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Departments of 1Neurology, 2Ophthalmology, and 4Neuroscience, Case Western Reserve University, Cleveland, Ohio 44106; and 3Department of Cell Biology, University of Potsdam, D-144171 Potsdam, Germany
Submitted 10 April 2003 ; accepted in final form 23 April 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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myomesin; M protein; adenylate kinase; oculomotor muscles
The internal organization of skeletal muscle also reflects coupling of different structural and functional compartments: the sarcolemma, sarcoplasmic reticulum, metabolic machinery, and contractile elements are linked in such a way as to optimize the mechanical response triggered by neural stimulation. This precise spatial and functional coupling of specific cellular components provides clues regarding specific functional requirements of skeletal muscles. For example, M lines are conspicuous macromolecular aggregates in the middle of the sarcomeric A band and are associated with the assembly and maintenance of the highly ordered myofibrillar thick-filament lattice and function in providing ATP to the actomyosin ATPase (31).
The M lines contain myomesin, M protein, titin, and the metabolic enzyme creatine kinase (CK) (12, 31, 36). Myomesin (185 kDa) and M protein (165 kDa) are closely related cytoskeletal proteins of the immunoglobulin superfamily, with unique head domains followed by multiple immunoglobulin-like and fibronectin type III domains arranged in the same pattern. Myomesin has binding sites for both myosin and titin, whereas only a myosin-binding site has been described in M protein (4, 5, 23, 24, 39). Whereas myomesin is found in cardiac and skeletal muscle fibers from early stages of myofibrillogenesis on, in adults, M protein is restricted to cardiac and fast skeletal muscle fibers (6, 12, 15). Embryonic heart myomesin (EH myomesin), an alternatively spliced isoform of myomesin bearing an additional exon in the central part of the molecule, was recently reported as expressed exclusively in embryonic hearts (2, 33). Alternative splicing of the C terminus of myomesin results in an isoform only found so far in avian hearts (2).
Another M line component, CK, belongs to a family of oligomeric isoenzymes with tissue-specific expression and isoform-specific cellular localization. Two cytosolic isoforms, ubiquitous "brain-type" (CK-B) and "muscle-type" (CK-M), and two mitochondrial isoforms, ubiquitous mitochondrial CK (uCK) and "sarcomeric" mitochondrial CK (sCK), are expressed in a tissue-specific manner. Both mitochondrial CKs are associated with the inner mitochondrial membrane (29, 30). In vivo, the cytosolic M and B subunits (43 kDa) form homo- and heterodimers, CK-MM, -MB, and -BB isoenzymes, whereas the mitochondrial subunits aggregate preferentially as homooctamers (30). CK catalyzes the reversible transfer of the phosphoryl group from phosphocreatine to ADP and is key for maintaining normal ATP levels in striated muscle and other tissues with intermittently high-energy requirements (16). In differentiated skeletal muscle, CK-MM and sCK are the predominant isoforms, although low levels of CK-MB heterodimers can be detected. A significant fraction of CK-MM is specifically bound to the M line (35). This arrangement couples the CK-dependent, ATP-generating system to the myofibrillar actin-activated Mg2+-ATPase and is important for normal contractile function (38, 41).
