ARTICLE |
Correspondence to: Fatima PedrosaDomellöf, Dept. of Integrative Medical Biology, Section of Anatomy, Umeå University, SE-901 87 Umeå, Sweden. E-mail: Fatima.Pedrosa-Domellof@anatomy.umu.se
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Summary |
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Data on the myosin heavy chain (MyHC) composition of human muscle spindles are scarce in spite of the well-known correlation between MyHC composition and functional properties of skeletal muscle fibers. The MyHC composition of intrafusal fibers from 36 spindles of human biceps brachii muscle was studied in detail by immunocytochemistry with a large battery of antibodies. The MyHC content of isolated muscle spindles was assessed with SDS-PAGE and immunoblots. Four major MyHC isoforms (MyHCI, IIa, embryonic, and intrafusal) were detected with SDS-PAGE. Immunocytochemistry revealed very complex staining patterns for each intrafusal fiber type. The bag1 fibers contained slow tonic MyHC along their entire fiber length and MyHCI, -cardiac, embryonic, and fetal isoforms along a variable part of their length. The bag2 fibers contained MyHC slow tonic, I,
-cardiac, embryonic, and fetal isoforms with regional variations. Chain fibers contained MyHCIIa, embryonic, and fetal isoforms throughout the fiber, and MyHCIIx at least in the juxtaequatorial region. Virtually each muscle spindle had a different allotment of numbers of bag1, bag2 and chain fibers. Taken together, the complexity in intrafusal fiber content and MyHC composition observed indicate that each muscle spindle in the human biceps has a unique identity. (J Histochem Cytochem 50:171183, 2002)
Key Words: human biceps brachii, muscle spindles, intrafusal fiber, bag1 fiber, bag2 fiber, chain fiber, MyHC
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Introduction |
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MUSCLE SPINDLES are intramuscular sensory receptors that relay information about the range and rate of muscle length changes. They consist of a bundle of encapsulated small-diameter muscle fibers, the intrafusal fibers, which receive both sensory and motor innervation and exhibit characteristic morphological and physiological features (for review see
Myosin ATPase activity resides in the head region of the myosin heavy chain (MyHC) molecule, and its relative activity determines the shortening velocity of muscle fibers (
Several lines of evidence suggest the existence of more than one slow MyHC (MyHCI) isoform, although only one corresponding gene has been identified to date (
Although there is an extensive body of knowledge concerning the MyHC composition of human extrafusal muscle fibers, corresponding information on human muscle spindles is rare (-cardiac MyHC (MyHC
) and, to a variable extent, with embryonic MyHC (MyHCemb). In addition, the nuclear bag2 fibers react with antibodies against fast-twitch MyHCII and neonatal MyHCneo, whereas the nuclear chain fibers mainly react with antibodies specific for MyHCneo and MyHCemb.
In this study we investigated the composition and distribution of MyHC isoforms in the muscle spindles of adult human biceps brachii muscle. This study is the first part of a large investigation aimed at thoroughly characterizing and comparing muscle spindles in human muscles with distinct architecture and functional demands.
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Materials and Methods |
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Materials
Twelve muscle samples were collected shortly after death from the biceps brachii muscle of four females (age 48, 38, 19, and 15, six specimens) and six males (age 38, 37, 27, 25, 22, and 19) without known muscle-related disorders. The samples were collected according to the ethical recommendation of the Swedish Transplantation Law, with the approval of the Medical Ethical Committee, Umeå University.
The biceps muscle samples were mounted, rapidly frozen in liquid propane chilled with liquid nitrogen, and stored at -81C until sectioned. Frozen specimens were serially sectioned at -25C using a Reichert Jung cryostat (Leica; Nussloch, Germany). Sections to be used for demonstration of myofibrillar ATPase activity after preincubation at pH 10.4, 4.6, and 4.3 (
Additional samples were collected shortly after death from adult extraocular, lumbrical, and heart muscles and frozen as above before SDS-PAGE analyses and immunoblotting. A sample of fetal (20 weeks of gestation) biceps brachii muscle (
Antibodies and Labeling
Monoclonal and polyclonal antibodies specific for different MyHC isoforms were used in the present study (Table 1). The specificity of these antibodies was carefully assessed for human muscles.
