|Molecular Vision 2006;
Received 7 September 2005 | Accepted 29 March 2006 | Published 30 March 2006
Postnatal development of myosin heavy chain isoforms in rat extraocular muscles
Sang Jin Lim,1 Hak Hyun Jung,2,3
Yoonae A. Cho1
Departments of 1Ophthalmology and 2Otolaryngology-Head and Neck Surgery and the 3Division of Biomedical Sciences, Korea University College of Medicine, Seoul, Republic of Korea
Correspondence to: Yoonae A. Cho, MD, Department of Ophthalmology, Korea University College of Medicine, 126-1, Anam-Dong 5Ka, Sungbuk-Ku, (136-705), Seoul, Republic of Korea; Phone: (82)-2-920-5220; FAX: (82)-2-924-6820; email: firstname.lastname@example.org
Purpose: To determine the composition of myosin heavy chain (MHC) isoforms in rat extraocular muscles (EOMs) during postnatal development.
Methods: The MHC composition of rat EOMs at postnatal day 0 (P0), postnatal day 14 (P14), and adults was evaluated at mRNA levels by competitive polymerase chain reaction and MHC composition of each six EOM in adult rats.
Results: EOMs at P0 revealed predominant expression of neonatal MHC (75.5%) with a lesser percentage of embryonic MHC (12.8%) and 2A MHC (11.5%). 2X MHC was expressed at low levels and other MHC isoforms were not detected. At P14, EOMs expressed mostly 2X MHC (42.4%) and 2A MHC (27.4%). Expression levels of neonatal MHC (14.1%) and embryonic MHC (4.9%) decreased. 2B MHC (8.2%), EOM MHC (1.9%), and β-cardiac MHC (1.1%) were detected at low levels. In the adult rats, EOMs contained over 80% of three fast MHC isoforms, such as 2X MHC (29.9%), 2A MHC (29.3%), and 2B MHC (24.5%). Each of six adult EOM showed slightly different expression levels of MHC composition.
Conclusions: A strong correlation exists between the composition of fast MHC isoforms and muscle development. MHC isoform followed a neonatal MHC-2X MHC-2B MHC isoform switching pattern after birth. Postnatal development of EOMs had a slightly different expression pattern for MHC isoforms and may have different regulatory roles related to their functional requirement.
Extraocular muscles (EOMs) have diverse functional characteristics ranging from fast saccadic movement to slow vergence movement. EOMs are known to be rapidly twitching muscles that contain predominant fast-contracting motor units and a small number of slow-contracting motor units [1,2].
Myosin, the most abundant protein in skeletal muscles, is directly responsible for muscle contraction. It is composed of two heavy chain subunits and four light chain subunits. Both myosin heavy chain (MHC) composition and alkali myosin light chain affect shortening velocity and muscle contractile properties, but MHC is a major functional domain of myosin protein [3,4].
MHC exists as multiple isoforms encoded by a family of genes. The rat skeletal MHC family contains at least eight MHC genes corresponding 2A MHC (MYH2A, MYH2), 2B MHC (MYH2B, MYH4), 2X MHC (MYH2X, MYH1), EOM MHC (MYHC-EO, MYH13), embryonic MHC (MYHC-EMB, MYH3), neonatal MHC (MYHC-PN, MYH8), α-cardiac MHC (MYHCA, MYH6), and β-cardiac MHC (MYHCB, MYH7) [5-14]. Identification of MHC isoforms is important in determining their functional characteristics since MHC isoforms differ in their functional properties [4,14]. MHC isoforms have been differentiated by ATPase activity, immunohistochemistry, and electrophoretical analysis. Some EOM fibers have multiple innervation and display acid-stable myosin ATPase activity . These muscles are fast muscles, predominantly composed of fast-contracting higher ATPase muscle fibers than other skeletal muscles . MHC composition in EOM was also reported at protein levels with the predominance of 2B MHC and EOM MHC isoforms .
MHC isoforms follow an embryonic->neonatal->adult transition pattern during mammalian skeletal muscle development [10,18]. EOMs also showed the same pattern of isoform transition during postnatal development, but it is supposed to be controlled by tissue-specific and/or environmental factors . Even though the mechanisms regulating isoform transitions are not well understood, coordinated oculomotor function at birth is not well developed and can be established during the perinatal period, and this could be related to their MHC isoform transitions [19,20].
