mean ± SEM greatest relative difference in respiration between the mitochondrial respiration was significantly greater two muscles (Fig. 6A). Meanwhile, correcting across all complexes and conditions when corrected to mitochondrial respiration to total cellular protein muscle dry weight and total protein, while complex I state 4 reduced the relative difference in respiration (Fig. 6C), leak was no longer significant when corrected to CS which was reduced further by correcting mitochondrial activity. CS activity was greater (p = 0.04) in red respiration to CS activity (Fig. 6D). Gastrocnemius gastrocnemius muscle compared to FDB muscle (Fig. 6E), Tarpey et al. Skeletal Muscle (2018) 8:14 Page 9 of 15 Fig. 5 CTX-induced muscle injury. Mice were injected with 10 μLof10 μM CTX in one FDB and 10 μL sterile PBS in the contralateral FDB. Mice were euthanized 4 days (n = 4) or 10 days (n = 2) post-injection. FDBs were dissected and force production was recorded for control and injured muscles using a force-frequency curve protocol. FDBs tested 10 days post-treatment were flash frozen in OCT, sectioned to 12 μm, and stained with H&E before imaging at ×20. A Four days post-injection absolute force (mN). B Four days post-injection specific force (N/cm ) corrected to PCSA. C Ten days post-injection absolute force (mN). D Ten days post-injection specific force (N/cm ) corrected to PCSA. E PBS-treated FDB muscle section stained with H&E. F CTX-treated FDB muscle section stained with H&E. *, p < 0.05. Data are mean ± SEM suggesting that the red gastrocnemius muscle has a higher shows a mouse being prepared for second harmonic mitochondrial content then the FDB muscle. generation microscopy. Our results demonstrate high- resolution label-free images where we were able to In vitro and in vivo microscopy image myosin/collagen and/or NAD(P)H at the single Here we demonstrate the FDB muscle is amenable to cell level while the muscle remained intact (Fig. 7F–K). multiple imaging approaches. First, we took advantage of the susceptibility of the FDB to cDNA electroporation to cDNA electroporation-induced elevation of Pgc1α demonstrate fluorescent imaging approaches using the increases mitochondrial respiration in intact FDB muscle whole FDB muscle (Fig. 7A, B) and single muscle fibers Fluorescent microscopy of whole FDB muscle confirmed isolated from the FDB (Fig. 7C). It is possible to deter- the effective expression of both GFP-tagged Pgc1α in mine localization of tagged proteins with in vitro im- one foot and YFP in the contralateral foot (Fig. 8A, B). aging options using commercially available dyes such as Additionally, we confirmed the successful upregulation DAPI and mitotracker deep red (MTDR) (Fig. 7D). of mitochondrial biogenesis by measuring CS activity. However, it is also possible to demonstrate cellular FDB muscle overexpressing Pgc1α demonstrated signifi- localization when imaging the whole FDB muscle by cantly greater CS activity when compared to FDB using second harmonic generation microscopy. Figure 7E muscle expressing YFP (Fig. 8C). Overexpression of Tarpey et al. Skeletal Muscle (2018) 8:14 Page 10 of 15 Fig. 6 Comparison of mitochondrial respiration between the FDB and red gastrocnemius muscle. Permeabilized FDB and gastrocnemius muscle bundles were prepared for high-resolution respirometry (n = 4). Substrates and inhibitors were added sequentially to measure CI leak and ADP- stimulated CI, CI + II, CII, and CIV mitochondrial respiration. A Comparison of mitochondrial respiration in intact permeabilized red gastrocnemius and FDB muscle bundles corrected to dry weight. B Inset of graph A showing FDB mitochondrial respiration only. C Comparison of mitochondrial respiration in intact permeabilized red gastrocnemius and FDB muscle bundles corrected to total protein content. D Comparison of mitochondrial respiration in intact permeabilized red gastrocnemius and FDB muscle bundles corrected to CS activity. E Comparison of CS activity between red state4 state3ADP state3ADP gastrocnemius and FDB muscle. CI leak, complex I leak ; CI, complex I respiration ; CI + II, complex I and II respiration ; CII, complex II state3ADP respiration ; CIV, super-complex respiration. *, p < 0.05. Data are mean ± SEM Pgc1α significantly increased mitochondrial respiration respiration observed in Pgc1α-treated FDBs was due in the FDB muscle compared to the contralateral FDB solely to increased mitochondrial content (Fig. 8E). electroporated with YFP (Fig. 8D). Specifically, complex I leak respiration (p = 0.002), ADP-stimulated complex I Discussion (p = 0.049), and complex I + II (p = 0.038) respiration was In these experiments, we demonstrate the broad versatil- higher in Pgc1α overexpressing FDB muscle compared ity of the FDB muscle as a potential tool for the assess- to YFP-expressing FDBs. ADP-stimulated complex II ment and investigation of skeletal muscle biology. Our (p = 0.080) respiration as well as complex IV (p = 0.069) findings validate the FDB muscle across a range of meth- respiration were not significantly elevated. Interestingly, odologies and highlight its inherent experimental advan- normalizing oxygen consumption to CS activity largely tages. The location of the FDB, beneath the skin, and abolished the differences between YFP and Pgc1α- relatively small size provide a means to accurately target treated FDBs, thereby confirming that increased and efficiently transfect cDNA for the manipulation of Tarpey et al. Skeletal Muscle (2018) 8:14 Page 11 of 15 Fig. 7 In vitro and In vivo imagining. In vitro imaging techniques were used to demonstrate the expression of YFP protein in whole FDB muscle 7 days post cDNA electroporation, and the retention of YFP post-myofiber isolation. A Whole FDB muscle expressing YFP; images were taken with a 488-nm filter cube and in phase contrast then merged to show transfection efficiency. B Whole FDB muscle YFP negative control; images were taken with a 488-nm filter cube and in phase contrast then merged. C Isolated FDB myofiber demonstrating retention of YFP. FDB myofibers were isolated and stained with mitotracker deep red and DAPI prior to imaging with a single photon confocal LSM and oil immersion ×60 objective. D Representative images of stained isolated FDB myofibers showing mitochondria in red and nuclei in blue. Two-photon excitation fluorescence microscopy and second generation harmonics were employed to generate high-resolution in vivo images of the FDB myosin structure (green) and nicotinamide-containing molecules (red) using an oil immersion ×60 objective. Representative images displayed. E Photograph of mouse being prepared for second harmonic generation microscopy of the FDB. The mouse has been tilted slightly to better show the fixing of the foot to the slide. Images were captured with the mouse in a prone position. F Myosin. G Nicotinamide. H Composite of F and G. I ×3 zoom inset of F. J ×3 zoom inset of G. K Composite of I and J protein expression. Importantly, the FDB can then be that electroporation of cDNA encoding PGC1α signifi- used across a variety of physiological and/or cantly increased mitochondrial respiration. biochemical-based assays. As proof of principle, in the To provide investigators a better understanding of experiments described here, we specifically demonstrate the FDB, we performed a comprehensive phenotypic Tarpey et al. Skeletal Muscle (2018) 8:14 Page 12 of 15 Fig. 8 Electroporation of PGC1α cDNA increased mitochondrial respiration. GFP-conjugated PGC1α and YFP cDNA were electroporated into one FDB and a contralateral control FDB, respectively (n = 7). Mice were euthanized 14 days later and whole FDB muscle was imaged using fluorescent microscopy to confirm protein expression before intact permeabilized FDB muscle bundles were prepared for assessment of mitochondrial respiration between FDBs overexpressing Pgc1α or YFP. A YFP expression in whole FDB 14 days post cDNA electroporation; images were taken with a 488-nm filter cube and in phase contrast then merged to show transfection efficiency. B GFP-conjugated Pgc1α expression in whole FDB 14 days post cDNA electroporation; images were taken with a 488-nm filter cube and in phase contrast then merged to show transfection efficiency. C Comparison of CS activity between YFP and Pgc1α overexpressing FDB muscle bundles. D Overview of mitochondrial respiration across ETC protein complexes between YFP and Pgc1α overexpressing FDB muscle bundles, corrected to muscle dry weight. E Overview of mitochondrial respiration across ETC protein state 4 complexes between YFP and Pgc1α overexpressing FDB muscle bundles, corrected to CS activity. CI leak, complex I leak ;CI, state3ADP state3ADP state3ADP complex I respiration ; CI + II, complex I and II respiration ; CII, complex II respiration ;CIV,super-complex respiration. * p < 0.05. Data are mean ± SEM description compared to the EDL and/or soleus length to muscle length ratios were previously known muscle. A novel aspect of the current study is that to [19]. In the past, and in the absence of a fiber length the knowledge of the authors’ this is the first study to ratio for murine FDB muscle, force has been normal- measure the muscle fiber length to muscle length ra- ized without accounting for muscle fiber lengths [18]. tio in the FDB of mice. This provides an important This method of normalization has been employed in advancement in the ability to accurately compare the other investigations across other muscles such as the force-producing capacity of the FDB with other mus- soleus and EDL. We have therefore normalized our cles, such as the EDL and soleus, for which fiber data using both approaches. However, it may be Tarpey et al. Skeletal Muscle (2018) 8:14 Page 13 of 15 important to account for the fiber length to muscle of fluorescently labeled cDNA and specific dyes or fluor- length ratio as any intervention that could affect ei- ophores, such as MTDR and DAPI, may be used to ther variable would alter force output. Examination of image protein localization. These techniques may be fur- the force frequency curves between FDB, EDL, and ther enhanced through in vivo imaging. We specifically soleus muscles, or the FDB and EDL muscles follow- demonstrate how two-photon confocal microscopy and ing passive stress exemplifies the substantial effect of principles of second harmonic generation allow for the accounting for the muscle fiber to muscle length ra- high-resolution imaging of non-centro-symmetrical ma- tio. Researchers should be mindful of the terials such as muscle myosin, nicotinamide-containing normalization method used when analyzing and inter- molecules, and collagen [23]. The FDB muscle provides preting data, and when comparing to data in the several advantages for using second harmonic generation literature. microscopy. The natural prone posture of the animal We observed an intermediate