Identification of Myosin Heavy Chain I, IIa and IIx in Canine Skeletal Muscles by an Electrophoretic and Immunoblotting Study

Abstract

To determine which myosin heavy chain (MHC) isoforms are expressed in canine skeletal muscles, different muscle samples of five mixed-breed dogs were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated MHC isoforms were identified by immunoblotting technique using a set of specific monoclonal antibodies. To compare the results of the electrophoretic and immunoblotting study, the pattern of MHC isoform expression and histochemical profiles of canine fibres were additionally demonstrated on serial muscle sections by immunohistochemistry and myofibrillar adenosine triphosphatase (mATPase) histochemistry. Not more than three MHC isoforms were demonstrated by SDS-PAGE in the analysed canine muscles. By the immunoblotting technique, the fastest migrating MHC band was identified as slow or MHC-I, the intermediate one as MHC-IIx and the slowest migrating band as MHC-IIa isoform. Since none of the three MHC bands and none of the analysed fibres were recognized by the antibody specific to MHC-IIb of rats, we concluded that MHC-IIb is not expressed in large skeletal muscles of dogs. Similarly, only three major fibre types, i.e. I, IIA and IIX, were revealed according to the pattern of MHC immunohistochemistry and mATPase reaction. Type IIA fibres were more alkali- and acid-stable than type IIX fibres after mATPase histochemistry; hence, the latter corresponded to type IIDog fibres. However, beside the three major fibre types, scarce hybrid fibres co-expressing two MHC isoforms (I/IIA and IIA/IIX) were demonstrated by immunohistochemistry.

Black wildebeest skeletal muscle exhibits high oxidative capacity and a high proportion of type IIx fibres

“…Western blotting and immunohistochemistry using previously validated antibodies (for both isoform specificity and species reactivity) showed that the black wildebeest expressed only MHC IIa, MHC IIx and MHC I (corresponding to the migratory pattern observed in Fig.1A) (Smerdu et al, 2005;Acevedo and Rivero, 2006;Kohn et al, 2011). This migratory profile is consistent with that from a number of large domestic and wild species, including cattle, horses, lions, dogs, tigers and cheetahs, whereas human MHC IIx migrates slower than its MHC IIa isoform (Toniolo et al, 2005;Acevedo and Rivero, 2006;Kohn et al, 2007a;Rivero et al, 2007;Hyatt et al, 2010).…”

Enzyme‐ and immunohistochemical aspects of skeletal muscle fibers in brown bear (Ursus arctos)

“…Both recognized slow or Type-I fibers. As these antibodies revealed Type-I fibers in skeletal muscles of different species (Rivero et al, 1996(Rivero et al, , 1999Argü ello et al, 2001;Lefaucheur et al, 2002;Š trbenc et al, 2004;Smerdu et al, 2005), it is clear that slow MHC-I isoform is highly conserved among species. On the contrary, fast MHC isoforms show species-dependent antigenic specificity.…”

Muscle Fibre Phenotyping from a Single Section: Is It as Informative as from Serial Sections?

“…Briefly slow fibres were demonstrated by BA-D5 antibody immunoreactive with /slow MyHC-I in rats (2) and humans (5) in a dilution 1:100; fast fibres were demonstrated by A4.74 antibody (former Alexis Biochemicals, now Enzo Life Sciences) immunoreactive according to the product data sheet in rats and humans with MyHC-IIA, but according to (16) with MyHC-IIA and MyHC-IIX in humans and in dogs (17). A dilution of A4.74 was 1:100.…”

“…Several distinct MyHC isoforms exist. Human limb and trunk muscles contain muscle fibres expressing skeletal muscle-specific MyHC isoform IIA (MyHC-IIA) encoded by MYH2 gene on chromosome 17, MyHC isoform IID/IIX (MyHC-IIX) (2-4) encoded by MYH1 gene on chromosome 17 and an isoform that is expressed in both skeletal and cardiac muscle MyHC-or MyHC-I encoded by MYH7 gene on chromosome 14 (5,6). Two developmental myosin heavy chain isoforms (MyHC-embryonic and MyHC-perinatal) may be present in adult muscles, particularly in regenerating muscle fibres.…”