We are grateful to the Boettcher Foundation and the Amyotrophic Lateral Sclerosis Association (#17-II-344) for their support of this research.

All procedures involving mice were approved by the University of Colorado-Anschutz Medical Campus Institutional Animal Care and Use Committee (91813(05)1D).
1. Day 1: Primary (1&#176;) Immunostaining with Bovine Serum Albumin (BSA) Blocking
<ol>
	<li>Air-dry frozen sections of mouse hindlimb muscle (e.g., tibialis anterior, soleus) mounted on charged slides for ~ 30 min11. Draw a border around the sections using a hydrophobic barrier PAP pen.</li>
	<li>Place ~ 250 &#181;L of 5% BSA/phosphate buffered saline (BSA/PBS) on each slide to block non-specific antibody binding. Incubate slides at room temperature for 1 h.</li>
	<li>While blocking, prepare 1:50 dilutions of the 1&#176; antibody supernatant in 5% BSA/PBS (all are mouse monoclonal antibodies; see Table 16,12); prepare 250 &#181;L of solution per slide. Keep 1&#176; antibody stocks and dilutions on ice.</li>
</ol>
<img alt="Table 1" src="/files/ftp_upload/56024/56024table1.jpg" /> 
Table 1: Primary Antibodies used to Distinguish MyHCs.
<ol start="4">
	<li>Following aspiration of the 5% BSA/PBS blocking solution, add 250 &#181;L of 1&#176; antibody dilution to the appropriate slides. Add 250 &#181;L of 5% BSA/PBS to secondary (2&#176;) antibody-only negative control slides.</li>
	<li>Incubate at 4 &#176;C for 24 - 48 h in a humid environment. 
	NOTE: A Petri dish covered in aluminum foil with dampened filter paper may serve as a suitable incubation chamber.</li>
</ol>
2. Day 2: 2&#176; Immunostaining with Fluorescent Antibodies
<ol>
	<li>Aspirate 1&#176; antibody solution or control 5% BSA/PBS solution. Wash all slides three times, 10 min with 5% BSA.</li>
	<li>While washing, prepare 1:200 dilutions of the purified, fluorophore-conjugated 2&#176; antibodies in 5% BSA/PBS (see Table 2, all are goat anti-mouse). Prepare 250 &#181;L of solution per slide. Keep 2&#176; fluorophore-conjugated antibodies in the dark on ice.</li>
</ol>
<img alt="Table 2" src="/files/ftp_upload/56024/56024table2.jpg" /> 
Table 2: Fluorophore-conjugated Secondary Antibodies used to Visualize Primary Antibody Recognition of MyHCs.
<ol start="3">
	<li>Following aspiration of the third application of 5% BSA/PBS blocking solution, add 250 &#181;L of 2&#176; antibody dilution to all slides. Incubate for 90 min at room temperature in a dark, humid environment.</li>
	<li>Aspirate 2&#176; antibody solution. Wash all slides three times, 10 min with 5% BSA/PBS.</li>
	<li>Rinse with PBS. Dry for 10 min.</li>
	<li>Mount the coverglass with a non-permanent, low-viscosity aqueous mounting medium.</li>
	<li>When dry, seal the slide edges with nail polish. Dry and store in a dark slidebox.</li>
</ol>
3. Imaging Slides with Epifluorescence Microscopy
<ol>
	<li>Clean the slides with a small, 70% ethanol-soaked lab-wipe.</li>
	<li>Obtain digital images of immunostained muscle sections using an epifluorescence microscope equipped with a photographic apparatus (see the Table of Materials).
	<ol>
		<li>Select an area of approximately 1 mm2 (10X objective, 1.4 NA).</li>
		<li>View Alexa 488-conjugated 2&#176; antibody fluorescence via a 505 nm long-pass filter. View the fluorescence generated by excitation of Alexa 594-conjugated 2&#176; antibodies via a 595 nm long-pass filter. Digitize images with a PC computer equipped with a compatible imaging software. Include the scale bar from imaging software for later analysis.</li>
	</ol>
	</li>
</ol>
4. Analysis of Images with Fiji
Note: Before proceeding with analysis, Fiji (available freely from the National Institutes of Health, Bethesda, MD) must be installed via https://imagej.net/Fiji/Downloads. Also, the macro used in this process is provided in the Supplemental File. Place the macro file in an easily accessible directory.
<ol>
	<li>Load a brightfield image of a selected section in Fiji. Navigate to Plugins &#8594; Segmentation&#160;&#8594; Trainable Weka Segmentation.</li>
	<li>In order to stipulate the cellular areas of the section, draw a line on a fiber, and click &#34;Add to class 1&#34;. Then, draw a line on the space between fibers, and click &#34;Add to class 2&#34; to mark the extracellular domains. Repeat until there are 5-10 labels in each class.
	<ol>
		<li>Click Train Classifier. On the resulting red and green overlay, add more labels manually (see step 4.2) to incorrectly segmented pieces of the image, if necessary. Repeat until fibers (red) are appropriately separated from the space between fibers (green).</li>
	</ol>
	</li>
	<li>Click &#34;Get Probability&#34; and save the image in a newly labeled folder.</li>
	<li>Repeat the above procedure (steps 4.2 - 4.3) with the fluorescent image taken from the same field. Label immunostained fibers as &#34;class 1&#34; and all non-fluorescent regions as &#34;class 2&#34;. Save the resulting probability map to the same folder where the processed brightfield image is stored.</li>
	<li>Open one of the previously saved probability maps in Fiji. Draw a straight line on the scale bar provided by the imaging software. Go to Analyze&#160;&#8594; Set Scale. Change the parameters (i.e., known distance, unit of length) to their appropriate values as given by the scale bar, then check the &#34;Global&#34; box to standardize the scale for each image.</li>
	<li>Navigate to Plugins&#160;&#8594; Macros&#160;&#8594; Run&#160;&#8594; Tyagi et al. fiber quantification macro.ijm (see the Supplemental File). Immediately, a navigation pane will appear. Open the image&#39;s host folder; a dialog box will appear. For each dialog box, change the &#34;Background&#34; dropdown value to &#34;Light.&#34; 
	NOTE: The folder will now be populated with images of the fiber outlines and spreadsheet software files of the results (CSA and Feret diameter are most pertinent to quantifying fiber morphology; measured parameters can be adjusted in Analyze Set Measurements). Fiber morphology data generated by the pore-analysis function of Fiji will be automatically transferred to spreadsheet software files for both fluorescent and bright field images.</li>
	<li>Calculate the fraction of fibers expressing a given MyHC in a field by dividing the number of fluorescent fibers in the field by the number of total fibers in the corresponding bright field image.</li>
</ol>

