Published online before print October 20, 2008, doi: 10.1161/CIRCULATIONAHA.108.788331
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Circulation. 2008;118:1979-1988.)
© 2008 American Heart Association, Inc.
Molecular Cardiology |
Cardiac-Specific Overexpression of Caveolin-3 Induces Endogenous Cardiac Protection by Mimicking Ischemic Preconditioning
From the Departments of Anesthesiology (Y.M.T., Y.T.H., M.M.J., M.W.K., I.R.N., B.P.H., P.M.P., H.H.P., D.M.R.), Pharmacology (U.Y., P.A.I.), and Medicine (P.A.I.) and Gene Therapy Program (A.M.), University of California, San Diego, La Jolla; National Institute of Neuroscience, Kodaira, Tokyo, Japan (Y.H.); Cardiovascular Research Institute, Yokohama City University School of Medicine, Yokohama, Kanazawa, Japan (Y.I.); Department of Cell Biology and Molecular Medicine (Y.I.) and Department of Medicine (Cardiology Division) (Y.I.), New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark; and Department of Anesthesiology, VA San Diego Healthcare Systems, San Diego, Calif (P.M.P., D.M.R.).
Correspondence to Dr D.M. Roth or Dr H.H. Patel, VA San Diego Healthcare System, 9125, 3350 La Jolla Village Dr, San Diego, CA 92161–9125. E-mail droth{at}ucsd.edu or hepatel@ucsd.edu
Received April 23, 2008; accepted August 29, 2008.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionConclusionsReferences |
|---|
Background— Caveolae, lipid-rich microdomains of the sarcolemma, localize and enrich cardiac-protective signaling molecules. Caveolin-3 (Cav-3), the dominant isoform in cardiac myocytes, is a determinant of caveolar formation. We hypothesized that cardiac myocyte–specific overexpression of Cav-3 would enhance the formation of caveolae and augment cardiac protection in vivo.
Methods and Results— Ischemic preconditioning in vivo increased the formation of caveolae. Adenovirus for Cav-3 increased caveolar formation and phosphorylation of survival kinases in cardiac myocytes. A transgenic mouse with cardiac myocyte–specific overexpression of Cav-3 (Cav-3 OE) showed enhanced formation of caveolae on the sarcolemma. Cav-3 OE mice subjected to ischemia/reperfusion injury had a significantly reduced infarct size relative to transgene-negative mice. Endogenous cardiac protection in Cav-3 OE mice was similar to wild-type mice undergoing ischemic preconditioning; no increased protection was observed in preconditioned Cav-3 OE mice. Cav-3 knockout mice did not show endogenous protection and showed no protection in response to ischemic preconditioning. Cav-3 OE mouse hearts had increased basal Akt and glycogen synthase kinase-3β phosphorylation comparable to wild-type mice exposed to ischemic preconditioning. Wortmannin, a phosphoinositide 3-kinase inhibitor, attenuated basal phosphorylation of Akt and glycogen synthase kinase-3β and blocked cardiac protection in Cav-3 OE mice. Cav-3 OE mice had improved functional recovery and reduced apoptosis at 24 hours of reperfusion.
Conclusions— Expression of caveolin-3 is both necessary and sufficient for cardiac protection, a conclusion that unites long-standing ultrastructural and molecular observations in the ischemic heart. The present results indicate that increased expression of caveolins, apparently via actions that depend on phosphoinositide 3-kinase, has the potential to protect hearts exposed to ischemia/reperfusion injury.
Key Words: caveolae • caveolins • heart • myocardial ischemia
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionsReferences |
|---|
The concept that an organ can develop tolerance to subsequent ischemic stress was initially suggested by studies of traumatic injury.1 In 1986, Murry et al2 found that nonlethal injury, called ischemic preconditioning (IPC), could protect the heart from lethal injury. Subsequent work has shown that IPC is a highly effective way to protect multiple organs from ischemic injury. Many studies have evaluated individual signaling molecules and pathways in IPC,3 but no single, unifying intervention explains the temporal efficiency (ie, rapid coupling of plasma membrane to intracellular signaling) and spatial 3-dimensionality (ie, simultaneous activation of numerous parallel pathways) of IPC. An emerging idea in signal transduction emphasizes the role of multiprotein complexes organized in discrete microenvironments in cell regulation and pathophysiology4,5; such organization might explain the temporal/spatial conundrum of IPC.
Clinical Perspective p 1988
Caveolae, cholesterol- and sphingolipid-enriched invaginations of the plasma membrane, a subset of lipid/membrane rafts, are 1 such microenvironment.6–8 Caveolins, structural proteins essential for caveolae formation, are present in 3 isoforms9: caveolin (Cav)-1 and Cav-2 are expressed in multiple cell types, and Cav-3 is found primarily in striated (skeletal and cardiac) muscle and certain smooth muscle cells.10 Caveolins have scaffolding domains that anchor and regulate the function of proteins that modulate a variety of cellular processes11 and signal transduction.4,5 Caveolins can function as scaffolds for multiple, interacting signaling molecules, thereby providing temporal and spatial regulation of cellular signal transduction.5
Disruption of caveolae attenuates protection of adult cardiac myocytes from ischemic damage,12 and Cav-3 knockout mice, which lack cardiac myocyte caveolae, are resistant to pharmacological preconditioning.13 Such findings imply that myocyte caveolae are a prerequisite for protection against ischemia/reperfusion injury. Because Cav-3 expression is essential for the formation of caveolae in cardiac myocytes,14 we hypothesized and provide evidence that cardiac myocyte–specific overexpression of Cav-3 increases the formation of caveolae and enhances protective signaling from ischemia, thus identifying cell-specific expression of caveolins and caveolae as a novel approach to achieve such protection.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionsReferences |
|---|
Antibodies
Sources of antibodies included the following: polyclonal antibody to Cav-1, Abcam Inc (Cambridge, Mass) and Cell Signaling Technology (Danvers, Mass); polyclonal antibody to Cav-2, Abcam; monoclonal and polyclonal antibody to Cav-3, BD Transduction, Abcam, and Santa Cruz Biotechnology (Santa Cruz, Calif); monoclonal inducible nitric oxide synthase (NOS), polyclonal neuronal NOS, and polyclonal antibodies to phospho-eNOS (Ser1177) and (Thr495), Cell Signaling Technology; polyclonal antibody to inhibitor of apoptosis protein-1, Abcam; monoclonal antibody to Fas, Upstate; and monoclonal to GAPDH, IMGENEX (San Diego, Calif).
