Characterisation of flexor digitorum profundus, flexor digitorum superficialis and extensor digitorum communis by electrophoresis and immunohistochemical analysis of myosin heavy chain isoforms in older men

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RESEARCH ARTICLE

Characterisation of flexor digitorum profundus, flexor digitorum superficialis and extensor digitorum communis by electrophoresis and immunohistochemical analysis of myosin heavy chain isoforms in older men 1 ˇ Marija Meznaric ∗ , Andrej Carni Institute of Anatomy, Faculty of Medicine, University of Ljubljana, Korytkova 2, 1000 Ljubljana, Slovenia

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Article history: Received 7 November 2018 Received in revised form 19 July 2019 Accepted 29 July 2019 Available online xxx Keywords: Myosin heavy chain isoforms SDS-PAGE Immunohistochemistry Fiber types Extrinsic finger muscles Extensor muscles Flexor muscles

a b s t r a c t Introduction: Few data exist on the fiber type composition of the extrinsic finger muscles. The aim of the present study was to describe myosin heavy chain (MyHC) composition of flexor digitorum profundus (FDP), flexor digitorum superficialis (FDS) and extensor digitorum communis (EDC). MyHC composition is relevant for whole muscle contractile performance and several studies on single muscle fibers demonstrated that fibers expressing only slow MyHC-1 develop less specific force than fibers expressing fast MyHCs. Since contraction force of finger extensors is smaller than of finger flexors a hypothesis was posited that the content of MyHC-1 is higher in EDC than in extrinsic finger flexors. Methods: Autopsy samples of FDP, FDS, and EDC in 27 healthy older men were analyzed and compared with each other and with biceps brachii (BB). MyHC isoforms were quantified on silver-stained 6% SDSPAGE. Muscle fibers were classified immunohistochemically according to the expression of adult MyHC isoforms and their morphometric parameters were determined. Results: EDC stood out for its higher proportion of slow MyHC-1 (50%) compared to FDP (37%), FDS (38%) and BB (35%) (p < 0.001 in all), and its lower proportion of fast MyHC-2x (13%) compared to FDP (26%, p = 0.001), FDS (22%, p = 0.028) and BB (29%, p < 0.001) detected on SDS-PAGE. Immunohistochemically, EDC had a higher area proportion of pure slow type-1 fibers (63%) than FDP (47%, p = 0.002), FDS (49%, p = 0.007) and BB (47%, p = 0.002), and lower area proportion of pure fast type-2x fibers (2%) than FDP (12%, p = 0.014), FDS (8%, p = 0.256) and BB (14%, p = 0.002). All muscles contained a similar area proportion of pure type-2a fibers and hybrid type-2a/2x fibers. Conclusions: The study results support the hypothesis that the content of MyHC-1 is higher in EDC than in extrinsic finger flexors, which is in agreement with the lower contraction force of finger extensors compared to finger flexors. © 2019 Elsevier GmbH. All rights reserved.

1. Introduction Flexor digitorum superficialis (FDS) is located in the superficial and flexor digitorum profundus (FDP) in the deep layer of the anterior compartment of the forearm; extensor digitorum communis (EDC) is located in the superficial layer of the posterior compart-

Abbreviations: BB, biceps brachii; EDC, extensor digitorum communis; FDP, flexor digitorum profundus; FDS, flexor digitorum superficialis; MyHC, myosin heavy chain. ∗ Corresponding author. E-mail address: [email protected] (M. Meznaric). 1 Present address: Gynecology and Obstetrics Hospital Kranj, Kidriˇceva 38a, 4000 Kranj, Slovenia.