contractile phenotype in may aid in reducing motion artifact due to breathing. the FDB that quantitatively places it between the EDL and Imaging the FDB in the fashion described is less invasive soleus muscles with respect to contractile kinetics. Not than alternative methods, such as excision of the surprisingly, the contractile kinetics of the FDB mirror the cremaster [32]. The amenable nature of the FDB to fiber type composition, which consists predominantly of electroporation with fluorescently tagged cDNA may types IIx and IIa with a small percentage of type I fibers. also allow for a combination of single- and two-photon When compared to the soleus muscle (type I =35%, IIa = microscopy, generating high-definition protein 53% and IIx = 11%, IIb = 1%) or the EDL (type I = 0%, type localization images. IIa = 3%, IIx = 25% and IIb = 72%) [26], the FDB presents In recent years, there has been a substantial increase as a more mixed phenotype. It is worth noting that the in the utilization of methods assessing mitochondrial re- greater fatigue resistance of the FDB muscle may not be spiratory function, particularly so within the field of the direct result of biochemical differences, but an indirect skeletal muscle-related metabolic disease. We demon- result of a reduced oxygen diffusion distance, resulting strate a novel approach, utilizing high-resolution respi- from the smaller size of the FDB relative to the soleus and rometry to measure mitochondrial respiration in the EDL. In conjunction with the EDL and soleus, the FDB FDB muscle. To validate the use of the FDB, we com- provides an additional muscle in which functional pared mitochondrial respiration rates between red outcomes can be assessed in response to experimental ma- gastrocnemius muscle and the FDB. As anticipated, res- nipulations. Due to the unique anatomical location, the piration rates were higher in red gastrocnemius muscle. FDB is amenable to direct injections of various small mol- This is in part due to differences in mitochondrial con- ecules and/or toxins. As proof of principle, we show that tent as demonstrated by greater CS activity, but also a the loss and recovery of isometric force production within result of the respective tissue preparation protocols. The the FDB following CTX-induced muscle injury is similar magnitude of the difference in respiration rates was to that previously reported in the EDL [27]. Together, dependent on whether O consumption was normalized these data suggest the FDB may be a valuable addition to, to muscle dry weight, total protein, or CS activity. Nor- or substitution for, the EDL and soleus muscles when malizing to dry weight or total protein altered respir- assessing skeletal muscle force production and contractile ation values as the FDB contains a relatively higher kinetics. content of connective tissue compared to the gastrocne- The FDB has a smaller fiber: muscle length ratio rela- mius, thus leading to potential underestimation of FDB tive to the EDL. This ratio makes the FDB amenable to O consumption rates. While adjusting the method of efficient single myofiber isolation, which is more chal- normalization cannot fully account for differences in lenging in the EDL and/or soleus muscles. These single connective tissue content, when rates were normalized myofibers can be cultured and imaged on multiple dif- to CS activity, this possible underestimation was elimi- ferent microscopy platforms. Skeletal muscle microscopy nated. This suggests that strong consideration to is a valuable tool for identifying protein localization [28], normalization must be given prior to starting any study muscle morphology [29], and the dynamic changes in with the FDB. CS activity was selected as an alternative molecules such as H O [10, 30] and calcium [29, 31]. In normalization factor for O consumption rates due to its 2 2 2 vitro imaging of isolated FDB myofibers is well- strong association with mitochondrial content [33]. Fur- established; however, our findings demonstrate that elec- ther normalization methods, such as mitochondrial pro- troporated cDNA tagged with fluorescent proteins is tein complex content, may be used, but interactions retained in myofibers following isolation. This provides between the normalization factor and treatment should numerous possibilities for the manipulation of protein be considered. Investigators should also consider the size expression and examination of subsequent alterations to of the FDB in their study designs as this represents a morphology and function. In addition, the combination limiting factor to the number of protocols and replicates Tarpey et al. Skeletal Muscle (2018) 8:14 Page 14 of 15 that may be tested. Additionally, the FDB is too sensitive Abbreviations cDNA: Complementary DNA; CS: Citrate synthase; CSA: Cross-sectional area; to standard mechanical separation of muscle fibers, so CTX: Cardiotoxin; EDL: Extensor digitorum longus; ETC: Electron transport this should be avoided to protect mitochondrial mem- chain; FDB: Flexor digitorum brevis; GFP: Green fluorescent protein; brane integrity. KRB: Krebs Ringer Buffer; MTDR: Mitotracker deep red; PCSA: Physiological cross-sectional area; Pgc1α: Peroxisome proliferator-activated receptor Finally, as an overall proof of concept that the FDB af- gamma coactivator 1-alpha; YFP: Yellow fluorescent protein fords the investigator a unique flexibility, we overex- pressed GFP-tagged Pgc1α or YFP protein in the FDB Acknowledgements We thank Dr. Jeffery Brault for providing us the cDNA for PGC-1alpha, and for a period of 14 days using cDNA electroporation. A Drs. R. M. Lovering and P.D. Neufer for technical discussions. fluorescence imaging-based approach in combination with a CS activity assay confirmed the successful overex- Funding This study was funded by NIH RO1AR06660 (EES), ADA 1-15-BS-170 (EES), NIH pressing of Pgc1α. The significantly greater complex RO1HL125695 (JMM), and F32HL129632 (TER). LEAK ADP ADP I , complex I , and complex I + II respiration rates in GFP-tagged Pgc1α-injected FDBs demonstrates Availability of data and materials The datasets used and/or analyzed during the current study are available that the electroporation of cDNA into FDBs is an effect- from the corresponding author on reasonable request. ive means of manipulating the phenotype or function of the muscle. Normalizing oxygen consumption rates to Authors’ contributions MDT wrote the manuscript, completed the experimental procedures, and CS activity resulted in no group differences compared to contributed to the experimental design. AJA and NPB completed the dry muscle weight normalization. These data suggest experimental procedures and read drafts of the manuscript. TER contributed that Pgc1α may have a larger influence on CS activity to designing and troubleshooting the described experimental procedures. CAS contributed to designing and troubleshooting the described relative to the protein complexes of the electron trans- experimental procedures. JMM provided intellectual insight and contributed port chain (ETC). More specifically, each complex of the to the project design and critical reading of the manuscript. EES contributed ETC is made up of numerous proteins that are likely not to the experimental analysis and design and provided critical reading revision of manuscript. All authors have read and approved the manuscript. all regulated by Pgc1α suggesting the necessity of add- itional regulators of mitochondrial function [34]. Ethics approval and consent to participate While there are practical advantages to using the EDL, Animal care and experimental procedures were approved by the Institutional Review Committee at East Carolina University and complied with the Guide soleus, or gastrocnemius muscles for assessing skeletal for the Care and Use of Laboratory Animals, Institute of Laboratory Animal muscle isometric force production or mitochondrial res- Resources, Commission on Life Sciences, National Research Council piration, these muscles are not effective targets for (Washington: National Academy Press, 1996). cDNA electroporation due primarily to their size and/or Competing interests anatomical location (which impedes cDNA distribution The authors declare that they have no competing interests. within the muscles). Conducting mechanistic studies into the relationships between protein expression and Publisher’sNote function using such muscles therefore requires the pro- Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. duction of muscle-specific transgenic mice. The FDB provides a time sensitive, non-invasive, and cost- Received: 17 January 2018 Accepted: 3 April 2018 effective method for assessing multiple functional indi- ces within skeletal muscle after deliberately altering pro- References tein expression through cDNA or shRNA delivery. This 1. Lynch GS, Hinkle RT, Chamberlain JS, Brooks SV, Faulkner JA. Force and combined utility provides advantages to investigators power output of fast and slow skeletal muscles from mdx mice 6-28 months old. J Physiol. 2001;535(Pt 2):591–600. https://doi.org/10.1111/J. pursuing mechanistic studies in the field of skeletal 1469-7793.2001.00591.X. muscle biology. 2. Pant M, Sopariwala DH, Bal NC, Lowe J, Delfín DA, Rafael-Fortney J, et al. Metabolic dysfunction and altered mitochondrial dynamics in the utrophin- dystrophin deficient mouse model of Duchenne muscular dystrophy. PLoS One. 2015;10:e0123875. https://doi.org/10.1371/journal.pone.0123875. Conclusions 3. Roy P, Rau F, Ochala J, Messéant J, Fraysse B, Lainé J, et al. Dystrophin The use of the FDB represents an expanded tool set to restoration therapy improves both the reduced excitability and the force drop induced by lengthening contractions in dystrophic mdx skeletal the mechanism-focused investigator. We demonstrate muscle. Skelet Muscle. 2016;6:23. https://doi.org/10.1186/s13395-016-0096-4. that the FDB may act as a substitute for a number of 4. Kim J, Wu H-H, Lander AD, Lyons KM, Matzuk MM, Calof AL, et al. GDF11 commonly assessed skeletal muscles across a range of controls the timing of progenitor cell competence in developing retina. Science (80- ). 2005;308:1927–30. https://doi.org/10.1126/science.1110175. methodologies. In addition, the versatility of the FDB 5. Moran AL, Warren GL, Lowe DA. Soleus and EDL muscle contractility across supports a number of complementary methodologies the lifespan of female C57BL/6 mice. Exp Gerontol. 2005;40:966–75. https:// that may advance the assessment of skeletal muscle doi.org/10.1016/j.exger.2005.09.005. 