<ol>
	<li>Engel, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essentiality+of+histo-+and+cytochemical+studies+of+skeletal+muscle+in+the+investigation+of+neuromuscular+disease.">The essentiality of histo- and cytochemical studies of skeletal muscle in the investigation of neuromuscular disease.</a> Neurol. 12, 778-784 (1962).</li><li>Dobrowolny, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+is+a+primary+target+of+SOD1G93A-mediated+toxicity.">Skeletal muscle is a primary target of SOD1G93A-mediated toxicity.</a> Cell Metab. 8, 425-436 (2008).</li><li>Sultana, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restricting+calcium+currents+is+required+for+correct+fiber+type+specification+in+skeletal+muscle.">Restricting calcium currents is required for correct fiber type specification in skeletal muscle.</a> Development. 143 (9), 1547-1559 (2016).</li><li>Guth, L., Samaha, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedure+for+the+histochemical+demonstration+of+actomyosin+ATPase.">Procedure for the histochemical demonstration of actomyosin ATPase.</a> Exp Neurol. 28 (2), 365-367 (1970).</li><li>Bloemberg, D., Quadrilatero, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+determination+of+myosin+heavy+chain+expression+in+rat,+mouse,+and+human+skeletal+muscle+using+multicolor+immunofluorescence+analysis.">Rapid determination of myosin heavy chain expression in rat, mouse, and human skeletal muscle using multicolor immunofluorescence analysis.</a> PLoS One. 7 (4), (2012).</li><li>Lucas, C. A., Kang, L. H., Hoh, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monospecific+antibodies+against+the+three+mammalian+fast+limb+myosin+heavy+chains.">Monospecific antibodies against the three mammalian fast limb myosin heavy chains.</a> Biochem Biophys Res Commun. 272 (1), 303-308 (2000).</li><li>Lee, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++permeation+and/or+binding+to+Cav1.1+fine-tunes+skeletal+muscle+Ca2++signaling+to+sustain+muscle+function.">Ca2+ permeation and/or binding to Cav1.1 fine-tunes skeletal muscle Ca2+ signaling to sustain muscle function.</a> Skelet Muscle. 5 (4), (2015).</li><li>Sawano, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+one-step+immunostaining+method+to+visualize+rodent+muscle+fiber+type+within+a+single+specimen.">A one-step immunostaining method to visualize rodent muscle fiber type within a single specimen.</a> PLoS One. 11 (11), (2016).</li><li>Meunier, B., Picard, B., Astruc, T., Labas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+image+analysis+tool+for+the+classification+of+muscle+fibre+type+using+immunohistochemical+staining.">Development of image analysis tool for the classification of muscle fibre type using immunohistochemical staining.</a> Histochem Cell Biol. 134 (3), 307-317 (2010).</li><li>Kammoun, M., Casser-Malek, I., Meunier, B., Picard, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+immunohistochemical+classification+of+skeletal+muscle+fibres+in+mouse.">A simplified immunohistochemical classification of skeletal muscle fibres in mouse.</a> Eur J Histochem. 58 (2), 2254 (2014).</li><li>Kumar, A., Accorsi, A., Rhee, Y., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do's+and+don'ts+in+the+preparation+of+muscle+cryosections+for+histological+analysis.">Do's and don'ts in the preparation of muscle cryosections for histological analysis.</a> J Vis Exp. (99), (2015).</li><li>Schiaffino, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+myosin+heavy+chain+isoforms+in+type+2+skeletal+muscle+fibres.">Three myosin heavy chain isoforms in type 2 skeletal muscle fibres.</a> J Muscle Res Cell Motil. 10 (197), (1989).</li><li>Augusto, V., Padovani, C. R., Campos, G. E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+fiber+types+in+C57Bl6j+mice.">Skeletal muscle fiber types in C57Bl6j mice.</a> Braz J Morphol Sci. 21 (2), 89-94 (2004).</li><li>Schiaffino, S., Reggiani, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiber+types+in+mammalian+skeletal+muscles.">Fiber types in mammalian skeletal muscles.</a> Physiol Rev. 91 (4), 1447-1531 (2011).</li><li>Allen, D. L., Harrison, B. C., Maass, A., Bell, M. L., Byrnes, W. C., Leinwand, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+and+skeletal+muscle+adaptations+to+voluntary+wheel+running+in+the+mouse.">Cardiac and skeletal muscle adaptations to voluntary wheel running in the mouse.</a> J Appl Physiol. 90 (5), 1900-1908 (2001).</li></ol>

