Intermuscular and intramuscular differences in myosin heavy chain composition of the human masticatory muscles

Journal of the Neurological Sciences 178 (2000) 95–106 www.elsevier.com / locate / jns

Intermuscular and intramuscular differences in myosin heavy chain composition of the human masticatory muscles J.A.M. Korfage*, P. Brugman, T.M.G.J. Van Eijden Department of Functional Anatomy, Academic Centre for Dentistry Amsterdam (ACTA), Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands Received 28 April 2000; received in revised form 6 July 2000; accepted 6 July 2000

Abstract Among and within the human masticatory muscles a large number of anatomical differences exists indicating that different muscles and muscle portions are specialized for certain functions. In the present study we investigated whether such a specialization is also reflected by intermuscular and intramuscular differences in fibre type composition and fibre cross-sectional area. Fibre type compositions and fibre cross-sectional areas of masticatory muscles were determined in eight cadavers using monoclonal antibodies against myosin heavy chain (MyHC). The temporalis, masseter and pterygoid muscles could be characterized by a relatively large number of fibres containing more than one MyHC isoform (hybrid fibres). In these muscles a large number of fibres expressed MyHC-I, MyHC-fetal and MyHC-cardiac a. Furthermore, in these muscles type I fibres had larger cross-sectional areas than type II fibres. In contrast, the mylohyoid, geniohyoid and digastric muscle were characterized by less hybrid fibres, and by less fibres expressing MyHC-I, MyHC-fetal, and MyHC-cardiac a, and by more fibres expressing MyHC-IIA; the cross-sectional areas of type I and type II fibres in these muscles did not differ significantly. Compared to the masseter and pterygoid muscles, the temporalis had significantly larger fibres and a notably different fibre type composition. The mylohyoid, geniohyoid, and digastric muscles did not differ significantly in their MyHC composition and fibre cross-sectional areas. Also intramuscular differences in fibre type composition were present, i.e., a regionally higher proportion of MyHC type I fibres was found in the anterior temporalis, the deep masseter, and the anterior medial pterygoid muscle portions; furthermore, significant differences were found between the bellies of the digastric.  2000 Elsevier Science B.V. All rights reserved. Keywords: Human; Muscle; Fibre type; Fibre diameter; Mastication; Myosin heavy chain; Jaw closers; Jaw openers

1. Introduction The myosin heavy chain (MyHC) content of muscle fibres mainly determines their force–velocity properties [1]. MyHC type I fibres are slower than MyHC type IIA fibres, which in turn are slower than MyHC type IIX fibres. In humans MyHC-IIB was never found although its gene has recently been cloned [2]. Muscle fibres can either contain one single MyHC or a combination of different MyHCs [3]. In these so-called hybrid fibres the physiological properties have been observed to change according to the relative amount of the different MyHC contents [4,5]. *Corresponding author. Tel.: 131-20-566-5357; fax: 131-20-6911856. E-mail address: [email protected] (J.A.M. Korfage).

Apart from the aforementioned MyHCs human masticatory muscle fibres can abundantly express two more MyHCs. Firstly, MyHC-fetal which is normally expressed in developing muscle fibres [6,7]. This MyHC isoform is even shown to increase in the masseter during ageing [7,8]. Secondly, MyHC-cardiac a which is normally expressed in the atrium of the heart [9]. The amount of force a muscle fibre is capable of producing is proportional to its cross-sectional area which is related to the fibre type and the amount of resistance that is experienced during contraction [10,11]. Human masticatory muscle fibre cross-sections are reported to be smaller than in limb and trunk muscles, and, in contrast to the latter muscles, in human masticatory muscles type II fibres are thinner than type I fibres [12,13]. Thus far, several studies are available that investigated

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the fibre type composition and / or fibre cross-sectional area in the human masticatory muscles, e.g., masseter [8,14,15,17,18], temporalis [16,19,20], pterygoid muscles [16,19,21,22], digastric [23], mylohyoid [19]. The majority of these studies concerned jaw-closing muscles using biopsies and ATPase as a marker. However, ATPase histochemistry gives an incomplete image of the fibre contents [8]. Furthermore, none of these studies have compared the fibre type composition and fibre cross-sectional areas between and within masticatory muscles obtained from the same series of subjects. Among and within the masticatory muscles a large number of anatomical and functional differences exists. For example, the length, spatial orientation and position of muscle fibres differ and therefore fibre and sarcomere excursions are not the same for various muscles and muscle portions [24,25]. As a consequence the maximum force and excursion range of muscles and muscle portions differ. This suggests that different muscles and muscle portions are specialized for certain functions. Indeed, electromyographic studies have demonstrated a differential activation of muscle groups (jaw closers versus jaw openers), muscles and muscle portions (e.g., Refs. [26– 34]). In an earlier study [35] samples were taken from various jaw-closing and jaw-opening muscles and we noticed a large difference in fibre type composition between these muscle groups. The results of this study indicated that jaw closers are more specialized to display a tonic activity whereas the jaw openers are more specialized to display a phasic activity. In the present study the hypothesis was tested whether the architectural and functional specialization of the masticatory muscles is also reflected by inter- and intramuscular differences in fibre type composition and fibre crosssectional area.