Paradoxically, the extraocular muscles, small muscles responsible for voluntary and reflexive movements of the eyes, do not present M lines, even though these muscles are extremely fast and constantly active (20). In consequence, our first hypothesis was that the absence of M lines in mouse extraocular muscle would reflect decreased expression of its constituents, myomesin, M protein, and CK-M, compared with the typical leg muscles, gastrocnemius and soleus. Because the extraocular muscles are characterized by abundant mitochondria and the presence of developmental and cardiac markers, our second hypothesis was that this muscle group would express CK-B and/or the mitochondrial CK isoforms at higher levels than in gastrocnemius and soleus. Our data confirmed that mouse extraocular muscles do not contain detectable levels of myomesin and M protein and that the expression of CK-M is lower than in gastrocnemius and soleus. Surprisingly, other cytosolic and mitochondrial CK isoforms are also expressed at lower levels in the extraocular muscles. Moreover, total CK activity in this muscle group is significantly less than in gastrocnemius and soleus. Instead, two adenylate kinase (AK) isoforms are expressed at higher levels in the extraocular muscles compared with the limb muscles and may provide the ATP buffering effect normally attributed to CK. In addition, increased AK expression may indicate the preferential use of AMP as a metabolic signal to stimulate glycolysis and augment blood flow. These results suggest that the functional requirements of the extraocular muscles impose mechanical and metabolic loads that are different from those typically seen by other skeletal muscles. Therefore, the extraocular muscles may employ AK as an alternative strategy to cope with these demands.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Electron microscopy. Perfusion-fixed muscle samples were postfixed in 1% osmium tetroxide, stained en bloc in uranyl acetate, dehydrated in a methanol series and propylene oxide, and embedded in epoxy resin. Thin (80-nm) sections were stained with uranyl acetate and lead citrate and examined and photographed with a JEOL 1200EX transmission electron microscope (JEOL USA, Peabody, MA).
RNA isolation. Tissues were homogenized in TRIzol (GIBCO-BRL, Rockville, MD), and total RNA was isolated, following the manufacturer's instructions. Reverse transcription was carried out by using Superscript II RNAse H- Reverse Transcriptase (Invitrogen, Carlsbad, CA), with oligo(dT)12–18 primers (for myomesin, M protein, and CK) or random hexamers (for AK).
Myomesin, M protein, and CK expression. PCR amplification of cDNA (33–40 cycles) used the PCR SuperMix kit (GIBCO-BRL) with specific primers. Aliquots of PCR products were run on a 1.5% agarose gel containing ethidium bromide (Sigma Chemical, St. Louis, MO) for visualization. The two RT-PCR products (687 and 395 bp) obtained from extraocular muscle with the myomesin primers (see below) were cut from the gels, purified by using the QIAEX II Gel Extraction kit (QIAGEN, Valencia, CA), and identified by sequencing. Alignment with the published myomesin sequence (GenBank accession no. AJ012072 [GenBank] ) was performed by using BLAST (NCBI, Bethesda, MD).
RT-PCR primers. Primers and predicted product sizes were as follows: myomesin (687 and 395 bp), forward 5'-GGC AAA ATC ATC CCA AGT AG-3', reverse 5'-ATA ATA GCC TGT AAT CTC TGC-3'; EH myomesin (250 bp), forward 5'-CAG ATG TGT GGC CTC AAC TGA-3', reverse 5'-TCG GAT TGA CTT TGC TCC T-3'; M protein (376 bp), forward 5'-TGT GGC GGG AAC AAA CAT-3', reverse 5'-ACC TTC CAC CGT TAA GAT CCT C-3'; CK-M (371 bp), forward 5'-CAA CAC CCA CAA CAA GTT CAA-3'', reverse 5'-AGG TGC TCG TTC CAC ATG AA-3'; CK-B (383 bp), forward 5-TCC AAC AGC CAT AAT ACG GCA-3', reverse 5'-TGT CAT TGT GCC ATA TGC CA-3'; sCK primer set 1 (430 bp), forward 5'-CAA GAA GAA GGA TGG CCA GT-3', reverse 5'-TTC ATC ACC CCT GGG TCA TA-3'; sCK primer set 2 (226 bp), forward 5'-AGG CAG AAG GTA TCT GCT GAT-3', reverse 5'-CCA TGC CCA CAG TCT TAA TGA-3'; uCK (942 bp), forward 5'-TTC TCC CGT CTG CTG TCT G-3', reverse 5'-TGG ACA GGT CAA GAT GTA GCC-3'.