The authors have no conflicting interests to disclose.

Here, we have provided useful direction for the identification of skeletal muscle fiber types. In doing so, we describe a novel algorithm for analysis of the data.
Since our results largely confirm those of previous reports5,8,10 and reflect our own manual measurements, the algorithm appears to be accurate. Still, we encountered some infrequent experimental pitfalls including unclear fiber borders resulting from sectioning artifact. The impact of these artifacts can be minimized through careful sectioning and vigilant examination of the processed section prior to analysis. In particular, less-than-optimal image segmentation may be circumvented through multiple rounds of reclassification on the same image to achieve sharper contrast.
The most crucial step in our protocol is step 4.2 in which we stipulate the cellular and extracellular areas of the section by drawing a line on a given fiber, thereby distinguishing a &#34;class 1&#34; or &#34;class 2&#34; area. Obviously, this manual distinction is critical because it determines the boundaries of the muscle fibers; an error in this step will adversely affect the assessment of the fiber dimensions.
In this study, we probed sections for only one MyHC in a given serial section. However, some fibers, such as hybrid fibers, express two types of MyHC, indicative of overlapping metabolic/functional profiles (e.g., type IIX and type IIB). Thus, the fractions of fiber-type summed to a value &#62; 1 for both the soleus and tibialis anterior (Figure 4A). Hybrid fibers can be identified with double-, or even triple-, labelling protocols such as those described by Lee et al.7and Bloemberg and Quadrilatero5, respectively. Importantly, our method of analysis can be applied to these more complex staining protocols. Another limitation of our protocol is that it is not fully automatic; however, we believe that this minor concession ensures that our method does not compromise rigor for the sake of speed.
While the MyHC compositions of both soleus and tibialis anterior muscles observed in this study reflect the results of previous studies5,6,7,10,14, there are some contrasts with earlier studies regarding the assessment of CSA. For example, our measurements of average CSA are somewhat smaller than those reported by either Bloemberg and Quadrilatero5 and Allen et al.15 In particular, our CSA measurements ranged from 539.86 &#177; 24.44 &#181;m2 to 1319.50 &#177; 57.47 &#181;m2 across fiber types whilst theirs spanned roughly 735.7 &#177; 31.2 &#181;m2 to 3073.8 &#177; 51.3 &#181;m2. However, our values more closely resemble those observed by Augusto et al.13 and Lucas et al.6 who reported average CSAs ranging from 436 &#177; 605 &#181;m2 to 2404 &#177; 412.5 &#181;m2 and 815 &#177; 221 &#181;m2 to 1904 &#177; 570 &#181;m2, respectively. Thus, some variability in morphometric analysis of mouse hindlimb fibers exists across the literature.
Immunohistochemistry presents an efficient way to label and classify muscle fibers on the basis of their MyHC content. Accurate quantification of immunohistochemical experiments is essential to making meaningful conclusions about the data; to this end, a method which can automate the tedious parts of analysis is a useful one, especially when hundreds, or even thousands, of fibers need to be measured and classified. In addition to providing measurements similar to manual observations, our algorithm provides an accurate assessment of these data without bias; this advantage over manual measurements ensures scientific rigor. In this regard, our methodology may be useful in the characterization of the effects of genetic and experimental manipulations that impact muscle integrity. Specifically, MyHC immunohistochemistry coupled with our method of rapid analysis may be employed to assess morphological and metabolic changes in muscle of disease models such as muscular dystrophies, ALS and cancer cachexia. Moreover, the efficacy of interventions which prevent atrophy or cause hypertrophy may also be assessed.