2. Materials and methods The masticatory muscles were used of eight Caucasian cadavers (five males and three females, mean age6S.D.5 71.6615.0 years). Six cadavers had upper and lower dental prostheses, two were partially dentate. The muscles were obtained within 12–36 h post mortem. After the muscles were exposed they were cut from their attachment sites. The following muscles or muscle portions were removed separately: anterior and posterior temporalis, masseter, medial pterygoid, superior and inferior head of the lateral pterygoid, mylohyoid, geniohyoid, anterior and posterior belly of the digastric, and stylohyoid. The muscles were rapidly frozen in liquid nitrogen-cooled isopentane and stored at 2808C until required for further processing.

2.1. Immunohistochemistry

cryomicrotome. They were obtained from the belly of the muscles perpendicular to the main direction of the muscle fibres. In case of the masseter the sections were taken at approximately 0.5 cm from the attachment to the zygomatic arch to ensure that the deep masseter was included. For the mylohyoid care was taken that the part of the muscle attaching to the hyoid bone was included. After overnight fixation at 2208C in a mixture of methanol:acetone:acetic acid:water (35:35:5:25) [36], the sections were incubated with monoclonal antibodies raised against purified myosin [9,37] (Table 1). The specificity and characterization of these monoclonal antibodies against human myosin heavy chain isoforms were demonstrated elsewhere [37–39]. These studies showed that human ATPase type I and type IIA muscle fibres reacted with antibodies against MyHC-I and MyHC-IIA, respectively, and that human ATPase defined type IIB muscle fibres contained a MyHC isoform which was homologous to the MyHC-IIX isoform of rodents. In the present study we classified the fibres according to the MyHCs they expressed. Thus, MyHC type I, MyHC type IIA, and MyHC type IIX fibres mentioned in this study were fibres expressing MyHC-I, MyHC-IIA, and MyHC-IIX, respectively. Anti-fetal MyHC was purchased (Novocastra Laboratories, UK). The indirect unconjugated immunoperoxidase technique (PAP-technique) was applied to detect the specific binding of the different antibodies. Nickel-DAB was used to visualize the staining [40].

2.2. Sample method, fibre type classification and crosssectional area measurements The following number of sample areas was taken from each muscle section: anterior temporalis, three; posterior temporalis, four; masseter, eight (four from the deep masseter and four from the superficial masseter); medial pterygoid, six (two from the anterior one-third, one superficial and one deep, and four from the posterior two-thirds, two superficial and two deep); superior head of the lateral pterygoid, three; inferior head of the lateral pterygoid, six (three from the deep half and three from the

Table 1 Monoclonal antibodies and their specific binding a MyHC antibody

219-1D1 249-5A4 332-3D4 333-7H1 anti-fetal a

Serial transverse sections of 10 mm were cut in a

Myosin heavy chain isoforms MyHCI

MyHCIIA

MyHCIIX

MyHCcardiac-a

MyHCfetal

1 2 2 2 2

2 2 1 1 2

2 2 1 2 2

2 1 2 2 2

2 2 2 2 1

1, positive reaction between MyHC and antibody; 2, negative reaction between MyHC and antibody.

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superficial half); mylohyoid, four (three from the part of the muscle that is attached to the mylohyoid raphe and one from the part that is attached to the hyoid bone); geniohyoid, four; anterior digastric, four; posterior digastric, four; stylohyoid, two. In each muscle portion the sample locations were at equal anteroposterior (temporalis, masseter, medial pterygoid, mylohyoid), mediolateral (superior head of the lateral pterygoid), and craniocaudal (inferior head of the lateral pterygoid) distances from each other; from the geniohyoid and the digastrics samples were taken from each muscle quadrant, and from the stylohyoid from each muscle half. In each sample area (about 0.6–0.4 mm) 50–450 fibres (average 178.6) were drawn, by means of a projection microscope (Carl Zeiss, Oberkochen, Germany) and a mirror table, onto a transparent sheet. Each fibre was classified by means of a series of six consecutive incubated sections. Fibres that were not recognized in each of the six sections were omitted. In addition, transition in staining properties was not always completely discontinuous. Therefore, in an earlier study [20] we determined the reproducibility of the classification method. It appeared that 6.6% of the fibres compared gave conflicting results. In 50% of these conflicted fibres MyHC-cardiac a was not reproducible. The cross-sectional area of the fibres was measured by reading the drawn sheets, together with a grade mark for correction of enlargement, via a flat-bed scanner (HewlettPackard, Scanjet 4c) into a personal computer. A custom made program computed the cross-sectional area of each muscle fibre from the reproduced image. In total more than 62 000 fibres were analysed in all muscles. The percentage of the total area of a muscle cross-section that was occupied by the fibres of a specific MyHC type, the relative area, was estimated according to the formula: (sum of cross-sectional areas of a specific fibre type) /(sum of cross-sectional areas of all fibres)3100%.