6. Renganathan M, Messi ML, Delbono O. Overexpression of IGF-1 exclusively function and aid in elucidating the underlying mecha- in skeletal muscle prevents age-related decline in the number of nisms central to understanding skeletal muscle-related dihydropyridine receptors. J Biol Chem. 1998;273:28845–51. http://www. genetic disorders and disease. ncbi.nlm.nih.gov/pubmed/9786885. Accessed 9 Apr 2018 Tarpey et al. Skeletal Muscle (2018) 8:14 Page 15 of 15 7. Anderson EJ, Lustig ME, Boyle KE, Woodlief TL, Kane DA, Lin C-T, et al. accumulation than EDL (fast) muscle: fiber type-specific analysis. J Muscle Mitochondrial H2O2 emission and cellular redox state link excess fat intake Res Cell Motil. 2017;38:163–73. https://doi.org/10.1007/s10974-017-9468-6. to insulin resistance in both rodents and humans. J Clin Invest. 2009;119: 27. Koh TJ, Bryer SC, Pucci AM, Sisson TH. Mice deficient in plasminogen 573–81. https://doi.org/10.1172/JCI37048. activator inhibitor-1 have improved skeletal muscle regeneration. AJP Cell 8. Fisher-Wellman KH, Ryan TE, Smith CD, Gilliam LAA, Lin C-T, Reese LR, et al. Physiol. 2005;289:C217–23. https://doi.org/10.1152/ajpcell.00555.2004. A direct comparison of metabolic responses to high-fat diet in C57BL/6J 28. Liu Y, Cseresnyés Z, Randall WR, Schneider MF. Activity-dependent nuclear and C57BL/6NJ mice. Diabetes. 2016;65:3249–61. https://doi.org/10.2337/ translocation and intranuclear distribution of NFATc in adult skeletal muscle db16-0291. fibers. J Cell Biol. 2001;155:27–39. https://doi.org/10.1083/jcb.200103020. 29. Brown LD, Rodney GG, Hernández-Ochoa E, Ward CW, Schneider MF. Ca2+ 9. Kane DA, Anderson EJ, Price JW, Woodlief TL, Lin C-T, Bikman BT, et al. sparks and T tubule reorganization in dedifferentiating adult mouse skeletal Metformin selectively attenuates mitochondrial H2O2 emission without muscle fibers. Am J Physiol Cell Physiol. 2007;292:1156–66. https://doi.org/ affecting respiratory capacity in skeletal muscle of obese rats. Free Radic Biol 10.1152/ajpcell.00397.2006. Med. 2010;49:1082–7. https://doi.org/10.1016/J.FREERADBIOMED.2010.06.022. 30. Wojtala A, Bonora M, Malinska D, Pinton P, Duszynski J, Wieckowski MR. 10. Pearson T, Kabayo T, Ng R, Chamberlain J, McArdle A, Jackson MJ. Skeletal Methods to monitor ROS production by fluorescence microscopy and muscle contractions induce acute changes in cytosolic superoxide, but slower Fluorometry. Methods Enzymol. 2014;542:243–62. https://doi.org/10.1016/ responses in mitochondrial superoxide and cellular hydrogen peroxide. PLoS B978-0-12-416618-9.00013-3. One. 2014;9:e96378. https://doi.org/10.1371/journal.pone.0096378. 31. Mckenzie M, Lim SC, Duchen MR. Simultaneous measurement of 11. Robison P, Hernández-Ochoa EO, Schneider MF. Atypical behavior of mitochondrial calcium and mitochondrial membrane potential in live cells NFATc1 in cultured intercostal myofibers. Skelet Muscle. 2014;4(1) https:// by fluorescent microscopy. Video Link J Vis Exp. 2017;11955166 https://doi. doi.org/10.1186/2044-5040-4-1. org/10.3791/55166. 12. Scarda A, Franzin C, Milan G, Sanna M, Dal Prà C, Pagano C, et al. Increased 32. Rehberg M, Krombach F, Pohl U, Dietzel S. Label-free 3D visualization of adipogenic conversion of muscle satellite cells in obese Zucker rats. Int J cellular and tissue structures in intact muscle with second and third Obes. 2010;34:1319–27. https://doi.org/10.1038/ijo.2010.47. harmonic generation microscopy. PLoS One. 2011;6:e28237. https://doi.org/ 13. Wozniak AC, Pilipowicz O, Yablonka-Reuveni Z, Greenway S, Craven S, Scott 10.1371/journal.pone.0028237. E, et al. C-met expression and mechanical activation of satellite cells on 33. Larsen S, Nielsen J, Hansen CN, Nielsen LB, Wibrand F, Stride N, et al. cultured muscle fibers. J Histochem Cytochem. 2003;51:1437–45. https://doi. Biomarkers of mitochondrial content in skeletal muscle of healthy young org/10.1177/002215540305101104. human subjects. J Physiol. 2012;590:3349–60. https://doi.org/10.1113/ 14. Wohlers LM, Powers BL, Chin ER, Spangenburg EE. Using a novel coculture jphysiol.2012.230185. model to dissect the role of intramuscular lipid load on skeletal muscle 34. Baar K, Song Z, Semenkovich CF, Jones TE, Han D-H, Nolte LA, et al. Skeletal insulin responsiveness under reduced estrogen conditions. Am J Physiol muscle overexpression of nuclear respiratory factor 1 increases glucose Endocrinol Metab. 2013;304:E1199–212. https://doi.org/10.1152/ajpendo. transport capacity. FASEB J. 2003;17:1666–73. https://doi.org/10.1096/fj. 00617.2012. 03-0049com. 15. Felder A, Ward SR, Lieber RL. Sarcomere length measurement permits high 35. Burkholder TJ, Fingado B, Baron S, Lieber RL. Relationship between muscle resolution normalization of muscle fiber length in architectural studies. J fiber types and sizes and muscle architectural properties in the mouse Exp Biol. 2005;208(Pt 17):3275–9. https://doi.org/10.1242/jeb.01763. hindlimb. J Morphol. 1994;221:177–90. https://doi.org/10.1002/jmor. 16. Schmidt CA, Ryan TE, Lin C-T, Inigo MMR, Green TD, Brault JJ, et al. Diminished force production and mitochondrial respiratory deficits are strain-dependent myopathies of subacute limb ischemia. J Vasc Surg. 2017; 65:1504–14.e11. https://doi.org/10.1016/j.jvs.2016.04.041. 17. Spangenburg EE, Le Roith D, Ward CW, Bodine SC. A functional insulin-like growth factor receptor is not necessary for load-induced skeletal muscle hypertrophy. J Physiol. 2008;586:283–91. https://doi.org/10.1113/jphysiol. 2007.141507. 18. Scott K, Benkhalti M, Calvert ND, Paquette M, Zhen L, Harper M-E, et al. K channel deficiency in mouse FDB causes an impairment of energy ATP metabolism during fatigue. Am J Physiol Physiol. 2016;311:C559–71. https:// doi.org/10.1152/ajpcell.00137.2015. 19. Brooks SV, Faulkner JA. Contractile properties of skeletal muscles from young, adult and aged mice. J Physiol. 1988;404:71–82. http://www.ncbi. nlm.nih.gov/pubmed/3253447. Accessed 9 Apr 2018 20. Hakim CH, Wasala NB, Duan D. Evaluation of muscle function of the extensor digitorum longus muscle ex vivo and tibialis anterior muscle in situ in mice. J Vis Exp. 2013; https://doi.org/10.3791/50183. 21. Lark DS, Torres MJ, Lin C-T, Ryan TE, Anderson EJ, Neufer PD. Direct real- time quantification of mitochondrial oxidative phosphorylation efficiency in permeabilized skeletal muscle myofibers. Am J Physiol Cell Physiol. 2016; 311:C239–45. https://doi.org/10.1152/ajpcell.00124.2016. 22. Ryan TE, Brophy P, Lin C-T, Hickner RC, Neufer PD. Assessment of in vivo skeletal muscle mitochondrial respiratory capacity in humans by near- infrared spectroscopy: a comparison with in situ measurements. J Physiol. 2014;592:3231–41. https://doi.org/10.1113/jphysiol.2014.274456. 23. Campagnola PJ, Dong C-Y. Second harmonic generation microscopy: principles and applications to disease diagnosis. Laser Photon Rev. 2011;5: 13–26. https://doi.org/10.1002/lpor.200910024. 24. Liu W, Raben N, Ralston E. Quantitative evaluation of skeletal muscle defects in second harmonic generation images. J Biomed Opt. 2013;18:26005. https://doi.org/10.1117/1.JBO.18.2.026005. 25. DiFranco M, Quinonez M, Capote J, Vergara J. DNA transfection of mammalian skeletal muscles using in vivo electroporation. J Vis Exp. 2009; https://doi.org/10.3791/1520. 26. Komiya Y, Sawano S, Mashima D, Ichitsubo R, Nakamura M, Tatsumi R, et al. Mouse soleus (slow) muscle shows greater intramyocellular lipid droplet http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

Characterization and utilization of the flexor digitorum brevis for assessing skeletal muscle function

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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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Abstract

Background: The ability to assess skeletal muscle function and delineate regulatory mechanisms is essential to uncovering therapeutic approaches that preserve functional independence in a disease state. Skeletal muscle provides distinct experimental challenges due to inherent differences across muscle groups, including fiber type and size that may limit experimental approaches. The flexor digitorum brevis (FDB) possesses numerous properties that offer the investigator a high degree of experimental flexibility to address specific hypotheses. To date, surprisingly few studies have taken advantage of the FDB to investigate mechanisms regulating skeletal muscle function. The purpose of this study was to characterize and experimentally demonstrate the value of the FDB muscle for scientific investigations. Methods: First, we characterized the FDB phenotype and provide reference comparisons to skeletal muscles commonly used in the field. We developed approaches allowing for experimental assessment of force production, in vitro and in vivo microscopy, and mitochondrial respiration to demonstrate the versatility of the FDB. As proof-of principle, we performed experiments to alter force production or mitochondrial respiration to validate the flexibility the FDB affords the investigator. Results: The FDB is made up of small predominantly type IIa and IIx fibers that collectively produce less peak isometric force than the extensor digitorum longus (EDL) or soleus muscles, but demonstrates a greater fatigue resistance than the EDL. Unlike the other muscles, inherent properties of the FDB muscle make it amenable to multiple in vitro- and in vivo-based microscopy methods. Due to its anatomical location, the FDB can be used in cardiotoxin-induced muscle injury protocols and is amenable to electroporation of cDNA with a high degree of efficiency allowing for an effective means of genetic manipulation. Using a novel approach, we also demonstrate methods for assessing mitochondrial respiration in the FDB, which are comparable to the commonly used gastrocnemius muscle. As proof of principle, short-term overexpression of Pgc1α in the FDB increased mitochondrial respiration rates. Conclusion: The results highlight the experimental flexibility afforded the investigator by using the FDB muscle to assess mechanisms that regulate skeletal muscle function. Keywords: cDNA electroporation, Mitochondrial respiration, Muscle stimulation, Skeletal muscle function * Correspondence: Spangenburge14@ecu.edu Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27834, USA © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tarpey et al. Skeletal Muscle (2018) 8:14 Page 2 of 15 Background investigators have generally limited use of the FDB to Skeletal muscle is susceptible to a number of genetic, envir- isolated fiber imaging-based approaches or satellite cell onmental, and age-related pathologies that impair the tis- function in culture [10–13]. In this study, we demon- sue’s normal mechanical and metabolic function. This often strate the utility of the FDB as a model for assessing leads to the development of comorbidities and sometimes skeletal muscle function across a range of methodologies death. Defining the mechanisms that regulate the develop- commonly used within the skeletal muscle research ment of skeletal muscle dysfunction is critical for designing community, including isometric force production, in therapeutic interventions. Investigators