Intravenous sphingosylphosphorylcholine protects ischemic and postischemic myocardial tissue in a mouse model of myocardial ischemia/reperfusion injury

Abstract

HDL, through sphingosine-1-phosphate (S1P), exerts direct cardioprotective effects on ischemic myocardium. It remains unclear whether other HDL-associated sphingophospholipids have similar effects. We therefore examined if HDL-associated sphingosylphosphorylcholine (SPC) reduces infarct size in a mouse model of transient myocardial ischemia/reperfusion. Intravenously administered SPC dose-dependently reduced infarct size after 30 minutes of myocardial ischemia and 24 hours reperfusion compared to controls. Infarct size was also reduced by postischemic, therapeutical administration of SPC. Immunohistochemistry revealed reduced polymorphonuclear neutrophil recruitment to the infarcted area after SPC treatment, and apoptosis was attenuated as measured by TUNEL. In vitro, SPC inhibited leukocyte adhesion to TNFα-activated endothelial cells and protected rat neonatal cardiomyocytes from apoptosis. S1P₃ was identified as the lysophospholipid receptor mediating the cardioprotection by SPC, since its effect was completely absent in S1P₃-deficient mice. We conclude that HDL-associated SPC directly protects against myocardial reperfusion injury in vivo via the S1P₃ receptor.

Figure 1

SPC protects against myocardial ischemia/reperfusion injury in vivo. SPC (0.625, 1.25 and 2.5 μg/g body weight) and 1% bovine serum albumin in PBS were injected intravenously 30 minutes before and after myocardial ischemia with reinstitution of reperfusion. Infarct size, after MI/R as a function of area at risk is reduced in SPC treated mice.

Figure 2

SPC inhibits PMN adhesion to activated endothelium under flow in vitro and PMN recruitment in the infarction area in vivo. (a) PMN adhesion to TNF-α-activated endothelial cells in vitro in the presence and absence of 10 μM SPC as quantified in a parallel-plate flow-chamber system. (b) Representative immunohistochemistry and morphometric quantification of PMN in infarcts of vehicle- and SPC-treated mice, respectively, 24 hours after ischemia/reperfusion.

Figure 3

SPC inhibits apoptosis of cardiomyocytes in vitro and in the infarction in vivo. (a) Quantification of TUNEL-positive nuclei in rat neonatal cardiomyocytes after simulated ischemia/reperfusion in the presence or absence of 10 μM SPC. (b) Representative terminal dUTP nick end-labeling (TUNEL) staining in the area at risk (outside the TTC-positive area) of control and SPC-treated mice 24 hours after ischemia/reperfusion. Morphometric quantification is presented above.

Figure 4

The S1P₃ lysophospholipid receptor is required for cardioprotection by SPC. Infarct size/area at risk was determined in S1P₃-deficient mice and their matching wild-type controls (C57BL/6) after treatment with SPC (1.25 μg/g body weight).