Animals
Animals were treated in compliance with the Guide for the Care and Use of Laboratory Animals (National Academy of Science), and protocols were approved by the VA San Diego Healthcare System Institutional Animal Care and Use Committee. Animals were kept on a 12-hour light-dark cycle in a temperature-controlled room with ad libitum access to food and water.
Transgenic (TG) mice with cardiac myocyte–specific overexpression of Cav-3 (Cav-3 OE) were produced in a C57BL/6 background. Full-length Cav-3 cDNA (455 bp) was cloned into a vector containing the 
-myosin heavy chain promoter and was used for microinjection (UCSD Transgenic Core; Figure I of the online Data Supplement). Cav-3 knockout mice were created as reported previously.15 Transgene-negative (TGneg) siblings served as controls for Cav-3 OE mice, and wild-type C57BL/6 mice served as controls for Cav-3 knockout mice.
Electron Microscopy
Whole hearts or cells were fixed with 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer for 2 hours, postfixed in 1% OsO4 in 0.1 mol/L cacodylate buffer (1 hour), and embedded as monolayers in LX-112 (Ladd Research, Williston, Vt). Sections were stained in uranyl acetate and lead citrate and were observed with an electron microscope (JEOL 1200 EX-II, JEOL USA, Peabody, Mass; or Philips CM-10, Philips Electronic Instruments, Mahwah, NY). Random sections were taken by an electron microscopy technician blinded to the treatments.
Sucrose Density Membrane Fractionation
Whole hearts were fractionated with sucrose density gradients as reported.16 Fractions 4 through 6 were buoyant membrane fractions (BFs) enriched in caveolins and proteins associated with caveolins. Fractions 9 through 12 were defined as nonbuoyant fractions (non-BFs).
Immunoblot
Whole tissue or cell lysates were separated by SDS-PAGE with 10% polyacrylamide precast gels (Invitrogen, Carlsbad, Calif) and transferred to polyvinylidene difluoride membranes by electroelution. Membranes were blocked in 20 mmol/L TBS Tween (1%) containing 4% BSA and incubated with primary antibody overnight at 4°C. Blots were visualized using secondary antibodies conjugated with horseradish peroxidase from Santa Cruz Biotechnology and enhanced chemiluminescence reagent from GE Healthcare (Waukesha, Wis).
Adult Cardiac Myocytes
Cardiac myocytes were isolated from male Sprague-Dawley rats as described.12 Myocytes were plated in 4% FBS on laminin (2 µg/cm2)- coated plates for 1 hour. Plating media was changed to serum-free media (1% BSA) to remove nonmyocytes, and cardiac myocytes were incubated at 37°C in 5% CO2.
Plasmid and Recombinant Adenovirus Production
A mouse Cav-3 cDNA (455 bp) was generated, cloned, and cotransfected with pJM170 (containing E1 region deletion). Plaques were expanded in HEK293 cells transformed with adenovirus E1. Adenovirus containing LacZ (Adv.LacZ) served as control. Cardiac myocytes were treated with Adv.LacZ or Adv.Cav-3 for 16 to 24 hours.
Immunofluorescence
Ventricular tissue was mounted on a cryostat (–23°C), and 10-µm sections were cut in long axis. Sections were prepared for immunofluorescence microscopy, and deconvolution images were obtained as described.17
Cholesterol and NOS Activity Assays
Cholesterol in fractions was measured with the Amplex Red Cholesterol Assay Kit (Invitrogen) as described by the manufacturer. Basal NOS activity was determined in TGneg and Cav-3 OE mice. Homogenized tissues were assayed with a NOS assay kit (Colorimetric, EMD Biosciences, Gibbstown, NJ) as described by the manufacturer. Additionally, TGneg and Cav-3 OE hearts were fractionated on a sucrose density gradient, and NOS activity was assessed in BF (caveolar) and non-BF (noncaveolar) with a [3H]-arginine NOS assay (EMD Bioscience) that measures conversion of [3H]-arginine to [3H]-citrulline as described by the manufacturer.
