Human plasma platelet-derived exosomes: effects of aspirin.

Platelet-derived exosomes mediate platelet atherogenic interactions with endothelial cells and monocytes. A new method for isolation of plasma platelet-derived exosomes is described and used to examine effects of aging and aspirin on exosome cargo proteins. Exosome secretion by purified platelets in vitro did not increase after exposure to thrombin or collagen, as assessed by exosome counts and quantification of the CD81 exosome marker. Thrombin and collagen increased exosome content of α-granule chemokines CXCL4 and CXCL7 and cytoplasmic high-mobility group box 1 (HMGB1) protein, but not membrane platelet glycoprotein VI (GPVI), with dependence on extracellular calcium. Aspirin consumption significantly blocked thrombin- and collagen-induced increases in exosome cargo levels of chemokines and HMGB1, without altering total exosome secretion or GPVI cargo. Plasma platelet-derived exosomes, enriched by absorption with mouse antihuman CD42b [platelet glycoprotein Ib (GPIb)] mAb, had sizes and cargo protein contents similar to those of exosomes from purified platelets. The plasma platelet-derived exosome number is lower and its chemokine and HMGB1 levels higher after age 65 yr. Aspirin consumption significantly suppressed cargo protein levels of plasma platelet-derived exosomes without altering total levels of exosomes. Cargo proteins of human plasma platelet-derived exosomes may biomark platelet abnormalities and in vivo effects of drugs.- Goetzl, E. J., Goetzl, L., Karliner, J. S., Tang, N., Pulliam, L. Human plasma platelet-derived exosomes: effects of aspirin.

Immunosuppression With FTY720 Reverses Cardiac Dysfunction in Hypomorphic ApoE Mice Deficient in SR-BI Expression That Survive Myocardial Infarction Caused by Coronary Atherosclerosis.

AIMS: We recently reported that immunosuppression with FTY720 improves cardiac function and extends longevity in Hypomorphic ApoE mice deficient in scavenger receptor Type-BI expression, also known as the HypoE/SR-BI(­/­) mouse model of diet-induced coronary atherosclerosis and myocardial infarction (MI). In this study, we tested the impact of FTY720 on cardiac dysfunction in HypoE/SR-BI(­/­) mice that survive MI and subsequently develop chronic heart failure. METHODS/RESULTS: HypoE/SR-BI(­/­) mice were bred to Mx1-Cre transgenic mice, and offspring were fed a high-fat diet (HFD) for 3.5 weeks to provoke hyperlipidemia, coronary atherosclerosis, and recurrent MIs. In contrast to our previous study, hyperlipidemia was rapidly reversed by inducible Cre-mediated gene repair of the HypoE allele and switching mice to a normal chow diet. Mice that survived the period of HFD were subsequently given oral FTY720 in drinking water or not, and left ventricular (LV) function was monitored using serial echocardiography for up to 15 weeks. In untreated mice, LV performance progressively deteriorated. Although FTY720 treatment did not initially prevent a decline of heart function among mice 6 weeks after Cre-mediated gene repair, it almost completely restored normal LV function in these mice by 15 weeks. Reversal of heart failure did not result from reduced atherosclerosis as the burden of aortic and coronary atherosclerosis actually increased to similar levels in both groups of mice. Rather, FTY720 caused systemic immunosuppression as assessed by reduced numbers of circulating T and B lymphocytes. In contrast, FTY720 did not enhance the loss of T cells or macrophages that accumulated in the heart during the HFD feeding period, but it did enhance the loss of B cells soon after plasma lipid lowering. Moreover, FTY720 potently reduced the expression of matrix metalloproteinase-2 and genes involved in innate immunity-associated inflammation in the heart. CONCLUSIONS: Our data demonstrate that immunosuppression with FTY720 prevents postinfarction myocardial remodeling and chronic heart failure.

IL-15: a novel prosurvival signaling pathway in cardiomyocytes.