ment of the forearm (Platzer, 2004; Biant, 2016). FDS, FDP and EDC provide tendons for the second to fifth finger. FDS attaches to the palmar surface of the centers of the middle phalanges and FDP to the palmar surface of the bases of the distal phalanges (Platzer, 2004; Biant, 2016). EDC tendons become laterally expanded over the dorsal aspect of the metacarpophalangeal joints to form the dorsal digital expansion to which the tendons of interossei and lumbrical muscles attach. At the distal end of the proximal phalanges of the second to fifth finger, the expansion trifurcates: the central slip is inserted into the dorsal surface of the base of the middle phalanx, the lateral slips unite over the body of the middle phalanx to be inserted into the dorsal surface of the base of the distal phalanx (Hall-Crags, 1990). FDS is anatomically more compartmentalized (Ohtani, 1979) than FDP and does not have a mechanical connec-

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ˇ A., Characterisation of flexor digitorum profundus, flexor digitorum superficialis Please cite this article in press as: Meznaric, M., Carni, and extensor digitorum communis by electrophoresis and immunohistochemical analysis of myosin heavy chain isoforms in older men. Ann. Anatomy (xxxx), https://doi.org/10.1016/j.aanat.2019.151412

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tion between the tendons in the carpal tunnel as FDP has (Leijnse et al., 1997) or as EDC at the dorsum of the hand has (von Schroeder and Botte, 2001). Extrinsic finger muscles produce large finger movements (van Beek et al., 2018): FDS flexes the proximal interphalangeal joints, FDP flexes the distal interphalangeal joints; FDP and FDS are also capable of flexing the wrist and metacarpholangeal joints and FDP also flexes proximal interphalangeal joints (Platzer, 2004; Biant, 2016). EDC extends the wrist, metacarpophalangeal and proximal and distal interphalangeal joints (Hall-Crags, 1990; Biant, 2016). These multitendon muscles have been extensively studied in humans from the perspective of rehabilitation protocols for tendon injuries (Kursa et al., 2005). Functional studies of finger independence have demonstrated that is not possible to selectively flex one distal interphalangeal joint without flexing adjacent digits (Kilbreath and Gandevia, 1994; Zatsiorsky et al., 2000; Reilly and Schieber, 2003), whereas index, middle and ring digital components of FDS appear to be activated by volition in a more selective way than for FDP (Butler et al., 2005). Finger extension is more independent than flexion at distal interphalangeal joints, but less independent than flexion at proximal interphalangeal joints (van Duinen et al., 2009). Both neural and mechanical constraints limit finger independence (Dupan et al., 2018). Extrinsic finger muscles also differ in the susceptibility to sporadic inclusion body myositis (Mastaglia, 2012), the most common acquired myopathy in people over the age of 50 (Dalakas, 2012): FDP is affected early in the course of the disease while FDS is spared at the onset and EDC is affected as the last of the extrinsic finger muscles. The cause of this selective muscle involvement in sporadic inclusion body myositis is not known. Some studies on transgenic mouse models and human biopsies of sporadic inclusion body myositis (Sugarman et al., 2006; Lünemann et al., 2007) show that slow–twitch fibers are less commonly affected than fast-twitch fibers. In spite of extrinsic finger muscles being an interesting object for research, few data exist on the fiber type composition of the extrinsic finger muscles and are based on myofibrillar ATPase histochemistry (Johnson et al., 1973; Polgar et al., 1973). Data on myosin heavy chain (MyHC) composition of these muscles are lacking. The focus of the present study was to characterize MyHC composition (and muscle fiber size) of the extrinsic finger muscles by electrophoresis and immunohistochemistry in healthy older men, which has never been studied before. MyHC composition co-determines whole muscle contractile performance (Harridge et al., 1996) and several studies on single muscle fibers in vitro demonstrated that muscle fibers expressing only slow MyHC-1 develop less specific force than muscle fibers expressing fast MyHCs (Bottinelli et al., 1996; Bottinelli and Reggiani, 2000; Schiaffino and Reggiani, 2011; Miller et al., 2015). Since the contraction force of finger extensors is smaller than of finger flexors (Suzuki et al., 1994; Brorsson et al., 2012), a hypothesis was posited that the content of MyHC-1 is higher in EDC than in extrinsic finger flexors.

