First published online July 23, 2003
Postnatal suppression of myomesin, muscle creatine kinase and the M-line in rat extraocular muscle
John D. Porter1,2,3,*,
Anita P. Merriam1,
Bendi Gong1,
Sriram Kasturi1,
Xiaohua Zhou1,
Kurt F. Hauser4,
Francisco H. Andrade2 and
Georgiana Cheng1
1 Department of Ophthalmology, Case Western Reserve University and
University Hospitals of Cleveland, Cleveland, OH 44106, USA
2 Department of Neurology, Case Western Reserve University and University
Hospitals of Cleveland, Cleveland, OH 44106, USA
3 Department of Neurosciences, Case Western Reserve University and
University Hospitals of Cleveland, Cleveland, OH 44106, USA
4 Department of Anatomy and Neurobiology, University of Kentucky Medical
Center, Lexington, KY 40536, USA
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Fig. 1. Schematic representation of myomesin sequencing strategy. Myomesin 1 cDNAs
were amplified and sequenced from E18 cardiac and P45 hindlimb muscle. The
approximate position of the four primers used for sequencing (pr1 to pr4) is
indicated. Myom1 is present in two splice variants, with and without
the EH (embryonic heart) domain. Primers pr1 and pr2 yielded a 393-bp fragment
from hindlimb muscle that lacked the EH domain. The alternatively spliced EH
domain is shown here relative to two of the conserved fibronectin III repeats
(My6 and My7 domains shown) that dominate the central portion of myomesin 1.
Primers pr1 and pr4 yielded a 542-bp fragment, and primers pr3 and pr2 yielded
a 389-bp fragment, from E18 heart muscle. Collectively, sequencing of these
fragments gave full-length coverage to the cDNA of the EH domain. The
assembled EH-myomesin 1 sequence was then used in a BLASTn search of
GenBank.
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Fig. 2. The deduced amino acid sequences of rat, mouse, human and chicken
EH-Myom1 exhibit significant divergence. ClustalW was used to align
the deduced amino acid sequence of the EH domain and surrounding residues for
rat with homologous sequences deduced from chicken (GenBank accession numbers
AF185572 and U58204), human (GenBank accession numbers AF185573 and NM_003803)
and mouse (GenBank accession number NM_010867) nucleotide sequences. The start
and end of the rat EH domain are indicated by arrowheads. Residues differing
from consensus by <6 distance units are shown in dotted boxes, while those
differing by <6 distance units (non-conservative substitutions) are shown
in white boxes.
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Fig. 3. Deduced secondary structure for myomesin 1 domains in rat and chicken. From
the deduced amino acid sequences, secondary structure features were determined
for the rat (A) and chicken (B) EH domains, the My2 immunoglobulin-like repeat
domain (C) and the My4 fibronectin III repeat domain (D). My2 and My4 were
deduced from a full-length rat Myom1 cDNA assembled from GenBank
AC103176.4 using full-length mouse sequence as a template. Algorithms used for
secondary structure predictions were from Lasergene software (DNASTAR, Inc.)
and included Garnier-Robson (G-R) and Chou-Fasman (C-F) for  -helix,
ß-sheet, turn and coil content, Eisenberg for - and
ß-amphipathic regions, Kyte-Doolittle for the hydrophilicity plot, and
Karpus-Shultz to identify flexible regions. The secondary structure pattern
for rat EH was highly conserved in mouse and human (data not shown), while the
chicken EH region had substantially higher -helical content and lower
turn and coil content, rendering it with a lower flexibility index. The
remainder of rat myomesin 1 is comprised of seven immunoglobulin-like repeats
(My2, My3, My9, My10, My11, My12 and My13), with My2 shown as representative
(C), and five fibronectin repeats (My4-My8), with My4 shown as representative
(D). Immunoglobulin and fibronectin repeats are principally comprised of
-helical and ß-sheet structure, respectively.
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Fig. 4. Myomesin isoforms are differentially regulated in muscle classes during
development. qPCR analysis of hindlimb, cardiac and extraocular muscle (EOM)
was performed with the Roche LightCycler and SYBR green reagent. Different
primer sets were used to evaluate expression levels of (A) myomesin 1, (B)
EH-myomesin 1 and (C) myomesin 2 in the three muscle groups between the ages
of E18 and P45. Data establish differential regulation patterns for each
transcript and muscle group during development.
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Fig. 5. M-lines are expressed early during in vivo development and
continuously in organotypic culture of extraocular muscle (EOM). The presence
or absence of a structural M-line was evaluated in EOMs of rats between the
ages of E17 and P45. A representative electron photomicrograph illustrating
the presence of an M-line in E17 eye muscle (A) is shown here. M-line
morphology was also assessed in EOM grown in organotypic co-culture with
oculomotor motoneurons after 20 days (B), 28 days (C) and 35 days (D) in
vitro. M-lines were present in newborn and prenatal EOM and in
organotypic co-cultures at all stages. M-lines are indicated by arrows or by
the letter M; arrowheads denote Z-lines.
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Fig. 6. Extraocular muscle (EOM) M-line expression is not preserved in dark-reared
rats. Dark rearing is known to alter eye movements and to delay development of
EOM-specific traits. We raised newborn rats either in complete darkness (DR)
or in a 12 h:12 h light:dark cycle (con). M-line morphology was evaluated by
electron microscopy. At P14 (A) and P56 (C), EOMs of normally reared rats lack
M-lines. M-lines were not preserved in rats dark reared for 14 days (B) or 56
days (D).
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Fig. 7. Immunocytochemical localization of myomesin 1 in adult skeletal muscle (A)
and absence from adult extraocular muscle (EOM) (B), consistent with low
Myom1 transcript levels in EOM.
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Fig. 8. Creatine kinase (CK) isoforms are differentially regulated in muscle
classes during development. qPCR analysis of hindlimb, cardiac and extraocular
muscle (EOM) was performed with the Roche LightCycler and SYBR green reagent.
qPCR analysis of E18 to P45 muscles used primers specific for CK-M (A), sCK
(B), CK-B (C) and ubiquitous mitochondrial CK (D) transcripts. These data
established tissue-specific regulation patterns for each isoform during
development.
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Fig. 9. Evaluation of total creatine kinase (CK) enzyme activity in hindlimb and
extraocular muscle (EOM). Total CK enzyme activity was evaluated in triplicate
for homogenates of hindlimb and EOM between P7 and P45. P7 activity levels
were not different among the two muscle groups and both showed postnatal
increases in activity. Activity levels, however, subsequently diverged, with
adult hindlimb muscle attaining values 3-fold higher than those of EOM. Values
are means + S.D.; * denotes P<0.01.
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© The Company of Biologists Ltd 2003
