Abstract
Skeletal muscles of the mammalian trunk and limbs comprise myofibers that express four types of myosin heavy-chain (MyHC) isoforms, each with distinct contractile and metabolic properties. Despite histochemical and immunohistochemical staining to identify myofiber types, all myofiber types cannot be identified simultaneously in vivo. In this study, we generated a novel knock-in mouse model, termed “MusColor,” that enables the simultaneous identification of individual MyHC isoforms through the expression of four fluorescent proteins. The identification of fibre types by fluorescent expression in MusColor mice was consistent with that achieved by immunostaining and had higher sensitivity. By studying the aging-associated changes in myofiber types using the MusColor mice, we were able to identify changes in hybrid myofibers that simultaneously express multiple MyHCs. Furthermore, by culturing satellite cells isolated from MusColor mice and treatment of thyroid hormone or rapamycin, changes in myofiber type and metabolic function could be analysed in living cells. The MusColor mouse proved useful for elucidating the mechanisms of muscle fibre changes caused by diseases such as sarcopenia, neuromuscular and metabolic diseases, as well as by exercise and nutritional environments.
Introduction
Based on their primary composition of slow-twitch or fast-twitch fibres, skeletal muscles are classified into two main groups: slow-twitch (red) muscle and fast-twitch (white) muscles. Each muscle contains myofibers that express a combination of myosin heavy chains (MyHC) alongside those with a singular MyHC profile1. Capillary-rich slow-twitch muscles primarily mediate activities that require prolonged exertion such as endurance exercises and maintenance of optimal body posture2. In contrast, fast-twitch muscles are engaged during resistance training and other activities that necessitate high strength levels. Skeletal muscles regulate body temperature through heat production and, following exercise-induced stimulation, secrete various cytokines (myokines) that contribute to metabolic regulation not only of skeletal muscles but also of other organs3,4,5,6,7.
In most mammals, skeletal muscle is composed of four types of myofibers; each type has different contractile and metabolic properties and expresses myosin heavy chain (MyHC) isoforms with different ATPase activities8. Type I (Myh7) is a slow-twitch myofiber that depends on oxidative metabolism with a slow-twitch contraction rate. Type IIB (Myh4) is a fast-twitch myofiber that depends on glycolytic metabolism with a fast-twitch contraction rate. Types IIA (Myh2) and IIID/X (Myh1) exhibit the characteristics of both type I and IIB myofibers. Human muscle, like that of other large mammals, contains only type 1, 2 A and 2X myofibers9. In vitro motility assays of single myofibers have demonstrated that the myosin composition of myofibers mediates the speed of fibre shortening and the power of contraction10,11. The characteristics of muscle stem cells (also referred to as satellite cells) that differentiate into fast- and slow-twitch myofibers are distinct and heterogeneous12,13. The specification of fibre types during postnatal development and in adults is subject to further modification by motor neuron activity and diverse hormones, particularly thyroid hormones, in addition to the nutritional environment1. Furthermore, muscle weakness and atrophy caused by various underlying pathological conditions, including sarcopenia, neuromuscular disorders, metabolic diseases, hormonal imbalances, and drug abuse, reduce fibre type plasticity, which leads to a specific ratio of fast-twitch-to-slow-twitch myofibers14. The precise causal relationship between muscle wasting and these fibre-type shifts remains unclear.
Although the signalling pathways and transcription factors that determine fibre types have been identified, their precise function remains unelucidated owing to the inability to visualize the conversion of myofiber types in living cells1,15. Two analytical methods have been employed for the examination of images of the type of myofibers in muscle Sect16. One method is a histochemical staining technique that identifies differences in ATPase activity among MyHC isoforms17. The other is immunohistochemical staining, which utilizes specific antibodies against each MyHC isoform to detect antigenic proteins in frozen transverse sections of skeletal muscle and isolated myofibers16,18. However, these methods cannot identify all four types of living muscle cells simultaneously, are incapable of quantitatively analysing myofiber running and MyHC expression changes within the muscle on three-dimensional images. Recently, two novel omics analysis-based methodologies—single-fibre proteomics and single-nucleotide transcriptomics19,20,21,22,23—have identified genes and proteins that distinguish among myofiber types. Although these analytical techniques evaluate myofiber status at the point of collection, they cannot assess concomitant metabolic function and fibre-type alterations in living muscle cells. To address these technical challenges, we used knock-in mice and developed an experimental system that permits the fluorescent protein-based visualization of all four fibre types.
