Role of lipid rafts in ceramide and nitric oxide signaling in the ischemic and preconditioned hearts

doi:10.1016/j.yjmcc.2005.10.005

Journal of Molecular and Cellular Cardiology

Volume 40, Issue 2, February 2006, Pages 313-320

https://doi.org/10.1016/j.yjmcc.2005.10.005 Get rights and content

Abstract

Nitric oxide plays a crucial role in myocardial ischemia reperfusion injury as well as in myocardial adaptation to ischemic stress. To understand the dichotomy of nitric oxide behavior in the ischemic myocardium, isolated rat hearts were subjected to ischemia/reperfusion protocol. The tissue contents of sphingomyelin (SM), ceramide and sphingosine were determined by high performance thin layer chromatography (HPTLC). The myocardial plasma proteins were immunoprecipitated with caveolin-1 specific antibody. Ischemia/reperfusion resulted in the breakdown of SM with corresponding accumulation of ceramide and sphingosine. Immunoprecipitation with eNOS-specific antibody revealed the association of eNOS with caveolin-1 fraction of the heart. Ischemia/reperfusion caused a depression of contractile function and an increased apoptotic cell death and myocardial infarct size, which were reversed by preperfusing the hearts with desipramine, an sphingomyelinase inhibitor that also prevented ceramide accumulation and eNOS association with caveolin-1. The similar results were obtained when the hearts were adapted to ischemic stress by subjecting them to repeated reversible ischemia and reperfusion. The results indicate that ischemia/reperfusion causes an increase in eNOS, which is unavailable to the ischemic heart because of its binding with caveolin-1. Ceramide plays a crucial role in this process, because prevention of ceramide formation either by myocardial adaptation to ischemia or with desipramine results in the inhibition of eNOS association with caveolin-1 thereby reducing myocardial ischemic reperfusion injury.

Introduction

In animal models of tissue ischemia, brief cyclic episodes of ischemia and reperfusion induce tolerance and protect the tissue from subsequent otherwise lethal ischemia. This phenomenon has been named as “myocardial adaptation to ischemia” or “ischemic preconditioning (PC)”, and PC remains a state-of-the-art technique for protection of many vital organs including brain, liver, kidney, lung and heart [1], [2], [3], [4], [5]. The mechanism of PC has been extensively studied in the heart [5], [6]. A series of events regulated by the intracellular mediators occurs immediately during the development of stress tolerance by PC [7], [8]. A cascade of signaling events initiated by intracellular mediators and/or reactive oxygen species (ROS) follow, resulting in a stress-resistant heart, which can protect itself from subsequent lethal ischemic injury [9], [10].

Recently, a role of the intracellular messenger ceramide has been indicated in PC [11], [12]. Ceramide generated from sphingomyelin (SM) during ischemia/reperfusion can induce cardiomyocyte death [13]. Ischemia/reperfusion can also generate another second messenger sn-1,2-diacylglycerol (DAG) that tends to inhibit the effects of ceramide [14]. In a recent study, PC was found to enhance DAG, which in turn neutralized the damaging effects of ceramide [15]. In another study, TNFα-mediated PC occurred through ceramide signaling [16]. Cell permeable exogenous ceramide reduced the infarct size of the brain supporting the role of ceramide in PC [17].

Evidence is rapidly accumulating suggesting that ceramide performs its signaling function from within the lipid rafts, ordered sphingolipid and cholesterol-rich lipid domains [18], which can function as an ordered support for receptor-mediated signaling events. Caveolae, 50–100 nm invaginations of the plasma membrane, are subset of lipid rafts enriched in sphingolipids and cholesterol. The caveolae can selectively sequestrate membrane-targeted proteins and create a unique signaling microdomain, thereby controlling transmembrane signaling [19]. Caveolae are characterized by the presence caveolins, which distinguishes caveolae from other lipid raft domains. At least three caveolins isoforms of molecular weights between 22 and 24 kDa have been identified of which caveolin-1 and caveolin-2 are abundant in most cell types while caveolin-3 is specific to muscle cells [20]. Caveolin-1, a substrate for nonreceptor tyrosine kinases including Fyn, Abl and Src, acts as a scaffolding protein and can be phosphorylated on tyrosine 14 by these kinases in response to external stimuli such as oxidative stress [21]. Such tyrosine phosphorylation activates the downstream signaling targets, and thus, serves as a crucial step for intracellular signaling occurring within caveolae.

