sphingosine-kinase and Myocardial-Infarction

Sphingolipids have recently emerged as a biomarker of recurrence and mortality after myocardial infarction (MI). The increased ceramide levels in mammalian heart tissues during acute MI, as demonstrated by several groups, is associated with higher cell death rates in the left ventricle and deteriorated cardiac function. Ceramidase, the only enzyme known to hydrolyze proapoptotic ceramide, generates sphingosine, which is then phosphorylated by sphingosine kinase to produce the prosurvival molecule sphingosine-1-phosphate. We hypothesized that Acid Ceramidase (AC) overexpression would counteract the negative effects of elevated ceramide and promote cell survival, thereby providing cardioprotection after MI.. We performed transcriptomic, sphingolipid, and protein analyses to evaluate sphingolipid metabolism and signaling post-MI. We investigated the effect of altering ceramide metabolism through a loss (chemical inhibitors) or gain (modified mRNA [modRNA]) of AC function post hypoxia or MI.. We found that several genes involved in de novo ceramide synthesis were upregulated and that ceramide (C16, C20, C20:1, and C24) levels had significantly increased 24 hours after MI. AC inhibition after hypoxia or MI resulted in reduced AC activity and increased cell death. By contrast, enhancing AC activity via AC modRNA treatment increased cell survival after hypoxia or MI. AC modRNA-treated mice had significantly better heart function, longer survival, and smaller scar size than control mice 28 days post-MI. We attributed the improvement in heart function post-MI after AC modRNA delivery to decreased ceramide levels, lower cell death rates, and changes in the composition of the immune cell population in the left ventricle manifested by lowered abundance of proinflammatory detrimental neutrophils.. Our findings suggest that transiently altering sphingolipid metabolism through AC overexpression is sufficient and necessary to induce cardioprotection post-MI, thereby highlighting the therapeutic potential of AC modRNA in ischemic heart disease.

Topics: Acid Ceramidase; Animals; Animals, Newborn; Apoptosis; Ceramides; Cicatrix; Embryoid Bodies; Enzyme Induction; Female; Genetic Therapy; Humans; Hypoxia; Induced Pluripotent Stem Cells; Inflammation; Male; Mice; Myocardial Infarction; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Recombinant Proteins; RNA, Messenger; Sphingolipids; Transfection; Up-Regulation

Sphingosine 1-phosphate (S1P) mediates multiple pathophysiological effects in the cardiovascular system. However, the role of S1P signaling in pathological cardiac remodeling following myocardial infarction (MI) remains controversial. In this study, we found that cardiac S1P greatly increased post-MI, accompanied with a significant upregulation of cardiac sphingosine kinase-1 (SphK1) and S1P receptor 1 (S1PR1) expression. In MI-operated mice, inhibition of S1P production by using PF543 (the SphK1 inhibitor) ameliorated cardiac remodeling and dysfunction. Conversely, interruption of S1P degradation by inhibiting S1P lyase augmented cardiac S1P accumulation and exacerbated cardiac remodeling and dysfunction. In the cardiomyocyte, S1P directly activated proinflammatory responses via a S1PR1-dependent manner. Furthermore, activation of SphK1/S1P/S1PR1 signaling attributed to β1-adrenergic receptor stimulation-induced proinflammatory responses in the cardiomyocyte. Administration of FTY720, a functional S1PR1 antagonist, obviously blocked cardiac SphK1/S1P/S1PR1 signaling, ameliorated chronic cardiac inflammation, and then improved cardiac remodeling and dysfunction in vivo post-MI. In conclusion, our results demonstrate that cardiac SphK1/S1P/S1PR1 signaling plays an important role in the regulation of proinflammatory responses in the cardiomyocyte and targeting cardiac S1P signaling is a novel therapeutic strategy to improve post-MI cardiac remodeling and dysfunction.