Results
Visualization of MyHC proteins in skeletal muscles of muscolor mice
To identify myofibers that express each of the four-myosin heavy-chain genes (MyHC; Myh7, Myh2, Myh1, and Myh4) in mice with different fluorescent proteins, knock-in mice expressing each of the four fusion proteins were generated. To the 5’ end of each MyHC gene (Myh7, Myh2, Myh1, and Myh4), we added a yellow fluorescent protein (EYFP)24, an ultramarine fluorescent protein (Sirius)25, an enhanced cyan fluorescent protein (Cerulean)26, and a red fluorescent protein (mCherry) gene27, respectively (Fig. 1A–D). Mice expressing EYFP-Myh7, Sirius-Myh2, Cerulean-Myh1, and mCherry-Myh4 at their native promoters were named “MusColor” mice. These mice were born with the expected Mendelian ratios and showed no obvious abnormalities. Plantaris (PLA) and soleus (SOL) skeletal muscles of the lower limb of MusColor mice were collected and four fluorescent proteins were identified under a fluorescence stereomicroscope. PLA is a fast-twitch muscle composed of type IIA, IID/X, and IIB myofibers, only type IIA-Sirius was positive EYFP-Myh7 / Sirius-Myh2 heterozygous mice (Fig. 2A), whereas in Cerulean-Myh1 / mCherry-Myh4 heterozygous mice both type Cerulean and mCherry were positive (Fig. 2B). In contrast, SOL is a slow-twitch muscle composed primarily of type I and type IIA myofibers. Both EYFP and Sirius are positive in EYFP-Myh7 / Sirius-Myh2 heterozygous mice (Fig. 2A). However, in SOL, the Cerulean-Myh1 / mCherry-Myh4 heterozygous mice tested locally positive for Cerulean and were negative for mCherry (Fig. 2B). Freshly isolated myofibers from the SOL muscle of EYFP-Myh7 / Sirius-Myh2/ Cerulean-Myh1 / mCherry-Myh4 heterozygous mice were distinguishable based on their fluorescent expression (Fig. 2C). As shown in Fig. 6D, type IIB myofibers constitute a minor percentage of soleus muscles. Figure 2C shows myofibers taken from this site, but as Fig. 2B shows, it is at the limit of detection with a fluorescence stereomicroscope. These observations are consistent with the expected results.
Fig. 1
figure 1
Generation of MyHC gene fusion alleles. Diagram of wild-type MyHC genes (top) and targeting constructs (bottom) for EYFP-Myh7 (A), Sirius-Myh2 (B), Cerulean- Myh1 (C), and mCherry- Myh4 (D) knock-in mice. Open boxes represent 5’ UTRs and solid boxes represent translated exons. The coding sequence of fluorescent proteins is shown as colored boxes. PGK-gb2-neomycin cassettes flanked by FRT sequences and PGK-puromycin cassettes flanked by loxP sequences were inserted into the targeting vectors for positive selection.
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Fig. 2
figure 2
Expression of MyHC gene fusion proteins in freshly isolated PLA and SOL muscles. The images shown are the observation of whole muscles (PLA and SOL) from (A) a Myh7–EYFP / Myh2–Sirius heterozygous mouse (female, 14 months old. n = 1) and (B) a Myh1-Celurean / Myh4-mCherry heterozygous mouse (female, 17 months old, n = 1) using a fluorescence stereomicroscope. Scale bar = 4 mm. (C) Freshly isolated myofibers from the SOL muscle of a Myh7-YFP/Myh2-Sirius/Myh1-Cerulean/Myh4-mCherry mouse (female, 17 months old, n = 1), observed under a fluorescence microscope. Scale bar = 300 μm.
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Protein signal- and antibody staining-stratified MyHC level
Next, frozen cross sections were prepared from skeletal muscles from MusColor mice and analysed to determine whether the knock-in fluorescent protein was expressed in each myofiber type. The gastrocnemius (GAS) muscle of EYFP-Myh7 / Sirius-Myh2 heterozygous mice and the tibialis anterior (TA) muscle of the Cerulean-Myh1 / mCherry-Myh4 heterozygous mice were immunohistochemically stained with primary antibodies specific for the four myosin heavy-chain isoforms as well as with secondary antibodies labelled with a fluorescent dye whose excitation wavelength differs from that of the knock-in fluorescent protein. An anti-myosin heavy-chain antibody was used to stain each muscle section. Consequently, myofibers of type I-EYFP (GAS), IIA-Sirius (GAS), and IIB-mCherry (TA) were identified with 100% probability by fluorescent immunostaining of each fluorescent protein (Fig. 3A,B,D,E,F,H). In contrast, the percentage of type IID/X identified by fluorescent immunostaining of type IID/X-Cerulean (TA) myofibers was 77% (Fig. 3C,G). The sensitivity of fluorescent protein (Cerulean) to identify myofiber type in MusColor mice was higher than that of the fluorescent antibody method. The amount of fluorescence expressed by fluorescent proteins is proportional to the amount of endogenous MyHC protein, so even low levels of expression can be observed down to the detection limit of light microscopy. On the other hand, when using the fluorescent antibody staining method, the detection limit varies depending on the staining conditions for sections or isolated myofibers. Previous studies have demonstrated the presence of hybrid-type myofibers using immunohistochemistry and in situ hybridization analysis of muscle Sects8,23. The expression of fluorescent proteins in each myofiber that tested positive in fluorescence immunostaining was analysed using frozen sections of MusColor mouse muscle. In IIA-Sirius (GAS) myofibers, 15.1% tested immunohistochemically positive for IIB/X (Fig. 3B,F), whereas in IIB/X-Cerulean (TA) myofibers, 37.5% were IIA-positive and 30% were IIB-positive (Fig. 3C,G). Type IID/X was positive in 11.5% of IIB-mCherry (TA) myofibers (Fig. 3D,H).
Fig. 3
figure 3
Comparison of MyHC expression by detection of fluorescent protein signals and antibody staining. (A, B) Cross sections of GAS muscle from EYFP-Myh7 / Sirius-Myh2 heterozygous male mice (15 months old) and (C, D) TA muscles from Cerulean- Myh1 / mCherry-Mhy4 heterozygous male mice (15 months old), respectively. Sections were stained with antibodies that recognize individual MyHC isoforms (I, IIA, IID/X and IIB). Myofibers positive for both fluorescent protein and antibody are indicated by *. Scale bar = 100 μm. (E–H) Ratios of antibody-stained fibres in EYFP (+), Sirius (+), Cerulean (+), and mCherry (+) fibres, respectively. Values are presented as mean ± SE (n = 3).
