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Keywords:

  • opossum;
  • tail;
  • muscle;
  • fiber;
  • MHC;
  • antibody;
  • marsupial;
  • immunospecificity

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

Muscle fiber type is a well studied property in limb muscles, however, much less is understood about myosin heavy chain (MHC) isoform expression in caudal muscles of mammalian tails. Didelphid marsupials are an interesting lineage in this context as all species have prehensile tails, but show a range of tail-function depending on either their arboreal or terrestrial locomotor habits. Differences in prehensility suggest that MHC isoform fiber types may also be different, in that terrestrial opossums may have a large distribution of oxidative fibers for object carrying tasks instead of faster, glycolytic fiber types expected in mammals with long tails. To test this hypothesis, MHC isoform fiber type and their regional distribution (proximal/transitional/distal) were determined in the tail of the Virginia opossum (Didelphis virginiana). Fiber types were determined by a combination of myosin-ATPase histochemistry, immunohistochemistry, and SDS-PAGE. Results indicate a predominance of the fast MHC-2A and -2X isoforms in each region of the tail. The presence of two fast isoforms, in addition to the slow MHC-1 isoform, was confirmed by SDS-PAGE analysis. The overall MHC isoform fiber type distribution for the tail was: 25% MHC-1, 71% MHC-2A/X hybrid, and 4% MHC-1/2A hybrid. Oxidative MHC-2A/X isoform fibers were found to be relatively large in cross-section compared to slow, oxidative MHC-1 and MHC-1/2A hybrid fibers. A large percentage of fast MHC-2A/X hybrids fibers may be suggestive of an evolutionary transition in MHC isoform distribution (fast-to-slow fiber type) in the tail musculature of an opossum with primarily a terrestrial locomotor habit and adaptive tail-function. Anat Rec, 2013. © 2012 Wiley Periodicals, Inc.

Muscle fiber type and fiber architecture are well studied properties in a variety of limb muscles from a large number of mammalian species (e.g., Smerdu et al., 1994, 2009; Rivero et al., 1993, 1999b; Hermanson et al., 1998; Zhong et al., 2001, 2008; Toniolo et al., 2004, 2005, 2007; Butcher et al., 2010; Hyatt et al., 2010), however, much less is known about these properties in paravertebral muscles (Schilling, 2005, 2009; Kohn et al., 2007; Moritz et al., 2007), and little is understood about the functional properties of caudal muscles (Ovalle, 1976; Wada et al., 1994; Spiegel et al., 2002). Interest in the evolution of tail-function has mainly centered around descriptions of myology, osteology, and muscle architecture in species of non-human primates displaying varying degrees of tail prehensility (Lemelin, 1995; Bezanson, 1999; Schmitt et al., 2005; Organ et al., 2009; Organ, 2010). Yet a number of mammalian lineages are specialized for arboreal habit and thus rely on their tail for rapid maneuvering (e.g., cantilevering) and suspension in arboreal locomotion (Youlatos, 2008). Didelphid marsupials are an interesting model of study in this context as they all have characteristically long and prehensile tails (Nowak, 1991), but show a range of tail-function depending on their degree of tail prehensility and locomotor habits (Lemelin, 1995, 1999). For example, in a highly arboreal genus such as Caluromys (Charles-Dominique, 1983), the tail is used during the postural behaviors foot hanging and tail hanging, often to acquire and manipulate food (Youlatos, 2008). The tail may also provide some degree of insurance against falling during walking, clambering, and bridging on small (terminal) branches, where the tail can rapidly grasp a branch for stabilization (Schmitt and Lemelin, 2002). In more terrestrial forms, nest building and object manipulation appear to be the primary roles of the prehensile tail (Smith, 1941; Layne 1951; Unger, 1982).

Previous studies (Peters et al., 1984, Hansen et al., 1987; Sciote and Rowlerson, 1998) have evaluated muscle fiber types in didelphids. Among these reports, only Hansen et al. (1987) analyzed muscle fiber type of the caudal musculature; however, muscle fiber typing was limited to myosin ATPase (mATPase) histochemistry to broadly classify fibers as either Type I (slow) or Type II (fast) (old nomenclature) based on their acid and alkaline reaction lability or stability (Brook and Kaiser, 1970; Guth and Samaha, 1970). Identification of subdivisions of fast myosin heavy chain (MHC) isoform fiber types by this technique is not clear, as difficulties are encountered with the use of acid-stable histochemistry due to differing pH sensitivities of mATPases in fast fiber types (Zhong et al., 2008). However, while histochemical properties are an important component of analyses of muscle structure–function because they correlate with mATPase activity of the muscle fibers (Rivero et al., 1999b), the exact MHC isoforms expressed in muscle fibers can only be inferred by conventional histochemistry. Identification of MHC isoforms is essential to fiber type classification as MHC isoforms are the primary determinants of muscle fiber contractile properties, and thus in vivo contractile performance of whole muscles (Reiser et al., 1985; Schiaffino and Reggiani, 1996; Bottinelli et al., 1999).

