Method for estimating type of human skeletal muscle fiber from body fluid

Abstract

To provide a noninvasive method for identifying the types of skeletal muscle fiber types in humans.SOLUTION: The method for estimating types of skeletal muscle fibers in humans has been established on the basis of the presence ratio of a plurality of biomarkers that leak into body fluid after eccentric exercise. Specifically, the present invention provides a method for estimating the composition ratio of types of skeletal muscle fiber in humans on the basis of the presence ratio of a plurality of biomarkers that leak into body fluid after eccentric exercise.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present invention relates to a method for estimating human skeletal muscle fiber types from body fluids. [Background technology]

[0002] Skeletal muscle is a large tissue that accounts for approximately 40% of human body weight, supporting the body and generating movement (Non-Patent Document 1). Muscle fibers that make up skeletal muscle are primarily classified as slow-twitch or fast-twitch based on their contractile and metabolic properties. The type of muscle fiber is determined primarily by the isoform of myosin heavy chain (MyHC), a contractile protein that makes up muscle tissue (Non-Patent Documents 2-4, Figure 1). Muscle fibers composed of MyHC I, a slow-twitch myosin, have a slow contraction rate, but are highly fatigue-resistant, rich in mitochondria, and have excellent oxidative metabolic properties. On the other hand, muscle fibers composed of MyHC II, a fast-twitch myosin, have a fast contraction rate, but are low fatigue-resistant, have few mitochondria, and have excellent glycolytic metabolic properties. Rodent skeletal muscle mainly contains MyHC I, IIa, IId / x, and IIb types (Non-Patent Document 5), and it has been reported that in human skeletal muscle, MyHC I, IIa, and IId / x types are primarily expressed in the limbs (Non-Patent Document 1).

[0003] Furthermore, several studies have reported that muscle fiber type composition in elite athletes varies significantly depending on their sporting characteristics (Non-Patent Documents 6-10). Therefore, since muscle fiber type composition is strongly related to athletic performance, understanding muscle composition could potentially be applied to talent discovery. Furthermore, if changes in muscle fiber types in athletes could be continuously monitored, it could be used to monitor training adaptation. However, current methods for identifying muscle fiber types require muscle biopsy, which is highly invasive and therefore cannot be applied in training settings. Therefore, a non-invasive method for identifying skeletal muscle fiber types is needed. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Schiaffino S, et al., Physiol Rev. 2011;91:1447 [Non-Patent Document 2] Reiser PJ, et al., J Biol Chem. 1985;260: 14403 [Non-Patent Document 3] Billeter R, et al., Histochemistry,1980;65:249 [Non-Patent Document 4] Schiaffino S, et al., Acta Physiol Scand. 1988;134:575 [Non-Patent Document 5] Hamalainen N, et al., J Histochem Cytochem. 1993;41:733 [Non-Patent Document 6] Staron RS, et al., J Histochem Cytochem. 1984;32: 146 [Non-Patent Document 7] Costill DL, et al,, J Appl Physiol. 1976;40:149 [Non-Patent Document 8] Fink WJ, et al., Ann N Y Acad Sci. 1977;301:323 [Non-Patent Document 9] Mackova E, et al, Int J Sports Med. 1986;7:295 [Non-Patent Document 10] Gerard ES, et al., Am J Sports Med. 1986;14: 77 [Summary of the Invention] [Problems to be Solved by the Invention]

[0005] The present invention aims to provide a method for non-invasively identifying skeletal muscle fiber types in humans. [Means for solving the problem]

[0006] The present inventors have conducted extensive research and established a method for estimating skeletal muscle fiber types in humans from the abundance ratios of multiple biomarkers that leak into body fluids after eccentric exercise.

[0007] Specifically, the present invention provides a method for estimating the composition ratio of skeletal muscle fiber types in humans from the abundance ratio of multiple biomarkers leaked into body fluids after eccentric exercise.

[0008] In the method of the present invention, the skeletal muscle fiber type may be at least one skeletal muscle fiber type selected from the group consisting of myosin heavy chain (MyHC) type I (hereinafter also referred to as "MyHC I"), type IIa (hereinafter also referred to as "MyHC IIa"), type IId / x (hereinafter also referred to as "MyHC IId / x"), and fast-twitch muscle-specific proteins.

[0009] In the methods of the present invention, the biomarker may be a muscle fiber-specific protein.

[0010] In the method of the present invention, the muscle fiber-specific protein is (i) Group: As a myofiber-specific protein for myofiber type I (MyHC I), ·MYL3(Myosin light chain 3), ·TNNC1 (Troponin C, slow skeletal and cardiac muscles), ·TNNI1(Troponin I, slow skeletal muscle), MYOM3 (Myomesin-3), and ·ANP32B(Acidic leucine-rich nuclear phosphoprotein 32 family member B); (ii) Group: Myofiber-specific proteins for myofiber type IIa (MyHC IIa) and myofiber type IIx / d (MyHC IId / x), ·TNNC2(Troponin C, skeletal muscle), ·TNNI2(Troponin I, fast skeletal muscle), ·TNNT3(Troponin T, fast skeletal muscle), ·MYL1(Myosin light chain 1 / 3, skeletal muscle isoform), ·TPM1 (Tropomyosin alpha-1 chain), ·ALDOA (Fructose-bisphosphate aldolase A), ·ENO3(Beta-enolase;Enolase), ·PYGM (Glycogen phosphorylase, muscle form), PKM (pyruvate kinase), ·TPI1 (Triosephosphate isomerase), ·PFKM (ATP-dependent 6-phosphofructokinase, muscle type), ·AK1 (Adenylate kinase isoenzyme 1), ·GPD1 (Glycerol-3-phosphate dehydrogenase [NAD(+)], cytoplasmic), ·ESD(S-formylglutathione hydrolase), ·ADSL (Adenylosuccinate lyase), ·GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) and ·RPS6KA3(Ribosomal protein S6 kinase alpha-3); (iii) Group: As myofiber-specific proteins for myofiber type IIx / d (MyHC IId / x), GPI (lucose-6-phosphate isomerase), and ·PGM1 (Phosphoglucomutase-1); and, (iv) Group: As muscle fiber-specific proteins for fast-twitch muscle, MYOM2 (Myomesin-2), and ·CK (Creatin kinase); may be selected from the group consisting of:

[0011] In the method of the present invention, at least two groups of muscle fiber-specific proteins are selected in combination from each of the muscle fiber-specific protein groups (i), (ii), (iii), and (iv), and the amount of leakage of at least one muscle fiber-specific protein and its fragment from each selected group into body fluids after eccentric exercise may be measured.

