Myostatin short interfering hairpin RNA gene transfer increases skeletal muscle mass.
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.
Magee
Thomas R
TR
Department of Urology, David Geffen School of Medicine, UCLA, Los Angeles, CA 90024, USA. tmagee@labiomed.org
Artaza
Jorge N
JN
Ferrini
Monica G
MG
Vernet
Dolores
D
Zuniga
Freddi I
FI
Cantini
Liliana
L
Reisz-Porszasz
Suzanne
S
Rajfer
Jacob
J
Gonzalez-Cadavid
Nestor F
NF
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D018274:Electroporation; D015870:Gene Expression; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D007763:Lac Operon; D008297:Male; D051379:Mice; D018482:Muscle, Skeletal / Q000254:growth & development* / Q000378:metabolism*; D055435:Myostatin; D010957:Plasmids / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D011916:Rats, Inbred F344; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors* / Q000235:genetics*
C525911:MSTN protein, human; C525912:Mstn protein, mouse; C525913:Mstn protein, rat; D055435:Myostatin; D034741:RNA, Small Interfering; D016212:Transforming Growth Factor beta