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Department of Anatomy and Cell Biology, School of Dental Medicine, and Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, Pennsylvania
Submitted 7 November 2005 ; accepted in final form 20 January 2006
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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adeno-associated virus; E peptide; IGF-I splicing
Although there is general agreement that the IGF-I protein is one of the major mediators of these hypertrophic actions, there is a growing body of evidence that suggests that the isoform from which IGF-I is produced can affect its potency. Through multiple splicing at both the 5' and 3' ends of the gene, multiple classes, or isoforms, arise in mammals and fish (1, 8, 20). In rodents, there are four possible classes of IGF-I resulting from alternative splicing events, where class 1 and 2 denote the use of exon 1 or 2, respectively, and class A and B denote the absence or presence of exon 5 (Fig. 1A). All classes of prepropeptides produce an identical IGF-I protein. However, IGF-IB contains exon 5 of the Igf-1 gene, which causes a frame shift and premature termination in exon 6 and results in a different E peptide extension (17, 20) in the IGF-I propeptide. IGF-IA is the predominant form expressed by muscle, liver, and several other tissues. The rodent IGF-IB is equivalent to human IGF-IC and has also been called mechano-growth factor (MGF) to indicate the increased expression of this isoform in response to stretch or damage (29, 30). Increased expression of human IGF-IC in response to eccentric contraction has been shown to diminish with age (18). MGF has been proposed to be a more potent form of IGF-I for promoting skeletal muscle hypertrophy after plasmid injection, to promote proliferation of muscle cells in culture, and to act through a novel receptor independent of the known IGF-I receptor (17, 31).
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To determine whether class 1 A and B IGF-I mediated different levels of skeletal muscle hypertrophy, viral-mediated gene transfer of each class was performed in young and adult mice. Young mice are assumed to have an active satellite pool as normal muscle growth progresses. In contrast, adult mice have ceased active growth, and so the satellite cell pool is dormant unless activated by damage. The use of two age groups provided the ability to distinguish the response of muscle to the IGF-I isoforms.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Injections. All experiments were approved by the university animal care committee. Animals were lightly anesthetized with ketamine-xylazine, and 1 x 1011 viral particles diluted in 75–100 µl of phosphate-buffered saline (PBS) were injected into the anterior compartment of one lower hindlimb of C57Bl/6 mice (male and female), targeting the tibialis anterior (TA) and extensor digitorum longus (EDL). The contralateral limb received an equal volume of PBS in the same manner as a control for the injection procedure. After injection, mice were allowed to recover from anesthesia and were housed in the animal facility until time of analysis. In one study, mice were 2–3 wk of age at the time of injection. Mice were killed at 1, 2, and 4 mo postinjection for analysis (n = 4–8 each time point and construct). In a second study, mice were injected at 6 mo of age and killed 4 mo postinjection for analysis (n = 7 each construct).
Functional analysis. Mice were anesthetized with ketamine-xylazine and exsanguinated. Blood samples were allowed to clot for 2 h at room temperature and then were centrifuged at 2,000 g for 20 min and then stored at –80°C for subsequent IGF-I content measurements. The TA and EDL muscles were removed and placed in a bath of Ringer solution gas equilibrated with 95% O2–5% CO2. The TA either was blotted, weighed, and rapidly frozen in liquid nitrogen for immunoblotting analysis or, after weighing, was homogenized in 1 ml of sterile PBS and frozen in liquid nitrogen for IGF-I content measurements. The EDL muscles were subjected to isolated muscle mechanical measurements by using a previously described apparatus (Aurora Scientific, Aurora, Ontario, Canada) (5). After determination of optimum length by single supramaximal twitch stimulation, maximum isometric tetanus was measured in the muscles from both limbs. On completion of mechanical measurements, EDL muscles were blotted, weighed, and rapidly frozen in melting isopentane.