Quantitative PCR. cDNA samples were also used for quantitative PCR (qPCR) by using the LightCycler (Roche, Mannheim, Germany) for M-protein, myomesin, and CK isoforms, and the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) for AK isoforms. Primer pairs specific to M-protein, myomesin, and CK isoforms were identical to those used for RT-PCR, except that uCK was not run because it was not detected by RT-PCR. For AK isoforms, we used the following primers: AK1 (product size, 70 bp), forward 5'-GTG AAA CAG GGA GAA GAA TTT GAA C-3', reverse 5'-GCG CCT GCG TCC ACA TAC-3'; AK2 (71 bp), forward 5'-TGG AGG CCT ACC ACA TCA GA-3', reverse 5'-CAA TGG CGC AGT GAA TGA-3'; AK3 (75 bp), forward 5'-ACC AGT TGT AGC TGG CTG TTG-3', reverse 5'-TCT GAT AAA CTC TCC AGG GCT TCT-3'; AK4 (87 bp), forward 5'-TCT CAG GCT ATT CAG TCT CAT GAT G-3', reverse 5'-TTG AAC CCA GTC ACC TAC TTC TCA-3'; AK5 (72 bp), forward 5'-ACA CCA AGG GCT TTC CTG ATT G-3', reverse 5'-CCG ATC CTT CGT CCA AAC TTT-3'. The corresponding manufacturer's recommended protocol was used for the LightCycler-DNA Master SYBR Green I kit or the ABI SYBR Green Master Mix kit. The relative abundance of target mRNAs in the different tissues was determined with the comparative cycle threshold method (13, 18).
Immunolocalization of myomesin isoforms. Fixed 5-µm-thick longitudinal cryosections from extraocular and soleus muscles were blocked with 0.1% bovine serum albumin in phosphate-buffered saline and incubated overnight in primary antibody at 4°C. The primary antibody was omitted in control slides. We used previously described monoclonal antibodies specific for M protein and myomesin (39). After washing with phosphate-buffered saline, immunoreactivity was visualized by incubation for 1 h in the appropriate Texas Red-conjugated secondary antibody (1:50; Molecular Probes, Eugene, OR). Sections were rinsed in phosphate-buffered saline, mounted in Immu-Mount (Shandon, Pittsburgh, PA), and viewed with an Olympus BX-50 microscope equipped for epifluorescence (Olympus, Melville, NY). Images were captured with a Dage DC-330 charge-coupled device camera (Dage-MTI, Michigan City, IN) and PowerPC G3 computer (Apple Computer, Cupertino, CA) equipped with a Scion CG-7 frame-grabber board (Scion, Frederick, MD) and Scion Image version 1.62 software (adapted from NIH Image, National Institutes of Health, Bethesda, MD).
Total CK activity in muscle. Gastrocnemius, soleus, and extraocular muscle samples were homogenized (1:1,000 wt/vol) in a Tris-buffered solution (26 mM Tris, 30 mM dithiothreitol, 0.3 M sucrose, and 1% Triton X-100, pH 8.0) and extracted in ice for 1 h. CK activity in the homogenates was determined in triplicate by a hexokinase-glucose-6-phosphate dehydrogenase coupled system, which yields NADH at a rate proportional to CK activity (Sigma Chemical) and was expressed as units per milligram of protein. Protein content was determined by the method of Lowry et al. (19).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Downregulation of myomesin and M-protein mRNA in extraocular muscle. Primers for myomesin amplified two products, a 395-bp fragment and a 687-bp fragment containing an additional previously reported exon (33). RT-PCR identified the presence of the 395-bp myomesin product in leg muscle (gastrocnemius and soleus), heart, embryonic heart, and extraocular muscle (Fig. 2A). By qPCR, myomesin expression levels were in the following order: leg muscle > heart > embryonic heart > extraocular muscle (Table 1). RT-PCR confirmed the presence of EH myomesin in E14 heart, but also identified transcripts in adult extraocular muscle. Quantification of EH myomesin by qPCR showed that expression levels were twofold greater in embryonic heart than in extraocular muscle; only trace levels of product were observed in adult leg muscle and heart. Alignment of the two sequencing products from myomesin and EH myomesin RT-PCR bands obtained from extraocular muscle against the published myomesin sequence (accession no. AJ012072 [GenBank] ) showed 100% correspondence, confirming the expression of these two alternatively spliced myomesin isoforms in the eye muscle group.
