Morphological, histochemical, and myosin isoform analysis of the diaphragm of adult horses, Equus caballus

Abstract

The horse provides an interesting model for study of the structure and function of the mammalian diaphragm. Multiple regions of diaphragm from seven adult horses were prepared for histochemistry, immunocytochemistry, myosin heavy chain electrophoresis, and native myosin electrophoresis. Two additional adults were dissected to demonstrate myofiber and central tendon morphology and stained for acetylcholinesterase to demonstrate motor endplates. All regions of the adult diaphragm were histochemically characterized by a preponderance of type I fibers with some type IIa fibers. Type IIb fibers were absent in all adult specimens. Myosin heavy chain electrophoresis supported the histochemical study: two isoform bands were present on SDS gels that comigrated at the same rate as rat type I and IIa myosin heavy chain isoforms. No isoform was determined to comigrate with rat type IIb heavy chain isoforms. Native myosin isoform analysis revealed two isoforms that comigrated with rat FM-4 and FM-3 (FM = fast myosin) and two isoforms that comigrated with rat SM-1 and SM-2 (SM = slow myosin) isoforms. In some samples, a third slow native myosin isoform was observed that comigrated at the same rate as the SM-3 of the equine biceps brachii muscle. This doublet (or "triplet") of slow isoforms is unique to some horse muscles compared with other adult animals studied. It is not known if these multiple slow native myosin isoforms confer some functional advantage to the equine muscles. The adult equine diaphragm also differs in its morphology by having a large central tendon compared to that in other mammals, and is predominantly slow in fiber type and myosin isoform composition.

Myosin heavy chain isoforms in adult equine skeletal muscle: An immunohistochemical and electrophoretic study

“…Later, electrophoretic methods similar to those used in the present study were applied on samples from equine biceps brachii (Hermanson et al, 1991) and diaphragm (Cobb et al, 19941, but only a single fast MyHC was observed. The difference between these two studies and the present results might be explained by the fact that horse biceps brachii (Hermanson et al, 1991) and diaphragm (Cobb et al, 1994) muscles are exclusively composed of type I and type IIA muscle fibers. Sosnicki et al (1989) identified fast and slow fiber types in horse skeletal muscle by differences in the mobility of their MyHCs.…”

“…In a more recent immunocytochemical study using anti-slow (5-4D), anti-fast (lAlO), and anti-fast red (5-2B) MAbs with cross reactivity for type I, all type I1 and type IIa MyHCs, respectively, in a number of species, did not enable subclassification of type I1 MyHCs in equine gluteus medius muscle (Sinha et al, 1992). Similarly, a clear discrimination between fibers containing either slow or fast MyHCs has been obtained by immunohistochemistry in both horse diaphragm (Cobb et al, 1994) and gluteus medius muscle (Serrano et al, 1996), but subdivision of type I1 fibers was not possible. Nevertheless, only MAbs directed against slow and all fast MyHC isoforms were tested in these latter studies.…”

“…Some studies have investigated the composition of MyHC isoforms in equine skeletal muscle by using immunohistochemistry (Snow et al, 1981;Sinha et al, 1992) or gel electrophoresis (Billeter et al, 1987;Sosnicki et al, 1989;Yamaguchi et al, 1993;Barrey et al, 1995) or a combination of both methods (Hermanson et al, 1991;Cobb et al, 1994). The content of MyHCs of five equine muscles has also been investigated by using enzyme-linked immunosorbent assay (Barrey et al, 1995).…”

The ontogeny of aerobic and diving capacity in the skeletal muscles of Weddell seals

“…To our knowledge, Weddell seals are the first reported example where there is a shift from aerobic Type I fibers towards an increase in Type IIA oxidative fibers with a significant decrease in mitochondrial density as the animals mature. By contrast, the locomotor muscles of precocial terrestrial mammals are similar in mass (as a percentage of total body mass) and fiber type composition across age classes (Cobb et al, 1994a;Cobb et al, 1994b;Dearolf et al, 2000;Schutt et al, 1994).…”

Myosin Heavy Chain Composition in Normal and Atrophic Equine Laryngeal Muscle

“…These electrophoresis parameters have previously been shown to provide optimal separation and imaging of the bands representing myosin isoforms. 8,32 Western blot An acrylamide gel containing separated protein from the laryngeal muscles of a normal horse was transferred onto a nitrocellulose membrane at 100 mA overnight at 4uC. The membrane was blocked and incubated with a primary monoclonal antibody against all MHC isoforms (mouse anti-human MHC; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, Iowa) for 1 hour (1 : 1000).…”