Expression analysis. Muscles were removed from liquid nitrogen storage and ground with a mortar and pestle cooled with dry ice, and total RNA was isolated from the resultant powdered tissue (Trizol, Invitrogen, Carlsbad, CA). Quantification of RNA was performed with Ribogreen quantitation kit (Molecular Probes, Eugene, OR), and RNA integrity was confirmed by gel electrophoresis. Total RNA (150 ng) from each sample was subjected to single-strand reverse transcription (Applied Biosystems, Foster City, CA). The resultant cDNA was utilized for real-time PCR with oligonucleotides that were specific for IGF-IA and IGF-IB using the Roche Lightcycler system, and reagents (LightCycler FastStart DNA MasterPLUS SYBRgreen I). The location of these oligonucleotides within the IGF-I sequences and the resultant band size of the products are depicted in Fig. 1, A and C. The exon 6 oligonucleotide could detect both IGF-IA and IGF-IB, whereas the exon 5 oligonucleotide could only detect IGF-IB. Real-time PCR reactions contained 6 µl of cDNA, SYBRgreen master mix, 0.5 µM of each primer and nuclease free water to a total volume of 20 µl. IGF-I mRNA levels were assessed by calculating the crossing point (Cp), where measured fluorescence rises above background, as the second derivative maximum of the reaction curve. Each sample was analyzed in duplicate, and the resulting data were averaged. Quantification of expressed viral transgene copies was achieved by using plasmids containing the respective cDNAs. Serial dilutions of the plasmids provided the expected changes in Cp such that a 10-fold decrease in plasmid copy number resulted in a Cp that was approximately three cycles longer than the previous dilution. Melting-point analysis of control plasmids and experimental samples confirmed that the primers were specific for IGF-I, where there was only one melting point for each experimental sample that was not different from the melting point with control plasmids. Controls included only RNA that was not subjected to reverse transcription and water.
Immunoblotting analysis. TA muscles were removed from liquid nitrogen storage and homogenized in 10 volumes/muscle wet weight of modified lysis buffer (50 mM Tris·HCl pH 7.4, 1% wt/vol Triton X-100, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM NaVO4, 1 mM NaF, 1 mM EGTA). Homogenates were centrifuged to pellet debris, and the total protein was measured in the supernatant. Equal amounts of protein from each muscle lysate were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Immobilon-P, Millipore, Bedford, MA). Membranes were incubated in a blocking buffer [5% nonfat dry milk in Tris-buffered saline (TBS) plus 0.1% Tween 20 (5% milk-TTBS)] and then incubated in primary antibody diluted in 5% milk-TTBS overnight at 4°C. The following antibodies were used: phospho-Akt (no. 9271), total Akt (no. 9272), phospho-ERK1/2 (no. 9101), total ERK1/2 (no. 9102) (Cell Signaling, Beverly, MA). Membranes were then washed in 5% milk-TTBS and incubated with horseradish peroxidase-conjugated secondary antibody. After a series of washes in 5% milk-TTBS, TTBS, and TBS, protein detection was performed using enhanced chemiluminescence and the Kodak mm4000 detection system. Analysis of band intensity was performed by use of the associated image analysis software. Membranes were stained with Coomassie brilliant blue R-250 after immunoblotting to confirm equal protein loading.
Immunoprecipitation of IGF-I receptor complexes. Frozen TA muscles were ground and homogenized in buffer [100 mM HEPES (pH = 7.4); 150 mM NaCl; 5 mM EDTA; 5 mM MgCl2; 2% NP-40; 1.5% Triton X-100, protease and phosphatase inhibitors (Sigma, St. Louis, MO)] and incubated at 4°C for 90 min with frequent vortexing. Samples were centrifuged at 10,000 g for 10 min at 4°C. Supernatants were transferred to new tubes and centrifuged at 150,000 g for 90 min at 4°C. The resultant supernatant was utilized for immunoprecipitation. A total of 500 µg of protein was incubated with protein A/G agarose (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min at 4°C. Samples were centrifuged for 2 min to collect the pellet, and the supernatant was exposed overnight to IGF-IR antibody (C-20, Santa Cruz Biotechnology) on a rocker at 4°C. Protein A/G agarose was then added for 6 h. The agarose pellet was collected by centrifugation and washed four times with PBS, then resuspended in 40 µl of 2x sample buffer, which was subjected to SDS-PAGE and subsequent immunoblotting (described in the previous section) with P-Tyr antibody (P-Tyr-100, no. 9411, Cell Signaling) to measure phosphorylated IGF-IR or IGF-IR antibody to measure total receptor.
Detection of IGF-I protein. The serum and TA homogenates were thawed, and the homogenates were centrifuged at 5,000 g for 5 min. The serum and supernatants were utilized for measurements of total IGF-I in a commercially available ELISA kit specific for rodent IGF-I (R&D Systems, Minneapolis, MN), and calculations of IGF-I content were based on a standard curve generated from recombinant mouse IGF-I. This kit detects total rodent IGF-I, and the measurements are not affected by the presence of IGF-I binding proteins or IGF-II. The assay can accurately detect IGF-I at 30–2,000 pg/ml with an intra-assay precision of 4.3% and an interassay precision of 5.9%. Preliminary experiments were performed on muscle samples from transgenic mice expressing rat IGF-IA under the MLC1/3 promoter/enhancer (21) to validate that the procedures used in the present study could accurately determine total IGF-I to the same degree as the acid-ethanol procedure used in a former study (3, 11). Preliminary experiments were also performed to verify that the TA and EDL from the same injection had similar IGF-I content. Measurements were acquired on a microtiter-plate reader (Dynatech Laboratories, Chantilly, VA) at 450 nm. All samples were measured in duplicate.
