Damaged muscle fibers might masquerade as hybrid fibers – a cautionary note on immunophenotyping mouse muscle with mouse monoclonal antibodies

We report that, labeling mouse muscle tissue, with mouse monoclonal antibodies specific to slow or fast myosin heavy chain (sMyHC and fMyHC, respectively), can lead to artefactual labeling of damaged muscle fibers, as hybrid fibers (sMyHC+ and fMyHC+). We demonstrate that such erroneous immunophenotyping of muscle may be avoided, by performing colabeling or serial-section-labeling, to identify damaged fibers. The quadriceps femoris muscle group (QF) in 7-month-old, male, C57BL/6J mice had: 1.21 ± 0.21%, 98.34 ± 1.06%, 0.07 ± 0.01%, and 0.53 ± 0.85% fibers, that were, sMyHC+, fMyHC+, hybrid, and damaged, respectively. All fibers in the tibialis anterior muscle (TA) of 3-month-old, male, C57BL/6J mice were fMyHC+; and at 3 days after injurious eccentric contractions, there was no fiber-type shift, but ~ 18% fibers were damaged.

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The Adaptive Potential of Skeletal Muscle Fibers

Canadian Journal of Applied Physiology ◽

10.1139/h02-023 ◽

2002 ◽

Vol 27 (4) ◽

pp. 423-448 ◽

Cited By ~ 109

Author(s):

Dirk Pette

Keyword(s):

Skeletal Muscle ◽

Fiber Type ◽

Muscle Fibers ◽

Protein Isoforms ◽

Mammalian Skeletal Muscle ◽

Adaptive Potential ◽

Hybrid Fibers ◽

Neuromuscular Activity ◽

Skeletal Muscle Fibers ◽

Initial Location

Mammalian skeletal muscle fibers display a great adaptive potential. This potential results from the ability of muscle fibers to adjust their molecular, functional, and metabolic properties in response to altered functional demands, such as changes in neuromuscular activity or mechanical loading. Adaptive changes in the expression of myofibrillar and other protein isoforms result in fiber type transitions. These transitions occur in a sequential order and encompass a spectrum of pure and hybrid fibers. Depending on the quality, intensity, and duration of the alterations in functional demand, muscle fibers may undergo functional transitions in the direction of slow or fast, as well as metabolic transitions in the direction of aerobic-oxidative or glycotytic. The maximum range of possible transitions in either direction depends on the fiber phenotype and is determined by its initial location in the fiber spectrum. Key words: Ca-sequestering proteins, energy metabolism, fiber type transition, myofibrillar protein isofonns, myosin, neuromuscular activity

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Phenotypic Modulation of Skeletal Muscle Fibers in LPIN1-Deficient Lipodystrophic (fld) Mice

Veterinary Pathology ◽

10.1177/0300985818809126 ◽

2018 ◽

Vol 56 (2) ◽

pp. 322-331

Author(s):

Rani S. Sellers ◽

S. Radma Mahmood ◽

Geoffrey S. Perumal ◽

Frank P. Macaluso ◽

Irwin J. Kurland

Keyword(s):

Electron Microscopy ◽

Skeletal Muscle ◽

Fiber Type ◽

Periodic Acid ◽

Muscle Fibers ◽

Myosin Atpase ◽

Hybrid Fibers ◽

Periodic Acid Schiff ◽

Skeletal Muscle Fibers ◽

Lipin 1

Lipin-1 ( Lpin1)–deficient lipodystrophic mice have scant and immature adipocytes and develop transient fatty liver early in life. Unlike normal mice, these mice cannot rely on stored triglycerides to generate adenosine triphosphate (ATP) from the β-oxidation of fatty acids during periods of fasting. To compensate, these mice store much higher amounts of glycogen in skeletal muscle and liver than wild-type mice in order to support energy needs during periods of fasting. Our studies demonstrated that there are phenotypic changes in skeletal muscle fibers that reflect an adaptation to this unique metabolic situation. The phenotype of skeletal muscle (soleus, gastrocnemius, plantaris, and extensor digitorum longus [EDL]) from Lpin1-/- was evaluated using various methods including immunohistochemistry for myosin heavy chains (Myh) 1, 2, 2a, 2b, and 2x; enzyme histochemistry for myosin ATPase, cytochrome-c oxidase (COX), and succinyl dehydrogenase (SDH); periodic acid–Schiff; and transmission electron microscopy. Fiber-type changes in the soleus muscle of Lpin1-/- mice were prominent and included decreased Myh1 expression with concomitant increases in Myh2 expression and myosin-ATPase activity; this change was associated with an increase in the presence of Myh1/2a or Myh1/2x hybrid fibers. Alterations in mitochondrial enzyme activity (COX and SDH) were apparent in the myofibers in the soleus, gastrocnemius, plantaris, and EDL muscles. Electron microscopy revealed increases in the subsarcolemmal mitochondrial mass in the muscles of Lpin1-/- mice. These data demonstrate that lipin-1 deficiency results in phenotypic fiber-specific modulation of skeletal muscle necessary for compensatory fuel utilization adaptations in lipodystrophy.

