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.

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Abstract 1394: Sublethal Levels of Aldehydes Augmented Cardiac Anti-Oxidant Defense through Activation of eIF2 α -ATF4 Pathway via GCN2 Kinase

Circulation ◽

10.1161/circ.118.suppl_18.s_320 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Takaharu Katayama ◽

Motoaki Sano ◽

Jin Endo ◽

Kentaro Hayashida ◽

Tomohiro Matsuhashi ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Metabolism ◽

Acid Metabolism ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Special Importance ◽

Dependent Manner ◽

Protein Levels

[Introduction] Despite an increase in the levels of aldehydes, the heart from aldehyde dehydrogenase ( ALDH ) 2*2 -transgenic (Tg) mice, loss of function model of ALDH, exhibited a greater tolerance to oxidative stress via activation of amino acid metabolism leading to glutathione biosynthesis. This study was designed to identify the signaling cascades responsible for the activation of amino acid metabolism by aldehydes. [Methods & Results] (1) Phosphorylation of α -subunit of eukaryotic translation initiation factor 2 (eIF2 α ) and subsequent translational activation of ATF4 have been shown to induce amino acid metabolism as a common response to a wide variety of stressors. Consistent with this, phosphorylation levels of eIF2 α and protein expression of ATF4 were increased in ALDH2*2 -Tg hearts. (2) Among four eIF2 α kinases, general control non-depressible (GCN)2 kinase, a sensor for amino acid insufficiency, was activated in ALDH2*2 -Tg heart. (3) Quantification of intracellular amino acid demonstrated that free histidine concentration in ALDH2*2 -Tg heart was selectively reduced by 50% compared to that in non-Tg littermates. (4) To clarify the functional significance of observed reduction in histidine, ALDH2*2 -Tg mice were fed a high histidine diet. The phosphorylation levels of eIF2 α and the protein levels of ATF4 were diminished by 50% in ALDH2*2 -Tg mice fed the high histidine diet, in agreement with the normalization of histidine concentration. Accordingly, both enhanced tolerance to ischemia-reperfusion injury and elevated levels of glutathione were partially diminished in the heart from ALDH2*2 -Tg mice fed the high histidine diet compared to ALDH2*2 -Tg mice fed normal chow. (5) In culture, exposure to 4-hydroxy-2-nonenal (4-HNE) phosphorylated GCN2 and eIF2 α and increased protein levels of ATF4 in a time-dependent manner. (6) siRNA-mediated knockdown of GCN2 abrogated 4-HNE-induced induction of amino acid metabolic genes. [Conclusions] Activation of eIF2 α -ATF4 pathway via GCN2 kinase might be of special importance in the transcriptional control that coordinately promotes amino acid metabolism in response to aldehydes. Intracellular depletion of free histidine is at least partly involved in the activation of GCN2 kinase by aldehydes.

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Abstract 14877: Native T1-based Cardiovascular Magnetic Resonance for Characterizing Chronic Myocardial Infarctions: Proof-of-Concept Study in Canines

Circulation ◽

10.1161/circ.130.suppl_2.14877 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Avinash Kali ◽

Ivan Cokic ◽

Richard L Tang ◽

Hsin J Yang ◽

Behzad Sharif ◽

...

Keyword(s):