2. Materials and methods 2.1. Subjects Muscle specimens were excised within 24 h post mortem from the FDS, FDP, EDC and biceps brachii (BB) muscles in the right upper limb of 27 presumably healthy older males aged 54–89 years, x ± SD = 71.6 ± 11.4 years, who suddenly died. BB was included to find out if flexors located in the proximal and distal compartments of upper limbs have similar MyHC composition. None had a history of neuromuscular disease and none exhibited signs of neuromuscular disease on histological sections. During dissection, the flexor

carpi ulnaris muscle and the ulnar nerve were located and gently pushed medially. The median nerve was located in the forearm and the muscle above it was identified as FDS and the muscle below it as FDP. Muscle samples were taken 5 cm below the medial epicondyle of the humerus from the humeroulnar head of the FDS and 8 cm below the medial epicondyle from the FDP in front of the ulna. Sampling of EDC muscle was performed 5 cm below the lateral epicondyle of the humerus in the central proximal part of the muscle. At the levels indicated it was not possible to distinguish muscular compartments related to individual fingers. BB samples were taken from the superficial part of the middle portion of the muscle belly which was a continuation of the long head of BB. The size of the muscle samples was 0.3 cm3 –0.5 cm3 . The muscle sampling was approved by the National Medical Ethics Committee of the Republic of Slovenia. 2.2. Electrophoresis Each muscle sample (on average about 6500 muscle fibers) was cut in a cryostat microtome. Ten to thirty 5 ␮m sections of each muscle sample were collected in Eppendorf tubes and dissolved in 100 ␮l of the extraction buffer (0.05 mol/L dithiothreitol, 0.1 mol/L ethylenediaminetetraacetic acid, 0.125 mol/L Tris, 4% sodium dodecyl sulfate) to prepare a total muscle homogenate as previously described (Fanin et al., 2003). Two quantities, 1.6 or 2.8 ␮g, of total protein were loaded per lane. Electrophoresis of MyHC isoforms (MyHC-1, MyHC-2a and MyHC-2x) was carried out on 6% 0.75 mm thick mini gels in Mini-Protean 3 Cell (Bio-Rad, Hercules, California, United States). We used a protocol by Talmadge and Roy (Talmadge and Roy, 1993) that was modified for human samples (Bamman et al., 1999). Gels were run at constant voltage of 150 V for 18 h at 4 ◦ C, silver stained (Blum et al., 1987) and scanned wet. Individual MyHC bands were identified according to the electrophoretic mobility: the fastest band between 250 and 200 kD was recognized as MyHC-1, the slowest as MyHC-2x and the intermediate as MyHC2a (Bamman et al., 1999; Lucas et al., 2000; Smerdu and Soukup, 2008; Gantelius et al., 2012). The size and intensity of individual MyHC bands were quantified with a computer programme for gel analysis “ELFO for Image Tool 2.00” (ViDiTo, Kosice, Slovakia) and expressed in %. 2.3. Immunohistochemical stainings The same muscle samples which were cut for electrophoresis were also cut in a cryostat microtome into successive 10 ␮m thick transverse sections which were processed to demonstrate the expression of MyHC isoforms. Immunohistochemical stainings were performed as previously described (Meznaric and Erˇzen, 2012; Meznaric et al., 2018): we used a BAD5 antibody immunoreactive with MyHC-1 (Smerdu et al., 1994), SC-71 antibody immunoreactive with both MyHC-2a and MyHC-2x (Smerdu and Soukup, 2008), and 6H1 antibody (Developmental Studies Hybridoma Bank, Iowa City, USA) immunoreactive with MyHC-2x (Lucas et al., 2000). The BAD-5 and SC-71 antibodies were produced in a local laboratory from the corresponding cell lines provided by Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). 2.4. Image acquisition, image analysis and statistics In each muscle section two areas were selected at random and bright field images of successive immunohistochemical sections stained with specific anti-MyHC antibodies were captured on a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan) at objective lens NIKON CFI Plan Fluor 10X. The 5.0 Mega Pixels Colour Digital Camera Head DS-Fi1 NIKON camera and NIS-Elements BR, Basic

