Published Online
on July 23, 2007

A more recent version of this article appeared on July 31, 2007

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Submitted on January 15, 2007
Accepted on May 29, 2007

Laminin-4 and Integrin-Linked Kinase Mutations Cause Human Cardiomyopathy Via Simultaneous Defects in Cardiomyocytes and Endothelial Cells

Ralph Knöll MD, PhD^*, Ruben Postel MSc, Jianming Wang PhD, Ralph Krätzner PhD, Gerrit Hennecke PhD, Andrei M. Vacaru MSc, Padmanabhan Vakeel PhD, Cornelia Schubert MD, Kenton Murthy; , Brinda K. Rana PhD, Dieter Kube MD, Gudrun Knöll; , Katrin Schäfer MD, Takeharu Hayashi MD, PhD, Torbjorn Holm MD, Akinori Kimura MD, PhD, Nicholas Schork MD, PhD, Mohammad Reza Toliat PhD, Peter Nürnberg MD, PhD, Heinz-Peter Schultheiss MD, Wolfgang Schaper MD, PhD, Jutta Schaper MD, PhD, Erik Bos MSc, Jeroen Den Hertog PhD, Fredericus J.M. van Eeden PhD, Peter J. Peters PhD, Gerd Hasenfuss MD, PhD, Kenneth R. Chien MD, PhD, and Jeroen Bakkers PhD

From the Institute of Molecular Medicine (R. Knöll, J.W., T. Holm, K.R.C.), Department of Medicine (K.R.C.), and Department of Psychiatry (K.M., B.K.R., N.S.), University of California at San Diego, La Jolla, Calif; Harvard Medical School (K.R.C.), Boston, Mass; Max-Planck Institute (W.S., J.S.), Bad Nauheim, Germany; Department of Molecular Pathogenesis (T. Hayashi, A.K.), Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan; Heart Center (R. Knöll, P.V., G.K., K.S., G. Hasenfuss), Hematology and Oncology (D.K.), Molecular Pharmacology and Institute of Molecular Genetics (R. Krätzner, G. Hennecke) and Abteilung Humangenetik (C.S.), University Hospital Göttingen, Göttingen, Germany; Universitätsklinikum Benjamin Franklin (H.-P.S.), Medizinische Klinik II, Freie Universität Berlin, Berlin, Germany; Cologne Center for Genomics and Institute for Genetics (M.R.T., P.N.), University of Cologne, Cologne, Germany; Hubrecht Laboratory (R.P., A.M.V., F.J.M.v.E., J.B.), Utrecht, the Netherlands; Interuniversity Cardiology Institute for the Netherlands (R.P., J.B.), Utrecht, the Netherlands; and Netherlands Cancer Institute (E.B., P.J.P.), Amsterdam, the Netherlands. Dr van Eeden is presently affiliated with the Department of Biomedical Science, Sheffield University, Sheffield, UK.

^* To whom correspondence should be addressed. E-mail: j.bakkers{at}niob.knaw.nl.

Background--Extracellular matrix proteins, such as laminins, and endothelial cells are known to influence cardiomyocyte performance; however, the underlying molecular mechanisms remain poorly understood.

Methods and Results--We used a forward genetic screen in zebrafish to identify novel genes required for myocardial function and were able to identify the lost-contact (loc) mutant, which encodes a nonsense mutation in the integrin-linked kinase (ilk) gene. This loc/ilk mutant is associated with a severe defect in cardiomyocytes and endothelial cells that leads to severe myocardial dysfunction. Additional experiments revealed the epistatic regulation between laminin-4 (Lama4), integrin, and Ilk, which led us to screen for mutations in the human ILK and LAMA4 genes in patients with severe dilated cardiomyopathy. We identified 2 novel amino acid residue-altering mutations (2828C>T [Pro943Leu] and 3217C>T [Arg1073X]) in the integrin-interacting domain of the LAMA4 gene and 1 mutation (785C>T [Ala262Val]) in the ILK gene. Biacore quantitative protein/protein interaction data, which have been used to determine the equilibrium dissociation constants, point to the loss of integrin-binding capacity in case of the Pro943Leu (K_d=5±3 µmol/L) and Arg1073X LAMA4 (K_d=1±0.2 µmol/L) mutants compared with the wild-type LAMA4 protein (K_d=440±20 nmol/L). Additional functional data point to the loss of endothelial cells in affected patients as a direct consequence of the mutant genes, which ultimately leads to heart failure.

Conclusions--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.

Key words: heart failure • signal transduction • cardiomyopathy • endocardium

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