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M-protein RT-PCR products were detected in leg muscle, heart, and E14 heart, but not in extraocular muscle (Fig. 2A). qPCR showed only trace levels of M protein in extraocular muscle; relative expression levels were in the following sequence: leg muscle > heart >> embryonic heart >> extraocular muscle (Table 1).
Myomesin and M protein are absent in extraocular muscle. With the use of immunocytochemistry, myomesin and M protein were not detected in extraocular muscle, but were clearly found in soleus muscle fibers in a stereotypical banding pattern (Fig. 3).
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Downregulation of CK isoforms in extraocular muscle. CK-M associates with the sarcomeric M line. Because the expression of the M-line proteins myomesin and M protein was low in adult extraocular and embryonic heart muscle, we identified and quantified all known CK isoforms in these muscles relative to adult heart and leg muscle (gastrocnemius and soleus). By RT-PCR, CK-M was present in leg muscle, heart, and extraocular muscle and absent in brain (Fig. 2B). Consistent with the low expression of myomesin and M protein in extraocular muscle, CK-M expression in this muscle group was nearly an order of magnitude lower than in leg muscle. The overall pattern of CK-M expression by qPCR was as follows: leg muscle >> heart > embryonic heart > extraocular muscle (Table 1). By contrast, whereas RT-PCR detected CK-B in all striated muscles studied (Fig. 2B), qPCR showed low levels in all muscles compared with brain (Table 1). Because extraocular muscle has particularly high mitochondrial content, sCK could potentially compensate for the decreased CK-M expression. However, whereas sCK was detected in extraocular muscle by RT-PCR (Fig. 2B), qPCR showed that only trace levels of this isoform were present in this muscle group (Table 1). Relative transcript abundance for sCK was in the following order: heart > embryonic heart > leg muscle >> extraocular muscle (Table 1). The other mitochondrial isoform, uCK, was detected by RT-PCR in brain, but not in striated muscle (Fig. 2B).
Lower CK activity in extraocular muscle. To confirm the finding of lower expression of CK isoforms, we determined total CK enzymatic activity in extraocular and gastrocnemius and soleus muscles. The CK activity in extraocular muscles was only 18% of that in the leg muscles (6.4 ± 2.9 vs. 34.5 ± 6.3 U/mg protein, respectively, n = 5, P < 0.001).
Upregulation of AK isoforms in extraocular muscle. The
"myokinase" reaction (2 ADP 
ATP + AMP), another ATP
buffering system in skeletal muscle, is catalyzed by AK, a family of cytosolic
and mitochondrial enzymes. There are five known AK isoforms in rodents, of
which two (AK1 and AK2) are expressed at relatively high levels in skeletal
muscle (22,
34). A previous study
indicated that another isoform, AK4, is preferentially expressed in the
extraocular muscles (26).
