AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 291: H344-H350, 2006. First published February 24, 2006; doi:10.1152/ajpheart.01100.2005
0363-6135/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/1/H344    most recent
01100.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (14)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Patel, H. H.
Right arrow Articles by Roth, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Patel, H. H.
Right arrow Articles by Roth, D. M.

Protection of adult rat cardiac myocytes from ischemic cell death: role of caveolar microdomains and {delta}-opioid receptors

Hemal H. Patel,1,2 Brian P. Head,1 Heidi N. Petersen,1 Ingrid R. Niesman,3 Diane Huang,2,4 Garrett J. Gross,5 Paul A. Insel,1,6,* and David M. Roth2,4,*

Departments of 1Pharmacology, 3Cellular and Molecular Medicine, 6Medicine, and 2Anesthesiology, University of California, San Diego, La Jolla; 4Veterans Affairs San Diego Healthcare System, San Diego, California; and 5Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin

Submitted 17 October 2005 ; accepted in final form 6 February 2006


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The role of caveolae, membrane microenvironments enriched in signaling molecules, in myocardial ischemia is poorly defined. In the current study, we used cardiac myocytes prepared from adult rats to test the hypothesis that opioid receptors (OR), which are capable of producing cardiac protection in vivo, promote cardiac protection in cardiac myocytes in a caveolae-dependent manner. We determined protein expression and localization of {delta}-OR (DOR) using coimmunohistochemistry, caveolar fractionation, and immunoprecipitations. DOR colocalized in fractions with caveolin-3 (Cav-3), a structural component of caveolae in muscle cells, and could be immunoprecipitated by a Cav-3 antibody. Immunohistochemistry confirmed plasma membrane colocalization of DOR with Cav-3. Cardiac myocytes were subjected to simulated ischemia (2 h) or an ischemic preconditioning (IPC) protocol (10 min ischemia, 30 min recovery, 2 h ischemia) in the presence and absence of methyl-beta-cyclodextrin (MbetaCD, 2 mM), which binds cholesterol and disrupts caveolae. We also assessed the cardiac protective effects of SNC-121 (SNC), a selective DOR agonist, on cardiac myocytes with or without MbetaCD and MbetaCD preloaded with cholesterol. Ischemia, simulated by mineral oil layering to inhibit gas exchange, promoted cardiac myocyte cell death (trypan blue staining), a response blunted by SNC (37 ± 3 vs. 59 ± 3% dead cells in the presence and absence of 1 µM SNC, respectively, P < 0.01) or by use of the IPC protocol (35 ± 4 vs. 62 ± 3% dead cells, P < 0.01). MbetaCD treatment, which disrupted caveolae (as detected by electron microscopy), fully attenuated the protective effects of IPC or SNC, resulting in cell death comparable to that of the ischemic group. By contrast, SNC-induced protection was not abrogated in cells incubated with cholesterol-saturated MbetaCD, which maintained caveolae structure and function. These findings suggest a key role for caveolae, perhaps through enrichment of signaling molecules, in contributing to protection of cardiac myocytes from ischemic damage.

opioids; caveolae; G proteins; ischemia; myocytes

MYOCARDIAL INFARCTION (MI) remains a major cause of death in industrialized countries. Ischemic preconditioning (IPC) is a beneficial experimental intervention for preventing MI, whereby exposure of the heart to brief periods of ischemia results in protection from subsequent prolonged ischemic insults. The signal transduction pathways that mediate IPC are diverse and complex (11). Putative triggers, mediators, and effectors have been described, but there is an imprecise understanding as to how these factors interact to produce cardiac protection.

Opioid receptor stimulation has provided a model to study how extracellular molecules can produce cardiac protection. Opioid peptides are expressed in the heart (16, 49) and the {delta}-opioid receptor (DOR), the dominant opioid receptor in the heart, facilitates acute IPC (36), thus suggesting a role in IPC for endogenously released opioid peptides. In addition, agonists or antagonists of DOR induce or attenuate cardiac protection, respectively (3, 12, 36–38). Although acute and delayed cardiac protection elicited by opioids has been characterized, mechanistic information regarding expression of opioid receptor subtypes and the role of their subcellular localization on signaling, in particular in IPC, is lacking.