The purpose of this study was to investigate the transition of MHC isoforms in the EOMs during postnatal development and the MHC composition in each of six adult EOM using a competitive PCR assay. This competitive polymerase chain reaction (PCR) can detect low expression levels of MHC isoforms and can be useful to quantify all known MHC mRNA transcripts during postnatal development, but conventional molecular techniques may have limitations to quantify all isoforms of MHC due to their similar homology among various MHC mRNA transcripts in rat skeletal muscles [5,7,14,21].
Design of polymerase chain reaction primers and construction of the competitor
The protocols for the construction of MHC competitor and PCR primers have been described in detail elsewhere in the literature [14,21]. Briefly, the consensus sense primer (CSP), a degenerate oligonucleotide from a highly conserved region from 620-660 bases from the 3'-end of all known rat MHC genes [5,7,14,21], was used to amplify all known MHC isoforms, but seven antisense primers specific for 3'-ends of known MHC isoforms of the rat skeletal muscle (2A MHC, 2B MHC, 2X MHC, EOM MHC, embryonic MHC, neonatal MHC, and β-cardiac MHC) were used (Table 1). One set of PCR primers specific for rat β-actin was also used for the internal control of transcription and amplification efficiency (Table 1).
Two competitors (β-actin and MHC) were used (Figure 1). For construction of the MHC competitor, one sense primer (CSP) and seven antisense primers (2A MHC, 2B MHC, 2X MHC, EOM MHC, embryonic MHC, neonatal MHC, and β-cardiac MHCs), designed to be 40-80 bp long, were ligated to the backbone of the 400 bp DNA fragments of 2A MHC clone (Genbank accession number L13606) by multiplex PCR (Figure 1). Another β-actin competitor was constructed by ligating β-actin primers to nonhomologous 178 bp DNA fragments by PCR (Figure 1). The competitors were ligated into the pGEM-T vector (Promega, Madison, WI) and sequenced using an ABI 373A automated DNA sequencing system. All of the primer sequences were arranged in the competitor molecule and product lengths of the competitor were different from the native reaction products (613-683 bp) by 119 to 168 bp when the competitor was used with these primer sets (Table 1).
RNA extraction and competitive polymerase chain reaction
Sprague-Dawley rats at postpartum day 0 (P0), days 14 (P14), and over nine weeks (adult, 150-250 g) were used for the extraction of total RNA. Three complete developmental series were analyzed in each group. For postnatal ages, pregnant rats were checked in the early morning and late afternoon, and the day on which pups were produced was recorded as postnatal day 0. Rats were anesthetized with ketamine (50 mg/kg), and all portions of six extraocular muscles (medial rectus, lateral rectus, superior rectus, inferior rectus, superior oblique, and inferior oblique) were removed using a surgical microscope. Tissues were immediately frozen in isopentane cooled by liquid nitrogen (-70 °C) and stored at -70 °C until later use for cDNA synthesis. Total RNA was isolated from frozen samples using Trizol Reagent (Life Technologies, Gaithersburg, MD). Total RNA from six muscles from one animal was extracted and 0.5 μg of total RNA per reaction was reverse-transcribed into cDNA using oligo-(dT)12-18 primers and the Superscript Preamplification System (Life Technologies). One μl from the resulting 80 μl of cDNA solution was amplified for competitive PCR. For initial determination of the existence of MHC isoforms, cDNA of each muscle group was amplified by traditional PCR, using the sense CSP and one of each of the eight specific antisense primers (2A MHC, 2B MHC, 2X MHC, EOM MHC, embryonic MHC, neonatal MHC, α-cardiac MHC, and β-cardiac MHC; Table 1). Rat heart muscle cDNA was used for PCR control template for α-cardiac MHC. Constant expression of β-actin was also evaluated in each sample by RT-PCR.
