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Summary
We studied the expression of myosin heavy chain isoforms at mRNA and protein levels as well as fiber type composition in the fast extensor digitorum longus (EDL) and slow soleus (SOL) twitch muscles of adult inbred Lewis strain rats. Comparison of the results from Real Time RT-PCR, SDS-PAGE and fiber type analysis showed corresponding proportions of MyHC transcripts {MyHC-1, -2a, -2x/d, -2b), protein isoforms (MyHC-1, -2a, -2x/d, -2b) and fiber types (type 1, 2A, 2X/D, 2B) in both muscles. Furthermore, we found that slow MyHC-1 mRNA expression in the SOL was up to three orders higher than that of fast MyHC transcripts. This finding can explain the predominance of MyHC-1 isoform and fiber type 1 and the absence of pure 2X/D and 2B fibers in the SOL muscle. Based on our data presenting quantitative evidence of corresponding proportions between mRNA level, protein content and fiber type composition, we suggest that the Real Time RT-PCR technique can be used as a routine method for analysis of muscle composition changes and could be advantageous for the analysis of scant biological samples such as muscle biopsies in humans.
Key words
Rat * Soleus * Extensor digitorum longus * Myosin heavy chain isoforms * Muscle gene expression * Real Time RT-PCR * SDS-PAGE * Fiber type analysis
Introduction
It is generally accepted that skeletal muscle fiber types are defined by myosin heavy chain (MyHC) isoform content, which is dependent on the level of expression of the specific mRNAs. Both fiber type composition, MyHC isoform content and mRNA expression can be determined by appropriate (semi) quantitative methods. The fiber type composition can be estimated e.g. by stereological analysis (Zacharova and Kubinova 1995), MyHC isoform content by SDS-PAGE (Talmage and Roy 1993), though some attempts to employ other approaches have been published (Ricny and Soukup 2011). MyHC transcript level determination was performed by in situ hybridization or by various modifications of RT-PCR (DeNardi et al. 1993, Esser et al. 1993, Lieber et al. 1993, Smerdu et al. 1994, Peuker and Pette 1995, 1997, Jänkälä et al. 1997, Wright et al. 1997, Jaschinski et al. 1998, Jung et al. 1998, Stevens et al. 1999a,b, Weiss et al. 1999, Huey et al. 2001, Serrano et al. 2001, Smerdu and Erzen 2001,...