Echocardiography
Echocardiography was performed in mice anesthetized with isoflurane using an echocardiograph and L15/6-MHz transducer (Sonos 5500, Philips Medical Systems, Andover, Mass) as described previously.18
In Vivo Ischemia/Reperfusion Experimental Protocol
Mice anesthetized with pentobarbital (80 mg/kg) were mechanically ventilated, and ischemia was produced by occluding the left coronary artery with a 7-0 silk suture on a tapered BV-1 needle (Ethicon, Johnson & Johnson, New Brunswick, NJ) for 30 minutes.18 After 30 minutes of occlusion, the ligature was released, and the heart was reperfused for 2 or 24 hours. IPC was induced by occlusion of the left coronary artery for 5 minutes, followed by 15 minutes of reperfusion just before ischemia. Some Cav-3 OE mice were treated with 5-hydroxydecanoate (5-HD; a mitochondrial KATP channel inhibitor; Sigma, St Louis, Mo; 10 mg/kg IV) 10 minutes before ischemia or wortmannin (a phosphoinositide 3-kinase [PI3K] inhibitor; 15 µg/kg) 15 minutes before ischemia.
Infarct Size
The area at risk (AAR) was determined by staining with 1% Evans blue (1.0 mL; Sigma).18 The heart was immediately excised and cut into 1-mm slices (McIlwain tissue chopper, Brinkmann Instruments, West Bury, NY). The left ventricle was counterstained with 1% 2,3,5,-triphenyltetrazolium chloride (Sigma). Images were analyzed by Image-Pro Plus (Media Cybernetics, Bethesda, Md), and infarct size was determined by planimetry. Cardiac troponin I levels in the serum were measured with a high-sensitivity mouse cardiac troponin-I ELISA kit (Life Diagnostics, West Chester, Pa).
Cardiac Function
Mice underwent surgery as described in the ischemia/reperfusion protocol and were allowed to recover for 24 hours. After 24 hours, mice were anesthetized with pentobarbital (80 mg/kg), and cardiac catheterization was performed with a high-fidelity 1.4F microtip pressure transducer (SPR-671, Millar Instruments Inc, Houston, Tex). The catheter was advanced via the right carotid artery into the left ventricle after measuring mean arterial pressure. Parameters were determined by an algorithm from EMKA Technologies (Falls Church, Va).
Apoptosis
For terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) assays, AAR was removed from the mice after 24 hours of reperfusion. The tissue was cut and fixed in 3.7% formalin, and sections (5 µm) were used for TUNEL assays with the Apoptosis Detection Kit (R&D Systems, Minneapolis, Minn) according to the manufacturer’s instructions. Real-time polymerase chain reaction analysis of gene expression of proapoptotic and antiapoptotic genes was performed on total RNA isolated from the heart after 24 hours of reperfusion with the RNeasy Mini Kit (Qiagen Inc, Valencia, Calif) as described previously.18 Immunoblots for Fas and inhibitor of apoptosis-1 were performed.
Statistical Analysis
Data analysis was performed by observers blinded to experimental groups. Group size to determine the primary outcome variable of infarct size was established by power analysis. The power analyses suggest that a sample size of 7 mice per experimental group was sufficient, assuming =0.05, 2 tailed at 90% power with a hypothetical mean difference of 20%. We performed statistical analysis with Prism 4.0 (GraphPad) by the unpaired Student t test or 1-way ANOVA followed by a posthoc test with Bonferroni correction for multiple comparisons. All data are expressed as mean±SEM. Statistical significance was defined as P<0.05.
All authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionsReferences |
|---|
IPC Modulates Membrane Caveolae and Cav-3 Expression
We assessed the effect of IPC on cardiac membrane caveolae by subjecting mice to IPC and performing electron microscopy. Representative electron microscopy images show that IPC increases formation of caveolae (arrows in Figure 1A). To verify these morphological findings with biochemical techniques, hearts from IPC and control animals were fractionated on a discontinuous sucrose gradient and analyzed for protein and cholesterol content and for distribution of caveolin. IPC increases the total protein and cholesterol content in fractions 4 through 6 (BFs), which are enriched in caveolin17 (Figure 1B and 1C), and increases the amount of Cav-3, but not Cav-1, in BF (Figure 1D and 1E). These findings are consistent with evidence that the formation of cardiac myocyte caveolae is dependent on Cav-3.14,19
|
Adenoviral Overexpression of Cav-3 in Adult Cardiac Myocytes In Vitro Increases Caveolar Formation and Phosphorylation of Survival Kinases
Cardiac myocytes incubated with a Adv.Cav-3 have increased expression of caveolae (Figure 2A) and increased levels of phosphorylated protein kinase B (Akt) and glycogen synthase kinase-3β (GSK3β), 2 enzymes associated with IPC-induced cardiac protection3 (Figure 2B).
|
Cardiac Myocyte–Specific Cav-3 OE Mice Have Increased Myocyte Caveolae but Unaltered NOS Expression and Activity
Using an -myosin heavy chain promoter, we generated mice with cardiac myocyte–specific overexpression of Cav-3 (supplementary Figure IA). Thirty-nine mouse lines were generated, 5 of which were TG positive (supplementary Figure IB). Two of these 5 lines were propagated, one of which had an 8-fold elevation in Cav-3 mRNA expression compared with TG-negative (TGneg) animals (Figure 3A). We developed and characterized this line and refer to it as the Cav-3 OE. Cav-3 OE mice have increased Cav-3 protein expression without a change in Cav-1 and Cav-2 expression (Figure 3B and 3C, red pixels). The increased protein expression results in an increased number of caveolae in cardiac myocytes (Figure 3D); increased expression of Cav-3 was not observed in lung, brain, liver, kidney, or skeletal muscle of these mice (data not shown).