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3. Results

3.1. Muscle compositions Examples of the fibre type composition in five consecutive sections of the temporalis and the masseter, incubated with the five antibodies used, are shown in Figs. 1 and 2. Note, for example, that both muscles contain a large number of hybrid fibres, that several fibres are stained with antibodies against MyHC-fetal and / or MyHC-cardiac a, and that fibres of the temporalis are larger than fibres of the masseter. Also note that MyHC type II fibres have smaller cross-sectional areas than MyHC type I fibres, and that this difference is more marked in the masseter. An example of three consecutive sections of the anterior and posterior belly of the digastric, incubated with antibodies against MyHC-I, MyHC-IIA, and the two fast MyHC isoforms, is shown in Fig. 3. No microphotos are shown of sections incubated with antibodies against MyHC-fetal and MyHC-cardiac a because they were negative for these two antibodies. Note, that the anterior belly contains more MyHC type IIX fibres than the posterior belly, and that fibres in the anterior belly were also larger. The total proportion of fibres, thus all pure plus hybrid fibres, expressing a particular MyHC isoform is shown in Fig. 4. Based on similarities in their fibre type composition two groups of muscles could be distinguished. One group consisted of the temporalis, masseter, and pterygoid muscles (further named the ‘jaw closers’); the other group consisted of the mylohyoid, geniohyoid, and digastric muscles (further named the ‘jaw openers’), and the stylohyoid. Compared to the jaw openers, the jaw closers were characterized by a larger number of hybrid fibres and a larger number of fibres expressing MyHC-I, MyHC-fetal, or MyHC-cardiac a, and a smaller number of fibres expressing MyHC-IIA.

3.2. Distribution of MyHC fibre types 2.3. Statistical analysis A distinction was made between pure fibre types that expressed only one MyHC isoform and hybrid fibre types which expressed more than one MyHC isoform. For each muscle, or muscle portion, the relative amount of the various pure and hybrid fibres was determined by averaging over all its sample areas. Also the cross-sectional area of these fibres was measured and related to their fibre type. Mean and standard deviation (S.D.) values were calculated over the muscles and muscle portions. Differences in fibre type distribution and in fibre cross-sectional area between muscles and muscle portions were analysed by the Friedman ranking test for paired data. In addition, differences among the samples within the muscles or muscle portions were also tested. The level of significance was set to P,0.05.

Table 2 lists the distribution of pure and hybrid MyHC fibre types in the different muscles and muscle portions; the standard deviation values are a measure for interindividual variability. MyHC-fetal and MyHC-cardiac a were only found in hybrid fibres, thus in combination with another MyHC isoform. Many different hybrid fibre types were found with various combinations of MyHC isoforms (data not shown). The most frequent hybrid fibre type in the jaw-closing muscles was MyHC-cardiac a1I1IIA, and in the jaw-opening muscles MyHC-cardiac a1IIA, although the proportion of a particular hybrid fibre type was seldom higher than 5% of the total amount of fibre types. The percentage of pure MyHC type I fibres did not differ significantly between the muscles. In contrast, several significant differences in the percentages of the

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Fig. 1. Example of a small area from the posterior temporalis incubated with antibodies against MyHC-I (A), MyHC-cardiac a (B), MyHC-IIA (C), MyHC-fetal (E), MyHC-IIA and IIX isoforms (F). Lower magnification of cross-sections in (A) is shown in (D). Drawing shows some of the fibre types. (1) MyHC type I, (2) MyHC type IIX, (3) MyHC type cardiac a1I1IIA, (4) MyHC type fetal1cardiac a1I1IIA.

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Fig. 2. Example of an area showing the anterodeep portion of the masseter incubated with antibodies against MyHC-I (A), MyHC-cardiac a (B), MyHC-IIA (C), MyHC-fetal (E), MyHC-IIA and IIX isoforms (F). Lower magnification of cross-sections in (F) is shown in (D). Drawing shows some of the fibre types. (1) MyHC type I, (2) MyHC type IIX, (5) MyHC type IIA, (6) MyHC type I1IIA, (7) MyHC type fetal1I, (8) MyHC type fetal1cardiac a1I. Note the difference in fibre cross-sectional areas compared to fibres in the temporalis (Fig. 1).

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Fig. 3. Example of an area from the anterior and posterior bellies of the digastric incubated with antibodies against MyHC-I (A,E), MyHC-IIA (B,F), and MyHC-IIA and IIX (C,G). Lower magnification of cross-sections in (A) and (E) are shown in (D) and (H), respectively. Note the smaller fibre cross-sectional areas in the posterior belly.

other pure and hybrid fibres were found (Tables 2 and 3). Within the group of jaw closers the temporalis contained more MyHC type IIA fibres and less hybrid fibres than the

masseter, more MyHC type I and type IIA fibres and less hybrid fibres than the medial pterygoid, and more MyHC type IIX fibres than the lateral pterygoid. The lateral

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Fig. 4. Total proportion of pure plus hybrid fibres expressing a particular MyHC isoform in the temporalis, masseter, and pterygoid (A) and the mylohyoid, geniohyoid, digastric, and stylohyoid (B).

pterygoid contained more MyHC type IIA and less MyHC type IIX fibres than the masseter and medial pterygoid muscles. Within the group of jaw-opening muscles, no significant differences were found with one exception, namely MyHC type IIX fibres which were more present in the digastric muscle than in other jaw openers. In some muscles significant intramuscular differences in fibre type composition were found (Table 2). The anterior temporalis contained more MyHC type I fibres than the posterior temporalis. Comparison of the various anteroposterior samples showed a gradual change in the proportion of MyHC type I fibres across the muscle [20]. The deep masseter contained more MyHC type I fibres and less fibres expressing MyHC-fetal (data not shown) than the superficial masseter. Furthermore, the posterior portion of

the superficial masseter contained less MyHC type I and more MyHC type IIX fibres than the anterior portion (data not shown). In the anterior medial pterygoid more MyHC type I fibres were present than in the posterior medial pterygoid. In the lateral samples of this muscle more MyHC type I fibres and less pure fast fibres were found than in the medial samples (data not shown). No significant difference in fibre type proportion was seen between the heads of the lateral pterygoid, nor among the samples of the superior head. Within the inferior head more MyHC type I fibres were found caudal than cranial [22]. The anterior and posterior belly of the digastric differed considerably in their percentages of MyHC type IIA and type IIX fibres: the anterior belly contained more IIX fibres, whereas the posterior belly contained more IIA

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Table 2 MyHC fibre type composition (%) in the masticatory muscles I

IIA

IIX

Hybrid

Mean

S.D.