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Absence of a growth hormone effect on rat soleus atrophy during a 4-day spaceflight

Journal of Applied Physiology ◽

10.1152/jappl.1993.74.2.527 ◽

1993 ◽

Vol 74 (2) ◽

pp. 527-531 ◽

Cited By ~ 26

Author(s):

B. Jiang ◽

R. R. Roy ◽

C. Navarro ◽

V. R. Edgerton

Keyword(s):

Growth Hormone ◽

Succinate Dehydrogenase ◽

Cross Sections ◽

Fiber Type ◽

Cross Sectional Area ◽

Sectional Area ◽

Cross Sectional ◽

Fast Myosin ◽

Hormone Effect ◽

Fast Myosin Heavy Chain

The objectives of the present study were to determine the size and enzyme properties of soleus fibers of rats subjected to a 4-day spaceflight (National Aeronautics and Space Administration, STS-41) and the effects of exogenous growth hormone (GH) on the atrophic response of the muscle. Four groups of rats were studied: 1) control (Con), 2) Con plus GH treated (Con + GH), 3) flight (Fl), and 4) F1 plus GH treated (Fl + GH). Cross-sectional area and the activities of succinate dehydrogenase and myofibrillar adenosinetriphosphatase (ATPase) were determined in fibers identified in frozen serial cross sections. Fibers were categorized immunohistochemically as slow, fast, or slow-fast on the basis of their reaction with slow and fast myosin heavy-chain (MHC) monoclonal antibodies. Fibers also were categorized as light or dark on the basis of their staining for ATPase at pH 8.6. After the 4-day flight, mean body weight was significantly decreased compared with control. The absolute and relative (muscle wt/body wt) soleus weights were significantly smaller in the Fl and Fl + GH rats compared with their respective ground-based controls. In both flight groups, the cross-sectional area of the light ATPase fibers was significantly smaller (approximately 30%) than control. Three of 11 flight rats had a higher proportion of fibers expressing both slow and fast MHCs than expected on the basis of the fiber type distribution in the 11 control rats. Mean fiber succinate dehydrogenase and ATPase activities were similar among the four groups.(ABSTRACT TRUNCATED AT 250 WORDS)

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Fast myosin heavy chains expressed in secondary mammalian muscle fibers at the time of their inception

Journal of Cell Science ◽

10.1242/jcs.107.9.2361 ◽

1994 ◽

Vol 107 (9) ◽

pp. 2361-2371 ◽

Cited By ~ 9

Author(s):

M. Cho ◽

S.M. Hughes ◽

I. Karsch-Mizrachi ◽

M. Travis ◽

L.A. Leinwand ◽

...

Keyword(s):

Muscle Development ◽

Muscle Fibers ◽

Fiber Formation ◽

Mammalian Skeletal Muscle ◽

Western Blots ◽

Mammalian Muscle ◽

Heavy Chains ◽

Fast Myosin ◽

Myhc Isoforms ◽

Fast Myosin Heavy Chain

Mammalian skeletal muscle is generated by two waves of fiber formation, resulting in primary and secondary fibers. These fibers mature to give rise to several classes of adult muscle fibers with distinct contractile properties. Here we describe fast myosin heavy chain (MyHC) isoforms that are expressed in nascent secondary, but not primary, fibers in the early development of rat and human muscle. These fast MyHCs are distinct from previously described embryonic and neonatal fast MyHCs. To identify these MyHCs, monoclonal antibodies were used whose specificity was determined in western blots of MyHCs on denaturing gels and reactivity with muscle tissue at various stages of development. To facilitate a comparison of our results with those of others obtained using different antibodies or species, we have identified cDNAs that encode the epitopes recognized by our antibodies wherever possible. The results suggest that epitopes characteristic of adult fast MyHCs are expressed very early in muscle fiber development and distinguish newly formed secondary fibers from primary fibers. This marker of secondary fibers, which is detectable at the time of their inception, should prove useful in future studies of the derivation of primary and secondary fibers in mammalian muscle development.

