Gene expression was normalized to expression of ACTN2 , which is present in all muscle fiber types. Results are expressed as fold change compared with expression of the gene in quadriceps muscles. The total SR fraction was isolated from flash-frozen muscle samples human orbicularis oculi, EOM, quadriceps muscles, and mouse heart as previously described Anderson et al.
Gels were stained with Coomassie brilliant blue. When more than two groups were compared, analysis was performed by the ANOVA test followed by the Bonferroni post hoc test, using Prism 4. OriginPro 8. Solution exchange was controlled by an electrically controlled rapid switch microperfuser VC8; Ala Scientific Instruments.
Excitation of fluo-3 was achieved at nm, and emission was collected between and nm. Line scans were acquired at a rate of 5. To acquire multiple line scans from each cell, the duration of the single line scan image was limited to 1, lines to avoid photobleaching and cell damage. Experiments were performed at constant room temperature. Images were analyzed using ImageJ National Institutes of Health and further processed together with current analysis in OriginPro software.
Four to five images were taken at different positions across each cell. Table S1 lists the sequence of primers used for qPCR. The expression levels of the major gene products involved in skeletal ECC are shown in Fig. The latter muscles were used as reference, and the expression level of different genes therein was set to 1.
JP45 levels remained unchanged. Total SR fractions were prepared and probed with antibodies against the major skeletal ECC proteins as well as Ca v 1. Surprisingly, at the protein level, the content of RyR1, Ca v 1.
Utrophin was highly expressed in both EOM and orbicularis oculi but less so in quadriceps fourfold in orbicularis oculi vs. In this context, it should also be mentioned that human myotubes are different from mouse myotubes or fibers in that they do not contract even after exposure to high concentrations of KCl, 4-chloro-m-cresol 4-cmc or ionomycin plus thapsigargin. S1 , a value similar to that previously reported in quadriceps-derived myotubes Rokach et al.
This is confirmed by results depicted in Fig. Although they lack a mature organization, RyR1 and Ca v 1. However, Ca v 1. A similar subcellular punctate distribution of Ca v 1. Because qPCR and Western blot analysis revealed that orbicularis oculi and EOM but not quadriceps muscle biopsies express high levels of utrophin, we investigated by confocal microscopy the expression and distribution of dystrophin and utrophin in the different kinds of myotubes.
Quadriceps-derived myotubes Fig. Orbicularis oculi—derived myotubes Fig. EOM-derived myotubes Fig. Interestingly, utrophin and dystrophin appeared to have a separate subcellular distribution that does not overlap within the myotubes Fig. In the present study, we investigated the biochemical and physiological characteristics of orbicularis oculi muscles, a group of facial muscles that are selectively spared or involved in different neuromuscular disorders.
As far as the expression of proteins involved in ECC is concerned, it appears that orbicularis oculi muscles are closer to quadriceps than to EOMs. Indeed, the content of skeletal muscle SR proteins was similar between quadriceps and orbicularis oculi muscles and differed from that of EOMs, as only the latter express proteins characteristic of both cardiac and skeletal muscle ECC Sekulic-Jablanovic et al.
Surprisingly, however, the expression of transcripts did not always match the actual protein content, highlighting the importance of validating arrays or qPCR experiments whenever possible. This was particularly relevant for the difference between the expression of Ca v 1. An intriguing result of this study is that although gene expression in orbicularis oculi is quite different from that in quadriceps, the expression of proteins involved in the ECC process RyR1, Ca v 1. In contrast, in our previous study, the differences in gene expression between EOM and quadriceps were positively correlated with differences in the expression of ECC proteins Sekulic-Jablanovic et al.
A limited overlap in genomic and proteomic data were reported by Khanna et al. Such a hypothesis is compatible with previous findings Fessenden et al. One of the physiological characteristics of EOMs is that they are fatigue resistant Fuchs and Binder, , and this may be functionally related to the high levels of expression of Ca v 1. As far as RYR3 expression is concerned, our results are enigmatic because, in general, RYR3 transcripts are expressed at low levels, if at all, in adult skeletal muscle Martin et al.
Our results concerning the expression of utrophin are interesting and most likely explain why in patients with Duchenne muscular dystrophy ocular and facial muscles are spared.
Utrophin and dystrophin share considerable sequence and structural similarity Tinsley et al. Interestingly, in cultured myotubes, the subcellular distribution of utrophin differs from that of dystrophin. Indeed, and as previously reported Trimarchi et al. The reason for this difference is unclear but may reflect distinct binding partners of the two proteins.