Skeletal muscles of marsupials are composed of four conventional MHC isoform fiber types homologous to those expressed in small Eutherians (Lucas et al., 2000): MHC-1 (slow, oxidative), MHC-2A (fast, oxidative), MHC-2X (fast, oxidative/glycolytic), and MHC-2B (fast, glycolytic). However, recent observations from limb muscles in Australian marsupials (Zhong et al., 2001, 2008) suggest that specification of the three fast MHC fiber types (and hybrid fibers) in marsupials by conventional mATPase histochemistry alone (Gorza, 1990; Rivero et al., 1999a) is inadequate. Correspondingly, previous analyses in Didelphis virginiana (Peters et al., 1984; Hansen et al., 1987) and Monodelphis domestica (Sciote and Rowlerson, 1998) have reported difficulties classifying fast fiber types (and hybrid fibers) by their histochemical reactivity. MHC isoforms and fiber types are most directly identified at the protein level by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunohistochemistry (IHC) techniques (Rivero et al., 1996, 1999a; Zhong et al., 2001, 2008; Kohn et al., 2007; Butcher et al., 2010; Hyatt et al., 2010). With respect to the latter, the commercial availability of monoclonal antibodies (mAbs) and their improved immunospecificity for reaction against MHC isoforms expressed in skeletal muscle fibers from a diversity of mammalian species (Lucas et al., 2000) has necessitated re-evaluation of MHC isoform fiber types by a combination of histochemistry, electrophoresis, and IHC methods (Rivero et al., 1999a,b; Toniolo et al., 2004, 2005, 2007; Zhong et al., 2008). At present, there has been no direct assessment of MHC isoform composition in tail musculature by a combination of these techniques. Therefore, it is not known whether the expression of all four MHC isoforms is conserved in the caudal muscles of South American didelphid marsupials as they are in the limb muscles of Australian marsupials (Zhong et al., 2001, 2008). Fiber type distributions typical of other arboreal mammals with prehensile tails are also unknown.

The objective of this study is to reinvestigate the tail of the Virginia opossum by (i) identifying MHC isoforms of a caudal muscle and (ii) determining the relative size of each MHC isoform fiber type in the opossum tail. It is hypothesized that the distribution of fast muscle fibers identified will contain a majority of the fast, MHC-2A isoform. D. virginiana has a terrestrial locomotor habit, and their prehensile tail may be used more for adaptive behaviors involving object carrying and manipulation (e.g., nest building: Smith, 1941). These tasks require periods of prolonged low force production typical of oxidative fibers. A large distribution of highly oxidative MHC-2A fibers is also consistent with a postural function of caudal muscles associated with locomotor balance. Collectively, this finding may be suggestive of an evolutionary shift in tail-function for the primarily terrestrial D. virginiana and a consequent transition to slower MHC isoform expression because of limited use in arboreal locomotion. Alternatively, fast muscle fibers of the tail may express a majority of the fast, MHC-2X or MHC-2B isoforms. This finding may represent an ancestral condition of predominately glycolytic fibers in the caudal muscles of mammals with long tails. As a secondary goal, this study will contribute toward improved understanding of mAbs specificity for MHC isoforms in marsupials and overall, add to the growing phylogenetic and functional diversity of MHC isoform muscle fiber typing studies.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

Animals and Muscle Samples

Caudal muscle tissue was sampled from the tails of five Virginia opossums (D. virginiana Kerr). Three adult male and two adult female opossums (body mass: 2.2 ± 0.5 kg; tail length: 29.6 ± 2.1 cm) were live captured in the field (Mahoning County, Ohio) using Havahart Easy Set traps (80 cm × 30 cm × 35 cm), and euthanized in the laboratory by an overdose IP injection of Euthasol (Virbac AH). Animal trapping (ODNR Wild Animal Permits: 10-252, 11-292) and all experimental procedures followed approved protocols (YSU, IACUC protocol: 03-09; PI: M.T. Butcher). Immediately post-mortem, the tails were removed and divided into “proximal,” “transitional,” and “distal” regions (Hansen et al., 1987; Organ et al., 2009) for muscle dissection (Fig. 1). Small blocks of muscle tissue were harvested from the m. flexor caudae longus (Lemelin, 1995) attached ventrally and laterally along the caudal vertebrae. Muscle blocks were mounted to cork with tragacanth mounting media (Sigma-Aldrich), frozen in isopentane, cooled in liquid nitrogen, and stored at −80°C until analysis.

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Figure 1. Diagram of the deep caudal muscles of D. virginiana. Muscles illustrated are m. flexor caudae longus (FCL), m. flexor caudae brevis (FCB), and m. extensor caudae longus (ECL). Shown is a left, lateral view of the tail indicating the “proximal,” “transitional,” and “distal” tail regions (by vertical dashed lines) from which muscle blocks were harvested from the FCL. Muscle harvesting began just distal to FCB (beginning of hairless tail region) and ventral to ECL using the transverse processes of the caudal vertebrae as landmarks. Two prominent tendons (gray lines and regions) each span the entire length of both the dorsal (extensor) and ventral (flexor) aspects of the tail. Muscle nomenclature for prehensile tails taken from Lemelin (1995). For veterinary reference, FCL relates to m. sacrocaudalis ventralis lateralis.

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Two additional muscle blocks from each tail region were dissected and prepared for electrophoresis by freezing in liquid nitrogen, grinding to powder, and homogenizing 50 mg of muscle powder in 800 μL (ratio 1:16) of Laemmli buffer with 62.5 mM Tris (pH 6.8), 10% glycerol, 5% β-mercaptoethanol, and 2.3% SDS (Laemmli, 1970). Protein samples were diluted (1:500) to a final protein concentration of ∼0.125 μg/μL with gel sample buffer containing 80 mM Tris (pH 6.8), 21.5% glycerol, 50 mM dithiothreitol (DTT), 2.0% SDS, and 0.1% bromophenol blue (Mizunoya et al., 2008). Samples were then heated (90°C) for 5 min, and stored at −20°C. To serve as a control standard for identifying MHC isoforms expressed in caudal muscle from D. virginiana, muscle blocks of rat tibialis anterior (TA) and extensor digitorum longus (EDL) were harvested and muscle samples were prepared by the described methods.