Nitric oxide plays a crucial role in myocardial PC [22], [23]. Both iNOS and eNOS have been found to be activated in the preconditioned heart [24], [25]. Because ceramide signaling may occur through NO and NOS can localize in lipid raft/caveolin-rich microdomains, we sought to determine if lipid rafts play any role in ceramide-NO signaling in the ischemic and preconditioned heart. The results of our study determined that sphingosine-1-phosphate derived from ceramide in the PC heart could reduce cardiomyocyte apoptosis. There was an increased association of eNOS with caveolin-1 in the ischemic reperfused heart, which was disrupted either by PC or with desipramine, both of which functioned by reducing ceramide formation in the ischemic reperfused myocardium.

Section snippets

Chemicals

SM, sphingosine, sphingosine-1-phosphate (sphingosine-1-P) and ceramide were obtained from Sigma, St. Louis, MO, Antibodies against caveolin-1, iNOS, eNOS and cSrc were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA.

Experimental protocol

The study used two different protocols using isolated working rat heart model: i) isolated working rat hearts preperfused with the Krebs Henseleit buffer (KHB) for 15 min in the absence or presence of desipramine followed by 30 min ischemia and 2 h of reperfusion and

Effects of ischemia/reperfusion and PC on caramide and spgingosine-1-P accumulation in caveolae

We estimated the amount of ceramide and sphingosine-1-P content in the isolated caveolae fraction. As shown in Fig. 1, significant amount of ceramide was associated with caveolae after ischemia/reperfusion. The amount of ceramide in caveolae fraction was significantly lower (threefold less) when the heart was subjected to PC. In the preconditioned heart, the reduction of ceramide content was associated with an enhancement in sphingosine-1-P content in the caveolae, suggesting that

Discussion

The results of the present study demonstrate that significant amount of ceramide is accumulated in the caveolin-rich membrane as a result of ischemia/reperfusion. An increased amount of eNOS becomes associated with caveolin-1 in these hearts. PC results in a reduction of ceramide with corresponding increase in its metabolite, sphingosine-1-P. The amount of caveolin-1-associated eNOS is reduced when the heart is subjected to PC. Inhibition of ceramide formation with desipramine also resulted in

Acknowledgments

This study was supported in part by NIH HL 34360, HL 22559 and HL 33889.

References (42)

P.L. Huang
Nitric oxide and cerebral ischemic preconditioning
Cell Calcium
(2004)
K.M. Park et al.
Inducible nitric-oxide synthase is an important contributor to prolonged protective effects of ischemic preconditioning in the mouse kidney
J. Biol. Chem.
(2003)
A. Eisen et al.
Ischemic preconditioning: nearly two decades of research. A comprehensive review
Atherosclerosis
(2004)
J. Cui et al.
Role of ceramide in ischemic preconditioning
J. Am. Coll. Surg.
(2004)
W.D.J. Jarvis et al.
Attenuation of ceramide-induced apoptosis by diglyceride in human myeloid leukemia cells
J. Biol. Chem.
(1994)
J. Couet et al.
Molecular and cellular biology of caveolae: paradoxes and plasticities
Trends Cardiovasc. Med.
(1997)
R.G. Parton
Caveolae and caveolins
Curr Opin Cell Biol
(1996)
R. Bolli
Cardioprotective function of inducible nitric oxide synthase and role of nitric oxide in myocardial ischemia and preconditioning: an overview of a decade of research
J. Mol. Cell. Cardiol.
(2001)
N. Maulik et al.
Regulation of cardiomyocyte apoptosis by redox-sensitive transcription factors
FEBS Lett.
(2000)
K.S. Song et al.
Co-purification and direct interaction of Ras with Caveolin, an integral membrane protein of caveolae microdomains
J. Biol. Chem.
(1996)

G.A. Cordis et al.

HPTLC analysis of sphingomylein, ceramide and sphingosine in ischemic/reperfused rat heart

J. Pharm. Biomed. Anal.

(1998)

P. Pagliaro

Differential biological effects of products of nitric oxide (NO) synthase: it is not enough to say NO

Life Sci.

(2003)

K.M. Park et al.

Inducible nitric oxide synthase is an important contributor to prolonged protective effects of ischemic preconditioning in the mouse kidney

J. Biol. Chem.

(2003)

D.T. Engelman et al.

L-Arginine reduces endothelial inflammation and myocardial stunning during ischemia/reperfusion

Ann Thorac Surg

(1995)

M. Salinas et al.

Inhibition of PKB/Akt1 by C2-ceramide involves activation of ceramide-activated protein phosphatase in PC12 cells

Mol. Cell. Neurosci.

(2000)

P.P. Ruvolo et al.