currently employ a vitro and in vivo imaging, and mitochondrial respiration. variety of established methods for answering such ques- The full utility of the FDB is best demonstrated in stud- tions, but are often experimentally hampered by unique in- ies combining these physiological tests with genetic ma- herent heterogeneity between muscle groups and cells nipulation (cDNA electroporation), thus allowing the within the same muscle tissue. Muscles commonly used for investigator to manipulate cellular protein levels effi- functional and mechanistic experiments include the exten- ciently in a setting that allows for accurate functional sor digitorum longus (EDL), soleus, plantaris, gastrocne- outcome analyses. mius, tibialis anterior (TA), and/or the quadriceps. These muscles each offer unique advantages across a host of Methods methodologies including measuring isometric force pro- Animals duction, susceptibility to muscle injury, mitochondrial res- All mice were 3–11-month-old C57BL/6 males at the time piration, protein content, and histology. For example, the of testing. Mice were purchased from Jackson laboratories, EDL is frequently used for measures of isometric force pro- housed in a temperature (22 °C) and light-controlled facil- duction or susceptibility to stretch-induced injury in order ity, and given free access to food and water. Mice were eu- to better understand and assess the efficacy of interventions thanized via isoflurane overdose. All animal procedures targeting Duchenne muscular dystrophy [1–3] and/or aging and usage were approved by the Institutional Review [4–6]. Meanwhile, studies investigating metabolic diseases Committee at East Carolina University. Animal care com- such as obesity and type II diabetes commonly measure plied with the Guide for the Care and Use of Laboratory skeletal muscle mitochondrial respiration using the red por- Animals, Institute of Laboratory Animal Resources, tion of the gastrocnemius muscle [7–9]. However, due to Commission on Life Sciences, National Research Council inherent differences across muscles, investigators are often (Washington: National Academy Press, 1996). forced to apply specific experimental approaches for each muscle. This specificity can limit the broad interpretation FDB dissection of the results. Critical to any procedure utilizing the FDB is careful dissec- Mechanistically driven research often utilizes DNA tion that prevents damage to the muscle (the muscle is sur- manipulation to alter protein expression in skeletal roundedbyanareaof dense connective tissue)(seeFig. 1). muscle. The electroporation of cDNA or shRNA into muscles often delivers inconsistent results due to both the size of the muscle and anatomical location, which each impede uniform distribution of cDNA. These fac- tors can lead to low transduction efficiencies, making physiological and biochemical assays unreliable. To over- come this technical limitation, investigators have tagged their gene of interest and used immunohistochemical procedures to determine changes in tagged fibers that are known to express their gene of interest. Unfortu- nately, this approach limits the assays that can be employed and rules out most functional assays. While transgenic models have proven a valuable resource, they remain a costly, time-consuming, and uncertain en- deavor. In contrast, the flexor digitorum brevis's (FDB) unique anatomical location coupled with its size makes the muscle amenable to cDNA electroporation allowing for high transduction efficiencies. Fig. 1 FDB dissection. A FDB prepared for dissection, displaying the The FDB is a skeletal muscle located in the base of the proximal tendon and tendons of the toes. B Increased magnification foot that has previously been used to isolate and culture of A with muscle outlined in black to define borders of muscle single muscle fibers. For reasons that are unclear, Tarpey et al. Skeletal Muscle (2018) 8:14 Page 3 of 15 To facilitate dissection, the toes were pinned to a cork- was used to normalize all measured myofiber lengths board, securing the foot in place with the sole of the foot fa- to optimal sarcomere length. cing upward. Next, the skin at the proximal end of the foot (above the calcaneus) was pinched allowing micro-scissors Individual fiber type and fiber size to make incisions along the lateral edges of the foot down Muscle fiber type analysis of the FDB was conducted on to the toes. Still pinching the skin above the calcaneus, samples from C57BL/6 mice, as previously described micro-scissors were used to separate the skin from the [16]. Sections were probed with primary antibodies underlying musculature. The remaining skin flap was against myosin heavy chain type I (BA-F8), IIa (SC-71), peeled back and removed to expose the FDB and tendons IIb (BF-F3) (Development Studies Hybridoma Bank, of the toes. The proximal tendon was then cut, and while Univ of Iowa), and anti-dystrophin (Rb-9024, Thermo holding the tendon with forceps, the FDB was cut away Fisher, Waltham, MA), then imaged using an EVOS FL from the underlying fascia. The toe tendons were then cut auto microscope and accompanying software (Life to free the FDB from the foot. Technologies, Bothell, WA). Fiber type and fiber cross- sectional area (CSA) were assessed using ImageJ, as Myofiber isolation previously described [16]. After dissection of the FDB, the muscles were placed in fresh culture media (DMEM with glutamine, 2% sterile- Isometric force production filtered FBS, 0.1% gentamycin) supplemented with 4 mg/ Isometric force production and fatigue was assessed in ml collagenase A (Roche – 11088793001) for 90– EDL (n = 5), soleus (n = 4), and FDB (n = 6) muscles of 120 min at 37 °C in 5% CO as previously described C57BL/6 mice, as previously described [17] with slight [14]. The FDB muscle was placed in 2 mL of culture modifications. The FDB was exposed and the proximal media without collagenase and gently triturated against tendon was secured using silk suture. Fine-tip forceps the wall of the dish to release the fibers from the bundle were placed under the three medial toe tendons and using the cut end of a P1000 pipette tip. Isolated myofi- pulled gently down toward the toes before the three ten- bers were adhered to glass bottom dishes that were dons (Fig. 1) were secured with silk suture. We tied the coated with entactin-collagen-laminin (ECL Cell attach- three medial tendons because it is not possible to tie one ment matrix, #08110 Millipore). Fibers were returned to of the toes due to anatomical location, and tying the 37 °C in 5% CO for several hours and subsequently fourth tendon does not, in our experience, result in a dif- imaged as described below. ferent absolute force (data not shown). Each tendon was then cut just above the knot, and the FDB was gently lifted FDB muscle fiber length away from the foot. Micro-scissors were used to remove The FDB muscles of male and female C57BL/6 mice were any remaining connective tissue, releasing it from the foot. tied at the proximal tendon and three medial toe tendons The muscle was then tied to a force transducer and sus- with silk suture and fastened to a metal clip to maintain pended in oxygenated Krebs Ringer Buffer (KRB—[mM] resting tension. Myofibers were isolated as described 115 NaCl, 2.5 KCl, 1.8 CaCl ,2.2 Na HPO ,0.85 2 2 4 above, but were not adhered to glass bottom plates. NaH PO ) at room temperature. The muscle length was 2 4 Myofibers were imaged using a ×4 objective and an EVOS then adjusted until the FDB produced a peak twitch force, XL core microscope and accompanying software (Life at which point the optimal resting tension (Lo) was set Technologies, Bothell, WA). The lengths of approximately and the muscle was allowed to equilibrate for 10 min. So- 1000 myofibers were measured in ImageJ (version 1.6.0, leus muscles were prepared by tying a double square knot NIH, Bethesda, MD). When isolated, the myofibers were at the distal soleus tendon. The tendon was cut above the no longer at tension and therefore did not represent opti- knot and the posterior muscles were gently pulled away mal length. To account for this change in myofiber length, from the leg revealing the proximal soleus tendon, which a conversion factor was utilized using sarcomere length, was tied with a double square knot. The EDL was dis- as previously described by Dr. Richard Lieber [15]. When sected and tied as previously described [17]. EDL and so- at optimal length, the assumed mouse muscle optimal leus muscles were equilibrated in oxygenated room sarcomere length of a myofiber is 2.5 μmacross [15]. To temperature KRB at resting tension for 10 min. Following normalize myofiber lengths to sarcomere length, we equilibration, muscle tension was optimized by perform- measured sarcomere length in a subset of 30 myofibers. ing maximal twitch stimulations and adjusting the muscle The distance between 10 sarcomeres of each myofiber was length until peak force was achieved. Twitch stimulations measured, and an average sarcomere length was calculated were performed 30 s apart to avoid fatiguing the muscle. across all 30 myofibers. The optimal sarcomere length Muscles were then stimulated 60 s apart at 10, 20, 40, 60, (2.5 μm) divided by the average measured sarcomere 80, 100, and 120 Hz to generate a force frequency curve. length produced a conversion factor of 1.14, which Muscles were rested for an additional 1 min before Tarpey et al. Skeletal Muscle (2018) 8:14 Page 4 of 15 completing a 10-min stimulation protocol to determine for sectioning and H&E staining, as previously described fatigue resistance. The fatigue protocol stimulated muscles [16]. at 30 Hz every 2 s for a period of 600 s for a total of 300 contractions. Optimal muscle length was recorded and Skeletal muscle high-resolution mitochondrial muscles were blotted to remove excess KRB before being respirometry weighed. An optimum voltage of 20 V was established Preparation of permeabilized gastrocnemius and FDB prior to the experiments to ensure maximal stimulation of muscle fiber bundles the FDB, EDL, and soleus (data not shown). Absolute Respirometry was conducted on isolated permeabilized muscle force data were converted to specific force (N/ gastrocnemius and FDB muscle bundles excised from cm ) using previously described equations for the the same limb of C57BL/6 mice (n = 4). A portion of the mathematical estimation of muscle CSA [18]and red gastrocnemius was dissected and used for the prep- physiological cross-sectional area (PCSA) [19]. The pri- aration of permeabilized fiber bundles, as previously de- mary difference between CSA and PCSA is the inclusion scribed [21]. Red gastrocnemius muscle was used as the of the muscle fiber length to muscle length ratio in the comparative tissue as it is commonly used to assess PCSA equation. We used both corrected methods to pro- murine skeletal muscle mitochondrial respiration [8, 21]. vide a wider compatibility with the literature. The protocol for preparing permeabilized FDB muscle fiber bundles was adapted from previously described Passive contractile properties methods on the permeabilization of red gastrocnemius Passive contractile properties were assessed in EDL and muscle bundles [21] and is outlined below. The FDBs FDB muscles from C57BL/6 male mice (n = 4), as previ- were dissected and immediately added to ice-cold buffer ously described [20], with slight modifications. The EDL X ([mM]—7.23 K EGTA, 2.77 CaK EGTA, 20 imidazole, 2 2 and FDB muscles dissected and tied to a force trans- 20 taurine, 5.7 ATP, 14.3 phosphocreatine, 6.56 ducer as described above. Muscles were equilibrated for MgCl ·6H O, 50 MES, pH 7.1, 295 mosmol/kgH O). 