Statistics. Data are presented as means ± SE. Paired t-tests were utilized for comparisons between adeno-associated virus (AAV)-injected and contralateral control samples. One-way ANOVA was utilized for comparisons between IGF-I isoform treatments. Statistical significance was accepted for P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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IGF-I can activate both the PI3K/Akt pathway and the MAPK pathways via IGF-I receptor binding and activation (6, 10, 23). To test for differences in activation of these pathways by the IGF-I isoforms, immunoblotting was performed on muscle homogenates 1 mo postinjection (n = 3 muscle pairs for each IGF-I isoform). Both IGF-I isoforms produced robust IGF-IR and Akt phosphorylation compared with sham-injected control limbs (Fig. 2). Injection of rAAVIGF-IB also resulted in an increase of phosphorylated ERK1/2, which was not evident in rAAVIGF-IA-injected muscles and also produced more phosphorylation of IGF-IR than IGF-IA. There was no change in the content of IGF-IR, Akt, or ERK1/2 in rAAV-injected limbs compared with sham-injected limbs.
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5% increase in muscle mass only in IGF-IA AAV- injected muscles, whereas no difference could be detected in IGF-IB AAV-injected muscles compared with contralateral control limbs. Tetanic force increased in concert with hypertrophy, and, as in young muscles, specific force was not changed due to injection of either isoform (Fig. 6B). Comparisons between young and mature animals at 4 mo postinjection showed that there were no statistical differences in mass increases.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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The recent discovery of new AAV serotypes has improved the ability for viral-mediated gene delivery to efficiently transduce target tissues. AAV serotype 8 (AAV-2 genomes pseudo-packaged into AAV-8 capsids) has very high tropism for skeletal muscle and an accelerated time course for gene expression (15, 26). As shown in Figs. 1D and 2A, high levels of IGF-I RNA and protein are present as early as 1 mo postinjection, which is more rapid than that achieved in former studies of IGF-I-mediated hypertrophy with different serotypes (4, 19). The deletion of the 5' untranslated region (UTR) in the constructs utilized in this study could also contribute to increased total IGF-I. Viral expression of class A and B IGF-I exceeded levels obtained by transgenic expression with the same promoter (3, 21) and approached those obtained by transgenic expression of human IGF-IA lacking the 5' UTR (9). The 5' UTR harbors multiple AUG translation initiation codons followed by in-frame termination codons that can impair the ability to initiate protein synthesis at the correct authentic start site (13, 14, 27). Therefore, the removal of these regulatory regions from the open reading frame of Igf-1 gene improved production of IGF-I from a strong skeletal muscle- specific promoter.
Even with these modifications, it is surprising that that rAAVIGF-IB produced significantly more IGF-I mRNA and protein than rAAVIGF-IA. Identical numbers of viral particles were introduced for each construct, so the difference cannot be explained by differences in viral titer. The sole difference between the construct is the inclusion of exon 5 in rAAVIGF-IB. In light of the lower endogenous expression of IGF-IB that has been observed in other studies (18), it is possible that there is enhanced transcription and translation of the Igf-1B that results in greater accumulation of IGF-I protein. This issue was not resolved in these experiments, and the effect that the E peptide has on the efficiency of transcription or translation will require testing in future studies.
The signaling pattern also differs between the two IGF-I isoforms. As former studies in vitro and in vivo have established, the PI-3K/Akt pathway has been linked predominantly to myogenic actions and the hypertrophic responses to IGF-I, whereas both the PI-3K/Akt and MAPK pathways have been implicated in mitogenic actions of IGF-I (6, 7, 10, 22, 23). The fact that only IGF-IB caused increased P-ERK1/2 was consistent with previous observations that the EB peptide promoted cell proliferation (24, 31). However, at the 1-mo time point in the study, rAAVIGF-IB produced approximately seven times more IGF-I than rAAVIGF-IA, and so it is not clear whether ERK1/2 phosphorylation is the result of differences in isoform specification to signal transduction pathways or merely that the increased level of IGF-I production by AAVIGF-IB reveals the dose-response sensitivity of these signaling pathways.
It is intriguing that, even with higher production of total IGF-I by rAAVIGF-IB, the level of hypertrophy achieved in young growing animals by either construct was indistinguishable. In both cases, the IGF-I levels must have been above the necessary threshold for hypertrophy to occur. Alternatively, the bioavailability of IGF-I could have been restricted by increased expression of binding proteins. Only total IGF-I was measured, and so it is not known how much free IGF-I was available to bind to IGF-I receptors. Whether IGF-IB affects binding protein expression or has greater affinity for existing binding proteins in the muscle matrix has yet to be determined but will be important to clarify in future studies.
