title: Fiber types in canine muscles: myosin isoform expression and functional characterization.
abstract: This study was aimed to achieve a definitive and unambiguous identification of fiber types in canine skeletal muscles and of myosin isoforms that are expressed therein. Correspondence of canine myosin isoforms with orthologs in other species as assessed by base sequence comparison was the basis for primer preparation and for expression analysis with RT-PCR. Expression was confirmed at protein level with histochemistry, immunohistochemistry, and SDS-PAGE combined together and showed that limb and trunk muscles of the dog express myosin heavy chain (MHC) type 1, 2A, and 2X isoforms and the so-called "type 2dog" fibers express the MHC-2X isoform. MHC-2A was found to be the most abundant isoform in the trunk and limb muscle. MHC-2X was expressed in most but not all muscles and more frequently in hybrid 2A-2X fibers than in pure 2X fibers. MHC-2B was restricted to specialized extraocular and laryngeal muscles, although 2B mRNA, but not 2B protein, was occasionally detected in the semimembranosus muscle. Isometric tension (P(o)) and maximum shortening velocity (V(o)) were measured in single fibers classified on the basis of their MHC isoform composition. Purified myosin isoforms were extracted from single muscle fibers and characterized by the speed (V(f)) of actin filament sliding on myosin in an in vitro motility assay. A close proportionality between V(o) and V(f) indicated that the diversity in V(o) was due to the different myosin isoform composition. V(o) increased progressively in the order 1/slow < 2A < 2X < 2B, thus confirming the identification of the myosin isoforms and providing their first functional characterization of canine muscle fibers.
authors.affiliation: Department of Anatomy and Physiology, University of Padova, Via Marzolo 3, 35131 Padova, Italy.
authors.forename: Luana
authors.initials: L
authors.lastname: Toniolo
authors.forename: Lisa
authors.initials: L
authors.lastname: Maccatrozzo
authors.forename: Marco
authors.initials: M
authors.lastname: Patruno
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Pavan
authors.forename: Francesca
authors.initials: F
authors.lastname: Caliaro
authors.forename: Rosetta
authors.initials: R
authors.lastname: Rossi
authors.forename: Chiara
authors.initials: C
authors.lastname: Rinaldi
authors.forename: Monica
authors.initials: M
authors.lastname: Canepari
authors.forename: Carlo
authors.initials: C
authors.lastname: Reggiani
authors.forename: Francesco
authors.initials: F
authors.lastname: Mascarello
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007821:Laryngeal Muscles / Q000737:chemistry; D009119:Muscle Contraction; D018656:Muscle Fibers, Fast-Twitch / Q000737:chemistry* / Q000378:metabolism; D018657:Muscle Fibers, Slow-Twitch / Q000737:chemistry* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000032:analysis; D020033:Protein Isoforms / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D024744:Skeletal Muscle Myosins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
chemical_list: D000199:Actins; D020033:Protein Isoforms; D012333:RNA, Messenger; D024744:Skeletal Muscle Myosins; D018995:Myosin Heavy Chains