SDS-PAGE

MHC isoforms were separated using established methods (Talmadge and Roy, 1993) performed with slight modifications (Mizunoya et al., 2008). The acrylamide-N,N′-methylenebisacrylamide (Bis) ratio of the gels was 50:1, with the total acrylamide percentage equaling 8% and 4% in the separating gel (35% glycerol) and stacking gel (30% glycerol), respectively. The basic formulation of the electrode buffer was 50 mM Tris (pH 8.3), 75 mM glycine, and 0.5% SDS; upper buffer was 6× the concentration of the lower buffer and also contained 0.12% β-mercaptoethanol (Mizunoya et al., 2008). Approximately, 1.0 μg of protein was loaded per gel lane, and electrophoresis was run on a mini-PROTEAN Tetra system (Bio-Rad) at constant low voltage (140 V) for 21 hr at 4°C (Talmadge and Roy, 1993; Mizunoya et al., 2008). Gels were stained with a Silver Stain Plus Kit (Bio-Rad) for visualization of MHC isoforms, and imaged using a laser-scan protein imaging system (Bio-Rad). MHC isoform identity was evaluated by resolution and comparative migration patterns of the protein bands.

Histochemistry and Immunohistochemistry

Transverse serial sections (10 μm) were cut on a Leica 1850 cryostat (Leica Microsystems) at −20°C and mounted on GOLDSEAL slides (Becton Dickinson) for histochemistry and charged Superfrost slides (Fisher Scientific) for IHC. Serial sections were stained for metabolic activity of nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) and α-glycerol phosphate dehydrogenase (GPD) to assess oxidative (Novikoff et al., 1961) and glycolytic (Wattenburg and Leong, 1960) capacity, respectively. Muscle sections were either incubated in 0.2 M phosphate buffer (pH 7.4) containing 9.3 mM GPD, 1.2 M nitroblue tetrazolium, and 2.3 mM menadione for 30–50 min at 35°C (body temperature for opossums) or in phosphate buffered saline (PBS) (pH 7.4) containing 1.4 mM NADH and 2.5 mM nitroblue tetrazolium for 10–15 min at 35°C. Sections were rinsed in dH2O (3 min), dehydrated in 50% acetone (3 min), and mounted with glass coverslips.

Serial sections were reacted for mATPase activity using modifications (Hermanson et al., 1998) of the methods derived from Brooke and Kaiser (1970). Briefly, muscle sections were pre-incubated at 35°C in either 0.2 M Na-acetate and 75 mM 5,5-diethyl barbituric acid sodium salt (pH 4.3, 4.4, 4.5: 5 min) or 20 mM glycine, 74 mM NaCl, and 38 mM CaCl2 (pH 10.3, 10.4: 10 min). Acid sections were rinsed in sodium barbital buffer containing 75 mM 5,5-diethyl barbituric acid sodium salt and 18 mM CaCl2, while alkaline sections were rinsed in dH2O (pH 8.6–9.0) (Hermanson et al., 1998). Next, all sections were incubated at 35°C (30 min) in sodium barbital buffer (pH 9.4) to which ATP (1.4 mg/ml) was added. After mATPase reaction, sections were alternately washed in 1% CaCl2 (3 min) and 2% CoCl2 (3 min) in between dH2O rinses. All sections were then stained in 1% (NH4)2S (1 min) and rinsed in tap water (5 min) before being dehydrated in ethanol (3 min), dried in xylenes (1 min), and mounted.

Other serial sections were reacted against a panel of mAbs specific to slow and fast MHC isoforms (see Table 1) used to identify MHC isoform fiber types by IHC. S58 was specific to the MHC-1 isoform in numerous vertebrate species (Miller et al., 1985), while three mAbs were specific to rat MHC isoforms (Schiaffino et al., 1998): SC71 specific to MHC-2A, BF-35 specific to all MHC isoforms except MHC-2X, and BF-F3 specific to MHC-2B. Each of these antibodies were purchased from the Developmental Studies Hybridoma Bank (DSHB, University of Iowa). In addition, serial sections were reacted against MY32 (Sigma-Aldrich) specific to all fast MHC isoforms in various species ranging from rat (Naumann and Pette, 1994) to horse (Butcher et al., 2010), and two mAbs also obtained from DSHB with known specificity to marsupial fast MHC isoforms (Lucas et al., 2000): 6H1 specific to MHC-2X isoform, and 10F5 specific to MHC-2B isoform. All mAbs (supernatant) were diluted in PBS (pH 7.4). MY32 was diluted according to the manufacturer's specifications (1:400). Antibodies from DSHB were diluted to working Ig concentrations of 2–5 μg/mL for IHC.

Table 1. Monoclonal Antibodies (mAbs) Used for IHC Analyses and Their MHC Isoform Reaction Specificity Against Caudal Muscle From D. virginiana
AntibodySlow, MHC-1Fast (MHC)MHC-2AMHC-2XMHC-2B
  1. (+) positive reaction against muscle MHC antigen.

  2. (++) strong positive reaction against muscle MHC antigen.

  3. (+/−) weak reaction against muscle MHC antigen.

  4. (−) no reaction against muscle MHC antigen.