It has been known for some time that skeletal muscle is composed of single fibers of many types1. Initially, two groups of fibers were characterized based on their contractile properties and named, appropriately, slow-twitch (type I) and fast-twitch (type II). These categories were further distinguished on the basis of fiber metabolism. Since type I fibers are rich in mitochondria and reliant on oxidative metabolism, they were elucidated by robustly positive nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR) diaphorase2 or succinate dehydrogenase (SDH)3 staining. By contrast, type II fibers exhibited lesser and variable degrees of NADH-TR diaphorase or SDH staining and were divided into two fast-twitch subgroups (type IIA and type IIB) somewhat crudely based on their relative oxidative capacities. These distinctions between fibers have been visualized more effectively by myosin-ATPase staining where type I fibers stain dark after a pre-incubation at pH 4.0 and type IIB fibers absorb precipitate following pre-incubation at pH 10.0 with type IIA fibers staining intermediately4.
More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type5. Type I, type IIA and type IIB fibers can be all identified with precision based on their MyHC profile. In addition, another fast-twitch metabolically-intermediate fiber type, type IIX, has been identified6. Hybrid fibers expressing more than one MyHC have also been confirmed5,7,8. Some species such as the cat and the baboon are known not to express Type IIB MyHCs6. Though MyHC immunostaining is currently the state-of-the-art assessment of muscle composition, the analysis of the data obtained via this technique is cumbersome and time-consuming without automated assistance. To this end, a handful of semi-automated methods to analyze these data have been developed5,9,10. Here, we present a relatively standard protocol for immunohistochemical identification of muscle fiber-type5,7,8,10, along with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology with accuracy.

<table><tbody><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>A9418-100G</td><td>5% in PBS</td></tr><tr><td>Hydrophobic Barrier Pap Pen</td><td>Scientific Device Laboratory</td><td>9804-02</td><td></td></tr><tr><td>Microscope Slides</td><td>Globe Scientific</td><td>1358W</td><td></td></tr><tr><td>Coverglass</td><td>Fisher Scientific</td><td>12-544-E</td><td></td></tr><tr><td>Immumount</td><td>Thermo Scientific</td><td>9990402</td><td></td></tr><tr><td>Nail Polish</td><td>L&#039;Oreal</td><td></td><td></td></tr><tr><td>Nikon Eclipse TE-200 Inverted Fluorescence and Brightfield Microscope</td><td></td><td>Discontinued</td><td></td></tr><tr><td>SPOT RT/KE</td><td>SPOT Imaging Solutions</td><td>RT940</td><td></td></tr><tr><td>Dell Optiplex</td><td></td><td></td><td></td></tr><tr><td>BA-F8 Primary Antibody</td><td>Developmental Studies Hybridoma Bank at the University of Iowa</td><td></td><td>monoclonal mouse IgG2b; 1:50</td></tr><tr><td>SC-71 Primary Antibody</td><td>Developmental Studies Hybridoma Bank at the University of Iowa</td><td></td><td>monclonal mouse IgG1; 1:50</td></tr><tr><td>BF-F3 Primary Antibody</td><td>Developmental Studies Hybridoma Bank at the University of Iowa</td><td></td><td>monoclonal mouse IgGM; 1:50</td></tr><tr><td>6H1 Primary Antibody</td><td>Developmental Studies Hybridoma Bank at the University of Iowa</td><td></td><td>monoclonal mouse IgGM; 1:50</td></tr><tr><td>Alexa Fluor 594 anti-IgG2b</td><td>Invitrogen</td><td>A21145</td><td>goat anti-mouse; 1:200</td></tr><tr><td>Alexa Fluor 488 anti-IgG1</td><td>Invitrogen</td><td>A21121</td><td>goat anti-mouse;1:200</td></tr><tr><td>Alexa Fluor 594 anti-IgGM</td><td>Invitrogen</td><td>A21044</td><td>goat anti-mouse;1:200</td></tr><tr><td>OCT</td><td>Sakura Finetek</td><td>4583</td><td></td></tr><tr><td>isopentane</td><td>Fisher Scientific</td><td>O3551-4</td><td>cool with liguid nitrogen</td></tr><tr><td>PBS</td><td>Fisher Bioreagents</td><td>BP665-1</td><td>10x, dilute to 1x</td></tr><tr><td>Kim wipes</td><td>Kimberly-Clark</td><td>06-666A</td><td></td></tr></tbody></table>