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Fig. 1. Electrophoresis of MyHC isoforms in the extrinsic finger muscles and biceps brachii in a 62-year-old male. Two quantities of total proteins, 1.6 ␮g or 2.8 ␮g, were loaded per lane. FDS – flexor digitorum superficialis; FDP—flexor digitorum profundus; EDC—extensor digitorum communis; BB—biceps brachii. Vertical lines mark the position of the empty deleted lane that marked the order of the samples in the original gel.

Fig. 2. Proportions of MyHC-1, MyHC-2x and MyHC-2a in the extrinsic finger muscles and biceps brachii determined by electrophoresis. EDC stood out with the highest content of MyHC-1 and the lowest content of MyHC-2x. Values are means ± SD. FDP—flexor digitorum profundus; FDS—flexor digitorum superficialis; EDC—extensor digitorum communis; BB—biceps brachii.

Research software were used for image acquisition. A computerassisted system (Karen et al., 2009) with manual delineation of fiber outlines in one muscle section and semi-automatic registration of muscle fibers on successive muscle sections was used to perform fiber classification and quantitative analysis for the following: (i) numerical proportions of fiber types, (ii) area proportions of fiber types, and (iii) fiber diameters. Muscle fibers were classified into pure fibers (type-1, -2a and -2x), expressing either MyHC-1, -2a or -2x and hybrid fibers co-expressing two or three adult MyHC isoforms (type-2a/2x, -1/2a, -1/2x and -1/2a/2x). On average about 250 muscle fibers were analyzed per each muscle sample. Statistical analyses were performed using IBM SPSS Statistics for Windows, Version 25. All data were expressed as mean ± SD. Differences in mean values were tested by one-way analysis of variance and the Bonferroni post hoc test. The level of significance was set at p < 0.05.

3. Results 3.1. Electrophoresis of MyHC isoforms EDC stood out for its significantly higher proportion of slow MyHC-1 (50 ± 10%) compared to FDP (37 ± 7%), FDS (38 ± 8%) and BB (35 ± 7%) (p < 0.001 in all), and its lower proportion of fast MyHC-2x (13 ± 11%) compared to FDP (26 ± 10%, p = 0.001), FDS (22 ± 10%, p = 0.028) and BB (29 ± 9%, p < 0.001). EDC had an identical or similar proportion of MyHC-2a (37 ± 7%) as FDP (37 ± 7%), FDS (40 ± 8%) and BB (36 ± 6%). Extrinsic finger flexors (FDP and FDS) and BB had similar proportions of MyHC-1, MyHC-2x and MyHC-2a (Figs. 1 and 2).

3.2. Immunohistochemical demonstration of MyHC isoforms EDC had a significantly higher area proportion of fibers expressing MyHC-1, i.e. pure slow type-1 fibers (63 ± 14%), than FDP (47 ± 12%, p = 0.002), FDS (49 ± 13%, p = 0.007) and BB (47 ± 14%, p = 0.002), as well as lower area proportion of fast fibers expressing MyHC-2x, i.e. pure fast type-2x fibrs (2 ± 3%), than FDP (12 ± 11%, p = 0.014) and BB (14 ± 11%, p = 0.002). The difference between EDC and FDS (2 ± 3% vs. 8 ± 9%) was not statistically significant. Area proportions of pure fast type-2a fibers were similar in all muscles, as were area proportions of hybrid type-2a/2 × . FDP, FDS and BB also did not differ in area proportions of type-1 and type-2x fibers (Figs. 3 and 4). EDC also had a higher numerical proportion of pure type1 fibers (62 ± 16%) and a lower numerical proportion of type-2x fibers (2 ± 3%) than other investigated muscles, but the differences were statistically significant only for type-1 fibers compared to BB (45 ± 13%, p = 0.002), and type-2x compared to FDP (11 ± 10%, p = 0.020) and BB (15 ± 11%, p < 0.001) (Fig. 5). Mean muscle fiber diameters of major fiber types were similar in all muscles except for type-2x fibers, which were statistically significantly smaller in EDC (38 ± 14 ␮m) than in FDP (55 ± 14 ␮m, p = 0.003) and FDS (53 ± 13 ␮m, p = 0.017) (Fig. 6). In all muscles, numerical proportions of hybrid type-2x/2a were significantly higher than numerical proportions of pure type-2x fibers (Fig. 7). Hybrid type-1 fibers expressing MyHC-1/2a or MyHC1/2x or MyHC-1/2a/2x were few in all muscles and represented a maximum of 2.5% of the total area on average. Area proportion varied considerably and did not differ significantly from one muscle to another (results not shown).