Cardiovascular disease is the leading cause of death in Western countries. A major limitation of current treatments is the inability to efficiently repair or replace dead myocardium. Recently, stem cell-based therapies have been explored as an avenue to circumvent current therapeutic limitations. Overall, these therapies seem to result in small improvements in the contractile function of the heart. The exact mechanism(s) of action that underlie these improvements remain unknown, and it is believed that paracrine effects play a significant role. Previously, we had reported that an extract derived from bone marrow cells, in the absence of any live cell, contained cardioprotective soluble factors. In this study, we identify IL-15 as a putative cardioprotectant within the bone marrow cells paracrine profile. Using an in vitro culture system, we assessed the ability of IL-15 to protect cardiomyocytes under hypoxic conditions. For the first time, we have identified IL-15 receptors on the surface of cardiomyocytes and delineated the signaling system by which hypoxic cardiomyocytes may be protected from cellular death and rescued from oxidative stress with IL-15 treatment.

The immunosuppressant FTY720 prolongs survival in a mouse model of diet-induced coronary atherosclerosis and myocardial infarction.

FTY720, an analogue of sphingosine-1-phosphate, is cardioprotective during acute injury. Whether long-term FTY720 affords cardioprotection is unknown. Here, we report the effects of oral FTY720 on ischemia/reperfusion injury and in hypomorphic apoE mice deficient in SR-BI receptor expression (ApoeR61(h/h)/SRB1(-/- mice), a model of diet-induced coronary atherosclerosis and heart failure. We added FTY720 (0.3 mg·kg(-1)·d(-1)) to the drinking water of C57BL/6J mice. After ex vivo cardiac ischemia/reperfusion injury, these mice had significantly improved left ventricular (LV) developed pressure and reduced infarct size compared with controls. Subsequently, ApoeR61(h/h)/SRB1(-/-) mice fed a high-fat diet for 4 weeks were treated or not with oral FTY720 (0.05 mg·kg(-1)·d(-1)). This sharply reduced mortality (P < 0.02) and resulted in better LV function and less LV remodeling compared with controls without reducing hypercholesterolemia and atherosclerosis. Oral FTY720 reduced the number of blood lymphocytes and increased the percentage of CD4+Foxp3+ regulatory T cells (Tregs) in the circulation, spleen, and lymph nodes. FTY720-treated mice exhibited increased TGF-ß and reduced IFN-γ expression in the heart. Also, CD4 expression was increased and strongly correlated with molecules involved in natural Treg activity, such as TGF-ß and GITR. Our data suggest that long-term FTY720 treatment enhances LV function and increases longevity in mice with heart failure. These benefits resulted not from atheroprotection but from systemic immunosuppression and a moderate reduction of inflammation in the heart.

N-terminal truncated intracellular matrix metalloproteinase-2 induces cardiomyocyte hypertrophy, inflammation and systolic heart failure.

Matrix metalloproteinase-2 (MMP-2) is increasingly recognized as a major contributor to progressive cardiac injury within the setting of ischemia-reperfusion injury and ischemic ventricular remodeling. A common feature of these conditions is an increase in oxidative stress, a process that engages multiple pro-inflammatory and innate immunity cascades. We recently reported on the identification and characterization of an intracellular isoform of MMP-2 generated by oxidative stress-mediated activation of an alternative promoter located within the first intron of the MMP-2 gene. Transcription from this site generates an N-terminal truncated 65 kDa isoform of MMP-2 (NTT-MMP-2) that lacks the secretory sequence and the inhibitory prodomain region. The NTT-MMP-2 isoform is intracellular, enzymatically active and localizes in part to mitochondria. Expression of the NTT-MMP-2 isoform triggers Nuclear Factor of Activated T-cell (NFAT) and NF-κB signaling with the expression of a highly defined innate immunity transcriptome, including Interleukin-6, MCP-1, IRF-7 and pro-apoptotic transcripts. To determine the functional significance of the NTT-MMP-2 isoform in vivo we generated cardiac-specific NTT-MMP-2 transgenic mice. These mice developed progressive cardiomyocyte and ventricular hypertrophy associated with systolic heart failure. Further, there was evidence for cardiomyocyte apoptosis and myocardial infiltration with mononuclear cells. The NTT-MMP-2 transgenic hearts also demonstrated more severe injury following ex vivo ischemia-reperfusion injury. We conclude that a novel intracellular MMP-2 isoform induced by oxidant stress directly contributes, in the absence of superimposed injury, to cardiomyocyte hypertrophy. inflammation, systolic heart failure and enhanced susceptibility to ischemia-reperfusion injury.