Using qPCR, we found that AK1, AK2, and AK3 are expressed at roughly
equivalent levels in the extraocular and gastrocnemius and soleus muscles;
differences were less than the twofold threshold for significance
(Fig. 4). However, mRNAs for
AK4 and AK5 were 5.9- and 12.9-fold more abundant in the extraocular than the
leg muscles.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Extraocular muscles lack myomesin and M protein. Present understanding of sarcomere organization proposes that the M lines are needed to support the thick-filament lattice and as binding sites for CK-M; in consequence, these structures tend to be more prominent in fast-twitch muscles (31). In addition, M-line components appear as part of the myofibrillogenesis program, without correlation to neuronal activity or expression of myosin heavy chains, suggesting that they are important in determining the structure of the thick-filament array (6, 31). This study shows the paradoxical absence of these scaffolding elements in a very fast and active muscle group, the extraocular muscles (Fig. 3). Whether this muscle group requires these structures at least transiently during development remains an open question. Interestingly, the extraocular muscles did express EH myomesin at levels comparable to those of embryonic heart, although EH myomesin apparently does not participate in the formation of M lines (2). It should be noted that the developmentally regulated expression of EH myomesin, which differs from myomesin by an additional exon, was previously shown to be restricted to embryonic heart (2). We do not know the cellular localization of EH myomesin protein in adult mouse extraocular muscles, if any. It is possible that this product is present only in those extraocular muscle fibers known to express other developmental and cardiac markers. However, the myomesin antibody used here targets an epitope located close to the carboxy end of the protein, away from the proposed location of the additional exon in EH myomesin (2, 39), and it failed to react with extraocular muscle fibers. This suggests that EH myomesin, if present, does not form macromolecular aggregates in extraocular muscle fibers. The absence of M-protein in the very fast extraocular muscle is also remarkable, because, in skeletal muscle, M protein is found exclusively in fast-twitch fibers (6, 15).
The absence of M lines in extraocular muscles is particularly striking in rodents, in which most muscle fibers do not present these sarcomeric structures, but is variable in other species (20). This morphological characteristic had not been previously correlated to the functional properties of this peculiar muscle group. Because the present model of sarcomeric organization includes the M lines as a stabilizer component, it is surprising that the mouse extraocular muscles do not include this feature. These muscles are arranged in antagonistic pairs and exhibit relatively high activity rates in their "off" direction (8). In other words, cocontraction of antagonistic extraocular muscle pairs leading to eccentric or lengthening contractions is a common occurrence. Because the extraocular muscles are obviously adapted to this situation, it is likely that the distribution of mechanical stresses in the extraocular muscles is different from that in other skeletal muscles. Coincidentally, loss of another important cytoskeletal element, dystrophin, does not result in overt extraocular muscle abnormalities (27).
CK downregulation in extraocular muscle. M lines are also
anchoring sites for a fraction of cytosolic CK-M activity, creating a ready
link between actomyosin ATPase activity and the phosphocreatine energy buffer
system (41). Because the M
lines do not exist in the mouse extraocular muscles, we expected that CK-M
mRNA would also be less abundant. This was confirmed by qPCR, although the
magnitude of the difference in CK-M expression between extraocular and limb
muscles was surprising, considering that 
5% of CK-M is normally found
bound to the M lines (41).
Some extraocular muscle fibers express developmental and cardiac markers
(25). In consequence, we
expected a relative increase in the expression of CK-B, because the CK-MB
heterodimer is particularly abundant in developing skeletal muscles and in
adult myocardium. Although CK-B expression in all muscles was dramatically
less than in brain, CK-B mRNA was detectable in the mouse extraocular muscles
at levels comparable to those in heart, suggesting that cytosolic CK in these
muscles is present in the CK-MB form, a finding that accounts for the measured
CK activity.
A typical compensatory response in genetically engineered CK-M knockout mice is increased mitochondrial content in fast-twitch skeletal muscle fibers, presumably to reduce metabolite diffusion distance between mitochondria and myofibrils (37). Extraocular muscles are characteristically mitochondria rich; however, of the mitochondria-associated CK isoforms, sCK mRNA was lower than in leg muscle, and uCK message was undetectable in the extraocular muscle group. These data, in conjunction with the lower CK activity measured in the extraocular muscle, suggest that this muscle group does not rely on the CK-dependent ATP buffering capacity. The abundant mitochondria in extraocular muscle fibers decrease the ATP diffusion distance, which is a major explanation for the need for phosphocreatine in biological systems. Then, the efficient movement of ATP from the site of production to the site of use in the extraocular muscle may not require the creatine-phosphocreatine system. The use of phosphocreatine in skeletal muscle energetics allows for high-power output and effective buffering of ATP concentration. However, maintenance of constant ATP concentration during contractile activity is neither universal nor absolutely required. On the other hand, the use of phosphocreatine as fuel increases the power output attainable by skeletal muscles; this function is the most perturbed in CK knockout mice (32, 37). Whereas CK activity is important for delaying or avoiding fatigue after the onset of high-intensity stimulation, recent data indicate that it may actually contribute to fatigue development during prolonged stimulation by increasing intracellular concentration of inorganic phosphate (9). Coincidentally, the functional properties of skeletal muscles with impaired CK activity resemble the typical contractile properties of extraocular muscles: low force and power, and resistance to fatigue (3, 7, 11, 14). Curiously, loss of CK activity also slows skeletal muscle relaxation, an effect probably due to altered cross-bridge kinetics; this could explain the presence of the extraocular muscle-specific myosin, with its fast kinetics, in some extraocular muscle fibers (28).
