Laminin-alpha4 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.


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-alpha4 (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 (Kd=5+/-3 micromol/L) and Arg1073X LAMA4 (Kd=1+/-0.2 micromol/L) mutants compared with the wild-type LAMA4 protein (Kd=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.
Knöll
Ralph
R
Institute of Molecular Medicine, University of California at San Diego, La Jolla, Calif, USA. rknoell@med.uni-goettingen.de
Postel
Ruben
R
Wang
Jianming
J
Krätzner
Ralph
R
Hennecke
Gerrit
G
Vacaru
Andrei M
AM
Vakeel
Padmanabhan
P
Schubert
Cornelia
C
Murthy
Kenton
K
Rana
Brinda K
BK
Kube
Dieter
D
Knöll
Gudrun
G
Schäfer
Katrin
K
Hayashi
Takeharu
T
Holm
Torbjorn
T
Kimura
Akinori
A
Schork
Nicholas
N
Toliat
Mohammad Reza
MR
Nürnberg
Peter
P
Schultheiss
Heinz-Peter
HP
Schaper
Wolfgang
W
Schaper
Jutta
J
Bos
Erik
E
Den Hertog
Jeroen
J
van Eeden
Fredericus J M
FJ
Peters
Peter J
PJ
Hasenfuss
Gerd
G
Chien
Kenneth R
KR
Bakkers
Jeroen
J
D000328:Adult; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002311:Cardiomyopathy, Dilated / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002448:Cell Adhesion; D002522:Chlorocebus aethiops; D002874:Chromosome Mapping; D018389:Codon, Nonsense; D004252:DNA Mutational Analysis; D004625:Embryo, Nonmammalian / Q000473:pathology; D042783:Endothelial Cells / Q000473:pathology*; D044127:Epigenesis, Genetic; D005109:Extracellular Matrix / Q000378:metabolism / Q000473:pathology; D005260:Female; D006321:Heart / Q000196:embryology; D006333:Heart Failure / Q000209:etiology / Q000473:pathology; D006801:Humans; D016023:Integrins / Q000378:metabolism; D007797:Laminin / Q000235:genetics* / Q000502:physiology; D008297:Male; D008875:Middle Aged; D008958:Models, Molecular; D020125:Mutation, Missense; D009206:Myocardium / Q000473:pathology; D032383:Myocytes, Cardiac / Q000473:pathology*; D016376:Oligonucleotides, Antisense / Q000633:toxicity; D010375:Pedigree; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000502:physiology; D017434:Protein Structure, Tertiary; D014162:Transfection; D015027:Zebrafish / Q000196:embryology / Q000235:genetics; D029961:Zebrafish Proteins / Q000235:genetics / Q000502:physiology; D000099200:Scaffold Protein ILK
D018389:Codon, Nonsense; D016023:Integrins; D007797:Laminin; D016376:Oligonucleotides, Antisense; D017346:Protein Serine-Threonine Kinases; D029961:Zebrafish Proteins; D000099200:Scaffold Protein ILK; C501369:LAMA4 protein, human; C522179:LAMA4 protein, zebrafish; C522180:ILK protein, zebrafish