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Muscle plasticity and hybrid fibers in the masticatory musculature. Literature review

International Journal of Medical and Surgical Sciences ◽

10.32457/ijmss.v7i3.583 ◽

2020 ◽

pp. 1-11

Author(s):

Felipe Inostroza

Keyword(s):

Speech Therapy ◽

Fiber Type ◽

Muscle Fibers ◽

Masticatory Muscles ◽

Craniofacial Morphology ◽

Type I ◽

Hybrid Fibers ◽

Muscle Plasticity ◽

Dental Prostheses ◽

The Relationship

The masticatory musculature is characterized by presenting hybrid fibers that in recent years have been related to the phenomenon of muscle plasticity. The objective of the study was to describe the relationship between muscle plasticity and the hybrid muscle fibers present in the masticatory muscles, through a narrative review. For this, an electronic search was conducted in PUBMED, ScienceDirect and BIREME, using the keywords: “Muscle Plasticity”, “Hybrid Muscle Fibers” and “Hybrid Fibers”. Documents that report the myosin heavy chain (MHC) isoforms present in the masticatory muscles of humans and other mammals were selected, along with the changes linked to functional demands. The presence of type I and type II pure fibers were described, in addition to other isoforms such as MHC-la, MHC-IIM, MHC-fetal and MHC-cardiac. However, a significant percentage of fibers in the masticatory muscles are hybrids, that is, they express more than one MHC isoform, which are also different at the intermuscular and intramuscular level. Local influences can contribute to the variation of fiber type expression. In the chewing muscles, weaning, the hardness of food, bruxism, craniofacial morphology and the use of dental prostheses generate changes at the level of the chewing muscles, where the presence of hybrid fibers is common. The important presence of hybrid fibers in the masticatory muscles and their relationship with muscle plasticity throughout the life cycle, due to functional and pathological changes, is concluded. It is important for Speech Therapy and Myofunctional Therapy to deepen their understanding of the physiology of oromyofunctional behavior.

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Enhancement of hybrid-fiber types in rat soleus muscle after clenbuterol administration during hindlimb unloading

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y04-034 ◽

2004 ◽

Vol 82 (5) ◽

pp. 311-318 ◽

Cited By ~ 5

Author(s):

F Picquet ◽

L De-Doncker ◽

M Falempin

Keyword(s):

Myosin Heavy Chain ◽

Heavy Chain ◽

Fiber Type ◽

Myosin Heavy Chain Isoforms ◽

Hindlimb Unloading ◽

Fiber Types ◽

Myosin Isoforms ◽

Fast Myosin ◽

Contractile Parameters ◽

Fast Myosin Heavy Chain

Our objective was to determine the effects of a clenbuterol (CB) treatment orally administered (2 mg per kg) to rats submitted to 14 days of hindlimb unloading (HU). The morphological and the contractile properties as well as the myosin heavy chain isoforms contained in each fiber type were determined in whole soleus muscles. As classically described after HU, a decrease in muscle wet weight and in body mass associated with a loss of muscular force, an evolution of the contractile parameters towards those of a fast muscle type, and the emergence of fast myosin heavy chain isoforms were observed. The CB treatment in the HU rats helped reduce the decrease in 1) muscle and body weights, 2) force and 3) the proportion of slow fibers, without preventing the emergence of fast myosin isoforms. Clenbuterol induced a complex remodelling of the muscle typing promoting the combination of both slow and fast myosin isoforms within one fiber. To conclude, our data demonstrate that CB administration partially counteracts the effects produced by HU, and they allow us to anticipate advances in the treatment of muscular atrophy.Key words: β2 agonist, clenbuterol, soleus, contractile parameters, myosin, immunohistochemistry, simulated microgravity, countermeasure.

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Evidence for three adult fast myosin heavy chain isoforms in type II skeletal muscle fibers in pigs.