semi-automated analysis of mouse skeletal muscle morphology and fiber-type composition

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p &#62; 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Hindlimb muscles (i.e., tibialis anterior, soleus) dissected from a male C57BL/6 mouse of unknown age were flash frozen by sinking a plastic mold containing the muscle in OCT compound in liquid nitrogen-cooled isopentane. Then, using a cryotome, 8-10 &#181;m serial sections were cut at -20 &#176;C and transferred to different positively-charged glass slides12.
We chose tibialis anterior and soleus because these muscles are predominantly composed of fast- and slow-twitch fibers, respectively13,14. Following immunohistochemical staining for individual type I, IIA, IIX and IIB MyHCs, brightfield and fluorescent images were captured. The left panels of Figure 1 show sections of a soleus muscle immunostained with the type I, IIA, IIX and IIB MyHC-specific antibodies listed in Table 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56024/56024fig1.jpg" /> 
Figure 1: Immunohistochemical staining of MyHC type I, IIA, IIX and IIB in mouse soleus muscle. Left panels show fluorescent images obtained from sections of a mouse soleus muscle probed with primary antibodies directed to type I (BA-F8; A), type IIA (SC-71; B), type IIX (6H1; C) and type IIB (BF-F3; D) MyHCs. Right panels show the sections in which the primary antibody used in the experiment depicted in the corresponding left panel was omitted. Bars= 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56024/56024fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Specifically, we observed substantial fractions of fibers expressing type I (Figure 1A, left) and type IIA MyHCs (Figure 1B, left) with a fair amount of the remaining fibers displaying positive staining for type IIX MyHCs (Figure 1C, left). By contrast, virtually no type IIB fibers were evident (Figure 1D, left). These observations are consistent with earlier reports that soleus muscles are composed mostly of type I, type IIA and type IIX fibers (less so) with almost no type IIB fibers5,10,12. Importantly, no staining was observed in sections in which the primary antibody had been omitted from the protocol, demonstrating the specificity of the antibodies (Figure 1A-D, right panels).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56024/56024fig2.jpg" /> 
Figure 2: Immunohistochemical staining of MyHC type IIB, IIX, IIA and I in mouse tibialis anterior muscle. Left panels show fluorescent images obtained from sections of a mouse tibialis anterior muscle probed with primary antibodies directed to type IIB (A), type IIX (B), type IIA (C) and type I (D) MyHCs. Right panels show the sections in which the primary antibody used in the experiment depicted in the adjacent left panel was omitted. Bars = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56024/56024fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
We also stained tibialis anterior muscles with the aforementioned antibodies. Tibialis anterior muscles were composed mostly of type IIB and type IIX MyHC-positive fibers (Figure 2A-B, left panels) with smaller contributions from fibers expressing type IIA MyHCs (Figure 2C, left); virtually no fibers expressing type I MyHC were observed (Figure 2D, left). Our data indicating that the mouse tibialis anterior is composed predominantly of fast twitch type IIA, type IIB and type IIX fibers are consistent with earlier studies5,10,13. Again, no staining was observed in the sections in which the primary antibody had been omitted from the protocol (Figure 2A-D, right panels).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56024/56024fig3.jpg" /> 