S58++    
MY32 +/−   
SC71  ++ 
6H1   +/− 
BF-35+ +  
BF-F3    
10F5    

IHC was conducted as previously described (Hermanson et al., 1998; Butcher et al., 2010). Muscle sections were blocked with 2% goat serum (Histostain Plus Kit: Invitrogen) at room temperature for 10 min. Sections were then incubated with mAbs in a humidified chamber for 12–16 hr at 4°C. The sections were washed in PBS, followed by incubation with a biotinylated rabbit (anti-mouse) secondary antibody for 1 hr at room temperature (washed with PBS), application of a streptavidin horseradish peroxidase (HRP) enzyme conjugate (10 min), and reaction with 3,3′-diaminobenzidine (DAB) chromagen (3–6 min), all using a Histostain Plus kit (Invitrogen). After a rinse in dH2O (10 min), muscle sections were counterstained with Mayer modified hematoxylin (Newcomer Supply) to better visualize fiber morphology. Several muscle sections from rat LG/SOL and TA were also incubated with mAbs to serve as a comparison standard for immunospecificity of reactivity. Experimental controls were muscle sections treated with PBS in place of mAbs.

Data Analysis

MHC isoform distributions were quantified from images of IHC-stained serial sections visualized on an Olympus CX31 microscope (Olympus Microscopes) and photographed with a SPOT Idea digital camera system (Diagnostic Instruments). Percentages of each MHC fiber type were calculated based on counts of at least 1,000 fibers per animal, from 3 to 4 sections of muscle from each tail region. Muscle fibers were classified as hybrids if they showed cross-reaction with two mAbs specific for different MHC isoforms. Fiber size was determined by measuring cross-sectional area (CSA: in μm2) and minimum diameter (in μm) of each MHC fiber type from section images imported into Image J (v.1.43: NIH) running on a Macintosh mini computer. Fiber size was assessed for at least 200 fibers (of each fiber type classified) in each tail region. Fiber type data are presented as whole percentages. Fiber CSA and diameter are presented as mean ± SD. Statistical differences in fiber type architecture were not tested in this study.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

SDS-PAGE Identification of MHC

Electrophoresis on protein homogenates from each tail region showed expression of three MHC isoform bands: MHC-1, 2A, and 2X (Fig. 2). Slow MHC-1 and both fast MHC-2A and MHC-2X bands were clearly resolved in the proximal samples, while clear separation of 2A and 2X bands was not obtained in all transitional and distal samples analyzed. The MHC-1 isoform was identified as the band that consistently migrated the furthest and fastest in the gel lanes, while the MHC-2X isoform occupied the position intermediate to the slow migrating MHC-2A isoform. The identity of the three isoforms in opossum caudal muscle was verified by similar band migration patterns of MHC isoforms in rat limb muscles (Fig. 2), although the mobility and alignment of 2A and 2X bands of the rat samples slightly differed. Additionally, the MHC-2B isoform band was present in each rat sample, but was not expressed in the opossum caudal muscle samples.

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Figure 2. Representative SDS-PAGE gels identifying three MHC bands in caudal muscle of D. virginiana. MHC isoform identity and band migration patterns were confirmed by comparison with muscle homogenates from rat TA (shown) and EDL. Composite of gel lanes from left to right: Lane 1 rat TA; Lanes 2–3 MHC isoforms expressed in proximal tail region; Lanes 4–5 MHC isoforms expressed in transitional tail region; Lane 6 MHC isoforms expressed in distal tail region.

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Histochemical Activity of Tail Muscle Fibers

Strong NADH-TR activity was evident in all muscle fibers from each tail region, indicating a high oxidative capacity of opossum caudal muscle. Moderately dark NADH-TR staining was uniform across all muscle sections analyzed (Fig. 3D). Conversely, muscle sections consistently stained lightly in reaction to GPD (Fig. 3E), indicating low glycolytic potential. No differences in both NADH-TR and GPD activity were observed regardless of sex or body mass of the opossums used for this study (N = 5).

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Figure 3. Representative fiber type reactivity for mATPase histochemistry, IHC, and metabolic activity in caudal muscle of D. virginiana. Serial cross-sections (taken from the proximal tail) reacted (A) after acid incubation (pH 4.4) and with a panel of mAbs specific to MHC isoforms: B: S58 (anti MHC-1) and (C) SC71 (anti MHC-2A, 2X). Fibers that stain darkly (highly acid stable) are slow, MHC-1 fibers. Fast, oxidative MHC-2A/X hybrid fibers are lightly stained, and slow/fast MHC-1/2A hybrids are moderately stained at acid incubation pH 4.4. mATPase fiber typing was confirmed by reaction against mAbs. Panels (D) NADH-TR and (E) α-GPD are an additional pair of serial sections showing representative metabolic profiles of opossum caudal muscle tissue. Fibers exhibited high NADH-TR activity, while α-GPD activity was very low in all sections analyzed. Fibers labeled with an asterisk (*) are the same fiber of reference in each set of serial sections. Scale bars = 100 μm.

