Activation of lectin-like oxidized low-density lipoprotein receptor-1 induces apoptosis in cultured neonatal rat cardiac myocytes.

BACKGROUND: Lectin-like oxidized LDL receptor-1 (LOX-1) was originally identified as a receptor expressed predominantly in endothelial cells. LOX-1 can also be expressed in other cell types, and the activation of the LOX-1 pathway has been implicated in apoptosis. There have been no reports, however, about LOX-1 expression in cardiac myocytes or regulation of myocardial cell apoptosis by LOX-1. METHODS AND RESULTS: In primary cardiac myocytes from neonatal rats, immunohistochemical analyses using a specific monoclonal antibody against LOX-1 demonstrated that LOX-1 expression was markedly induced by stimulation with norepinephrine and endothelin-1. LOX-1 expression was upregulated in cardiac myocytes as well as in vessel walls of failing rat hearts in vivo. In the presence of a low concentration of oxidized LDL that did not induce apoptosis by itself, artificial overexpression of LOX-1 in cardiac myocytes in culture resulted in apoptosis. LOX-1 overexpression induced activation of p38 mitogen-activated protein kinase (MAPK) and oxidative stress in cardiac myocytes, as demonstrated by an increase in positive immunostaining for 8-hydroxy-2'-deoxyguanosine. Inhibition of p38 MAPK by cotransfection of a dominant-negative form of MKK6 as well as by administration of a specific inhibitor, SB203580 or FR167653, almost completely blocked the induction of apoptosis by LOX-1 activation. Antioxidant catalase also blocked LOX-1-induced apoptosis as well as activation of p38 MAPK. CONCLUSIONS: These findings demonstrate that LOX-1 expression in cardiac myocytes is induced by neurohormonal factors activated in heart failure and that LOX-1-dependent apoptosis in these cells requires p38 MAPK, a component of oxidant stress-sensitive signaling pathways.

Calcineurin-GATA4 pathway is involved in beta-adrenergic agonist-responsive endothelin-1 transcription in cardiac myocytes.

Increases in the expression of endothelin-1 (ET-1) in cardiac myocytes play a critical role in the development of heart failure in vivo. Whereas norepinephrine (NE) is a potent inducer of ET-1 expression in cardiac myocytes, the signaling pathways that link NE to inducible cardiac ET-1 expression are unknown. Adrenergic stimulation results in an increase in intracellular calcium levels, which in turn activates calcineurin. Here, we have shown that stimulation with NE markedly increased the expression of the ET-1 gene in primary cardiac myocytes from neonatal rats. This increase was severely attenuated by a beta-adrenergic antagonist, metoprolol, but not by an alpha-adrenergic antagonist, prazosin. Consistent with these data, the beta-adrenergic agonist isoproterenol (ISO) activated the rat ET-1 promoter activity to an extent that was similar to NE. The ISO-stimulated increase in promoter activity was significantly inhibited by a Ca(2+)-antagonist, nifedipine, and an immunosuppressant, cyclosporin A, which blocks calcineurin. Mutation analysis indicated that the GATA4 binding site is required for ISO-responsive ET-1 transcription. Stimulation with ISO enhanced the interaction between NFATc and GATA4 in cardiac myocytes. Consistent with this interaction, overexpression of GATA4 and NFATc synergistically activated the ET-1 promoter. These findings demonstrate that NE-stimulated ET-1 expression in cardiac myocytes is mediated predominantly via a beta-adrenergic pathway, and that calcium-activated calcineurin-GATA4 plays a role in this process.

Calcineurin pathway is required for endothelin-1-mediated protection against oxidant stress-induced apoptosis in cardiac myocytes.