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Immunocytochemistry
Immunocytochemistry was performed using the indirect peroxidase-anti-peroxidase (PAP) technique (
All sections were examined and photographed with a Zeiss Axiophot microscope (Carl Zeiss; Oberkochen, Germany) equipped with a 3CCD camera (Dage-MTI; Michigan City, MI), using the Adobe Photoshop (version 5.5) software program.
Biochemistry
SDS-PAGE.
Individual muscle spindles were dissected from freeze-dried sections under a stereomicroscope (Wild; Heerbrugg, Switzerland) and lysed in Laemmli sample buffer (Bio-Rad Laboratories; Hercules, CA) containing 5% ß-mercaptoethanol. Whole muscle extracts were prepared as previously described (
Western Blotting Analysis
After SDS-PAGE, proteins were transferred to 0.45-µm nitrocellulose membrane (Bio-Rad) in a Trisglycine buffer system using a Mini Trans-Blot apparatus (Bio-Rad) surrounded by a 15C water bath unit for 17 hr at 30-V constant voltage. The membranes were washed twice for 5 min with 20 ml distilled water to remove gel and transfer buffer components and weakly bound proteins. Immmunostaining was performed with WesternBreeze kit (Novex; Carlsbad, CA) according to the manufacturer's instructions (WesternBreeze Chromogenic Immunodetection Protocol) with the following primary antibodies (diluted in blocking solution): A4.840 (1:20), A4.951 (1:100), A4.74 (1:250), 2B6 (1:500), VII1H3 (1:10,000), ALD19 (1:500), N2.261 (1:250), and BF35 (1:3000).
Survey
A total of 36 muscle spindles (25 from females, 11 from males) were examined, 20 of them in consecutive serial transverse sections covering 13 mm of the length of the fibers. The spindles were divided into three regions: the A region, including equator (EQ) and juxtaequatorial parts (Aj), containing the periaxial fluid space; the B region, extending from the end of the periaxial fluid space to the end of the capsule; and the extracapsular C region (
Statistical Analyses
The StatView software (Abacus Concepts; Berkeley, CA) was used for statistical analysis. Statistical comparison between males and females for the fiber numbers was performed with an unpaired t-test. Differences were considered significant at p<0.05. Group (gender) data were presented as mean and standard deviations (SD).
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Results |
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Biochemistry
SDS-PAGE revealed the presence of four MyHC bands in muscle spindles (Fig 1) similar to our previous findings (
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The specificity of A4.840 and A4.951 to MyHCI, A4.74 to MyHCIIa, 2B6 to MyHCemb, and VII1H3 to MyHCI+IIa+IIx was confirmed for human muscles (data not shown). In addition, MAb ALD19 showed strong affinity to MyHCsto in chicken muscle (data not shown). MAb N2.261 (Fig 2) recognized MyHCI and MyHCIIa as previously described (-c band and the MyHCeom (eom = extraocular muscle), a band unique to the extraocular muscles (unpublished results). The specificity of MAb BF35 was further assessed for human muscle. It labeled MyHCI, MyHCIIa, MyHCeom, MyHC
-c, and MyHCfet (fet = fetal). This MAb also recognized the chicken slow-tonic MyHC, MyHCsto.
Intrafusal Fiber Type Composition
All the spindles investigated in the present study occurred as a single isolated receptor. In the 36 spindles, a total of 235 intrafusal fibers (183 from females, 52 from males) were examined and classified as 59 bag1 fibers, 46 bag2 fibers, and 127 chain fibers on the basis of their ATPase activity. The remaining three intrafusal fibers did not fit into any of these categories because of their atypical mATPase activity (see below). The fiber type composition of the muscle spindles varied considerably (Table 2). On average, there were 9.1 (range 416) intrafusal fibers per spindle, 2.2 (range 14) nuclear bag1, 1.3 (range 04) nuclear bag2, and 5.7 (range 212) nuclear chain fibers in each spindle. Intrafusal fibers occurring only in the C region were not counted. Strikingly, among 22 muscle spindles examined in the A or B regions, where the full complement of intrafusal fibers is present, only two spindles had exactly the same fiber composition (Table 2). There were no statistically significant differences either in the total number of intrafusal fibers (p=0.20) per spindle or the number of bag1 (p=0.54), bag2 (p=0.84), or chain (p=0.24) fibers per spindle between females and males. Furthermore, there were no distinct differences in staining patterns between males and females, so they will be described together.
