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Human extraocular muscles: molecular diversity of a unique muscle allotype
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Introduction: The extraocular muscles (EOMs) are considered a separate class of skeletal muscle, allotype. Myosin is the major contractile protein in muscle. The myosin heavy chain (MyHC) isoforms are the best molecular markers of functional heterogeneity of muscle fibers. The relaxation rate, reflects the rate at which Ca2+ is transported back into the sarcoplasmic reticulum (SR) mostly by SR Ca2+ATPase (SERCA). Myosin binding protein C (MyBP-C), plays a physiological role in regulating contraction. The laminins (Ln) are the major non-collagenous components of the basement membrane (BM) surrounding muscle fibers and are important for muscle fiber integrity.

Methods: Adult human EOMs were studied with SDS-PAGE, immunoblots and immunocytochemistry, the latter with antibodies against six MyHC, 2 SERCA, 2 MyBP-C and 8 laminin chain isoforms. The capillary density was also determined.

Results: Most fibers contained a mixture of MyHC isoforms. Three major groups of fibers could be distinguished. Fast fibers that stained with anti-MyHCIIa, slow fibers that stained with anti-MyHCI and MyHCeompos/MyHCIIaneg-fibers that stained with neither of these antibodies but with anti-MyHCI+IIa+eom and anti-MyHCeom. A majority of the fibers contained both SERCA1 and 2 whereas 1% were unstained with both antibodies. Biochemically SERCA2 was more abundant than SERCA1. MyBP-Cfast was not present in the EOMs and MyBP-Cslow was only detected immunocytochemically. The extrasynaptical BM of the EOM muscle fibers contained Lna2, b1, b2, g1, a4 and a5 chains. The capillary density in the EOMs was very high (1050 +/-190 capillaries/mm2) and significantly (p<0.05) higher in the orbital than in the global layer.

Conclusions: The co-existence of complex mixtures of several crucial protein isoforms provide the human EOMs with a unique molecular portfolio that a) allows a highly specific fine-tuning regime of contraction and relaxation, and b) imparts structural properties that are likely to contribute to protection against certain neuromuscular diseases.

Place, publisher, year, edition, pages
Umeå: Umeå university , 2004. , 51 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 890
Keyword [en]
Ophtalmology, extraocular, muscle, myosin, protein c, laminin, serca, immunocytochemistry, human
Keyword [sv]
Oftalmiatrik
National Category
Ophthalmology
Research subject
Ophtalmology
Identifiers
URN: urn:nbn:se:umu:diva-260ISBN: 91-7305-638-3 (print)OAI: oai:DiVA.org:umu-260DiVA: diva2:142853
Public defence
2004-05-28, E04, 6E, Norrlands Universitetssjukhus, Umeå, 13:15
Opponent
Supervisors
Available from: 2004-05-05 Created: 2004-05-05 Last updated: 2011-04-08Bibliographically approved
List of papers
1. Myosin heavy chain isoforms in human extraocular muscle
Open this publication in new window or tab >>Myosin heavy chain isoforms in human extraocular muscle
2003 (English)In: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, Vol. 44, no 4, 1419-1425 p.Article in journal (Refereed) Published
Abstract [en]

PURPOSE: To investigate the myosin heavy chain (MyHC) composition of human extraocular (EOM) and levator palpebrae (LP) muscle fibers. METHODS: Adult human EOMs and LP were studied with SDS-PAGE, immunoblots, and immunocytochemistry, with antibodies against six MyHC isoforms. Myofibrillar adenosine triphosphatase (mATPase) and reduced nicotinamide adenine dinucleotide (NADH)-TR activity and fiber area were also determined. RESULTS: Most of the fibers in both layers stained strongly with anti-MyHCIIa. Approximately 14% of the fibers in the global layer and 16% in the orbital layer were labeled with anti-MyHCI. The remaining 24% of the fibers in the global layer and 3% in the orbital layer were not stained with either of these two antibodies, but were reactive to anti-MyHCeom (MyHCeom(pos)/MyHCIIa(neg) fibers). The fibers stained with anti-MyHCI had acid-stable mATPase activity, and the remainder of the fibers had alkaline-stable mATPase activity. Almost all the slow fibers stained with both anti-MyHCI and anti-MyHCslow tonic in both layers. Anti-MyHCalpha-cardiac stained approximately 26% of these slow fibers in the orbital layer and 7% in the global layer. Some slow fibers in both layers lacked staining with anti-MyHCslow tonic or with anti-MyHCalpha-cardiac. MyHCemb and/or MyHCeom were also present in some of the fibers of all the groups. The LP did not stain with anti-MyHCslow tonic. CONCLUSIONS: The present study revealed that the human EOMs have a very complex fiber type and MyHC composition and differ significantly from the EOMs of other species. The features of the LP were distinct from those of the four recti, the obliquus superior, and the limb muscles.