[0012] In the method of the present invention, the body fluid may be at least one selected from the group consisting of serum, plasma, whole blood, saliva, and urine.

[0013] In the method of the present invention, the percentage (%) of MyHC I fibers was used as the increase in MYOM3, MYOM2, MYL3, and CK after eccentric exercise, and was calculated by the following formula:

number

[0014] The present invention provides a non-invasive method for identifying skeletal muscle fiber types in humans. [Brief explanation of the drawings]

[0015] [Figure 1] Diagram showing the changes and characteristics of muscle fiber types. [Figure 2] Diagram outlining a calf raise exercise using a Smith machine, involving repeated eccentric contractions. [Figure 3] Diagram showing the muscle fiber type composition of the subject. Human vastus lateralis muscle was immunohistochemically stained according to muscle fiber type. Different colors are used for each myosin heavy chain type: blue indicates MyHC I, green indicates MyHC IIa, and black indicates MyHC IId / x. The subject's name is listed in the upper left of the image. The scale bar represents 100 μm. [Figure 4] This graph shows changes in serum myomesin-3 (MYOM3) and its fragments before eccentric exercise and 1, 2, 3, and 4 days after exercise. Top: Western blot images of serum MYOM3 and its fragments before eccentric exercise (Pre), and 1 day (1d), 2 days (2d), 3 days (3d), and 4 days (4d) after. The subject's name is shown on the left of each Western blot image. Bottom: Quantitative results of MYOM3. [Figure 5] Changes in blood biomarkers before and 4 days after eccentric exercise. Western blot image of myomesin-3 (MYOM3) and its fragments (top). Western blot image of myomesin-2 (MYOM2) and its fragments (center). Western blot image of myosin light chain 3 (MYL3) and its fragments (bottom). Lysate from human vastus lateralis muscle was used as a positive control (VL). Pre: before eccentric exercise, Post: 4 days after eccentric exercise. [Figure 6]Changes in blood troponin T type 3 (TNNT3) levels before and four days after eccentric exercise. Western blot image (top) using anti-TNNT3 antibody (HPA056909; ATLAS ANTIBODIES, 1:100). Western blot image (bottom) using anti-TNNT3 antibody (HPA037810; ATLAS ANTIBODIES, 1:100). Lysate from vastus lateralis muscle was used as a positive control (VL). Pre: before eccentric exercise, Post: 4 days after eccentric exercise. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention establishes a method for estimating skeletal muscle fiber type in humans based on the abundance ratios of multiple biomarkers leaked into body fluids after eccentric exercise. Specifically, the present invention is a method for estimating the composition ratio of skeletal muscle fiber types in humans based on the abundance ratios of multiple biomarkers leaked into body fluids after eccentric exercise.

[0017] In this specification, the term "eccentric exercise" is used in the general sense of "eccentric exercise." That is, "eccentric exercise" is also called "eccentric muscle contraction exercise," and refers to exercise in which muscles are stretched from a contracted state to exert force.

[0018] In the method of the present invention, the skeletal muscle fiber type can be at least one skeletal muscle fiber type selected from the group consisting of myosin heavy chain (MyHC) type I (hereinafter also referred to as "MyHC I"), type IIa (hereinafter also referred to as "MyHC IIa"), type IId / x (hereinafter also referred to as "MyHC IId / x"), and fast-twitch muscle-specific proteins.

[0019] In the methods of the present invention, the biomarker can be a muscle fiber-specific protein.

[0020] In the method of the present invention, the muscle fiber-specific protein is (i) Group: As a myofiber-specific protein for myofiber type I (MyHC I), ·MYL3(Myosin light chain 3), ·TNNC1 (Troponin C, slow skeletal and cardiac muscles), ·TNNI1(Troponin I, slow skeletal muscle), MYOM3 (Myomesin-3), and ·ANP32B(Acidic leucine-rich nuclear phosphoprotein 32 family member B); (ii) Group: Myofiber-specific proteins for myofiber type IIa (MyHC IIa) and myofiber type IIx / d (MyHC IId / x), ·TNNC2(Troponin C, skeletal muscle), ·TNNI2(Troponin I, fast skeletal muscle), ·TNNT3(Troponin T, fast skeletal muscle), ·MYL1(Myosin light chain 1 / 3, skeletal muscle isoform), ·TPM1 (Tropomyosin alpha-1 chain), ·ALDOA (Fructose-bisphosphate aldolase A; Fructose-bisphosphate aldolase), ·ENO3 (Beta-enolase; Enolase), ·PYGM (Glycogen phosphorylase, muscle form), PKM (pyruvate kinase), ·TPI1 (Triosephosphate isomerase), ·PFKM (ATP-dependent 6-phosphofructokinase, muscle type), ·AK1 (Adenylate kinase isoenzyme 1), ·GPD1 (Glycerol-3-phosphate dehydrogenase [NAD(+)], cytoplasmic), ·ESD(S-formylglutathione hydrolase), ·ADSL (Adenylosuccinate lyase), ·GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) and ·RPS6KA3(Ribosomal protein S6 kinase alpha-3); (iii) Group: As myofiber-specific proteins for myofiber type IIx / d (MyHC IId / x), GPI (lucose-6-phosphate isomerase), and ·PGM1 (Phosphoglucomutase-1); and, (iv) Group: As muscle fiber-specific proteins for fast-twitch muscle, MYOM2 (Myomesin-2), and ·CK (Creatin kinase); can be selected from the group consisting of:

[0021] In this specification, for convenience, the gene name of the protein may be used as an abbreviation for the protein name.

[0022] In the method of the present invention, at least two groups are selected in combination from each of the muscle fiber-specific protein groups (i), (ii), (iii), and (iv), and the amount of leakage of at least one muscle fiber-specific protein and its fragment from each selected group into body fluids after eccentric exercise can be measured.

[0023] In the method of the present invention, the body fluid can be at least one selected from the group consisting of serum, plasma, whole blood, saliva, and urine.