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Fig. 3. Expression of MyHC isoforms in successive cross-sections of the extrinsic finger muscles and biceps brachii. While FDP, FDS and BB are similar, EDC stood out with respect to the highest content of type-1 fibers expressing MyHC-1 and lowest content of type-2x fibers expressing MyHC-2x. FDP—flexor digitorum profundus; FDS—flexor digitorum superficialis; EDC—extensor digitorum communis; BB—biceps brachii. Scale bar 100 ␮m.

Fig. 4. Area proportions of major fiber types classified on the basis of adult myosin heavy chain isoform expression in the extrinsic finger muscles and biceps brachii. EDC contained the highest area proportion of type-1 fibers and the lowest area proportion of type-2x fibers. Values are means ± SD. FDP—flexor digitorum profundus; FDS—flexor digitorum superficialis; EDC—extensor digitorum communis; BB—biceps brachii.

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Fig. 5. Numerical proportions of major fiber types classified on the basis of adult myosin heavy chain isoform expression in the extrinsic finger muscles and biceps brachii. EDC contained the highest numerical proportion of type-1 fibers and the lowest numerical proportion of type-2x fibers. Values are means ± SD. FDP—flexor digitorum profundus; FDS—flexor digitorum superficialis; EDC—extensor digitorum communis; BB—biceps brachii.

Fig. 6. Diameters of type-1, type-2x, type-2a and type-2a/2x fibers in the extrinsic finger muscles and biceps brachii. EDC had significantly smaller-sized type-2x fibers with respect to FDP and FDS. Values are means ± SD. FDP—flexor digitorum profundus; FDS—flexor digitorum superficialis; EDC—extensor digitorum communis; BB—biceps brachii.

Fig. 7. Numerical proportions of pure fast fibers (type-2x, type-2a) and hybrid fast fibers (type-2a/2x) in the extrinsic finger muscles and biceps brachii. In all muscles, numerical proportions of hybrid type-2x/2a were significantly higher than numerical proportions of pure type-2x fibers. Values are means ± SD. FDP—flexor digitorum profundus; FDS—flexor digitorum superficialis; EDC—extensor digitorum communis; BB—biceps brachii.