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Three fiber types could be distinguished based on mATPase reactions in an alkaline (10.3) and acidic incubations (4.4, 4.5). Fast fibers retained their mATPase activity during alkaline incubations, and each muscle section analyzed showed large numbers of darkly stained fibers, while slow fibers were unstained or labile. After acid incubations, three different staining intensities of the mATPase activity were commonly observed across muscle sections from each tail region. Images of histochemically stained (in acid incubation pH 4.4) caudal muscle tissue are shown in Figs. 3A and 4A. Fibers darkly stained in acid incubations pH 4.4 and 4.5 (highly acid stable) were classified as slow, MHC-1 fibers. Moderately acid stable fibers (Fig. 4) were classified as slow/fast hybrid fibers. Fibers lightly stained in acid incubations were best classified as fast (MHC-2A/X) hybrid fibers (Rivero et al., 1996, 1999b). This was due to inconsistent staining intensities that could lead to classification as either pure MHC-2X fibers or fast hybrids over the range of acid incubations, and the inability to clearly distinguish pure MHC-2A fibers (often unstained or acid labile at pH 4.4) based on acid mATPase histochemistry (Peters et al., 1984; Sciote and Rowlerson, 1998). The best resolution for mATPase histochemistry for all animals studied occurred at acid pre-incubation pH 4.4.

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Figure 4. Representative fiber type reactivity for mATPase histochemistry and IHC in the distal tail region of D. virginiana. Serial cross-sections reacted (A) for mATPase after acid incubation (pH 4.4) and against (B) SC71. Panels (C) S58 and (D) SC71 are additional serial sections from a separate experiment showing hybrid fibers (+) that reacted with both S58 and SC71. Fibers labeled with an asterisk (*) are the same fiber of reference in each set of serial sections. Scale bars = 100 μm.

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Immunohistochemical Determination of MHC Fiber Types

Three distinct MHC isoform fiber types were clearly identified using select mAbs specific for MHC isoforms, matching well with results of mATPase histochemistry. Table 1 shows a summary reaction specificity of mAbs with caudal muscle tissue from D. virginiana. Slow MHC-1 fibers were identified by strong reactivity against the antibody S58 (Figs. 3B, 4B, 5B,E). S58 consistently identified MHC-1 isoform fibers in each tail region and animal studied. By the principle of exclusion, serial sections of fibers of that did not react against S58 were either fast fiber types or hybrids, composed of the MHC-2A and MHC-2X isoforms. An abundance of MHC-2A/X hybrid fibers were identified by strong reaction against the antibody SC71 (Figs. 3C, 4B, 4D, 5C,F), which is specific to the MHC-2A isoform in rats, and now indicated to recognize both 2A and 2X isoforms in D. virginiana (Table 1). The third fiber type was confirmed as MHC-1/2A hybrids by matching fibers with moderate acid stability and reactions against both S58 and SC71 (Fig. 4C,D). Low numbers of slow/fast hybrid fibers were found in all three regions of the tail, and reaction intensities against S58 and SC71 for the same fiber varied slightly with each tail region.

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Figure 5. Monoclonal antibody reactivity in two sets of serial sections of caudal muscle from D. virginiana. Left: representative muscle sections (taken from the transitional tail) were reacted against mAbs (A) BF-35 (anti MHC-1, 2A, 2B), (B) S58, and (C) SC71. BF-35 works on the principle of exclusion by reacting against all MHC isoforms except 2X. All fibers in sections analyzed reacted against the BF-35 antibody. Right: serial sections from a separate experiment showing representative reactivity against mAbs (D) 10F5 (anti MHC-2B), (E) S58, and (F) SC71. Reaction against 10F5 was negative confirming the lack of the MHC-2B isoform fibers. Fibers labeled with an asterisk (*) are the same fiber of reference in each set of serial sections. Scale bars = 100 μm.

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Reaction against 6H1 showed no clear ability to identify pure MHC-2X fibers in the tail of D. virginiana (data not shown). 6H1 was found to react weakly against the same fibers reactive with SC71; however, often it was difficult to distinguish between positive reactions and background staining, while at other times, 6H1 did not appear to react with any fibers. BF-35, specific for all isoforms except MHC-2X (old formulation; Schiaffino et al., 1989; Toniolo et al., 2007), moderately reacted against all muscle fibers in each tail section studied (Fig. 5A). Reaction with all fibers further indicated a lack of pure MHC-2X fibers and verified the presence of the 2X isoform in fibers classified as MHC-2A/X hybrids. MY32 showed weak reaction against fibers that did not react against S58 and thus was only effective in isolating the same fast fibers reactive against SC71. Lastly, BF-F3 and 10F5 showed no reaction against muscle fibers from any section of the tail (Fig. 5D) in accordance with a lack of MHC-2B expression in SDS-PAGE gels and low glycolytic capacity observed in caudal muscle fibers.

Fiber Type Distributions and Fiber Size

MHC isoform fiber type distribution was determined from a total of N = 6,640 fibers. In all five animals studied, there was a majority of fast, oxidative MHC-2A/X hybrid fibers, though their percent distribution varied with tail region across each animal studied (Table 2). In the proximal tail, nearly 84% of muscle fibers counted were positively identified as MHC-2A/X hybrids by reaction against the panel of mAbs used. The distribution of MHC-2A/X hybrids in the transitional and distal tail regions was relatively lower at 61% and 73%, respectively. Overall, 70.6% of muscle fibers studied in the tail of D. virginiana were found to be composed of MHC-2A/X isoforms (Table 3). MHC-1 fibers were the only pure isoform fiber type to be identified. Slow, oxidative MHC-1 fibers also varied in their percent distribution with tail region, though the percentages of slow fibers were very consistent in each tail region across each animal studied. In the proximal tail, just over 10% of MHC-1 fibers were found, compared to the transitional and distal tail regions which showed percentage distributions of 36% and 23%, respectively (Table 2). Collectively, the tail was composed of 25.4% MHC-1 fibers (Table 3). Slow/fast, oxidative MHC-1/2A hybrid fibers were present in low percentages and showed slightly different distributions in each tail region. The percentage distribution of MHC-1/2A hybrids was just over 4% for the entire tail (Table 3).