[0024] In the method of the present invention, the percentage (%) of MyHC I fibers was used as the increase in MYOM3, MYOM2, MYL3, and CK after eccentric exercise, and was calculated by the following formula:

number

[0025] In the present invention, methods for measuring the concentration of muscle fiber-specific proteins in body fluids include, but are not limited to, Western blotting, ELISA (Enzyme-linked enzyme immune sorbent assay), gel filtration column chromatography, and high-performance liquid chromatography, and quantitative analysis can be performed according to standard methods for these analytical methods.

[0026] The significance of the method of the present invention will be explained in detail below.

[0027] Several reports have shown that muscle fiber types vary significantly depending on the characteristics of the sport in high-level athletes (Staron et al., 1984; Costill et al., 1976; Fink et al., 1977; Mackova et al., 1986; Gerard et al., 1986). When the vastus lateralis muscles of healthy individuals, weightlifters, and long-distance runners were examined, the proportion of MyHC I fibers was significantly higher in long-distance runners than in the other two groups (Staron et al., 1984).

[0028] Furthermore, when the proportion of MyHC I fibers in the gastrocnemius muscles of competitive track and field athletes and untrained subjects was examined, it was found to be approximately 30% in sprinters (100m), approximately 60% in middle-distance runners (800m), approximately 70% in long-distance runners (5000m), and approximately 50% in untrained subjects (Costill, et al., 1976). Another study also reported that the proportion of slow-twitch muscle fibers in the gastrocnemius muscles was approximately 50% in non-exercise groups, compared to approximately 80% in elite long-distance track and field runners (Fink, et al., 1977). When the proportion of slow-twitch muscles in the vastus lateralis of male and female swimmers was examined, it was reported that it was 60.0% in swimmers specializing in distances of 400m or more, 43.8% in men and 58.0% in women specializing in distances of 200m, and 47.8% in men and 52.0% in women specializing in distances of 100m or less (Gerard, et al., 1986).When the vastus lateralis of able-bodied individuals and national-level cyclists was examined, it was found that the able-bodied individuals had approximately 60% fast-twitch muscles, while the elite cyclists had only 40% fast-twitch muscles (Mackova, et al., 1986).

[0029] Muscle fiber type composition has long been thought to be genetically determined (Komi, et al., 1977). However, Bouchard et al. reported that although the proportion of Type I and Type IId / x fibers in genetically identical monozygotic twins was consistent, a test for heterogeneity using the statistical analysis of Christian et al. (Christian, et al., 1979) revealed no significant correlation (Bouchard, et al., 1986). Furthermore, a recent systematic review and meta-analysis of the heritability of athletic performance found that the heritability of maximal oxygen uptake was 56% and that of muscle strength and power was 52% (Miyamoto, et al., 2018). Therefore, genetic influences are not dominant in muscle fiber type.

[0030] Numerous studies using experimental animals have demonstrated that training alters muscle fiber types (Fitzsimons et al., 1990; Guezennec et al., 1990; Demirel et al., 1999; Allen et al., 2001). In the fast-twitch gastrocnemius and tibialis anterior muscles of mice, the proportion of MyHC IIa-expressing fibers significantly increased and the proportion of MyHC IIb-expressing fibers decreased after 4 weeks of voluntary running (Allen et al., 2001). Furthermore, endurance treadmill exercise in rats decreased the proportion of MyHC IIb fibers and increased the proportion of MyHC IIa fibers in the extensor digitorum longus, while simultaneously decreasing the proportion of MyHC IIa fibers and increasing MyHC I fibers in the soleus (Demirel et al., 1999).

[0031] A few studies in humans have shown that muscle activity, particularly exercise training, can alter muscle fiber types (Andersen et al., 1977; Green et al., 1979; Simoueau et al., 1985; Staron et al., 1990; Andersen et al., 1994). For example, the proportion of MyHC II fibers in the vastus lateralis increased after 8 weeks of high-intensity bicycle training (Andersen et al., 1977). The proportion of MyHC I fibers increased and the proportion of MyHC IId / x decreased after 15 weeks of high-intensity interval training (Simoueau et al., 1985). Furthermore, the proportion of MyHC IIa fibers increased and the proportion of MyHC IId / x decreased after high-intensity lower limb training (Staron et al., 1990). It has been reported that after 3 months of sprint training, the proportion of MyHC IIa fibers in the vastus lateralis increased, the proportion of MyHC I and MyHC IId / x decreased, and 20m and 30m running times significantly improved (Andersen, et al., 1994). On the other hand, there are many reports in which no changes in muscle fiber type were observed before and after training (Gollnick, et al., 1973; Andersen, et al., 1977; Thorstensson, et al., 1975; Henriksson, et al., 1976; Bylund, et al., 1977; Costill, et al., 1976; Dons, et al., 1979; Houston, et al., 1979; Constable, et al., 1980; Orlander, et al., 1980; Coyle, et al., 1981; Alen, et al., 1984). Many of the training methods, durations, and intensities used in these studies may not have resulted in changes in muscle fiber type, and different fiber type identification methods may have influenced the results of these studies (Plotkin, et al., 2021).

[0032] As mentioned above, the muscle fiber type composition of elite athletes varies depending on their sporting characteristics, so for example, athletes who genetically have more slow-twitch muscle fibers are expected to be able to demonstrate higher athletic performance in endurance sports. Therefore, if we can understand an athlete's genetic muscle composition, we may be able to suggest sports that are suitable for that athlete, and this method may also be applicable to talent discovery.

[0033] Furthermore, as mentioned above, muscle fiber type composition is strongly related to athletic performance, so if we could continuously understand changes in muscle fiber type during training, it could be used to monitor training adaptation. In other words, this would allow athletes and coaches to confirm whether their current training is effective for them, which could have a significant impact on changing and improving current and future training plans.

[0034] The only currently established method for identifying muscle fiber types is to perform histological staining of muscle tissue obtained by muscle biopsy (Johnson, et al., 1973; Shanely, et al., 2014; Ekblom, 2017). Muscle biopsy involves surgically extracting muscle tissue through an incision in the skin, which is highly invasive and causes pain around the surgical site for several days after the procedure. Therefore, training must be suspended after the biopsy, making it unsuitable for athletes currently undergoing training or prior to a match. If this invention could identify muscle fiber types noninvasively, muscle fiber type composition could potentially be used in sports for the purposes of talent identification and training adaptation monitoring.