Mean

S.D.

Mean

S.D.

Mean

S.D.

Temporalis Anterior Posterior Total

51.2 37.9* 45.5

9.7 12.1 7.5

9.8 19.5 13.7

8.7 12.4 8.3

10.0 9.9 10.8

15.4 11.4 10.4

28.9 32.5 30.0

9.7 14.4 10.1

Masseter Superficial Deep Total

26.8 47.8* 35.0

8.5 14.5 10.9

9.8 6.4 8.3

12.0 4.6 8.6

19.3 5.4 14.4

16.6 4.5 12.0

44.1 40.4 42.4

17.9 14.4 15.5

Medial pterygoid Anterior Posterior Total

39.5 29.2* 32.3

12.2 4.9 6.2

4.1 6.1 5.4

5.2 7.2 6.3

6.3 12.8 10.6

11.5 12.8 12.0

50.1 51.7 51.5

16.8 17.4 15.4

Lateral pterygoid Superior Inferior Total

33.9 36.1 35.5

11.9 8.4 9.2

22.9 15.0 17.2

16.8 12.6 13.1

3.6 3.5 3.5

5.9 6.7 6.4

39.6 45.3 43.7

13.2 12.2 11.8

Mylohyoid Raphe Hyoid Total

41.0 47.7 42.7

12.6 9.4 11.2

51.2 47.8 50.5

10.1 10.3 9.5

2.2 0.7 1.7

2.5 1.8 1.8

5.6 3.8 5.1

3.6 2.2 2.8

Geniohyoid

34.9

9.6

44.5

7.9

8.3

2.8

12.2

8.5

Digastric Anterior Posterior Total

28.7 30.1 29.7

4.8 7.0 4.2

31.5 55.0* 44.2

10.1 8.9 6.9

31.1 6.8* 17.8

16.4 9.4 11.1

8.6 8.0 8.2

13.3 9.3 7.4

Stylohyoid

41.2

16.3

45.9

11.1

0.8

1.0

12.1

8.6

*

Significant difference (P,0.05) between the two muscle portions.

fibres. No differences were found between the mylohyoid muscle portions.

3.3. Fibre cross-sectional areas Table 4 lists the fibre cross-sectional area for the various fibre types; the standard deviation values are a measure for interindividual variability. Within the group of jaw-closing muscles significant differences in fibre cross-sectional

Table 3 Significant difference (P,0.05) between the percentage of MyHC fibre types among the jaw-closing muscles a Temporalis Temporalis Masseter Medial pterygoid Lateral pterygoid a

– IIA, hyb I, IIA, hyb IIX

Masseter

Medial pterygoid

Lateral pterygoid

– IIA, IIX

–

– IIA, IIX

I, MyHC type I; IIA, MyHC type IIA; IIX, MyHC type IIX; hyb, hybrid fibre types.

areas were found (Table 5). In the masseter and the pterygoids MyHC type II fibres had very small crosssectional areas compared to their MyHC type I fibres. In the temporalis MyHC type I fibres had approximately 30% larger cross-sectional areas, and MyHC-IIA fibres approximately 100% larger cross-sectional areas than the same fibre types of other jaw-closing muscles. Within the group of jaw-opening muscles no significant differences in the cross-sectional areas of the pure fibre types were found, but hybrid fibres in the geniohyoid muscle were significantly larger than in the digastric muscle. Intramuscular differences in fibre cross-sectional area were only found for MyHC type IIA fibres in the medial pterygoid, lateral pterygoid and digastric, and for MyHC type IIX fibres in the digastric (Table 4). The relative area of pure MyHC type I fibres was larger (approximately 50%) in the jaw closers than in the jaw openers (approximately 40%). A significant difference was found in the relative area of pure MyHC type IIA and hybrid fibres. In the jaw openers a larger part of the muscle was occupied by pure MyHC type IIA fibres (642%) and a smaller part by hybrid fibres (67%) than in the jaw closers (respectively, 69 and 633%). In jaw closers and

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Table 4 Fibre cross-sectional areas (mm 2 ) in the masticatory muscles I

IIA

IIX

Hybrid

Mean

S.D.

Mean

S.D.

Mean

S.D.

Mean

S.D.

