Abstract
BackgroundIntegrin-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.Methodology/Principal FindingsPrimary 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 (ILKR211A) resulted in much greater increase in the number of new cell aggregates as compared to overexpression of wild-type ILK (ILKWT). The cardiomyogenic effects of ILKR211A and ILKWT 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 ILKR211A and ILKWT. 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/SignificanceIn 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.
Highlights
Integrin-linked kinase (ILK) is a multidomain integrin adaptor protein that possesses widely conserved structural and signal transduction functions [1,2]
35% of cells were positive for the early cardiac lineage marker nk62.5, and,20% of cells were positive for muscle marker a-actin and cardiomyocyte-specific sarcomeric protein ß-myosin heavy chain (ß-MHC) and were Ki-67 positive (Figure 1A)
Data presented in this study indicate for the first time that experimental overexpression of the multifunctional serine/threonine kinase ILK promotes, in vitro, recruitment of human fetal heart cells to a cardiomyogenic fate
Summary
Integrin-linked kinase (ILK) is a multidomain integrin adaptor protein that possesses widely conserved structural and signal transduction functions [1,2]. Adaptor complexes centered around ILK comprise a signaling platform that, in response to distinct signal inputs from integrins and growth factor receptor tyrosine kinases, activates signaling pathways regulating growth, survival, cell cycle progression, epithelial-mesenchymal transition, and cellular differentiation [1,3]. Specific localization of ILK to costameric and Z-disc structures implies a functional role in the integration of cardiac mechanoreception and contractility [8]. 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
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