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Analysis of myofiber types that change with age
Although many pathological studies have shown that aging induces changes in the proportion of myofiber types in skeletal muscle, the simultaneous observation of all four myofiber types with different fluorescent protein tags has not been reported before14. Therefore, MusColor mice were crossed to obtain male mice heterozygous for expressing EYFP-Myh7 / Sirius-Myh2 / Cerulean-Myh1 / mCherry-Myh4. We collected SOL and PLA from young (6 months old), middle-aged (14 months old) and old (26–29 months old) MusColor mice and prepared frozen cross sections. Using fluorescence microscopy, the ratio of each myofiber type relative to the total number of myofibers was quantified through a quantitative analysis of fluorescence image data of four types of myofibers obtained simultaneously (Figs. 4, 5 and 6). Figure 6 shows the statistical analysis of the four mice, including the mouse used in Figs. 4 and 5. A comparison of myofiber composition in SOL from the old group versus those from the middle-aged group revealed a statistically significant increase in type I-EYFP myofibers in the former (from 34.8 to 48.8%; Fig. 6A), accompanied by a corresponding decrease in type IIA-Sirius myofibers (from 61.4 to 48.7%; Fig. 6B). In contrast, the type IID/X-Cerulean and IIB-mCherry myofibers did not demonstrate a significant change in the three groups (Fig. 6C,D). These results were consistent with our previous results obtained by immunohistochemistry28. In PLA, the ratio of type IID/X-Cerulean myofibers increased significantly (Fig. 6G) in the middle-aged group (30.1%) and old group (32.8%), as compared to the young group (0.6%). The ratio of type IIB-mCherry myofibers was higher in the young group (65.2%) than in the middle-aged group (39%). Conversely, the old group demonstrated a considerable degree of variability and exhibited no statistically significant difference in comparison to the young and middle-aged groups (Fig. 6H). For type I-EYFP and type IIA-Sirius myofibers, no statistically significant difference in variation was observed among the three groups (Fig. 6E,F).
Fig. 4
figure 4
Cross sections of whole muscles from four-color MusColor mice. Fluorescent images of cross sections of (A) SOL and (B) PLA muscles from EYFP-Myh7 / Sirius-Myh2 / Cerulean-Myh1 / mCherry-Myh4 heterozygous male mice. Young (6 months old, n = 1); Middle (14 months old, n = 1); Old (26–29 months old, n = 1). (Scale bar = 400 μm)
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Fig. 5
figure 5
Fluorescence superimposed images of all myofiber types. Magnified fluorescence images of cross sections of SOL (A, B and C) and PLA (D, E and F) muscles from EYFP-Myh7 / Sirius-Myh2 / Cerulean-Myh1 / mCherry-Myh4 heterozygous male mice. (A, B and C) Hybrid myofibers are indicated with *. (D, E and F) Type IID/X / IIB hybrid myofibers are indicated with #, and Type IIA / IID/X hybrid myofibers are indicated with *. Young (6 months old, n = 1); Middle (14 months old, n = 1); Old (26–29 months old, n = 1). (Scale bar = 100 μm)
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The change in the ratio of hybrid myofibers expressing multiple MyHC simultaneously in SOL and PLA with age was examined using the identical imaging data set (Fig. 6I -N). In SOL, compared to the young (0.2%) and middle-aged group (0.8%), the old group (2.7%) showed an increase in type I-EYFP / IIA-Sirius / IID/X-Cerulean triple-hybrid myofibers (Fig. 6I). However, there was no significant intergroup difference in the percentage of type I-EFYP / IIA-Sirius, type I-EFYP / IID/X-Cerulean, and type IIA-Sirius / IID/X-Cerulean hybrid myofibers (Fig. 6J–L). In PLA, the ratio of hybrid myofibers expressing type IID/X-Cerulean / IIB-mCherry was higher in the old groups (12%) compared to the young group (0%) (Fig. 6M). Furthermore, the ratio of type IIA-Sirius / IID/X-Cerulean hybrid-type myofibers increased in the old (3.3%) group compared to the young group (0%; Fig. 6N). The total number of myofibers in each age group remained unchanged in the SOL (Fig. 6O), whereas, in the PLA, there was a significant increase in the middle-aged (841 fibres) and old (806 fibres) groups, as compared to the young (543 fibres) group (Fig. 6P). These results indicate that the MusColor mice can be used for sensitive and quantitative analyses of aging-associated changes in the myofiber types in skeletal muscles, including the hybrid myofibers.
Fig. 6
figure 6
Changes in expression ration of myofiber types in four-color MusColor mice with aging. The ratio of (A) type I, (B) type IIA, (C) type IID/X and (D) type IIB, (I) type I / IIA / IID/X triple-hybrid, (J) type I / IIA hybrid, (K) type I / IID/X hybrid and (L) type IIA / IID/X hybrid myofiber to the total number of myofiber in SOL muscles. The ratio of (E) type I, (F) type IIA, (G) type IID/X and (H) type IIB fiber, (M) type IID/X / IIB hybrid, (N) type IIA / IID/X hybrid myofiber to the total number of myofibers in PLA muscles. Number of total myofibers in (O) SOL and (P) muscles. Young (male, 6 months old, n = 4); Middle (male, 14 months old, n = 4); Old (male, 26–29 months old, n = 4). One-way ANOVA followed by Tukey’s multiple tests was performed for statical analysis. *P < 0.05, **P < 0.01, ****P < 0.0001. Values are presented as means ± SE.