Endothelin-1 (ET-1) acts not only as a growth-promoting peptide but also as a potent survival factor against myocardial cell apoptosis. However, the signaling pathways leading to myocardial cell protection by ET-1 are poorly understood. Using a culture system of primary cardiac myocytes derived from neonatal rats, we show in the present study that ET-1 almost completely blocked the hydrogen peroxide-induced increase in the percentage of TdT-mediated dUTP-biotin nick-end labeling-positive myocytes. Apoptosis inhibition by ET-1 was confirmed by cytofluorometric analysis as well as by examination of the ladder formation, morphological features, and caspase-3 cleavage. We have found that ET-1 converts the nuclear factor of activated T lymphocytes (NFATc) in cardiac myocytes into high-mobility forms and translocates cytoplasmic NFATc to the nuclei. In addition, ET-1 stimulates the interaction between NFATc and the cardiac-restricted zinc-finger protein GATA4 in these cells. The immunosuppressants cyclosporin A and FK506, which antagonize calcineurin, negated the inhibitory effect of ET-1 on apoptosis. Calcineurin activation de novo was sufficient to inhibit hydrogen peroxide-induced apoptosis. ET-1 induced the expression of an antiapoptotic protein bcl-2 in cardiac myocytes in a cyclosporin A-dependent manner, but it did not alter the expression of bax. Cyclosporin A also attenuated the ET-1-stimulated transcription of the bcl-2 gene in these cells. These findings demonstrate that the calcineurin pathway is required for the inhibitory effect of ET-1 on oxidant stress-induced apoptosis in cardiac myocytes.

A p300 protein as a coactivator of GATA-6 in the transcription of the smooth muscle-myosin heavy chain gene.

The mechanisms that regulate smooth muscle development and differentiation are poorly understood. Although recent studies have suggested the possible role of a zinc finger transcription factor, GATA-6, in the differentiation of vascular smooth muscle cells (VSMCs), the downstream gene targeted by GATA-6 is unknown. The expression of smooth muscle-myosin heavy chain (Sm-MHC) provides a highly specific marker for the differentiated phenotype of VSMCs as well as the smooth muscle cell lineage. Here, we show that GATA-6 bound to a GATA-like motif (-810/-805) within the rat Sm-MHC promoter in a sequence-specific manner and activated this promoter through this site. In addition, we show that the transcriptional coactivator p300 associated with GATA-6 during the transcription of the Sm-MHC gene. A p300/GATA-6 complex in VSMCs was up-regulated by induction of the quiescent phenotype. A wild-type E1A, which interferes with endogenous p300, but not a mutant E1A defective for p300 binding, markedly down-regulated the expression of endogenous Sm-MHC in quiescent-phenotype VSMCs. These studies provide the first identification of a functionally important GATA-6 binding site within a smooth muscle-specific promoter and suggest a role for p300 in the maintenance of the differentiated phenotype in VSMCs as a coactivator of GATA-6.

Phosphorylation of GATA-4 is involved in alpha 1-adrenergic agonist-responsive transcription of the endothelin-1 gene in cardiac myocytes.

The expression of endothelin-1 (ET-1) in cardiac myocytes is markedly induced during the development of heart failure in vivo and by stimulation with the alpha(1)-adrenergic agonist phenylephrine in culture. Although recent studies have suggested a role for cardiac-specific zinc finger GATA factors in the transcriptional pathways that modulate cardiac hypertrophy, it is unknown whether these factors are also involved in cardiac ET-1 transcription and if so, how these factors are modulated during this process. Using transient transfection assays in primary cardiac myocytes from neonatal rats, we show here that the GATA element in the rat ET-1 promoter was required for phenylephrine-stimulated ET-1 transcription. Cardiac GATA-4 bound the ET-1 GATA element and activated the ET-1 promoter in a sequence-specific manner. Stimulation by phenylephrine caused serine phosphorylation of GATA-4 and increased its ability to bind the ET-1 GATA element. Inhibition of the extracellularly responsive kinase cascade with PD098059 blocked the phenylephrine-induced increase in the DNA binding ability and the phosphorylation of GATA-4. These findings demonstrate that serine phosphorylation of GATA-4 is involved in alpha(1)-adrenergic agonist-responsive transcription of the ET-1 gene in cardiac myocytes and that extracellularly responsive kinase 1/2 activation plays a role upstream of GATA-4.