Expression of insulin growth factor-1 splice variants and structural genes in rabbit skeletal muscle induced by stretch and stimulation

Abstract

1. Skeletal muscle is a major source of circulating insulin growth factor-1 (IGF-1), particularly during exercise. It expresses two main isoforms. One of the muscle IGF-1 isoforms (muscle L.IGF-1) is similar to the main liver IGF-1 and presumably has an endocrine action. The other muscle isoform as a result of alternative splicing has a different 3' exon sequence and is apparently designed for an autocrine/paracrine action (mechano-growth factor, MGF). Using RNase protection assays with a probe that distinguishes these differently spliced forms of IGF-1, their expression and also the expression of two structural genes was measured in rabbit extensor digitorum longus muscles subjected to different mechanical signals. 2. Within 4 days, stretch using plaster cast immobilization with the limb in the plantar flexed position resulted in marked upregulation of both forms of IGF-1 mRNA. Electrical stimulation at 10 Hz combined with stretch (overload) resulted in an even greater increase of both types of IGF-1 transcript, whereas electrical stimulation alone, i.e. without stretch, resulted in no significant increase over muscle from sham-operated controls. Previously, it was shown that stretch combined with electrical stimulation of the dorsiflexor muscles in the adult rabbit results in a marked increase in muscle mass involving increases in both length and girth, within a few days. The expression of both systemic and autocrine IGF-1 growth factors provides a link between the mechanical signal and the marked increase in the structural gene expression involved in tissue remodelling and repair. 3. The expression of the beta actin gene was seen to be markedly upregulated in the stretched and stretched/stimulated muscles. It was concluded that the increased expression of this cytoskeletal protein gene is an indication that the production of IGF-1 may initially be a response to local damage. 4. Switches in muscle fibre phenotype were studied using a specific gene probe for the 2X myosin heavy chain gene. Type 2X expression was found to decrease markedly with stimulation alone and when electrical stimulation was combined with stretch. Unlike the induction of IGF-1 and beta actin, the decreased expression of the 2X myosin mRNA was less marked in the 'stretch only' muscles. This indicates that the interconversion of fibre type 2X to 2A may in some situations be commensurate with, but not under the control of IGF-1.

Figure 1. IGF-1 mRNA expression in skeletal muscle after 4 days of stretch only, electrical stimulation only, and stretch combined with electrical stimulation presented in triplicate together with controls

A, RNase protection assay after 2 days exposure of alternatively spliced IGF-1 mRNA transcripts expressed in rabbit EDL muscle after 4 days of stretch and/or electrical stimulation. The RNA probe used was complementary to exons 4-5-6. The probe fully protected the mRNA of the MGF isoform but resulted in two bands from the muscle L.IGF-1 mRNA. A 10 μg sample of total RNA from EDL muscle from each individual rabbit was used for each lane. Yeast tRNA was used to determine the specificity of the RNA probe. The radiolabelled RNA marker was generated by in vitro transcription using the RNA century marker template set (Ambion) and T7 RNA polymerase. Stretch, left hindlimb subjected to stretch in the plantar flexed position using a plaster cast; Stim, stimulation at 10 Hz continuously; St/St, stretch combined with electrical stimulation at 10 Hz continuously. B, quantification of RNase protection assay results by PhosphoImage analysis in triplicate. Data are expressed with s.e.m. and significance levels are given in the text.

Figure 2. In situ hybridization showing the localization of expression of the IGF-1 gene in muscles that were subjected to stretch

The probe used was DIG-labelled cRNA from a 280 bp that covers exon 4, which is common to the two forms of IGF-1 (Yang et al. 1996). Left, control muscle; right, muscle subjected to stretch by plastercast immobilization for 4 days. Scale bars, 100 μm.

Figure 3. Results of RNase protection assays for β actin mRNA expression in rabbit muscle after 4 days of stretch and/or electrical stimulation

A, autoradiograph of the protected bands. Samples were separated on a 5 % denaturing polyacrylamide gel and the dried gel was exposed to X-ray film at room temperature for 30 min. B, quantification of the RNase protection assay results (in triplicate) by scanning densitometry. Data are expressed with s.e.m. Significance values are given in the text.

Figure 4. 2X myosin heavy chain mRNA expression in rabbit muscle after 4 days of stretch and/or electrical stimulation

A, autoradiograph of the protected bands. Samples were separated on a 5 % denaturing polyacrylamide gel and the dried gel was exposed to X-ray film at room temperature for l h. B, quantification of the RNase assay results (in triplicate) by PhosphoImage analysis. Data are expressed with s.e.m. Significance values are given in the text.