Topics: Animals; Animals, Newborn; Cytokines; Fingolimod Hydrochloride; Heart Diseases; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myocarditis; Myocytes, Cardiac; Phosphotransferases (Alcohol Group Acceptor); Rats, Sprague-Dawley; Receptors, Lysosphingolipid; RNA, Small Interfering; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Transfection; Ultrasonography

In animal models platelet P2Y12 receptor antagonists put the heart into a protected state, not as a result of suppressed thrombosis but rather through protective signaling, similar to that for ischemic postconditioning. While both ischemic postconditioning and the P2Y12 blocker cangrelor protect blood-perfused hearts, only the former protects buffer-perfused hearts indicating that the blocker requires a blood-borne constituent or factor to protect. We used an anti-platelet antibody to make thrombocytopenic rats to test if that factor resides within the platelet. Infarct size was measured in open-chest rats subjected to 30-min ischemia/2-h reperfusion. Infarct size was not different in thrombocytopenic rats showing that preventing aggregation alone is not protective. While ischemic preconditioning could reduce infarct size in thrombocytopenic rats, the P2Y12 inhibitor cangrelor could not, indicating that it protects by interacting with some factor in the platelet. Ischemic preconditioning is known to require phosphorylation of sphingosine. In rats treated with dimethylsphingosine to block sphingosine kinase, cangrelor was no longer protective. Thus cangrelor's protective mechanism appears to also involve sphingosine kinase revealing yet another similarity to conditioning's mechanism.

Topics: Adenosine Monophosphate; Animals; Blood Platelets; Cardiotonic Agents; Heart; Ischemic Postconditioning; Ischemic Preconditioning, Myocardial; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protective Agents; Purinergic P2Y Receptor Antagonists; Rats; Rats, Sprague-Dawley; Sphingosine

Three bioactive sphingolipids, namely sphingosine-1-phosphate (S1P), ceramide (CER) and sphingosine (SPH) were shown to be involved in ischemia/reperfusion injury of the heart. S1P is a powerful cardioprotectant, CER activates apoptosis and SPH in a low dose is cardioprotective whereas in a high dose is cardiotoxic. The aim of the present study was to examine effects of experimental myocardial infarction on the level of selected sphingolipids in plasma, erythrocytes and platelets in the rat. Myocardial infarction was produced in male Wistar rats by ligation of the left coronary artery. Blood was taken from the abdominal aorta at 1, 6 and 24 h after the ligation. Plasma, erythrocytes and platelets were isolated and S1P, dihydrosphingosine-1-phosphate (DHS1P), SPH, dihydrosphingosine (DHS) and CER were quantified by means of an Agilent 6460 triple quadrupole mass spectrometer using positive ion electrospray ionization source with multiple reaction monitoring. The infarction reduced the plasma level of S1P, DHS1P, SPH and DHS but increased the level of total CER. In erythrocytes, there was a sharp elevation in the level of SPH and DHS early after the infarction and a reduction after 24 h whereas the level of S1P, DHS1P and total CER gradually increased. In platelets, the level of each of the examined compounds profoundly decreased 1 and 6 h after the infarction and partially normalized in 24 h. The results obtained clearly show that experimental heart infarction in rats produces deep changes in metabolism of sphingolipids in the plasma, platelets and erythrocytes.

Topics: Anesthesia; Animals; Ceramides; Coronary Vessels; Erythrocyte Count; Femoral Artery; Ligation; Lysophospholipids; Male; Myocardial Infarction; Phosphotransferases (Alcohol Group Acceptor); Platelet Count; Rats; Rats, Wistar; Sphingolipids; Sphingosine; Troponin T