2 2 2 10 min in oxygenated KRB at room temperature. Fol- Using a dissecting microscope, connective tissue, fat, lowing equilibration, muscle tension was optimized by and blood vessels were removed carefully to avoid performing maximal twitch stimulations and adjusting muscle loss. FDBs were cut into bundles and divided the muscle length until peak force was achieved. Twitch into groups of three to four bundles weighing 1.5–2. stimulations were performed 30 s apart to avoid fa- 0 mg wet weight. Bundle groups were then perme- tiguing the muscle. The muscle reference length was abilized in buffer X containing 22.5 μg/ml saponin with measured as the Lo before undergoing a passive stretch continuous rotation at 4 °C for 5 min. Muscle bundles of 105, 110, 115, 120, 125, and 130% of Lo. Muscles were were promptly transferred to ice-cold buffer Z ([mM]— blotted to remove excess KRB and then weighed. Data 110 K-MES, 35 KCl, 1 EGTA, 5 K HPO ,3 2 4 were corrected to CSA and PCSA. MgCl ·6H O, 5 mg/ml BSA, pH 7.4, 295 mosmol/ 2 2 kgH O) and washed with continuous rotation at 4 °C for Cardiotoxin (CTX) injury 15 min. Comparisons were made in C57BL/6 mice between a CTX-treated FDB (Naja nigricollis, #02152238, MP Bio- Mitochondrial respiration medicals, Santa Ana, CA) and PBS-treated contralateral Measurements of high-resolution O consumption were FDB 4 days (n = 4) and 10 days post-treatment (n = 2). made using the OROBOROS Oxygraph-2K (Oroboros Sterile 8-mm-long 31G syringes were prepared with Instruments, Innsbruck, Austria) at 37 °C with a starting 10 μLof 10 μM CTX or 10 μL of sterile 1× PBS, as pre- oxygen concentration of ~ 300–350 μM as previously viously described [16]. Following isoflurane-induced described [22]. Experiments were conducted in buffer Z anesthesia, the base of the feet of four mice was cleaned containing 20 mM creatine monohydrate and 25 μM with alcohol wipes. CTX was injected at the proximal blebbistatin. Mitochondrial respiration was assessed by portion of the foot with the needle positioned under the the sequential addition of substrates at a final concentra- skin and toward the toes. PBS was injected into the tion of pyruvate 4 mM, malate 0.5 mM, glutamate contralateral foot. Mice were sacrificed at the appropri- 5 mM, ADP 2.5 mM, succinate 5 mM, cytochrome c ate time, and FDBs were dissected for the measurement 5 μM, rotenone 10 μM, antimycin A 5 μM, ascorbic acid of force production, as described above. Data were cor- 2 mM, and TMPD 0.5 mM (N,N,N′,N′-tetramethyl-p- rected to PCSA. phenylenediamine dihydrochloride). Integrity of the mitochondrial membrane was confirmed by excluding Hematoxylin and eosin staining gastrocnemius muscle bundles and FDB muscle bundles FDB muscles subjected to 10 days CTX treatment were that produced a > 10 or > 20% increase in respiration, re- flash frozen in optimal cutting temperature (OCT) solution spectively, following exogenous cytochrome c addition. Tarpey et al. Skeletal Muscle (2018) 8:14 Page 5 of 15 A different exclusion criteria was used for the gastrocne- NA 1.35) and an Olympus FV1000 LSM operating mius and FDB muscle bundles as preliminary testing in- FV10-ASW 4.2 acquisition software. dicated a greater percent increase in respiration was common in FDB fiber bundles compared to gastrocne- cDNA electroporation and high-resolution respirometry of mius fiber bundles, following the addition of cytochrome skeletal muscle overexpressing Pgc1α c. Upon completion of the protocol, muscle bundles Electroporation of cDNA into FDBs was performed as were rinsed in distilled H O, freeze-dried (Labconco, previously described [25]. Briefly, the feet of seven Kansas City, MO), and weighed (Orion Cahn C-35, C57BL/6 male and female anesthetized mice were Thermo Electron, Beverly, MA). Respiration rates for in- cleaned with an alcohol wipe and the footpads were tact gastrocnemius muscle bundles are commonly cor- injected with 10 μl of 2 mg/mL hyaluronidase suspended rected to dry weight; however, due to differences in in sterile-filtered KRB using an 8-mm-long 31 gauge connective tissue content of the two muscle groups, JO sterile needle. Approximately 1 h, later mice underwent values were also corrected to total protein and citrate anesthesia for a second time. Feet were again cleaned synthase (CS) activity. CS activity was measured using with an alcohol wipe and one foot received 30 μgof kit CS0720. Chemicals and reagents were purchased green fluorescent protein (GFP)-tagged PGC1α plasmid from Sigma Aldrich. and the contralateral foot was injected with YFP cDNA. Following a full recovery from anesthesia, mice were Microscopy anesthetized for a third time ~ 10 min later. Platinum In vitro electrodes were inserted under the skin and positioned FDB muscle fibers were isolated from C57BL/6 mice perpendicular to the FDB and parallel to one another at and attached to 35-mm glass bottom dishes coated with the heel and footpad beneath the toes. The FDB was ECL, as previously discussed. Isolated myofibers were stimulated with 20 pulses of 20 ms duration at 1 Hz and stained with mitotracker deep red (M2246, Thermo 100 V [25]. Mice were sacrificed 14 days later, and FDBs Fisher) and NucBlue (R37605, Thermo Fisher) in DMEM were dissected and imaged under a fluorescent micro- for 30 min. Fibers were washed three times with 2 mL scope to confirm plasmid expression. Intact FDB muscle KRB. Fibers were imaged using a single photon confocal samples were then prepared and measured for mito- laser scanning microscope with a ×60 oil immersion ob- chondrial respiration as outline above. jective (Olympus, Plan Apochromat, NA 1.35) and exci- tation was achieved using the 405- and 488-nm lines of Statistics a multiline argon laser. Data distributions were assessed and data that did not conform to a normal distribution were log base 10 trans- In vivo formed. Force frequency contractile data were analyzed Second harmonic generation describes the optical effect via two-way ANOVA with Tukey multiple comparisons. produced from the passage of laser pulses through Muscle mass, fiber type, time to max and half- highly polarized, non-centro-symmetrical materials such relaxation, and fatigue data were analyzed via one-way as myosin. When polarized at the appropriate wave- ANOVA with Tukey multiple comparisons. In cases length, these materials emit light at half the wavelength where data could not be transformed to a normal distri- of that entering, producing high-resolution images with- bution, a Kruskal-Wallis test with Dunn multiple com- out the need for fluorescent probes that are subject to parisons was performed. ANOVAs were completed photobleaching and phototoxicity. Furthermore, the near using GraphPad Prism 7.03. Respiratory data and CS ac- infrared wavelengths used allow for deep tissue penetra- tivity were analyzed via paired two-tailed t-tests with an tion without the need for invasive procedures [23]. alpha level of 0.05 using Microsoft Excel. All data are C57BL/6 mice were anesthetized before having the skin presented as mean ± SEM. covering the FDB removed, exposing the FDB muscle. Once exposed, the FDB was hydrated with sterile KRB Results and the mouse was laid prone on a glass cover slip (#1.5, FDB fiber type and comparative characteristics Leica), as previously described [24] (Fig. 1C). Myosin Phenotypic characterization of the FDB demonstrates it and nicotinamide-containing molecules were excited at is a smaller muscle than the EDL and soleus and dis- 900 and 720 nm using a mode locked Ti:Sapphire pulsed plays a smaller fiber length to muscle length ratio rela- laser (Mai Tai Deep See HP series, Spectra-Physics, tive to the EDL and soleus, as measured by Brooks and Santa Clasa, CA), and emission was recorded using non- Faulkner [19] (Table 1). The FDB myofiber length is descanned detection with a FV10 MRV/G filter set at highly variable (Fig. 2A, B), although an analysis of the 450 and 420 nm, respectively. All images were taken myofiber length frequency indicates a majority of the using a ×60 oil immersion objective (Plan Apochromat, myofibers are approximately 500–700 μm (Fig. 2C). The Tarpey et al. Skeletal Muscle (2018) 8:14 Page 6 of 15 Table 1 Characteristics of the FDB compared to the EDL and soleus muscles FDB EDL Soleus Muscle mass (mg) 6.7 ± 0.4* 11.2 ± 0.7 10.0 ± 0.4 Optimal muscle length (mm) 9.5 ± 0.2* 14.2 ± 0.3 13.4 ± 0.3 Fiber length (mm) 0.744 ± 0.01 5.44 ± 0.12 [19] 7.84 ± 0.22 [19] Fiber to muscle length ratio 0.079 ± 0.002 0.45 ± 0.004 [19] 0.69 ± 0.006 [19] Fiber type % - Type I 4.4 ± 2.9 0 35 - Type IIa 43.9 ± 1.9 3 53 - Type IIx 51.6 ± 4.8 25 11 - Type IIb 0 72 1 [26][26] CSA (μm ± SD) - Type I 488 ± 67 – 943 ± 198 - Type IIa 927 ± 314 634 ± 142‡ 790 ± 149‡ - Type IIx 1267 ± 462 –– - Type IIb – 772 ± 120 – [35][35] Numbers in [brackets] represent the data citation *FDB v.s. EDL; p < 0.05 ‡Figure represents a combination of type IIa and type IIx muscle fibers. Data are mean ± SEM unless otherwise stated FDB exhibits a mixed muscle fiber type comprising of Isometric contractions and force production predominantly type IIa fibers (43.9 ± 1.9%) and type IIx The respective whole muscle length and contractile kin- fibers (51.6 ± 4.8%) with a small population of type I etics of the FDB, EDL, and soleus are demonstrated in fibers (4.4 ± 2.9%) (Fig. 2D). The cross-sectional area Fig. 3A–F. The FDB produces less peak force than either (CSA) of the individual muscle fiber types were all sig- the EDL or soleus muscle (Fig. 4A–C). The comparative nificantly different, with the largest CSA found in type relationship between the FDB, EDL, and soleus specific IIx fibers, followed by IIa and I, respectively (Fig. 2E, F). force is highly dependent on whether force is normalized Fig. 2 FDB fiber size, type, and cross-sectional area (CSA). A, B Example images of single myofibers isolated from FDB muscle. Blue arrows indicate viable myofibers. Red arrows indicate non-viable myofibers. Only viable myofiber lengths were measured. C Relative frequency of FDB muscle fiber lengths. D Fiber type percentage of whole FDB muscles (n =4). E Average individual CSA (μm ) of each muscle fiber type in the FDB. F Representative FDB cross section stained for type I (blue), type IIa (green), type IIx (no stain), type IIb (red), and dystrophin (purple). *, p < 0.05. Data are mean ± SEM Tarpey et al. Skeletal Muscle (2018) 8:14 Page 7 of 15 Fig. 3 Comparison of FDB, EDL, and soleus muscle length, and in vitro isometric contraction profile. Example images of A FDB, B EDL, and C soleus muscle after the tendons were secured with silk suture for subsequent isometric force assessment. Example tetanic (100 Hz) tracings for each muscle: D FDB, E EDL, F soleus to CSA (Fig. 4B) or PCSA (Fig. 4C). The FDB is more fa- CTX-induced muscle injury prevents force production tigue resistant than the EDL and the soleus (Fig. 4D). FDB muscles injected with CTX failed to produce meas- The FDB exhibits differing contractile kinetics compared urable force at 4 days post injection (Fig. 5A, B). to the EDL. Relative to the soleus, the FDB displays a However, force began to recover by 10 days post injec- statistically similar time to max force and half-relaxation tion, although force production still remained impaired time (Fig. 4E, F). relative to contralateral control limbs (Fig. 5C, D). H&E- stained muscle sections showed a greater number of nu- Passive contractile properties clei, which were predominantly centrally located, in FDB Using a previously described protocol [20] with slight muscle injected with CTX, compared to PBS-injected modifications, we assessed the passive mechanical prop- FDB muscle sections, following 10 days of recovery erties of the FDB and EDL muscles. In response to a (Fig. 5E, F). graded increase in muscle length (strain), both the FDB and EDL recorded increasing force (mN/mm )ateach Comparison of mitochondrial respiration between red length up to 125% of Lo. At 130% Lo, both the FDB and gastrocnemius and FDB muscle state 4 EDL did not demonstrate any further increases in force Mitochondrial respiration rates for complex I leak , state3ADP state3ADP output compared to 125% Lo. The relationship between complex I ,complex Iand II ,complex state3ADP the FDB and EDL in response to muscle lengthening II , and complex IV were consistently higher in was substantially altered by the method of force red gastrocnemius muscle compared to FDB muscle normalization. When normalized to CSA, the FDB (Fig. 6A). FDB mitochondrial respiratory data, consistently exhibited significantly more force than the normalized to muscle dry weight, is displayed alone in EDL (Fig. 4G). However, when normalized to PCSA Fig. 6B. Themagnitudeof thedifferencein respiration (accounting for the respective fiber length to muscle between red gastrocnemius and FDB muscle was length ratios), the EDL exhibited significantly more force dependent on the corrective method employed. than the FDB (Fig. 4H). Correcting to muscle dry weight demonstrated the Tarpey et al. Skeletal Muscle (2018) 8:14 Page 8 of 15 Fig. 4 Comparison of in vitro force production characteristics and fatigue resistance. Force frequency curves were generated from FDB (n = 6), 2 2 EDL (n = 5), and soleus muscles (n = 4). A Absolute force (mN). B Specific force (N/cm ) corrected to CSA. C Specific force (N/cm ) corrected to PCSA. D Percent decline in baseline force during a 10-min fatigue resistance protocol. E Time (s) to maximum force. F Half-relaxation time (s). Time to maximum force and half-relaxation times for each muscle were averaged from 100 Hz contractions. F–H Comparison of passive stress lengthening contractions between the FDB and EDL (mN/mm ) corrected to CSA and PCSA, respectively. *, FDB v.s. EDL p < 0.05; †, FDB v.s. soleus p < 0.05; #, EDL v.s. soleus p < 0.05. Data are mean ± SEM greatest relative difference in respiration between the mitochondrial respiration was significantly greater two muscles (Fig. 6A). Meanwhile, correcting across all complexes and conditions when corrected to mitochondrial respiration to total cellular protein muscle dry weight and total protein, while complex I state 4 reduced the relative difference in respiration (Fig. 6C), leak was no longer significant when corrected to CS which was reduced further by correcting mitochondrial activity. CS activity was greater (p = 0.04) in red respiration to CS activity (Fig. 6D). Gastrocnemius gastrocnemius muscle compared to FDB muscle (Fig. 6E), Tarpey et al. Skeletal Muscle (2018) 8:14 Page 9 of 15 Fig. 5 CTX-induced muscle injury. Mice were injected with 10 μLof10 μM CTX in one FDB and 10 μL sterile PBS in the contralateral FDB. Mice were euthanized 4 days (n = 4) or 10 days (n = 2) post-injection. FDBs were dissected and force production was recorded for control and injured muscles using a force-frequency curve protocol. FDBs tested 10 days post-treatment were flash frozen in OCT, sectioned to 12 μm, and stained with H&E before imaging at ×20. A Four days post-injection absolute force (mN). B Four days post-injection specific force (N/cm ) corrected to PCSA. C Ten days post-injection absolute force (mN). D Ten days post-injection specific force (N/cm ) corrected to PCSA. E PBS-treated FDB muscle section stained with H&E. F CTX-treated FDB muscle section stained with H&E. *, p < 0.05. Data are mean ± SEM suggesting that the red gastrocnemius muscle has a higher shows a mouse being prepared for second harmonic mitochondrial content then the FDB muscle. generation microscopy. Our results demonstrate high- resolution label-free images where we were able to In vitro and in vivo microscopy image myosin/collagen and/or NAD(P)H at the single Here we demonstrate the FDB muscle is amenable to cell level while the muscle remained intact (Fig. 7F–K). multiple imaging approaches. First, we took advantage of the susceptibility of the FDB to cDNA electroporation to cDNA electroporation-induced elevation of Pgc1α demonstrate fluorescent imaging approaches using the increases mitochondrial respiration in intact FDB muscle whole FDB muscle (Fig. 7A, B) and single muscle fibers Fluorescent microscopy of whole FDB muscle confirmed isolated from the FDB (Fig. 7C). It is possible to deter- the effective expression of both GFP-tagged Pgc1α in mine localization of tagged proteins with in vitro im- one foot and YFP in the contralateral foot (Fig. 8A, B). aging options using commercially available dyes such as Additionally, we confirmed the successful upregulation DAPI and mitotracker deep red (MTDR) (Fig. 7D). of mitochondrial biogenesis by measuring CS activity. However, it is also possible to demonstrate cellular FDB muscle overexpressing Pgc1α demonstrated signifi- localization when imaging the whole FDB muscle by cantly greater CS activity when compared to FDB using second harmonic generation microscopy. Figure 7E muscle expressing YFP (Fig. 8C). Overexpression of Tarpey et al. Skeletal Muscle (2018) 8:14 Page 10 of 15 Fig. 6 Comparison of mitochondrial respiration between the FDB and red gastrocnemius muscle. Permeabilized FDB and gastrocnemius muscle bundles were prepared for high-resolution respirometry (n = 4). Substrates and inhibitors were added sequentially to measure CI leak and ADP- stimulated CI, CI + II, CII, and CIV mitochondrial respiration. A Comparison of mitochondrial respiration in intact permeabilized red gastrocnemius and FDB muscle bundles corrected to dry weight. B Inset of graph A showing FDB mitochondrial respiration only. C Comparison of mitochondrial respiration in intact permeabilized red gastrocnemius and FDB muscle bundles corrected to total protein content. D Comparison of mitochondrial respiration in intact permeabilized red gastrocnemius and FDB muscle bundles corrected to CS activity. E Comparison of CS activity between red state4 state3ADP state3ADP gastrocnemius and FDB muscle. CI leak, complex I leak ; CI, complex I respiration ; CI + II, complex I and II respiration ; CII, complex II state3ADP respiration ; CIV, super-complex respiration. *, p < 0.05. Data are mean ± SEM Pgc1α significantly increased mitochondrial respiration respiration observed in Pgc1α-treated FDBs was due in the FDB muscle compared to the contralateral FDB solely to increased mitochondrial content (Fig. 8E). electroporated with YFP (Fig. 8D). Specifically, complex I leak respiration (p = 0.002), ADP-stimulated complex I Discussion (p = 0.049), and complex I + II (p = 0.038) respiration was In these experiments, we demonstrate the broad versatil- higher in Pgc1α overexpressing FDB muscle compared ity of the FDB muscle as a potential tool for the assess- to YFP-expressing FDBs. ADP-stimulated complex II ment and investigation of skeletal muscle biology. Our (p = 0.080) respiration as well as complex IV (p = 0.069) findings validate the FDB muscle across a range of meth- respiration were not significantly elevated. Interestingly, odologies and highlight its inherent experimental advan- normalizing oxygen consumption to CS activity largely tages. The location of the FDB, beneath the skin, and abolished the differences between YFP and Pgc1α- relatively small size provide a means to accurately target treated FDBs, thereby confirming that increased and efficiently transfect cDNA for the manipulation of Tarpey et al. Skeletal Muscle (2018) 8:14 Page 11 of 15 Fig. 7 In vitro and In vivo imagining. In vitro imaging techniques were used to demonstrate the expression of YFP protein in whole FDB muscle 7 days post cDNA electroporation, and the retention of YFP post-myofiber isolation. A Whole FDB muscle expressing YFP; images were taken with a 488-nm filter cube and in phase contrast then merged to show transfection efficiency. B Whole FDB muscle YFP negative control; images were taken with a 488-nm filter cube and in phase contrast then merged. C Isolated FDB myofiber demonstrating retention of YFP. FDB myofibers were isolated and stained with mitotracker deep red and DAPI prior to imaging with a single photon confocal LSM and oil immersion ×60 objective. D Representative images of stained isolated FDB myofibers showing mitochondria in red and nuclei in blue. Two-photon excitation fluorescence microscopy and second generation harmonics were employed to generate high-resolution in vivo images of the FDB myosin structure (green) and nicotinamide-containing molecules (red) using an oil immersion ×60 objective. Representative images displayed. E Photograph of mouse being prepared for second harmonic generation microscopy of the FDB. The mouse has been tilted slightly to better show the fixing of the foot to the slide. Images were captured with the mouse in a prone position. F Myosin. G Nicotinamide. H Composite of F and G. I ×3 zoom inset of F. J ×3 zoom inset of G. K Composite of I and J protein expression. Importantly, the FDB can then be that electroporation of cDNA encoding PGC1α signifi- used across a variety of physiological and/or cantly increased mitochondrial respiration. biochemical-based