Laminin-α4 and Integrin-Linked Kinase Mutations Cause Human Cardiomyopathy Via Simultaneous Defects in Cardiomyocytes and Endothelial Cells

Knöll, Ralph; Postel, Ruben; Wang, Jianming; Krätzner, Ralph; Hennecke, Gerrit; Vacaru, Andrei M.; Vakeel, Padmanabhan; Schubert, Cornelia; Murthy, K M K; Rana, Brinda K.; Kube, Dieter; Knöll, Gudrun; Schäfer, Katrin; Hayashi, Takeharu; Holm, Torbjörn; Kimura, Akinori; Schork, Nicholas J.; Toliat, Mohammad Reza; Nürnberg, Peter; Schultheiss, Heinz‐Peter; Schäper, Wolfgang; Schaper, Jutta; Bos, Erik; Hertog, Jeroen den; Eeden, Fredericus J.M. van; Peters, Peter J.; Hasenfuß, Gerd; Chien, Kenneth R.; Bakkers, Jeroen

doi:10.1161/circulationaha.107.689984

Cited by 208 publications

(150 citation statements)

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“…renal agenesis during kidney development (21). One can similarly explain two other phenotypic single amino acid substitution mutations identified in ILK KD, L308P that causes a lethal heart failure in zebrafish (8) and A262V that causes a severe dilated cardiomyopathy in humans (9). As shown in supplemental Fig.…”

Section: Discussionmentioning

confidence: 78%

Biochemical, Proteomic, Structural, and Thermodynamic Characterizations of Integrin-linked Kinase (ILK)

Fukuda

Knight

Piszczek

et al. 2011

Journal of Biological Chemistry

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Integrin-linked kinase (ILK) is one of the few evolutionarily conserved focal adhesion proteins involved in diverse cell adhesion-dependent physiological and pathological responses. Despite more than a decade of studies and extensive literature, the kinase function of ILK is controversial. ILK contains a highly degraded kinase active site but it has been argued that ILK may be an unusual manganese (Mn)-dependent serine-threonine kinase that targets specific substrates such as glycogen synthase kinase-3␤ (GSK-3␤). In this study, we have tackled this issue by a systematic bottom-up biochemical, proteomic, structural, and thermodynamic analysis of ILK. We show that recombinant ILK from either bacteria or mammalian cells exhibits no kinase activity on GSK-3␤ in the presence of either Mn 2؉ or the conventional kinase co-factor Mg 2؉ . A comprehensive and unbiased whole cell-based kinase assay using entire mammalian CG-4 and C2C12 cell lysate did not identify any specific ILK substrates. High resolution crystallographic structure analysis further confirmed that the Mn-bound ILK adopts the same pseudo active site conformation as that of the Mg-bound ILK. More importantly, thermodynamic analysis revealed that the K220M mutation, previously thought to inactivate ILK by disrupting ATP binding, significantly impairs the structural integrity and stability of ILK, which provides a new basis for understanding how this mutation caused renal agenesis, a failure of fetal kidney development. Collectively, our data provide strong evidence that ILK lacks intrinsic kinase function. It is a bona fide pseudokinase that likely evolved from an ancestral catalytic counterpart to act as a distinct scaffold to mediate protein-protein interactions during focal adhesion assembly and many other cellular events.The adhesion of cells to the extracellular matrix (ECM) 3 is primarily mediated by heterodimeric integrin transmembrane receptors. Upon activation, the integrin extracellular domain adheres to the ECM by binding to numerous ligands such as fibronectin and fibrinogen. However, for cells to firmly adhere to the ECM, integrins must connect to the backbone of the cell, the actin cytoskeleton, via its cytoplasmic tails (CTs) (1-2). Such connection is mediated by the supramolecular focal adhesion (FAs) complex. The reassembly/disassembly of FAs can lead to dynamic cell adhesion processes such as cell migration and spreading, which are fundamental for a variety of physiological responses. Over the past decades, major effort has been made to search for proteins involved in FA assembly and regulation. Integrin-linked kinase (ILK) was discovered in 1996 as a binding partner of integrin ␤ CTs (3) and was found to critically regulate FAs (4). Genetic and genomic analyses have established that ILK is one of the few essential and evolutionarily conserved proteins coupling integrins to actin (5-7), and its dysregulation has been linked to major human diseases such as heart failure (8 -10) and cancer (11). ILK has a molecular mass of ϳ51 kDa containi...