[0035] Therefore, if muscle proteins specifically expressed in specific muscle fiber types could be captured in body fluids, it would be possible to identify skeletal muscle fiber types without muscle biopsy. It has long been reported that skeletal muscle proteins leak into tissue fluids such as blood after high-intensity or eccentric exercise (Sorichter, et al., 1997; Sorichter, et al., 2001; Martinez, et al., 2007; Thorpe, et al., 2012). For example, plasma analysis after eccentric exercise such as downhill running showed elevated levels of skeletal muscle troponin I (Sorichter, et al., 1997). Plasma myosin levels increased after eccentric exercise in the quadriceps femoris (Sorichter, et al., 2001). Serum analysis of athletes with and without muscle injury showed significantly higher levels of α-actin (Martinez, et al., 2007). Additionally, increases in salivary CK and myoglobin have been reported after soccer matches, suggesting that proteins contained in skeletal muscle may leak into saliva (Thorpe, et al., 2012).

[0036] Based on these findings, it is thought that if similar muscle damage is caused to individuals with different muscle fiber compositions, muscle proteins associated with that individual's muscle fiber type composition will leak into the blood. In other words, it is expected that individuals with a muscle composition predominantly consisting of fast twitch muscle will have more fast twitch muscle proteins migrate into the blood, while individuals with a muscle composition predominantly consisting of slow twitch muscle proteins will have more slow twitch muscle proteins migrate into the blood. This raises the possibility that muscle fiber type may be indirectly estimated by using muscle proteins released into the blood specifically in muscle fiber types as biomarkers.

[0037] The purpose of this study was to explore blood biomarkers that reflect skeletal muscle fiber type and to examine whether these biomarkers reflect skeletal muscle fiber type in humans.

[0038] Specifically, we will identify muscle fiber type-specific molecules using an existing dataset that comprehensively analyzes proteins leaked into the blood of patients with muscular dystrophy, which involves muscle necrosis and regeneration.

[0039] Next, we will examine whether these biomarkers can be applied to humans and their validity through human trials. Specifically, we will target multiple individuals with different exercise characteristics, induce muscle damage through eccentric exercise (Kanda, et al., 2013), and measure changes in these biomarkers. At the same time, we will collect muscle tissue from the vastus lateralis and confirm the muscle fiber type. By comparing these, we aim to establish biomarkers that can estimate muscle fiber type in humans. [Example]

[0040] The present invention will be described in more detail below with reference to examples, but modifications can be made as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited to the specific examples shown below.

[0041] 1. Method 1.1 Search for muscle fiber type-specific proteins that leak into body fluids upon muscle injury To identify muscle fiber type-specific proteins leaked into body fluids following muscle injury, we used PubMed (URL: https: / / pubmed.ncbi.nlm.nih.gov / ), a literature database published by the National Center for Biotechnology Information, to extract proteins that are increased in the serum of patients with muscle diseases characterized by necrosis and regeneration of skeletal muscle, such as Duchenne muscular dystrophy (DMD), from a comprehensive comparison dataset of serum proteins between patients and healthy controls (Ayoglu, et al., 2014; Rouillon, et al., 2014; Oonk, et al., 2016; Palolo, et al., 2018; Spitali, et al., 2018; Hathout, et al., 2019; Alayi, et al., 2020; Strandberg, et al., 2020).

[0042] On the other hand, using a dataset that comprehensively analyzed the proteins expressed in each single muscle fiber using quantitative proteomics (Murgia, et al., 2021), we searched for proteins specific to each muscle fiber type and extracted proteins that were increased in the serum of patients with muscle diseases accompanied by muscle damage compared to healthy individuals and that were expressed in a muscle fiber type-specific manner.

[0043] 1.2 Human subject experiments This study respected the Declaration of Helsinki, and the research plan was designed with consideration for protecting the human rights and interests of the subjects. It was approved by the Waseda University Research Ethics Committee (approval number: 2023-038). Furthermore, all subjects were fully informed in advance of the study's purpose, methods, and the pain and risks associated with the experiment, and their written consent to participate was obtained. Furthermore, participation was voluntary, and participants were informed of their right to withdraw at any time during the study period. This study was pre-registered with the University Hospital Medical Information Network Center (UMIN), a clinical trial registration system, and was conducted (ID: UMIN000049576).

[0044] 1.3 Subjects The subjects were six healthy Japanese men (age: 22.5±1.7 yr, height: 173.9±5.1 cm, weight: 71.8±13.2 kg, mean±standard deviation). Exclusion criteria included those with a history of smoking, those unable to perform low-intensity exercise, those with medical or surgical diseases as determined by a health check within the past year, and those planning to lose weight during the study period.

[0045] For three days before and four days after the eccentric exercise, the subjects were instructed to refrain from strenuous exercise or resistance training, as well as from eating a significantly unbalanced diet or taking supplements, and to avoid massaging or stretching the area where muscle pain occurred, or applying hot or cold stimulation for long periods of time, and to go about their daily lives as usual except during measurement times.

[0046] 1.4 Eccentric exercise In this experiment, the eccentric exercise was a modified version of the single-leg calf raise described by Kanda et al. (Kanda, et al., 2013). Because the triceps surae, the primary muscle, is a biarticular muscle that spans both the knee and ankle joints, the movement during exercise had to be limited to the ankle joint. Subjects arrived at the training room at 8:30 AM, where their height and weight were measured. They then warmed up by performing submaximal strength exercises and stretching. Prior to exercise, the starting position, maximum plantar flexion, and maximum dorsiflexion of the ankle were measured in an unloaded position. The subject was then placed on the Smith machine with a load of half their measured body weight. Exercise began at 9:00 AM, with the subject standing with both legs on a platform approximately 20 cm high. The starting position was determined by weighting the forefoot without placing the heel on the platform, and the ankle was maintained in maximum dorsiflexion (Figure 2(C)). During the exercise, subjects were given verbal instructions to maintain the knee in an extended position, to keep the plantar load midway between the second and third toes, and to perform the task using the full range of motion of the ankle. From the starting position, the ankle was plantar-flexed to maximum plantar flexion while resisting the load over one second, and then returned to maximum dorsiflexion while supporting the load over one second. The movement speed was synchronized to the metronome tempo (0.5 Hz) (plantar flexion: 1 second, dorsiflexion: 1 second), and 40 repetitions were repeated in 10 sets for a total of 400 repetitions, with a three-minute rest between sets (Figure 2).

