Expression and splicing of the insulin-like growth factor gene in rodent muscle is associated with muscle satellite (stem) cell activation following local tissue damage

Abstract

Muscle satellite cells are mononuclear cells that remain in a quiescent state until activated when they proliferate and fuse with muscle fibres to donate nuclei, a process necessary for post-embryonic growth, hypertrophy and tissue repair in this post-mitotic tissue. These processes have been associated with expression of the insulin-like growth factor (IGF-I) gene that can undergo alternative splicing to generate different gene products with varying functions. To gain insight into the cellular mechanisms involved in local tissue repair, the time courses of expression of two IGF-I splice variants produced in muscle were determined together with marker genes for satellite cell activation following local muscle damage. Using real-time RT-PCR with specific primers, the mRNA transcripts in rat tibialis anterior muscles were measured at different time intervals following either mechanical damage imposed by electrical stimulation of the stretched muscle or damage caused by injection with bupivacaine. It was found that the autocrine splice variant mechano growth factor (MGF) was rapidly expressed and then declined within a few days following both types of damage. Systemic IGF-IEa was more slowly upregulated and its increase was commensurate with the rate of decline in MGF expression. Satellite cell activation as measured by M-cadherin and one of the muscle regulatory factors MyoD and the sequence of expression suggests that the initial pulse of MGF is responsible for satellite cell activation, as the systemic IGF-IEa mRNA expression peaks after the expression of these markers, including M-cadherin protein. Later splicing of the IGF-I gene away from MGF but towards IGF-IEa seems physiologically appropriate as IGF-IEa is the main source of mature IGF-I for upregulation of protein synthesis required to complete the repair.

Figure 1. Real-time RT-PCR analysis

A, analysis screen (top panel) using the Fit Point method in the Roche LightCycler protocol for quantification of specific gene expression. The software uses the selected number of points to calculate a ‘best fit’ long, linear fluorescence curve for each sample. The intersection (marked with a red cross) of this line with the Crossing Line (green horizontal line) is the so-called Crossing Point (expressed as a fractional cycle number, i.e. a cycle number that is calculated to two decimal places). All the Crossing Points of the standards are used to plot a graph of cycle number vs. log concentration (lower panel). This standard curve is used to estimate the concentration of each sample. B, example of a melting curve for MGF LightCycler PCR product: the melting temperature of MGF PCR product was 89.7 °C (blue vertical line) and those of the primer-dimers were lower than 82 °C (yellow and red vertical lines). An extra window with a temperature approximately 10 °C higher than that of the elongation temperature was set during the amplification program; this melted the unspecific LightCycler products at 89 °C and eliminated any non-specific fluorescence signals.

Figure 2. Quantification of MGF and IGF-IEa transcript levels following mechanical damage

Here and in subsequent figures: s/s, stretch and stimulation. A, there was a significant increase (^**P < 0.001) in MGF level in the stretched and stimulated muscle compared to those in the sham and contralateral controls. MGF peaked at 1 day after treatment and decreased thereafter. B, a significant increase (**P < 0.001) in IGF-IEa level, showing maximum expression at day 7, was also demonstrated in the stretched and stimulated muscle compared to those in the sham-operated and normal control muscle groups. Interestingly, the contralateral control (right) muscles, which received increased use in these animals, expressed almost as much IGF-IEa as the damaged muscles. □, left TA, experimental; , right TA, contralateral. For all groups n = 6. Values are expressed as means ±s.e.m.

Figure 3. Quantification of MGF and IGF-IEa transcript levels normalised against GAPDH, following bupivacaine injection

A, MGF levels in the bupivacaine-injected group were significantly higher (**P < 0.001) compared with those of their contralateral muscle and of the saline-injected and non-injected groups from day 4 to day 14 and significantly higher (*P < 0.05) at day 24. MGF levels in the bupivacaine-injected group peaked at day 4 and decreased thereafter. B, IGF-IEa levels were also significantly higher (**P < 0.001) in the bupivacaine-injected group compared to those of their contralateral muscle and of the saline-injected and non-injected groups at 4, 7, 11 and 24 days. IGF-IEa expression was seen to peak 11 days after myotoxin injury, by which time MGF expression had declined considerably. For all groups n = 6. Values are expressed as normalised means ± normalised standard error of the mean (s.e.m.).

Figure 4. Quantification of MyoD transcript levels following stretch and stimulation and bupivacaine injection

A, there was a significant increase (**P < 0.001) in MyoD levels (pg (μg total RNA)⁻¹)in the stretched and stimulated tibialis anterior (TA) muscle at all time points compared to the levels in sham and normal control groups. B, MyoD levels in the bupivacaine-damaged TA and control groups were normalised against GAPDH expression. There was a gradual decrease in MyoD mRNA levels in the bupivacaine-injected TA muscle from day 4 to day 14, with significant differences from control values (**P < 0.001). Orange asterisks indicate statistically significant increases in levels of MyoD in the contralateral and normal muscles compared to those in bupivacaine-injected muscle. For all groups n = 6. Values are expressed as normalised means ± normalised s.e.m.

Figure 5. Quantification of M-cad mRNA following stretch and stimulation and bupivacaine injection

A, there was a significant increase (**P < 0.001) in levels of M-cadherin (M-cad) RNA (pg (μg total RNA)⁻¹) in the stretched and stimulated TA muscle compared to those in the sham control and normal groups at all time points. M-cad showed maximal expression at day 5. B, changes in M-cad protein density levels with time following bupivacaine injection and after mechanical damage. Note that, at the protein level, M-cad gene expression also reaches a maximum after only 5 days. Thereafter it declines. This maximum is preceded by expression of MGF but not IGF-IEa. For all groups n = 6. Values are in arbitrary units as normalised means ± normalised s.e.m.

Figure 6

M-cad staining in cross-sections of a non-injected control TA muscle (A) and a bupivacaine-injected TA muscle 4 days after recovery (B) where M-cad-positive rings can be seen staining the satellite cells underneath the basal lamina in the small regenerating fibres (arrows). Scale bar = 50 μm.