|
Because caveolins negatively regulate activity of NOS isoforms,11,20 we quantified basal NOS expression and activity in the Cav-3 OE mice. Given the well-known interaction of NOS with caveolins, we were surprised to find similar expression of NOS and phosphorylated endothelial NOS (eNOS) isoforms and similar NOS activity in the whole hearts of TGneg and Cav-3 OE mice (Figure 4A and 4B). To determine whether activity of NOS in caveolin-enriched membrane fractions may be altered by cardiac myocyte–specific caveolin-3 overexpression, we subjected TGneg and Cav-3 OE hearts to sucrose density fractionation and assessed NOS activity in BF and non-BF using a [3H]-arginine assay. No difference in NOS activity was observed in BF and non-BF in TGneg versus Cav-3 OE mice (Figure 4C). Rat cerebellum homogenate was used as a positive control ([3H]-citrulline, 42 000 cpm/µL sample).
|
Cav-3 OE Mice Are Protected From Ischemia/Reperfusion Injury
Cav-3 OE mice exposed to 30 minutes of cardiac ischemia and 2 hours of reperfusion have a substantial reduction in infarct size compared with TGneg mice (23.4±3.0% versus 43.0±3.9% AAR; n=11; P<0.001; Figure 5A), even though these mice show no differences in preocclusion hemodynamics (heart rate, mean arterial pressure, and rate-pressure product; supplementary Table I) or in the cardiac AAR (supplementary Figure II). This "endogenous protection" in Cav-3 OE mice is similar to that produced in TGneg mice subjected to IPC (26.3±2.4% risk area; n=8; P<0.05) and was not enhanced by IPC (21.1±2.8% risk area; n=8) but was attenuated by pretreatment with 5-HD, a mitochondrial ATP-sensitive potassium (KATP) channel inhibitor (38.1±4.4% risk area; n=8; Figure 5A). Akin to the results with 5-HD, IPC does not protect Cav-3 knockout mice from ischemic damage (Figure 5A). Cardiac troponin I confirmed the infarct size measurements (Figure 5B).
|
Role of Survival Kinases in Endogenous Protection
Hearts excised from Cav-3 OE mice had an 
3-fold increase in basal phosphorylation of Akt and GSK3β (P<0.05; n=6; Figure 6A), signaling molecules involved in cardiac protection.21 The level of basal elevation in phosphorylated Akt and GSK3β in the hearts of Cav-3 OE mice was comparable to the elevation seen after IPC in vivo (Figure 6A). To determine the role of PI3K/Akt/GSK3β in this endogenous protection, Cav-3 OE mice were treated with wortmannin, a PI3K inhibitor, or dimethyl sulfoxide (vehicle). Wortmannin reduced the phosphorylation of both Akt and GSK3β in Cav-3 OE mice relative to vehicle control (Figure 6B). Vehicle-treated Cav-3 OE mice showed a cardiac-protected phenotype (as in Figure 5A) with reduced infarct size and cardiac troponin I (23.3±2.0% risk area; n=7; Figure 6C and 6D). Wortmannin attenuated the endogenous cardiac protection observed in vehicle-treated Cav-3 OE mice (45.0±3.0% risk area; n=7; Figure 6C and 6D).
|
Cav-3 OE Mice Have Preserved Ultrastructure After Ischemia/Reperfusion Injury
We also used electron microscopy analysis to examine the AAR after ischemia/reperfusion (30 minutes/2 hours) in Cav-3 OE and TGneg mice (Figure 7). After injury, the ischemia/reperfusion TGneg groups displayed highly disorganized patterns of cardiac myocytes and their mitochondria. In addition, the sarcolemma exhibited evidence of damage, including disrupted Z lines and myofibrillar stretching. Mitochondria were swollen and contained amorphous matrix densities, indicating that injury was in an irreversible phase22 (Figure 7C). In contrast, hearts of Cav-3 OE mice subjected to ischemia/reperfusion showed limited ultrastructural change relative to sham, in particular, no mitochondrial swelling, myofibril stretching, or Z-line deformation (Figures 7D).
|
Cav-3 OE Mice Have Preserved Cardiac Function and Reduced Apoptosis After Ischemia/Reperfusion
We assessed cardiac function of TGneg and Cav-3 OE mice during cardiac catheterization after 30 minutes of ischemia and 24 hours of reperfusion. Left ventricular systolic function (dP/dtmax) was greater and cardiac troponin I levels were significantly lower in the Cav-3 OE mice (Figure 8A and 8B).
|
Cav-3 OE mice also had reduced apoptosis, assayed by TUNEL-positive cells (arrows in Figure 8C, left), as the percent of total nuclei after 24 hours of reperfusion (Figure 8C, right). Hearts of Cav-3 OE animals had decreased proapoptotic and increased antiapoptotic gene (Figure 8D) and protein (Figure 8E) expression.
Total-body overexpression of Cav-3 results in cardiomyopathy in 6-month-old mice.23 Accordingly, we assessed morphology, echocardiography, and hemodynamics in 6- to 9-month-old cardiac myocyte–specific Cav-3 OE mice. We found that the ratios of heart weight to tibia length were similar in Cav-3 OE and age-matched TGneg mice (supplementary Table II). In addition, echocardiographic parameters, heart rate, mean arterial pressure, and rate-pressure product were similar between Cav-3 OE and TGneg mice (supplementary Table II).