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ATPase Activity
In general, the myofibrillar ATPase activities of the bag fibers but not of chain fibers showed regional variation (Fig 3). Nuclear bag1 fibers displayed low alkaline ATPase activity over their entire fiber length, low acid ATPase activity along the A and B regions, and an intermediate acid ATPase activity in the C region. Nuclear bag2 fibers showed very weak acid ATPase activity in the equator, moderate in the remaining A region, and tended to become strong along the B and C regions. The alkaline ATPase activity of the nuclear bag2 fibers was very low in the equator but moderate along the remaining A region and most of the B region. Towards the end of the B region and in the C region, the activity was weak to moderate. In general, nuclear chain fibers showed high alkaline and low acid ATPase activity along the entire fiber length. Differentiation between bag2 and bag1 fibers in the C region could be carried out easily and precisely at pH 4.6, since very high activity appeared on bag2 fibers and moderate or low on bag1 fibers (see Fig 7B).
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Although most intrafusal fibers conformed to the general ATPase activity described above, exceptions were observed. Three fibers exhibited atypical ATPase activity, showing strong or moderate alkaline but no acid ATPase activity. All these fibers were strongly and consistently stained with anti-MyHCsto.
Immunocytochemistry
Three intrafusal fiber types exhibited complex staining patterns with the different antibodies used (Fig 4).
Nuclear Bag1 Fibers.
All the nuclear bag1 fibers were strongly and evenly stained with anti-MyHCsto along their entire fiber length (Fig 5B, and Fig 7C). Bag1 fibers showed two different staining patterns with two different antibodies against MyHCI. They were weakly stained with anti-MyHCI/1st in the outer encapsulated pole and C region and were unstained along the remaining of their length (Fig 5C). However, bag1 fibers were moderately stained with anti-MyHCI/2nd in the C and the outer B region, tended to become less stained from the inner B to the outer A region, and lost their staining towards the equator (Fig 5D, and Fig 7D). MAb N2.261 labeled bag1 fibers in the C region only (Fig 5E, and Fig 7E) and so did MAb BF35 (Fig 5H, and Fig 7G), except that in one spindle two fibers showed low reactivity for a short distance in the A region. In general, the bag1 fibers were moderately stained with MAb VII1H3 in the extracapsular region, weakly in the B region, and unstained in the A region (Fig 5G). All bag1 fibers stained with MAb VII1H3 were also stained with anti-MyHCI/2nd in the same region. The bag1 fibers showed weak staining with anti-MyHCemb in the A and B regions (Fig 5I). In the C region, more than half of the fibers were weakly to moderately stained (Fig 7H); the remainder were unstained with this antibody (not shown). Although the bag1 fibers were generally weakly stained by anti-MyHCfet, fibers that were unstained or moderately stained for a short distance in the A or B region were also encountered (Fig 8). The staining intensity of bag1 fibers with anti-MyHC-c varied among different fibers and different spindles. At the equator, bag1 fibers showed a wide variation of staining intensity, from strong to weak. Bag1 fibers were often strongly to moderately stained in the juxtaequatorial region, less stained along the inner B region, and became negative in the remaining B and C regions (Fig 5J). However, the staining patterns of four bag1 fibers from one spindle were rather peculiar. These fibers were strongly stained in the juxtaequatorial region and unstained both in the B region and the equator. There were no fibers extending into the C region in this particular muscle spindle. None of the bag1 fibers reacted with anti-MyHCIIa (Fig 5F, and Fig 7F).
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Nuclear Bag2 Fibers.