In mdx mice, it is believed that utrophin compensates for the lack of dystrophin Wakefield et al. Based on our results and on the fact that mdx knocked out also for utrophin show EOM involvement Baker et al. Collectively, these studies show that subspecialization of skeletal muscles occurs through multiple factors; although it is true that muscle innervation plays a prominent role, the distribution of a specific muscle within a niche devoted to a precise physiological function is also important.
Indeed, the term muscle allotype was proposed to describe the different capacities of myogenic cells of different lineages to express a different subset of myofibrillar genes. Because EOMs, orbicularis oculi, and levator palpebrae and the retractor bulbi which is present in some mammals but not in humans are derived from cells from a different lineage than those giving rise to limb muscles, their myogenic precursors must be programmed to express different subsets of proteins. The present study substantiates the validity of the muscle allotype hypothesis because we show that satellite cells derived from different muscles are primed and will follow the developmental characteristics of their muscle of origin, a property that can be exploited in laboratories devoted to tissue engineering.
Expression of major ECC transcripts and proteins in human orbicularis oculi muscle biopsies. Each reaction was performed in triplicate, in pooled muscle samples from five biopsies from different individuals. Expression levels were normalized to ACTN2 expression. Results are expressed as mean fold change of transcripts in orbicularis oculi compared with quadriceps; the latter was set as 1. Results are expressed as mean fold change of transcripts in orbicularis oculi OO compared with quadriceps QU ; the latter were set as 1.
Gels and conditions were as described in Materials and methods. C Western blot analysis of total SR proteins in human orbicularis oculi and quadriceps muscles. Blots were probed with the indicated antibodies. Calcium homeostasis of orbicularis oculi—derived myotubes compared with quadriceps- and EOM-derived myotubes.
Continuous line, quadriceps-derived myotubes; dashed line, orbicularis oculi—derived myotubes; dotted line, EOM-derived myotubes. Closed circles and continuous lines, quadriceps-derived myotubes; open circles and dashed lines, orbicularis oculi—derived myotubes; open squares and dotted lines, EOM-derived myotubes.
For quadriceps QU -derived myotubes, cells from six donors were measured; for orbicularis oculi OO —derived myotubes, cells from five donors were measured; and for EOM-derived myotubes, cells from four donors were measured. The black bar indicates the addition of 60 mM KCl. The lacrimal portion compresses the lacrimal sac, which receives tears from the lacrimal ducts and conveys them into the nasolacrimal duct.
Facial paralysis often affects the orbicularis oculi muscle. Inability to close the eye causes it to dry out, resulting in pain or even blindness in extreme cases. The superior oblique is a fusiform spindle-shaped muscle belonging to the extraocular group of muscles. It originates near the nose. Along with the…. The ophthalmic artery branches off from a major group of blood vessels in the head and neck known as the internal carotid arteries.
The ophthalmic…. The optic chiasm or optic chiasma is an X-shaped space, located in the forebrain, directly in front of the hypothalamus. Crucial to vision, the left…. The optic nerve is located in the back of the eye. It is also called the second cranial nerve or cranial nerve II. It is the second of several pairs…. The jejunum is one of three sections that make up the small intestine. The orbicularis oculi muscle is a muscle of facial expression , a ring-like muscle functioning in a number of eyelid movements.
The orbicularis oculi is subdivided into orbital, palpebral and lacrimal parts. Each has defined actions. The orbicularis oculi is secured to the medial and lateral palpebral ligament forming a ring in the eyelid tissue centered about the anterior eye. It also inserts on the eyelid 'skeleton' the tarsal plate.
It forms connections between the superficial muscular aponeurotic system SMAS of the face and frontalis muscle , the temple and the cheek. Laterally the fibers are pierced by the zygomaticofacial nerve which supplies the skin over the zygoma.
The orbital part forms the bulk of the muscle, can squeeze the eyelid closed tightly and is used in bright light. Forms a small slip originating adjacent to the lacrimal gland to insert on the lateral eyelid.
It is thought to promote flow within the gland. Please Note: You can also scroll through stacks with your mouse wheel or the keyboard arrow keys. Updating… Please wait. Unable to process the form. Check for errors and try again. Thank you for updating your details. Log In. Sign Up.
0コメント