Induction of beta-MHC transgene in overloaded skeletal muscle is not eliminated by mutation of conserved elements

Abstract

Mechanical overload leads to hypertrophy, increased type I fiber composition, and beta-myosin heavy chain (beta-MHC) induction in the fast-twitch plantaris muscle. To better understand the mechanism(s) involved in beta-MHC induction, we have examined inducible expression of transgenes carrying the simultaneous mutation of three DNA regulatory subregions [muscle CAT (MCAT), C-rich, and beta e3] in the context of either 5,600-base pair (bp; beta 5.6mut3) or 600-bp (beta 0.6mut3) beta-MHC promoter in overloaded plantaris muscles of transgenic mice. Protein extract from mechanically overloaded plantaris muscle of mice, harboring either mutant transgene beta 5.6mut3 or beta 0.6mut3, showed an unexpected 2.8- to 4.5-fold increase in chloramphenicol acetyltransferase (CAT) specific activity relative to their respective controls. Similar results were obtained with wild-type (wt) beta-MHC transgenes (beta 5.6wt, beta 0.6wt). Histochemical staining for both myofibrillar ATPase and CAT activity and CAT immunohistochemistry revealed a striking increase in type I fibers and that CAT expression was restricted to these fibers in overloaded plantaris muscle of beta 5.6mut3 transgenic mice. Our transgenic data suggest that beta-MHC transgenes, and perhaps the endogenous beta-MHC gene, are induced by mechanical overload via a mechanism(s) that does not exclusively require the MCAT, C-rich, or beta e3 subregions.

Functional overload increases β-MHC promoter activity in rodent fast muscle via the proximal MCAT (βe3) site

“…5) a significant increase in the level of specific binding of the ␤e3 probe was detected with the extracts from overloaded plantaris muscles compared with normal control plantaris extracts. However, this conclusion conflicts with a transgenic mouse plantaris overload study using a mouse ␤-MHC promoter-CAT reporter transgene that harbored simultaneous mutations of ␤e2, CACC, and ␤e3 elements (30). That report found that, despite the mutations, reporter activity increased in response to 8 wk of bilateral plantaris overload similar to that of the wild type.…”

“…Our 1-wk model explored the regulatory processes that are involved during the adaptive phase of the overload response. In contrast, their 8-wk study (30) assessed the promoter activity at the steady-state level of the overload stimulus. It is possible that the mechanisms regulating promoter activity are different during the steady state compared with the adaptive phase such that, after such a long period of overload perturbation, the involvement of other elements may override the ␤e3 mutation and induce an increase in promoter activity.…”

“…In this model, the overload stress is induced by the removal of synergistic muscles along with normal weight-bearing activity, which collectively causes compensatory hypertrophy of muscle fibers (1,26). Adaptation to the overload stimulus also prompts a shift in the fiber-type profile toward a slow fiber-type pattern, characterized by a lower myofibrillar ATPase activity, reduced glycogenolytic enzyme activities, decreased expression of fast MHC isoforms IIx and IIb, and increased expression of slow myosin light chain isoforms and slower MHC isoforms ␤ (type I) and IIa (2,28,30,34).We are interested in determining the molecular mechanisms responsible for the increase in the slow, ␤-MHC gene expression in overloaded plantaris. It is believed that the phenotypic adaptations are mediated in some way by the interaction of transcriptional regulatory proteins with specific cis elements within the ␤-MHC gene's promoter sequence.…”

“…Unlike previous studies (20,30,32), the present report involves a comprehensive mutation analysis of all of the putative regulatory elements in the rat ␤-MHC proximal promoter to resolve their role in the load responsiveness of the ␤-MHC gene in vivo. Herein, we describe a direct gene transfer approach whereby deleted or mutated promoters are linked to a firefly luciferase reporter gene and injected directly in rat plantaris muscles.…”

Changes in muscle mass and phenotype and the expression of autocrine and systemic growth factors by muscle in response to stretch and overload

“…Experiments have shown that without this 5h sequence expression levels are very low. When spliced to reporter gene sequences and introduced into transgenic animals, the 5h regulatory sequence is important for the stretch response (Tsika et al 1996). Therefore one must assume that there is a stretch response element in the promoter region but what transactivating factor or factors are involved needs to be elucidated.…”