Sequencing of the 2a, 2x, and slow isoforms of the bovine myosin heavy chain and the different expression among muscles.

Abstract

Myosin is a major structural protein of the thick filament of the sarcomere. It constitutes 40% of myofibrillar protein. Each myosin molecule consists of two identical heavy chains (MyHC, 220 kDa each) and two pairs of non-identical myosin light chains (20 kDa each; Warrick and Spudich 1987). There are four major sarcomeric MyHC isoforms in adult mammalian skeletal muscle, MyHC-2a, -2b, -2x (also referred to as -2d), and ‘‘-slow’’. Each MyHC isoform is encoded by a distinct gene (Schiaffino and Reggiani 1996). Individual muscle fibers are characterized according to their MyHC content: type II A fiber (containing MyHC-2a), type II B fiber (containing MyHC-2b), type II X fiber (containing MyHC2x), and type I fiber (containing MyHC-slow; Pette and Staron 1990). Skeletal muscle fibers can alter their MyHC isoform composition in response to electrical stimuli and mechanical factors such as stretch (Ausoni et al. 1990; Loughna et al. 1990; Termin et al. 1989). However, the molecular mechanisms determining genespecific activation of these MyHC genes are not well defined (Schiaffino and Reggiani 1996). Expression of the four major MyHC isoform genes was reported in rat muscles (Schiaffino and Reggiani 1996). Expressions of these four MyHC isoforms in the porcine skeletal muscles were also detected by in situ hybridization (Chang and Fernandes 1997) and RT-PCR (Tanabe et al. 1999). Expression pattern of a fetal bovine MyHC gene was reported (Young et al. 1994); however, expression pattern MyHC isoform genes in bovine skeletal muscles has not been reported. Cattle are large ruminant mammals, whose physiology is different from that of non-ruminant mammals; therefore, bovine MyHC isoform composition would differ from that of other mammals. Moreover, MyHC isoform composition would also affect the quality of the meat originating from bovine muscles (Xiong 1994). In this report, we determined the sequences containing 58 non-coding region of bovine MyHC-2a, -2x and -slow isoforms, which differed among the isoforms. Based on these sequences, we developed a procedure to detect the expression of these three MyHC isoforms in bovine muscles by the RT-PCR method with the use of the multiplex PCR technique. Published sequences of porcine MyHCs (Chang et al. 1993, 1995; Tanabe et al. 1999) were used to amplify the bovine cDNA fragments by PCR. From the reported sequences, three sense primers (MYO1, MYO103, MYO401) and four antisense primers (MYO2, MYO6, MYO102, MYO404) were synthesized. The sequences of these primers are described in Table 1. We obtained first-strand cDNAs from M. biceps femoris of 7-month-old cattle and diaphragm of 3-month-old cattle by using CapFinderTM PCR cDNA Library Construction Kit (CLONTECH, Calif.). This kit contained a primer named 58PCR (the sequence is described in Table 1) for cDNA amplification. Two PCR fragments were obtained from the first-strand cDNAs of M. biceps femoris by using the primer pairs MYO1/MYO2 and 58PCR/MYO6 and then were directly sequenced. The resulting sequence was highly different from the porcine MyHC-2a sequence, especially on the non-coding region; therefore, two sense primers (MYO201, MYO203) and one antisense primer (MYO202) on the bovine non-coding region were synthesized to confirm the bovine sequence. The sequences of these primers are described in Table 1. With MYO201, MYO202, MYO203, MYO2, and 58PCR, a cDNA fragment of the M. biceps femoris was amplified again. Consequently, this bovine cDNA fragment was confirmed to be homologous to the porcine MyHC2x isoform reported by Chang and Fernandes (1997). We amplified and sequenced a cDNA fragment of the bovine MyHC-2a isoform from the cDNAs of M. biceps femoris with the use of MYO102, MYO103, MYO2, and 58PCR. We amplified and seCorrespondence to: R. Tanabe