Figure 3: Iterated computerized fiber segmentation. Brightfield image of a mouse tibialis anterior section (A). Transformation of the same image using our semi-automated algorithm (B). Fluorescent image of the same section showing immunostaining of type IIA MyHC (C). Transformation image showing only type IIA MyHC-expressing fibers (D). Bars = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56024/56024fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For the purpose of morphometric analysis, brightfield images of the immunostained fields were also obtained. The example shown in Figure 3A is from a tibialis anterior muscle section probed with SC-71 antibodies directed to type IIA MyHCs13. Using our original semi-automated algorithm, several image transformations were used to generate probability maps from the brightfield images. A typical image transformation derived from the brightfield image shown in Figure 3A is presented in Figure 3B. Using the transformation and the pore analysis function of Fiji, we measured the mean CSA (891.4 &#177; 45.0 &#181;m2), maximum (46.9 &#177; 1.3 &#181;m) and minimum (26.0 &#177; 0.8 &#181;m) mean Feret diameters of each fiber in the field. To test the fidelity of our measurements, we also assessed these parameters in the brightfield image manually. The mean CSA (867.0 &#177; 52.0 &#181;m2), maximum (45.7 &#177; 3.5 &#181;m) and minimum (25.6 &#177; 1.9 &#181;m) mean Feret diameters obtained using the more tedious manual acquisition method were not significantly different from the semi-automated method (all p &#62; 0.05, unpaired t-tests; Table 3).
<img alt="Table 3" src="/files/ftp_upload/56024/56024table3.jpg" /> 
Table 3: Fiber morphology and composition of tibialis anterior muscle immunostained for type I, IIA, IIB and IIX MyHCs as determined by manual and semi-automatic analytic methods. Mean fiber CSA, mean Feret diameters and MyHC type in a mouse tibialis anterior quantified via traditional manual evaluation versus the presented algorithm. Please note that combined fractions exceed a value of 1 due to the presence of fibers expressing more than one MyHC type.
Figure 3C shows a fluorescent image of the same field immunostained with antibodies directed to type IIA MyHCs. Positively-stained fibers in the field were selected by number (Figure 3D) and used to determine the fraction of type IIA MyHC-expressing fibers by dividing by the total number of fibers in the brightfield transformation field (Table 3). The average CSA, maximum and minimum Feret diameters of type IIA fibers were then assessed (Table 3). The proportion of type I, type IIX and type IIB fibers, as well as their dimensions, were determined by repeating this protocol in serial sections immunostained with BA-F8, 6H1 and BF-F3 antibodies, respectively6,12(Table 4; Figure 4A-D).
<img alt="Table 4" src="/files/ftp_upload/56024/56024table4.jpg" /> 
Table 4: MyHC content and morphology in mouse soleus and tibialis anterior. Fiber type compositions of representative sections of the soleus and tibialis anterior. Quantification of average CSA, minimal and maximal Feret diameters of type I, type IIA, type IIX and type IIB in both the soleus and tibialis anterior are shown. Data are given as mean &#177;SEM.
We also assessed these parameters in a field obtained from the soleus muscle (Table 4; Figure 4A-D). 3,052 and 2,876 individual fibers were counted for the tibialis anterior and soleus muscles, respectively. Taken together, these data demonstrate the feasibility of our method in fiber-type and morphometric analysis.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56024/56024fig4.jpg" /> 
Figure 4: Morphometric analysis data summary. Fiber-type compositions of representative sections of the tibialis anterior and soleus muscles (A). Quantification of average CSA, minimimal and maximal Feret diameters of type I, type IIA, type IIX and type IIB fivers in both the tibialis anteriort and soleus are shown in (B-D). Data are given as mean &#177;SEM. Significant differences between the tibialis anterior and soleus are indicated (*denotes p &#60; 0.05: ** denotes p&#160;&#60; 0.01; *** denotes p&#160;&#60; 0.005; t-test). 3,052 and 2,876 individual fibers were counted for the tibialis anterior and soleus muscles, respectively. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56024/56024fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental File" src="/files/ftp_upload/56024/56024supfile.jpg" /> 
Supplemental File: Fiber quantification macro. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56024/Tyagi_etal_fiber_quantification_macro" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition at JoVE.com

