Immunohistochemical identification and fiber type specific localization of protein kinase C isoforms in equine skeletal muscle

Mireille M.E. van Ginneken Department of Equine Sciences, Discipline of Internal Medicine, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands

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Hans A. Keizer Department of Movement Sciences, Faculty of Health Sciences, Maastricht University, Maastricht, the Netherlands.

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Inge D. Wijnberg Department of Equine Sciences, Discipline of Internal Medicine, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands.

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Klien G. van Dam Department of Equine Sciences, Discipline of Internal Medicine, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands.

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Gert Schaart Department of Movement Sciences, Faculty of Health Sciences, Maastricht University, Maastricht, the Netherlands.

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Ellen de Graaf-Roelfsema Department of Equine Sciences, Discipline of Internal Medicine, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands.

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Johannes H. van der Kolk Department of Equine Sciences, Discipline of Internal Medicine, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands.

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Eric van Breda Department of Movement Sciences, Faculty of Health Sciences, Maastricht University, Maastricht, the Netherlands.

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Abstract

Objective—To investigate whether protein kinase C (PKC) isoforms are expressed in equine skeletal muscle and determine their distribution in various types of fibers by use of immunofluorescence microscopy.

Animals—5 healthy adult Dutch Warmblood horses.

Procedure—In each horse, 2 biopsy specimens were obtained from the vastus lateralis muscle. Cryosections of equine muscle were stained with PKC isoform (α, β1, β2, δ, ξ, or ζ)-specific polyclonal antibodies and examined by use of a fluorescence microscope. Homogenized muscle samples were evaluated via western blot analysis.

Results—The PKC α, β1, β2, δ, ξ, and ζ isoforms were localized within the fibers of equine skeletal muscle. In addition, PKC α and β2 were detected near or in the plasma membrane of muscle cells. For some PKC isoforms, distribution was specific for fiber type. Staining of cell membranes for PKC α was observed predominantly in fibers that reacted positively with myosin heavy chain (MHC)-IIa; PKC δ and ξ staining were more pronounced in MHC-I-positive fibers. In contrast, MHC-I negative fibers contained more PKC ζ than MHC-I-positive fibers. Distribution of PKC β1 was equal among the different fiber types.

Conclusions and Clinical Relevance—Results indicated that PKC isoforms are expressed in equine skeletal muscle in a fiber type-specific manner. Therefore, the involvement of PKC isoforms in signal transduction in equine skeletal muscle might be dependent on fiber type. ( Am J Vet Res 2004; 65:69–73)

Abstract

Objective—To investigate whether protein kinase C (PKC) isoforms are expressed in equine skeletal muscle and determine their distribution in various types of fibers by use of immunofluorescence microscopy.

Animals—5 healthy adult Dutch Warmblood horses.

Procedure—In each horse, 2 biopsy specimens were obtained from the vastus lateralis muscle. Cryosections of equine muscle were stained with PKC isoform (α, β1, β2, δ, ξ, or ζ)-specific polyclonal antibodies and examined by use of a fluorescence microscope. Homogenized muscle samples were evaluated via western blot analysis.

Results—The PKC α, β1, β2, δ, ξ, and ζ isoforms were localized within the fibers of equine skeletal muscle. In addition, PKC α and β2 were detected near or in the plasma membrane of muscle cells. For some PKC isoforms, distribution was specific for fiber type. Staining of cell membranes for PKC α was observed predominantly in fibers that reacted positively with myosin heavy chain (MHC)-IIa; PKC δ and ξ staining were more pronounced in MHC-I-positive fibers. In contrast, MHC-I negative fibers contained more PKC ζ than MHC-I-positive fibers. Distribution of PKC β1 was equal among the different fiber types.

Conclusions and Clinical Relevance—Results indicated that PKC isoforms are expressed in equine skeletal muscle in a fiber type-specific manner. Therefore, the involvement of PKC isoforms in signal transduction in equine skeletal muscle might be dependent on fiber type. ( Am J Vet Res 2004; 65:69–73)

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