Abstract 1103: Integrin-linked Kinase and Laminin alpha 4 Mutations Cause Human Cardiomyopathy

Abstract

Background - Dilated Cardiomyopathy (DCM) is a syndrome characterized by ventricular dilation, contractile dysfunction and symptoms of congestive heart failure. Extracellular matrix proteins such as laminins as well as endothelial cells are known to influence cardiomyocyte performance, however a molecular link between mutations in the ECM and DCM has not been provided. Methods and Results - Using a forward genetic screen in zebrafish to identify novel genes required for myocardial function we were able to identify the lost-contact (loc) mutant, encoding a nonsense mutation in the integrin-linked kinase (ILK) gene. This loc/ilk mutant is associated with both, a severe defect in cardiomyocytes as well as in endothelial cells leading to severe myocardial dysfunction. Additional experiments revealed the epistatic regulation between the ECM component laminin alpha 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 (DCM). We identified two novel amino acid residue altering mutations (2828C>T (Pro943Leu), 3217C>T (Arg1073X)) in the integrin interacting domain of the LAMA4 gene and one 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 (KD=5 +/− 3 uM) and Arg1073X lama 4 (KD=1 +/− 0.2 uM) mutants in comparison to the wildtype lama 4 protein (KD = 440 +/− 20 nM). Cell attachment assays point to a defect in endothelial cell adhesion when LAMA4 mutants have been expressed in vitro and used as a substrate. 785C>T (Ala262Val) ILK did not change its affinity, as measured by GST pull down assays as well as by a yeast 2 hybrid analysis, to a well known interacting protein such as b-parvin but revealed a 63% decrease in kinase activity. Additional functional data point to the loss of endothelial cells in affected patients as a direct consequence of the mutant genes, leading ultimately to heart failure. Conclusions - We report the first human mutations in the LAMA4, integrin and ILK system and provide a new mechanistic basis for DCM in man, which involves endothelial cells as well as cardiomyocytes.