When a reaction product of the predicted length was obtained, competitive PCR was performed for that isoform using 3 fold serial dilutions of the competitor consisting of 1 fmole, 0.333 fmole, 0.111 fmole, 0.037 fmole, 0.0123 fmole, 0.00412 fmole, 0.00137 fmole, 0.000457 fmole, 0.000152 fmole, and 0.000051 fmole (0.051 amole). Competitive PCR was performed three times for each isoform present in each individual muscle sample. The PCR cycle protocol consisted of the following: 94 °C for 30 s; 55 °C for 30 s; and 72 °C for 1 min 30 s. This protocol was repeated for 35 cycles. Plasmid DNA containing β-actin and each MHCs, and cDNA without the competitor were also amplified for positive controls. PCR reactions containing taq polymerase and the primer combination, but no cDNA or competitor, were used as negative controls. Competitive PCR was repeated three times using three rats.
Quantification of reaction products of polymerase chain reaction
All PCR products were separated on a 1.5% agarose gel and visualized by ethidium bromide staining. Polaroid photographs were optically scanned at 300 dots/inch and analyzed using the National Institutes of Health Image Software (version 1.60) with the available gel macros. For quantification, two PCR products of each lane corresponding to target and competitor were outlined serially and generated to peaks. Each peak was defined by manual editing, through identification of the peak baseline and the nadir between the two peaks. Each peak defined was converted to optical density. The logarithm of optical density of target/competitor (t/c) was plotted as a function of the logarithm of known competitor concentration, and the resulting linear relationship was analyzed by linear regression analysis. The regression equation was solved for the condition target=competitor to yield the target concentration.
The method to estimate quantification errors due to each step of the analysis was described previously in the literature . Briefly, we performed three competitive PCR reactions on each sample. We scanned gels once and selected the peaks three times, since the majority of the error within the analysis of a muscle consisted of peak selection. All data are reported in the text as means±SD.
In initial PCR screening of three muscle groups (P0, P14, adult), P0 EOMs revealed the expression of 2A MHC, 2X MHC, embryonic MHC, and neonatal MHC, but other MHCs were not detected. P-14 EOMs and adult EOMs demonstrated the existence of all MHC isoforms (2A MHC, 2B MHC, 2X MHC, EOM MHC, embryonic MHC, neonatal MHC, and β-cardiac MHC; Figure 2). The PCR products of β-actin were identified in all samples and were expressed at nearly constant levels. α-Cardiac MHC was not detected in all samples (data not shown).
For competitive PCR, the initial amount of cDNA was estimated from any competitor-to-cDNA ratio, as described previously. MHC mRNA expression in the P0 EOMs was characterized by predominant neonatal MHC (75.5±9.9%) with a lesser percentage of embryonic MHC (12.8±2.0%) and 2A MHC (11.5±1.0%) and much lower proportions of 2X MHC (<1%). 2B MHC, EOM MHC, and β-cardiac MHC transcripts were not detected (Figure 2, Table 2).
P14 EOMs contained predominant type 2X MHC (42.4±2.8%) and 2A MHC (27.4±5.0%) with a lesser percentage of 2B MHC (8.2±1.3%). Expression levels of neonatal MHC transcript and embryonic MHC transcript were markedly decreased to 14.1±0.8% and 4.9±0.8%. Expression levels of EOM MHC (1.9±0.9%) and β-cardiac MHC (1.1±0.5%) were low (Figure 2, Table 2).
Adult EOMs were composed of approximately equal proportions of 2X MHC (29.9±4.5%), 2A MHC (29.3±4.4%), and 2B MHC (24.5±4.4%). Expression levels of neonatal MHC (11.3±1.7%) and embryonic MHC remained when compared with that of P14 EOMs. Expression levels of β-cardiac MHC (1.1±0.7%) and EOM MHC (1.3±0.3%) were low (Figure 2, Table 2).
In adult EOMs, the MHC composition in each of six EOMs was separately identified. The MHC composition in each EOM was slightly different (Figure 3, Table 3). 2X MHC ranged 18.5-37.1%, 2A MHC ranged 27.4-36.7%, and 2B MHC ranged 14.7-30.7% in each of the six EOM. Expression levels of neonatal MHC in each EOM (ranging 10.7-13.7%) were nearly constant. Embryonic MHC, β-cardiac MHC, and EOM MHC transcripts ranged 0.3-3.5%, <0.1-6.5%, and <0.1-4.2%.