Cardiovascular Magnetic Resonance ◽

Magnetic Resonance ◽

Infarct Size ◽

T1 Mapping ◽

Ischemia Reperfusion Injury ◽

Ex Vivo ◽

Ischemia Reperfusion ◽

Infarcted Myocardium ◽

Native T1 ◽

Replacement Fibrosis

Introduction: Gadolinium infusion required for Late Gadolinium Enhancement (LGE) Cardiovascular Magnetic Resonance (CMR) imaging is contraindicated in nearly 20% of myocardial infarction (MI) patients due to chronic end-stage kidney disease. Hypothesis: Using a canine model of MI, we investigated whether native T1 mapping at 3T could be an alternative to LGE CMR for characterizing chronic MIs (CMIs). Methods: Canines (n=29) were subjected to ischemia-reperfusion injury. Native T1 maps, native T2 maps and LGE images were acquired at 7 days (acute, AMI) and 4 months (CMI) post-MI at 1.5T and 3T. Infarct location, size and transmurality, measured using Mean + 5 Standard Deviations criterion, were compared between T1 maps and LGE images. Native T2 maps were used to examine the resolution of edema between AMI and CMI. Following the CMR studies, animals were euthanized and ex-vivo histology was performed. Results: T1 maps and LGE images were not different for measuring infarct size (p=0.61) and transmurality (p=0.81) in CMI at 3T. In AMI at 3T, T1 maps overestimated both infarct size (p=0.007) and transmurality (p=0.007) relative to LGE images. At 1.5T, T1 maps underestimated both infarct size and transmurality relative to LGE images in both AMI and CMI (p<0.001 for all cases). Relative to the remote territories, T1 of the infarcted myocardium was elevated in AMI (3T: p<0.001; 1.5T: p<0.001) and CMI (3T: p<0.001; 1.5T: p=0.037). T2 of the infarcted myocardium was elevated in AMI (p<0.001 at both 3T and 1.5T), but not in CMI (3T: p=0.19, 1.5T: p=0.55) indicating that myocardial edema resolved by 4 months post-MI. Masson’s trichrome staining showed extensive replacement fibrosis within CMIs. Sensitivity and specificity of T1 maps to detect CMI were 95% and 97% respectively at 3T, and 58% and 78% respectively at 1.5T. Conclusions: Native T1 mapping at 3T can characterize CMIs with high diagnostic accuracy. T1 elongations in CMI appear to arise predominantly from replacement fibrosis.

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PPARβ/δ Is Required for Mesenchymal Stem Cell Cardioprotective Effects Independently of Their Anti-inflammatory Properties in Myocardial Ischemia-Reperfusion Injury

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2021.681002 ◽

2021 ◽

Vol 8 ◽

Author(s):

Nitirut Nernpermpisooth ◽

Charlotte Sarre ◽

Christian Barrere ◽

Rafaël Contreras ◽

Patricia Luz-Crawford ◽

...

Keyword(s):

Infarct Size ◽

Ischemia Reperfusion Injury ◽

Ex Vivo ◽

Ischemia Reperfusion ◽

Therapeutic Effects ◽

Beneficial Effects ◽

Adult Mesenchymal Stem Cells ◽

Myocardial Ischemia Reperfusion Injury ◽

Anti Inflammatory ◽

Myocardial Ischemia Reperfusion

Myocardial infarction ranks first for the mortality worldwide. Because the adult heart is unable to regenerate, fibrosis develops to compensate for the loss of contractile tissue after infarction, leading to cardiac remodeling and heart failure. Adult mesenchymal stem cells (MSC) regenerative properties, as well as their safety and efficacy, have been demonstrated in preclinical models. However, in clinical trials, their beneficial effects are controversial. In an experimental model of arthritis, we have previously shown that PPARβ/δ deficiency enhanced the therapeutic effect of MSC. The aim of the present study was to compare the therapeutic effects of wild-type MSC (MSC) and MSC deficient for PPARβ/δ (KO MSC) perfused in an ex vivo mouse model of ischemia-reperfusion (IR) injury. For this purpose, hearts from C57BL/6J mice were subjected ex vivo to 30 min ischemia followed by 1-h reperfusion. MSC and KO MSC were injected into the Langendorff system during reperfusion. After 1 h of reperfusion, the TTC method was used to assess infarct size. Coronary effluents collected in basal condition (before ischemia) and after ischemia at 1 h of reperfusion were analyzed for their cytokine profiles. The dose-response curve for the cardioprotection was established ex vivo using different doses of MSC (3.105, 6.105, and 24.105 cells/heart) and the dose of 6.105 MSC was found to be the optimal concentration. We showed that the cardioprotective effect of MSC was PPARβ/δ-dependent since it was lost using KO MSC. Moreover, cytokine profiling of the coronary effluents collected in the eluates after 60 min of reperfusion revealed that MSC treatment decreases CXCL1 chemokine and interleukin-6 release compared with untreated hearts. This anti-inflammatory effect of MSC was also observed when hearts were treated with PPARβ/δ-deficient MSC. In conclusion, our study revealed that the acute cardioprotective properties of MSC in an ex vivo model of IR injury, assessed by a decreased infarct size at 1 h of reperfusion, are PPARβ/δ-dependent but not related to their anti-inflammatory effects.

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Abstract 314: Infarct Size Reduction by Statins Requires Parkin and Mitophagy

Circulation Research ◽

10.1161/res.111.suppl_1.a314 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Allen M Andres ◽

Pamela Lee ◽

Genaro Hernandez ◽

Chengqun Huang ◽

Eric P Ratliff ◽

...

