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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 3, 2003 as doi:10.1096/fj.03-0293fje. |
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Basic Veterinary Sciences, School of Veterinary Medicine, Irbid-Jordan;
* Departments of Basic Medical Sciences, School of Veterinary Medicine,
Department of Animal Sciences and
Department of Food Sciences, Lipid Chemistry and Molecular Biology Laboratory, Purdue University, West Lafayette, Indiana, USA
2Correspondence: Department of Basic Medical Sciences, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907, USA. E-mail: kmh{at}vet.purdue.edu
SPECIFIC AIM
The specific aim of our study was to identify anabolic proteins that can inhibit disuse atrophy in skeletal muscle and bone when electroporated and ectopically expressed in skeletal muscle in vivo.
PRINCIPAL FINDINGS
1. Electroporation into and ectopic expression of IGF-I and/or Shh in gastrocnemius/soleus muscles of 15-wk-old mice resulted in significantly enlarged muscle fibers, increased muscle mass, increased tibia/fibula bone mineral density (BMD), and bone mineral content (BMC) 7, 14, and 30 days post-DNA injection
IGF-I, Shh, or IGF-I+Shh expression plasmids were injected and electroporated into the gastrocnemius/soleus muscles of 15-wk-old mice. Compared with the contralateral control injected muscle fibers, muscles fibers ectopically expressing IGF-I were on average 46, 51, and 41% larger 7, 14, and 30 days post-DNA injection, respectively (all differences P<0.05). Muscles fibers ectopically expressing Shh were 41, 53, and 47% larger than contralateral control LacZ-injected muscle fibers 7, 14, and 30 days post-DNA injection and electroporation, respectively (all differences P<0.05). Muscles fibers ectopically expressing IGF-I and Shh were 71, 76, and 62% larger than contralateral control injected muscle fibers 7, 14, and 30 days post-DNA injection and electroporation, respectively (all differences P<0.05). Ectopic expression of IGF-I or Shh+IGF-I in the gastrocnemius/soleus significantly increased BMD 5.3 and 5.0%, respectively, in weighted animals (all differences P<0.05). Expression of Shh had no effect on BMD. Expression of Shh+IGF-I increased BMC 7.2% (P<0.05); Shh or IGF-I alone had no effect.
2. Electroporation and ectopic expression of IGF-I and/or Shh within the gastrocnemius/soleus muscle significantly attenuates the loss of muscle fiber area, muscle mass, and muscle mass density that normally occurs during disuse muscle atrophy
IGF-I and/or Shh expression plasmids were electroporated and ectopically expressed in gastrocnemius/soleus muscles of 15-wk-old mice, which were then suspended by the tail for 7 days to induce disuse atrophy. Ten days post-DNA injection (7 days post-hind limb suspension), gastrocnemius/soleus muscles were collected and analyzed. As expected, hind limb suspension caused a progressive loss of muscle mass density, muscle weight, and muscle fiber area. Compared with the contralateral control injected muscles, ectopic expression of IGF-I and/or Shh significantly attenuated the lost muscle mass density associated with suspension. The loss of muscle mass density associated with 7 days of hind limb suspension was inhibited an average 46, 41, and 51% in IGF-I-, Shh-, and IGF-I+Shh-injected suspended muscles, respectively, vs. those of contralateral control injected suspended muscle (all differences P<0.05). Compared with the contralateral control injected suspended muscle, muscle weights of IGF-I-, Shh-, and IGF-I+Shh-injected muscles were an average 13, 14, and 18% larger, respectively, 7 days post-hind limb suspension (Fig. 1
; all differences P<0.05). Finally, muscle fiber areas of IGF-I-, Shh-, and IGF-I+Shh-injected suspended muscles were on average 36, 27, and 40% larger, respectively, than those of contralateral injected suspended muscle 7 days post-hind limb suspension (all differences P<0.05).
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3. Electroporation and ectopic expression of IGF-I and Shh in the gastrocnemius/soleus muscle inhibits parameters of osteopenia in the tibia and fibula associated with hind limb unloading
IGF-I and/or Shh expression plasmids were electroporated and ectopically expressed in gastrocnemius/soleus muscles of 15-wk-old mice and the mice were suspended by the tail for 7 days. Ten days post-DNA injection (7 days post-hind limb suspension), hind limbs were subjected to DXA analysis. Hind limb suspension significantly decreased BMD and BMC. In comparison to the contralateral control limb, ectopic expression of Shh, IGF-I, or Shh + IGF-I within the gastrocnemius/soleus significantly attenuated loss in bone mineral content associated with hind limb suspension (Fig. 2
, P<0.05). While there was a trend for inhibition, ectopic expression of Shh, IGF-I, or Shh + IGF-I had no significant effect on the loss bone mineral density associated with hind limb suspension.
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CONCLUSIONS AND SIGNIFICANCE
The loss of normal weight-bearing activity that occurs during bed rest, limb immobilization, and spaceflight stimulates a catabolic response within the musculoskeletal system that results in loss of skeletal muscle mass and bone mineral. The mechanism by which loading of muscle and bone is sensed and translated into signals controlling tissue formation remains a major question in the field of musculoskeletal research. No universally effective treatments are currently available for musculoskeletal atrophy because of the dearth of information to advance the knowledge of muscle and bone relationships. To our knowledge, no studies have analyzed the relationship and interactions between muscle and bone during loading and unloading in vivo. Muscle-derived cells have been used as a source of inducible osteoprogenitor cells for bone healing. However, the concept of muscle regulating bone homeostasis, or vice versa, during unloading has not been rigorously investigated. Recent advances in DNA electroporation into skeletal muscle in vivo allow for sustained, high-level expression of cDNA constructs within muscle myofibers. This technique offers an efficient alternative method for production of therapeutic proteins from skeletal muscle. We have examined the ability of two potentially anti-atrophic proteins, IGF-I and Shh, to inhibit disuse atrophy within muscle and bone when electroporated into skeletal muscle. We found that electroporation and ectopic expression of IGF-I and/or Shh significantly attenuated the lost of muscle atrophy and osteopenia associated with hind limb unloading. This data would be the first to demonstrate that growth factors produced by skeletal muscle following electroporation appear to regulate the maintenance of adjacent bones during periods of disuse. It will be important to determine the exact cellular and molecular mechanism(s) by which muscle-derived IGF-I and Shh inhibit decreases in both muscle size and bone mineral during disuse. Our results support the theory that skeletal muscle can regulate bone maintenance and could potentially offer novel and efficient therapeutic options for attenuating muscle and bone atrophy during aging, illness, and spaceflight.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0293fje 
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