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Section: Discussionmentioning

confidence: 78%

Biochemical, Proteomic, Structural, and Thermodynamic Characterizations of Integrin-linked Kinase (ILK)

Fukuda

Knight

Piszczek

et al. 2011

Journal of Biological Chemistry

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“…8 Phenotypic characterization has been limited: increased mortality, cardiac hypertrophy, ventricular dilation, and reduced LVEF were described in mice with targeted ILK deletion, 8 whereas reduced integrin-binding capacity and loss of endothelial cells were reported with clinical ILK mutationassociated DCM. 9 This study constitutes the first detailed phenotypic characterization of the cardiomyopathy caused by ILK deletion. A schematic representation of our findings is provided in Figure 8.…”

Section: Ilk-dysfunction Cardiomyopathymentioning

confidence: 97%

Loss of Cardiomyocyte Integrin-Linked Kinase Produces an Arrhythmogenic Cardiomyopathy in Mice

Quang

Maguy

et al. 2015

Circ: Arrhythmia and Electrophysiology

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Background— Integrin-linked kinase (ILK), a serine/threonine protein kinase, has roles in cell signaling and molecular scaffolding. ILK mutation/deletion causes cardiomyopathic phenotypes, but the functional and electrophysiological features have not been characterized. This study investigated the structural, functional, ion channel, and electrophysiological changes associated with cardiomyocyte-directed ILK deletion in mice. Methods and Results— Adult mice with cardiomyocyte-directed ILK knockout were compared with littermate controls. Knockout mice showed markedly increased mortality, with sudden death beginning after 5 weeks and 100% mortality at 18 weeks. In 10-week-old knockout mice, spontaneous and inducible ventricular tachyarrhythmias were common, occurring in 60% and 86%, respectively, and absent in controls ( P <0.001, P <0.05 versus knockout mice). Ventricular refractoriness was prolonged, along with both QRS and QT interval. Action potentials were prolonged and displayed triggered activity. A wide range of ion currents were downregulated, including total, fast and slow components of transient outward K + current and inward rectifier K + current, along with corresponding ion channel subunit genes, providing a plausible explanation of action potential prolongation. At 5 weeks, only voltage-dependent K + currents were reduced, possibly related to direct ILK-Kv4.2 subunit interactions. Action potentials were prolonged, but no arrhythmias or cardiac dysfunction were noted. Structural remodeling was prominent at 10 weeks: connexin-43 was downregulated and redistributed to lateral cell margins, and left ventricular fibrosis occurred, with a strong regional distribution (predominating in the basal left ventricle). Conduction was slowed. High-throughput quantitative polymerase reaction gene-expression studies in 10-week-old ILK knockout showed upregulation of structural, remodeling and fibrosis-related genes, and downregulation of a wide range of ion channel and transporter subunits. Conclusions— Cardiomyocyte ILK deletion produces a lethal arrhythmogenic cardiomyopathy associated with important ion channel and structural remodeling.

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“…In addition to DGC, integrins (a and b) are concentrated at the costemeres that overly Z-lines in striated muscles, and the integrin complex also has a function in mechanical links of power transmission. 58 laminin a4 gene (LMNA4), 60 and integrin-linked kinase gene (ILK) 60 were found to cause DCM of autosomal dominant inheritance (Table 1). It was proposed that DCM was the disease of cytoskeleton or its interacting proteins.…”

Section: Membranous and Cytoskeletal Mutations In Dcmmentioning

confidence: 99%

Molecular basis of hereditary cardiomyopathy: abnormalities in calcium sensitivity, stretch response, stress response and beyond

Kimura

2010

J Hum Genet

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Cardiomyopathy is caused by functional abnormality of cardiac muscle. The functional abnormality involved in its etiology includes both extrinsic and intrinsic factors, and cardiomyopathy caused by the intrinsic factors is called as idiopathic or primary cardiomyopathy. There are several clinical types of primary cardiomyopathy including hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). Linkage studies and candidate gene approaches have explored the disease genes for hereditary primary cardiomyopathy. The most notable finding was that mutations in the same disease gene can be found in different clinical types of cardiomyopathy. Functional analyses of disease-related mutations have revealed that characteristic functional alterations are associated with the clinical types, such that increased and decreased Ca 2+ sensitivity due to sarcomere mutations are associated with HCM and DCM, respectively. In addition, our recent studies have suggested that mutations in the Z-disc components found in HCM and DCM may result in increased and decreased stiffness of sarcomere; that is, stiff sarcomere and loose sarcomere, respectively, and hence altered stretch response. More recently, mutations in the components of I region were found in hereditary cardiomyopathy and the functional analyses of the mutations suggested that the altered stress response was associated with cardiomyopathy, further complicating the etiology and pathogenesis. However, elucidation of genetic etiology and functional alterations caused by the mutations shed lights on the new therapeutic approaches to hereditary cardiomyopathy, such that treatment of DCM with a Ca 2+ sensitizer prevented the disease in a mouse model.

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Laminin-α4 and Integrin-Linked Kinase Mutations Cause Human Cardiomyopathy Via Simultaneous Defects in Cardiomyocytes and Endothelial Cells

Abstract: This is the first report on mutations in the laminin, integrin, and ILK system in human cardiomyopathy, which has consequences for endothelial cells as well as for cardiomyocytes, thus providing a new genetic basis for dilated cardiomyopathy in humans.

Cited by 208 publications

References 33 publications

Biochemical, Proteomic, Structural, and Thermodynamic Characterizations of Integrin-linked Kinase (ILK)

Biochemical, Proteomic, Structural, and Thermodynamic Characterizations of Integrin-linked Kinase (ILK)

Loss of Cardiomyocyte Integrin-Linked Kinase Produces an Arrhythmogenic Cardiomyopathy in Mice

Molecular basis of hereditary cardiomyopathy: abnormalities in calcium sensitivity, stretch response, stress response and beyond

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