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. 2014 Jan;7(1):184-93.
doi: 10.1161/CIRCHEARTFAILURE.113.000649. Epub 2013 Dec 6.

Deep Sequence Analysis of Gene Expression Identifies Osteopontin as a Downstream Effector of Integrin-Linked Kinase (ILK) in Cardiac-Specific ILK Knockout Mice

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Deep Sequence Analysis of Gene Expression Identifies Osteopontin as a Downstream Effector of Integrin-Linked Kinase (ILK) in Cardiac-Specific ILK Knockout Mice

Jing Dai et al. Circ Heart Fail. .
Free PMC article

Abstract

Background: Integrin-linked kinase (ILK) is a serine/threonine kinase that has been linked to human and experimental heart failure, but its role in the heart is not fully understood.

Methods and results: To define the role of cardiomyocyte ILK, we generated cardiac-specific ILK knockout mice using α-myosin heavy chain-driven Cre expression. Cardiac-specific ILK knockout mice spontaneously developed lethal dilated cardiomyopathy and heart failure with an early increase in apoptosis, fibrosis, and cardiac inflammation. To identify downstream effectors, we used deep sequence analysis of gene expression to compare comprehensive transcriptional profiles of cardiac-specific ILK knockout and wild-type hearts from 10-day-old mice before the development of cardiac dysfunction. Approximately 2×10(6) cDNA clones from each genotype were sequenced, corresponding to 33 274 independent transcripts. A total of 93 genes were altered, using nominal thresholds of >1.4-fold change and P<0.001. The most highly upregulated gene was osteopontin (47-fold increase; P=9.6×10(-45)), an inflammatory chemokine implicated in heart failure pathophysiology. ILK also regulated osteopontin expression in cardiomyocytes in vitro. Importantly, blocking antibodies to osteopontin mitigated but did not fully rescue the functional decline in cardiac-specific ILK knockout mice.

Conclusions: Cardiomyocyte-specific ILK deletion leads to a lethal cardiomyopathy characterized by cardiomyocyte death, fibrosis, and inflammation. Comprehensive profiling identifies ILK-dependent transcriptional effects and implicates osteopontin as a contributor to these phenotypes.

Keywords: cardiomyopathy, dilated; gene expression profiling; integrin-linked kinase; osteopontin.

Figures

Figure 1
Figure 1. Cardiac Specific Deletion of ILK
Total RNA and protein were isolated from whole ventricles of 10 day old CSILK-KO and control littermates. (A) ILK mRNA expression was determined by QRT-PCR, *p < 0.05, n=5 in each genotype. (B) ILK protein expression was determined by immunoblotting. Results shown are representative of three independent experiments. (C) Immunofluorescence confocal microscopy shows virtually absent ILK staining in CSILK-KO cardiomyocytes identified by α-actinin staining but preserved vascular expression. Representative results from three independent experiments are shown.
Figure 2
Figure 2. CSILK-KO cardiac phenotoype
(A) Kaplan-Meier survival curves show early postnatal mortality among CSILK-KO which died by 3-6 weeks of age (n=12). (B) Both heart and lung weight normalized to tibial length were significantly increased compared with littermate controls, *p < 0.05, n=6. (C) Morphological and histological analyses of 21 day old CSILK-KO hearts show right and left ventricular chamber enlargement and wall thinning compared to control mice. Representative M-Mode echocardiograms are shown.
Figure 3
Figure 3. Increased apoptosis, fibrosis, and inflammation in CSILK-KO hearts
Representative TUNEL-stained (A), Masson’s Trichrome-stained (C) or CD45-stained (E) cryosections of the heart from CSILK-KO mice and littermate controls at the indicated ages are shown, n=5 for each. Corresponding quantitative data are shown on the right panel, *p < 0.05, ‡ p < 0.001 vs. WT at each time point, n=5 (B, D, F).
Figure 4
Figure 4. DSAGE analysis and validation
25 transcripts identified by DSAGE were independently assessed by QRT-PCR, and 16 of these DSAGE candidates have been confirmed at *p<0.05, †p<0.01, n=5.
Figure 5
Figure 5. Cardiac OPN expression in CSILK-KO mice
OPN mRNA expression increased dramatically in CSILK-KO ventricles before the development of cardiac dysfunction, and continued to rise at post-natal days 15 and 21, †p<0.01, ‡ p < 0.001 vs. WT at each time point, n=5. (A). OPN protein level also increased early, significantly by 15 days and further at 21 days post-natal age in CSILK-KO hearts, †p<0.01, ‡ p < 0.001 vs. WT at each time point, n=5 in each group (B). siRNA-mediated depletion of ILK in rat neonatal cardiomyocytes leads to OPN mRNA (C) and protein, (D) expression increase, †p<0.01, ‡ p < 0.001 vs. control, n=5.
Figure 6
Figure 6. Anti-OPN antibody mitigates the functional decline in ILK-KO mice
Mice were treated with a neutralizing anti-mouse OPN IgG or control goat IgG every other day by intraperitoneal injection, starting from FS% is approximately 40%. All mice received echocardiographic assessment before each injection. M-mode echocardiographic analyses showed that inhibiting OPN mitigates the functional decline in ILK-KO mice. Representative M-mode echocardiograms from five independent experiments are shown. Corresponding quantitative data are shown on the lower panel, *p < 0.05, ‡p<0.001 vs. control goat IgG injection at each time point, n=5.
Figure 7
Figure 7. Akt1 does not regulate cardiac OPN expression
OPN mRNA (A) and protein expression (B) were not increased in Akt1 knockout hearts, n=3.

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