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology.

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

semi-automated-analysis-mouse-skeletal-muscle-morphology-fiber-type

Nanyang Technological University

Research

JoVE Journal

Medicine

10.4K Views.  University of Colorado School of Medicine. Immunohistochemische kleuring van myosin zware ketting isoforms heeft ontpopt als de state-of-the-art-discriminator van skeletale spiervezel-type (d.w.z., typ ik typ IIA, typ IIX, type IIB). Hier presenteren we een kleuring protocol samen met een nieuwe semi-automatische algoritme, dat vergemakkelijkt de snelle beoordeling van de morfologie van het type vezel en vezels.

Video: Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

MyDoBID- A Novel Technique for Identifying Muscle Fiber Types Efficiently

Fiber Type Identification of Human Skeletal Muscle

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot blotting, hereafter referred to as Myosin heavy chain detection by Dot Blotting for IDentification of muscle fiber type (MyDoBID). This protocol describes the process of freeze-drying human skeletal muscle and isolating segments of single muscle fibers. Using MyDoBID, type I and II fibers are classified with MHCI- and IIa-specific antibodies, respectively. Classified fibers are then combined into fiber type-specific samples for each biopsy.
The total protein in each sample is determined by Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and UV-activated gel technology. The fiber type of samples is validated using western blotting. The importance of performing protein loading normalization to enhance target protein detection across multiple western blots is also described. The benefits of consolidating classified fibers into fiber type-specific samples compared to single-fiber western blots, include sample versatility, increased sample throughput, shorter time investment, and cost-saving measures, all while retaining valuable fiber type-specific information that is frequently overlooked using homogenized muscle samples. The purpose of the protocol is to achieve accurate and efficient identification of type I and type II fibers isolated from freeze-dried human skeletal muscle samples.
These individual fibers are subsequently combined to create type I and type II fiber type-specific samples. Furthermore, the protocol is extended to include the identification of type IIx fibers, using Actin as a marker for fibers that were negative for MHCI and MHCIIa, which are confirmed as IIx fibers by western blotting. Each fiber type-specific sample is then used to quantify the expression of various target proteins using western blotting techniques.

Author Spotlight: Deciphering the Mysteries of Skeletal Muscle Fiber Types Using the MyDoBID Technique 

This protocol demonstrates single-fiber isolation from freeze-dried human skeletal muscle and fiber-type classification according to Myosin heavy chain (MHC) isoform using the dot blotting technique. Identified MHC I and II fiber samples can then be further analyzed for fiber type-specific differences in protein expression using western blotting.

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot ...

Biochemistry

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

Neuroscience

Fiber Type and Subcellular-Specific Analysis of Lipid Droplet Content in Skeletal Muscle

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative prognostic factor for several other disorders such as cardiovascular disease and cancer. Excessive lipid infiltration decreases muscle mass and strength. It also results in lipotoxicity and insulin resistance depending on total intramyocellular lipid content, lipid droplet (LD) morphology, and subcellular distribution. Fiber type (oxidative vs glycolytic) is also important, since oxidative fibers have a greater capacity to utilize lipids. Because of their crucial implications in pathophysiology, in-depth studies on LD dynamics and function in a fiber type-specific manner are warranted.
Herein, a complete protocol is presented for the quantification of intramyocellular lipid content and analysis of LD morphology and subcellular distribution in a fiber type-specific manner. To this end, serial muscle cryosections were stained with the fluorescent dye Bodipy and antibodies against myosin heavy chain isoforms. This protocol enables the simultaneous processing of different muscles, saving time and avoiding possible artifacts and, thanks to a personalized macro created in Fiji, the automatization of LD analysis is also possible.

Increasing evidence indicates that excessive infiltration of lipids inside skeletal muscle results in lipotoxicity and diabetes. Here, we present a complete protocol, including tissue processing, staining with Bodipy, image acquisition, and analysis, to quantify the size, density, and subcellular distribution of lipid droplets in a fiber-type specific manner.

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Biology

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches involve either differentiating myogenic progenitor cells into immature myotubes or the short-term ex vivo culture of isolated individual muscle fibers. A key benefit of ex vivo culture over in vitro is the retention of the complex cellular architecture and contractile characteristics. Here, we detail an experimental protocol for the isolation of intact flexor digitorum brevis muscle fibers from mice and their subsequent ex vivo culture. In this protocol, muscle fibers are embedded in a fibrin-based and basement membrane matrix hydrogel to immobilize the fibers and maintain their contractile function. We then describe methods to assess the muscle fiber contractile function using an optics-based, high-throughput contractility system. The embedded muscle fibers are electrically stimulated to induce contractions, after which their functional properties, such as sarcomere shortening and contractile velocity, are assessed using optics-based quantification. Coupling muscle fiber culture with this&#160;system allows for high-throughput testing of the effects of pharmacological agents on contractile function and ex vivo studies of genetic muscle disorders. Finally, this protocol can also be adapted to study dynamic cellular processes in muscle fibers using live-cell microscopy.

Skeletal muscle function can be assessed by quantifying the contractility of isolated muscle fibers, traditionally using laborious, low-throughput approaches. Here, we describe an optics-based, high-throughput method to quantify the contractility of hydrogel-embedded muscle fibers. This approach has applications for drug screening and therapeutic development.

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches ...

