Laminin-α4 and integrin-linked kinase mutations cause human cardiomyopathy via simultaneous defects in cardiomyocytes and endothelial cells

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-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. 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 mu mol/L) and Arg1073X LAMA4 (K-d=1 +/- 0.2 mu mol/L) mutants compared with the wild-type LAMA4 protein (K-d=440 +/- 20nmol/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.