Clenbuterol, a beta2-agonist, administration results in hypertrophy of fast fibres and an increase in the fast myosin heavy chain (MHC) composition of both fast and slow muscles. The present study was designed to determine the phenotypic response at the single fibre level. Clenbuterol was added to the drinking water (30 mg L(-1)) of adult male Wistar rats for 4 weeks. Single fibres from the soleus muscle of control (10 rats; 555 fibres) and clenbuterol-treated (10 rats; 577 fibres) were dissected and their MHC isoform composition was determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis. Body, heart, and soleus weights were 9, 24, and 27% higher in clenbuterol-treated than control rats. The mean cross-sectional areas of fast and slow/fast hybrid fibres were approximately 64 and approximately 74% larger in the clenbuterol-treated than control rats, whereas the size of the slow fibres were similar in the two groups. Fibres from control soleus showed three MHC patterns: pure type I (84%), pure type IIa (4%), and type I + IIa (12%) MHC. Some fibres from clenbuterol-treated soleus showed a de novo expression of type IIx MHC resulting in the following fibre type proportions: pure type I (62%), pure type IIa (2%), type I + IIa (26%), type I + IIa + IIx (6%), and type IIa + IIx (1%). In those fibres containing multiple MHCs, there was a shift towards the faster MHC isoforms after clenbuterol treatment. These data indicate that clenbuterol results in muscle fibre hypertrophy, stimulates a de novo expression of type IIx MHC and increases the percentage of fibres containing multiple MHC isoforms in the rat soleus muscle. These phenotypic changes at the single fibre level are consistent with a clenbuterol-related shift in the functional properties of the soleus towards those observed in a faster muscle.

To examine the changes in heat shock proteins (HSPs) and calcineurin (CaN), a calcium/calmodulin regulated protein phosphatase, in hypertrophied rat skeletal muscles, adult male Wistar rats were administered clenbuterol (CLB, 30 mg l(-1) in drinking water), a beta 2-agonist, for 4 weeks. Compared to controls, CLB-treated rats had significantly larger body (10%) and relative (to body weight) soleus (Sol, 16%), plantaris (Plt, 32%) and gastrocnemius (Gast, 27%) weights. Immunohistochemically classified fast fibers were hypertrophied in the Sol (64%), Plt (62%), and deep (d, 70%) and superficial (s, 44%) regions of the Gast, whereas slow fibers were hypertrophied only in the Plt (47%). The percentage of fast fibers in the Sol increased from 10% to 21%. The myosin heavy chain (MHC) isoform composition shifted from slow to fast in the Sol (increase in the percentage of type IIa MHC and de novo synthesis of type IIx MHC) and Gast-d (de novo synthesis of type IIb MHC) and to the faster isoforms in the Plt (increase in the percentage of type IIb MHC). Hsp72 and Hsp90 levels in CLB-treated rats were 52% and 50% lower in the Sol and 44 and 41% lower in the Gast-d, respectively, than in control rats. In control rats, the relative content of CaN was: Sol>Gast-d>Plt>Gast-s, and CLB treatment enhanced the CaN content by 1.4-, 1.2-, 5.0-, and 3.3-fold, respectively. These results indicate that the down-regulation of HSPs in the Sol and Gast-d was closely related to a decrease in the slow phenotype, and that the relative up-regulation of CaN among the muscles/regions was closely related to the selective hypertrophy of fast fibers in the CLB-treated rats.