Downregulation of Cytoskeletal Muscle LIM Protein by Nitric Oxide

Abstract

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. Methods and 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 cytoskeletonassociated 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 (Ϫ49%, PϽ0.01) and protein (Ϫ52%, PϽ0.01) abundance in ET-1-treated cardiomyocytes via cGMPdependent protein kinase and superoxide/peroxynitrite-dependent signaling pathways. Treatment of cardiac myocytes with IL-1␤ and IFN-␥ 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 (Ϫ52%, PϽ0.01) 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.

Attenuation of cardiac remodeling after myocardial infarction by muscle LIM protein-calcineurin signaling at the sarcomeric Z-disc

“…To further test this concept, MLP protein expression was down-regulated by Ϸ50% in cultured cardiomyocytes by a specific antisense oligonucleotide (AS4) (10). Transfection with AS4 prevented the increases in NFAT transcriptional activity induced by endothelin-1 or mechanical stretch (Fig.…”

“…Muscle LIM protein (MLP), which is tethered to the Z-disk via its interacting partners, ␣-actinin and telethonin, has been proposed to be an essential part of the mechanical stretch sensor machinery (9) and to be involved in the transmission of humoral growth signals in cardiomyocytes (10). Intriguingly, myocardial MLP levels are reduced by Ϸ50% in patients with heart failure after MI (11).…”

NO Balance

“…Finally, antisense MLP downregulation suppressed cardiac myocyte hypertrophy and, in turn, MLP overexpression was sufficient for some aspects of cardiomyocyte hypertrophy. 19 These data provide the first molecular link between NO/ ONOO Ϫ and a component of the cardiac myocyte cytoskeleton crucial for a normal function of the heart. Together with in vivo results, 18 these data support the concept that the adverse effects of ONOO Ϫ on cardiac myocytes are, at least in part, mediated through the cardiac myocyte cytoskeleton.…”

“…However, cGMP-dependent activation of PKG also contributed to the phenotype, as demonstrated by the use of cardiac myocytes adenovirally transduced with a dominantnegative PKG mutant. 19 Importantly, the expression levels of NOSII and MLP displayed an inverse correlation in human hearts with congestive heart failure due to ischemic or dilated cardiomyopathy. Finally, antisense MLP downregulation suppressed cardiac myocyte hypertrophy and, in turn, MLP overexpression was sufficient for some aspects of cardiomyocyte hypertrophy.…”

“…Indeed, in the conditional iNOS transgenic animals, where a phenotype was observed, drastic increases in ONOO Ϫ were demonstrated. 18 In the current issue of Circulation, Heineke et al 19 report that addition of the NO donor S-Nitro-N-AcetylPenicillamine to rat neonatal ventricular cardiac myocytes reduced the endothelin-1-induced MLP upregulation by approximately 50%. Similarly, induction of NOSII in cardiac myocytes also significantly reduced the endothelin-1-induced MLP upregulation.…”