Ceramide induces Bcl-2 dephos-phorylation via a mechanism involving mitochondrial PP2A

J. Biol. Chem.

(1999)

X. Xu et al.

Effect of the Structure of Natural Sterols and Sphingolipids on the Formation of Ordered Sphingolipid/Sterol Domains (Rafts). Comparison of cholesterol to plant, fungal, and disease-associated sterols and comparison of sphingomyelin, cerebrosides, and ceramide

J. Biol. Chem.

(2001)

P.A. Clavien et al.

A prospective randomized study in 100 consecutive patients undergoing major liver resection with versus without ischemic preconditioning

Annal Surgery.

(2003)

K. Ito et al.

Pharmacological preconditioning protects lung injury induced by intestinal ischemia/reperfusion in rat

Shock

(2003)

M. Sato et al.

SAPKs regulation of ischemic preconditioning

Am. J. Physiol.

(2000)

T. Yoshida et al.

Hmox-1 constitutes an adaptive response to effect antioxidant cardioprotection: a study with transgenic mice heterozygous for targeted disruption of the HO-1 gene

Circulation

(2001)

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Activation of ASMase is often an early response to various cellular stresses and precedes ceramide production, which accelerates signaling pathways involved in cell death (Marchesini and Hannun, 2004). Previous studies have shown that elevation of ceramide levels occurs in several in vivo ischemia models, including heart (Bielawska et al., 1997; Cordis et al., 1998; Cui et al., 2004; Der et al., 2006; Zhang et al., 2001), liver (Bradham et al., 1997; Kukimoto et al., 1995), kidney (Zager et al., 1998), brain (Herr et al., 1999; Kubota et al., 1989, 1996; Nakane et al., 2000; Takahashi et al., 2004; Yu et al., 2000) and retinas (Fan et al., 2016). In the brain, the upregulation of ASMase activity is responsible for the ischemia-induced rise in astroglial ceramide levels (Ohtani et al., 2004).
Sphingomyelinases (SMase), enzymes that catalyze the hydrolysis of sphingomyelin to ceramide, are important sensors for inflammatory cytokines and apoptotic signaling. Studies have provided evidence that increased SMase activity can contribute to retinal injury. In most tissues, two major SMases are responsible for stress-induced increases in ceramide: acid sphingomyelinase (ASMase) and Mg²⁺-dependent neutral sphingomyelinase (NSMase). The purposes of the current study were to determine the localization of SMases and their substrates in the retina and optic nerve head and to investigate the effects of ocular hypertension and ischemia on ASMase and NSMase activities.
Tissue and cellular localization of ASMase and NSMase were determined by immunofluorescence imaging. Tissue localization of sphingomyelin in retinas was further determined by Matrix-Assisted Laser Desorption/Ionization mass spectrometry imaging. Tissue levels of sphingomyelins and ceramide were determined by liquid chromatography with tandem mass spectrometry. Sphingomyelinase activities under basal conditions and following acute ischemic and ocular hypotensive stress were measured using the Amplex Red Sphingomyelinase Assay Kit. Our data show that ASMase is in the optic nerve head and the retinal ganglion cell layer. NSMase is in the optic nerve head, photoreceptor and retinal ganglion cell layers. Both ASMase and NSMase were identified in human induced pluripotent stem cell-derived retinal ganglion cells and optic nerve head astrocytes. The retina and optic nerve head each exhibited unique distribution of sphingomyelins with the abundance of very long chain species being higher in the optic nerve head than in the retina. Basal activities for ASMase in retinas and optic nerve heads were 54.98 ± 2.5 and 95.6 ± 19.5 mU/mg protein, respectively. Ocular ischemia significantly increased ASMase activity to 86.2 ± 15.3 mU/mg protein in retinas (P = 0.03) but not in optic nerve heads (81.1 ± 15.3 mU/mg protein). Ocular hypertension significantly increased ASMase activity to 121.6 ± 7.3 mU/mg protein in retinas (P < 0.001) and 267.0 ± 66.3 mU/mg protein in optic nerve heads (P = 0.03). Basal activities for NSMase in retinas and optic nerve heads were 12.3 ± 2.1 and 37.9 ± 8.7 mU/mg protein, respectively. No significant change in NSMase activity was measured following ocular ischemia or hypertension.
Our results provide evidence that both ASMase and NSMase are expressed in retinas and optic nerve heads; however, basal ASMase activity is significantly higher than NSMase activity in retinas and optic nerve heads. In addition, only ASMase activity was significantly increased in ocular ischemia or hypertension. These data support a role for ASMase-mediated sphingolipid metabolism in the development of retinal ischemic and hypertensive injuries.
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This process is dependent on hydrolysis of ASMase [122]. Numerous models assessing ischaemia and re-perfusion in vivo have evidenced the role of Cer as a pro-apoptotic agent in the heart, kidney and brain when bearing this pathological burden [123–127]. Furthermore, Takahashi et al. have also demonstrated that S1PR1 agonism is associated with collateral retinal vessel formation in laser-induced, retinal vein occlusion mouse models [128].
The sphingolipids ceramide (Cer), ceramide-1-phosphate (C1P), sphingosine (Sph), and sphingosine-1-phosphate (S1P)) are key signaling molecules that regulate many patho-biological processes. During the last decade, they have gained increasing attention since they may participate in important and numerous retinal processes, such as neuronal survival and death, proliferation and migration of neuronal and vascular cells, inflammation, and neovascularization. Cer for instance has emerged as a key mediator of inflammation and death of neuronal and retinal pigment epithelium cells in experimental models of retinopathies such as glaucoma, age-related macular degeneration (AMD), and retinitis pigmentosa. S1P may have opposite biological actions, preventing photoreceptor and ganglion cell degeneration but also promoting inflammation, fibrosis, and neovascularization in AMD, glaucoma, and pro-fibrotic disorders. Alterations in Cer, S1P, and ceramide 1- phosphate may also contribute to uveitis. Furthermore, use of inhibitors that either prevent Cer increase or modulate S1P signaling, such as Myriocin, desipramine, and Fingolimod (FTY720), have been shown to preserve neuronal viability and retinal function. Collectively, the expanding role for these sphingolipids in the modulation of vital processes in retina cell types and in their dysregulation in retinal degenerations makes them attractive therapeutic targets.
Role of sphingomyelinase in mitochondrial ceramide accumulation during reperfusion