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4. Discussion We demonstrated, according to the hypothesis, that EDC expressed a higher proportion of MyHC-1 than extrinsic finger flexors; a smaller proportion of MyHC-2x in EDC compared to extrinsic finger flexors was also found. MyHC composition co-determines the whole muscle contractile performance (Harridge et al., 1996) and several studies on single muscle fibers in vitro demonstrated that muscle fibers expressing only MyHC-1 develop less specific force than muscle fibers expressing MyHC-2a and muscle fibers expressing MyHC-2x, i.e. specific force of fibers expressing individual MyHC isoforms increases in the direction MyHC-1 < MyHC-2a < MyHC-2x (Bottinelli et al., 1996; Bottinelli and Reggiani, 2000; Schiaffino and Reggiani, 2011; Miller et al., 2015). Higher content of MyHC-1 and lower content of MyHC-2x in EDC compared to extrinsic finger flexors, are in agreement with the smaller power of finger extensors compared to finger flexors (Suzuki et al., 1994; Brorsson et al., 2012). A study by Gantelius et al. (2012) has revealed that differences in MyHC composition similar to these also exist between wrist extensors and wrist flexors in healthy subjects. These observations lead us to conclude that in the forearm, the proportion of fast fibers is higher in flexors, while the proportion of slow fibers is higher in extensors. Long finger flexors also had a similar composition of MyHCs as BB i.e. flexors located in the proximal and distal compartments of upper limbs had similar MyHC composition. Postural and endurance muscles contain more MyHC-1 while muscles responsible for rapid, high-power, short duration movements consist of more MyHC-2a and MyHC-2x (Bottinelli et al., 1996; Schiaffino and Reggiani, 2011). Electromyographic studies using wire electrodes during power grip (Johanson et al., 1998), one type of prehensile movements used in the activities of daily life (Napier, 1956), revealed phasic activity of the FDP and FDS during grip, while EDC was active during grip and during release i.e. had continuous activity. Continuous activity of EDC is compatible with the higher content of slow fibers in EDC which are oxidative, fatigue resistant and have more endurance (Blaauw et al., 2013). Gripping, which activates the flexor muscles, creates a flexion moment around the wrist joint and as a result the extensor muscles, including EDC, are co-activated (simultaneously finger flexors are contracting); co-contraction of EDC contributes to an extension moment that stabilizes the wrist joint (Basmajian and De Luca, 1985; Snijders et al., 1987; van Elk et al., 2004). The position of the wrist is one of the most important determinants that influences grip strength and 25◦ –30◦ of wrist extension is required for optimum grip strength (O’Driscoll et al., 1992; Li, 2002; Mitsukane et al., 2015). MyHC electrophoresis and MyHC immunohistochemistry provided similar information on MyHC composition of individual muscles: e.g. EDC was characterised as the muscle with the highest content of slow MyHC-1 by electrophoresis and the highest content of pure type-1 fibers by immunohistochemistry. However, absolute numbers of MyHC proportion and area proportion of type-1 fibers differed: i.e. the proportion of MyHC-1 in EDC was 50%, whereas area proportion of pure type-1 fibers in EDC was 63%. This difference might be related to sampling: total muscle homogenate for MyHC electrophoresis was prepared from whole muscle sample sections (on average about 6500 muscle fibres) whereas with immunohistochemistry two randomly selected regions per muscle sample section (on average about 250 muscle fibers) were analyzed. Considering regional differences in the MyHC composition in individual parts of muscle sample sections (Hesse et al., 2013), bias can be easily found in immunohistochemistry. When analyzing sampling errors in biopsy techniques using data from whole muscle cross sections of vastus lateralis (Lexell et al., 1985) it was concluded that a substantial reduction in sampling error is achieved taking biopsies with at least 600 fibers, ideally from sev-

