Transcriptional Control of Myosin Heavy Chain IIB Gene Expression in Normal and Overloaded Rat Plantaris

Abstract

1038 The rat plantaris (P) muscle predominantly expresses fast-contracting isoforms of myosin heavy chain (MHC) proteins. During hypertrophy induced by chronic mechanical overload, the expression of the fastest type IIb MHC isoform decreases as the muscle shifts phenotype toward expression of slower-contracting MHC isoforms. PURPOSE: To determine the transcriptional control elements that reside downstream of the transcription start site (tss) and regulate MHC IIb expression in normal (Con) and functional overload (FO) rat P. METHODS: Female SD rats (N = 8/group) underwent 7 d FO of the left P. MHC IIb pre-mRNA and mRNA expression were measured by RT-PCR. The transcriptional activities of five MHC IIb promoters were tested by direct gene transfer into the P. Each promoter fragment began −1.431 kb upstream of the tss, and extended into either exon 1 (+0.013 kb), intron 2 (+1.008, +1.568, +2.054 kb), or exon 3 (+2.814 kb). Promoter activity was measured by a firefly luciferase (fLuc) reporter assay 7 d after bilateral intramuscular injection of plasmid constructs containing the fLuc gene controlled by the inserted IIb promoter fragment. To control for plasmid uptake efficiency, the fLuc reporter activity was corrected to the renilla luciferase activity of a co-injected alpha-actin promoter-reporter plasmid. RESULTS: After 7 d FO, the wet weight of the P increased ∼31% (P < 0.05). IIb pre-mRNA and mRNA each decreased by ∼60% after FO (P < 0.05). Activities of the +2.054 and +2.814 kb promoters were similar, and decreased ∼50% after FO (P < 0.05). The three promoters that extended downstream between +0.013 and +1.568 kb were 80–99% less active (P < 0.05) than the +2.814 kb promoter, and did not decrease after FO. CONCLUSION: MHC IIb expression in Con and FO P is controlled by transacting factor(s) located downstream of the tss, and specifically within the intron 2 region between +1.568 and +2.054 kb. Further studies are aimed at identifying the trans-acting factors that bind this region of DNA to control transcription of the IIb gene in the normal rat P and its down-regulation during hypertrophy. Supported by NIH grant AR30346