Recent data emphasize a potentially important role for signaling microdomains [such as lipid rafts, and a subset of lipid rafts, caveolae, cholesterol- and sphingolipid-enriched 50- to 100-nm invaginations of the plasma membrane (28, 52)] as sites that localize G protein-coupled receptors (GPCR), heterotrimeric G proteins, and G protein-regulated effector molecules in a confined region via lipid-protein and protein-protein interaction (17, 24). The latter interaction occurs in caveolae via caveolins, structural proteins that contain scaffolding domains; such localization facilitates coordinated, precise, and rapid regulation of cell function (21, 23, 25–27, 44, 50). There are three isoforms of caveolin: caveolin-1 (Cav-1) and -2 are expressed in multiple cell types, whereas caveolin-3 (Cav-3) is found exclusively in striated (i.e., skeletal and cardiac) myocytes and certain smooth muscle cells, in which it forms oligomeric complexes (43, 47). Compartmentation and spatial organization of GPCR signaling molecules in caveolae appears to play a key role in physiological and pharmacological responses (6, 9, 21, 27, 45). Little is known, however, regarding opioid receptor localization in caveolae or how caveolae might impact cardiac myocyte function during ischemia. In the present study, we therefore sought to 1) characterize the expression, localization, and cardiac protective role of DOR in isolated adult rat cardiac myocytes and 2) determine whether intact caveolae are important for cardiac protection produced by DOR activation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Antibodies to DOR were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cav-3 (monoclonal) antibody was obtained from BD Biosciences (San Jose, CA). SNC-121 was obtained from Tocris. Unless otherwise stated, all other chemicals and reagents were obtained from Sigma.

Preparation of adult rat ventricular myocytes. Cardiac myocytes were isolated from adult Sprague-Dawley rats (250–300 g, male). All animal use protocols were approved by the University of California, San Diego, Institutional Animal Care and Use Committee. The investigations conform with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Animals were heparinized (1,000–2,000 units ip) 5 min before being anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg), and the hearts were removed and placed in ice-cold cardioplegic (20 mM KCl) heart media solution (in mmol/l: 112 NaCl, 5.4 KCl, 1 MgCl2, 9 NaH2PO4, and 11.1 D-glucose; supplemented with 10 HEPES, 30 taurine, 2 DL-carnitine, and 2 creatine, pH. 7.4). The hearts were retrograde perfused on a Langendorff apparatus with Ca2+-free heart media for 5 min at 5 ml/min at 37°C, followed by perfusion with Ca2+-free heart media containing collagenase II (210 U/mg; Worthington) for 20 min. After perfusion, both ventricles were removed from the heart and minced in collagenase II-containing heart media for 10–15 min. The cell solution was then washed several times to remove collagenase II and reexposed to 1.2 mM Ca2+ over 25 min to produce Ca2+-tolerant cardiac myocytes. Myocytes were then plated in 4% FBS on laminin (2 µg/cm2)-coated plates for 1 h. Plating medium was changed to serum-free medium (1% BSA) to remove all nonmyocytes, and cardiac myocytes were incubated at 37°C in 5% CO2 for 12–24 h before experiments. Additionally, experiments were performed on freshly isolated myocytes.

Membrane fractionation. Cardiac myocytes were fractionated to isolate caveolin-rich domains using detergent-free methods (41, 43). Cells from a 15-cm plate were washed two times in ice-cold PBS, scraped in 3 ml of 500 mM Na2CO3 (pH 11.0), homogenized with a tissue grinder with three 10-s bursts, and then sonicated with three cycles of 20-s bursts followed by 1 min of incubation on ice. Approximately 2 ml of homogenate were mixed with 2 ml of 90% sucrose in 150 mM MES-buffered saline (MBS, pH 6.5) to form 45% sucrose and loaded at the bottom of an ultracentrifuge tube. A discontinuous sucrose gradient was generated by layering 4 ml of 35% sucrose prepared in MBS-250 mM Na2CO3 (MBS-Na2CO3) and then 4 ml of 5% sucrose in MBS-Na2CO3. The gradient was centrifuged at 280,000 g using a SW41Ti rotor (Beckman Instruments) for 16–20 h at 4°C. Samples were removed in 1-ml aliquots to yield 12 fractions, which were analyzed for protein content. We define fractions 4 and 5 as buoyant caveolin-associated membrane fractions (BF) enriched in lipid-rich caveolae and proteins associated with caveolae, whereas fractions 9–12 were termed heavy membrane fractions containing nonbuoyant membranes and organelles.