Keyword(s):

Infarct Size ◽

Cardiac Tissue ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Statin Treatment ◽

Mtor Signaling ◽

Wild Type ◽

Area At Risk ◽

Hmg Coa Reductase ◽

Coa Reductase

The cardioprotective effects of statins are well known yet the mechanism is unclear. Previously we showed that autophagy is required for cardioprotection from ischemia/reperfusion injury. More recently, we reported that ischemic preconditioning involves Parkin-mediated mitophagy. We hypothesized that the molecular basis of statin-mediated cardioprotection may involve mitochondrial quality control through mitophagy. HL-1 cardiomyocytes treated with simvastatin for 24hr exhibited diminished Akt/mTOR signaling, increased activation of ULK1, and upregulation of autophagy (n=3, p<0.05). Similar findings were obtained in cardiac tissue in mice 4hr after i.p. administration of simvastatin. Mevalonate addition abolished statin’s effects on Akt/mTOR signaling and autophagy induction in HL-1 cells, indicating that the effects are mediated through inhibition of HMG-CoA reductase. Statin treatment in HL-1 cells triggered mitochondrial fragmentation, translocation of Parkin and p62/SQSTM1 to the mitochondria followed by mitophagy. To establish the requirement for statin-mediated mitophagy in cardioprotection, we investigated the ability of statins to reduce infarct size in Parkin knockout (KO) mice. While statin treatment reduced infarct size from 55% of area at risk to 30% in wild type mice, it had no protective benefit in Parkin KO mice (n=4-6, p<0.05). These findings indicate that cardioprotection by HMG-CoA reductase inhibitors involves suppression of mTOR signaling and induction of Parkin-dependent mitophagy. Figure: Statin-induced cardioprotection against I/R injury: solid bars/diamonds = wild-type; open bars/diamonds = Parkin knockout mice.

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Intralipid, a Clinically Safe Compound, Protects the Heart Against Ischemia-Reperfusion Injury More Efficiently Than Cyclosporine-A

Anesthesiology ◽

10.1097/aln.0b013e3182655e73 ◽

2012 ◽

Vol 117 (4) ◽

pp. 836-846 ◽

Cited By ~ 52

Author(s):

Jingyuan Li ◽

Andrea Iorga ◽

Salil Sharma ◽

Ji-Youn Youn ◽

Rod Partow-Navid ◽

...

Keyword(s):

Infarct Size ◽

Cyclosporine A ◽

Mitochondrial Permeability Transition Pore ◽

Mitochondrial Permeability Transition ◽

Ischemia Reperfusion Injury ◽

Ex Vivo ◽

Ischemia Reperfusion ◽

Permeability Transition ◽

Mitochondrial Permeability

Background We have recently shown that postischemic administration of intralipid protects the heart against ischemia-reperfusion injury. Here we compared the cardioprotective effects of intralipid with cyclosporine-A, a potent inhibitor of the mitochondrial permeability transition pore opening. Methods In vivo rat hearts or isolated Langendorff-perfused mouse hearts were subjected to ischemia followed by reperfusion with intralipid (0.5%, 1% and 2% ex-vivo, and 20% in vivo), cyclosporine-A (0.2 μM, 0.8 μM, and 1.5 μM ex- vivo and 10 mg/kg in vivo), or vehicle. The hemodynamic function, infarct size, calcium retention capacity, mitochondrial superoxide production, and phosphorylation levels of protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β) were measured. The values are mean ± SEM. Results Administration of intralipid at reperfusion significantly reduced myocardial infarct size compared with cyclosporine-A in vivo (infarct size/area at risk)%: 22.9 ± 2.5% vs. 35.2 ± 3.5%; P = 0.030, n = 7/group). Postischemic administration of intralipid at its optimal dose (1%) was more effective than cyclosporine-A (0.8 μM) in protecting the ex vivo heart against ischemia-reperfusion injury, as the rate pressure product at the end of reperfusion was significantly higher (mmHg · beats/min: 12,740 ± 675 [n = 7] vs. 9,203 ± 10,781 [n = 5], P = 0.024), and the infarct size was markedly smaller (17.3 ± 2.9 [n = 7] vs. 29.2 ± 2.7 [n = 5], P = 0.014). Intralipid was as efficient as cyclosporine-A in inhibiting the mitochondrial permeability transition pore opening (calcium retention capacity = 280 ± 8.2 vs. 260.3 ± 2.9 nmol/mg mitochondria protein in cyclosporine-A, P = 0.454, n = 6) and in reducing cardiac mitochondrial superoxide production. Unlike intralipid, which increased phosphorylation of Akt (6-fold) and GSK-3β (5-fold), cyclosporine-A had no effect on the activation of these prosurvival kinases. Conclusions Although intralipid inhibits the opening of the mitochondrial permeability transition pore as efficiently as cyclosporine-A, intralipid is more effective in reducing the infarct size and improving the cardiac functional recovery.

