Expression of IGF-I splice variants in young and old human skeletal muscle after high resistance exercise

Abstract

The mRNA expression of two splice variants of the insulin-like growth factor-I (IGF-I) gene, IGF-IEa and mechano growth factor (MGF), were studied in human skeletal muscle. Subjects (eight young, aged 25-36 years, and seven elderly, aged 70-82 years) completed 10 sets of six repetitions of single legged knee extensor exercise at 80 % of their one repetition maximum. Muscle biopsy samples were obtained from the quadriceps muscle of both the control and exercised legs 2.5 h after completion of the exercise bout. Expression levels of the IGF-I mRNA transcripts were determined using real-time quantitative RT-PCR with specific primers. The resting levels of MGF were significantly (approximately 100-fold) lower than those of the IGF-IEa isoform. No difference was observed between the resting levels of the two isoforms between the two subject groups. High resistance exercise resulted in a significant increase in MGF mRNA in the young, but not in the elderly subjects. No changes in IGF-IEa mRNA levels were observed as a result of exercise in either group. The mRNA levels of the transcription factor MyoD were greater at rest in the older subjects (P < 0.05), but there was no significant effect of the exercise bout. Electrophoretic separation of myosin heavy chain (MHC) isoforms showed the older subjects to have a lower (P < 0.05) percentage of MHC-II isoforms than the young subjects. However, no association was observed between the composition of the muscle and changes in the IGF-I isoforms with exercise. The data from this study show an attenuated MGF response to high resistance exercise in the older subjects, indicative of age-related desensitivity to mechanical loading. The data in young subjects indicate that the MGF and IGF-IEa isoforms are differentially regulated in human skeletal muscle.

Figures

Figure 1. IGF-I gene

A schematic representation of the IGF-I gene and its splice variants expressed in muscle. The black boxes denote the insert in exon 5 (49 bp), which gives rise to the alternatively spliced MGF isoform.

Figure 2. Measurement of muscle gene expression using real time RT-PCR

A, amplification profile of standards and a sample show similar amplification efficiency. B, the standard curve generated by the LightCycler software ranging from 1 × 10−2 to 1 × 10−7 ng of MGF DNA. For each sample the crossing point was plotted against the known concentration of the standard. The resulting standard curve is shown as a graph of cycle number vs. log concentration. All calculated ‘unknown’ sample values must fall within the limits of the standards used to generate this curve. C, the melting curve profile of MGF PCR products. After the end of the PCR all products are gradually melted down in 0.1 °C increments until all products have been denatured. The melting curve profile of a specific product is easily identified as a sharp, narrow peak. Non-specific amplifications, such as primer dimers tend to melt at much lower temperatures and over a broader range and can therefore be detected with the melt curve analysis. Only runs that after careful inspection did not show evidence of non-specific binding were used. The area under the overall melting peaks relates to the total amount of amplification products. In this instance it can be seen that the specific MGF product melts at 80 °C. D, 2 % agarose gel picture showing PCR products for each of the targets measured. There are 4 products shown for each target - these correspond to a young subject (pre- and post-exercise) and an old subject (pre- and post-exercise). A single peak in the melting curve profile (C) corresponds to a single band of the predicted size. Lanes 1 and 10: 100 bp DNA size marker; lanes 2–5: IGF-IEa; lanes 6–9: MGF; lanes 11–14: α-actin; and lanes 15–18: MyoD.

Figure 3. Example of electrophoretic separation of myosin heavy chain isoforms

Separation of MHC isoforms using SDS-PAGE. Lane 2 shows subject RS with a total of 33 % MHC-IIX. None of the other subjects showed any other detectable amounts of the MHC-IIX isoform.

Figure 4. Change in MGF and IGF-IEa in young and old muscles after exercise

A, MGF mRNA levels in young and old subjects before (control) and after (test) exercise. Values are expressed as ng of mRNA per µg total RNA (mean ± s.e.m.). There is a significant increase in MGF mRNA 2.5 h after exercise in the young subjects (P < 0.05). The subject represented by the dotted line showed the most dramatic response to the exercise and was also the only subject to express a high proportion of MHC-IIX isoforms in his quadriceps muscle. B, IGF-IEa mRNA levels in young and old subjects before (control) and after (test) exercise. Values are expressed as ng of mRNA per µg total RNA (means ± s.e.m.).

Figure 5. MyoD mRNA levels in young and old subjects before (control) and after (test) exercise

Values are expressed as ng of mRNA per µg total RNA (mean ± s.e.m.). Older subjects express significantly higher baseline levels of MyoD mRNA when compared with young subjects (*P < 0.05).