Downregulation of cytoskeletal muscle LIM protein by nitric oxide: impact on cardiac myocyte hypertrophy.
BACKGROUND
In chronic heart failure, myocardial expression of the inducible isoform of nitric oxide (NO) synthase (NOS2) is enhanced, leading to a sustained production of NO. We postulated that NO modulates expression of genes in cardiac myocytes that may be functionally important in the context of cardiac hypertrophy and failure.


RESULTS
As revealed by cDNA expression array analyses, the NO donor SNAP, which has been shown previously to inhibit agonist-induced cardiac myocyte hypertrophy, downregulates expression of the cytoskeleton-associated muscle LIM protein (MLP) in endothelin-1 (ET-1)-stimulated neonatal rat cardiac myocytes. Northern blotting and immunoblotting experiments confirmed this finding and established that SNAP negatively controls MLP mRNA (-49%, P<0.01) and protein (-52%, P<0.01) abundance in ET-1-treated cardiomyocytes via cGMP-dependent protein kinase and superoxide/peroxynitrite-dependent signaling pathways. Treatment of cardiac myocytes with IL-1beta and IFN-gamma downregulated MLP expression levels via induction of NOS2. Moreover, expression levels of NOS2 and MLP were inversely correlated in the failing human heart, indicating that NOS2 may regulate MLP abundance in vitro and in vivo. Antisense oligonucleotides were used to explore the functional consequences of reduced MLP expression levels in cardiac myocytes. Like SNAP, antisense downregulation of MLP protein expression (-52%, P<0.01) blunted the increases in protein synthesis, cell size, and sarcomere organization in response to ET-1 stimulation. Conversely, overexpression of MLP augmented cell size and sarcomere organization in cardiac myocytes.


CONCLUSIONS
NO negatively controls MLP expression in cardiac myocytes. Because MLP is necessary and sufficient for hypertrophy and sarcomere assembly, MLP downregulation may restrain hypertrophic growth in pathophysiological situations with increased cardiac NO production.
Heineke
Jörg
J
Department of Cardiology and Angiology, Hannover Medical School, Hannover, Germany.
Kempf
Tibor
T
Kraft
Theresia
T
Hilfiker
Andres
A
Morawietz
Henning
H
Scheubel
Robert J
RJ
Caroni
Pico
P
Lohmann
Suzanne M
SM
Drexler
Helmut
H
Wollert
Kai C
KC
D000818:Animals; D048429:Cell Size; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000502:physiology; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000502:physiology; D016207:Cytokines / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D015536:Down-Regulation; D019332:Endothelin-1 / Q000494:pharmacology; D005786:Gene Expression Regulation; D006333:Heart Failure / Q000378:metabolism*; D006801:Humans; D060588:LIM Domain Proteins; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D016376:Oligonucleotides, Antisense / Q000235:genetics; D030421:Peroxynitrous Acid / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012518:Sarcomeres / Q000648:ultrastructure; D013481:Superoxides / Q000378:metabolism
D016207:Cytokines; D003598:Cytoskeletal Proteins; D019332:Endothelin-1; D060588:LIM Domain Proteins; D009124:Muscle Proteins; D020030:Nitric Oxide Donors; D016376:Oligonucleotides, Antisense; C090114:cysteine and glycine-rich protein 3; D013481:Superoxides; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; C569718:PRKG1 protein, human; D006152:Cyclic GMP