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Fibre type and metabolic analysis of muscolor muscle cells
We hypothesized that muscle cells from MusColor mice could help visualize the changes in myofiber types in vitro. To confirm this possibility, satellite cells were isolated by fluorescence-activated cell sorting (FACS) from the skeletal muscle of male mice heterozygously expressing type I-EYFP / IIA-Sirius / IIX/D-Cerulean / IIB-mCherry and simian virus 40 (SV40) large T antigen (tsA58) genes. The SV40 large T antigen (tsA58) protein remains active under 33 °C cell-culture conditions, and thus immortalizes the expressing cells. However, when the culture temperature is increased to 37 °C, the tsA58 protein is inactivated, which results in cell growth arrest and the induction of differentiation29. The collected satellite cells were cultivated in growth medium at 33 °C and subsequently differentiated in differentiation medium at 37 °C for 72 h. Thereafter, images were recorded in a time-lapse sequence, 25 times every 2 h, with a fluorescence microscope with fluorescent filters that separate the respective fluorescent colours. Myotubes expressing type I-EYFP, IIX/D-Cerulean, and IIB-mCherry upon differentiation induction were successfully observed alive; however, no myotubes expressing Type IIA-Sirius were observed (Supplementary movies).
Previous studies have demonstrated that the administration of rapamycin or the thyroid hormone T3 (3,3′,5-triiodo-L-thyronine) to mice increases the proportion of Type I and Type IIB myofibers, respectively30,31. Accordingly, we investigated whether rapamycin or T3 induce type I and IIB differentiation of myotubes derived from MusColor mice, respectively. For this study, satellite cells were isolated by FACS from skeletal muscles of male mice heterozygously expressing type I-EYFP or IIB-mCherry with simian virus 40 (SV40) large T antigen (tsA58) genes. After induction of myotubes derived from the MusColor mice at 37 °C in vitro, rapamycin or T3 were added to the culture medium and fluorescent images were acquired using a microscope to analyse fibre-type changes in myotubes (Fig. 7A). The results showed that in myotubes treated with rapamycin, the myotube area of type I-EYFP increased (from 55.4 to 69.8%), whereas that of IIB-mCherry decreased (from 3.4 to 1.1%; Fig. 7B and D). In contrast, T3 significantly increased the myotube cell area of type IIB-mCherry (from 3.4 to 10.2%), while it decreased the area of I-EYFP myotubes (from 55.4 to 23.9%) (Fig. 7C,D). These findings are consistent with previous observations of myofiber type alterations in mice subjected to rapamycin and T3 treatment in vivo30,31. The Extracellular Flux Analyzer was then used to assess changes in mitochondrial function and glycolytic capacity in cultured myotubes by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) (Fig. 7E,K). For this study, satellite cells were isolated by FACS from skeletal muscles of male mice heterozygously expressing SV40 large T antigen (tsA58) gene. The results showed that rapamycin significantly reduced mitochondrial functions, including basal respiration, maximal respiration, ATP production, and reserve respiratory capacity, compared to the control group (Fig. 7E–G,I,J). In contrast, there was no significant change in proton leak (Fig. 7H). Regarding glucose metabolism, rapamycin was found to significantly decrease glycolysis, glycolytic capacity, and glycolytic reserve (Fig. 7K–N). The same analysis was performed for T3, which did not cause significant changes in mitochondrial and glycolytic function (Fig. 7E–N). The extracellular flux analyzer demonstrated that rapamycin reduced mitochondrial and glycolytic functions. The number of IIB myofibers increased when T3 was added; however, contrary to expectations, it did not induce any changes in mitochondrial functions and glycolytic metabolism. In this study, we used a standard culture medium recommended by the measurement kit, which consists of a culture medium containing a certain amount of glucose, pyruvate, and glutamine, but it is possible that changes will be observed if the concentrations of fatty acids, glucose, and glutamine are changed32,33. Mesenchymal stem cells and immune cells may be necessary for the metabolic changes of type IIB myofibers.
Fig. 7
figure 7
Fibre-type changes and metabolic shift of cultured myotubes by rapamycin and T3. (A) Study design for induction of myotube differentiation and metabolic analysis by addition of rapamycin and T3 to the cultures. Percentage of area of (B) type I (EYFP) and (C) type IIB (mCherry) myotubes to total cell area after addition of rapamycin or T3. (D) Fluorescence images of myotube cells after addition of rapamycin or T3 (Scale bar = 200 mm). A statistical analysis was conducted on eight to ten image data sets obtained from three wells in each experimental group. The same analysis was performed three times to confirm the reproducibility. (E) The effect of rapamycin or T3 on oxygen consumption rate (OCR) in myotube cells over time was examined using the Extracellular Flux Analyzer. The following parameters were calculated from the OCR values [shown in (E)]: (F) basal respiration, (G) maximal respiration, (H) proton leak, (I) ATP production, and (J) reserve respiratory capacity. (K) The effect of rapamycin or T3 on extracellular acidification rate (ECAR) in myotube cells over time was examined using the Extracellular Flux Analyzer. (L) glycolysis (basal glycolysis), (M) glycolytic capacity (maximal glycolysis) and (N) glycolytic reserve were calculated from the ECAR values shown in (K). (E–N) Statistical analysis was performed on the data sets obtained from 6–7 wells in each experimental group. The same analysis was performed three times to confirm reproducibility. One-way ANOVA followed by Tukey’s multiple tests was performed for statical analysis. **p < 0.01, ***p < 0.001, ****p < 0.0001. Values are presented as means ± SE.