Temporalis Anterior Posterior Total

1768.6 1981.3 1854.0

469.5 354.3 386.5

1181.7 1480.4 1334.7

822.9 1007.7 722.9

1127.1 1451.5 1205.1

617.6 1226.2 587.0

1162.8 1442.8 1293.9

488.2 646.2 549.7

Masseter Superficial Deep Total

1135.6 1278.5 1210.4

425.7 575.4 491.8

494.0 562.9 520.0

310.9 365.0 248.3

444.3 474.6 437.1

281.8 251.3 245.2

548.0 676.3 597.0

233.7 269.4 243.5

Medial pterygoid Anterior Posterior Total

1353.5 1421.3 1387.9

288.7 351.3 306.7

422.0 874.6* 677.6

303.2 576.5 386.2

825.4 690.3 726.8

762.2 574.0 600.8

783.3 737.3 741.5

299.3 255.3 257.5

Lateral pterygoid Superior Inferior Total

1389.8 1166.8 1220.8

219.0 259.8 232.1

1082.1 647.1* 766.7

356.5 185.9 229.1

1133.3 722.9 793.9

622.1 248.3 349.3

955.7 669.1 736.4

357.2 177.0 209.4

Mylohyoid Raphe Hyoid Total

1025.8 1219.5 1086.7

338.1 423.0 317.3

1015.7 1013.3 1004.2

381.6 417.4 357.6

1035.7 1362.6 1090.2

834.9 0.0 771.9

968.7 830.1 986.3

275.2 499.2 278.0

Geniohyoid

1069.7

313.8

1063.9

303.6

808.5

410.4

1064.9

499.1

Digastric Anterior Posterior Total

1075.0 852.2 941.7

301.5 130.7 155.0

982.3 678.8* 762.3

409.4 325.8 329.3

929.9 684.3* 853.6

515.4 422.3 439.8

624.8 608.2 639.6

255.5 213.4 171.9

Stylohyoid

922.7

186.7

802.2

441.5

639.1

205.0

823.5

339.2

*

Significant difference (P,0.05) between the two muscle portions.

openers the relative area occupied by pure MyHC type IIX fibres was not different (68 and 7%).

4. Discussion To our knowledge this is the first study which compares fibre type composition and fibre cross-sectional areas between and within all human masticatory muscles from one group of individuals by using antibodies against

Table 5 Significant difference (P,0.05) between the fibre cross-sectional areas of MyHC fibre types among the jaw-closing muscles a

Temporalis Masseter Medial pterygoid Lateral pterygoid a

Temporalis

Masseter

– I, IIA, IIX, hyb hyb I, IIA, hyb

– IIA, hyb IIA

Medial pterygoid

Lateral pterygoid

– –

I, MyHC type I; IIA, MyHC type IIA; IIX, MyHC type IIX; hyb, hybrid fibre types.

MyHC isoforms. The fibre type distributions and fibre cross-sectional areas of the human masticatory muscles reported in the present study and in other studies [15– 17,19,21] are different from what is commonly found in limb and trunk muscles [12,41]. The masticatory muscles are featured by having more hybrid fibres, many of them coexpressing MyHC-fetal and / or MyHC-cardiac a. Furthermore, masticatory muscle fibres have smaller crosssectional areas. It was also noticed that type I fibres in human masticatory muscles were larger than type II fibres, while in limb and trunk muscle the opposite is true [13]. As indicated by the present results, these differences are more pronounced for jaw closers than for jaw openers, because we found less hybrid fibres in the jaw openers than in the jaw closers, and type I and type II fibres in the openers were equal in size. Several suggestions have been made to explain this difference between masticatory muscles and limb and trunk muscles, including differences in their function and / or nerve supply [6,42–44], and differences in their genetic pathways to express muscle specific proteins during embryogenesis [45] and in their chronological or spatial activation of some transcription factors [46].

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In an earlier study we demonstrated that the jaw closers have architectural features that suit them for force production, whereas the jaw openers are better designed to produce velocity and displacement [25]. The jaw closers are contracting more slowly and experience more resistance during trituration of food than the jaw openers. This is reflected in the fibre type composition of both muscle groups, as indicated by the present results. The jaw closers have more fibres (pure plus hybrid) expressing the slow MyHC type I, whereas the jaw openers have more fibres expressing the fast MyHC type IIA. Furthermore, in the jaw closers all fibres expressing MyHC-I occupy the largest relative area, and in the jaw openers all fibres expressing MyHC-IIA occupy the largest relative area. Therefore, the jaw closers can be considered as being slower than the jaw openers. According to the size principle [47] type I motor units are recruited first in a motor task, whereas type II units are recruited when larger velocities or forces are required. As the jaw closers possess more fibres expressing MyHC-I and the jaw openers possess more type II fibres, this suggests that the jaw closers will display a more tonic, or prolonged, activity, whereas the jaw openers will display a more phasic, or short-lived, activity [22]. Furthermore, type I motor units have smaller innervation ratios than type II units [48]. Therefore, it can be expected that the jaw closers are better equipped to regulate the magnitude of the produced force during chewing or biting than the jaw openers. The higher proportion of hybrid fibres in the jaw closers might be another factor that contributes to this better force regulating capacity of the jaw closers. Studies on the contractile properties of single muscle fibres and motor units expressing multiple MyHCs have shown that such a coexpression yields a greater variance in shorting velocities and maximal force generation [5,49,50]. The results of the present study show that the individual jaw closers differ in fibre type composition and in fibre cross-sectional area. The most deviant pattern was observed for the temporalis. Compared to the other jaw closers, this muscle contained more pure MyHC type I fibres and less hybrid fibres, and all fibre types had considerable larger cross-sectional areas. In other jaw closers MyHC type I fibres were also larger than MyHC type II and hybrid fibres, which confirms findings in other studies [16,19,51]. These larger cross-sectional areas of MyHC type I fibres might be the result of the larger resistance that is experienced by the jaw closers during chewing. It was demonstrated that training against a resistance caused an increase in fibre cross-sectional area [11], while weightlessness caused a decrease in fibre crosssectional area [10]. The observed differences in fibre type proportion and fibre cross-sectional areas suggest that the temporalis is slower than the other jaw closers and that it experiences more resistance during chewing or biting. The latter might be due to the length of its moment arm, which is shorter than that of the masseter and medial pterygoid