β-Actin levels remained constant in each of the cDNA transcripts, but differed in different muscles and in different transcripts of the same muscles, possibly due to different transcription efficiency and quantification error on peak selection. To validate the competitive PCR assay, the logarithm of target to competitor ratio was calculated and compared with the logarithm of 3 fold serial dilution (1 fmol to 0.05 amol) of competitor DNAs. The linearity (r was 0.98-0.99 for all muscles) and consistent slopes of standard curves demonstrated that these competitive PCR assays could be used for quantitative analysis.
The present study clearly demonstrates that a strong correlation between the composition of fast MHC isoforms and muscle development exists. The EOMs of rats at P0 contained predominant perinatal MHCs, such as neonatal MHC (75.5%) and embryonic MHC (12.8%). In P14 rats, the EOMs expressed mostly 2X MHC (42.4%) and 2A MHC (27.4%) and low levels of 2B MHC (8.2%), whereas adult EOM contained over 80% of three fast MHC isoforms, such as 2X MHC (29.9%), 2A MHC (29.3%), and 2B MHC (24.5%). This suggests that a neonatal MHC-2X MHC-2B MHC switching pattern exists during postnatal development and this transition may be related to their functional requirement.
In the present study, the expression levels of both neonatal MHC (14.1% at P14 and 11.3% at adult) and embryonic MHC (4.9% at P14 and 2.7% at adult) were constant from P14 EOMs to adult EOMs. These expression levels in EOMs were much higher than in any other skeletal muscle. Among somatic skeletal muscles, neonatal MHC and embryonic MHC were either expressed at low levels or not expressed . These isoforms have been considered as developmental isoforms that were only expressed in the early postnatal period, and can be expressed again in adult muscles in case of muscle injury and regeneration [22,23]. Persistence of neonatal myosin from birth to adulthood could be explained by an incomplete development of the muscle fibers . However, branchial cleft muscles, such as intrinsic laryngeal muscles (0.1-3.3%) and masseter (1%), contained relatively higher neonatal MHC transcripts. Even though their roles are not clear yet, it is possible that the presence of both MHC isoforms in adult EOMs at relatively high levels is related to an additional functional role in muscle contraction, since EOMs are usually small in volume and provide precise control of ocular movement. Interestingly, as the development of a muscle fiber into its final subtype is influenced by many factors, including neurotrophic hormones, sex hormones, thyroid hormones, and nerve injury with reinnervation [25-27], understanding the regulatory mechanism of MHC isoform transition may be important to future researche.
Adult EOMs contained over 80% of fast isoforms, mainly 2X MHC (29.9%), 2A MHC (29.3%), and 2B MHC (24.5%). The contraction speeds of three fast isoforms are arranged in the order 2B->2X->2A on physiological study. Type 2B MHC is fast fatigable, whereas type 2A MHC is fatigue-resistant . Most fast-twitching muscles, such as intrinsic laryngeal muscles, contained over 80% fast isoforms. Thyroarytenoid muscle (TA) is the fastest laryngeal muscle with contraction times of 6.5-14 msec [28,29]. The ventral TA functions in laryngeal closure during laryngeal reflexes which need rapid muscle contraction and contains 52.1% of type 2B MHC transcript, whereas vocalis functions in phonation and contains 80.6% of type 2X MHC transcript [21,29]. This may suggest the proportion of fast isoforms is important in their functional requirement. Adult EOMs could be considered as fast-twitching and intermediate fatigue-resistant muscles. Alternatively, P14 EOMs contained mostly 2X MHC (42.4%) and 2A MHC (27.4%) with a lesser percentage of 2B MHC (8.2%). This suggests that P14 EOMs cannot perform fast movement well, and precise muscular function improves during postnatal development.