Nuclear bag2 fibers showed strong and homogeneous staining with anti-MyHCsto (Fig 5B, and Fig 7C). They were strongly labeled by both anti-MyHCI/1st and anti-MyHCI/2nd over their entire length except at the inner juxtaequatorial and the equatorial regions, where they were moderately and faintly stained, respectively (Fig 5C, Fig 5D, and Fig 7D). Bag2 fibers were stained identically with MAbs N2.261 and BF35, moderately along the C and B regions, weakly in the juxtaequatorial region, and tended to become very weakly stained or unstained at the equator (Fig 5E, Fig 7E, Fig 5H, and Fig 7G). These fibers were strongly stained with MAb VII1H3 in the C and outer B regions, moderately in the inner B and A regions (Fig 5G). Two bag2 fibers, however, showed weak or negative staining in the equator and the latter fiber was unstained with MAbs A4.951, N2.261, A4.74, and BF35. Bag2 fibers showed the same staining patterns as bag1 fibers did with anti-MyHCemb, although two bag2 fibers in one spindle showed high activity in the A region (Fig 5I, and Fig 7H). The bag2 fibers showed a heterogeneous staining pattern with anti-MyHCfet. In some of the spindles, the bag2 fibers were moderately to strongly stained (Fig 8), whereas in other spindles they were weakly stained. In general, bag2 fibers were strongly to moderately stained with anti-MyHC-c in the A region, less stained in the inner B region, and became unstained in the remaining portion (Fig 5J). Bag2 fibers generally did not react with anti-MyHCIIa (Fig 5F, and Fig 7F), although two fibers showed weak staining in the equatorial region (not shown).
Nuclear Chain Fibers.
Nuclear chain fibers were definitely unstained with anti-MyHCsto, anti-MyHCI/1st, anti-MyHCI/2nd, and anti-MyHC-c, whereas they were strongly and uniformly stained with MAbs VII1H3 and BF35 and with anti-MyHCemb and anti-MyHCfet. All nuclear chain fibers were strongly stained with MAb N2.261, except that in the juxtaequatorial region almost one third of the fibers were moderately or even weakly stained (Fig 5E). Nuclear chain fibers were generally strongly stained with anti-MyHCIIa in the equatorial region and in the outer B and C regions. The staining intensity varied in the juxtaequatorial region, and variability was observed not only along the individual fibers but also between spindles. Most chain fibers were strongly stained, but some were weakly stained or even unstained (Fig 5F). These MyHCIIa-negative chain fibers, however, were always positive with both VII1H3 and N2.261 MAbs.
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Discussion |
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The present investigation yielded major results on the specificity of MAbs N2.261, BF35, and the MyHC composition of human biceps muscle spindles. Taken together, the data showed that the muscle spindles in the biceps are by far more complex than anticipated.
A thorough characterization of the Abs used was a prerequisite for interpreting the immunocytochemical data. The present study further extended our knowledge of the MyHC affinity for MAbs N2.261 and BF35 in human muscle to include MyHC-c and MyHCeom (N2.261 and BF35) and MyHCsto (BF35).