Table 2. Regional Distributions (%) of MHC Isoform Fiber Types in the Tails of Individual Opossums
AnimalProximalTransitionalDistal
MHC-1MHC-2A/XMHC-1/2AMHC-1MHC-2A/XMHC-1/2AMHC-1MHC-2A/XMHC-1/2A
  1. In parentheses is the total number of fibers counted for calculation of percentage MHC isoform fiber type

Op5–17 (N = 970)11.282.76.134.462.13.420.572.76.8
Op6–25 (N = 1,773)10.683.75.635.462.42.228.167.84.1
Op7–6 (N = 1,287)8.785.85.538.658.72.720.275.34.5
Op7–7 (N = 1,252)11.182.46.535.661.92.421.974.53.6
Op9–8 (N = 1,358)10.184.25.738.158.03.921.774.73.6

Means of fiber CSA and diameter for each MHC isoform fiber type identified are shown in Fig. 6. Along the tail in the direction cranial to caudal, fiber size (for each fiber type) progressively decreased (see Supporting Information Tables), remaining in following order by size: MHC-2A/X >MHC-1 >MHC-1/2A. Overall, MHC-2A/X hybrid fibers had a mean CSA of 4,888.1 ± 1 566.2 μm2 and were substantially larger than MHC-1 fibers (3 596.6 μm2), and nearly two-fold larger in cross-section than MHC-1/2A hybrids (Fig. 6A). Architectural measurements for diameter of muscle fibers were consistent with those of fiber CSA, indicating a good correlation between fiber CSA and diameter. MHC-2A/X hybrid fibers again had largest minimum diameter, followed by MHC-1 fibers and MHC-1/2A hybrids. However, unlike fiber CSA, means of 81.4 μm and 74.6 μm for MHC-2A/X hybrids and MHC-1 fibers, respectively, were more similar, and both were substantially larger than the mean diameter of MHC-1/2A hybrids (Fig. 6B).

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Figure 6. Mean fiber CSA (A) and fiber diameter (B) of MHC isoform fiber types in the tail of the D. virginiana. Means of CSA were calculated from a total N = 1,725 fibers measured across the proximal, transitional, and distal tail regions. Means of fiber diameter were calculated from a total N = 1,825 fibers measured across the proximal, transitional, and distal tail regions. Error bars are standard deviations (SD). Fiber size was not tested for statistical significance.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

The major findings of this investigation are the identification of three MHC isoform fiber types (MHC-1, MHC-1/2A, and MHC-2A/X) and their regional distributions in the tail of D. virginiana. Results indicate a predominance of relatively large, fast MHC-2A/X hybrid fibers in the proximal and distal regions of the tail. Alternatively, the transitional tail region had a more similar distribution of fast MHC-2A/X hybrids and slow MHC-1 isoform fibers, and each tail region showed a low percentage of relatively small and highly oxidative MHC-1/2A hybrid fibers. An overall distribution of 25.4% for slow MHC-1 fibers and 70.6% fast MHC-2A/X hybrids matches well with percentages of slow Type I (25%) and fast Type II (75%) fibers previously reported for the tail of the Virginia opossum (Hansen et al., 1987). However, the former mATPase histochemistry analysis was not sensitive enough to distinguish the fast MHC isoforms present, and it is recognized that the 4.1% MHC-1/2A hybrid fibers we found would have been classified as fast Type II fibers by histochemical staining. Determination of specific fast MHC isoform fiber types and hybrids in caudal muscle by SDS-PAGE and IHC in this study improves upon past analyses in D. virginiana, and provides a starting point for future evaluation of muscle structure–function in the prehensile tails of didelphid marsupials.

The observation that the m. flexor caudae longus (FCL) in the tail of D. virginiana contains the MHC-2A isoform co-expressed in fast, oxidative hybrid fibers supports our first hypothesis and may be consistent with an evolutionary change in tail-function (Hansen et al., 1987), and a consequent shift in muscle fiber phenotype to slower-contracting fiber types. The common ancestor of D. virginiana was arboreal (Cozzuol et al., 2006; Voss and Jansa, 2009), and the tail likely evolved to function as an additional appendage for arboreal locomotion and suspensory behaviors (Charles-Dominique, 1983; Lemelin, 1999). Arboreal habits have been linked to prehensile tails with caudal flexor muscles capable of both high force and appreciable shortening (Organ et al., 2009), thus the muscles are specialized for work performance. Muscles well-suited for work performance must also have appreciable shortening velocity which is primarily determined by MHC isoform expression. A number of arboreal locomotor behaviors, such as bridging and cantilevering (Youlatos, 2008), may require rapid tail movements of short duration which can best be performed by caudal muscles composed of fast MHC-2X and 2B fibers (moderately oxidative/glycolytic). Although MHC isoforms have not been previously identified in prehensile tails, histochemical analyses in a number of small mammals with long tails (including Monodelphis) indicate a composition of predominately fast, glycolytic fiber types in the paravertebral muscles about the base of the tail (Moritz et al., 2007; Schilling 2005, 2009), suggesting fast muscles may be an ancestral feature of mammal tails. In particular, D. virginiana adapted a terrestrial locomotor habit (McManus, 1970; Jenkins, 1971; Hunsaker, 1977) and prehensility of the tail for climbing function has been reduced to what may be defined as semi-prehensile (Emmons and Gentry, 1983), but has diversified for adaptive behaviors such as foraging and carrying materials (e.g., leaves) for nest building (Pray, 1921; Layne, 1951). Behaviors using the tail for carrying objects require prolonged low force production, which would best be performed by slower, oxidative muscle fibers. Moreover, D. virginiana may rely less on their long tails for balance and agile maneuvering over ground as they are slow-moving terrestrial scamperers that commonly walk (McManus, 1970; White, 1990). Therefore, changes in tail-function throughout their evolution and convergence to slow terrestrial habits may have led to the observed hybrid MHC isoform distribution.

