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

Ankyrin-B dysfunction predisposes to arrhythmogenic cardiomyopathy and is amenable to therapy

Arrhythmogenic cardiomyopathy (ACM) is an inherited arrhythmia syndrome characterized by severe structural and electrical cardiac phenotypes, including myocardial fibrofatty replacement and sudden cardiac death. Clinical management of ACM is largely palliative, owing to an absence of therapies that target its underlying pathophysiology, which stems partially from our limited insight into the condition. Following identification of deceased ACM probands possessing ANK2 rare variants and evidence of ankyrin-B loss of function on cardiac tissue analysis, an ANK2 mouse model was found to develop dramatic structural abnormalities reflective of human ACM, including biventricular dilation, reduced ejection fraction, cardiac fibrosis, and premature death. Desmosomal structure and function appeared preserved in diseased human and murine specimens in the presence of markedly abnormal \u3b2-catenin expression and patterning, leading to identification of a previously unknown interaction between ankyrin-B and \u3b2-catenin. A pharmacological activator of the WNT/\u3b2-catenin pathway, SB-216763, successfully prevented and partially reversed the murine ACM phenotypes. Our findings introduce what we believe to be a new pathway for ACM, a role of ankyrin-B in cardiac structure and signaling, a molecular link between ankyrin-B and \u3b2-catenin, and evidence for targeted activation of the WNT/\u3b2-catenin pathway as a potential treatment for this disease

ILK^<i>R211A</i> induces Hsp70 expression in the infarct zone of LAD-ligated mice.

<p>Western blot analysis of the protein levels of ILK, Hsp70 and Hsp90 in the lysates of myocardial infarct zone (MI) and control, viable, non infarcted myocardial zone (C) obtained from the hearts of two 28 days post LAD ligated Tg^<i>R211A</i> mice (+) and their littermate controls (-). GAPDH served as a loading control.</p

Mutation in Integrin-Linked Kinase (ILK^<i>R211A</i>) and Heat-Shock Protein 70 Comprise a Broadly Cardioprotective Complex

<div><p>Rationale</p><p>Integrin-linked kinase (ILK) has been proposed as a novel molecular target that has translational potential in diverse cardiac diseases, since its upregulation promotes a broadly cardioprotective phenotype. However, ILK has been implicated as both a cardioprotective and oncogenic target, which imposes therapeutic constraints that are generally relevant to the translational potential of many kinases.</p> <p>Objective</p><p>To study the cardioprotective properties of the activation-resistant, non-oncogenic, mutation of ILK (ILK^<i>R211A</i>) against experimental MI <i>in</i><i>vivo</i> and Doxorubicin induced apoptosis <i>in</i><i>vitro</i> and it’s relationships to stress induced heat shock proteins.</p> <p>Methods/Results</p><p>The transgenic mouse heart over-expressing a point mutation in the ILK pleckstrin homology (PH) domain (Tg^<i>R211A</i>) exhibits a highly cardioprotective phenotype based on LAD-ligation-induced MI reduction <i>in</i><i>vivo</i>, and on protection against doxorubicin (DOX)-induced cardiomyocyte apoptosis when overexpressed in human induced pluripotent stem cell (iPS)-derived cardiomyocytes <i>in</i><i>vitro</i>. Intriguingly, the degree of cardioprotection seen with the ILK^<i>R211A</i> mutation exceeded that with the ILK^<i>S343D</i> mutation. Microarray and immunoprecipitation analyses revealed upregulation of expression levels and specific binding of ILK^<i>WT</i>, ILK^<i>S343D</i> and ILK^<i>R211A</i> to both constitutively active heat-shock protein 70 (Hsc70) and inducible Hsp70 in response to MI, and to acute ILK overexpression in iPSC-cardiomyocytes. ILK-mediated cardioprotection was shown to depend upon Hsp70 ATPase activity.</p> <p>Conclusions</p><p>These findings indicate that wild type ILK and the non-oncogenic ILK^<i>R211A</i> mutation comprise a cardioprotective module with Hsp/c70. These results advance a novel target discovery theme in which kinase mutations can be safely engineered to enhance cardioprotective effects.</p> </div

ILK forms a complex with Hsc70.

