Developmental expression of myostatin in cardiomyocytes and its effect on foetal and neonatal rat cardiomyocyte proliferation.
OBJECTIVE
Myostatin, a member of the transforming growth factor-beta (TGF-beta) family, plays a key role in skeletal muscle myogenesis by limiting hyperplastic and hypertrophic muscle growth. In cardiac muscle, myostatin has been shown to limit agonist-induced cardiac hypertrophic growth. However, its role in cardiac hyperplastic growth remains undetermined. The aim of this study was to characterise the expression of myostatin in developing myocardium, determine its effect on cardiomyocyte proliferation, and explore the signalling mechanisms affected by myostatin in dividing cardiomyocytes.


METHODS
We used quantitative PCR and Western blotting to study the expression of myostatin in cardiomyocytes isolated from rat myocardium at different developmental ages. We determined the effect of recombinant myostatin on proliferation and cell viability in dividing cardiomyocytes in culture. We analysed myostatin's effect on cardiomyocyte cell cycle progression by flow cytometry and used Western blotting to explore the signalling mechanisms involved.


RESULTS
Myostatin is expressed differentially in cardiomyocytes during cardiac development such that increasing expression correlated with a low cardiomyocyte proliferation index. Proliferating foetal cardiomyocytes, from embryos at 18 days of gestation, expressed low levels of myostatin mRNA and protein, whereas isolated cardiomyocytes from postnatal day 10 hearts, wherein the majority of cardiomyocytes have lost their ability to proliferate, displayed a 6-fold increase in myostatin expression. Our in vitro studies demonstrated that myostatin inhibited proliferation of dividing foetal and neonatal cardiomyocytes. Flow cytometric analysis showed that this inhibition occurs mainly via a block in the G1-S phase transition of the cardiomyocyte cell cycle. Western blot analysis showed that part of the mechanism underpinning the inhibition of cardiomyocyte proliferation by myostatin involves phosphorylation of SMAD2 and altered expressions of the cell cycle proteins p21 and CDK2.


CONCLUSIONS
We conclude that myostatin is an inhibitor of cardiomyocyte proliferation with the potential to limit cardiomyocyte hyperplastic growth by altering cardiac cell cycle progression.
McKoy
Godfrina
G
Cardiovascular Research Group, School of Pharmacy, The University of Reading, PO Box 226, Whiteknights, Reading, RG6 6AP, UK.
Bicknell
Katrina A
KA
Patel
Ketan
K
Brooks
Gavin
G
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western / Q000379:methods; D002453:Cell Cycle / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D047108:Embryonic Development / Q000502:physiology; D005260:Female; D005434:Flow Cytometry; D015870:Gene Expression; D018507:Gene Expression Regulation, Developmental; D032383:Myocytes, Cardiac / Q000378:metabolism*; D055435:Myostatin; D010766:Phosphorylation; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051899:Smad2 Protein / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
C525913:Mstn protein, rat; D055435:Myostatin; D012333:RNA, Messenger; D051899:Smad2 Protein; C497784:Smad2 protein, rat; D016212:Transforming Growth Factor beta