Although mitochondria are key determinants of myocardial injury during ischemia-reperfusion (I/R), their interaction with critical cytoprotective signaling systems is not fully understood. Sphingosine-1-phosphate (S1P) produced by sphingosine kinase-1 protects the heart from I/R damage. Recently a new role for mitochondrial S1P produced by a second isoform of sphingosine kinase, SphK2, was described to regulate complex IV assembly and respiration via interaction with mitochondrial prohibitin-2. Here we investigated the role of SphK2 in cardioprotection by preconditioning. Littermate (WT) and sphk2 (-/-) mice underwent 45 min of in vivo ischemia and 24 h reperfusion. Mice received no intervention (I/R) or preconditioning (PC) via 5 min I/R before the index ischemia. Despite the activation of PC-cytoprotective signaling pathways in both groups, infarct size in sphk2 (-/-) mice was not reduced by PC (42 ± 3% PC vs. 43 ± 4% I/R, p = ns) versus WT (24 ± 3% PC vs. 43 ± 3% I/R, p < 0.05). sphk2 (-/-) mitochondria exhibited decreased oxidative phosphorylation and increased susceptibility to permeability transition (PTP). Unlike WT, PC did not prevent ischemic damage to electron transport or the increased susceptibility to PTP. To evaluate the direct contribution to the resistance of mitochondria to cytoprotection, SphK2, PHB2 or cytochrome oxidase subunit IV was depleted in cardiomyoblasts. PC protection was abolished by each knockdown concomitant with decreased PTP resistance. These results point to a new action of S1P in cardioprotection and suggest that the mitochondrial S1P produced by SphK2 is required for the downstream protective modulation of PTP as an effector of preconditioning protection.

Topics: Animals; Blotting, Western; Ischemic Preconditioning; Lysophospholipids; Male; Mice; Mice, Transgenic; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocardial Infarction; Phosphotransferases (Alcohol Group Acceptor); Prohibitins; RNA, Small Interfering; Signal Transduction; Sphingosine; Transfection

The sphingosine kinase (SphK)/sphingosine 1-phosphate (S1P) pathway, known to determine the fate and growth of various cell types, can enhance cardiac myocyte survival in vitro and provide cardioprotection in acute ex vivo heart preparations. However, the relevance of these findings to chronic cardiac pathology has never been demonstrated. We hypothesized that S1P signaling is impaired during chronic remodeling of the uninfarcted ventricle during the evolution of post-myocardial infarction (MI) cardiomyopathy and that a therapeutic enhancement of S1P signaling would ameliorate ventricular dysfunction. SphK expression and activity were measured in the remote, uninfarcted myocardium (RM) of C57Bl/6 mice subjected to coronary artery ligation. The mRNA expression of S1P receptor isoforms was also measured, as was the activation of the downstream S1P receptor mediators. A cardioprotective role for S1P(1) receptor agonism was tested via the administration of the S1P(1)-selective agonist SEW2871 during and after MI. As a result, the expression data suggested that a dramatic reduction in SphK activity in the RM early after MI may reflect a combination of posttranscriptional and posttranslational modulation. SphK activity continued to decline gradually during chronic post-MI remodeling, when S1P(1) receptor mRNA also fell below baseline. The S1P(1)-specific agonism with oral SEW2871 during the first 2-wk after MI reduced apoptosis in the RM and resulted in improved myocardial function, as reflected in the echocardiographic measurement of fractional shortening. In conclusion, these results provide the first documentation of alterations in S1P-mediated signaling during the in situ development of cardiomyopathy and suggest a possible therapeutic role for the pharmacological S1P receptor agonism in the post-MI heart.

Topics: Animals; Disease Models, Animal; Male; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myocytes, Cardiac; Oxadiazoles; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; RNA, Messenger; Signal Transduction; Sphingolipids; Thiophenes; Ventricular Remodeling

Sphingosine-1-phosphate (S1P) plays a vital role in cytoskeletal rearrangement, development, and apoptosis. Sphingosine kinase-1 (SphK1), the key enzyme catalyzing the formation of S1P, mediates ischaemic preconditioning. Ischaemic postconditioning (POST) has been shown to protect hearts against ischaemia/reperfusion injury (IR). To date, no studies have examined the role of SphK1 in POST.. Wild-type (WT) and SphK1 null (KO) mouse hearts were subjected to IR (45 min of global ischaemia and 45 min of reperfusion) in a Langendorff apparatus. Left ventricular developed pressure (LVDP), maximum velocity of increase or decrease of LV pressure (+/-dP/dtmax), and LV end-diastolic pressure (LVEDP) were recorded. Infarction size was measured by 1% triphenyltetrazolium chloride staining. POST, consisting of 5 s of ischaemia and 5 s of reperfusion for three cycles after the index ischaemia, protected hearts against IR: recovery of LVDP and +/-dP/dtmax were elevated; LVEDP was decreased; infarction size (% of risk area) was reduced from 40 +/- 2% in the control group to 29 +/- 2% of the risk area in the POST group (P < 0.05, n = 4 per group). Phosphorylation of Akt and extracellular signal-regulated kinases detected by Western blotting was increased at 10 min of reperfusion. The protection induced by POST was abolished in KO hearts. Infarction size in KO hearts (57 +/- 5%) was not different from the KO control group (53 +/- 5% of risk area, n = 4, P = NS).. A short period of ischaemic POST protected WT mouse hearts against IR. The cardiac protection induced by POST was abrogated in SphK1-KO mouse hearts. Thus, SphK1 is critical for successful ischaemic POST.