[0047] 1.5 Taking blood samples Blood samples were collected at 9:00 AM on the day before exercise and on the 1st, 2nd, 3rd, and 4th days after exercise. Venous blood was collected into blood collection tubes (VJ-AS076A001) containing a coagulation activator to isolate serum. The collected blood was allowed to clot at room temperature for 30 minutes, then centrifuged at 2,000 rpm for 10 minutes at 4°C. The serum was then dispensed into storage microtubes and frozen at -80°C until further analysis.

[0048] 1.6 Muscle tissue collection The biopsy site was determined to be the center of the imaginary line between the greater trochanter and the superior border of the patella of the subject's right leg, where the vastus lateralis muscle was at its thickest point. An ultrasound diagnostic device (229ACBZX00025000, Canon, Tokyo, Japan) was used to confirm the absence of large blood vessels at the biopsy site. Afterwards, a physician performed palpation to further ensure safety before the muscle biopsy was performed.

[0049] Local anesthesia was administered by subcutaneous injection of 1% xylocaine (487614431009, SANDOZ, Tokyo, Japan) at the biopsy site, and a 1-1.5 cm skin incision was made. The subcutaneous tissue was bluntly dissected, and the fascia was incised with a scalpel while grasping it with hooked forceps. The incision was extended longitudinally, and a silk thread was placed perpendicular to the direction of the muscle fibers to mark the muscle. An appropriate amount of muscle was lifted with ophthalmic scissors, and a portion of the muscle was then excised. This was then cut proximally using ophthalmic scissors, and a superficial sample of the vastus lateralis muscle tissue measuring approximately 6 mm x 6 mm x 10 mm was obtained. The skin and fascia were then sutured. Patients were instructed to maintain wound care for two weeks after muscle biopsy and to avoid getting the tissue wet. The collected muscles were divided into those for histological analysis and those for biochemical analysis. Samples for histological analysis were embedded in OCT Compound, frozen in isopentane cooled on liquid nitrogen, and stored at -80°C until analysis.

[0050] 1.7 Identification of muscle fiber type composition by immunohistochemical staining Frozen sections were cut at 10 μm using a cryostat and fixed on ice in 4% paraformaldehyde / PBS (Fujifilm Wako). After two 5-minute washes with PBS, they were permeabilized with 0.3% Triton X-100 / PBS for 10 minutes and then washed with PBS. To prevent nonspecific antibody reactions, they were blocked for 30 minutes with 5% normal goat serum (NGS, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) / PBS. The primary antibody, anti-MyHC IIb antibody (BF-F3, DSMZ, Brunswick, DEU), was diluted 1:25 in 5% NGS / PBS and applied to the sections for overnight incubation at 4°C. After washing twice with PBS for 5 minutes, the sections were incubated with Goat anti-mouse IgG-Dy549 (Jackson Immunoresearch Laboratory) at a 1:100 dilution for 1 hour at room temperature. After washing twice with PBS for 5 minutes, the sections were incubated with Anti-MyHC I antibody (BA-F8, DSHB, Iowa City, IA, USA) at a 1:25 dilution in 5% NGS / PBS for 2 hours at room temperature. After washing twice with PBS for 5 minutes, the sections were incubated with Goat anti-mouse IgG2b-Dy405 (Jackson Immunoresearch Laboratory) at a 1:100 dilution for 30 minutes at room temperature. After washing twice with PBS for 5 minutes, anti-MyHC IIa antibody (SC-71, DSHB) diluted 1:250 and anti-dystrophin antibody (7A10, DSHB) diluted 1:100 to visualize muscle fiber contours were added to each section and incubated for 2 hours at room temperature. After washing twice with PBS for 5 minutes, goat-anti-mouse IgG-Alexa488 (Jackson Immunoresearch Laboratory) diluted 1:100 was added to each sample and incubated for 30 minutes at room temperature. After washing three times with PBS for 5 minutes, images were captured using a digital camera attached to a fluorescence microscope (IX-70, OLYMPUS). The images were merged in Adobe Photoshop 10.Muscle fiber type composition, muscle fiber cross-sectional area, and total muscle tissue area were measured using ImageJ.

[0051] 1.8 Determination of total protein concentration Total protein concentration was quantified using a DC Protein Assay kit (Biorad, Hercury, CA, USA). 5 μl of each of the following standard protein solutions was added to a 96-well microplate (208004, BMBio, Tokyo, Japan): 0 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.4 mg / ml, 0.8 mg / ml, and 1.6 mg / ml of albumin from bovine serum (BSA) fatty acid free (013-15143, Fujifilm). Human serum was diluted 1 / 100 with distilled water and added to each well in 5 μl portions. Next, 25 μl of DC Protein Assay Reagent A (5000113, Biorad) was added to each well. After vortexing for 1 minute, 200 μl of DC Protein Assay Reagent B (5000114, Biorad) was added. The plate was vortexed for 1 minute and then allowed to stand at room temperature for 15 minutes. The absorbance at 650 nm was then measured using an EMax Plus Microplate Reader (Molecular Devices, CA, USA). The total serum protein concentration was calculated from a standard curve plotting the concentration of the standard protein solution on the horizontal axis and the absorbance on the vertical axis.

[0052] 1.9 Measurement of specific proteins by Western blot Serum was mixed with 2x sample buffer (Tris-HCl, pH 6.8, 100 mM, SDS 2%, glycerol 20%, dithiothreitol (DTT) 80 mM, 2-mercaptoethanol 285 mM, 0.01% bromophenol blue, complete protease inhibitor cocktail tablets complete mini (Roche, Basel, Switzerland)). For detection with anti-myomesin 3 antibody (17692-1-AP; Proteintech, IL, USA, 1:1000), samples were incubated at 95°C for 1 min. For SDS-polyacrylamide gel electrophoresis (SDS-PAGE), 7.5% or 15% separation gels were used. 20–100 μg of protein was applied to each well and electrophoresis was performed at 100 V for 90 min.