[25]. A shorter moment arm requires a larger force output to produce a particular chewing or bite force. Within the jaw closers intramuscular differences in fibre type composition were also observed, particularly with respect to the relative amount of MyHC type I fibres, which differed in the temporalis (anterior more than posterior), masseter (deep more than superficial), and medial pterygoid (anterior more than posterior). These regional differences confirm the results of earlier studies of the temporalis [20], the masseter [7,16], and the medial pterygoid [16,22]. The differences are in line with results of electromyographic studies which indicate that the anterior temporalis is more intensively used than the posterior temporalis [32] and that the deep masseter is more intensively used than the superficial masseter [30,35,52]. It has been demonstrated for cat hindlimb muscles [53] and for various muscles in man [54] that the percentage of type I fibres is higher in muscles or muscle portions which are more often and / or longer recruited during the day. Based on the proportions of the pure and hybrid fibres the lateral pterygoid had more similarities with the jaw closers than with the jaw openers. Therefore, we have included this muscle among the group of jaw closers although the total amount of fibres expressing MyHC-IIA, pure plus hybrid, in this muscle is more a characteristic of the jaw openers. The inferior head is supposed to be concentrically active during jaw opening movements and the superior head is supposed to be excentrically active during jaw closing movements and during clenching [26,29,55]. We did not find a difference in fibre type proportion between the superior and inferior head of the muscle, but type II and hybrid fibres were larger in the superior than in the inferior head. This might indicate an influence by resistance of the more powerful fibres in the superior head during clenching. Why type I fibres are not also larger in the superior head is not clear. In contrast to the jaw closers, differences in fibre composition among the jaw openers were relatively small. Only the bellies of the digastric were significantly different from each other. The anterior belly had more MyHC type IIX fibres than the posterior belly which had more MyHC type IIA fibres. Also, the fibres in the anterior belly had a larger cross-sectional area than in the posterior belly. Differences in fibre type composition and fibre diameter between the two bellies have also been reported in the literature [23]. These differences indicate that the bellies have the potential to act independently. Indeed, it was found [56] that the anterior belly was not only connected to the posterior belly but also to the hyoid bone by a fleshy or aponeurotic insertion. Both bellies also displayed a differential activity during chewing [57]. In earlier studies we have examined whether a finer regional difference in fibre type distribution could be observed within the main portions of the temporalis [20] and pterygoid muscles [22]. This was indeed the case,

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except for the superior head of the lateral pterygoid. In the temporalis a gradual change in MyHC type I fibres could be observed from the anteriormost to the posteriormost muscle portions, in the medial pterygoid more MyHC type I fibres and less MyHC type IIA fibres were found in the anterolateral than in the posteromedial muscle portion, and in the inferior head of the lateral pterygoid more MyHC type I fibres were found in the caudalmost than in the cranialmost muscle portion. The present study also shows that within the masseter less MyHC type I fibres were found in the posterior portion of the superficial muscle portion than in the other masseter muscle portions, which confirms a study into the masseter of young individuals [16]. This intramuscular heterogeneity partly explains the large variability in fibre type distributions reported in literature [8,15,16,18,19,21]. In most investigations a limited number of sample areas have been taken from different intramuscular locations. Another factor that should be mentioned is the effect of ageing. During ageing the proportion and fibre crosssectional area of the different fibre types change [58]. Since we used material of elderly individuals we have compared our data with data from other studies in which masticatory muscles of either young or old individuals have been examined. From this comparison it appears that young individuals have more type I fibres than elderly individuals, suggesting that the proportion of type I fibres decreases with age [8,16,19,21]. In the present study and in that of Monemi et al. [8] less fast fibre types and more hybrid fibres were observed than in other studies. This difference can be explained by the difference in the methods used, namely immunohistochemistry versus ATPase histochemistry, but possibly also be a difference in age. In elderly individuals an increase in the proportion of hybrid fibres was observed [8]. Other factors which might be of influence to the fibre type variability are gender, activity and / or genetic factors.

[2]

[3]

[4] [5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13] [14]

[15]

Acknowledgements

[16]

This research was institutionally supported by the Interuniversitary Research School of Dentistry, through the Academic Centre of Dentistry Amsterdam. We would like to express our gratitude L.J. van Ruijven for the technical support, J. Ruijter for statistical advice, Professor Dr. A.F.M. Moorman for the donation of the antibodies, and Dr. J.H. Koolstra and Dr. G.E.J. Langenbach for the critical reading of the manuscript.