Four MyHC isoforms were present in intrafusal fibers in amounts sufficient to be detected by SDS-PAGE. The present study confirmed the presence of MyHCI, MyHCif, and MyHCemb (
We have performed a number of immunoblots (data not shown) with MAbs known to react specifically with intrafusal fibers, but neither of those MAbs labeled the MyHCif band or any of the other three bands. In particular, MAbs ALD19 and ALD58, specific against chicken MyHCsto, failed to label any muscle spindle band, although they reacted with extracts of chicken Ald muscle. These are intriguing results because MAbs against MyHCsto generally label bag fibers strongly and specifically (
When sufficient spindles from the biceps or from the first lumbrical muscle were pooled together for SDS-PAGE, similar migration patterns were observed, indicating that MyHCIIa, MyHCemb, MyHCif, and MyHCI are the major isoforms present in human muscle spindles. However, it should be noted that clear differences in the relative amounts of these four isoforms are detectable by SDS-PAGE when distinct single muscle spindles or the different regions of a single muscle spindle are compared (
MyHCsto, as interpreted by staining after incubation with MAb ALD19, was present in both bag1 and bag2 fibers along their entire length. The staining intensity of bag2 fibers with MAb ALD19 was generally as strong as that of bag1 fibers along their entire length. In rat hindlimb muscle spindles, however, bag2 fibers are typically less stained in the inner B region and unstained in the outer B and C regions (
It is obvious from the current results that nuclear chain fibers lacked MyHCsto, MyHC-c, and MyHCI but contained MyHCIIa. Although immunocytochemistry is not a quantitative method, the binding patterns of MAb A4.74 indicate that the amount of MyHCIIa varied along the fiber length, being considerably less in the juxtaequatorial region. Therefore, in the juxtaequatorial region, chain fibers likely contained the MyHCIIx isoform according to their strong and even binding with MAb VII1H3, absent staining with the pure anti-MyHCI, and their weaker staining/unstaining with MAb A4.74. Confirmation of whether MyHCIIx was present only in the juxtaequatorial region or in other regions as well was not possible in this study because of the unavailability of a pure anti-MyHCIIx antibody. The presence of MyHCIIa and MyHCIIx in chain fibers in the present study is in contrast to reports that neither of these two MyHCs were found in rat chain fibers (
Two developmental MyHC isoforms, MyHCemb and MyHCfet, were found in all three intrafusal fiber types, although reactivity in bag fibers was lower than that in chain fibers, or might even be absent for a short distance.
MyHC-c was present in both bag1 and bag2 fibers but lacking in chain fibers, as previously reported for both human and rat muscle spindles (
-c in intrafusal fibers of human biceps was distinct from that of rat, and it remains to be determined whether motor innervation regulates the expression of this isoform (
During development, immunoreactivity to different anti-MyHCI antibodies (MAbs A4.840, A4.951 and N2.261) appears sequentially, suggesting that the three antibodies recognize different MyHCI epitopes in developing human and rat muscles (
Monoclonal antibody BF35 reacted strongly with MyHCsto from chicken Ald muscle in immunoblots (Fig 2), indicating that this bovine-derived antibody also recognizes MyHCsto epitopes. On immunoblots, however, MAb BF35 reacted only with the muscle spindle bands corresponding to MyHCIIa and MyHCI (Fig 2). With immunocytochemistry, no bag1 fibers were stained with MAb BF35 in the intracapsular region, and the staining seen with this MAb in the extracapsular region corresponded to the presence of MyHCI. Therefore, MyHCsto identified by BF35 MAb on immunoblots was lacking in bag1 fibers based on both immunostaining and Western blotting. Because bag1 fibers were strongly stained along their entire length with MAb ALD19 but were unreactive to MAb BF35, at least in the intracapsular region, ALD19 does not appear to recognize the same epitopes on MyHCsto as BF35 MAb does, perhaps because of their different sources (chicken/bovine). The staining seen with MAb BF35 on bag2 and chain fibers revealed the presence of MyHCemb and MyHCfet in chain fibers and MyHCI, MyHCemb, and MyHCfet in bag2 fibers rather than MyHCsto.
It is noteworthy that human spindles are far less stereotypical than spindles of small animals in terms of numbers of intrafusal fibers and their patterns of MyHC expression. Muscle spindles usually contain four or five fibers in the rat hind limb and six to nine in the cat, with no more than four nuclear bag fibers (
The biceps brachii muscle has a relatively ordered architecture in which the muscle fibers run parallel to the muscle's force-generating axis (
In a rather large muscle with low muscle spindle density, such as the biceps (
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Acknowledgments |
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Supported by grants from the Medical Faculty, Umeå University, The Swedish Society for Medical Research, The Swedish Medical Research Council (12X-03934), and the Magnus Bergvall Foundation.
We thank Drs A. Kelly, D.A. Fischman, G.S. ButlerBrowne, J.J. Leger, and S. Schiaffino for kindly providing antibodies. We also thank Mona Lindström and Inga Johansson for excellent technical assistance.
Received for publication May 30, 2001; accepted October 9, 2001.
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