<p><b>A</b>, (upper panel) SDS gel of ILK immunoprecipitates (IP) stained with Coomassie brilliant blue identified a major band at the size of 70 kD, which was confirmed by mass spectrometry as the mouse orthologue of heat-shock cognate protein 70 (Hsc70, also known as Hsp73). IP of ILK^<i>R211A</i> binds specifically to Hsc70 in 2D Isoelectric Focused (IEF)/SDS gel (lower panel). Myocardial lysates obtained from Tg^<i>R211A</i> hearts were immunoprecipitated using anti ILK rabbit Ab (IP) and control rabbit IgG (C) and then loaded into wells of an (IEF; pH 3.0-6.3) gel. ILK IP and C lanes were cut and inserted into wells of SDS gel, transferred and stained in Western blot using anti-Hsc70 goat Ab. <b>B</b>, ILK IPs derived from myocardial lysates of transgenic mice with cardiac-specific over-expression of ILK wild type (WT+), constitutively-active (S343D+) or activation resistant R211A+ mutations and their littermate controls (WT-, S343D- and R211A-) were probed with anti-Hsc70 antibodies. <b>C</b>, Western blot analysis demonstrating ILK IPs derived from myocardial lysates of transgenic mice showing gradient binding of ILK with Hsc70 which was the highest in R211A+, intermediate in S343D+, and lowest in WT+. <b>D</b>, Reciprocal analysis showing ILK binding in Hsc70 IP. <b>E</b>, ILK does not Co-IP with Hsp90. Co-IPs was performed in triplicate on independent samples and representative blots are shown.</p

ILK^<i>R211A</i> Improves Post-Infarct Remodeling.

<p><b>A</b>, H & E staining of axial sections 28 days post LAD ligation in transgenic (Tg) mice with cardiac-specific over-expression of constitutively-active (S343D+) or R211A+ mutation and littermate controls (S343D- and R211A-). Scale bar, 200 µm (high magnification), 2000 µm (low magnification). <b>B</b>, Planimetric calculations indicate a significant reduction in infarct size in R211A genotype, p(ANOVA) <0.01, and a similar trend in the S343D genotype. <b>C</b>, Echocardiographic measurements show a reduction in infarct size and reciprocal increase in region of viable myocardium in favor of R211A (p=0.04; p=0.001, respectively) and S343D (p=0.12; p=0.05) genotypes as compared to their littermate controls. Detailed measurements are provided in Tables S1 and S2, Online Supplement. <b>D</b>, Western blot showing the protein levels of phosphorylated Akt (pAkt) and Gsk-3β and total amounts of Akt and Gsk-3β in post-MI border zone myocardial lysates derived from two Tg^<i>S343D</i> and two Tg^<i>R211A</i> mice (+) and their littermate controls (-). GAPDH was used as a loading control. </p

ILK protects against DOX-induced apoptosis.

<p><b>A</b>, (top panel) Immunohistochemical staining for TUNEL-positive apoptotic nuclei in Hu iPSC-CMs transduced with ad-ILK<i>WT</i>, ad-ILK<i>R211A</i>, ad-GFP and in non-transduced cells following exposure to 1µM DOX for 24 hours. Scale bar, 100 µm. Arrows point to TUNEL-positive apoptotic nuclei. (bottom panel) Quantitative analysis showing the percentage of TUNEL-positive cell in Hu iPS-CMs after adenoviral infection and exposure to DOX. Bar graphs represent mean values ±SD, n=10 (random fields). * <i>P</i> < 0.01, ** <i>P</i> <0.02 by student <i>t</i> test. <b>B</b>, Western blots indicating the expression levels of ILK (on the left) and quantitative analysis showing the percentage of TUNEL-positive cell (on the right) in Hu iPS-CMs treated with ILK siRNA (siILK) or scrambled siRNA (siControl) following exposure to 1µM DOX for 24 hours (+DOX). Bar graphs represent mean values ±SD, n=10 (random fields). * <i>P</i> < 0.01 by student <i>t</i> test. <b>C</b>, Immunoblot showing the expression levels of ILK, Hsp70, Hsc70 and Hsp90 in Hu iPS-CM transduced with ILK siRNA (siILK) or scrambled siRNA (siC) following exposure to 1µM DOX for 24 hours (+DOX) as compared to DOX untreated cells (-DOX).</p

Hsc/p70 activity is required for cardioprotective effect of ILK (WT and R211A) against DOX-induced apoptosis.

<p><b>A</b>, Percentage of TUNEL-labelled apoptotic nuclei in Hu iPS-CMs transduced with ad-GFP, ad-ILK^<i>WT</i> and ad-ILK^<i>R211A</i> and then treated for 24 hours with 40 µM of Hsc/p70-ATPase inhibitor Ver-155008 in the presence or absence of 1 µM DOX. Ver-155008 was added one hour prior to exposure to DOX. Bar graphs represent mean values ±SD, n=10 (random fields). The apoptotic rates following exposure to Ver-155008 plus DOX are not statistically significantly different from one to another. <b>B</b>, Western blot analysis of human iPS-CMs transduced with ad-GFP, ad-ILK^<i>WT</i> and ad-ILK^<i>R211A</i> and then treated for 24 hours with 40 µM of Ver-155008 versus untreated cells in the presence of 1µM DOX. The blot was probed for ILK, Hsp70, Hsc70, Hsp90, pGsk-3β, total Gsk-3β, pAkt, total Akt, total PLN, and GAPDH was used as a loading control. </p