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionsReferences |
|---|
Numerous mechanisms have been proposed to explain the ability of an organ to develop tolerance to subsequent lethal ischemia/reperfusion injury.24 IPC, an intervention that was first shown >20 years ago to prevent such injury, has lacked a unifying hypothesis to account for the diverse pathways by which it affects the heart and other organs. We show here that IPC alters the morphology and composition of the plasma membrane of cardiac myocytes, increasing the number of caveolae. Moreover, we find that the protein Cav-3 is both necessary and sufficient to protect the heart from ischemia/reperfusion injury. Spatial organization of signaling molecules within caveolar microdomains and the interaction of signaling molecules with caveolins thus help to determine the protection of the heart from ischemia/reperfusion injury. The current data imply that expression of Cav-3 and caveolae represents a unifying mechanism for IPC.
Caveolae were first identified by electron microscopy in the 1950s by Palade6 and Yamada7 in endothelium and epithelium, respectively, and in the sarcolemma of cardiac myocytes in 1975 by McNutt.25 Early research on myocardial ischemia focused on ischemia-induced changes in the sarcolemma26,27 and provided evidence that caveolae in cardiac myocytes can be rapidly affected by perturbation of oxygen tension and tonicity.28,29 Despite such results, a role for caveolae and caveolins in the setting of IPC has not been explored.
An emerging concept emphasizes the organization of signaling molecules in multiprotein complexes, "signalosomes," that form and dissociate under basal and stimulated conditions.11 Caveolins play an integral role in the dynamics of these multiprotein complexes in caveolae by interacting with a wide range of signaling molecules that include multiple G-protein–coupled receptors, G subunits of heterotrimeric G proteins, Src kinases, PI3K, eNOS, protein kinase C isoforms, extracellular signal-regulated kinase 1 and 2 (ERK1/2), and superoxide dismutase. Many of these proteins can bind to the scaffolding domain of caveolin and be regulated by caveolin.30 A number of proteins that bind caveolin have been overexpressed in cardiac myocytes and shown to produce tolerance to myocardial ischemia/reperfusion injury; these include adenosine receptors,31 1-adrenergic receptors,32 protein kinase C isoforms,33 mitogen-activated protein kinases,34 eNOS,35,36 and proteins involved in the scavenging of free radicals.37 Overexpression of heat shock proteins (B crystallin, heat shock protein 60 to 10 complex, and H11 kinase38–40) also confers protection from ischemic damage; conceivably, caveolins contribute to such interactions.41 In silico analysis of the protein sequence of B crystallin reveals a putative caveolin binding motif (aa167wvcyqypgy), suggesting future avenues of investigation.
Caveolar microdomains are enriched in cholesterol. We show that IPC increases caveolar microdomains and total cholesterol primarily in BFs enriched in caveolin. The mechanism by which IPC increases total cholesterol is not known. Class B scavenger receptors (CD36) localize to caveolae and regulate cholesterol homeostasis.42 Coexpression of CD36 and caveolin has been shown to enhance the uptake of cholesterol.43 It is possible that the increase in cholesterol helps drive caveolar formation, or alternatively, an increase in caveolar formation may drive the influx of cholesterol. Defining this distinction will be an interesting avenue for future investigation.
Of particular importance for ischemia is the evidence that Cav-3 is an activator of PI3K/Akt/GSK3β signaling,44 a prosurvival pathway that can contribute to cardiac IPC.3,24 These findings are pertinent to the current data indicating that Adv.Cav-3–mediated overexpression in myocytes and Cav-3 OE mice shows increased Cav-3 expression, formation of caveolae, and phosphorylation of Akt and GSK3β. In addition, our results show that mice engineered to overexpress Cav-3 in cardiac myocytes are protected from ischemia/reperfusion injury to an extent comparable to that induced by IPC and in a manner that depends on mitochondrial KATP channel and PI3K activity, putative effectors of cardiac protection from ischemia/reperfusion injury.24,45 Cav-3 OE mice also showed a preserved sarcomeric ultrastructure after ischemia/reperfusion injury, suggesting that Cav-3 OE mice are resistant to membrane damage. This preserved ultrastructure may result from reduced injury or perhaps is a direct consequence of Cav-3 overexpression.
The precise molecular mechanism by which Cav-3 in myocytes protects the heart from ischemia/reperfusion injury remains to be determined. Caveolins can inhibit activity of signaling proteins such as eNOS and ERK1/246,47 by interaction of the scaffolding domain of caveolin with the binding motif of such partners. In addition, caveolins can promote signaling via enhanced receptor-effector coupling or enhanced receptor affinity.11,48 On the basis of data showing that infusion of a peptide with the scaffolding domain of caveolin sequence can protect the heart from ischemia/reperfusion injury, Young et al49 proposed that the scaffolding domain of caveolin peptide produces ischemic tolerance by enhancing release of endothelium-derived NO.49 Additional studies showed that ischemia/reperfusion injury activates a redistribution of Cav-3 and a downregulation of Cav-1 association with ERK,50 whereas IPC leads to a translocation of eNOS to caveolae.51 The present results, in which overexpressed Cav-3 in cardiac myocytes yields no change in basal expression of NOS isoforms or in NO generation, lead us to conclude that the ischemic tolerance produced by Cav-3 expression in myocytes does not result from changes in NO production and that cardiac myocyte eNOS is not a major source of cardiac NO. The latter idea is supported by evidence that reexpression of Cav-1 exclusively within the endothelium of Cav-1 knockout mice rescues cardiac defects.52
The phenotype of cardiac myocyte–specific Cav-3 OE mice is strikingly different from that of mice that have a total-body overexpression of Cav-3 in brain, fat, liver, lung, and spleen, as well as smooth, skeletal, and cardiac muscle.53 Such mice develop a muscular dystrophy phenotype at 3 to 4 weeks of age and after 6 months show cardiac degeneration, fibrosis, and reduced cardiac NOS activity and cardiac function.23 In contrast, cardiac myocyte–specific overexpression of Cav-3 does not result in cardiomyopathy in 6- to 8-month-old mice or a reduction in NOS activity. We conclude that increased cardiac myocyte expression of Cav-3 is not responsible for the late-appearing cardiac changes observed in the total-body Cav-3 OE mice.