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Ceramide accumulation in mitochondria has been associated with reperfusion damage, but the underlying mechanisms are not clearly elucidated. In this work we investigate the role of sphingomyelinases in mitochondrial ceramide accumulation, its effect on reactive oxygen species production, as well as on mitochondrial function by using the sphingomyelinase inhibitor, tricyclodecan-9-yl-xanthogenate (D609). Correlation between neutral sphingomyelinase (nSMase) activity and changes in ceramide content were performed in whole tissue and in isolated mitochondria from reperfused hearts. Overall results demonstrated that D609 treatment attenuates cardiac dysfuncion, mitochondrial injury and oxidative stress. Ceramide was accumulated in mitochondria, but not in the microsomal fraction of the ischemic-reperfused (I/R) group. In close association, the activity of nSMase increased, whereas glutathione (GSH) levels diminished in mitochondria after reperfusion. On the other hand, reduction of ceramide levels in mitochondria from I/R + D609 hearts correlated with diminished nSMase activity, coupling of oxidative phosphorylation and with mitochondrial integrity maintenance. These results suggest that mitochondrial nSMase activity contributes to compartmentation and further accumulation of ceramide in mitochondria, deregulating their function during reperfusion.
Lipid partitioning during cardiac stress
2016, Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
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Several research groups have identified an increase in ceramide content during ischemia and/or ischemia–reperfusion in animal models of coronary artery occlusion or Langendorff isolated heart preparations [39,71,99–101]. Furthermore, reducing ceramide accumulation via ischemic preconditioning attenuates measures of apoptosis [99,100,102]. Interestingly, an increase in specific ceramide species has been noted with profound increases in the C18:2 and C20:4 ceramides, which are typically in low abundance [71].
It is well documented that fatty acids serve as the primary fuel substrate for the contracting myocardium. However, extensive research has identified significant changes in the myocardial oxidation of fatty acids during acute or chronic cardiac stress. As a result, the redistribution or partitioning of fatty acids due to metabolic derangements could have biological implications. Fatty acids can be stored as triacylglycerols, serve as critical components for biosynthesis of phospholipid membranes, and form the potent signaling molecules, diacylglycerol and ceramides. Therefore, the contribution of lipid metabolism to health and disease is more intricate than a balance of uptake and oxidation. In this review, the available data regarding alterations that occur in endogenous cardiac lipid pathways during the pathological stressors of ischemia–reperfusion and pathological hypertrophy/heart failure are highlighted. In addition, changes in endogenous lipids observed in exercise training models are presented for comparison. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.
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Fatty acids (FAs) are essential components of the body that help maintain a normal physiological function and a protective response to pathological stimuli. Small changes in the dietary intake of FAs can influence membrane integrity and cellular signaling which can affect cardiovascular disease (CVD) outcomes. Myocardial damage resulting from ischemia/reperfusion injury has a profound effect in both the short- and long-term survivability of the individual. Relatively little mechanistic information is available regarding a molecular basis for the cardioprotective and cardiotoxic effects of FAs on ischemia/reperfusion injury. However, clearly, based on experimental data, the beneficial effects of polyunsaturated fatty acids (PUFAs) include the prevention of cardiac arrhythmias, an increased oxygen efficiency, the preservation of mitochondrial function, an altered membrane lipid composition, and a membrane-domain functionality. Thus, PUFAs can favorably modulate intracellular-signaling events in response to extracellular stress stimuli. Further mechanistic studies are needed to clarify the role of FAs in ischemia/reperfusion injury. However, while saturated fatty acids and trans fatty acids tend to be detrimental, both n-3 and n-6 PUFAs play distinct roles in mediating ischemic injury. As such, maintaining the balance between n-3 and n-6 PUFAs is a critical factor influencing a CVD outcome.
Enhanced glucose uptake via GLUT4 fuels recovery from calcium overload after ischaemiareperfusion injury in sevoflurane- but not propofol-treated hearts
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In our study, sevoflurane also decreased the formation of ceramides, which are known to mediate insulin resistance by inhibiting protein kinase B.28 Inhibition of serine palmitoyltransferase, the rate-limiting enzyme in ceramide synthesis, reduces FOX but concomitantly increases GOX.29 During ischaemia–reperfusion, ceramides also accumulate in lipid rafts,30 which might affect GLUT4 function. Using microdialysis probes, Carles and colleagues31 showed a better availability of glucose in the skeletal muscle of patients during ischaemia and reperfusion after sevoflurane exposure.
So far, no study has explored the effects of sevoflurane, propofol, and Intralipid on metabolic flux rates of fatty acid oxidation (FOX) and glucose oxidation (GOX) in hearts exposed to ischaemia–reperfusion.
Isolated paced working rat hearts were exposed to 20 min of ischaemia and 30 min of reperfusion. Peri-ischaemic sevoflurane (2 vol%) and propofol (100 µM) in the formulation of 1% Diprivan^® were assessed for their effects on oxidative energy metabolism and intracellular diastolic and systolic Ca²⁺ concentrations. Substrate flux was measured using [³H]palmitate and [¹⁴C]glucose and [Ca²⁺] using indo-1AM. Western blotting was used to determine the expression of the sarcolemmal glucose transporter GLUT4 in lipid rafts. Biochemical analyses of nucleotides, ceramides, and 32 acylcarnitines were also performed.
Sevoflurane, but not propofol, improved the recovery of left ventricular work (P=0.008) and myocardial efficiency (P=0.008) compared with untreated ischaemic hearts. This functional improvement was accompanied by reduced increases in post-ischaemic diastolic and systolic intracellular Ca²⁺ concentrations (P=0.008). Sevoflurane, but not propofol, increased GOX (P=0.009) and decreased FOX (P=0.019) in hearts exposed to ischaemia–reperfusion. GLUT4 expression was markedly increased in lipid rafts of sevoflurane-treated hearts (P=0.016). Increased GOX closely correlated with reduced Ca²⁺ overload. Intralipid alone decreased energy charge and increased long-chain and hydroxyacylcarnitine tissue levels, whereas sevoflurane decreased toxic ceramide formation.
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Original articleRole of lipid rafts in ceramide and nitric oxide signaling in the ischemic and preconditioned hearts

