β-MHC transgene expression in suspended and mechanically overloaded/suspended soleus muscle of transgenic mice

Non-weight-bearing (NWB) activity [space flight and hindlimb suspension (HS)] results in the loss of soleus muscle mass, a slow-to-fast fiber-type conversion, and decreased β-myosin heavy chain (β-MHC) protein and mRNA expression. To identify β-MHC promoter sequences required for decreased β- MHC expression in response to HS, we have modified an existing noninvasive hindlimb unweighting model to accommodate the use of (transgenic) mice. After 2 wk of HS, body and muscle (soleus > gastrocnemius > plantaris) weights were decreased as was the proportion of histochemically classified type I fibers in HS soleus muscle. Northern blot analysis revealed decreases in endogenous mRNA representing β-MHC, slow myosin light chain 1 and 2, and cardiac/slow troponin C, whereas those representing skeletal troponin C, muscle creatine kinase, and glyceraldehyde-3-phosphate dehydrogenase increased. Protein extracts prepared from HS soleus (SS) muscle of mice harboring transgenes comprised of 5.6 or 0.6 kilobase of wild type (wt) mouse β-MHC promoter (β5.6wt, β0.6wt) and those carrying the simultaneous mutation (mut) of the MCAT, C-rich, and βe3 subregions (β5.6mut3, β0.6mut3) revealed decreases in chloramphenicol acetyltransferase (CAT) specific activity relative to respective controls. Decreased CAT mRNA was observed for transgene β5.6mut3, line 85. Two weeks of the simultaneous imposition of mechanical overload (synergist ablation) and HS (MOV/HS) countermanded the loss in absolute and normalized SS weight but did not decrease β0.6wt transgene expression. These transgenic results demonstrate that regulatory sequences within a 600-base pair β-MHC promoter are sufficient to direct decreased transcription of β- MHC transgenes after 2 wk of HS.