[17]

[18]

[19] [20]

[21]

References [22] [1] Bottinelli R, Caneparri M, Pellegrino MA, Reggiani C. Forcevelocity properties of human skeletal muscle fibres: myosin heavy

105

chain isoforms and temperature dependence. J Physiol 1996;495:573–86. Weiss A, Schiaffino S, Leinwand LA. Comparative sequence analysis of the complete human sarcomeric myosin heavy chain family: implications for functional diversity. J Mol Biol 1999;290:61–75. Pette D, Staron RS. Cellular and molecular diversities of mammalian skeletal muscle fibres. Rev Physiol Biochem Pharmacol 1990;116:2–76. Schiaffino S, Reggiani C. Myosin isoforms in mammalian skeletal muscle. J Appl Physiol 1994;77:493–501. ¨ Kwa SHS, Weijs WA, Juch PJW. Contraction characteristics and myosin heavy chain composition of rabbit masseter motor units. J Neurophysiol 1995;73:538–49. Butler-Brown GS, Eriksson P-O, Laurent C, Thornell L-E. Adult human masseter muscle fibres express myosin isozymes characteristic of development. Muscle Nerve 1988;11:610–20. Monemi M, Eriksson P-O, Dubail I, Butler-Browne GS, Thornell L-E. Fetal myosin heavy chain increases in the human masseter muscle during aging. FEBS Lett 1996;386:87–90. Monemi M, Eriksson P-O, Kadi F, Butler-Browne GS, Thornell L-E. Opposite changes in myosin heavy chain composition of human masseter and biceps brachii muscles during aging. J Muscle Res Cell Motil 1999;20:351–61. Bredman JJ, Wessels A, Weijs WA, Korfage JAM, Soffers CAS, Moorman AFM. Demonstration of ‘cardiac-specific’ myosin heavy chain in masticatory muscles of human and rabbit. Histochem J 1991;23:160–70. Edgerton VR, Zhou M-Y, Ohira Y, Klitgaard H, Jiang B, Bell G, Harris B, Saltin B, Gollnick PD, Roy RR, Day MK, Greenisen M. Human fiber size and enzymatic properties after 5 and 11 days of spaceflight. J Appl Physiol 1995;78:1733–9. McCall GE, Byrnes WC, Dickinson A, Pattany PM, Fleck SJ. Muscle fiber hypertrophy, hyperplasia, and capillary density in college men after resistance training. J Appl Physiol 1996;81:2004– 12. Polgar J, Johnson MA, Weightman D, Appleton D. Data on fibre size in thirty-six human muscles. An autopsy study. J Neurol Sci 1973;19:307–18. Ringqvist M. Fiber types in human masticatory muscles. Relation to function. Scand J Dent Res 1974;82:333–55. Serratrice G, Pellisier JF, Vignon C, Baret J. The histochemical profile of the human masseter, an autopsy and biopsy study. J Neurol Sci 1976;30:189–200. Ringqvist M, Ringqvist I, Eriksson P-O, Thornell L-E. Histochemical fibre-type profile in the human masseter muscle. J Neurol Sci 1982;53:273–82. Eriksson P-O, Thornell L-E. Histochemical and morphological muscle-fibre characteristics of the human masseter, the medial pterygoid and the temporal muscle. Arch Oral Biol 1983;28:781–95. Monemi M, Eriksson P-O, Eriksson A, Thornell L-E. Adverse changes in fibre type composition of the human masseter versus biceps brachii muscle during aging. J Neurol Sci 1998;154:35–48. Sciote JJ, Rowlerson AM, Hopper C, Hunt NP. Fibre type classification and myosin isoforms in the human masseter muscle. J Neurol Sci 1994;126:15–24. Vignon C, Pellissier JF, Serratrice G. Further histochemical studies on masticatory muscles. J Neurol Sci 1980;45:157–76. Korfage JAM, Van Eijden TMGJ. Regional differences in fibre type composition in the human temporalis muscle. J Anat 1999;194:355– 62. Eriksson P-O, Eriksson A, Ringqvist M, Thornell L-E. Special histochemical muscle-fibre characteristics of the human lateral pterygoid muscle. Arch Oral Biol 1981;26:495–507. Korfage JAM, Van Eijden TMGJ. Myosin isoform composition of the human medial and lateral pterygoid muscles. J Dent Res 2000 (in press).