Sphingolipids in cardiovascular and cerebrovascular systems: Pathological implications and potential therapeutic targets.

The sphingolipid metabolites ceramide, sphingosine, and sphingosine-1-phosphate (S1P) and its enzyme sphingosine kinase (SphK) play an important role in the regulation of cell proliferation, survival, inflammation, and cell death. Ceramide and sphingosine usually inhibit proliferation and promote apoptosis, while its metabolite S1P phosphorylated by SphK stimulates growth and suppresses apoptosis. Because these metabolites are interconvertible, it has been proposed that it is not the absolute amounts of these metabolites but rather their relative levels that determine cell fate. The relevance of this "sphingolipid rheostat" and its role in regulating cell fate has been borne out by work in many labs using many different cell types and experimental manipulations. A central finding of these studies is that SphK is a critical regulator of the sphingolipid rheostat, as it not only produces the pro-growth, anti-apoptotic messenger S1P, but also decreases levels of pro-apoptotic ceramide and sphingosine. Activation of bioactive sphingolipid S1P signaling has emerged as a critical protective pathway in response to acute ischemic injury in both cardiac and cerebrovascular disease, and these observations have considerable relevance for future potential therapeutic targets.

FTY720 postconditions isolated perfused heart by a mechanism independent of sphingosine kinase 2 and different from S1P or ischemic postconditioning.

BACKGROUND: We investigated the hypothesis that postconditioning by FTY720 (FTY) in isolated perfused mouse hearts is independent of the sphingosine 1-phosphate (S1P) pathway. MATERIAL AND METHODS: Ex vivo hearts were exposed to postconditioning (POST) by either ischemia or FTY720. Protection against ischemia/reperfusion (IR) injury was measured by recovery of left ventricular developed pressure (LVDP) and infarct size. RESULTS: FTY effectively postconditioned (POST) ex vivo hearts against ischemia/reperfusion (IR) injury as measured by recovery of LVDP and a low infarct size. FTY protection, unlike S1P but like sphingosine (Sph), was insensitive to inhibition of S1P G-Protein Coupled Receptors (GPCRs) or inhibition of PI3 kinase. Protection by FTY and Sph was however blocked by inhibitors of PKA and PKG. Thus, FTY follows the same cardioprotective pathway as Sph. This was further supported by studies of FTY POST in knockout (KO) mice lacking the SphK2 form of Sph kinase that is needed for phosphorylation of FTY to an S1P analog. In the absence of SphK2, FTY (and Sph) POST was still cardioprotective. This differed from the effect of SphK2 KO on protection by ischemic POST (IPOST). IPOST was not effective in KO hearts. To see if the GPCR signaling pathway to protection is normal in KO hearts, we looked at POST by GPCR agonists S1P and adenosine. Both provided effective protection even in KO hearts suggesting that the problem with IPOST in KO hearts is a low level of S1P available for release during IPOST. Thus, pharmacologic POST with FTY or Sph, like adenosine and S1P, is unaffected in the KO. CONCLUSIONS: FTY720 administered in vivo might behave in a dual manner showing both S1P-like effects and sphingosine-like effects. It appears that the latter may have been overlooked and may be the more important in aging hearts.

Sphingosine kinase and sphingosine 1-phosphate in the heart: a decade of progress.

Activation of sphingosine kinase/sphingosine 1-phosphate (SK/S1P)-mediated signaling has emerged as a critical cardioprotective pathway in response to acute ischemia/reperfusion injury. S1P is released in both ischemic pre- and post-conditioning. Application of exogenous S1P to cultured cardiac myocytes subjected to hypoxia or treatment of isolated hearts either before ischemia or at the onset of reperfusion exerts prosurvival effects. Synthetic congeners of S1P such as FTY720 mimic these responses. Gene targeted mice null for the SK1 isoform whose hearts are subjected to ischemia/reperfusion injury exhibit increased infarct size and respond poorly either to ischemic pre- or postconditioning. Measurements of cardiac SK activity and S1P parallel these observations. Experiments in SK2 knockout mice have revealed that this isoform is necessary for survival in the heart. High density lipoprotein (HDL) is a major carrier of S1P, and studies of hearts in which selected S1P receptors have been inhibited implicate the S1P cargo of HDL in cardioprotection. Inhibition of S1P lyase, an endogenous enzyme that degrades S1P, also leads to cardioprotection. These observations have considerable relevance for future therapeutic approaches to acute and chronic myocardial injury. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

