Suzanne Reisz-Porszasz

Myostatin short interfering hairpin RNA gene transfer increases skeletal muscle mass

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.

Alterations in myostatin expression are associated with changes in cardiac left ventricular mass but not ejection fraction in the mouse.

Myostatin (Mst) is a negative regulator of skeletal muscle in humans and animals. It is moderately expressed in the heart of sheep and cattle, increasing considerably after infarction. Genetic blockade of Mst expression increases cardiomyocyte growth. We determined whether Mst overexpression in the heart of transgenic mice reduces left ventricular size and function, and inhibits in vitro cardiomyocyte proliferation. Young transgenic mice overexpressing Mst in the heart (Mst transgenic mice (TG) under a muscle creatine kinase (MCK) promoter active in cardiac and skeletal muscle, and Mst knockout (Mst (-/-)) mice were used. Xiscan angiography revealed that the left ventricular ejection fraction did not differ between the Mst TG and the Mst (-/-) mice, when compared with their respective wild-type strains, despite the decrease in whole heart and left ventricular size in Mst TG mice, and their increase in Mst (-/-) animals. The expected changes in cardiac Mst were measured by RT-PCR and western blot. Mst and its receptor (ActRIIb) were detected by RT-PCR in rat H9c2 cardiomyocytes. Transfection of H9c2 with plasmids expressing Mst under muscle-specific creatine kinase promoter, or cytomegalovirus promoter, enhanced p21 and reduced cdk2 expression, when assessed by western blot. A decrease in cell number occurred by incubation with recombinant Mst (formazan assay), without affecting apoptosis or cardiomyocyte size. Anti-Mst antibody increased cardiomyocyte replication, whereas transfection with the Mst-expressing plasmids inhibited it. In conclusion, Mst does not affect cardiac systolic function in mice overexpressing or lacking the active protein, but it reduces cardiac mass and cardiomyocyte proliferation.

MYOSTATIN OVEREXPRESSION REDUCES HEART MASS IN THE MOUSE BUT DOES NOT AFFECT CARDIAC FUNCTION.: 62

Myostatin (Mst) is an endogenous negative regulator of skeletal muscle mass in humans, rodents, cattle, and other animals. Mst has been found at low levels in normal sheep and cattle heart tissue, which increase considerably after infarction. Genetic blockade of Mst expression increases cardiomyocyte growth, but this also occurs in heart hypertrophy, possibly as a compensatory mechanism. We aimed here to compare cardiac size and function in young (7 weeks old) transgenic mice overexpressing Mst in the heart (Mst TG) under a muscle specific creatine kinase (MCK) promoter that is activated in both cardiac and skeletal muscle versus the Mst knockout (Mst [−/−]) mice, and to determine whether Mst modulation of cardiomyocyte growth occurs at the level of cell proliferation. Cardiac angiography revealed that the left ventricular ejection fraction (LVEF) did not differ significantly between the Mst TG and the Mst (−/−) mice compared with their respective wild-type strains. This occurred despite a significant decrease in whole heart and left ventricular size in Mst TG mice as opposed to the respective increase observed in Mst (−/−) animals, associated with the expected higher or nil expression of cardiac Mst, respectively, estimated by RT/PCR and Western blot. Mst and its receptor, the ActRIIb, were found to be expressed in H9c2 cardiomyocyte cell cultures by RT/PCR, and transfections with plasmids expressing Mst full-length protein under muscle-specific MCK (pMCK/Mst), or general CMV (pCMV/Mst) promoters, enhanced p21 and reduced cdk2 expression, assessed by Western blot. This translated into a significant decrease in cell proliferation with increasing concentrations of Mst recombinant protein, as determined by the formazan assay, whereas anti-Mst antibody exerted the opposite effects. Transfection of H9c2 cells with the pMCK/Mst, or pCMV/Mst plasmids, inhibited cell proliferation more effectively. In conclusion, Mst does not affect directly global cardiac systolic function in TG mice overexpressing or lacking the active protein, despite Mst9s inhibitory effects on cardiac mass and cardiomyocyte proliferation.

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.