assays. As proof of principle, in the To provide investigators a better understanding of experiments described here, we specifically demonstrate the FDB, we performed a comprehensive phenotypic Tarpey et al. Skeletal Muscle (2018) 8:14 Page 12 of 15 Fig. 8 Electroporation of PGC1α cDNA increased mitochondrial respiration. GFP-conjugated PGC1α and YFP cDNA were electroporated into one FDB and a contralateral control FDB, respectively (n = 7). Mice were euthanized 14 days later and whole FDB muscle was imaged using fluorescent microscopy to confirm protein expression before intact permeabilized FDB muscle bundles were prepared for assessment of mitochondrial respiration between FDBs overexpressing Pgc1α or YFP. A YFP expression in whole FDB 14 days post cDNA electroporation; images were taken with a 488-nm filter cube and in phase contrast then merged to show transfection efficiency. B GFP-conjugated Pgc1α expression in whole FDB 14 days post cDNA electroporation; images were taken with a 488-nm filter cube and in phase contrast then merged to show transfection efficiency. C Comparison of CS activity between YFP and Pgc1α overexpressing FDB muscle bundles. D Overview of mitochondrial respiration across ETC protein complexes between YFP and Pgc1α overexpressing FDB muscle bundles, corrected to muscle dry weight. E Overview of mitochondrial respiration across ETC protein state 4 complexes between YFP and Pgc1α overexpressing FDB muscle bundles, corrected to CS activity. CI leak, complex I leak ;CI, state3ADP state3ADP state3ADP complex I respiration ; CI + II, complex I and II respiration ; CII, complex II respiration ;CIV,super-complex respiration. * p < 0.05. Data are mean ± SEM description compared to the EDL and/or soleus length to muscle length ratios were previously known muscle. A novel aspect of the current study is that to [19]. In the past, and in the absence of a fiber length the knowledge of the authors’ this is the first study to ratio for murine FDB muscle, force has been normal- measure the muscle fiber length to muscle length ra- ized without accounting for muscle fiber lengths [18]. tio in the FDB of mice. This provides an important This method of normalization has been employed in advancement in the ability to accurately compare the other investigations across other muscles such as the force-producing capacity of the FDB with other mus- soleus and EDL. We have therefore normalized our cles, such as the EDL and soleus, for which fiber data using both approaches. However, it may be Tarpey et al. Skeletal Muscle (2018) 8:14 Page 13 of 15 important to account for the fiber length to muscle of fluorescently labeled cDNA and specific dyes or fluor- length ratio as any intervention that could affect ei- ophores, such as MTDR and DAPI, may be used to ther variable would alter force output. Examination of image protein localization. These techniques may be fur- the force frequency curves between FDB, EDL, and ther enhanced through in vivo imaging. We specifically soleus muscles, or the FDB and EDL muscles follow- demonstrate how two-photon confocal microscopy and ing passive stress exemplifies the substantial effect of principles of second harmonic generation allow for the accounting for the muscle fiber to muscle length ra- high-resolution imaging of non-centro-symmetrical ma- tio. Researchers should be mindful of the terials such as muscle myosin, nicotinamide-containing normalization method used when analyzing and inter- molecules, and collagen [23]. The FDB muscle provides preting data, and when comparing to data in the several advantages for using second harmonic generation literature. microscopy. The natural prone posture of the animal We observed an intermediate contractile phenotype in may aid in reducing motion artifact due to breathing. the FDB that quantitatively places it between the EDL and Imaging the FDB in the fashion described is less invasive soleus muscles with respect to contractile kinetics. Not than alternative methods, such as excision of the surprisingly, the contractile kinetics of the FDB mirror the cremaster [32]. The amenable nature of the FDB to fiber type composition, which consists predominantly of electroporation with fluorescently tagged cDNA may types IIx and IIa with a small percentage of type I fibers. also allow for a combination of single- and two-photon When compared to the soleus muscle (type I =35%, IIa = microscopy, generating high-definition protein 53% and IIx = 11%, IIb = 1%) or the EDL (type I = 0%, type localization images. IIa = 3%, IIx = 25% and IIb = 72%) [26], the FDB presents In recent years, there has been a substantial increase as a more mixed phenotype. It is worth noting that the in the utilization of methods assessing mitochondrial re- greater fatigue resistance of the FDB muscle may not be spiratory function, particularly so within the field of the direct result of biochemical differences, but an indirect skeletal muscle-related metabolic disease. We demon- result of a reduced oxygen diffusion distance, resulting strate a novel approach, utilizing high-resolution respi- from the smaller size of the FDB relative to the soleus and rometry to measure mitochondrial respiration in the EDL. In conjunction with the EDL and soleus, the FDB FDB muscle. To validate the use of the FDB, we com- provides an additional muscle in which functional pared mitochondrial respiration rates between red outcomes can be assessed in response to experimental ma- gastrocnemius muscle and the FDB. As anticipated, res- nipulations. Due to the unique anatomical location, the piration rates were higher in red gastrocnemius muscle. FDB is amenable to direct injections of various small mol- This is in part due to differences in mitochondrial con- ecules and/or toxins. As proof of principle, we show that tent as demonstrated by greater CS activity, but also a the loss and recovery of isometric force production within result of the respective tissue preparation protocols. The the FDB following CTX-induced muscle injury is similar magnitude of the difference in respiration rates was to that previously reported in the EDL [27]. Together, dependent on whether O consumption was normalized these data suggest the FDB may be a valuable addition to, to muscle dry weight, total protein, or CS activity. Nor- or substitution for, the EDL and soleus muscles when malizing to dry weight or total protein altered respir- assessing skeletal muscle force production and contractile ation values as the FDB contains a relatively higher kinetics. content of connective tissue compared to the gastrocne- The FDB has a smaller fiber: muscle length ratio rela- mius, thus leading to potential underestimation of FDB tive to the EDL. This ratio makes the FDB amenable to O consumption rates. While adjusting the method of efficient single myofiber isolation, which is more chal- normalization cannot fully account for differences in lenging in the EDL and/or soleus muscles. These single connective tissue content, when rates were normalized myofibers can be cultured and imaged on multiple dif- to CS activity, this possible underestimation was elimi- ferent microscopy platforms. Skeletal muscle microscopy nated. This suggests that strong consideration to is a valuable tool for identifying protein localization [28], normalization must be given prior to starting any study muscle morphology [29], and the dynamic changes in with the FDB. CS activity was selected as an alternative molecules such as H O [10, 30] and calcium [29, 31]. In normalization factor for O consumption rates due to its 2 2 2 vitro imaging of isolated FDB myofibers is well- strong association with mitochondrial content [33]. Fur- established; however, our findings demonstrate that elec- ther normalization methods, such as mitochondrial pro- troporated cDNA tagged with fluorescent proteins is tein complex content, may be used, but interactions retained in myofibers following isolation. This provides between the normalization factor and treatment should numerous possibilities for the manipulation of protein be considered. Investigators should also consider the size expression and examination of subsequent alterations to of the FDB in their study designs as this represents a morphology and function. In addition, the combination limiting factor to the number of protocols and replicates Tarpey et al. Skeletal Muscle (2018) 8:14 Page 14 of 15 that may be tested. Additionally, the FDB is too sensitive Abbreviations cDNA: Complementary DNA; CS: Citrate synthase; CSA: Cross-sectional area; to standard mechanical separation of muscle fibers, so CTX: Cardiotoxin; EDL: Extensor digitorum longus; ETC: Electron transport this should be avoided to protect mitochondrial mem- chain; FDB: Flexor digitorum brevis; GFP: Green fluorescent protein; brane integrity. KRB: Krebs Ringer Buffer; MTDR: Mitotracker deep red; PCSA: Physiological cross-sectional area; Pgc1α: Peroxisome proliferator-activated receptor Finally, as an overall proof of concept that the FDB af- gamma coactivator 1-alpha; YFP: Yellow fluorescent protein fords the investigator a unique flexibility, we overex- pressed GFP-tagged Pgc1α or YFP protein in the FDB Acknowledgements We thank Dr. Jeffery Brault for providing us the cDNA for PGC-1alpha, and for a period of 14 days using cDNA electroporation. A Drs. R. M. Lovering and P.D. Neufer for technical discussions. fluorescence imaging-based approach in combination with a CS activity assay confirmed the successful overex- Funding This study was funded by NIH RO1AR06660 (EES), ADA 1-15-BS-170 (EES), NIH pressing of Pgc1α. The significantly greater complex RO1HL125695 (JMM), and F32HL129632 (TER). LEAK ADP ADP I , complex I , and complex I + II respiration rates in GFP-tagged Pgc1α-injected FDBs demonstrates Availability of data and materials The datasets used and/or analyzed during the current study are available that the electroporation of cDNA into FDBs is an effect- from the corresponding author on reasonable request. ive means of manipulating the phenotype or function of the muscle. Normalizing oxygen consumption rates to Authors’ contributions MDT wrote the manuscript, completed the experimental procedures, and CS activity resulted in no group differences compared to contributed to the experimental design. AJA and NPB completed the dry muscle weight normalization. These data suggest experimental procedures and read drafts of the manuscript. TER contributed that Pgc1α may have a larger influence on CS activity to designing and troubleshooting the described experimental procedures. CAS contributed to designing and troubleshooting the described relative to the protein complexes of the electron trans- experimental procedures. JMM provided intellectual insight and contributed port chain (ETC). More specifically, each complex of the to the project design and critical reading