Peculiar staining patterns in the distal tail region previously observed (Hansen et al., 1987), and seen in both the transitional and distal tail regions of the FCL in our study, are consistent with changes in muscle fiber structure–function from an ancestral condition. A large distribution of fast, oxidative hybrid fibers provides evidence of a MHC transition (Pette et al., 1999) that may be related to a primarily terrestrial lifestyle and reduced tail prehensility. Hybrid fibers enable a muscle to tune its contractile efficiency for a wide range of forces, velocities, and levels of fatigue resistance (Bottinelli et al., 1994a,b; Pette and Staron, 2000). Intermediate contractile properties of hybrid fibers may be important for object manipulation where fine motor movements like grasping food items or leaves are required. Though high percentages of MHC hybrid fiber types are not typically found in limb muscles (Toniolo et al., 2005; Smerdu et al., 2009; Butcher et al., 2010), a relatively large distribution of fast hybrid fibers (MHC-2AX/B and MHC-2X/B) has also been shown in a tail extensor muscle of kangaroos (Spiegel et al., 2010), thus a high percentage of MHC-2A/X hybrids may not be atypical for tail flexor muscles with prehensile function. While fast MHC isoform identity in the caudal muscles of other mammals are not available for comparison, 70.6% of MHC-2A/X hybrid fibers distributed throughout the tail of D. virginiana (Table 3) appears to be consistent with relatively high percentages of fibers histochemically classified as either Type II in the tail of adult rats (72%: Ovalle, 1976) or both Type IIa (∼25%) and IIb (∼50%) in the tails of cats (Wada et al., 1994). Without knowing the exact fast MHC isoforms, we may only speculate that a primary composition of MHC-2X fibers may be ancestral to the caudal muscles of mammals and functionally, be important to tail posture and rapid movements in locomotion (Wada et al., 1993). Evaluation of MHC isoforms and function in the prehensile tails of arboreal specialists and non-prehensile tails of terrestrial locomotors are needed to start testing these possibilities.

Table 3. Percentage Distributions (%) of MHC Isoform Fiber Types in the Tail of D. virginiana
Tail regionMHC-1MHC-2A/XMHC-1/2AN
  1. n = total number of fibers counted for each tail region across all animals

  2. Percent fiber type data are presented a whole percentages

Proximal10.383.75.92,244
Transitional36.360.92.83,304
Distal23.372.83.91,092
Distribution %:25.470.64.1 

In addition to MHC isoform expression, function is also related to muscle fiber size. A smaller cross-sectional area provides a shorter capillary diffusion distance for more efficient uptake of oxygen, thus pure MHC-1 and 2A fibers typically have smaller CSA and diameters, correlating with their high oxidative capacity and sustained mechanical work performance (Hepple et al., 2000). These metabolic characteristics, in addition to muscle fiber architecture, can be meaningful for evaluating the relative size of oxidative hybrid fibers we measured in the tail of D. virginiana. First, a mean fiber CSA of 4881 μm2 for MHC-2A/X hybrid fibers (Fig. 6) is consistent with the large size measured for all fast MHC isoform fiber types in the tail of kangaroos (Spiegel et al., 2010). Second, relatively large fiber CSA appears to be coupled with tail length. Limited comparative data available from small Eutherian mammals (e.g., mouse, rat, tree-shrew, vole) indicate a large fiber CSA relative to long tail length (Schilling, 2005), which may simply reflect an ancestral feature of basal mammals that retain long tails (Schilling, 2009). Furthermore, well-developed musculature (i.e., high mass per unit length) has been consistently observed in prehensile tails (Lemelin, 1995; Organ et al., 2009), showing an overall greater investment of flexor muscle mass along the entire tail length as was seen in D. virginiana. Correlating with higher muscle mass and fiber size, the intertransversarii caudae muscles (flexors) of prehensile-tailed platyrrhines (e.g., Ateles, Cebus) and procyonids (Potos) have also been shown to have relatively large physiological cross-sectional area (PCSA). Thus, fiber size is not only related to oxidative vs. glycolytic properties that influence capacity for mechanical work, but is also important for muscle force production (i.e., large PCSA = high force). Large muscle fibers (and PCSA) may therefore represent an important functional feature of prehensile tails in particular, for production of high forces to support the body weight during arboreal behaviors (Organ et al., 2009). Similar architectural properties may then be expected to be retained in the FCL of the Virginia opossum. Additionally, this raises interesting questions about diversity in caudal muscle fiber types and architecture that exists across didelphids. Many opossums are small and arboreal (or semi-arboreal: Delciellos and Vieira, 2006, 2009), and maneuver the complex rainforests canopies of Central and South America. Differences in locomotor habits, and possibly suspensory use of the prehensile tail, suggest MHC isoforms and fiber size may also be different. The results of this study allow us to begin testing this hypothesis.

