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63 MYOSTATIN SHORT INTERFERING HAIRPIN RIBONUCLEIC ACID GENE TRANSFER INCREASES MUSCLE MASS AND REDUCES INTERSTITIAL CONNECTIVE TISSUE.

Purpose Myostatin negatively regulates skeletal muscle growth. Myostatin-knockout mice exhibit muscle hypertrophy and decreased interstitial fibrosis. We investigated whether a plasmid expressing a short hairpin interfering RNA (shRNA) against myostatin and transduced using electroporation would increase local skeletal muscle mass and reduce interstitial connective tissue. Methods Short interfering RNAs (siRNAs) targeting myostatin were transfected into HEK293 cells and identified for myostatin silencing by Western blot. Corresponding shRNAs were cloned into plasmid shRNA expression vectors. Myostatin or a randomer negative control shRNA plasmid were injected and electroporated into the tibialis anterior or its contralateral muscle, respectively, of 10 rats that were sacrificed after 2 weeks. Six other rats received a beta-galactosidase reporter plasmid and were sacrificed at 1, 2, and 4 weeks. Uptake of plasmid was examined by beta-galactosidase expression, whereas myostatin expression was determined by real-time PCR and Western blotting. Muscle fiber size and collagen expression were determined by histochemistry. Satellite cell proliferation was determined by PAX7 immunohistochemistry. Myosin heavy chain type II expression was determined by Western blot. Results Beta-galactosidase reporter plasmid was expressed at 1 and 2 weeks but diminished by 4 weeks. Myostatin shRNA reduced myostatin mRNA and protein expression by 27 and 48%, respectively. Tibialis weight, fiber size, and myosin heavy chain II increased by 10, 34, and 38%, respectively. Satellite cell number was increased by 64%. Interstitial collagen decreased by 40%. Conclusions This is the first demonstration that myostatin shRNA gene transfer is a potential strategy to increase muscle mass while reducing interstitial connective tissue.