The effects of rAAVIGF-IA in mature animals, where a 5% increase in mass was achieved, are reminiscent of the amount of hypertrophy achieved after gamma radiation (5). The presence of viral IGF-I in irradiated muscle produced a 5% increase in muscle mass compared with irradiation alone. In both cases, the satellite cell pool is quiescent, either by inhibition of proliferation by irradiation in the former study, or because there is little active growth in 6-mo-old animals utilized in the present study. The ability for rAAVIGF-IA to promote modest hypertrophy in the absence of an active satellite cell pool suggests that this isoform can bind to and activate IGF-I receptors on the muscle fibers.
Although the serotype and exact sequence injected differs between the present and former studies (4, 5, 19), the increased IGF-IA expression promoted equivalent levels of hypertrophy in young growing animals. However, the effect of IGF-IA in mature animals may be quite different than that observed in aging animals. As described above, increased IGF-IA could promote modest hypertrophy in mature animals, whereas muscle mass was maintained and not increased as the animals aged from 18 to 27 mo (4). Whether the 18-mo-old animals exhibited hypertrophy to the same extent as 6-mo-old animals at 4 mo postinjection or whether by 27 mo of age the 6-mo-old treated animals still exhibit hypertrophy has not been addressed. These comparisons will help to clarify the importance of an activated satellite cell pool vs. muscle fiber receptor activation in promotion of muscle hypertrophy and maintenance of muscle mass.
In contrast, the introduction of rAAVIGF-IB did not affect muscle mass at all in mature animals, even though there was more total IGF-I present. In addition, unlike plasmid delivery of IGF-IB (MGF) to young animals in which hypertrophy was observed 2 wk postinjection (16), viral delivery of IGF-IB did not promote hypertrophy until 2 mo after injection. Again, these differences might be explained by the presence of an activated satellite cell population subjected to IGF-IB. Plasmid delivery provides rapid, although inefficient expression, such that transgene expression is concurrent with the damage inevitably caused by the injection itself and with satellite cells that have been activated to repair the muscle tissue (28). If the target for IGF-IB is an activated satellite cell, there is a pool of them surrounding the injection site for the IGF-IB plasmid. Unlike plasmid DNA, rAAV DNA is single stranded and requires complementation before expression, therefore delaying expression until 2 wk postinjection. At this point, the muscle repair process is well underway, and the pool of activated satellite cells is reduced as they fuse to muscle fibers. The temporal separation between damage due to injection and expression of IGF-IB limits the population of activated satellite cells to those involved in normal growth.
These results suggest three possibilities. First, more of the IGF-I produced by rAAVIGF-IB could be bound to IGF-I binding proteins and is therefore unavailable for activating the IGF-I receptors on the muscle fibers. Second, the IGF-I receptors on the muscle fibers might have a lower affinity for IGF-I produced by rAAVIGF-IB. Third, IGF-I produced by rAAVIGF-IB might only bind to receptors on activated satellite cells. Because the IGF-I protein produced by any isoform is identical, these potential mechanisms would require that the remaining E peptide extension be involved in dictating the responses observed in this study. The generation of constructs harboring only the E peptide without the sequence encoding IGF-I as well as constructs lacking the sequence for the E peptide will be required to test how important the E peptide extensions are for IGF-I effects in skeletal muscle.
Previous studies have identified the EB peptide as a key effector of the mitogenic actions of IGF-I. The results described here are, in part, consistent with this hypothesis, for the proliferative effects in skeletal muscle rely on activated satellite cells. When there is an active satellite cell pool, such as in young growing muscle, IGF-IB can promote hypertrophy to an extent similar to that achieved by IGF-IA. When active satellite cells are diminished, such as in the 6-mo-old animals, IGF-IB cannot promote hypertrophy. Alternative proposals have suggested the presence of a novel receptor to which only IGF-IB can bind (24, 31). Although the present study cannot rule out this possibility, it is clear that both isoforms can activate the IGF-IR. However, the parameters tested here indicate that the proposed novel receptor must only be available in young growing animals, and so identification might be achieved in comparisons of receptor populations of young and old muscle.
In summary, a comparison of the effects of class I IGF-IA and IGF-IB in skeletal muscle reveal that the isoforms are not identical. Production of IGF-I is achieved more efficiently by IGF-IB. However, the ability of this isoform to promote muscle hypertrophy is not greater than IGF-IA in young growing muscle, and it is virtually nonexistent in adult muscle. These observations are counter to the implication that this isoform is a more potent form of IGF-I, yet there are clear differences in the accumulation and bioavailability between the classes of IGF-I, which should be explored further.
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GRANTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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P < 0.05).