Journal of Animal Science ◽

10.2527/1998.7661584x ◽

1998 ◽

Vol 76 (6) ◽

pp. 1584 ◽

Cited By ~ 58

Author(s):

L Lefaucheur ◽

R K Hoffman ◽

D E Gerrard ◽

C S Okamura ◽

N Rubinstein ◽

...

Keyword(s):

Skeletal Muscle ◽

Myosin Heavy Chain ◽

Heavy Chain ◽

Muscle Fibers ◽

Myosin Heavy Chain Isoforms ◽

Type Ii ◽

Fast Myosin ◽

Skeletal Muscle Fibers ◽

Fast Myosin Heavy Chain

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Sarcomere lesion damage occurs mainly in slow fibers of reloaded rat adductor longus muscles

Journal of Applied Physiology ◽

10.1152/jappl.1998.85.3.1017 ◽

1998 ◽

Vol 85 (3) ◽

pp. 1017-1023 ◽

Cited By ~ 36

Author(s):

Kalpana Vijayan ◽

Joyce L. Thompson ◽

Danny A. Riley

Keyword(s):

Fiber Type ◽

Hindlimb Unloading ◽

Fiber Types ◽

Hybrid Fiber ◽

Hybrid Fibers ◽

Fast Myosin ◽

Slow Fiber ◽

Fast Fiber ◽

Damage Susceptibility ◽

Adductor Longus

Sarcomere lesions were previously observed with reloading of rat adductor longus muscles after spaceflight and hindlimb unloading (HU). Spaceflown rats displayed more lesioned fibers in the “slow-fiber” region, suggesting a damage-susceptible fiber type. Unloading induces fast myosin expression in some slow fibers, generating hybrid fibers. We examined whether lesion damage differed among slow-, hybrid-, and fast-fiber types in HU-reloaded adductor longus muscles. Temporal HU for 5, 8, 11, 14, and 17 days revealed that hybrid fiber percent, detected by antimyosin immunostaining, peaked at 29 ± 12% by 14 days. A 14-day HU followed by 12–14 h of voluntary reloading was performed to induce lesions. χ2 analysis showed that slow fibers were preferentially damaged, accounting for 92 ± 5% of lesioned fibers; hybrid and fast fibers accounted for 7 ± 4 and <0.5%, respectively. Atrophy did not explain differential lesion damage across fiber types, as slow and hybrid fibers atrophied to a similar extent. Because active myofiber contractions are requisite for lesion formation, selective recruitment of slow fibers most likely explains their damage susceptibility.

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Use of type-specific antimyosins to demonstrate the transformation of individual fibers in chronically stimulated rabbit fast muscles.

The Journal of Cell Biology ◽

10.1083/jcb.79.1.252 ◽

1978 ◽

Vol 79 (1) ◽

pp. 252-261 ◽

Cited By ~ 99

Author(s):

N Rubinstein ◽

K Mabuchi ◽

F Pepe ◽

S Salmons ◽

J Gergely ◽

...

Keyword(s):

De Novo ◽

Fiber Type ◽

Muscle Fibers ◽

Staining Intensity ◽

Progressive Increase ◽

Fast Myosin ◽

Chicken Muscle ◽

Longus Muscle ◽

Fast Muscles ◽

Physiological And Biochemical

Continuous stimulation of a rabbit fast muscle at 10 Hz changes its physiological and biochemical parameters to those of a slow muscle. These transformations include the replacement of myosin of one type by myosin of another type. Two hypotheses could explain the cellular basis of these changes. First, if fibers were permanently programmed to be fast or slow, but not both, a change from one muscle type to another would involve atrophy of one fiber type accompanied by de novo appearance of the other type. Alternatively, preexisting muscle fibers could be changing from the expression of one set of genes to the expression of another. Fluorescein-labeled antibodies against fast (AF) and slow (AS) muscle myosins of rabbits have been prepared by procedures originally applied to chicken muscle. In the unstimulated fast peroneus longus muscle, most fibers stained only with AF; a small percentage stained only with AS; and no fibers stained with both antibodies. In stimulated muscles, most fibers stained with both AF and AS; with increasing time of stimulation, there was a progressive decrease in staining intensity with AF and a progressive increase in staining intensity with AS within the same fibers. These results are consistent with a theory that individual preexisting muscle fibers can actually switch from the synthesis of fast myosin to the synthesis of slow myosin.

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