of the manuscript. EES contributed ETC is made up of numerous proteins that are likely not to the experimental analysis and design and provided critical reading revision of manuscript. All authors have read and approved the manuscript. all regulated by Pgc1α suggesting the necessity of add- itional regulators of mitochondrial function [34]. Ethics approval and consent to participate While there are practical advantages to using the EDL, Animal care and experimental procedures were approved by the Institutional Review Committee at East Carolina University and complied with the Guide soleus, or gastrocnemius muscles for assessing skeletal for the Care and Use of Laboratory Animals, Institute of Laboratory Animal muscle isometric force production or mitochondrial res- Resources, Commission on Life Sciences, National Research Council piration, these muscles are not effective targets for (Washington: National Academy Press, 1996). cDNA electroporation due primarily to their size and/or Competing interests anatomical location (which impedes cDNA distribution The authors declare that they have no competing interests. within the muscles). Conducting mechanistic studies into the relationships between protein expression and Publisher’sNote function using such muscles therefore requires the pro- Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. duction of muscle-specific transgenic mice. The FDB provides a time sensitive, non-invasive, and cost- Received: 17 January 2018 Accepted: 3 April 2018 effective method for assessing multiple functional indi- ces within skeletal muscle after deliberately altering pro- References tein expression through cDNA or shRNA delivery. This 1. Lynch GS, Hinkle RT, Chamberlain JS, Brooks SV, Faulkner JA. Force and combined utility provides advantages to investigators power output of fast and slow skeletal muscles from mdx mice 6-28 months old. J Physiol. 2001;535(Pt 2):591–600. https://doi.org/10.1111/J. pursuing mechanistic studies in the field of skeletal 1469-7793.2001.00591.X. muscle biology. 2. Pant M, Sopariwala DH, Bal NC, Lowe J, Delfín DA, Rafael-Fortney J, et al. Metabolic dysfunction and altered mitochondrial dynamics in the utrophin- dystrophin deficient mouse model of Duchenne muscular dystrophy. PLoS One. 2015;10:e0123875. https://doi.org/10.1371/journal.pone.0123875. Conclusions 3. Roy P, Rau F, Ochala J, Messéant J, Fraysse B, Lainé J, et al. Dystrophin The use of the FDB represents an expanded tool set to restoration therapy improves both the reduced excitability and the force drop induced by lengthening contractions in dystrophic mdx skeletal the mechanism-focused investigator. We demonstrate muscle. Skelet Muscle. 2016;6:23. https://doi.org/10.1186/s13395-016-0096-4. that the FDB may act as a substitute for a number of 4. Kim J, Wu H-H, Lander AD, Lyons KM, Matzuk MM, Calof AL, et al. GDF11 commonly assessed skeletal muscles across a range of controls the timing of progenitor cell competence in developing retina. Science (80- ). 2005;308:1927–30. https://doi.org/10.1126/science.1110175. methodologies. In addition, the versatility of the FDB 5. Moran AL, Warren GL, Lowe DA. Soleus and EDL muscle contractility across supports a number of complementary methodologies the lifespan of female C57BL/6 mice. Exp Gerontol. 2005;40:966–75. https:// that may advance the assessment of skeletal muscle doi.org/10.1016/j.exger.2005.09.005. 6. Renganathan M, Messi ML, Delbono O. Overexpression of IGF-1 exclusively function and aid in elucidating the underlying mecha- in skeletal muscle prevents age-related decline in the number of nisms central to understanding skeletal muscle-related dihydropyridine receptors. J Biol Chem. 1998;273:28845–51. http://www. genetic disorders and disease. ncbi.nlm.nih.gov/pubmed/9786885. Accessed 9 Apr 2018 Tarpey et al. Skeletal Muscle (2018) 8:14 Page 15 of 15 7. Anderson EJ, Lustig ME, Boyle KE, Woodlief TL, Kane DA, Lin C-T, et al. accumulation than EDL (fast) muscle: fiber type-specific analysis. J Muscle Mitochondrial H2O2 emission and cellular redox state link excess fat intake Res Cell Motil. 2017;38:163–73. https://doi.org/10.1007/s10974-017-9468-6. to insulin resistance in both rodents and humans. J Clin Invest. 2009;119: 27. Koh TJ, Bryer SC, Pucci AM, Sisson TH. Mice deficient in plasminogen 573–81. https://doi.org/10.1172/JCI37048. activator inhibitor-1 have improved skeletal muscle regeneration. AJP Cell 8. Fisher-Wellman KH, Ryan TE, Smith CD, Gilliam LAA, Lin C-T, Reese LR, et al. Physiol. 2005;289:C217–23. https://doi.org/10.1152/ajpcell.00555.2004. A direct comparison of metabolic responses to high-fat diet in C57BL/6J 28. Liu Y, Cseresnyés Z, Randall WR, Schneider MF. Activity-dependent nuclear and C57BL/6NJ mice. Diabetes. 2016;65:3249–61. https://doi.org/10.2337/ translocation and intranuclear distribution of NFATc in adult skeletal muscle db16-0291. fibers. J Cell Biol. 2001;155:27–39. https://doi.org/10.1083/jcb.200103020. 29. Brown LD, Rodney GG, Hernández-Ochoa E, Ward CW, Schneider MF. Ca2+ 9. Kane DA, Anderson EJ, Price JW, Woodlief TL, Lin C-T, Bikman BT, et al. sparks and T tubule reorganization in dedifferentiating adult mouse skeletal Metformin selectively attenuates mitochondrial H2O2 emission without muscle fibers. Am J Physiol Cell Physiol. 2007;292:1156–66. https://doi.org/ affecting respiratory capacity in skeletal muscle of obese rats. Free Radic Biol 10.1152/ajpcell.00397.2006. Med. 2010;49:1082–7. https://doi.org/10.1016/J.FREERADBIOMED.2010.06.022. 30. Wojtala A, Bonora M, Malinska D, Pinton P, Duszynski J, Wieckowski MR. 10. Pearson T, Kabayo T, Ng R, Chamberlain J, McArdle A, Jackson MJ. Skeletal Methods to monitor ROS production by fluorescence microscopy and muscle contractions induce acute changes in cytosolic superoxide, but slower Fluorometry. Methods Enzymol. 2014;542:243–62. https://doi.org/10.1016/ responses in mitochondrial superoxide and cellular hydrogen peroxide. PLoS B978-0-12-416618-9.00013-3. One. 2014;9:e96378. https://doi.org/10.1371/journal.pone.0096378. 31. Mckenzie M, Lim SC, Duchen MR. Simultaneous measurement of 11. Robison P, Hernández-Ochoa EO, Schneider MF. Atypical behavior of mitochondrial calcium and mitochondrial membrane potential in live cells NFATc1 in cultured intercostal myofibers. Skelet Muscle. 2014;4(1) https:// by fluorescent microscopy. Video Link J Vis Exp. 2017;11955166 https://doi. doi.org/10.1186/2044-5040-4-1. org/10.3791/55166. 12. Scarda A, Franzin C, Milan G, Sanna M, Dal Prà C, Pagano C, et al. Increased 32. Rehberg M, Krombach F, Pohl U, Dietzel S. Label-free 3D visualization of adipogenic conversion of muscle satellite cells in obese Zucker rats. Int J cellular and tissue structures in intact muscle with second and third Obes. 2010;34:1319–27. https://doi.org/10.1038/ijo.2010.47. harmonic generation microscopy. PLoS One. 2011;6:e28237. https://doi.org/ 13. Wozniak AC, Pilipowicz O, Yablonka-Reuveni Z, Greenway S, Craven S, Scott 10.1371/journal.pone.0028237. E, et al. C-met expression and mechanical activation of satellite cells on 33. Larsen S, Nielsen J, Hansen CN, Nielsen LB, Wibrand F, Stride N, et al. cultured muscle fibers. J Histochem Cytochem. 2003;51:1437–45. https://doi. Biomarkers of mitochondrial content in skeletal muscle of healthy young org/10.1177/002215540305101104. human subjects. J Physiol. 2012;590:3349–60. https://doi.org/10.1113/ 14. Wohlers LM, Powers BL, Chin ER, Spangenburg EE. Using a novel coculture jphysiol.2012.230185. model to dissect the role of intramuscular lipid load on skeletal muscle 34. Baar K, Song Z, Semenkovich CF, Jones TE, Han D-H, Nolte LA, et al. Skeletal insulin responsiveness under reduced estrogen conditions. Am J Physiol muscle overexpression of nuclear respiratory factor 1 increases glucose Endocrinol Metab. 2013;304:E1199–212. https://doi.org/10.1152/ajpendo. transport capacity. FASEB J. 2003;17:1666–73. https://doi.org/10.1096/fj. 00617.2012. 03-0049com. 15. Felder A, Ward SR, Lieber RL. Sarcomere length measurement permits high 35. Burkholder TJ, Fingado B, Baron S, Lieber RL. Relationship between muscle resolution normalization of muscle fiber length in architectural studies. J fiber types and sizes and muscle architectural properties in the mouse Exp Biol. 2005;208(Pt 17):3275–9. https://doi.org/10.1242/jeb.01763. hindlimb. J Morphol. 1994;221:177–90. https://doi.org/10.1002/jmor. 16. Schmidt CA, Ryan TE, Lin C-T, Inigo MMR, Green TD, Brault JJ, et al. Diminished force production and mitochondrial respiratory deficits are strain-dependent myopathies of subacute limb ischemia. J Vasc Surg. 2017; 65:1504–14.e11. https://doi.org/10.1016/j.jvs.2016.04.041. 17. Spangenburg EE, Le Roith D, Ward CW, Bodine SC. A functional insulin-like growth factor receptor is not necessary for load-induced skeletal muscle hypertrophy. J Physiol. 2008;586:283–91. https://doi.org/10.1113/jphysiol. 2007.141507. 18. Scott K, Benkhalti M, Calvert ND, Paquette M, Zhen L, Harper M-E, et al. K channel deficiency in mouse FDB causes an impairment of energy ATP metabolism during fatigue. Am J Physiol Physiol. 2016;311:C559–71. https:// doi.org/10.1152/ajpcell.00137.2015. 19. Brooks SV, Faulkner JA. Contractile properties of skeletal muscles from young, adult and aged mice. J Physiol. 1988;404:71–82. http://www.ncbi. nlm.nih.gov/pubmed/3253447. Accessed 9 Apr 2018 20. Hakim CH, Wasala NB, Duan D. Evaluation of muscle function of the extensor digitorum longus muscle ex vivo and tibialis anterior muscle in situ in mice. J Vis Exp. 2013; https://doi.org/10.3791/50183. 21. Lark DS, Torres MJ, Lin C-T, Ryan TE, Anderson EJ, Neufer PD. Direct real- time quantification of mitochondrial oxidative phosphorylation efficiency in permeabilized skeletal muscle myofibers. Am J Physiol Cell Physiol. 2016; 311:C239–45. https://doi.org/10.1152/ajpcell.00124.2016. 22. Ryan TE, Brophy P, Lin C-T, Hickner RC, Neufer PD. Assessment of in vivo skeletal muscle mitochondrial respiratory capacity in humans by near- infrared spectroscopy: a comparison with in situ measurements. J Physiol. 2014;592:3231–41. https://doi.org/10.1113/jphysiol.2014.274456. 23. Campagnola PJ, Dong C-Y. Second harmonic generation microscopy: principles and applications to disease diagnosis. Laser Photon Rev. 2011;5: 13–26. https://doi.org/10.1002/lpor.200910024. 24. Liu W, Raben N, Ralston E. Quantitative evaluation of skeletal muscle defects in second harmonic generation images. J Biomed Opt. 2013;18:26005. https://doi.org/10.1117/1.JBO.18.2.026005. 25. DiFranco M, Quinonez M, Capote J, Vergara J. DNA transfection of mammalian skeletal muscles using in vivo electroporation. J Vis Exp. 2009; https://doi.org/10.3791/1520. 26. Komiya Y, Sawano S, Mashima D, Ichitsubo R, Nakamura M, Tatsumi R, et al. Mouse soleus (slow) muscle shows greater intramyocellular lipid droplet

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