A novel intracellular isoform of matrix metalloproteinase-2 induced by oxidative stress activates innate immunity.

BACKGROUND: Experimental and clinical evidence has pinpointed a critical role for matrix metalloproteinase-2 (MMP-2) in ischemic ventricular remodeling and systolic heart failure. Prior studies have demonstrated that transgenic expression of the full-length, 68 kDa, secreted form of MMP-2 induces severe systolic failure. These mice also had unexpected and severe mitochondrial structural abnormalities and dysfunction. We hypothesized that an additional intracellular isoform of MMP-2, which affects mitochondrial function is induced under conditions of systolic failure-associated oxidative stress. METHODOLOGY AND PRINCIPAL FINDINGS: Western blots of cardiac mitochondria from the full length MMP-2 transgenics, ageing mice and a model of accelerated atherogenesis revealed a smaller 65 kDa MMP-2 isoform. Cultured cardiomyoblasts subjected to transient oxidative stress generated the 65 kDa MMP-2 isoform. The 65 kDa MMP-2 isoform was also induced by hypoxic culture of cardiomyoblasts. Genomic database analysis of the MMP-2 gene mapped transcriptional start sites and RNA transcripts induced by hypoxia or epigenetic modifiers within the first intron of the MMP-2 gene. Translation of these transcripts yields a 65 kDa N-terminal truncated isoform beginning at M(77), thereby deleting the signal sequence and inhibitory prodomain. Cellular trafficking studies demonstrated that the 65 kDa MMP-2 isoform is not secreted and is present in cytosolic and mitochondrial fractions, while the full length 68 kDa isoform was found only in the extracellular space. Expression of the 65 kDa MMP-2 isoform induced mitochondrial-nuclear stress signaling with activation of the pro-inflammatory NF-κB, NFAT and IRF transcriptional pathways. By microarray, the 65 kDa MMP-2 induces an innate immunity transcriptome, including viral stress response genes, innate immunity transcription factor IRF7, chemokines and pro-apoptosis genes. CONCLUSION: A novel N-terminal truncated intracellular isoform of MMP-2 is induced by oxidative stress. This isoform initiates a primary innate immune response that may contribute to progressive cardiac dysfunction in the setting of ischemia and systolic failure.

A sphingosine kinase form 2 knockout sensitizes mouse myocardium to ischemia/reoxygenation injury and diminishes responsiveness to ischemic preconditioning.

Sphingosine kinase (SphK) exhibits two isoforms, SphK1 and SphK2. Both forms catalyze the synthesis of sphingosine 1-phosphate (S1P), a sphingolipid involved in ischemic preconditioning (IPC). Since the ratio of SphK1:SphK2 changes dramatically with aging, it is important to assess the role of SphK2 in IR injury and IPC. Langendorff mouse hearts were subjected to IR (30 min equilibration, 50 min global ischemia, and 40 min reperfusion). IPC consisted of 2 min of ischemia and 2 min of reperfusion for two cycles. At baseline, there were no differences in left ventricular developed pressure (LVDP), ± dP/dtmax, and heart rate between SphK2 null (KO) and wild-type (WT) hearts. In KO hearts, SphK2 activity was undetectable, and SphK1 activity was unchanged compared to WT. Total SphK activity was reduced by 53%. SphK2 KO hearts subjected to IR exhibited significantly more cardiac damage (37 ± 1% infarct size) compared with WT (28 ± 1% infarct size); postischemic recovery of LVDP was lower in KO hearts. IPC exerted cardioprotection in WT hearts. The protective effect of IPC against IR was diminished in KO hearts which had much higher infarction sizes (35 ± 2%) compared to the IPC/IR group in control hearts (12 ± 1%). Western analysis revealed that KO hearts had substantial levels of phosphorylated p38 which could predispose the heart to IR injury. Thus, deletion of the SphK2 gene sensitizes the myocardium to IR injury and diminishes the protective effect of IPC.