In the present study, adult EOMs of the rat contained over 80% of three fast MHC isoforms (2X MHC, 2A MHC, and 2B MHC) at the mRNA level. However, in the previous reports at the protein expression level of MHC isoforms, predominant MHC isoforms expressed in adult EOMs were 2B MHC (50-75%) and EOM MHC (14-25%) when analyzed by SDS-PAGE [17,30]. Discrepancy in the MHC composition of EOMs between mRNA and protein levels may exist, possibly due to protein stability, translational control, large spatial heterogeneity of MHC expression, different species , or the role of antisense RNAs . However, this competitive PCR can estimate actual numbers of mRNA molecules and even detect low expression levels of MHC isoforms that are not detected by SDS-PAGE. It also allows a comparison between the relative values of mRNA expression of the genes of interest within a tissue . An important advantage of competitive PCR is that it is not necessary to obtain data before the reaction reaches the plateau phase since the ratio of target to standard remains constant during the amplification. In EOM, it was also recently reported that longitudinal variation exists; the amount of 2B and EOM MHC is highest near the neuromuscular junction, and extremely low in the proximal and distal thirds of the muscle, where 2A, 2X, and developmental isoforms predominate [30,33,34]. The MHC ratios for EOM in the present study is different from the EOMs in a previous report , since we used all portions of six extraocular muscles for competitive PCR in the present study while the previous report  used some portion of six EOMs.
The existence of EOM MHC in the TA muscles and EOMs has been reported at protein or mRNA levels. EOM MHC was considered as a laryngeal/extraocular muscle specific gene related to functional characteristics of both muscles including rapid contraction time [8,14,35-37]. In the present study, P-14 EOMs and adult EOMs expressed low levels of EOM MHC (1.9% and 1.3%) and this suggests that EOM is not a major MHC isoform present in EOMs at mRNA levels. Expression of EOM MHC was observed in early stage of birth. This may be related to functional roles. Co-expression of neonatal MHC and EOM MHC transcripts in both intrinsic laryngeal muscles and EOMs may suggest that this MHC isoform could be related to developmental variant.
It was reported that α-cardiac MHC was expressed in all EOMs in rabbit , but we could not detect α-cardiac MHC in the present study, even though it was observed in rat hearts at high levels (data not shown). Expression levels of β-cardiac MHC was very low during postnatal development and this was also related to inverse relationship between the expression of β-cardiac MHC and 2B MHC transcripts in some skeletal muscles . The soleus muscle, one of the slowest muscles, has high expression levels of β-cardiac MHC and no detectable 2B MHC . Since the masseter and intrinsic laryngeal muscles, very rapidly contracting muscles, had highest proportion of 2B MHC and no detectable β-cardiac MHC , EOMs, as fast twitching muscles, may contain mostly three fast MHC isoforms (2A/2B/2X MHC) with very low levels of β-cardiac MHC in the present study.
The introduction of microarray technology allsws us to see gene expression levels for thousands of genes simultaneously and provides a powerful tool to identify large-scale mRNA differential expression and analyze changes in more than ten thousand of genes. Recently, it was reported that differential gene expression profiles of mice EOM in comparison to limb and/or masticatory muscles, using oligonucleotide microarray, identified up to 400 genes as having an EOM-specific expression pattern and it suggests that genes differentially expressed in EOM reflect key aspects of muscle biology . Gene expression profiles underlying the novel EOM phenotype were also examined using serial analysis of gene expression (SAGE)  and ologonucleotide microarray . These allotype-specific regulatory mechanisms in EOM may explain differential muscle group sensitivity to a variety of metabolic and neuromuscular diseases. Laser-capture microscopy coupled with microarray-based expression profiling was also used to identify transcriptional differences between the orbital layer and global layer of monkey EOMs  and rat EOMs . These suggest that differential gene expression profiles between the EOM layers may be correlated with the different loads and usage patterns, and evolutionary mechanisms [42,43]. This microarray technology allows one to identify how EOMs achieve such specialization or a differential response to diseases. Visual deprivation in rat (dark rearing) and monkey (monocular deprivation) in early developmental period may change EOM maturation identified microarray analysis . To identify a unique, group-specific pattern of gene expression in human EOMs using microarray, gene expression profiles of human EOMs were also studied and it may provide insights into how human EOMs achieve their unique structural, metabolic, and pathophysiological properties [45,46].
The authors wish to thank Seo Jin Kim for technical assistance and Dr. Jae Ku Cho for help. This work was supported by the Brain Korea 21 Project, BMS Korea, and Abott Korea.
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