Antibody Specificity in Opossum Tail Muscle

Immunoreactivity against S58 consistently identified the slow MHC-1 fiber type in the tail of D. virginiana. The MHC-1 isoform is highly conserved across vertebrate taxa (Miller et al., 1985) and the monospecific reactivity of S58 with MHC-1 is high (Crow and Stockdale, 1986), thus it can be used to reliably identify MHC-1 isoform fibers. We showed that S58 unambiguously identifies slow MHC-1 fibers (Figs. 3, 4, 5, 3–5) and verified its monospecific reactivity against marsupial MHC-1 in a caudal muscle of D. virginiana. In contrast to MHC-1, fast MHC isoforms show stronger species-dependent immunoreactivity or antigenic specificity. MHC-2A and 2X were the fast isoforms found in the tail, primarily identified by SDS-PAGE, and secondarily by strong reaction against SC71. Reactivity with SC71 has also been used to identify fast isoform fiber types in hindlimb muscles of the terrestrial M. domestica, and similar to our findings, fibers identified by SC71 as either MHC-2A or 2X also showed strong NADH-TR activity (Sciote and Rowlerson, 1998). Reactivity with SC71 is also consistent with Western blot analyses on other marsupial limb muscle tissue (Zhong et al., 2008), indicating SC71 reacts with both the 2A and 2X isoforms in a number of species. Considering all the available evidence, SC71 is indicated to have immunoreactivity against both the 2A and 2X isoforms expressed in caudal muscle of D. virginiana, but limited specificity for identification of pure MHC-2A fibers, unlike when SC71 is applied in small Eutherians (e.g., rats) (Schiaffino et al., 1989).

Determination of a distribution of pure MHC-2A and 2X fibers was not possible despite the combination of fiber typing techniques used to verify our panel of mAbs as monospecific against MHC in D. virginiana. A high percentage of MHC-2A/X hybrid fibers is additionally indicated by moderate reactivity of BF-35 with all fibers (Fig. 5). Similar patterns of moderate intensity reaction with BF-35 with all fibers in a tissue cross-section were also observed in the limb muscles of M. domestica (Sciote and Rowlerson, 1998). SDS-PAGE did show some regional variation in 2A and 2X band resolution and intensity in the transitional and distal tail regions (Fig. 2), although densoimetric analysis was not performed on the gels to determine the relative amounts of protein for each isoform (Toniolo et al., 2005, 2007). Furthermore, Western blots were not performed to validate monospecific reactivity of mAbs against opossum MHC, but are needed for future studies of caudal muscle MHC isoform fiber types. Irrespective of these additional analyses, we are confident the caudal muscle of D. virginiana is primarily composed of relatively large (CSA and diameter) and oxidative, fast hybrid fibers, and expect the numbers of any pure fast fiber types in the tail to be low.

The distribution of MHC hybrids and their molecular complexity (i.e., pattern of co-expression) is higher in muscles undergoing molecular and functional transformation (see review by Pette et al., 1999). We believe this phenomenon may explain the incomplete staining patterns for mATPase histochemistry that were previously observed in the distal tail region of D. virginiana (Hansen et al., 1987) as well as incomplete mATPase staining and gradients of anti-fast mAbs reactions within individual fibers noticed in this study. Arguably, MHC-2A/X hybrids in a terrestrial opossum that uses its prehensile tail for sustained grasping behaviors represent a transition from fast, MHC-2X fibers to highly oxidative, MHC-2A fibers. Evidence of fast, glycolytic MHC-2X/B hybrids in kangaroo tails (Spiegel et al., 2010) supports this argument. However, in this case, extensive limb modifications for saltatory locomotion in kangaroos have diminished the functional role of the tail causing a shift to fast MHC isoforms (i.e., ancestral condition). Similar transitions to fast MHC isoform expression have been shown in studies involving limb immobilization and or disuse (Grossman et al., 1998; Oishi et al., 1998; D'Antona et al., 2003). Shifts in MHC isoforms have also been shown to correlate with age and muscle maturation in Eutherian mammals (Rivero et al., 1993; Smerdu et al., 2009). While the exact ages of opossums used in this study were not known, and correlation of caudal muscle fiber type with age and ontogeny was beyond the scope of this investigation, future study of this relationship is warranted. This analysis may be particularly insightful given that joey Virginia opossums have the ability to suspend their body mass, but lose that ability as adults (McManus, 1970). The low degree of tail prehensility in D. virginiana adults is likely to be strongly associated with both developmental changes in MHC expression and low dependence on the tail for arboreal locomotor behaviors. Our future work is aimed at comprehensive and comparative analyses of MHC isoform fiber type in caudal muscles from arboreal opossums with high degrees of tail prehensility.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

A special thanks to Dr. Gary Segal at Northside Hospital in Youngstown, OH for access to the cryostat and the pathology staff for their assistance. Thanks to Ron Ollis at the Ohio Department of Natural Resources for coordinating permissions for animal collections, and D. Bakelar for assistance with local animal collection. Authors also thank M. Womble, J. Krontiris-Litowitz, J. Dearolf, and J. Organ for guidance, critical comments and discussions of this work. They are also grateful for the dedication of Alyson Cadman for help with data analysis. The monoclonal antibodies developed by F. Stockdale (S58), S. Schiaffino (SC71, BF-35, BF-F3), and C. Lucas (6H1, 10F5) were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. Portions of this work were submitted as a Masters Thesis by P.J. Hazimihalis.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
AR_22614_sm_SuppTab1.doc28KS1. Mean fiber CSA (mm2) of MHC isoform fiber types in the tail of D. virginiana.
AR_22614_sm_SuppTab2.doc28KS2. Mean fiber diameter (mm) of MHC isoform fiber types in the tail of D. virginiana.

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