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Discussion
In this study, we have generated MusColor mice, which are knock-in mice that express EYFP-Myh7, Sirius-Myh2, Cerulean-Myh1, and mCherry-Myh4 in their myofibers. Whole skeletal muscle and muscle sections from MusColor mice provide sensitive and quantitative analysis of fibre types without fixation or staining. Our findings demonstrate that the fusion genes accurately reflect the expression of wild-type MyHC in myofibers, in accordance with the anticipated regulatory patterns observed in fast-twitch and slow-twitch muscles. Furthermore, satellite cells derived from MusColor mice proved useful for the analysis of myofiber types and metabolic functions during differentiation into myotubes.
In previous studies, three transgenic mice were developed for identifying MyHC-expressing skeletal myofibers15. The use of cyan and red fluorescent proteins enabled the distinction of type I fibres from IIA and IIDX/IIB myofiber types. The transgenic mouse expresses a fluorescent protein that has been inserted into the start codon of MyHC in the introduced bacterial artificial chromosome (BAC). Therefore, this expression is superfluous regarding endogenous MyHC genes. Instead, knock-in of the YFP gene into the β-myosin heavy-chain gene (β-Myh) results in the expression of the transgene in cardiac muscle as normal β-MyHC, without any impairment of function, in mice34,35. Given that β-Myh and Myh7 are identical gene, it was hypothesized that other knock-in mice with fluorescent proteins for Myh2, Myh1 and Myh4 could be generated using a similar approach. It has been reported that mice with fluorescent proteins in the Myh7 or Myh1 genes can be used to observe the expression of myosin isoforms in living cells36. However, there have been no reports of mice that are able to distinguish between Myh1, Myh2, Myh4 and Myh7 using fluorescent proteins. EYFP-Myh7 is localized to chromosome 14, whereas Sirius-Myh2, Cerulean-Myh1, and mCherry-Myh4 are closely clustered on chromosome 17 (< 10-kb interval)23. As we expected, the double knock-in mice for Cerulean-Myh1 and mCherry-Myh4 had the same expression phenotype as the wild type mice. Over 16 generations, no phenotypic abnormalities were detected in MusColor mice; however, comparisons with the wild-type mice should be made to analyse distinct phenotypes in intervention studies. Although crossing multiple MusColor mice is a time-consuming process, establishing and freezing suitable cell lines for use in vitro is a convenient alternative.
Sarcopenia, which is defined as age-related skeletal muscle wasting and muscle weakness, can severely impair the quality of life of older people37. Qualitative changes in muscle mass occur in the early stages before muscle mass is reduced. Studies in humans and laboratory animals have shown that aging-related muscle weakness is associated with changes in myofiber composition along with the metabolic changes20. Using the MusColor mice, we were able to detect characteristic age-related changes in myofiber type that were undetectable with conventional methods. Type II/DX-Cerulean myofibers are particularly sensitive compared to detection by immunostaining. Single type II/DX-Cerulean myofibers or hybrid myofibers with IIA-Sirius or IIB-mCherry increase with age in PLA, while I-EYFP/IIA-Sirius/IID/X-Cerulean triple-hybrid myofibers increase with age in SOL. A previous study using immunohistochemistry revealed no significant change in the ratio of type IID myofibers in the PLA, compared to that in the 24-week young and 87-week old group38. Recently, RNA sequencing technology has made it possible to analyse gene expression at the single nucleus level in multi-nucleated myofibers1,23. The myofiber itself is a syncytium that is composed of hundreds of post-mitotic myonuclei that share the same cytoplasm and are formed by the fusion of myoblasts. In young mouse, most myonuclei express only one MyHC gene in all analysed muscles; however, a portion of the myonuclei of the fast-twitch extensor digitorum longus (EDL) muscle and the slow-twitch soleus muscle expressed the MyHC genes in different combinations23. It is possible that the increase in hybrid myofibers in aging muscles is attributable to a change in the proportion of myonuclei expressing a single MyHC gene, or to myonuclei expressing the gene simultaneously, or a mixture of the two. In this study, transverse sections were used for identification of hybrid myofibers; however, more detailed analysis would be possible if longitudinal sections and isolated myofibers were used. Not only aging, but also exercise alters myofiber types and metabolism-related genes, and both involve epigenetic mechanisms39,40,41.
Recently, multi-omics technologies of single fibre proteomics and single fibre and single nucleus transcriptomics have made it possible to analyse the expression of proteins and genes involved in energy metabolism, such as glycolytic and mitochondrial proteins1,20,23. The use of MusColor mice, which can help identify the type of myofibers in situ, may facilitate the elucidation of the mechanism of metabolic changes in vivo. Using cultured muscle cells derived from MusColor mice, it is possible to monitor changes in myofiber type by using fluorescent proteins over time and to test many experimental conditions on multiple specimens at once. This technology may be used to search for factors that induce changes in myofiber types and the metabolic functions (e.g., mitochondrial respiration ↔ glycolysis) and to elucidate the mechanisms. Nonetheless, considering the inability to simultaneously analyse the expression of embryonic and perinatal MyHC isoforms using imaging techniques, the use of cultured muscle cells from MusColor mice to analyse metabolic functions is associated with some limitations. However, developing novel methods to simultaneously measure changes in myosin heavy-chain isoforms could facilitate the evaluation of changes in metabolic functions.
