Sphingosine Mediates the Immediate Negative Inotropic Effects of Tumor Necrosis Factor-α in the Adult Mammalian Cardiac Myocyte

Abstract

To determine whether activation of the neutral sphingomyelinase pathway was responsible for the immediate (<30 min) negative inotropic effects of tumor necrosis factor-␣ (TNF-␣), we examined sphingosine levels in diluent and TNF-␣-stimulated cardiac myocytes. TNF-␣ stimulation of adult feline cardiac myocytes provoked a rapid (<15 min) increase in the hydrolysis of [ 14 C]sphingomyelin in cell-free extracts, as well as an increase in ceramide mass, consistent with cytokine-induced activation of the neutral sphingomyelinase pathway. High performance liquid chromatographic analysis of lipid extracts from TNF-␣-stimulated cardiac myocytes showed that TNF-␣ stimulation produced a rapid (<30 min) increase in free sphingosine levels. Moreover, exogenous D-sphingosine mimicked the effects of TNF-␣ on intracellular calcium homeostasis, as well as the negative inotropic effects of TNF-␣ in isolated contracting myocytes; time course studies showed that exogenous D-sphingosine produced abnormalities in cell shortening that were maximal at 5 min. Finally, blocking sphingosine production using an inhibitor of ceramidase, noleoylethanolamine, completely abrogated the negative inotropic effects of TNF-␣ in isolated contracting cardiac myocytes. Additional studies employing biologically active ceramide analogs and sphingosine 1-phosphate suggested that neither the immediate precursor of sphingosine nor the immediate metabolite of sphingosine, respectively, were likely to be responsible for the immediate negative inotropic effects of TNF-␣. Thus, these studies suggest that sphingosine mediates the immediate negative inotropic effects of TNF-␣ in isolated cardiac myocytes.

Oxidative Stress Mediates Tumor Necrosis Factor-α–Induced Mitochondrial DNA Damage and Dysfunction in Cardiac Myocytes

“…7,8 Our findings support the predominant role of sphingomyelinceramide signaling pathways in TNF-␣-induced ROS generation and mtDNA decline in cardiac myocytes as well. This is in line with the observation by Oral et al 15 that TNF-␣ may cause a rapid activation of sphingomyelinase, with subsequent sphingomyelin hydrolysis and sphingosine production in cardiac myocytes. Ceramide has been shown to result in an inhibition of electron transport at the level of complex III followed by an increased generation of ROS.…”

“…15 Previous studies in various cell types have demonstrated that TNF-␣ exerts a direct effect on mitochondrial function and ROS generation via involvement of sphingomyelin hydrolysis to produce ceramide as a lipid second messenger. 7,8 Our findings support the predominant role of sphingomyelinceramide signaling pathways in TNF-␣-induced ROS generation and mtDNA decline in cardiac myocytes as well.…”

Neutral sphingomyelinase inhibition participates to the benefits of N-acetylcysteine treatment in post-myocardial infarction failing heart rats

“…In its both reduced (GSH) and oxidized (GSSG) forms, glutathione serves as a natural inhibitor of N-SMase [25], which hydrolyzes sphingomyelin into ceramide, controlling the sphingolipid signaling cascade [26][27][28] and mediating TNF-α apoptotic [29] and negative inotropic effects in cardiomyocytes [11,30,31]. Downstream ceramide generation, the ordered cascade of events in N-SMase pathway comprises downregulation of the prosurvival factor Bcl-2, resulting into a decrease of the proapoptotic Bax/ prosurvival Bcl-2 protein ratio that in turn elicits activation of caspase-3 [32].…”

Myocardial Hibernation

“…The negative inotropic effects of TNF-␣ and the role of iNOS-derived nitric oxide in the control of cardiac function have been well characterized both in vitro and in vivo. TNF-␣ depresses myocardial contractility by two mechanisms: Within minutes, it causes acute contractile dysfunction by decreasing calcium transients through a sphingosine signaling intermediate 14 ; within hours, it induces iNOS gene expression, which increases nitric oxide production, leading to sustained contractile dysfunction. 15 iNOS generates nitric oxide at high flux rates, leading to concentrations in the steady state that are 100-to 1000-fold greater than those produced by endothelial or neuronal NOS.…”