Targeted ablation of ILK from the murine heart results in dilated cardiomyopathy and spontaneous heart failure - PubMed (original) (raw)

Targeted ablation of ILK from the murine heart results in dilated cardiomyopathy and spontaneous heart failure

Donald E White et al. Genes Dev. 2006.

Abstract

A requirement for integrin-mediated adhesion in cardiac physiology is revealed through targeted deletion of integrin-associated genes in the murine heart. Here we show that targeted ablation of the integrin-linked kinase (ILK) expression results in spontaneous cardiomyopathy and heart failure by 6 wk of age. Deletion of ILK results in disaggregation of cardiomyocytes, associated with disruption of adhesion signaling through the beta1-integrin/FAK (focal adhesion kinase) complex. Importantly, the loss of ILK is accompanied by a reduction in cardiac Akt phosphorylation, which normally provides a protective response against stress. Together, these results suggest that ILK plays a central role in protecting the mammalian heart against cardiomyopathy and failure.

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Figures

Figure 1.

Figure 1.

Left ventricular dilation and impaired contraction in ILK-null hearts. (A) Mice of the mckCRE ILKfl/fl genetic combination (black squares) succumbed to sudden death between 6 and 12 wk of age (median = 8 wk). Control littermate animals are represented by open circles. (B) Hearts from mckCRE ILKfl/fl mice (top row) appear enlarged relative to control mice (bottom row). Hearts are representative of moribund mckCRE ILKfl/fl animals. (C) Hearts from mckCRE ILKfl/fl animals (blue diamonds) have an average mass twofold greater than those from control animals (pink triangles). Mass was determined for seven representative animals from mckCRE ILKfl/fl and control genotypes, and is presented as a ratio to overall body mass to correct for mouse size. (D) Sections of cardiac tissue were prepared from control (left panels) and mckCRE ILKfl/fl (right panels) animals, and stained with trichrome stain. Samples were prepared as cross (top panels) and longitudinal (bottom panels) sections. A dilated left ventricle is visible in each section prepared from mckCRE ILKfl/fl mice. (E) End diastolic (EDA) and end systolic (ESA) areas were increased in mckCRE ILKfl/fl animals (black bars), relative to controls (white bars). The ejection fraction (EF) was correspondingly reduced in mckCRE ILKfl/fl animals. These data are indicative of left ventricular dilation and impaired pumping capacity of hearts from mckCRE ILKfl/fl mice. Error bars represent SEM. (**) p < 0.01, (***) p < 0.001, Student's unpaired _t_-test.

Figure 2.

Figure 2.

ILK is required for cardiac muscle integrity. (A) Frozen section of heart from a wild-type FVB mouse that was incubated with an antibody specific for ILK, followed by a Cy3-conjugated secondary antibody. Note the expression of ILK (red) in the costa-meric regions of the murine cardiac myocytes. Inset shows the expression pattern in a cross-section of a wild-type heart, revealing ILK expression in the sarcolemma. Phalloidin stain appears green. (B) Frozen section of heart from an mckCRE ILKfl/fl mouse that was subjected to the same immunostaining protocol as in A. Note the absence of Cy3 signal in the heart tissue from this animal, indicating an absence of ILK expression. Cardiomyocytes from hearts of this genotype appear disaggregated following the loss of ILK. Hearts of this genetic combination were prepared from moribund animals exhibiting a dilated phenotype. (C,D) Hearts from control (C) and mckCRE ILKfl/fl (D) mice were sectioned and stained with trichrome stain. Note again the disaggregated tissue in the mckCRE ILKfl/fl genotype, in addition to large amounts of interstitial fibrosis (blue stain).

Figure 3.

Figure 3.

Loss of ILK impacts β1-integrin/FAK signaling in mckCRE ILKfl/fl mice. (A) Pooled protein lysates from control (lane 1) and mckCRE ILKfl/fl (lanes 2,3) mice were subjected to immunoblot analysis, by probing with antibodies to β1-integrin (top panel) and ILK (bottom panel). Mice of the mckCRE ILKfl/fl genotype were selected at two time points: young, healthy animals not yet exhibiting evidence of cardiomyopathy (lane 2) and older mice showing evidence of morbidity due to cardiac failure (lane 3). Note that loss of ILK protein is accompanied by a corresponding reduction in β1-integrin protein levels. (B) Pooled lysates from A were subjected to immunoblot analysis for levels of FAK phospho-Tyr 576 (top panel) and phospho-Tyr 379 (bottom panel).

Figure 4.

Figure 4.

Loss of ILK in murine hearts results in impaired molecular stress response. (A, top panel) Pooled cardiac protein lysates from control (lane 1) and mckCRE ILKfl/fl animals (lanes 2,3) were probed for phospho-ERK1/2 levels. mckCRE ILKfl/fl mice were chosen at a young, healthy stage (lane 2) and an older moribund stage (lane 3). Bottom panel shows levels of total ERK1/2 protein. Elevated levels of phosphor-ERK1/2 reflect a molecular response to cardiac stress. (B, top panel) Lysates from A were subjected to immunoblot analysis for levels of Akt phospho-Tyr 473. Bottom panel shows total Akt protein levels. Note the reduction in Akt phosphorylation, which normally provides a protective role by promoting hypertrophy during cardiac stress.

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