In conclusion, the MusColor mice expresses four types of MyHC as fluorescent fusion proteins, thereby enabling the distinction of myofiber types in muscle tissue and cultured myotubes in a living state. Furthermore, the muscle fibre type of muscle tissue sections can be analysed without any staining. The MusColor mouse may be useful for elucidating the mechanisms underlying muscle fibre changes that are caused by diseases such as sarcopenia, neuromuscular and metabolic diseases, as well as by exercise and the nutritional environment.
Methods
Animals
All animal procedures were performed in accordance with the Basic Animal Care and Experimental Guidelines of the Ministry of Health, Labour and Welfare of Japan and were approved by the Experimental Animal Care and Use Committee of the Tokyo Metropolitan Institute of Gerontology (License No. 23019). The authors confirm that animal experiment procedures complied with the ARRIVE 2.0 guidelines42. C57BL/6 mice (6- to 8-week-old) were purchased from Japan SLC (Hamamatsu, Japan). Immorto mice® expressing the temperature-sensitive SV40 large T antigen (tsA58) gene29, which is regulated by the IFN-g inducible H-2 kb promoter, were obtained from Charles River Laboratories International, Inc. (Wilmington, MA, USA). B6.129S4-Gt(ROSA)26Sortm1(FLP1)Dym/RainJ mice and C57BL/6-Tg(CAG-cre)13Miya mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). All mice were housed and bred according to standard procedures.
Generation of targeting vectors and knock-in mice
Knock-in mice were generated using the previously reported method of expressing the β-MyHC isoform by fusion with YFP in the cardiac muscle34. To prepare targeting vectors for the knock-in mice, bacterial artificial chromosome (BAC) libraries for male C57BL/6J mice (RP24-374D24 for Myh1 and Myh2, RP24-149P22 for Myh4, and RP24-317F19 for Myh7) were obtained from BACPAC Resources (Oakland, CA, USA). The target genomic sequences of the Myh7, Myh2, Myh1 and Myh4 genes were subcloned from the BAC clones using a Red/ET recombination BAC subcloning kit (Gene Bridges, Heidelberg, Germany). Genomic DNA was introduced into PL253 plasmid in which a negative selection marker had been inserted (Gene Bridges). The cDNA sequences of EYFP24, Sirius25, Cerulean26, and mCherry27, were cloned by PCR and subsequently inserted into the 5’-terminus sides of the Myh7, Myh2, Myh1, and Myh4 genes, respectively, in frame with the translation initiation codon (ATG) (Fig. 1A–D). A PGK-gb2-neomycin cassette (Gene Bridges) was inserted into each targeting vector of Myh2, Myh4, and Myh7 as a positive selection marker, sandwiched by FRT sequences (Fig. 1A,B,D). A PGK-puromycin cassette (Gene Bridges) flanked by loxP sequences was inserted into the targeting vector of Myh1 (Fig. 1C). Given the proximity of Myh1, Myh2, and Myh4 on chromosome 1123, it is possible to produce only heterozygous mice that express two of the Cerulean-Myh1, Sirius-Myh2, and mCherry-Myh4 genes by mating. Therefore, we generated double knock-in (Cerulean-Myh1 / mCherry-Myh4) ES cells on the same chromosome 11 by introducing the Cerulean-Myh1 gene into ES cells that already carried the mCherry-Myh4 gene. The linearized targeting vectors were introduced into M1 ES cells, which were established from a male F1 blastocyst between 129SvJ and C57BL6/N43. Following positive selection using G418 for Myh2, Myh4, and Myh7, homologous recombinant ES clones were identified by PCR and Southern blotting. To generate double knock-in (Type IID/X-Cerulean/IIB-mCherry) ES cells, the Myh1 targeting vector was electroporated into ES clones into which the mCherry-Myh4 gene had been introduced. After selection with puromycin and G418, double-positive clones were identified. Subsequently, a chimeric mouse was generated through the aggregation of the identified ES clone, and the integration of the fusion gene into germline cells was verified by genetic typing of pups obtained via crossbreeding the chimeric mouse with a male C57BL/6 mouse. The knock-in mice were subsequently mated to B6.129S4-Gt (ROSA)26Sortm1(FLP1) Dym / RainJ mice or C57BL/6-Tg (CAG-cre) 13Miya mice, to remove the neomycin and puromycin selection cassettes, respectively. The mice were backcrossed more than eight times with C57BL/6J mice.
Genotyping of knock-in mice
An approximately 5-mm tail portion of the mouse that was to be analysed was excised and DNA extracted using DirectPCR Lysis Reagents (Viagen Biotech, USA). PCR was performed according to the protocol of Quick Taq HS Dyemix (TOYOBO, Japan). The primer sequences and annealing temperatures employed are presented in supplementary Table 1. The PCR products were subjected to capillary electrophoresis using the QIAxcel Connect apparatus (QIAGEN, USA).
Fluorescence stereomicroscopy of muscolor skeletal muscle
Mice were euthanized via cervical dislocation. The soleus (SOL) and plantaris (PLA) muscles were subsequently harvested without any fixations. Fluorescence proteins expressed in the SOL and PLA were observed via a Leica 205 F fluorescence stereomicroscope (Leica, Germany) and a Leica K5s CMOS camera, with fluorescent images acquired at 1x objective magnification. The excitation and absorption wavelengths of the fluorescence filter sets utilized in fluorescence stereomicroscopy are shown in Supplementary Table 2.
