Myostatin short interfering hairpin RNA gene transfer increases skeletal muscle

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J Gene Med. 2006 Jun 29

Myostatin short interfering hairpin RNA gene transfer increases

skeletal muscle mass.

Magee TR, Artaza JN, Ferrini MG, Vernet D, Zuniga FI, Cantini L,

Reisz-Porszasz S, Rajfer J, -Cadavid NF.

Department of Urology, Geffen School of Medicine, UCLA, Los

Angeles, CA 90024, USA.

BACKGROUND: 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.

METHODS: Short interfering RNAs (siRNAs) targeting myostatin were co-

transfected with a myostatin-expressing plasmid 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 was injected and

electroporated into the tibialis anterior or its contralateral

muscle, respectively, of nine 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 polymerase chain reaction

(PCR) and Western blotting. Muscle fiber size was determined by

histochemistry. Satellite cell proliferation was determined by PAX7

immunohistochemistry. Myosin heavy chain type II (MHCII) expression

was determined by Western blot.

RESULTS: beta-Galactosidase reporter plasmid was expressed at 1 and 2

weeks but diminished by 4 weeks in tibialis anterior skeletal muscle.

Myostatin shRNA reduced myostatin mRNA and protein expression by 27

and 48%, respectively. Tibialis anterior weight, fiber size, and

MHCII increased by 10, 34, and 38%, respectively. Satellite cell

number was increased by over 2-fold.

CONCLUSIONS: This is the first demonstration that myostatin shRNA

gene transfer is a potential strategy to increase muscle mass.