Expression of MyHC-15 and -2x in human muscle spindles: An immunohistochemical study

Abstract

To build on the existing data on the pattern of myosin heavy chain (MyHC) isoforms expression in the human muscle spindles, we aimed to verify whether the 'novel' MyHC-15, -2x and -2b isoforms are co-expressed with the other known isoforms in the human intrafusal fibres. Using a set of antibodies, we attempted to demonstrate nine isoforms (15, slow-tonic, 1, α, 2a, 2x, 2b, embryonic, neonatal) in different regions of intrafusal fibres in the biceps brachii and flexor digitorum profundus muscles. The reactivity of some antibodies with the extrafusal fibres was also tested in the masseter and laryngeal cricothyreoid muscles. In both upper limb muscles, the expression of slow-tonic isoform was a reliable marker for differentiating positive bag fibres from negative chain fibres. Generally, bag1 and bag2 fibres were distinguished in isoform 1 expression; the latter consistently expressed this isoform over their entire length. Although isoform 15 was not abundantly expressed in intrafusal fibres, its expression was pronounced in the extracapsular region of bag fibres. Using a 2x isoform-specific antibody, this isoform was demonstrated in the intracapsular regions of some intrafusal fibres, particularly chain fibres. To the best of our knowledge, this study is the first to demonstrate 15 and 2x isoforms in human intrafusal fibres. However, whether the labelling with an antibody specific for rat 2b isoform reflects the expression of this isoform in bag fibres and some extrafusal ones in the specialised cranial muscles requires further evaluation. The revealed pattern of isoform co-expression only partially agrees with the results of previous, more extensive studies. Nevertheless, it may be inferred that MyHC isoform expression in intrafusal fibres varies along their length, across different muscle spindles and muscles. Furthermore, the estimation of expression may also depend on the antibodies utilised, which may also react differently with intrafusal and extrafusal fibres.

Keywords: immunohistochemistry; intrafusal fibre; muscle spindle; myosin heavy chain isoforms (MyHC); skeletal muscle.

FIGURE 1

MyHC isoform expression in the B region of a muscle spindle in the biceps brachii of a 21‐year‐old patient. The muscle sections are labelled by: a. hematoxylin‐eosin staining (h & e) and antibodies specific for: b. MyHC‐st, c. MyHC‐1, d. MyHC‐15, e. MyHC‐2a+2x, f. MyHC‐2x, g. MyHC‐2b (10F5), h. MyHC‐2b (BF‐F3), i. MyHC‐emb, j. MyHC‐neo, k. MyHC‐α (MYH6) and l. MyHC‐α (BA‐G5). In a. intrafusal bag1 (b1), bag2 (b2) and chain (c), fibres are labelled. Scale bar: 50 μm.

FIGURE 2

MyHC isoform expression in the B region of a muscle spindle in the biceps brachii of a 41‐year‐old patient. The muscle sections are labelled by antibodies specific for: a. MyHC‐st, b. MyHC‐1, c. MyHC‐15, d. MyHC‐2a+2x, e. MyHC‐2x, f. MyHC‐2b (10F5), g. MyHC‐2b (BF‐F3), h. MyHC‐emb, i. MyHC‐neo and j. MyHC‐α (MYH6). In a. intrafusal bag1 (b1), bag2 (b2) and chain (c), fibres are labelled. Scale bar: 50 μm.

FIGURE 3

MyHC isoform expression in the C region of bag fibres in the biceps brachii of a 41‐year‐old patient. The muscle sections are labelled by antibodies specific for: a. MyHC‐st, b. MyHC‐2b (10F5), c. MyHC‐15 and d. MyHC‐2a+2x. In a. two bag (b), fibres are labelled. Note that both bag fibres express MyHC‐15. Scale bar: 50 μm.

FIGURE 4

MyHC isoform expression in the B region of a muscle spindle in the biceps brachii of a 63‐year‐old patient. The muscle sections are labelled by antibodies specific for: a. MyHC‐st, b. MyHC‐1, c. MyHC‐15, d. MyHC‐2a+2x, e. MyHC‐2x, f. MyHC‐2b (10F5), g. MyHC‐emb, h. MyHC‐neo and i. MyHC‐α (BA‐G5). In a. intrafusal bag1 (b1), bag2 (b2) and chain (c), fibres are labelled. Scale bar: 50 μm.

FIGURE 5

MyHC isoform expression in the B region of a muscle spindle in the flexor digitorum profundus. The muscle sections are labelled by antibodies specific for: a. MyHC‐st, b. MyHC‐1, c. MyHC‐15, d. MyHC‐2a+2x, e. MyHC‐2x, f. MyHC‐2b (10F5), g. MyHC‐emb, h. MyHC‐neo and i. MyHC‐15 (immunofluorescence). In a. intrafusal bag1 (b1), bag2 (b2) and chain (c) fibres are labelled. Scale bar: 50 μm.

FIGURE 6

Reactivity of antibodies specific for MyHC‐2b and ‐α in the masseter and laryngeal cricothyreoideus muscle. The staining pattern of 10F5 and BF‐F3 (rat MyHC‐2b), BA‐G5 and MYH6 (MyHC‐α) in masseter (a–d, k, l) and cricothyreoideus muscles (e–j). The muscle sections are labelled by antibodies specific for: a. MyHC‐st, b. MyHC‐2b (10F5), c. MyHC‐2a+2x, d. MyHC‐2x, e. MyHC‐st, f. MyHC‐2b (10F5), g. MyHC‐2a+2x, h. MyHC‐2b (BF‐F3), i. MyHC‐1, j. MyHC‐α (BA‐G5), k. MyHC‐α (BA‐G5) and l. MyHC‐α (MYH6). Note first, that in the masseter (a–d) all fast fibres, labelled by antibodies specific for fast MyHC‐2a and ‐2x isoforms (SC‐71, 6H1), are reactive to antibody 10F5. Note also that in the cricothyreoideus muscle (e–g), 10F5 did not stain the fibre expressing MyHC‐st (asterisk) but labelled all fast fibres, whereas antibody BF‐F3, also specific for rat MyHC‐2b, did not react with fast fibres (h). Note that antibody BA‐G5 (j), specific for MyHC‐α, cross‐reacted with MyHC‐1 (i) in the cricothyreoideus muscle, and in the masseter did not label (k) fibres positive with MYH6 (l, arrows), also specific for MyHC‐α. Scale bar: 25 μm.