[0053] After electrophoresis, the gel was transferred to a 0.45 μm Nitrocellulose Blotting Membrane (Product No. 10600002, GE Healthcare, Chicago, IL, USA) at 100 V constant pressure for 1 hour, or to a 0.20 μm PVDF Blotting Membrane (Product No. 10600021, GE Healthcare) for detection with anti-TNNT3 antibody (HPA056909; ATLAS ANTIBODIES, Stockholm, SWE, 1:100) or anti-TNNT3 antibody (HPA037810; ATLAS ANTIBODIES, Stockholm, SWE, 1:100) at 100 V constant pressure for 1 hour. To prevent nonspecific reactions, the membrane was blocked with 5% skim milk / Tris-buffered saline containing 0.05% Tween-20 (167-11515, Fujifilm Wako) (TBST) for 1 hour at room temperature, and then the transferred proteins were reacted with primary antibodies overnight at 4°C.Anti-myomesin 3 antibody (1:1000), Anti-myomesin 2 antibody (STJ94312; St John's Laboratory, London, UK, 1:1000), Anti-Myosin light chain 3 antibody (HPA016564; ATLAS ANTIBODIES, Stockholm, SWE, 1:200), Anti-TNNT3 antibody (HPA056909; ATLAS ANTIBODIES, Stockholm, SWE, 1:100), Anti-TNNT3 antibody (HPA037810; ATLAS ANTIBODIES, Stockholm, SWE, 1:100), Anti-myosin heavy chain antibody (A4 74, DSHB, 1:250), Anti-myosin heavy chain antibody (BF-35, DSHB, The following antibodies were used: anti-myosin heavy chain antibody (MF-20, DSHB, 1:500), anti-myosin heavy chain antibody (F59, DSHB, 1:500), anti-myosin heavy chain I antibody (BA-F8, DSHB, 1:50), anti-myosin heavy chain IIa antibody (SC-71, DSHB, 1:50), anti-myosin heavy chain IId / x antibody (6H1, DSHB, 1:500), and anti-myosin heavy chain IIb antibody (BF-F3, DSMZ, 1:50). The cells were then incubated with the secondary antibody for 1 hour at room temperature. The secondary antibodies used were Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (NA9340V, Cytiva, Tokyo, Japan, 1:5000), Mouse IgG HRP Linked F(ab′)2 Fragment (NA9310V, Cytiva, 1:10000), and Rabbit Anti-Mouse Immunoglobulins / HRP (P0161, Dako, 1:2000).The luminescence signal of Amsham ECL Prime Western Blotting Detection Reagent (GE Healthcare, MA, USA) was photographed using an LAS-3000 (Fujifilm Wako), and band intensity was measured using ImageJ.

[0054] 1.10 Statistical Processing and Analysis Data are shown as mean ± standard error. Statistical analysis was performed using SPSS ver. 28 (IBM, New York, NY, USA). One-way analysis of variance was used to test for significance. P < 0.05 was considered statistically significant.

[0055] To calculate the muscle fiber type estimation equation, multiple regression analysis was performed, with the proportion of muscle fiber types (Yi) as the objective variable and the increase in MYOM3, MYOM2, MYL3, and CK as the explanatory variables (X1, X2, X3, X4). The relationship with the measured values ​​(n=6) was formulated as a linear model Yβi = α0 + α1X1i + α2X2i + α3X3i + α4X4i, and multiple regression analysis was performed to find α0, α1, α2, α3, and α4 that minimized the "sum of squares of the difference between the estimated value Yβi and the actual measured value Yi."

[0056] 2. Results 2.1 Search for muscle fiber type-specific proteins leaked into the blood upon muscle injury Studies comparing the body fluids of patients with muscle-damaging muscle diseases, such as Duchenne muscular dystrophy (DMD), and healthy controls (Ayoglu, et al., 2014; Rouillon, et al., 2014; Oonk, et al., 2016; Palolo, et al., 2018; Spitali, et al., 2018; Hathout, et al., 2019; Alayi, et al., 2020; Strandberg, et al., 2020) have comprehensively analyzed the body fluids of patients with muscle diseases and healthy controls using mass spectrometry, SomaScan assay (Somalogic Inc., Boulder, CO), antibody bead arrays, and enzyme-linked immunosorbent assays (ELISA). The raw data from these studies revealed that a total of 323 proteins were significantly increased in the body fluids of patients compared to healthy controls.

[0057] Meanwhile, Dr. Schiaffino (Venetian Institute for Molecular Medicine) and colleagues reported comprehensive quantification of proteins expressed in single muscle fibers using quantitative proteomics (Murgia, et al., 2021). Murgia et al. collected muscle tissue from the vastus lateralis muscles of four young men aged 22-27 years and performed proteomic analysis on relatively pure muscle fibers containing at least 80% MyHC I, MyHC IIa, or MyHC IId / x. To evaluate the relative expression levels of each protein in the proteomic analysis, the obtained values ​​were normalized to the expression levels of actin, alpha I, and skeletal muscle (ACTA1). As a result, 3,746 proteins expressed in muscle fibers were detected, and of these 3,746 proteins, 404 proteins were identified that showed statistically significant expression differences (p<0.05) between fiber types. Of these 404 proteins, 147 proteins with expression levels three times higher than those of other muscle fiber types were identified as muscle fiber type-specific proteins.

[0058] In this study, we used these two datasets to extract proteins that increased in the patient's body fluids and were expressed by muscle fiber type. We found that 24 proteins were expressed specifically in muscle fiber types and may leak into body fluids following muscle damage (Table 1). Of the 24 proteins, four were specific to MyHC I fibers. Eighteen were specific to MyHC IIa fibers and MyHC IId / x fibers. Specific extraction results are as follows:

[0059] Ayoglu et al. used antibody bead arrays to identify four proteins that were increased in the serum and plasma of DMD patients and Becker muscular dystrophy patients compared to healthy controls (Ayoglu, et al., 2014). Of these four proteins, one was found to be expressed in a muscle fiber type-specific manner. Rouillon et al. used liquid chromatography / mass spectrometry to analyze urine samples from DMD patients aged 4-10 years (n=5) and healthy controls aged 3-13 years (n=5). They identified eight proteins that were increased in DMD patients compared to healthy controls as DMD biomarkers (Rouillon, et al., 2014). Of these eight proteins, none was found to be expressed in a muscle fiber type-specific manner. Oonk et al. comprehensively analyzed the serum of ambulatory DMD patients (8.4 ± 1.1 years, n = 4), non-ambulatory DMD patients (16.4 ± 2.7 years, n = 7), and age-matched pediatric controls (8.3 ± 2.0 years, n = 4), and adult controls (33.6 ± 2.3 years, n = 5) using 2D-HPLC and liquid chromatography / mass spectrometry. The candidate proteins were confirmed by ELISA, and two proteins were identified (Oonk, et al., 2016). None of the two proteins were found to be specifically expressed in muscle fiber types. Palolo et al. analyzed the serum of DMD patients (n = 42) and age-matched healthy controls (n = 28) using the SomaScan assay and identified 52 proteins (Palolo, et al., 2018). Of the 52 proteins, three were specifically expressed in muscle fiber types. Spitali et al. analyzed the sera of DMD patients (n = 15) with a median age of 9 years and age-matched healthy controls (n = 9) using the same SomaScan assay and identified 110 proteins (Spitali, et al., 2018). Of these 110 proteins, 11 were muscle fiber type-specific. Hathout et al. analyzed the sera of DMD patients (n = 31) aged 4 to 10 years and age-matched healthy controls (n = 12) using the SomaScan assay and identified 108 proteins (Hathout, et al., 2019).Of these 108 proteins, two were found to be myofiber type-specific. Alayi et al. analyzed serum from DMD patients (age: 4.38 ± 0.24 yr, n = 9) and healthy controls (age: 4.47 ± 0.32 yr, n = 9) using liquid chromatography / mass spectrometry and identified 35 proteins (Alayi, et al., 2020). Of these 35 proteins, 12 were found to be myofiber type-specific. Strandberg et al. analyzed serum or plasma from DMD patients (n = 285) and healthy controls (n = 37) using antibody bead arrays and identified nine proteins (Strandberg, et al., 2020). Of these nine proteins, two were found to be myofiber type-specific.

[0060] Since quantitative proteomic analysis of single muscle fibers allows for comparison of relative expression levels, in this study we investigated highly expressed proteins as muscle fiber type-specific biomarkers.Of the 24 proteins that are expressed specifically in muscle fiber types, five proteins are specifically expressed in MyHC I fibers, and the top three proteins with the highest expression levels were myosin light chain 3 (MYL3), troponin C, and troponin I.

[0061] Of these three proteins, MYL3 was investigated as a candidate MyHC I-specific biomarker, as an antibody with an epitope specific to a protein leaked into the blood of DMD patients was reported in an antibody bead array study (Strandberg et al., 2020). Furthermore, of the MyHC I-specific proteins myomesin-3 (MYOM3) and phosphoglucomutase-1, a MyHC IIa- and MyHC IId / x-specific protein, MYOM3, which is highly expressed, was selected as a candidate biomarker. Furthermore, myomesin-2 (MYOM2), an isoform of MYOM3, was reported to be elevated in the serum of DMD patients (Alayi et al., 2020), and MYOM2 has been reported to be highly expressed in fast-twitch muscle fibers (Agarkova et al., 2004). Therefore, MYOM2 was investigated as a candidate fast-twitch muscle biomarker. The top three proteins specifically expressed in MyHC IIa and MyHC IId / x were Troponin C, skeletal muscle (TNNC2), Troponin I, fast skeletal muscle (TNNI2), and Troponin T, fast skeletal muscle (TNNT3). Of these three proteins, TNNT3 was investigated as a candidate MyHC IIa- and MyHC IId / x-specific biomarker, since an antibody with an epitope against a protein leaked into the blood of DMD patients was reported in an antibody bead array study (Strandberg et al., 2020). Creatine kinase (CK), a long-standing marker of muscle damage, was also included as a candidate fast-twitch fiber-specific biomarker because it has been reported to be highly expressed in fast-twitch muscle fibers (Yamashita et al., 1991).

[0062] [Table 1]

[0063] 2.2 Human muscle damage model induced by eccentric exercise We examined whether MYOM3, MYOM2, MYL3, TNNT3, and CK, which are candidate blood biomarkers that reflect muscle fiber type, reflect muscle fiber type.

[0064] To estimate the degree of muscle damage caused by eccentric exercise, blood creatine kinase (CK) activity was measured before and after eccentric exercise (Table 2). Four of the six subjects, A, B, E, and F, showed levels that were approximately four times higher than before exercise, peaking four days after exercise. In particular, subject F's level increased by approximately 838 times four days after exercise compared to before exercise. On the other hand, subjects C and D showed an increase one day after exercise, but no change was observed thereafter.

[0065] [Table 2]

[0066] 2.3 Muscle fiber type composition of the human vastus lateralis and changes in blood biomarkers following eccentric exercise The muscle fiber type composition of human vastus lateralis was examined using immunohistochemical staining (Table 3, Figure 3). To identify muscle fiber type composition (% fibers), the % fibers were calculated from the number of MyHC I fibers, MyHC IIa fibers, and MyHC IId / x fibers stained by immunohistochemical staining and the total number. Subject B had 79.0% MyHC I fibers. Subjects A, C, and D had approximately 20-40% MyHC I fibers, approximately 40% MyHC IIa fibers, and approximately 15-25% MyHC IId / x fibers. Subjects E and F had approximately 10% MyHC I fibers.

[0067] To quantify the relative proportion of the cross-sectional area of ​​each fiber type to the total area of ​​muscle tissue (% Area Fibers), the areas of MyHC I fibers, MyHC IIa fibers, and MyHC IId / x fibers were measured using ImageJ and calculated from the area of ​​each muscle fiber type and the total area. Except for subject B, the other five subjects had a higher proportion of MyHC IIa fibers and a lower proportion of MyHC IId / x fibers in % Area Fibers compared to % Fibers. This indicates that one subject had a predominance of MyHC I fibers over MyHC II fibers, and five subjects had a predominance of MyHC II fibers over MyHC I fibers.

[0068] [Table 3]

[0069] To investigate the pattern of muscle protein leakage into the blood due to muscle damage, we examined changes in blood levels of MYOM3 and its fragments by Western blot using serum samples taken before eccentric exercise and 1, 2, 3, and 4 days after exercise (Figure 4). The highest levels of leakage were observed 4 days after exercise compared to before exercise. Therefore, we examined changes in MYOM3, MYOM2, MYL3, TNNT3, and their fragments, which are candidate biomarkers reflecting muscle fiber type, using serum samples taken before and 4 days after exercise (Figures 5 and 6).