Topics: Animals; Extracellular Signal-Regulated MAP Kinases; Ischemic Preconditioning, Myocardial; Isoenzymes; Male; Mice; Mice, Knockout; Models, Animal; Mutation; Myocardial Infarction; Myocardium; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; Reperfusion Injury

Sphingosine kinase (SKase) has been implicated in the protection of hearts from ischemia/reperfusion injury. This hypothesis was further examined.. Changes in SKase activity and cardiac function (left ventricular developed pressure, LVDP, and infarct size) in response to ischemia and reperfusion were studied in adult rat hearts by the ex vivo Langendorff method. Following initial equilibration or preconditioning, there was 45 min no-flow ischemia and then 45 min of reperfusion.. SKase activity declined 61% during ischemia and did not recover upon reperfusion. LVDP also did not recover upon reperfusion and the infarct size was 47%. A short 30 min period of ischemia was associated with variable recovery of SKase activity that directly correlated with LVDP recovery. Preconditioning of hearts reduced the decrease in SKase activity during ischemia by half, and upon reperfusion activity returned to normal. The LVDP recovered 79% and infarct size was small. Preconditioned hearts had higher S-1-P levels after ischemia/reperfusion relative to non-preconditioned hearts. The decline in SKase activity during ischemia of preconditioned hearts could not be mimicked in vitro by treatment with protein phosphatases. Attempts to alter activity of SKase from control, preconditioned, ischemic, or reperfused hearts by phosphorylation with ERK1/2 were unsuccessful. Treatment of non-preconditioned hearts at reperfusion with 100 nM S-1-P improved recovery of LVDP. The SKase inhibitor dimethylsphingosine blocked hemodynamic recovery in preconditioned hearts.. The data support a role for SKase activity in recovery of hemodynamic function after ischemic injury and also in the cardioprotective effect of preconditioning.

Topics: Animals; In Vitro Techniques; Ischemic Preconditioning, Myocardial; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Phosphotransferases (Alcohol Group Acceptor); Rats; Subcellular Fractions; Ventricular Pressure

Phosphorylation of sphingosine by sphingosine kinase (SK) is the rate-limiting step in the cellular synthesis of sphingosine 1-phosphate (S1P). The monoganglioside GM1, which stimulates SK, is cardioprotective in part through increased generation of S1P that protects myocytes by diverse mechanisms. Because protein kinase C (PKC)epsilon activation is necessary for myocardial ischemic preconditioning (IPC) and PKC activators increase SK activity, we tested the hypothesis that SK may be a central mediator of IPC.. In adult murine hearts, IPC sufficient to reduce infarct size significantly increased cardiac SK activity, induced translocation of SK protein from the cytosol to membranes, and enhanced cardiac myocyte survival. IPC did not increase SK activity in PKCepsilon-null mice. The SK antagonist N,N-dimethylsphingosine inhibited PKCepsilon activation and directly abolished the protective effects of IPC and the enhanced SK activity induced by IPC.. These findings demonstrate that PKCepsilon is thus recruited by IPC and induces activation of SK that then mediates IPC-induced cardioprotection in murine heart.

Topics: Animals; Cardiotonic Agents; Enzyme Activation; G(M1) Ganglioside; Ischemic Preconditioning, Myocardial; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Cardiovascular; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Protein Kinase C-epsilon; Protein Transport; Signal Transduction; Sphingosine