Immunohistochemistry
Mice were euthanized via cervical dislocation. The gastrocnemius (GAS), tibialis anterior (TA), soleus (SOL), and plantaris (PLA) muscles were mounted on cork pieces with tragacanth gum and rapidly frozen in isopentane cooled in liquid nitrogen, then sliced into 14-µm cross sections. The sections were fixed in acetone at − 20 °C for 5 min and subsequently blocked in phosphate-buffered saline (PBS) containing 5% goat serum and 1% bovine serum albumin (BSA) for 1 h at room temperature. Primary antibody reactions were conducted using one each of anti–MyHC-I (clone BA-F8, IgG1), anti–MyHC-IIA (clone SC-71, IgG2b), anti–MyHC-IID/X (clone 6H1, IgM), and anti–MyHC-IIB (clone BF-F3, IgM) on a single muscle section per muscle. Antigen-antibody reactions were carried out over night at 4 °C. All primary antibodies were antibodies purchased from the Developmental Studies Hybridoma Bank (USA). Antibodies labelled with Alexa Fluor® 568, Alexa Fluor® 594, or Alexa Fluor® 488 were purchase from Invitrogen (USA) and used for the secondary antibodies, respectively. The dilution concentrations for primary and secondary antibodies are presented in Supplementary Table 3. To ensure that there was no fluorescence leakage or background using fluorescent filters, muscle sections expressing fluorescent proteins or stained with fluorescent dyes were used for confirmation. The images were acquired with a Leica TUNDER Imaging system (Leica, Wetzlar, Germany) using the fluorescence filters shown in Supplementary Table 4 with a 20x objective lens.
The SOL and PLA of young (6-month-old), middle-aged (14-month-old), and old (26–29-month-old) MusColor mice were mounted on cork pieces with tragacanth gum and rapidly frozen in isopentane cooled in liquid nitrogen. Frozen muscle Sects. (8–14 μm) on glass slides of young, middle-aged, and old MusColor mice were sealed with mounting medium (K048, Diagnostic BioSystems, USA) and imaged using a fluorescent upright microscope (Leica THUNDER Imaging system, Germany) at 20× objective magnification. To ensure that there was no fluorescence leakage or background using fluorescent filters, muscle sections expressing other fluorescent proteins were used for confirmation and imaged. Myofibers expressing multiple fluorescent proteins were defined as hybrid fibers. The number of myofibers from MusColor mice was counted by creating a macro in the MetaMorph Meta Imaging Series Version 7.10 image-analysis software (Molecular Devices, USA).
Isolation of muscle satellite cells
Muscle satellite cells were isolated using a previously reported method with some modifications44. Skeletal muscle was harvested from the right and left thighs and lower legs of mice euthanized by cervical dislocation and immersed in Dulbecco’s phosphate-buffered saline (D-PBS) (045-29795, Wako, Japan). Following the removal of the tendons, fascia, and adipose tissue from the harvested skeletal muscle, the muscle tissue was shredded; 4 mL HBSS (14175-095, Gibco, USA) containing 0.2% type 2 collagenase (LS004202, Worthington, USA) were added to the mouse muscle tissue per animal, and the tissue was treated with enzymes at 37 °C for 50 min. The enzyme-treated sample was then passed through an 18G needle (NN-1838R, Terumo, Japan) attached to a syringe (SS-10SZ, Terumo, Japan) to further subdivide the tissue, followed by enzyme treatment at 37 °C for 20 min. The enzyme-treated solution was diluted with D-PBS (-) to a total volume of 50 mL and sequentially filtered through a cell strainer with mesh sizes of 100 μm (352360, Corning, USA) and 40 μm (352340, Corning). The supernatant was removed after centrifugation at 600×g for 5 min at room temperature, and the precipitate was mixed with D-PBS(-) containing 2.5% fetal bovine serum (FBS; 10270-106, Gibco), CD45 Monoclonal Antibody as primary antibody (30-F11 ), APC-eFluor™ 780, eBioscience™ (47-0451-82, Invitrogen, USA), PE/Cyanine7 anti-mouse CD31 Antibody (102418, BioLegend, USA), Mouse PDGFR alpha PE-conjugated Antibody (FAB1062P, R&D systems, USA), and anti-Muscle Satellite Cells Antibody, clone SM C-2.6 (MABT857, Sigma-Aldrich, USA). The dilution concentrations of the antibodies utilized are presented in Supplementary Table 5. The antigen-antibody reaction was performed under light-shielded conditions at 4 °C for 30 min. Following centrifugation at 400 × g for 5 min at room temperature, the supernatant was removed, and the precipitate was mixed with D-PBS (-) containing 2.5% FBS. Subsequently, BD Horizon BV421 Goat Anti-Rat Ig (565013, BD Biosciences, USA) was added as a secondary antibody, and an antigen-antibody reaction was performed at 4 °C for 30 min under light-shielding conditions. The dilution concentrations of the antibodies utilized are presented in Supplementary Table 5. After the antibody reaction, the supernatant was removed by centrifugation at 400 × g for 5 min at room temperature, and the precipitate was mixed with D-PBS (-) containing 2.5% FBS. The solution was filtered through a tube with a 35 mm mesh cell strainer (352235, Corning, USA), and muscle satellite cells were selectively isolated using a BD FACS ARIA II cell sorter (Becton, Dickinson and Company, USA).