[0070] MYOM3 was detected at approximately 160 kDa in samples using human vastus lateralis muscle lysate, and its fragments in blood were detected at approximately 100 kDa and 130 kDa (Figure 5). On the other hand, MYOM2 was detected at approximately 160 kDa in human vastus lateralis muscle lysate, and its fragments in blood were detected at approximately 130 kDa in the serum of subject F 4 days after exercise (Figure 5). Because the background in the detection of blood MYOM2 was high in subjects B and D, Western blots were performed using membranes different from those used in the other subjects. Blood MYL3 was detected in the serum of subject F 4 days after exercise at approximately 20 kDa, the same molecular weight as human vastus lateralis muscle lysate, and its fragment, approximately 17 kDa (Figure 5). Bands above 20 kDa were considered to be nonspecific signals. The amount of TNNT3 in the blood was examined using anti-TNNT3 antibody (HPA056909; ATLAS ANTIBODIES, 1:100) and anti-TNNT3 antibody (HPA037810; ATLAS ANTIBODIES, 1:100), but was not detectable (Figure 6).

[0071] Using the increase in MYOM3, MYL3, MYOM2, CK, and their fragments obtained by Western blotting from before exercise to 4 days after eccentric exercise, we attempted to calculate a muscle fiber type prediction equation. Here, we performed a multiple regression analysis using the percentage (%) of MyHC I fibers as the dependent variable and MYOM3, MYL3, MYOM2, MYL3, and CK as the four explanatory variables. The results of the multiple regression analysis are shown in Table 4. From these results, we derived the following muscle fiber type prediction equation. R 2 =0.992, showing good regression.

number

[0072] [Table 4]

[0073] 3. Summary Because proteins highly expressed in skeletal muscle leak into the bloodstream after muscle injury, we first attempted to identify proteins for which antibodies with known epitopes were identified among the top three highly expressed proteins among 24 muscle fiber type-specific biomarker candidates. Myomesin is a structural protein that binds to myosin in the M-band to maintain sarcomere structure. The myomesin isoform MYOM3 is localized to M6 / M6' in the M-band, and MYOM2 is localized to M1 (Lamber, et al., 2022). MYL3 is a subunit of the myosin light chain. TNNT3 is an isoform of troponin T, a subunit of the troponin complex. CK catalyzes the reaction of creatine phosphate and adenosine diphosphate from creatine and adenosine triphosphate. CKM is a CK isozyme specifically expressed in skeletal muscle (Yamashita, et al., 1991).

[0074] In this study, calf raises were performed to induce muscle damage in the gastrocnemius, and muscle tissue samples were collected from the vastus lateralis. In this study, exercise-induced muscle damage was induced through eccentric exercise, and muscle proteins expressed in specific muscle fiber types were captured in the body fluid. A multiple regression analysis was performed using the percentage of MyHC I fibers as the objective variable and the increases in MYOM3, MYL3, MYOM2, and CK before and after eccentric exercise as the explanatory variables, to derive a muscle fiber type estimation equation.

[0075] The results of this study provide a non-invasive method for identifying skeletal muscle fiber types in humans.

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Claims

1. A method for estimating the composition of skeletal muscle fiber types in humans based on the abundance ratio of multiple biomarkers leaked into body fluids after eccentric exercise.

2. 2. The method of claim 1, wherein the skeletal muscle fiber type is at least one skeletal muscle fiber type selected from the group consisting of myosin heavy chain (MyHC) type I (hereinafter also referred to as "MyHC I"), type IIa (hereinafter also referred to as "MyHC IIa"), type IId / x (hereinafter also referred to as "MyHC IId / x"), and fast-twitch muscle-specific proteins.

3. 2. The method of claim 1, wherein the biomarker is a muscle fiber-specific protein.

4. The muscle fiber-specific protein (i) Group: Myofiber-specific proteins for myofiber type I (MyHC I), ・MYL3 (Myosin light chain 3), ・TNNC1 (Troponin C, slow skeletal and cardiac muscles), ・TNNI1 (Troponin I, slow skeletal muscle), ・MYOM3 (Myomesin-3), and ・ANP32B(Acidic leucine-rich nuclear phosphoprotein 32 family member B); (ii) Group: Myofiber-specific proteins for myofiber type IIa (MyHC IIa) and myofiber type IIx / d (MyHC IId / x), ・TNNC2(Troponin C, skeletal muscle), ・TNNI2 (Troponin I, fast skeletal muscle), ・TNNT3(Troponin T, fast skeletal muscle), ・MYL1 (Myosin light chain 1 / 3, skeletal muscle isoform), ・TPM1 (Tropomyosin alpha-1 chain), ・ALDOA (Fructose-bisphosphate aldolase A; Fructose-bisphosphate aldolase), ・ENO3 (Beta-enolase; Enolase), ・PYGM (Glycogen phosphorylase, muscle form), ・PKM (Pyruvate kinase), ・TPI1 (Triosephosphate isomerase), ・PFKM (ATP-dependent 6-phosphofructokinase, muscle type), ・AK1 (Adenylate kinase isoenzyme 1), ・GPD1 (Glycerol-3-phosphate dehydrogenase [NAD(+)], cytoplasmic), ・ESD (S-formylglutathione hydrolase), ・ADSL (Adenylosuccinate lyase), ・GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), and ・RPS6KA3 (Ribosomal protein S6 kinase alpha-3); (iii) Group: Myofiber-specific proteins for myofiber type IIx / d (MyHC IId / x), GPI (lucose-6-phosphate isomerase), and ・PGM1 (Phosphoglucomutase-1); and, (iv) Group: As muscle fiber-specific proteins for fast-twitch muscle, ・MYOM2 (Myomesin-2), and ・CK (Creatin kinase); 4. The method of claim 3, wherein the compound is selected from the group consisting of:

5. The method of claim 4, characterized in that at least two groups are selected in combination from each of the muscle fiber-specific protein groups (i), (ii), (iii), and (iv), and the amount of leakage of at least one muscle fiber-specific protein and its fragment from each selected group into body fluids after eccentric exercise is measured.

6. The method according to any one of claims 1 to 5, wherein the body fluid is at least one selected from the group consisting of serum, plasma, whole blood, saliva, and urine.

7. The percentage (%) of MyHC I fibers was used as the increase in MYOM3, MYOM2, MYL3, and CK after eccentric exercise and calculated using the following formula: [Equation 1] The method of claim 6, wherein the calculated

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