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Abstract P339: The Mechanistic Relationship Between GRK2 and eNOS During Cardiac Ischemia/Reperfusion Injury

Circulation Research ◽

10.1161/res.109.suppl_1.ap339 ◽

2011 ◽

Vol 109 (suppl_1) ◽

Author(s):

Zheng M Huang ◽

Erhe Gao ◽

Xiying Shang ◽

Xufan Tian ◽

Gang Qiu ◽

...

Keyword(s):

Cardiac Function ◽

Infarct Size ◽

Cardiac Tissue ◽

Ventricular Myocytes ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Coronary Artery Ligation ◽

Protein Activity ◽

Artery Ligation ◽

Cardiac Phenotype

Background: Previous studies have shown that both cardiac-specific GRK2 transgenic (TG) (αMHC-GRK2) mice and eNOS knockout (KO) mice have larger infarcts after I/R compared to control mice. In contrast, cardiac-targeted eNOS TG mice and cardiac specific GRK2 KO mice show cardioprotection after I/R, suggesting a dynamic interaction between the two proteins. Mechanistically, GRK2 can be inhibited by cellular NO through S-nitrosylation with Cys340 being the major site; meanwhile GRK2 has been shown to directly bind to and inhibit Akt, which is a strong activator of eNOS. Aim: to investigate the potential novel interaction between GRK2 and eNOS, and the consequent functional impact on the protein activity and cardiac phenotype after I/R injury. Methods: αMHC-GRK2 mice were crossed with either eNOS TG or eNOS KO mice. All mice were subjected to sham or 30min myocardial ischemia via coronary artery ligation followed by 24hrs of reperfusion. Infarct size, cardiac function, and tissue apoptosis were examined. Co-IP was used to test the interaction between GRK2 and eNOS, and phosphorylation of eNOS was studied in neonatal myocytes. Results: 1).αMHC-GRK2/eNOS TG hybrid mice showed a significantly reduced infarct size after I/R compared to αMHC-GRK2 mice, accompanied by improved cardiac function measured by echocardiography and hemodynamics, and significantly less apoptosis tested by TUNEL assay, implying a rescue effect by eNOS. 2). αMHC-GRK2/eNOS KO mice exhibited a bigger infarct size compared to either αMHC-GRK2 mice or eNOS KO mice. 3). CO-IP confirmed the interaction between GRK2 and eNOS in cardiac tissue, which was increased upon β-AR agonist treatment. 4). In neonatal ventricular myocytes, GRK2 overexpression significantly decreased eNOS phosphorylation at Ser1177 after exposure to H2O2, while GRK2 knockdown by siRNA led to slight increase in pSer1177. Conclusions: eNOS interacts with and may be a downstream target of GRK2 in the heart. Decreased activation of eNOS may mediate the deleterious effect of GRK2 overexpression during cardiac I/R injury.

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Sphingosine Protects Aging Hearts from Ischemia/Reperfusion Injury: Superiority to Sphingosine 1-Phosphate and Ischemic Pre- and Post-Conditioning

Oxidative Medicine and Cellular Longevity ◽

10.4161/oxim.2.3.8622 ◽

2009 ◽

Vol 2 (3) ◽

pp. 146-151 ◽

Cited By ~ 30

Author(s):

Donald A. Vessey ◽

Michael Kelley ◽

Luyi Li ◽

Yong Huang

Keyword(s):

