Impact of viral-mediated IGF-I gene transfer on skeletal muscle following cast immobilization

Abstract

Insulin-like growth factor I (IGF-I) is a potent myogenic factor that plays a critical role in muscle regeneration and muscle hypertrophy. The purpose of this study was to evaluate the effect of IGF-I overexpression on the recovery of muscle size and function during reloading/reambulation after a period of cast immobilization in predominantly fast twitch muscles. In addition, we investigated concomitant molecular responses in IGF-I receptor and binding proteins (BPs). Recombinant adeno-associated virus vector for IGF-I (rAAV-IGF-IA) was injected into the anterior compartment of one of the hindlimbs of young (3 wk) C57BL6 female mice. At 20 wk of age, both hindlimbs were cast immobilized in a shortened position for 2 wk to unload the tibialis anterior (TA) and extensor longus digitorum (EDL) muscles. The TA and EDL muscles were removed bilaterally after 2 wk of cast immobilization and after 1 and 3 wk of free cage reambulation. Increases in IGF-I mRNA and protein levels with IGF-I overexpression were associated with significant increases in muscle wet weight, fiber size, and tetanic force, although overexpression did not protect against cast immobilization-induced muscle atrophy. After 1 wk of reambulation, evidence of enhanced muscle regeneration was noted in IGF-I-overexpressing muscles with an increased prevalence of central nuclei, embryonic myosin, and Pax7 positive fibers. We also observed larger relative gains in muscle size (wet weight and fiber area), but not force, during the 3-wk reambulation period in hindlimb muscles overexpressing IGF-I compared with contralateral control legs. Changes in IGFBP-5 mRNA expression during cast immobilization and reambulation paralleled those of IGF-I, whereas IGFBP-3 expression changed inversely to IGFBP-5.

Fig. 1.

Tibialis anterior (TA) IGF-I protein levels. Values are means ± SE. *Significant difference within each loading time point between IGF-I-injected and PBS-injected muscles. †Value at this loading time point was significantly different from noncasted group in the same injection group. An interaction of group (IGF-I injected vs. PBS injected) and time was detected where IGF-I-injected muscles had a significantly higher increase in IGF-I protein (not indicated on figure). P < 0.05. 2 wk Immob, 2 wk of cast immobilization; 1 wk Reamb, 1 wk of free cage reambulation; 3 wk Reamb, 3 wk of free cage reambulation.

Fig. 2.

A: extensor digitorum longus (EDL) wet weights. B: EDL tetanic force. C: EDL specific force. Values are means ± SE. *Significant difference within each loading time point between IGF-I-injected and PBS-injected muscles. †Value at this loading time point was significantly different from noncasted group in the same injection group (P < 0.05).

Fig. 3.

A: EDL fiber cross-sectional area (μm²). Fiber type size distributions as a percentage of total fibers before casting (B), after casting (C), after 1 wk Reamb (D), and after 3 wk Reamb (E). Note the fiber size shift in muscle injected with IGF-I by 3 wk Reamb. Values are means ± SE. *Significant difference within each loading time point between IGF-I-injected and PBS-injected muscles. †Value at this loading time point was significantly different from noncasted group in the same injection group (P < 0.05).

Fig. 4.

Presence of central nuclei (%) in IGF-I-injected and PBS-injected EDL muscles. A: cross-sections of EDL muscle stained with hematoxylin and eosin. Central nuclei (arrows) were most apparent after 1 wk Reamb in IGF-I-injected muscle. B: cross-sections of EDL muscle stained with monoclonal antibody against embryonic myosin isoform (green). Muscle fibers expressing embryonic myosin were most notable after 1 wk Reamb in IGF-I-injected muscle. C: %central nuclei at each time point (means ± SE). D: no. of fibers expressing embryonic myosin per 100 fibers. Values are means ± SE. *Significant difference within each loading time point between IGF-I-injected and PBS-injected muscles. †Value at this loading time point was significantly different from noncasted group in the same injection group. An interaction of group (IGF-I injected vs. PBS injected) and time was detected where IGF-I-injected muscles had a significantly higher increase in central nuclei and embryonic myosin-positive fibers (not indicated on figure). P < 0.05. Bar, 50 μm.

Fig. 5.

Satellite cell activity in muscle fibers for IGF-I-injected and PBS-injected EDL muscles. A: cross-sections of EDL muscle stained with laminin + 4,6-diamidino-2-phenylindole + paired box transcription factor 7 (Pax7). Pax7-positive fibers (arrows) are most apparent after 1 wk Reamb in IGF-I-injected muscle. B: percentage of Pax7-positive fibers at each time point. Values are means ± SE. *Significant difference within each loading time point between IGF-I injected and PBS injected muscles. †Value at this loading time point was significantly different from noncasted group in the same injection group. An interaction of group (IGF-I injected vs. PBS injected) and time was detected where IGF-I-injected muscles had a significantly higher increase in Pax7-positive fibers (not indicated on figure). P < 0.05. Bar, 25 μm.

Fig. 6.

EDL protein synthesis (%/day). Values are means ± SE. †Value at this loading time point was significantly different from noncasted group in the same injection group (P < 0.05).

Fig. 7.

%Change in EDL muscle wet weight, tetanic force, and fiber cross-sectional area (CSA). A: normalization of 2 wk Immob values to baseline. B: normalization of 3 wk Reamb values to 2 wk Immob. *Significant difference between IGF-I-injected and PBS-injected groups (P < 0.05).