106

J. A.M. Korfage et al. / Journal of the Neurological Sciences 178 (2000) 95 – 106

[23] Eriksson P-O, Eriksson A, Ringqvist M, Thornell L-E. Histochemical fibre composition of the human digastric muscle. Arch Oral Biol 1982;27:207–15. [24] Van Eijden TMGJ, Koolstra JH, Brugman P. Architecture of the human pterygoid muscles. J Dent Res 1995;74:1489–95. [25] Van Eijden TMGJ, Korfage JAM, Brugman P. Architecture of the human jaw-closing and jaw-opening muscles. Anat Rec 1997;248:464–74. [26] McNamara JA. The independent functions of the two heads of the lateral pterygoid muscle. Am J Anat 1973;138:197–206. [27] Møller E. Action of the muscles of mastication. Front Oral Physiol 1974;1:121–58. [28] Vitti M, Basmajian JV. Integrated actions of masticatory muscles: simultaneous EMG from eight intramuscular electrodes. Anat Rec 1977;187:173–89. [29] Mahan PE, Wilkinson TM, Gibbs CH, Mauderli A, Brannon LS. Superior and inferior bellies of the lateral pterygoid muscle EMG activity at basic jaw positions. J Prosth Dent 1983;50:710–8. [30] Belser UC, Hannam AG. The contribution of the deep fibers of the masseter muscle to selected tooth-clenching and chewing tasks. J Prosth Dent 1986;56:629–35. [31] Blanksma NG, Van Eijden TMGJ. Electromyographic heterogeneity in the human temporalis muscle. J Dent Res 1990;69:1686–90. [32] Blanksma NG, Van Eijden TMGJ. Electromyographic heterogeneity in the human temporalis and masseter muscles during static biting, open / close excursions, and chewing. J Dent Res 1995;74:1318–27. [33] Van Eijden TMGJ, Brugman P, Weijs WA, Oosting J. Coactivation of jaw muscles: recruitment order and level as a function of bite force direction and magnitude. J Biomech 1990;23:475–85. [34] Van Eijden TMGJ, Blanksma NG, Brugman P. Amplitude and timing of EMG activity in the human masseter muscle during selected motor tasks. J Dent Res 1993;72:599–606. [35] Korfage JAM, Schueler YT, Brugman P, Van Eijden TMGJ. Myosin heavy chain composition of the human jaw-closing and jaw-opening muscles. J Dent Res (submitted). [36] Wessels A, Vermeulen JLM, Moorman AFM, Becker AE. Immunohistochemical detection of myosin heavy chain isoforms in large sections of whole human hearts. In: Sarcomeric and non-sarcomeric muscles: basic and applied research prospects for the 90’s, Padova: Unipress, 1988, pp. 311–6. [37] Sant’Ana Pereira JAA, Wessels A, Nijtmans L, Moorman AFM, Sargeant AJ. New method for the accurate characterization of single human skeletal muscle fibres demonstrates a relation between mATPase and MyHC expression in pure and hybrid fibre types. J Muscle Res Cell Motil 1995;16:21–34. ´ ´ F, Lamers WH, [38] Wessels A, Vermeulen JLM, Viragh Sz, Kalman Moorman AFM. Spatial distribution of ‘tissue specific’ antigens in the developing human heart and skeletal muscle. II. An immunohistochemical analysis of myosin heavy chain isoform expression patterns in the embryonic heart. Anat Rec 1991;229:355–68. [39] Sant’Ana Pereira JAA, Moorman AFM. Do type IIB fibres of human muscle correspond to the IIX / D or to the IIB of rats? J Physiol 1994;479P:161P–2P.

[40] Hancock MB. A serotonin-immunoreactive fiber system in the dorsal columns of the spinal cord. Neurosci Lett 1982;31:247–52. [41] Johnson MA, Polgar J, Weightman D, Appleton D. Data on the distribution of fibre types in thirty-six human muscles. J Neurol Sci 1973;18:111–29. [42] Soussi-Yanicostas N, Barbet JP, Laurent-Winter C, Barton P, ButlerBrowne GS. Transition of myosin isozymes during development of human masseter muscle. Persistence of developmental isoforms during postnatal stage. Development 1990;108:239–49. [43] Eriksson P-O, Butler-Browne GS, Thornell L-E. Immunohistochemical characterization of human masseter muscle spindles. Muscle Nerve 1994;17:31–41. ˚ P, Eriksson P-O, Schiaffino S, Butler-Browne GS, Thornell L-E. [44] Stal Differences in myosin composition between human oro-facial, masticatory, and limb muscles: enzyme-, immunohisto- and biochemical studies. J Muscle Res Cell Motil 1994;15:517–34. [45] Noden DM. The embryonic origins of avian cephalic and cervical muscles and associated connective tissues. Am J Anat 1983;168:257–76. [46] Marcucio RS, Noden DM. Myotube heterogeneity in developing chick craniofacial skeletal muscles. Dev Dyn 1999;214:178–94. [47] Henneman E, Somjen G, Carpenter DO. Functional significance of cell size in spinal motoneurons. J Neurophysiol 1965;28:560–80. [48] Burke RE, Tsairis P. Anatomy and innervation ratios in motor units of cat gastrocnemius. J Physiol 1973;234:749–65. [49] Galler S, Schmitt TL, Pette D. Stretch activation, unloaded shortening velocity, and myosin heavy chain isoforms of rat skeletal muscle fibres. J Physiol 1994;478:513–21. [50] Johnson BD, Wilson LE, Zhan WZ, Watchko JF, Daood MJ, Sieck GC. Contractile properties of the developing diaphragm correlate with myosin heavy chain phenotype. J Appl Physiol 1994;77:481–7. [51] Ringqvist M. Fibre sizes of human masseter muscle in relation to bite force. J Neurol Sci 1973;19:297–305. [52] Blanksma NG, Weijs WA, Van Eijden TMGJ. Electromyographic heterogeneity in the human masseter muscle. J Dent Res 1992;71:47–52. [53] Kernell D, Hensbergen E, Lind A, Eerbeek O. Relation between fibre composition and daily duration of spontaneous activity in ankle muscles of the cat. Arch Ital Biol 1998;136:191–203. [54] Monster AW, Chan HC, O’Conner D. Activity patterns of human skeletal muscles: relation to muscle fiber type composition. Science 1978;200:314–7. [55] Juniper RP. The superior pterygoid muscle? Br J Oral Surg 1981;19:121–8. [56] Munro RR. Structure and functions of the digastric muscle. J Anat 1973;114:156. [57] Munro RR. Coordination of activity of the two bellies of the digastric muscle in basic jaw movements. J Dent Res 1974;51:1663–7. [58] Lexell J. Ageing and human muscle: observations from Sweden. Can J Appl Physiol 1993;18:2–18.