Infarct Size ◽

Ischemia Reperfusion Injury ◽

Ex Vivo ◽

Ischemia Reperfusion ◽

Left Ventricular ◽

Diminished Capacity ◽

Sphingosine 1 Phosphate ◽

Phosphate Treatment ◽

Developed Pressure ◽

Large Infarct

Aging hearts are known to have diminished capacity to be protected against reoxygenation ischemia/reperfusion (IR) injury provided by various cardioprotective regimens. In search of a more successful regimen, we have studied the response of aged hearts to preconditioning (PC) and postconditioning (POST) elicited by sphingosine or sphingosine 1-phosphate treatment.An ex vivo rat heart model was used to study the ability of PC and POST to protect old hearts (27 month) against I/R injury generated by 40 minutes (min) of index ischemia followed by 40 min of reperfusion. The response to ischemic PC was reduced in 27 month old hearts relative to 3–6 month (young) hearts as noted by a poor recovery of left ventricular developed pressure (LVDP) upon reperfusion (45% vs. 74% in young hearts) and a large infarct size after 40 min of reperfusion (37% versus 8% in young hearts). PC with sphingosine 1-phosphate (S1P) was also poor in old hearts yielding only 49% recovery of LVDP and a 27% infarct size. In contrast, PC with sphingosine was unaffected by aging; the 78% recovery of LVDP and 8% infarct size were not different from young hearts. Ischemic POST was less affected by aging than ischemic PC, but the old hearts still experienced infarct sizes of 28%. POST of old hearts with S1P was also associated with a substantial infarct size (24%). However, POST of old hearts with sphingosine was superior to the other forms of POST in that it reduced the infarct size to 12%. S1P levels were found to be lower in old hearts which may contribute to the decreased effectiveness of ischemic PC and POST. Further, phospho-Akt levels and distribution were altered in response to cardioprotection in the old hearts. In conclusion, POST was less affected by aging than PC; and sphingosine is a uniquely effective agent for both PC and POST of aging hearts.

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Decorin Protects Cardiac Myocytes against Simulated Ischemia/Reperfusion Injury

Molecules ◽

10.3390/molecules25153426 ◽

2020 ◽

Vol 25 (15) ◽

pp. 3426 ◽

Cited By ~ 1

Author(s):

Renáta Gáspár ◽

Kamilla Gömöri ◽

Bernadett Kiss ◽

Ágnes Szántai ◽

János Pálóczi ◽

...

Keyword(s):

Cardiac Myocytes ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Eukaryotic Translation ◽

Downstream Signaling ◽

Adult Rat ◽

Simulated Ischemia ◽

Survival Pathways

Search for new cardioprotective therapies is of great importance since no cardioprotective drugs are available on the market. In line with this need, several natural biomolecules have been extensively tested for their potential cardioprotective effects. Previously, we have shown that biglycan, a member of a diverse group of small leucine-rich proteoglycans, enhanced the expression of cardioprotective genes and decreased ischemia/reperfusion-induced cardiomyocyte death via a TLR-4 dependent mechanism. Therefore, in the present study we aimed to test whether decorin, a small leucine-rich proteoglycan closely related to biglycan, could exert cardiocytoprotection and to reveal possible downstream signaling pathways. Methods: Primary cardiomyocytes isolated from neonatal and adult rat hearts were treated with 0 (Vehicle), 1, 3, 10, 30 and 100 nM decorin as 20 h pretreatment and maintained throughout simulated ischemia and reperfusion (SI/R). In separate experiments, to test the mechanism of decorin-induced cardio protection, 3 nM decorin was applied in combination with inhibitors of known survival pathways, that is, the NOS inhibitor L-NAME, the PKG inhibitor KT-5823 and the TLR-4 inhibitor TAK-242, respectively. mRNA expression changes were measured after SI/R injury. Results: Cell viability of both neonatal and adult cardiomyocytes was significantly decreased due to SI/R injury. Decorin at 1, 3 and 10 nM concentrations significantly increased the survival of both neonatal and adult myocytes after SI/R. At 3nM (the most pronounced protective concentration), it had no effect on apoptotic rate of neonatal cardiac myocytes. No one of the inhibitors of survival pathways (L-NAME, KT-5823, TAK-242) influenced the cardiocytoprotective effect of decorin. MYND-type containing 19 (Zmynd19) and eukaryotic translation initiation factor 4E nuclear import factor 1 (Eif4enif1) were significantly upregulated due to the decorin treatment. In conclusion, this is the first demonstration that decorin exerts a direct cardiocytoprotective effect possibly independent of NO-cGMP-PKG and TLR-4 dependent survival signaling.

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