ILK Induces Cardiomyogenesis in the Human Heart

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.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).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

Dataset of integrin-linked kinase protein: Protein interactions in cardiomyocytes identified by mass spectrometry

Using hearts from mice overexpressing integrin linked kinase (ILK) behind the cardiac specific promoter αMHC, we have performed immunoprecipitation and mass spectrometry to identify novel ILK protein:protein interactions that regulate cardiomyocyte activity and calcium flux. Integrin linked kinase complexes were captured from mouse heart lysates using a commercial antibody, with subsequent liquid chromatography tandem mass spectral analysis. Interacting partners were identified using the MASCOT server, and important interactions verified using reverse immunoprecipitation and mass spectrometry. All ILK interacting proteins were identified in a non-biased manner, and are stored in the ProteomeXchange Consortium via the PRIDE partner repository (reference ID PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD001053). The functional role of identified ILK interactions in cardiomyocyte function and arrhythmia were subsequently confirmed in human iPSC-cardiomyocytes. Keywords: Cardiology, Protein:protein interactions, Calcium regulation, Mass spectrometr

Correction: Cardiac regenerative capacity is age- and disease-dependent in childhood heart disease.

[This corrects the article DOI: 10.1371/journal.pone.0200342.]

Over-expression of ILK induces <i>Isl1</i> expression and β-catenin stabilization <i>in vitro</i> and <i>in vivo.</i>

<p>(A) Semi-quantitative RT-PCR analysis showing the <i>Isl1</i> expression in adherent (AC) and non-adherent (NAC) cells derived from fetal myocardium transduced with ad-ILK<i>^WT</i>, ad-ILK<i>^R211A</i> or ad-GFP. GAPDH expression was also tested in all experimental groups. (B) Western Blot analysis of protein levels of ILK and <i>Isl1</i> in myocardial lysates derived from transgenic mice with cardiac-restricted expression of constitutively active ILK (Tg<i>^S343D</i>) or mutant ILK (Tg<i>^R211A</i>) and their littermate controls. (C) Semi-quantitative RT-PCR analysis demonstrating the <i>Isl1</i> expression in hearts of transgenic mice with cardiac-restricted expression of constitutively active ILK (Tg<i>^S343D</i>) (+) compared to their littermate controls (−). (D) Western blot analysis showing the protein levels of stabilized, dephosphorylated β-catenin and total amount of β-catenin in adherent and non adherent fetal cardiac fractions infected with ad-ILK<i>^WT</i>, ad-ILK<i>^R211A</i> or ad-GFP. Each experiment was performed at least three times on independent samples and one representative blot with its corresponding expression ratios (active/total β-catenin expression) is shown at the top. The bar graph at the bottom summarizes the quantitative analysis of stabilized β-catenin expression in cells derived from fetal myocardium transduced with ad-ILK<i>^WT</i>, ad-ILK<i>^R211A</i> or ad-GFP. Data are mean ± SD, *p&lt;0.007, **p&lt;0.001, ***p&lt;0.05.</p

Cellular phenotypes in primary cultures of human fetal myocardium.

<p>(A) Cells freshly isolated from 22 week fetal myocardium were cultured for 2 days and then immunostained with anti-nk×2.5 (red) and anti-vimentin (green) (top panel) or with anti α-actin (red) and anti ki-67 (green) (bottom panel) antibodies to determine the percentage of the cells with cardiomyocyte or fibroblast phenotypes. Nuclei were marked with DAPI staining (blue). Scale bar, 30 µm. (B) Transmission electron microscopy showing that cultures of cells freshly isolated from human fetal myocardium at day 2 contain primitive cardioblasts with nascent sarcomeres (s) and mitochondrial clusters (m) (left) and cells with the transitional features containing both nascent sarcomeres and deep invaginations containing collagen fibers (cf) (right). (C) Only a subset of cardioblasts expressed cardiac troponin T (cTnT). (D) Phase contrast images (upper panel) and fluorescent images (lower panel) showing the adherent (AC) and non-adherent (NAC) cells 2 days after isolation. Immunostaining with anti-β-MHC antibody demonstrates that non-adherent clusters consist mainly of β-MHC positive cardioblasts. Scale bar, 80 µm (for phase) and 25 µm (for fluorescent imaging).</p

Cellular aggregates in ILK over-expressing cultures are mostly comprised by cardiooblasts.