|
Conclusions |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionsReferences |
|---|
The present results show that IPC increases the number of plasma membrane caveolae in cardiac myocytes and that Cav-3 OE mice are protected from ischemic injury in a manner that mimics the cardiac protection produced by IPC. The ability to recapitulate or block the alterations of the plasma membrane produced by IPC by the respective overexpression or knockout of Cav-3 implies that Cav-3 is necessary and sufficient for IPC-induced cardiac protection. This protection may depend on PI3K and mitochondrial KATP channels. Our results define a molecular mechanism to explain aspects of sarcolemmal ultrastructure, caveolae, and IPC that have been incompletely understood for many years. Cardiac myocyte–targeted overexpression of Cav-3 may provide a novel means to protect the heart from ischemia/reperfusion injury. More generally, our results imply that cell type–selective expression of caveolins may offer a means to augment mechanisms of preservation of the heart and perhaps other organs.
|
Acknowledgments |
|---|
Sources of Funding
This work was supported by American Heart Association Beginning Grant in Aid 0765076Y (Dr Tsutsumi), American Heart Association Predoctoral Fellowship 06150217Y (Y.T. Horikawa), American Heart Association Scientist Development Grant 0630039N (Dr H. Patel), NIH 1PO1 HL66941 (Drs Insel and Roth), Senior Scholar Award from Ellison Foundation (Dr Insel), Merit Award from the Department of Veterans Affairs (Dr Roth), and NIH RO1 HL081400 (Dr Roth).
Disclosures
Drs Tsutsumi, Patel, Head, Insel, Patel, and Roth have a patent pending with the University of California, San Diego: "Molecular Scaffolds for Treating Cardiac Injury." The other authors report no conflicts.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionsReferences |
|---|
1. Noble R. The development of resistance by rats and guinea pigs to amounts of trauma usually fatal. Am J Physiol. 1943; 38: 346–351.
2. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986; 74: 1124–1136.
3. Hausenloy DJ, Tsang A, Yellon DM. The reperfusion injury salvage kinase pathway: a common target for both ischemic preconditioning and postconditioning. Trends Cardiovasc Med. 2005; 15: 69–75.[CrossRef][Medline] [Order article via Infotrieve]
4. Steinberg SF, Brunton LL. Compartmentation of G protein-coupled signaling pathways in cardiac myocytes. Annu Rev Pharmacol Toxicol. 2001; 41: 751–773.[CrossRef][Medline] [Order article via Infotrieve]
5. Williams TM, Lisanti MP. The caveolin proteins. Genome Biol. 2004; 5: 214.[CrossRef][Medline] [Order article via Infotrieve]
6. Palade G. Fine structure of blood capillaries. J Appl Physics. 1953; 24: 1424. Abstract.
7. Yamada E. The fine structure of the gall bladder epithelium of the mouse. J Biophys Biochem Cytol. 1955; 1: 445–458.[Medline] [Order article via Infotrieve]
8. Pike LJ. Lipid rafts: bringing order to chaos. J Lipid Res. 2003; 44: 655–667.
9. Rothberg KG, Heuser JE, Donzell WC, Ying YS, Glenney JR, Anderson RG. Caveolin, a protein component of caveolae membrane coats. Cell. 1992; 68: 673–682.[CrossRef][Medline] [Order article via Infotrieve]
10. Song KS, Scherer PE, Tang Z, Okamoto T, Li S, Chafel M, Chu C, Kohtz DS, Lisanti MP. Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells: caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins. J Biol Chem. 1996; 271: 15160–15165.
11. Feron O, Balligand JL. Caveolins and the regulation of endothelial nitric oxide synthase in the heart. Cardiovasc Res. 2006; 69: 788–797.
12. Patel HH, Head BP, Petersen HN, Niesman IR, Huang D, Gross GJ, Insel PA, Roth DM. Protection of adult rat cardiac myocytes from ischemic cell death: role of caveolar microdomains and delta-opioid receptors. Am J Physiol Heart Circ Physiol. 2006; 291: H344–H350.
13. Horikawa YT, Patel HH, Tsutsumi YM, Jennings MM, Kidd MW, Hagiwara Y, Ishikawa Y, Insel PA, Roth DM. Caveolin-3 expression and caveolae are required for isoflurane-induced cardiac protection from hypoxia and ischemia/reperfusion injury. J Mol Cell Cardiol. 2008; 44: 123–130.[CrossRef][Medline] [Order article via Infotrieve]
14. Woodman SE, Park DS, Cohen AW, Cheung M, Chandra M, Shirani J, Tang B, Jelicks LA, Kitsis RN, Christ GJ, Factor SM, Tanowitz HB, Lisanti MP. Caveolin-3 knock-out mice develop a progressive cardiomyopathy and show hyperactivation of the p42/44 MAP kinase cascade. J Biol Chem. 2002; 277: 38988–38997.
15. Hagiwara Y, Sasaoka T, Araishi K, Imamura M, Yorifuji H, Nonaka I, Ozawa E, Kikuchi T. Caveolin-3 deficiency causes muscle degeneration in mice. Hum Mol Genet. 2000; 9: 3047–3054.
