N, N‐Dimethylsphingosine protects murine heart and activates cytosolic sphingosine kinase by a PKCε dependent mechanism

Abstract

Sphingosine kinase (SphK) is a key enzyme responsible for formation of sphingosine-1-phosphate (S1P). N, N-Dimethylsphingosine (DMS) is recognized as a SphK inhibitor. However, the effect of DMS itself on cardiac function remains unknown. In Langendorff-perfused mouse hearts, cardiac systolic and diastolic functions were recorded. Infarction size was measured by TTC staining. SphK activity was measured by radioassay of [3H]sphingosine conversion to [3H]S1P. Pretreatment with 1 μM DMS for 10 min protected mouse hearts against global ischemia and reperfusion injury: cardiac functions were improved and infarction size was reduced. The cardiac protection induced by DMS was abolished in PKCε-null mouse hearts. Pretreatment with DMS increased cytosolic Akt phosphorylation (Ser 473) and Akt translocation from a triton-insoluble fraction to the cytosol. Administration of 1 μM DMS increased cytosolic SK activity (ratio of [3H]S1P/[3H]Sphingosine) from 6.1+/−1.1 in the control group to 10.3+/−1.4 in the DMS group (n=4, P<0.05). This enhanced SK activity was abolished in PKCε-null mouse hearts (n=4, P<0.05). DMS also increased PKCε translocation from the particulate to the cytosolic fraction with no effect on PKC alpha distribution. Co-immunoprecipitation showed that SK1 interacted with PKCε and phosphorylation of PKCε on Ser729. Physiological concentration of DMS protects murine hearts against ischemia/ reperfusion injury. DMS activates SphK in the cytosol via a PKCε dependent mechanism. The PKCε-SphK-S1P pathway is involved in the cardioprotection induced by DMS. Supported by NIH 1P01HL068738-01A1 to JSK.