<p>(A) Immunocytochemistry indicates that cellular aggregates present in the ILK<i>^WT</i> -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.</p

Over-expression of ILK in human fetal cardiac cells induces production of β-MHC.

<p>(A) Immunofluorescent staining for β-MHC (red) in human fetal heart-derived cells (21 weeks gestation) infected with adenovirus encoding ad-ILK<i>^WT</i>, ad-ILK<i>^R211A</i> and ad-GFP. Nuclei were detected with DAPI staining (blue). Scale bar, 30 µm. (B) Quantification of the number of β-MHC positive cells detected by immunostaining in adherent fetal cardiomyocytes infected with adenovirus encoding ad-ILK<i>^WT</i>, ad-ILK<i>^R211A</i> and ad-GFP. Bar graphs represent mean values ± SD, n = 14 (random fields), *p&lt;0.02, **p&lt;0.001, ***p&lt;0.03. (C) Western Blot analysis for ILK, cardiac specific α-MHC and β-MHC expression levels in adherent (AC) and non-adherent (NAC) fetal cardiac fractions infected with ad-ILK<i>^WT</i>, ad-ILK<i>^R211A</i> and ad-GFP. Each experiment was performed at least three times on independent samples and one representative blot is shown.</p

Response of cardioblast aggregates to electrical stimulation.

<p>(A) Upper panel shows electrical activity elicited by an increasing rate field electrical stimulation. Lower panel shows another aggregate subject to a constant-rate electrical stimulation. Optical voltage mapping was performed using fluorophore DI-4-ANEPPS as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037802#s4" target="_blank">Methods</a>. (B) Cells were stained with flouresence-labelled antibodies to β-MHC, C×43 and cTNT. Scale bar, 30 µm.</p

Over-expression of ILK induces robust cellular aggregation.

<p>(A) Fluorescent microscopy images and their composites with the phase contrast images identify the cells transduced with GFP-linked vectors (ad-GFP), wild type ILK vector (ad-ILK<i>^WT</i>) and mutant ILK vector (ad-ILK<i>^R211A</i>). Scale bar, 80 µm. (B) Quantification of cell aggregates in AC (right panel) and NAC (left panel) infected with ad-ILK<i>^WT</i>, ad-ILK<i>^R211A</i>, ad-GFP and non-infected cells. Bar graphs represent mean values ±SD, n = 10 (random fields), *p&lt;0.02, **p&lt;0.02, ***p&lt;0.03 (for AC) and *p&lt;0.001, **p&lt;0.0001, ***p&lt;0.001 (for NAC). Note that ad-ILKWT and ad-ILKR211A are significantly different from ad-GFP and that ad-ILKR211A is significant different from ad-ILKWT. (C) Western-blot analysis demonstrates a progressive increase in the level of ILK expression in AC and NAC transduced with ad-ILK<i>^WT</i>, ad-ILK<i>^R211A</i> as compared to non-transduced cells and cells transduced with the vector bearing the GFP-encoding message only. GAPDH was used as a loading control. (D) The number of aggregates was enumerated in human fetal cardiac cells cultures treated with ILK siRNA or scrambled siRNA. 2-tailed p&lt;0.05 at 24 (*) and 48 (**) hours post-transduction. (E) ILK (WT and R211A) overexpression increases the coexpression of ILK and C×43 (left panel); ILK siRNA reduces the endogenous expression levels of these proteins (right panel).</p

Endogenous ILK is expressed during human embryonic stem cell (hESC) cardiogenesis.

<p>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.</p