Immunoblot analysis. Proteins in individual fractions and whole cell lysates were separated by SDS-PAGE using 10% polyacrylamide precast gels (Invitrogen) and transferred to a polyvinylidene difluoride membrane by electroelution. Membranes were blocked in 20 mM PBS-Tween (1%) containing 1.5% nonfat dry milk and incubated with primary antibody overnight at 4°C. Bound primary antibodies were visualized using secondary antibodies conjugated with horseradish peroxidase from Santa Cruz Biotechnology and enhanced chemiluminescence reagent from Amersham Pharmacia Biotechnology (Piscataway, NJ). All displayed bands migrated at the appropriate size, as determined by comparison with molecular weight standards (Santa Cruz Biotechnology).

Immunoprecipitation. Immunoprecipitations were performed using either protein A or protein G-agarose. Whole cell lysates (isolated cells from 4 animals) were incubated with primary antibody for 1–3 h at 4°C and were subsequently immunoprecipitated with protein agarose overnight on a rocking platform at 4°C and centrifuged at 13,000 g for 5 min. Protein agarose pellets were washed one time in lysis buffer followed by subsequent washes in wash buffer 2 (50 mM Tris·HCl, pH 7.5, 500 mM NaCl, 0.2% IGEPAL CA-630) and wash buffer 3 (10 mM Tris·HCl, pH 7.5, and 0.2% IGEPAL CA-630). Immunoprecipitated proteins were analyzed by immunoblotting.

Immunofluorescence. Cells were prepared for immunofluorescence as previously described (39). Isolated cardiac myocytes were plated on precoated laminin (2 µg/cm2) glass coverslips, grown for 24 h, and fixed with 2% buffered paraformaldehyde for 10 min at room temperature. The fixed cells were quenched with 100 mM glycine (pH. 7.4) for an additional 5 min to remove aldehyde groups, permeabilized in buffered Triton X-100 (0.1%) for 10 min, and then blocked with 1% BSA-PBS-0.05% Tween for 20 min. Cells were incubated with primary antibodies (1:100) in 1% BSA-PBS-Tween (0.05%) for 24 h at 4°C. Excess antibody was removed by treatment with PBS-Tween (0.1%) three times at 5-min intervals. The cells were then incubated with FITC or Alexa-conjugated [F'(ab) fragment] secondary antibody (1:250) for 1 h. To remove excess secondary antibody, the cells were washed six times at 5-min intervals with PBS-Tween (0.1%) and incubated with 4',6-diamidino-2-phenylindole (1:5,000) diluted in PBS for 20 min. Cells were then washed for 10 min with PBS and mounted in gelvatol for microscopic imaging.

Deconvolution image analysis. Deconvolution images were obtained as previously described (1, 2). Images (3 cells each from 4 separate myocyte isolations) were captured with a DeltaVision deconvolution microscope system (Applied Precision, Issaquah, WA). The system includes a Photometrics charge-coupled device mounted on a Nikon TE-200 inverted epifluorescence microscope. In general, between 20 and 50 optical sections spaced by 0.2 µm were taken. Exposure times were set such that the camera response was in the linear range for each fluorophore. Lenses included x100 [numeric aperture (NA) 1.4], x60 (NA 1.4), and x40 (NA 1.3). The data sets were deconvolved and analyzed using SoftWorx software (Applied Precision) on a Silicon Graphics Octane workstation. When applicable, image quantitation was performed with the Data Inspector program in SoftWorx. Maximal projection volume views or single optical sections are shown as indicated. Colocalization was assessed by CoLocalizer Pro 1.3 analysis software.

