Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function

Abstract

During the aging process, mammals lose up to a third of their skeletal muscle mass and strength. Although the mechanisms underlying this loss are not entirely understood, we attempted to moderate the loss by increasing the regenerative capacity of muscle. This involved the injection of a recombinant adeno-associated virus directing overexpression of insulin-like growth factor I (IGF-I) in differentiated muscle fibers. We demonstrate that the IGF-I expression promotes an average increase of 15% in muscle mass and a 14% increase in strength in young adult mice, and remarkably, prevents aging-related muscle changes in old adult mice, resulting in a 27% increase in strength as compared with uninjected old muscles. Muscle mass and fiber type distributions were maintained at levels similar to those in young adults. We propose that these effects are primarily due to stimulation of muscle regeneration via the activation of satellite cells by IGF-I. This supports the hypothesis that the primary cause of aging-related impairment of muscle function is a cumulative failure to repair damage sustained during muscle utilization. Our results suggest that gene transfer of IGF-I into muscle could form the basis of a human gene therapy for preventing the loss of muscle function associated with aging and may be of benefit in diseases where the rate of damage to skeletal muscle is accelerated.

Figure 1

(a) Schematic of the rAAV)/IGF-I gene construct. Rat IGF-I cDNA expression was driven by a fast muscle specific promoter/enhancer (myosin light chain 1/3) and stabilized by simian virus 40 polyadenylation sequence (SV40pA). The gene construct was inserted into an rAAV transfer plasmid (pSUB201) between the inverted terminal repeats (ITRs). (b) (Top) Persistence of IGF-I expression shown by reverse transcription–PCR. Lane 1, 27-month-old EDL, 9 months post-AAV injection; lane 2, 6 month old EDL, 4 months post-AAV injection; lane 3, uninjected control EDL; lane 4, positive control using plasmid shown in a. (Middle) Constitutive expression of β-cytoplasmic actin in EDL muscles. Lanes 1–3, as described above; Lane 4, positive control using template supplied with oligonucleotides (CLONTECH). (Bottom) Integrity of RNA isolated from EDL muscles. Equal volume RNA samples were run on a nondenaturing agarose gel (Invitrogen). Lanes 1–3, as described above.

Figure 2

(A) Effect of IGF-I expression in EDL muscles of young adult (6 month) mice. Cross-sections stained with hematoxylin/eosin of control and AAV-injected EDL muscles from young adult mouse, 4 months post-AAV injection. AAV-injected EDLs (Upper Right) displayed significant increases in cross-sectional area compared with contralateral control EDLs (Upper Left). Muscle fiber regeneration was evident in injected EDLs (Lower Left) by the presence of central nuclei (arrow marks a central nucleus), which were absent in control sections (Lower Right). [Bars = 100 μm (Upper); 25 μm (Lower).] (B) Effect of IGF-I expression on muscle mass and force generation. The uninjected leg of each animal served as the control (defined as a value of 1) for the injected leg (expressed relative to the control value). Thus muscle mass (wet weight) and isometric tetanic force of AAV-injected muscles were expressed relative to the same measurements in the contralateral control muscle from the same animal. The average relative value (n = 5) for the injected limbs is plotted (±SEM). Asterisks denote a P < 0.05 for paired comparisons (Student’s t tests) between AAV-injected and control muscles.

Figure 3

Effect of IGF-I expression in EDL muscles of old (27 month) mice. (Left) Average (±SEM) muscle mass (n = 7); (Center) average (±SEM) tetanic force (n = 7); (Right) average (±SEM) specific force (n = 7). For determination of force per cross-sectional area (specific force), cross-sectional area was estimated using muscle mass and optimum muscle length (5). Asterisks denote a P < 0.05 for comparisons (Student’s t tests) to young adult control muscles.

Figure 4

Maintenance of IIb fibers in muscle of old (27 month) mice by IGF-I expression. The loss of IIb fibers normally associated with aging was prevented by IGF-I expression in 27-month-old mice (n = 7). No effect on fiber type distribution by IGF-I expression was observed in muscles of 6-month-old mice (n = 5). Asterisks denote a P < 0.05 for comparisons (Student’s t tests) to young adult control muscles. Fiber types were determined as described by Schiaffino and Salviati (16).