High-density lipoprotein determines adult mouse cardiomyocyte fate after hypoxia-reoxygenation through lipoprotein-associated sphingosine 1-phosphate.

The lipid mediator sphingosine 1-phosphate (S1P) confers survival benefits in cardiomyocytes and isolated hearts subjected to oxidative stress. High-density lipoprotein (HDL) is a major carrier of S1P in the serum, but whether HDL-associated S1P directly mediates survival in a preparation composed exclusively of cardiomyocytes has not been demonstrated. Accordingly, we tested the hypothesis that signal activation and survival during simulated ischemia-reperfusion injury in response to HDL require lipoprotein-associated S1P. As a model, we used adult mouse cardiomyocytes subjected to hypoxia-reoxygenation. Cells were treated or not with autologous mouse HDL, which significantly increased myocyte viability as measured by trypan blue exclusion. This survival effect was abrogated by the S1P(1) and SIP(3) receptor antagonist VPC 23019. The selective S1P(3) antagonist CAY10444, the G(i) antagonist pertussis toxin, the MEK (MAPK/ERK) kinase inhibitor PD-98059, and the phosphoinositide-3 kinase inhibitor wortmannin also inhibited the prosurvival effect of HDL. We observed that HDL activated both Akt (protein kinase B) and the MEK1/2-ERK1/2 pathway and also stimulated phosphorylation of glycogen synthase kinase-3beta. ERK1/2 activation was through an S1P(1) subtype receptor-G(i) protein-dependent pathway, whereas the activation of Akt was inhibited by CAY10444, indicating mediation by S1P(3) subtype receptors. We conclude that HDL, via its cargo of S1P, can directly protect cardiomyocytes against simulated oxidative injury in the absence of vascular effects and that prosurvival signal activation is dependent on both S1P(1) and S1P(3) subtype receptors.

Vincristine attenuates N-methyl-N'-nitro-N-nitrosoguanidine-induced poly-(ADP) ribose polymerase activity in cardiomyocytes.

The DNA-damaging agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) causes cardiomyocyte death as a result of energy loss from excessive activation of poly-(ADP) ribose polymerase-1 (PARP-1) resulting in depletion of its substrates nicotinamide adenine dinucleotide (NAD) and ATP. Previously we showed that the chemotherapeutic agent vincristine (VCR) is cardioprotective. Here we tested the hypothesis that VCR inhibits MNNG-induced PARP activation. Adult mouse cardiomyocytes were incubated with 100 micromol/L MNNG with or without concurrent VCR (20 micromol/L) for 2 to 4 hours. Cardiomyocyte survival was measured using the trypan blue exclusion assay. Western blots were used to measure signaling responses. MNNG-induced cardiomyocyte damage was time- and concentration-dependent. MNNG activated PARP-1 and depleted NAD and ATP. VCR completely protected cardiomyocytes from MNNG-induced cell damage and maintained intracellular levels of NAD and ATP. VCR increased phosphorylation of the prosurvival signals Akt, GSK-3beta, Erk1/2, and p70S6 kinase. VCR delayed PARP activation as evidenced by Western blot and by immunofluorescence staining of poly (ADP)-ribose, but without directly inhibiting PARP-1 itself. Known PARP-1 inhibitors also protected cardiomyocytes from MNNG-induced death. Repletion of ATP, NAD, pyruvate, and glutamine had effects similar to PARP-1 inhibitors. We conclude that VCR protects cardiomyocytes from MNNG toxicity by regulating PARP-1 activation, intracellular energy metabolism, and prosurvival signaling.