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ILK induces cardiomyogenesis in the human heart.

Traister A, Aafaqi S, Masse S, Dai X, Li M, Hinek A, Nanthakumar K, Hannigan G, Coles JG - PLoS ONE (2012)

Bottom Line: 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).

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiovascular Research, Hospital for Sick Children, Toronto, Canada.

ABSTRACT

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.

Methodology/principal findings: 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/significance: 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.

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Endogenous ILK is expressed during human embryonic stem cell (hESC) cardiogenesis.The induction of ILK is coincident with increasing expression of cardiomyocyte-specific sarcoplasmic endoplasmic reticulum calcium ATPase, isoform 2a (SERCA2a) and α-MHC. T0−20 marks the time in days during differentiation of cells from embryoid bodies to cardiomyocytes.
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pone-0037802-g008: Endogenous ILK is expressed during human embryonic stem cell (hESC) cardiogenesis.The induction of ILK is coincident with increasing expression of cardiomyocyte-specific sarcoplasmic endoplasmic reticulum calcium ATPase, isoform 2a (SERCA2a) and α-MHC. T0−20 marks the time in days during differentiation of cells from embryoid bodies to cardiomyocytes.

Mentions: Increasing expression of endogenous ILK level was also confirmed during growth factor-induced early cardiogenesis using an optimized combination of Activin A, Nodal and Bone Morphogenic Protein-4 in human embryonic stem cells (hESC) (Figure 8) [30]. Here, ILK expression coincided with that of the cardiac isoform of sarco(endo)plasmic reticulum calcium ATPase (SERCA2a) and α-MHC, indicating that endogenous ILK is upregulated during induction of the cardiomyogenic program in hESCs.


ILK induces cardiomyogenesis in the human heart.

Traister A, Aafaqi S, Masse S, Dai X, Li M, Hinek A, Nanthakumar K, Hannigan G, Coles JG - PLoS ONE (2012)

Endogenous ILK is expressed during human embryonic stem cell (hESC) cardiogenesis.The induction of ILK is coincident with increasing expression of cardiomyocyte-specific sarcoplasmic endoplasmic reticulum calcium ATPase, isoform 2a (SERCA2a) and α-MHC. T0−20 marks the time in days during differentiation of cells from embryoid bodies to cardiomyocytes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3362604&req=5

pone-0037802-g008: Endogenous ILK is expressed during human embryonic stem cell (hESC) cardiogenesis.The induction of ILK is coincident with increasing expression of cardiomyocyte-specific sarcoplasmic endoplasmic reticulum calcium ATPase, isoform 2a (SERCA2a) and α-MHC. T0−20 marks the time in days during differentiation of cells from embryoid bodies to cardiomyocytes.
Mentions: Increasing expression of endogenous ILK level was also confirmed during growth factor-induced early cardiogenesis using an optimized combination of Activin A, Nodal and Bone Morphogenic Protein-4 in human embryonic stem cells (hESC) (Figure 8) [30]. Here, ILK expression coincided with that of the cardiac isoform of sarco(endo)plasmic reticulum calcium ATPase (SERCA2a) and α-MHC, indicating that endogenous ILK is upregulated during induction of the cardiomyogenic program in hESCs.

Bottom Line: 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).

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiovascular Research, Hospital for Sick Children, Toronto, Canada.

ABSTRACT

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.

Methodology/principal findings: 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/significance: 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.

Show MeSH
Related in: MedlinePlus