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., J. Žurmanová, T. Soukup., and Obsahuje seznam literatury
In order to re-evaluate the presence and relative quantity of 2b and 2x/d myosin heavy chain (MyHC) transcripts in rat slow soleus muscle by using real time RT-PCR we have compared the available relevant cDNA sequences and designed a new set of primers having similar melting temperatures, matching separate MyHC exons in the regions of maximal differences in MyHC coding sequences, and containing G or C at the 3́-end. These also yielded PCR products of corresponding length, which is an important requirement for real time RT-PCR quantification. The experiments were performed on 8- month-old inbred female Lewis strain rats used in our current study of regenerating transplanted muscles. The real time RT-PCR measurement confirmed the expression of all four MyHC mRNAs (type 1, 2a, 2x/d and 2b) in both fast extensor digitorum longus and slow soleus muscles, although in the soleus muscle of adult rats, only type 1 and 2a protein isoforms can be usually detected, J. Žurmanová, F. Půta, R. Stopková, T. Soukup., and Obsahuje bibliografii a bibliografické odkazy