16. Patel HH, Tsutsumi YM, Head BP, Niesman IR, Jennings M, Horikawa Y, Huang D, Moreno AL, Patel PM, Insel PA, Roth DM. Mechanisms of cardiac protection from ischemia/reperfusion injury: a role for caveolae and caveolin-1. FASEB J. 2007; 21: 1565–1574.
17. Head BP, Patel HH, Roth DM, Lai NC, Niesman IR, Farquhar MG, Insel PA. G-protein coupled receptor signaling components localize in both sarcolemmal and intracellular caveolin-3-associated microdomains in adult cardiac myocytes. J Biol Chem. 2005; 280: 31036–31044.
18. Tsutsumi YM, Patel HH, Lai NC, Takahashi T, Head BP, Roth DM. Isoflurane produces sustained cardiac protection after ischemia-reperfusion injury in mice. Anesthesiology. 2006; 104: 495–502.[CrossRef][Medline] [Order article via Infotrieve]
19. Park DS, Cohen AW, Frank PG, Razani B, Lee H, Williams TM, Chandra M, Shirani J, Souza APD, Tang B, Jelicks LA, Factor SM, Weiss LM, Tanowitz HB, Lisanti MP. Caveolin-1 null (–/–) mice show dramatic reductions in life span. Biochemistry. 2003; 42: 15124–15131.[CrossRef][Medline] [Order article via Infotrieve]
20. Feron O, Kelly RA. The caveolar paradox: suppressing, inducing, and terminating eNOS signaling. Circ Res. 2001; 88: 129–131.
21. Juhaszova M, Zorov DB, Kim SH, Pepe S, Fu Q, Fishbein KW, Ziman BD, Wang S, Ytrehus K, Antos CL, Olson EN, Sollott SJ. Glycogen synthase kinase-3beta mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore. J Clin Invest. 2004; 113: 1535–1549.[CrossRef][Medline] [Order article via Infotrieve]
22. Murry CE, Richard VJ, Reimer KA, Jennings RB. Ischemic preconditioning slows energy metabolism and delays ultrastructural damage during a sustained ischemic episode. Circ Res. 1990; 66: 913–931.
23. Aravamudan B, Volonte D, Ramani R, Gursoy E, Lisanti MP, London B, Galbiati F. Transgenic overexpression of caveolin-3 in the heart induces a cardiomyopathic phenotype. Hum Mol Genet. 2003; 12: 2777–2788.
24. Hausenloy DJ, Yellon DM. Survival kinases in ischemic preconditioning and postconditioning. Cardiovasc Res. 2006; 70: 240–253.
25. McNutt NS. Ultrastructure of the myocardial sarcolemma. Circ Res. 1975; 37: 1–13.
26. Ashraf M, Halverson CA. Structural changes in the freeze-fractured sarcolemma of ischemic myocardium. Am J Pathol. 1977; 88: 583–594.[Abstract]
27. Sybers HD, Maroko PR, Ashraf M, Libby P, Braunwald E. The effect of glucose-insulin-potassium on cardiac ultrastructure following acute experimental coronary occlusion. Am J Pathol. 1973; 70: 401–420.[Medline] [Order article via Infotrieve]
28. Frank JS, Beydler S, Kreman M, Rau EE. Structure of the freeze-fractured sarcolemma in the normal and anoxic rabbit myocardium. Circ Res. 1980; 47: 131–143.
29. Kordylewski L, Goings GE, Page E. Rat atrial myocyte plasmalemmal caveolae in situ: reversible experimental increases in caveolar size and in surface density of caveolar necks. Circ Res. 1993; 73: 135–146.[Abstract]
30. Krajewska WM, Maslowska I. Caveolins: structure and function in signal transduction. Cell Mol Biol Lett. 2004; 9: 195–220.[Medline] [Order article via Infotrieve]
31. Matherne GP, Linden J, Byford AM, Gauthier NS, Headrick JP. Transgenic A1 adenosine receptor overexpression increases myocardial resistance to ischemia. Proc Natl Acad Sci U S A. 1997; 94: 6541–6546.
32. Rorabaugh BR, Ross SA, Gaivin RJ, Papay RS, McCune DF, Simpson PC, Perez DM. Alpha1A- but not alpha1B-adrenergic receptors precondition the ischemic heart by a staurosporine-sensitive, chelerythrine-insensitive mechanism. Cardiovasc Res. 2005; 65: 436–445.
33. Ping P, Song C, Zhang J, Guo Y, Cao X, Li RC, Wu W, Vondriska TM, Pass JM, Tang XL, Pierce WM, Bolli R. Formation of protein kinase C(epsilon)-Lck signaling modules confers cardioprotection. J Clin Invest. 2002; 109: 499–507.[CrossRef][Medline] [Order article via Infotrieve]
34. Wall JA, Wei J, Ly M, Belmont P, Martindale JJ, Tran D, Sun J, Chen WJ, Yu W, Oeller P, Briggs S, Gustafsson AB, Sayen MR, Gottlieb RA, Glembotski CC. Alterations in oxidative phosphorylation complex proteins in the hearts of transgenic mice that overexpress the p38 MAP kinase activator, MAP kinase kinase 6. Am J Physiol Heart Circ Physiol. 2006; 291: H2462–H2472.
35. Brunner F, Maier R, Andrew P, Wolkart G, Zechner R, Mayer B. Attenuation of myocardial ischemia/reperfusion injury in mice with myocyte-specific overexpression of endothelial nitric oxide synthase. Cardiovasc Res. 2003; 57: 55–62.