Place, publisher, year, edition, pages
Association for Research in Vision and Ophthalmology, 2003
Keyword
extraocular musles
National Category
Ophthalmology Cell and Molecular Biology
Research subject
Human Anatomy; Ophtalmology
Identifiers
urn:nbn:se:umu:diva-3935 (URN)10.1167/iovs.02-0638 (DOI)12657575 (PubMedID)744 (Local ID)744 (Archive number)744 (OAI)
Available from: 2004-05-05 Created: 2004-05-05 Last updated: 2013-12-12Bibliographically approved
2. Sarco(endo)plasmic reticulum ca2+ATPases (SERCA1 and 2) in human extraocular muscles
Open this publication in new window or tab >>Sarco(endo)plasmic reticulum ca2+ATPases (SERCA1 and 2) in human extraocular muscles
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2003 (English)In: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, Vol. 44, no 12, 5057-5062 p.Article in journal (Refereed) Published
Abstract [en]

PURPOSE: To investigate the composition of the fibers in human extraocular muscles (EOMs) with respect to the sarco(endo)plasmic reticulum Ca(2+)ATPases (SERCA)-1 and -2 and to investigate possible correlations between SERCA and myosin heavy chain (MyHC) composition. METHODS: EOM samples were processed for immunocytochemistry with monoclonal antibodies specific against SERCA1 (fast isoform), SERCA2 (slow isoform), or different MyHCs. A total of 1571 fibers were analyzed. Microsomal EOM fractions were analyzed with SDS-PAGE and immunoblots. RESULTS: The fast fibers, containing MyHCIIa, accounted for 79% of the fibers in the orbital layer (OL) and 74% in the global layer (GL). More than 99% of these fibers contained SERCA1, and 86% of them coexpressed SERCA1 and -2. Almost all slow fibers stained with SERCA2; 54% of those in the GL and all in the OL coexpressed SERCA1 and -2. Fifteen percent of the fibers in the GL and less than 1% in the OL were MyHCeom(pos)/MyHCIIa(neg) fibers. All these contained SERCA1 and in the OL also stained strongly with anti-SERCA2. Biochemically SERCA2 was more abundant than SERCA1. CONCLUSIONS: The human EOMs had a very complex pattern of expression of the major protein regulating fiber relaxation rate. The coexistence of SERCA1 and -2, together with complex mixtures of MyHCs in most of the fibers provide the human EOMs with a unique molecular portfolio that allows a highly specific fine-tuning regimen of contraction and relaxation.

Place, publisher, year, edition, pages
Association for Research in Vision and Ophthalmology, 2003
National Category
Ophthalmology
Identifiers
urn:nbn:se:umu:diva-3936 (URN)10.1167/iovs.03-0218 (DOI)14638697 (PubMedID)
Available from: 2004-05-05 Created: 2004-05-05 Last updated: 2013-12-12Bibliographically approved
3. Uncoordinated expression of myosin heavy chains and myosin-binding protein C isoforms in human extraocular muscles
Open this publication in new window or tab >>Uncoordinated expression of myosin heavy chains and myosin-binding protein C isoforms in human extraocular muscles
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2006 (English)In: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, Vol. 47, no 10, 4188-4193 p.Article in journal (Refereed) Published
Abstract [en]

PURPOSE: To examine the distribution of myosin-binding protein C (MyBP-C) in human extraocular muscles (EOMs) and to correlate the myosin heavy chain (MyHC) and the MyBP-C composition of the fibers. METHODS: Samples from 17 EOMs, 3 levator palpebrae (LP), and 6 limb muscles were analyzed with SDS-PAGE and immunoblot or processed for immunocytochemistry with monoclonal antibodies (mAbs) against MyBP-C-fast, MyBP-C-slow, MyHCIIa, MyHCI, MyHCsto, MyHCalpha-cardiac, and MyHCemb. RESULTS: In the limb muscle samples, fast fibers were labeled with anti-MyBP-C-fast and anti-MyBP-C-slow, whereas the slow fibers were immunostained with anti-MyBP-C-slow only, in accordance with previous studies. In 11 EOM samples MyBP-C-fast was not detected, and weak staining with anti-MyBP-C-fast was seen only in a few fibers in the proximal part of 2 muscles. The mAb against MyBP-C-slow labeled all fibers, but fibers containing MyHCI were generally more strongly stained. In the levator palpebrae, immunostaining with anti-MyBP-C-fast was present in some fibers labeled with anti-MyHCIIa and/or anti-MyHCeom. MyBP-C-fast and -intermediate were not detected biochemically in the EOMs. CONCLUSIONS: The lack of MyBP-C-fast and intermediate is an additional feature of the human EOM allotype. The true EOMs have a unique myofibrillar protein isoform composition reflecting their special structural and functional properties. The levator palpebrae muscle phenotype is intermediate between that of the EOMs and the limb muscles.