Enzymatic Isolation and Characterization of Variable Length Mouse Muscle Fibers

Intact Short, Intermediate, and Long Skeletal Muscle Fibers Obtained by Enzymatic Dissociation of Six Hindlimb Muscles of Mice: Beyond Flexor Digitorum Brevis

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal with the short fibers of the flexor digitorum brevis (FDB), which restrains the scope of results dealing with fiber types, limits the amount of biological material available, and impedes a clear connection between cellular physiological phenomena and previous biochemical and dynamical knowledge obtained in other muscles.
This paper describes how to obtain intact fibers from six muscles with different fiber type profiles and lengths. Using C57BL/6 adult mice, we show the muscle dissection and fiber isolation protocol and demonstrate the suitability of the fibers for Ca2+ transient studies and their morphometric characterization. The fiber type composition of the muscles is also presented. When dissociated, all muscles rendered intact, living fibers that contract briskly for more than 24 h. FDB gave short (&#60;1 mm), peroneus digiti quarti (PDQA) and peroneus longus (PL) gave intermediate (1-3 mm), while extensor digitorum longus (EDL), extensor hallucis longus (EHL), and soleus muscles released long (3-6 mm) fibers.
When recorded with the fast dye Mag-Fluo-4, Ca2+ transients of PDQA, PL, and EHL fibers showed the fast, narrow kinetics reminiscent of the morphology type II (MT-II), known to correspond to type IIX and IIB fibers. This is consistent with the fact that these muscles have over 90% of type II fibers compared with FDB (~80%) and soleus (~65%). Moving beyond FDB, we demonstrate for the first time the dissociation of several muscles, which render fibers spanning a range of lengths between 1 and 6 mm. These fibers are viable and give fast Ca2+ transients, indicating that the MT-II can be generalized to IIX and IIB fast fibers, regardless of their muscle source. These results increase the availability of models for mature skeletal muscle studies.

Author Spotlight: Isolation of Long Muscle Fibers from Mouse Hindlimb Muscles for Studying Excitation-Contraction Coupling Across Fiber Types

We describe a protocol to obtain enzymatically dissociated fibers of different lengths and types from six muscles of adult mice: three of them already described (flexor digitorum brevis, extensor digitorum longus, soleus) and three of them successfully dissociated for the first time (extensor hallucis longus, peroneus longus, peroneus digiti quarti).

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal ...

Real-Time Quantification of Mitochondrial Respiration in Muscle Fibers

Measurement of Mitochondrial Respiration in Human and Mouse Skeletal Muscle Fibers by High-Resolution Respirometry

Mitochondrial function, a cornerstone of cellular energy production, is critical for maintaining metabolic homeostasis. Its dysfunction in skeletal muscle is linked to prevalent metabolic disorders (e.g., diabetes and obesity), muscular dystrophies, and sarcopenia. While there are many techniques to evaluate mitochondrial content and morphology, the hallmark method to assess mitochondrial function is the measurement of mitochondrial oxidative phosphorylation (OXPHOS) by respirometry. Quantification of mitochondrial OXPHOS provides insight into the efficiency of mitochondrial oxidative energy production and cellular bioenergetics. A high-resolution respirometer provides highly sensitive, robust measurements of mitochondrial OXPHOS in permeabilized muscle fibers by measuring real-time changes in mitochondrial oxygen consumption rate. The use of permeabilized muscle fibers, as opposed to isolated mitochondria, preserves mitochondrial networks, maintains mitochondrial membrane integrity, and ultimately allows for more physiologically relevant measurements. This system also allows for the measurement of fuel preference and metabolic flexibility - dynamic aspects of muscle energy metabolism. Here, we provide a comprehensive guide for mitochondrial OXPHOS measurements in human and mouse skeletal muscle fibers using a high-resolution respirometer. Skeletal muscle groups are composed of different fiber types that vary in their mitochondrial fuel preference and bioenergetics. Using a high-resolution respirometer, we describe methods for evaluating both aerobic glycolytic and fatty acid substrates to assess fuel preference and metabolic flexibility in a fiber-type-dependent manner. The protocol is versatile and applicable to both human and rodent muscle fibers. The goal is to enhance the reproducibility and accuracy of mitochondrial function assessments, which will improve our understanding of an organelle important to muscle health.

Author Spotlight: Unveiling Mitochondrial Function and Cellular Metabolic Adaptation in Metabolic Diseases

Here, we describe a comprehensive method for measuring mitochondrial oxidative phosphorylation in fresh permeabilized skeletal muscle fibers from either human or mouse muscle. This method allows for the real-time quantification of mitochondrial respiration and the assessment of fuel preference and metabolic flexibility while preserving existing mitochondrial networks and membrane integrity.

Mitochondrial function, a cornerstone of cellular energy production, is critical for maintaining metabolic homeostasis. Its dysfunction in skeletal muscle ...

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

Samenvatting

Meer video's verkennen