Altering pyrroloquinoline quinone nutritional status modulates mitochondrial, lipid, and energy metabolism in rats.

We have reported that pyrroloquinoline quinone (PQQ) improves reproduction, neonatal development, and mitochondrial function in animals by mechanisms that involve mitochondrial related cell signaling pathways. To extend these observations, the influence of PQQ on energy and lipid relationships and apparent protection against ischemia reperfusion injury are described herein. Sprague-Dawley rats were fed a nutritionally complete diet with PQQ added at either 0 (PQQ-) or 2 mg PQQ/Kg diet (PQQ+). Measurements included: 1) serum glucose and insulin, 2) total energy expenditure per metabolic body size (Wt(3/4)), 3) respiratory quotients (in the fed and fasted states), 4) changes in plasma lipids, 5) the relative mitochondrial amount in liver and heart, and 6) indices related to cardiac ischemia. For the latter, rats (PQQ- or PQQ+) were subjected to left anterior descending occlusions followed by 2 h of reperfusion to determine PQQ's influence on infarct size and myocardial tissue levels of malondialdehyde, an indicator of lipid peroxidation. Although no striking differences in serum glucose, insulin, and free fatty acid levels were observed, energy expenditure was lower in PQQ- vs. PQQ+ rats and energy expenditure (fed state) was correlated with the hepatic mitochondrial content. Elevations in plasma di- and triacylglyceride and ß-hydroxybutryic acid concentrations were also observed in PQQ- rats vs. PQQ+ rats. Moreover, PQQ administration (i.p. at 4.5 mg/kg BW for 3 days) resulted in a greater than 2-fold decrease in plasma triglycerides during a 6-hour fast than saline administration in a rat model of type 2 diabetes. Cardiac injury resulting from ischemia/reperfusion was more pronounced in PQQ- rats than in PQQ+ rats. Collectively, these data demonstrate that PQQ deficiency impacts a number of parameters related to normal mitochondrial function.

S1P lyase: a novel therapeutic target for ischemia-reperfusion injury of the heart.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that promotes cardiomyocyte survival and contributes to ischemic preconditioning. S1P lyase (SPL) is a stress-activated enzyme responsible for irreversible S1P catabolism. We hypothesized that SPL contributes to oxidative stress by depleting S1P pools available for cardioprotective signaling. Accordingly, we evaluated SPL inhibition as a strategy for reducing cardiac ischemia-reperfusion (I/R) injury. We measured SPL expression and enzyme activity in murine hearts. Basal SPL activity was low in wild-type cardiac tissue but was activated in response to 50 min of ischemia (n = 5, P < 0.01). Hearts of heterozygous SPL knockout mice exhibited reduced SPL activity, elevated S1P levels, smaller infarct size, and increased functional recovery after I/R compared with littermate controls (n = 5, P < 0.01). The small molecule tetrahydroxybutylimidazole (THI) is a Federal Drug Administration-approved food additive that inhibits SPL. When given overnight at 25 mg/l in drinking water, THI raised S1P levels and reduced SPL activity (n = 5, P < 0.01). THI reduced infarct size and enhanced hemodynamic recovery in response to 50 min of ischemia and to 40 min of reperfusion in ex vivo hearts (n = 7, P < .01). These data correlated with an increase in MAP kinase-interacting serine/threonine kinase 1, eukaryotic translation initiation factor 4E, and ribosomal protein S6 phosphorylation levels after I/R, suggesting that SPL inhibition enhances protein translation. Pretreatment with an S1P1 and S1P3 receptor antagonist partially reversed the effects of THI. These results reveal, for the first time, that SPL is an ischemia-induced enzyme that can be targeted as a novel strategy for preventing cardiac I/R injury.

Transgenic expression of matrix metalloproteinase-2 induces coronary artery ectasia.

