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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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Cellular aggregates in ILK over-expressing cultures are mostly comprised by cardiooblasts.(A) Immunocytochemistry indicates that cellular aggregates present in the ILKWT -transduced cultures contain numerous cardioblasts displaying the presence of α-actin, β-MHC and nk×2.5 (all marked with red rhodamine). Nuclei were identified with blue DAPI and expression of ILK was marked with green GFP. In top and middle panels, scale bars represent 80 µm; in the bottom panel, scale bar represents 25 µm. (B) Transmission electron microscopy showing clusters of mitochondria (m) in the cytoplasm of the primitive cardioblast (left). The more differentiated cells (right panel) contained similar mitochondrial clusters located in close proximity to the nascent sarcomeres (s). (C) Cardioblasts transduced with ILK (WT and R211A) and control vector were analyzed by RT-PCR for expression of cTNT, GATA-4 and MEF-2c transcripts.
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pone-0037802-g004: Cellular aggregates in ILK over-expressing cultures are mostly comprised by cardiooblasts.(A) Immunocytochemistry indicates that cellular aggregates present in the ILKWT -transduced cultures contain numerous cardioblasts displaying the presence of α-actin, β-MHC and nk×2.5 (all marked with red rhodamine). Nuclei were identified with blue DAPI and expression of ILK was marked with green GFP. In top and middle panels, scale bars represent 80 µm; in the bottom panel, scale bar represents 25 µm. (B) Transmission electron microscopy showing clusters of mitochondria (m) in the cytoplasm of the primitive cardioblast (left). The more differentiated cells (right panel) contained similar mitochondrial clusters located in close proximity to the nascent sarcomeres (s). (C) Cardioblasts transduced with ILK (WT and R211A) and control vector were analyzed by RT-PCR for expression of cTNT, GATA-4 and MEF-2c transcripts.

Mentions: Nk×2.5 is the primordial homeodomain transcription factor required for cardiac gene expression and is specifically essential for left ventricular development [21]. To further characterize the content of cell aggregates in response to ILK upregulation, we probed our cell cultures with antibodies to the early cardiac lineage marker nk×2.5, the cardiomyocyte markers α-actin and sarcomeric protein β-MHC and to α-SMA, a smooth muscle actin-specific marker. We have established that aggregates induced by ad-ILKWT (Figure 4A) prevalently consisted of cells demonstrating the presence of cardioblast marker nk×2.5 and β-MHC. The same cellular features were also observed in aggregates induced by ad-ILKR211A and even those sparse aggregates in ad-GFP and non-infected control cells. EM of constituent cells revealed sarcomeric structures of variable size and degrees of organization (Figure 4B). These aggregates also contained scattered endothelial cells and other α-SMA-negative cells that did not display any GFP and thus were non ILK-transduced cells (data not shown). The cardiomyogenic effects of ILK (WT and R211A) were further evident by the increased expression of cardiomyogenic transcription factors MEF-2C and GATA-4 as determined by reverse transcriptase RT-PCR (Figure 4C).


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)

Cellular aggregates in ILK over-expressing cultures are mostly comprised by cardiooblasts.(A) Immunocytochemistry indicates that cellular aggregates present in the ILKWT -transduced cultures contain numerous cardioblasts displaying the presence of α-actin, β-MHC and nk×2.5 (all marked with red rhodamine). Nuclei were identified with blue DAPI and expression of ILK was marked with green GFP. In top and middle panels, scale bars represent 80 µm; in the bottom panel, scale bar represents 25 µm. (B) Transmission electron microscopy showing clusters of mitochondria (m) in the cytoplasm of the primitive cardioblast (left). The more differentiated cells (right panel) contained similar mitochondrial clusters located in close proximity to the nascent sarcomeres (s). (C) Cardioblasts transduced with ILK (WT and R211A) and control vector were analyzed by RT-PCR for expression of cTNT, GATA-4 and MEF-2c transcripts.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037802-g004: Cellular aggregates in ILK over-expressing cultures are mostly comprised by cardiooblasts.(A) Immunocytochemistry indicates that cellular aggregates present in the ILKWT -transduced cultures contain numerous cardioblasts displaying the presence of α-actin, β-MHC and nk×2.5 (all marked with red rhodamine). Nuclei were identified with blue DAPI and expression of ILK was marked with green GFP. In top and middle panels, scale bars represent 80 µm; in the bottom panel, scale bar represents 25 µm. (B) Transmission electron microscopy showing clusters of mitochondria (m) in the cytoplasm of the primitive cardioblast (left). The more differentiated cells (right panel) contained similar mitochondrial clusters located in close proximity to the nascent sarcomeres (s). (C) Cardioblasts transduced with ILK (WT and R211A) and control vector were analyzed by RT-PCR for expression of cTNT, GATA-4 and MEF-2c transcripts.
Mentions: Nk×2.5 is the primordial homeodomain transcription factor required for cardiac gene expression and is specifically essential for left ventricular development [21]. To further characterize the content of cell aggregates in response to ILK upregulation, we probed our cell cultures with antibodies to the early cardiac lineage marker nk×2.5, the cardiomyocyte markers α-actin and sarcomeric protein β-MHC and to α-SMA, a smooth muscle actin-specific marker. We have established that aggregates induced by ad-ILKWT (Figure 4A) prevalently consisted of cells demonstrating the presence of cardioblast marker nk×2.5 and β-MHC. The same cellular features were also observed in aggregates induced by ad-ILKR211A and even those sparse aggregates in ad-GFP and non-infected control cells. EM of constituent cells revealed sarcomeric structures of variable size and degrees of organization (Figure 4B). These aggregates also contained scattered endothelial cells and other α-SMA-negative cells that did not display any GFP and thus were non ILK-transduced cells (data not shown). The cardiomyogenic effects of ILK (WT and R211A) were further evident by the increased expression of cardiomyogenic transcription factors MEF-2C and GATA-4 as determined by reverse transcriptase RT-PCR (Figure 4C).

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