eral sampling sites. The potential limitation of the present study is a single spot sampling data. However, the sample size for electrophoresis was about ten times larger than recommended (Lexell et al., 1985), which should also reduce the sampling error. In our opinion the relative abundance of individual MyHCs determined by electrophoresis was a more faithful representation of muscle composition, due to a larger sample size, than when it was determined from immunohistochemical sections. Harridge et al. (1996) also considered MyHC separation by electrophoresis and subsequent densitometry potentially more representative of muscle MyHC composition than the information provided by analyzing muscle fiber types on immunohistochemical sections. When electrophoresis is not available, determination of MyHC composition by stereological analysis of whole muscle sample sections seems to be a good alternative (Cvetko et al., 2012). Nevertheless, immunohistochemistry provides complementary information to electrophoresis: data on pure fibers (fibers expressing only one MyHC) and data on hybrid fibers (fibres co-expressing several MyHCs). By combining the results of electrophoresis and immunohistochemistry, we have concluded that the differences in MyHC content observed by electrophoresis were caused by different contents of pure type-1 and pure type-2x fibers, which were most and least abundant in the EDC muscle respectively. Originally, hybrid fibers were linked to conditions of muscle remodelling, but it is now recognized that they can also be present in stable conditions (Williamson et al., 2000). Hybrid fast contracting fibers (type-2a/2x) co-expressing both fast MyHC isoforms were more numerous than pure fast type-2x fibers in all muscles. Hybrid fibers augment the span of contractile velocities of muscle fibers and are more numerous in muscles with complex functions (Hoh, 2005). The high proportion of hybrid fibers in extrinsic finger muscles reflects the variety of different tasks these muscles perform. Interestingly enough, hybrid type-1 fibers were infrequent in extrinsic finger muscles. It seems that contraction velocity is more extensively fine-tuned in fast than in slow fibers. Previous studies of extrinsic finger muscles described the composition of FDP and EDC based on myofibrillar ATPase histochemistry in six young subjects, but did not investigate FDS (Johnson et al., 1973; Polgar et al., 1973). They reported on similar proportions of slow type-1 fibers in EDC and FDP (approximately 47%), which is in contrast to the difference between EDC and FDP observed in the present study. Though different methods were used to estimate contractile properties of fibers (demonstration of ATPase activity and of MyHCs), the results should nevertheless be comparable (Smerdu and Soukup, 2008). However, subjects who participated in the two studies were of different age, and agerelated changes in muscles toward the slower phenotype are well documented (Lexell, 1995). Whether the difference between EDC and FDP observed in this study is, in fact, age-related remains to be determined. In addition, the older study (Johnson et al., 1973) used a smaller number of subjects than the present study and might therefore be less representative. Regarding the size of the muscle fibers, the study by Polgar et al. (1973) did not report on differences in muscle fiber size between FDP and EDC. They also did not subclassify fast fibers, as was done in the present study. To the best of our knowledge, our study is unique in using a panel of MyHC antibodies against major adult MyHC isoforms, which also provides the information about pure and hybrid fibers, and it is the first study on MyHC electrophoresis of extrinsic finger muscles.

5. Conclusions EDC has a different MyHC composition than FDP, FDS and BB: it expresses a relative higher proportion of MyHC-1 and lower pro-

ˇ A., Characterisation of flexor digitorum profundus, flexor digitorum superficialis Please cite this article in press as: Meznaric, M., Carni, and extensor digitorum communis by electrophoresis and immunohistochemical analysis of myosin heavy chain isoforms in older men. Ann. Anatomy (xxxx), https://doi.org/10.1016/j.aanat.2019.151412