Upregulation of AK4 and AK5 in extraocular muscle. The preferential localization of CK activity in specific cellular compartments is theoretically not necessary for spatial ATP buffering, because the overall flux through CK is mostly due to its cytosolic activity. Then myofibrillar and mitochondrial targeting merely localizes CK activity to where it is needed, thus economizing enzyme distribution. This would be particularly important in skeletal muscle fibers, in which sparsely and nonuniformly distributed mitochondria impose large diffusion distances (21), but would be less relevant in the mitochondria-rich extraocular muscles. In addition, the increased expression of two AK isoforms in the extraocular muscles may reflect the use of the myokinase reaction as an alternative ATP buffering system. Prior studies showed that mRNAs for AK1 and AK2 are present at high levels in skeletal muscle, whereas AK3 message is preferentially detected in heart (22, 34). AK4 and AK5 mRNAs were originally found in neurons, not in skeletal muscle. In this study, the expression of AK1, AK2, and AK3 was found to be equivalent in the extraocular and limb skeletal muscle groups. However, AK4 and AK5 mRNAs were present at higher levels in the extraocular muscles; this confirms an earlier gene expression profile study that found AK4 at higher levels in these muscles (26). AK activity is especially abundant in cells with high rates of ATP synthesis and utilization. Whether the subcellular localization of AK4 (potentially mitochondrial) and AK5 (cytosolic) serves to replace CK activity in the extraocular muscle remains to be determined. Interestingly, in CK-deficient mice, AK activity is increased, and inhibition of CK activity in intact skeletal muscle results in an increased phosphoryl transfer by AK (10, 17).
Clearly, the downregulation of mitochondrial CK in extraocular muscles would diminish the capacity for fine control and amplification of the energy state signal from the cytoplasm, as proposed for mitochondrial CK-containing systems (40). The increased expression of AK isoforms in these muscles may be an alternative strategy for metabolic regulation, providing AMP as the metabolic signal. In this scenario, AK becomes the main ATP buffer system and produces AMP during periods of increased activity. AMP is a strong signal to activate glycolysis via a positive allosteric effect on 6-phosphofructo-1-kinase and allosteric inhibition of fructose 1,6-bisphosphatase. Gene expression profiling and previous morphological findings indicated that glycogen breakdown and gluconeogenesis are less important in mouse extraocular muscles; apparently these muscles rely on glucose transport and fast glycolysis to maintain normal ATP levels (26). In addition, AMP may be further metabolized to adenosine, a potent vasodilator of skeletal muscle microcirculation, which serves to couple blood flow to metabolic rate. Interestingly, a recent study demonstrated that differential response of vascular beds to adenosine is not predicted by fiber type or oxidative capacity; rather, it may be determined by other functional and/or metabolic parameters (1). Then it is possible that blood flow to the extraocular muscles may be predominantly under local control.
Conclusion. The lack of structural and metabolic elements hitherto considered necessary for fast skeletal muscles suggests that the functional requirements of the extraocular muscles, an extremely fast contracting muscle group, impose mechanical and metabolic loads that are very different from those seen in other skeletal muscles.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Ct method.
Methods 25:
402-408, 2001.[Web of Science][Medline]
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