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 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.

Neurohormonal regulation of myocardial cell apoptosis during the development of heart failure.

Adult cardiac myocytes are terminally differentiated cells that are no longer able to divide. Accumulating data support the idea that apoptosis in these cells is involved in the transition from cardiac compensation to decompensated heart failure. Since a number of neurohormonal factors are activated in this state, these factors may be involved in the positive and negative regulation of apoptosis in cardiac myocytes. beta1-Adrenergic receptor and angiotensin type 1 receptor pathways, nitric oxide and natriuretic peptides are involved in the induction of apoptosis in these cells, while alpha1- and beta2-adrenergic receptor and endothelin-1 type A receptor pathways and gp130-related cytokines are antiapoptotic. The myocardial protection of the latter is mediated, at least in part, through mitogen-activated protein kinase-dependent pathways, compatible with the findings in other cell types. In contrast, signaling pathways leading to apoptosis in cardiac myocytes are distinct from those in other cell types. The cAMP/PKA pathway induces apoptosis in cardiac myocytes and blocks apoptosis in other cell types. The p300 protein, a coactivator of p53, mediates apoptosis in fibroblasts but appears to play a protective role in differentiated cardiac myocytes. The inhibition of myocardial cell apoptosis in heart failure may be achieved by directly blocking apoptosis signaling pathways or by modulating neurohormonal factors involved in their regulation. These may provide novel therapeutic strategies in some forms of heart failure.

Endothelin-1 as a protective factor against beta-adrenergic agonist-induced apoptosis in cardiac myocytes.

OBJECTIVES: The purpose of this study was to investigate the regulation of beta-adrenergic agonist-induced apoptosis by endothelin-1 (ET-1) in cardiac myocytes. BACKGROUND: Numerous hormonal factors including norepinephrine and ET-1 are activated in patients with heart failure. These factors may be involved in the positive and negative regulation of myocardial cell apoptosis observed in failing hearts. Recently, it has been shown that norepinephrine can induce myocardial cell apoptosis via a beta-adrenergic receptor-dependent pathway. METHODS: Primary cardiac myocytes were prepared from neonatal rats. These cells were stimulated with the beta-adrenergic agonist isoproterenol (ISO) in the presence or absence of ET-1. RESULTS: The administration of 10(-7) mol/liter of ET-1 completely blocked Iso-induced apoptosis. An endothelin type A receptor antagonist, FR139317, negated the inhibitory effect of ET-1 on apoptosis, while the endothelin type B receptor antagonist BQ788 did not show such a negation. Endothelin-1 also inhibited apoptosis induced by a membrane-permeable cAMP analogue (8-Br-cAMP), which bypassed Gi. The effect of ET-1 was neutralized by an MEK-1-specific inhibitor (PD098059), a phosphatidylinositol 3'-kinase inhibitor (wortmannin) and its downstream pp70 S6-kinase inhibitor, rapamycin. CONCLUSIONS: These findings suggest that ET-1 represents a protective factor against myocardial cell apoptosis in heart failure and that this effect is mediated mainly through endothelin type A receptor-dependent pathways involving multiple downstream signalings in cardiac myocytes.

alpha- and beta-adrenergic pathways differentially regulate cell type-specific apoptosis in rat cardiac myocytes.

BACKGROUND: The apoptosis of cardiac myocytes may play a role in the development of heart failure. Norepinephrine is one of the factors activated in heart failure and can induce myocardial cell apoptosis in culture. However, it is unknown if alpha- and beta-adrenergic pathways coordinately or differentially regulate apoptosis and if this apoptotic pathway uses common or cell type-specific apoptotic signals. METHODS AND RESULTS: We stimulated cultured neonatal rat cardiac myocytes with an alpha(1)-adrenergic agonist (PE, phenylephrine), a beta-adrenergic agonist (isoproterenol [Iso]) or a membrane-permeable cAMP analogue (8-Br-cAMP) in serum-free conditions for 48 hours. Iso and 8-Br-cAMP markedly increased the number of TUNEL-positive cells (%TUNEL-positive nuclei >40%) compared with saline stimulation (<10%). DNA fragmentation was also confirmed by ladder formation in agarose gels. Apoptotic myocytes were characterized by cell shrinkage and nuclear condensation, consistent with morphological features of apoptosis. The Iso-induced apoptosis was almost completely inhibited by the protein kinase A-specific inhibitor KT5720. In contrast, PE inhibited 8-Br-cAMP-induced myocardial cell apoptosis. The apoptosis-inhibitory effect by PE was negated by the alpha(1)-adrenergic receptor antagonist prazosin and the MEK-1-specific inhibitor PD098059. Interestingly, although 8-Br-cAMP markedly induced apoptosis in cardiac myocytes, it completely blocked serum depletion-induced apoptosis in PC12 cells, a rat pheochromocytoma cell line. CONCLUSIONS: These findings indicate that alpha- and beta-adrenergic pathways differentially regulate myocardial cell apoptosis. The results also suggest that a cAMP- protein kinase A pathway is necessary and sufficient for beta-adrenergic agonist-induced apoptosis and that this apoptotic pathway is not functional in other cell types, for example, PC12 cells.