Cell culture
Isolated muscle satellite cells were plated in dishes or plates (3513, 430196, 430167, Corning) coated with Matrigel (356234, Corning), diluted to 1 mg/mL in DMEM (043-30085, Wako), and grown at 33 °C. The proliferated cells were frozen at − 80 °C using CELLBANKER 1 (11910, Zenogen Pharma, Japan), thawed, and cultured again for use in experiments. After thawing, cells were grown in growth medium at 33 °C. After 1 d, the growth medium was replaced with a differentiation medium at 37 °C. Three days after the induction of differentiation, rapamycin (BML-A275, Enzo Life Sciences, USA) (final concentration 10 nM) or 3,3’,5-Triiodo-L-thyronine (T3) (T6397, Sigma-Aldrich, USA) (final concentration 30 nM) was added to the differentiation medium for 3 days. The cells were washed with serum-free DMEM (040-30095, Wako) and Mildform® 10 N (133-10311, Wako) was added to fix the cells for 30 min. The cells were washed with serum-free DMEM (040-30095, Wako) and fixed by adding mild form (133-10031, Wako) for 30 min, then replaced with Live cell imaging solution (A14291DJ, Gibco, USA). Images were acquired using a fluorescence inverted microscope (Leica THUNDER Imaging system, Germany) with a 10x objective lens. Analysis was performed using MetaMorph Meta Imaging Series Version 7.10 to quantify the cell area for each myofiber type. The composition of the medium used for the cell culture is shown in Supplementary Table 6.
Functional assays of mitochondrial respiration and Glycolysis
Myotube cells were induced to differentiate by plating myoblasts isolated from Immorto mice® at a cell density of 2.0 × 104 cells/cm2 in a 24-well plate for analysis (100777-004, Agilent Technologies, USA). The XF Mito Stress Kit (103015-100, Agilent Technologies) was used to analyse mitochondrial function and the XF Glycolysis Stress Kit (103020-100, Agilent Technologies) was used to analyse glycolytic activity. Analysis was performed according to the protocol for each analysis kit. After the addition of rapamycin or T3, the differentiation medium was replaced with analysis medium (Seahorse XF DMEM medium, PH7.4, 10 mM Glucose, 1 mM Pyruvate, 2 mM L-Glutamine, Agilent Technologies) and placed in an incubator set at 37 °C for 60 min without CO2 control. The sensor cartridges, which were pre-hydrated and loaded with substrates and inhibitors to be added during the analysis, were then attached to 24-well plates and analysed using an XFe24 extracellular flux analyzer (Agilent Technologies). Mitochondrial function was measured by the oxygen consumption rate (OCR) three times after the sequential injection of oligomycin (final concentration: 2 mM), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; 1 mM), and rotenone/antimycin A (1 mM). Glycolytic activity was determined by incubating the cells in the analysis medium (Seahorse XF DMEM medium, PH 7.4, 2 mM L-glutamine) for 60 min at 37 °C without CO2 control, followed by sequential addition of glucose, oligomycin, and 2-deoxy-D-glucose (2-DG), and extracellular acidification rate (ECAR) was measured.
Statistical analysis
All statistical analyses were performed using Excel (Microsoft, USA) and Prism 9 (GraphPad Software, USA). One-way ANOVA, followed by Sidak’s multiple comparisons test, was used to compare multiple groups.
Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
We thank the TMIG animal facility staff for their help with the animal experiments. We thank Editage for editing the English text.
Funding
This work was supported by the Grants-in-Aid for Scientific Research on Innovative Area (21200023), Grant-in-Aid for Scientific Research (25670437, 16H03266, 19H04064, 20800079, 20K19737), AMED Leap (JP21mg00100007) and Takeda Science Foundation. These funding sources had no role in the design of this study and will not have any role during its execution, analyses, interpretation of the data, or decision to submit results.
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Department of Geriatric Medicine, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo, 173- 0015, Japan
Shuuichi Mori, Takuya Omura, Mako Kono, Taichi Fukunaga & Kazuhiro Shigemoto
Laboratory for Developmental Genetics, RIKEN Center for Integrative Medical Sciences (IMS), Yokohama City, Kanagawa, 230-0045, Japan
Haruhiko Koseki
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Shuuichi Mori
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Contributions
Conceptualization, K.S.; Methodology, K.S., S.M., T.O, M.K., T.F., A.K.; Investigation, K.S., S.M., T.O., M.K., T.F., A.K.; Data curation, K.S., S.M., M.K.; Writing-Original Draft, K.S., M.K.; Writing-Review & Editing, K.S., M.K.; Funding Acquisition, K.S., S.M., T.O.
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KS is an inventor of three issued patents titled “Muscle stem cell or myoblast, method for screening substances that participate in metabolic conversion using same, and pharmaceutical composition comprising substance obtained from said screening method” (JP6159930, US9618502B2 and EP3061810B1). TF is a current employee of Daiichi-Sankyo Co., Ltd. SM, TO, MK and HK have no competing interests.
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Mori, S., Omura, T., Kono, M. et al. Creation of knockin mice for the fluorescence protein based in vivo identification of skeletal myofiber types. Sci Rep 15, 11389 (2025). https://doi.org/10.1038/s41598-025-96118-z
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Received:28 October 2024
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DOI:https://doi.org/10.1038/s41598-025-96118-z
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Keywords
Myofiber type
Myosin heavy chain
Knock-in mouse
Aging
Satellite cell
Metabolism