ILK induces cardiomyogenesis in the human heart.
BACKGROUND
Integrin-linked kinase (ILK) is a widely conserved serine/threonine kinase that regulates diverse signal transduction pathways implicated in cardiac hypertrophy and contractility. In this study we explored whether experimental overexpression of ILK would up-regulate morphogenesis in the human fetal heart.


RESULTS
Primary cultures of human fetal myocardial cells (19-22 weeks gestation) yielded scattered aggregates of cardioblasts positive for the early cardiac lineage marker nk × 2.5 and containing nascent sarcomeres. Cardiac cells in colonies uniformly expressed the gap junction protein connexin 43 (C × 43) and displayed a spectrum of differentiation with only a subset of cells exhibiting the late cardiomyogenic marker troponin T (cTnT) and evidence of electrical excitability. Adenovirus-mediated overexpression of ILK potently increased the number of new aggregates of primitive cardioblasts (p<0.001). The number of cardioblast colonies was significantly decreased (p<0.05) when ILK expression was knocked down with ILK targeted siRNA. Interestingly, overexpression of the activation resistant ILK mutant (ILK(R211A)) resulted in much greater increase in the number of new cell aggregates as compared to overexpression of wild-type ILK (ILK(WT)). The cardiomyogenic effects of ILK(R211A) and ILK(WT) were accompanied by concurrent activation of β-catenin (p<0.001) and increase expression of progenitor cell marker islet-1, which was also observed in lysates of transgenic mice with cardiac-specific over-expression of ILK(R211A) and ILK(WT). Finally, endogenous ILK expression was shown to increase in concert with those of cardiomyogenic markers during directed cardiomyogenic differentiation in human embryonic stem cells (hESCs).


CONCLUSIONS
In the human fetal heart ILK activation is instructive to the specification of mesodermal precursor cells towards a cardiomyogenic lineage. Induction of cardiomyogenesis by ILK overexpression bypasses the requirement of proximal PI3K activation for transduction of growth factor- and β1-integrin-mediated differentiation signals. Altogether, our data indicate that ILK represents a novel regulatory checkpoint during human cardiomyogenesis.
Division of Cardiovascular Research, Hospital for Sick Children, Toronto, Canada.
Alexandra
A
Traister
Shabana
S
Aafaqi
Stephane
S
Masse
Xiaojing
X
Dai
Mark
M
Li
Aleksander
A
Hinek
Kumaraswamy
K
Nanthakumar
Gregory
G
Hannigan
John G
JG
Coles
D024722:Cardiac Myosins; D002449:Cell Aggregation; D055724:Electrophysiological Phenomena; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D004789:Enzyme Activation; D005260:Female; D005333:Fetus / Q000166:cytology; D005786:Gene Expression Regulation; D006801:Humans; D060850:LIM-Homeodomain Proteins / Q000235:genetics; D032386:Myoblasts, Cardiac / Q000166:cytology / Q000378:metabolism; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011247:Pregnancy; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021381:Protein Transport; D012518:Sarcomeres / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics; D000099200:Scaffold Protein ILK
D024722:Cardiac Myosins; D060850:LIM-Homeodomain Proteins; D018995:Myosin Heavy Chains; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D014157:Transcription Factors; D000099200:Scaffold Protein ILK; C501641:MYH7 protein, human; C063843:insulin gene enhancer binding protein Isl-1