Sphingosine-1-phosphate activates I_Cl,swell in ventricular myocytes via mitochondrial reactive oxygen production

We previously demonstrated that bacterial sphingomyelinase (SMase), which converts plasmalemmal sphingomyelin to long-chain ceramides, activates the swelling-activated chloride current (I_Cl,swell) in rabbit ventricular myocytes in a reactive oxygen (ROS)-dependent manner under isosmotic conditions. Ceramides can be converted to sphingosine by ceramidase and, in turn, phosphorylated by sphingosine kinase to yield sphingosine-1-phosphate (S1P), which binds to multiple cytoplasmic targets and activates S1P receptors via inside-out transport. This study was designed to determine the cellular source of ROS production elicited by SMase, the sphingolipid species responsible, and thereby, the mechanism of activation of I_Cl,swell by sphingolipids.

Whole-cell patch clamp experiments were conducted using freshly isolated rabbit ventricular myocytes. Inhibition of ceramidase with D-erythro-MAPP, which increases the concentration of endogenous ceramides in the cell membrane, prevented activation of I_Cl,swell upon exposure to SMase. Similarly, inhibition of sphingosine kinase with DL-threo-dihydrosphingosine to prevent SIP formation by phosphorylation of sphingosine also completely inhibited SMase-induced Cl^- current. In contrast, addition of S1P to the bath solution elicited I_Cl,swell. ROS generated by both NADPH oxidase 2 (NOX2) and mitochondria previously were implicated in triggering I_Cl,swell. SMase-induced I_Cl,swell activation was abrogated by blocking mitochondrial electron transport at Complex I with rotenone but was insensitive to blockade of NOX2 with either apocynin or gp91ds-tat. Moreover, diazoxide, which augments mitochondrial ROS production, evoked I_Cl,swell, and 5-HD, an inhibitor of this pathway, reversed the SMase and diazoxide-induced currents. Flow cytometry using C-H₂DCFDA-AM to assess cytoplasmic ROS in HL-1 myocytes confirmed the effects of the interventions on ROS production.

Taken together, these data suggest S1P is the sphingolipid that triggers I_Cl,swell in cardiomyocyte, and activation of I_Cl,swell by SMase and S1P is due to ROS produced by mitochondria and appears independent of NOX2.

I_Cl,swell modulates apoptosis, cell volume, action potential duration, and participation in mechanoelectrical feedback in cardiomyocytes. Persistent activation of I_Cl,swell is seen in several forms of cardiac disease, including dilated cardiomyopathy [1] and models of heart failure [2]. Additionally, it has been implicated in metabolic syndrome and subsequent development of type 2 diabetes (DM2) [3]. This implies a complex relationship, in which there may be both direct effects on damaged cardiomyocytes and indirect effects on the cardiovascular system in general leading to chronic cellular stress such as that seen in DM2. This report demonstrates, for the first time, that S1P augments I_Cl,swell activation in cardiomyocytes via disruption of the mitochondrial respiratory chain resulting in ROS release. This presents potential therapeutic targets for the treatment of cardiovascular diseases, such as dilated cardiomyopathy or metabolic syndrome, characterized by alterations in sphingolipid metabolism.