36. Jones SP, Greer JJ, Kakkar AK, Ware PD, Turnage RH, Hicks M, van Haperen R, de Crom R, Kawashima S, Yokoyama M, Lefer DJ. Endothelial nitric oxide synthase overexpression attenuates myocardial reperfusion injury. Am J Physiol Heart Circ Physiol. 2004; 286: H276–H282.
37. Li G, Chen Y, Saari JT, Kang YJ. Catalase-overexpressing transgenic mouse heart is resistant to ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol. 1997; 273: H1090–H1095.
38. Depre C, Wang L, Sui X, Qiu H, Hong C, Hedhli N, Ginion A, Shah A, Pelat M, Bertrand L, Wagner T, Gaussin V, Vatner SF. H11 kinase prevents myocardial infarction by preemptive preconditioning of the heart. Circ Res. 2006; 98: 280–288.
39. Lau S, Patnaik N, Sayen R, Mestril R. Simultaneous overexpression of two stress proteins in rat cardiomyocytes and myogenic cells confers protection against ischemia-induced injury. Circulation. 1997; 96: 2287–2294.
40. Ray PS, Martin JL, Swanson EA, Otani H, Dillmann WH, Das DK. Transgene overexpression of alphaB crystallin confers simultaneous protection against cardiomyocyte apoptosis and necrosis during myocardial ischemia and reperfusion. FASEB J. 2001; 15: 393–402.
41. Gupta S, Knowlton AA. HSP60 trafficking in adult cardiac myocytes: role of the exosomal pathway. Am J Physiol Heart Circ Physiol. 2007; 292: H3052–H3056.
42. Graf GA, Matveev SV, Smart EJ. Class B scavenger receptors, caveolae and cholesterol homeostasis. Trends Cardiovasc Med. 1999; 9: 221–225.[CrossRef][Medline] [Order article via Infotrieve]
43. Matveev S, van der Westhuyzen DR, Smart EJ. Co-expression of scavenger receptor-BI and caveolin-1 is associated with enhanced selective cholesteryl ester uptake in THP-1 macrophages. J Lipid Res. 1999; 40: 1647–1654.
44. Fecchi K, Volonte D, Hezel MP, Schmeck K, Galbiati F. Spatial and temporal regulation of GLUT4 translocation by flotillin-1 and caveolin-3 in skeletal muscle cells. FASEB J. 2006; 20: 705–707.
45. Liu Y, Sato T, O'Rourke B, Marban E. Mitochondrial ATP-dependent potassium channels: novel effectors of cardioprotection? Circulation. 1998; 97: 2463–2469.
46. Engelman JA, Chu C, Lin A, Jo H, Ikezu T, Okamoto T, Kohtz DS, Lisanti MP. Caveolin-mediated regulation of signaling along the p42/44 MAP kinase cascade in vivo: a role for the caveolin-scaffolding domain. FEBS Lett. 1998; 428: 205–211.[CrossRef][Medline] [Order article via Infotrieve]
47. Feron O, Dessy C, Opel DJ, Arstall MA, Kelly RA, Michel T. Modulation of the endothelial nitric-oxide synthase-caveolin interaction in cardiac myocytes: implications for the autonomic regulation of heart rate. J Biol Chem. 1998; 273: 30249–30254.
48. Raikar LS, Vallejo J, Lloyd PG, Hardin CD. Overexpression of caveolin-1 results in increased plasma membrane targeting of glycolytic enzymes: the structural basis for a membrane associated metabolic compartment. J Cell Biochem. 2006; 98: 861–871.[CrossRef][Medline] [Order article via Infotrieve]
49. Young LH, Ikeda Y, Lefer AM. Caveolin-1 peptide exerts cardioprotective effects in myocardial ischemia-reperfusion via nitric oxide mechanism. Am J Physiol Heart Circ Physiol. 2001; 280: H2489–H2495.
50. Ballard-Croft C, Locklar AC, Kristo G, Lasley RD. Regional myocardial ischemia induced activation of MAPKs is associated with subcellular redistribution of caveolin and cholesterol. Am J Physiol Heart Circ Physiol. 2006; 291: H658–H667.
51. Koaneru S, Penumathsa SV, Thirunavukkarasu M, Samuel SM, Zhan L, Han Z, Maulik G, Das DK, Maulik N. Redox regulation of ischemic preconditioning is mediated by the differential activation of caveolins and their association with eNOS and GLUT-4. Am J Physiol Heart Circ Physiol. 2007; 292: H2060–H2072.
52. Murata T, Lin MI, Huang Y, Yu J, Bauer PM, Giordano FJ, Sessa WC. Reexpression of caveolin-1 in endothelium rescues the vascular, cardiac, and pulmonary defects in global caveolin-1 knockout mice. J Exp Med. 2007; 204: 2373–2382.
53. Galbiati F, Volonte D, Chu JB, Li M, Fine SW, Fu M, Bermudez J, Pedemonte M, Weidenheim KM, Pestell RG, Minetti C, Lisanti MP. Transgenic overexpression of caveolin-3 in skeletal muscle fibers induces a Duchenne-like muscular dystrophy phenotype. Proc Natl Acad Sci U S A. 2000; 97: 9689–9694.
|
Footnotes |
|---|
*The last 2 authors share equal senior authorship.
The online Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.788331/DC1.
Related Article:
Circulation 2008 118: 1911-1912.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||