Coronary artery ectasia (CAE) is generally diagnosed in patients undergoing arteriography for presumptive atherosclerotic coronary artery disease. CAE is commonly considered as a variant of atherosclerotic disease; however, recent studies suggest that CAE is the result of a systemic vascular disorder. There is increasing evidence that aneurysmal vascular disease is a systemic disorder characterized by enhanced expression of pro-inflammatory cytokines and increased synthesis of enzymes capable of degrading elastin and other components of the vascular wall. Matrix metalloproteinase-2 degrades a number of extracellular substrates, including elastin and has been shown to play a critical role in the development of abdominal aortic aneurysms. This study characterizes the development of CAE in a unique murine transgenic model with cardiac-specific expression of active MMP-2. Transgenic mice were engineered to express an active form of MMP-2 under control of the α-myosin heavy chain promoter. Coronary artery diameters were quantified, along with studies of arterial structure, elastin integrity and vascular expression of the MMP-2 transgene. Latex casts quantified total coronary artery volumes and arterial branching. Mid-ventricular coronary luminal areas were increased in the MMP-2 transgenics, coupled with foci of aneurysmal dilation, ectasia and perivascular fibrosis. There was no evidence for atherogenesis. Coronary vascular elastin integrity was compromised and coupled with inflammatory cell infiltration. Latex casts of the coronary arteries displayed ectasia with fusiform dilatation. The MMP-2 transgenic closely replicates human CAE and supports a critical and initiating role for this enzyme in the pathogenesis of this disorder.

Minocycline protects cardiac myocytes against simulated ischemia­reperfusion injury by inhibiting poly(ADP-ribose) polymerase-1.

There is an increase in reactive oxygen and nitrogen species in cardiomyocytes during myocardial ischemia/reperfusion injury. This leads to oxidative DNA damage and activation of nuclear repair enzymes such as poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 activation promotes DNA repair under normal conditions. However, excessive activation of PARP-1 leads to cell death. We report that PARP-1 enzymatic activity is directly inhibited by minocycline, and we propose that one mechanism of minocycline cardioprotection is the result of PARP-1 inhibition. Using cultured adult rat cardiac myocytes, we evaluated the mechanism of minocycline protection in which PARP-1 activation was induced by simulated ischemia/reperfusion injury using oxygen­glucose deprivation.We found an increase in reactive oxygen species production, PARP-1 activation, and PARP-1-mediated cell death after simulated ischemia/reperfusion. Cell death was significantly reduced by the PARP inhibitors 3, 4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (10 µM) and PJ-34 (500 nM) or by minocycline (500 nM). Cellular NAD(+) depletion and poly(ADP-ribose) formation, which are biochemical markers of PARP-1 activation, were also blocked by minocycline. Finally, simulated ischemia/reperfusion led to induction of the mitochondrial permeability transition, which was prevented by minocycline. Therefore, we propose that the protective effect of minocycline on cardiac myocyte survival is the result of inhibition of PARP-1 activity.

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.

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.

Cardioprotective activity of a novel and potent competitive inhibitor of lactate dehydrogenase.

Alkaline incubation of NADH results in the formation of a very potent inhibitor of lactate dehydrogenase. High resolution mass spectroscopy along with NMR characterization clearly showed that the inhibitor is derived from attachment of a glycolic acid moiety to the 4-position of the dihydronicotinamide ring of NADH. The very potent inhibitor is competitive with respect to NADH. The inhibitor added in submicromolar concentrations to cardiomyocytes protects them from damage caused by hypoxia/reoxygenation stress. In isolated mouse hearts, addition of the inhibitor results in a substantial reduction of myocardial infarct size caused by global ischemia/reperfusion injury.

Doxorubicin cardiomyopathy.

Established doxorubicin cardiomyopathy is a lethal disease. When congestive heart failure develops, mortality is approximately 50%. Extensive research has been done to understand the mechanism and pathophysiology of doxorubicin cardiomyopathy, and considerable knowledge and experience has been gained. Unfortunately, no effective treatment for established doxorubicin cardiomyopathy is presently available. Extensive research has been done and is being done to discover preventive treatments. However an effective and clinically applicable preventive treatment is yet to be discovered.