Place, publisher, year, edition, pages
Association for Research in Vision and Ophthalmology, 2006
Keyword
Adolescent, Adult, Aged; 80 and over, Antibodies; Monoclonal, Carrier Proteins/*metabolism, Electrophoresis; Polyacrylamide Gel, Female, Humans, Immunoblotting, Immunohistochemistry, Male, Muscle; Skeletal/metabolism, Myosin Heavy Chains/*metabolism, Myosins/metabolism, Oculomotor Muscles/*metabolism, Protein Isoforms
National Category
Ophthalmology
Identifiers
urn:nbn:se:umu:diva-12672 (URN)10.1167/iovs.05-1496 (DOI)17003405 (PubMedID)
Available from: 2008-01-11 Created: 2008-01-11 Last updated: 2013-12-12Bibliographically approved
4. Laminin isoforms in human extraocular muscles
Open this publication in new window or tab >>Laminin isoforms in human extraocular muscles
2004 (English)In: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, Vol. 45, no 12, 4233-4239 p.Article in journal (Refereed) Published
Abstract [en]

PURPOSE: To determine the laminin isoform composition of the basement membranes (BMs) in the human extraocular muscles (EOMs) and relate it to the fact that EOMs are spared in laminin alpha2-chain-deficient congenital muscular dystrophy. METHODS: Samples from adult human EOMs and limb muscle were processed for immunocytochemistry, with monoclonal antibodies against laminin chains (Ln) alpha1 to -5, beta1 and -2, and gamma1. Neuromuscular junctions (NMJs) were identified with acetylcholinesterase reaction. The capillary density was measured in sections stained with anti-Lnalpha5. RESULTS: The extrasynaptic BM of the EOM muscle fibers contained Lnalpha2, -beta1, -beta2, and -gamma1, and, in contrast to limb muscle, it also contained Lnalpha4 and -alpha5, to some extent. The distinct laminin composition of the EOMs was confirmed by the presence of Lutheran protein, an alpha5-chain-specific receptor not found in limb muscle. At the NMJs, there was increased expression of Lnalpha4 and expression of Lnalpha2, -alpha5, -beta1, -beta2, and -gamma1 was also maintained. The capillary density was very high (1050 +/- 190 capillaries/mm(2)) in the EOMs and significantly (P < 0.05) higher in the orbital (1170 +/- 180 capillaries/mm(2)) than in the global (930 +/- 110 capillaries/mm(2)) layer. CONCLUSIONS: The human EOMs showed important differences in laminin isoform composition and capillary density when compared with human limb muscle and muscles of other species. The presence of additional laminin isoforms other than laminin-2 in the BM of the extrasynaptic sarcolemma could partly explain the sparing of the EOMs in Lnalpha2-deficient congenital muscular dystrophy.

Place, publisher, year, edition, pages
Association for Research in Vision and Ophthalmology, 2004
Keyword
Adolescent, Adult, Aged, Aged; 80 and over, Basement Membrane/metabolism, Capillaries/anatomy & histology, Extremities, Histocytochemistry, Humans, Immunohistochemistry, Infant; Newborn, Laminin/*metabolism, Middle Aged, Muscle Fibers/metabolism, Muscle; Skeletal/blood supply/metabolism, Oculomotor Muscles/blood supply/*metabolism, Protein Isoforms/metabolism, Receptors; Laminin/metabolism, Sarcolemma/metabolism, Tissue Distribution
National Category
Ophthalmology
Identifiers
urn:nbn:se:umu:diva-12678 (URN)10.1167/iovs.04-0456 (DOI)15557425 (PubMedID)
Available from: 2008-01-10 Created: 2008-01-10 Last updated: 2013-12-12Bibliographically approved

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