Quantifying the Temporospatial Expression of Postnatal Porcine Skeletal Myosin Heavy Chain Genes

Abstract

S U M M A R Y Postnatal skeletal muscle fiber type is commonly defined by one of four major myosin heavy chain (MyHC) gene isoforms (slow/I, 2a, 2x, and 2b) that are expressed. We report on the novel use of combined TaqMan quantitative real-time RT-PCR and image analysis of serial porcine muscle sections, subjected to in situ hybridization (ISH) and immunocytochemistry (IHC), to quantify the mRNA expression of each MyHC isoform within its corresponding fiber type, termed relative fiber type-restricted expression. This versatile approach will allow quantitative temporospatial comparisons of each MyHC isoform among muscles from the same or different individuals. Using this approach on porcine skeletal muscles, we found that the relative fiber type-restricted expression of each postnatal MyHC gene showed wide spatial and temporal variation within a given muscle and between muscles. Marked differences were also observed among pig breeds. Notably, of the four postnatal MyHC isoforms, the 2a MyHC gene showed the highest relative fiber typerestricted expression in each muscle examined, regardless of age, breed, or muscle type. This suggests that although 2a fibers are a minor fiber type, they may be disproportionately more important as a determinant of overall muscle function than was previously believed.

Compensatory growth feeding strategy does not overcome negative effects on growth and carcass composition of low birth weight pigs

“…Each cDNA template for RT-PCR was prepared from mRNA extracted from 50 mg of LM tissue collected within 30 min after exsanguination (Trifast, Peqlab Biotechnologie GmbH, Erlangen, Gemany), by first-strand reverse transcription using 0.50 μg of oligo-d(T) (Improm-II reverse transcriptase, Promega, Mannheim, Germany) in a final volume of 20 µl. All used primers and taqman probes (Supplementary Table S1) are published by da Costa et al (2002). The TaqMan PCR assays for each target gene were performed in triplicate on cDNA templates with a Rotor-Gene 6500 QRT-PCR thermocycler analyser (Corbett Life Science, Sydney, Australia) according to the manufacturer's recommendations.…”

“…In the present study, the mean slope of the six genes was − 3.37 ± 0.25. The relative standard curve method was used to quantify the relative gene expression of embryonic, perinatal, slow/I, IIA, IIX, IIB MyHC isoforms and of β-actin as described by da Costa et al (2002). To correct for variations in the quality and quantity of cDNA templates, all MyHC isoform expression results were normalized to corresponding endogenous β-actin mRNA levels, which within each muscle were not affected by treatment (P ⩾ 0.42).…”

Expression of eight distinct MHC isoforms in bovine striated muscles:evidence for MHC-2B presence only in extraocular muscles

“…A recent study suggests that in the genome of some species, such as the horse, only a pseudogene with a sequence similar to that of the MHC-2B gene is present (Chikuni et al, 2004a). There are, however, exceptions: in pigs, the 2B isoform is expressed at the protein level in several skeletal muscles, generally in hybrid 2X/B fibres (Da Costa et al, 2002;Toniolo et al, 2004), and in cattle we have recently shown the expression of MHC-2B at the mRNA level in extraocular muscles .…”

Advances in research on the prenatal development of skeletal muscle in animals in relation to the quality of muscle-based food. II – Genetic factors related to animal performance and advances in methodology

“…Four out of eight isoforms of MyHC genes known in mammals have been identified in porcine muscle (Chang and Fernandes, 1997). Immunohistochemistry (Graziotti et al, 2001;da Costa et al, 2002;Lefaucheur et al, 2004), in situ hybridization (Graziotti et al, 2001;da Costa et al, 2002;Lefaucheur et al, 2004) and electrophoresis (Bee et al, 1999) have been used to examine muscle fibre typing essentially based on the discrimination of MyHC isoforms. Newly developed real-time PCR assays have been reported to distinguish four isoforms in pig skeletal muscle (da Costa et al, 2002;Lefaucheur et al, 2004).…”

“…Immunohistochemistry (Graziotti et al, 2001;da Costa et al, 2002;Lefaucheur et al, 2004), in situ hybridization (Graziotti et al, 2001;da Costa et al, 2002;Lefaucheur et al, 2004) and electrophoresis (Bee et al, 1999) have been used to examine muscle fibre typing essentially based on the discrimination of MyHC isoforms. Newly developed real-time PCR assays have been reported to distinguish four isoforms in pig skeletal muscle (da Costa et al, 2002;Lefaucheur et al, 2004). Frequencies of fibres determined by muscle fibre staining for ATPase and relative abundance of MyHC isoforms determined by quantitative reverse transcription-PCR of corresponding pairs of type I, IIa and IIx/IIb were correlated (r 5 0.71, 0.67 and 0.52, respectively) Wimmers et al, 2008).…”