Abstract

Introduction

Section snippets

Chemicals

Experimental protocol

Effects of ischemia/reperfusion and PC on caramide and spgingosine-1-P accumulation in caveolae

Discussion

Acknowledgments

Cell Calcium

J. Biol. Chem.

Atherosclerosis

J. Am. Coll. Surg.

J. Biol. Chem.

Trends Cardiovasc. Med.

Curr Opin Cell Biol

J. Mol. Cell. Cardiol.

FEBS Lett.

J. Biol. Chem.

J. Pharm. Biomed. Anal.

Life Sci.

J. Biol. Chem.

Ann Thorac Surg

Mol. Cell. Neurosci.

J. Biol. Chem.

J. Biol. Chem.

A prospective randomized study in 100 consecutive patients undergoing major liver resection with versus without ischemic preconditioning

Annal Surgery.

Pharmacological preconditioning protects lung injury induced by intestinal ischemia/reperfusion in rat

Shock

SAPKs regulation of ischemic preconditioning

Am. J. Physiol.

Hmox-1 constitutes an adaptive response to effect antioxidant cardioprotection: a study with transgenic mice heterozygous for targeted disruption of the HO-1 gene

Circulation

Original article
Role of lipid rafts in ceramide and nitric oxide signaling in the ischemic and preconditioned hearts