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portion of MyHC-2x. These differences refer to pure type-1 and pure type-2x fibers. Proportions of fast and slow MyHCs determined by electrophoresis and immunohistochemistry differed for 10%–13%. Relative abundance of individual MyHCs determined by electrophoresis was a more faithful representation of muscle composition, due to a larger sample size, than when it was determined from immunohistochemical sections. Higher proportion of MyHC-1 and lower proportion of MyHC2x in EDC compared to extrinsic finger flexors are in an agreement with the lower contraction force of finger extensors compared to finger flexors. Funding statement The study was supported by the Slovenian Research Agency, programme P3-0043. Ethical statement The muscle sampling was approved by the National Medical Ethics Committee of the Republic of Slovenia. Acknowledgements The excellent technical assistance of Ivan Blazinoviˇc, Majda ˇ ˇ Crnak-Maasarani, Friderik Stendler, Milan Stevanec, Nataˇsa Pollak Kristl, Marko Slak and Andreja Vidmar is gratefully acknowledged. References Bamman, M.M., Clarke, M.S., Talmadge, R.J., Feeback, D.L., 1999. Enhanced protein electrophoresis technique for separating human skeletal muscle myosin heavy chain isoforms. Electrophoresis 20, 466–468, http://dx.doi.org/10.1002/ (SICI)1522-2683(19990301)20:3<466::AID-ELPS466>3.0.CO;2-7. Basmajian, J.V., De Luca, C.J., 1985. Wrist, hand, and fingers. In: Butler, J. (Ed.), Muscles Alive. Their Functions Revealed by Electromyograph. , fifth ed. Williams & Wilkins, Baltimore, pp. 290–309. Biant, L.C., 2016. Elbow and forearm. In: Standring, S. (Ed.), Gray’s Anatomy. , fortyfirst ed. Elsevier, Philadelphia, pp. 837–861. Blaauw, B., Schiaffino, S., Reggiani, C., 2013. Mechanisms modulating skeletal muscle phenotype. Compr. Physiol. 3, 1645–1687, http://dx.doi.org/10.1002/cphy. c130009. Blum, H., Beier, H., Gross, H., 1987. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93–99, http://dx.doi.org/10. 1002/elps.1150080203. Bottinelli, R., Canepari, M., Pellegrino, M.A., Reggiani, C., 1996. Force-velocity properties of human skeletal muscle fibres: myosin heavy chain isoform and temperature dependence. J. Physiol 495 (Pt. 2), 573–586, http://dx.doi.org/10. 1113/jphysiol.1996.sp021617. Bottinelli, R., Reggiani, C., 2000. Human skeletal muscle fibres: molecular and functional diversity. Prog. Biophys. Mol. Biol. 73, 195–262. Brorsson, S., Nilsdotter, A., Pedersen, E., Bremander, A., Thorstensson, C., 2012. Relationship between finger flexion and extension force in healthy women and women with rheumatoid arthritis. J. Rehabil. Med. 44, 605–608, http://dx.doi. org/10.2340/16501977-0986. Butler, T.J., Kilbreath, S.L., Gorman, R.B., Gandevia, S.C., 2005. Selective recruitment of single motor units in human flexor digitorum superficialis muscle during flexion of individual fingers. J. Physiol. 567, 301–309, http://dx.doi.org/10.1113/ jphysiol.2005.089201. Cvetko, E., Karen, P., Erzen, I., 2012. Myosin heavy chain composition of the human sternocleidomastoid muscle. Ann. Anat. 194, 467–472, http://dx.doi.org/10. 1016/j.aanat.2012.05.001. Dalakas, M., 2012. Inflammatory and autoimmune features of inclusion-body myositis. In: Engel, W.K., Askanas, V. (Eds.), Muscle Aging, Inclusion-Body Myositis and Myopathies. Wiley-Blackwell, Chichester, pp. 146–158. Dupan, S.S.G., Stegeman, D.F., Maas, H., 2018. Distinct neural control of intrinsic and extrinsic muscles of the hand during single finger pressing. Hum. Mov. Sci. 59, 223–233, http://dx.doi.org/10.1016/j.humov.2018.04.012. Fanin, M., Nascimbeni, A.C., Fulizio, L., Trevisan, C.P., Meznaric-Petrusa, M., Angelini, C., 2003. Loss of calpain-3 autocatalytic activity in LGMD2A patients with normal protein expression. Am. J. Pathol. 163, 1929–1936, http://dx.doi.org/10.1016/ S0002-9440(10)63551-1. Gantelius, S., Hedström, Y., Pontén, E., 2012. Higher expression of myosin chain IIx in wrist flexors in cerebral palsy. Clin. Orthop. Relat. Res. 470, 1272–1277, http:// dx.doi.org/10.1007/s11999-011-2035-3.

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ˇ A., Characterisation of flexor digitorum profundus, flexor digitorum superficialis Please cite this article in press as: Meznaric, M., Carni, and extensor digitorum communis by electrophoresis and immunohistochemical analysis of myosin heavy chain isoforms in older men. Ann. Anatomy (xxxx), https://doi.org/10.1016/j.aanat.2019.151412