Electron microscopy. Cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 2 h at room temperature, postfixed in 1% OsO4 in 0.1 M cacodylate buffer (1 h), and embedded as monolayers in LX-112 (Ladd Research, Williston, VT), as described previously (7). Sections were stained in uranyl acetate and lead citrate and observed with an electron microscope (JEOL 1200 EX-II or Philips CM-10). Caveolae were quantitated on random images per micrometer of membrane.

Simulated ischemia in isolated adult cardiac myocytes. Isolated cardiac myocytes were plated on laminin-coated 24-well plates and allowed to incubate for 24 h or were used freshly isolated. Cells were then washed in PBS at room temperature and subjected to various experimental conditions. "Ischemia/hypoxia" ("simulated ischemia") was induced by layering mineral oil (0.5 ml for 120 min) over a thin film of media covering the cells followed by 60 min of "reperfusion" in normal media in the 24-well plate. "IPC" was performed by a 10-min period of incubation of cells under mineral oil followed by a 30-min recovery period before the 120-min "ischemic insult" and reperfusion. In other experiments, various concentrations of the DOR-selective agonist SNC-121 were administered 15 min before the ischemic insult. Cell death was quantified by counting trypan blue-stained cells and expressed as a percentage of the total cells counted. All experiments were carried out at 37°C (myocytes isolated from n = 6–9 animals/group).

To determine the impact of intact caveolae on cardiac protective events, we used methyl-beta-cyclodextrin (MbetaCD), which removes cellular cholesterol and thereby disrupts cholesterol-rich caveolae.(5, 14). Cells were incubated in MbetaCD (1 mM)-containing media for 1 h before the ischemia, IPC, or SNC-121 treatment protocols. Control experiments were conducted using MbetaCD preloaded with cholesterol, which results in a cyclodextrin not capable of removing cellular cholesterol (5, 33).

Statistical analysis. Statistical analyses were performed by one-way ANOVA followed by the Bonferroni post hoc test. All data are expressed as means ± SE. Statistical significance was defined as P < 0.05.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of IPC and exogenous opioid stimulation on cardiac protection in isolated cardiac myocytes. We exposed cardiac myocytes to simulated ischemia-reperfusion (SI/R) and determined cell death by trypan blue staining. A preconditioning protocol (10 min of mineral oil layering followed by 30 min recovery in maintenance media) before sustained SI/R decreased cell death compared with SI/R alone (35 ± 3 vs. 59 ± 3, P < 0.05, Fig. 1). Treatment with the DOR agonist SNC-121 (0.1–1 µM) 15 min before sustained SI/R resulted in a concentration-dependent reduction in cell death compared with SI/R alone (Fig. 1). We obtained similar results for the preconditioning protocol in freshly isolated cells (data not shown).


Figure 1
View larger version (25K):
[in this window]
[in a new window]
  		Fig. 1. In vitro cardiac protection: role of opioid receptors. Adult rat cardiac myocytes were subjected to simulated ischemia. Control cells were incubated in culture media for the duration of the experiment. Simulated ischemia-reperfusion significantly increased trypan blue-positive cells, indicative of cell death. Ischemic preconditioning (PC) and SNC-121 (SNC) pretreatment for 15 min reduced cell death induced by simulated ischemia. P < 0.05 vs. basal (*) and vs. control ischemia (+).

 
Biochemical characterization of caveolin distribution in cardiac myocytes. Subcellular fractionation after Na2CO3 buffer treatment of cardiac myocytes was performed on a discontinuous sucrose gradient (42). Immunoblot analysis of the resulting fractions demonstrated enrichment of Cav-3, the muscle-specific marker of caveolae (47), in BF 4 and 5 (Fig. 2A), which also predominantly localized DOR (Fig. 2B). Confirmation of these results was obtained by immunoprecipitation; an antibody to Cav-3 immunoprecipitated DOR and an antibody to DOR immunoprecipitated Cav-3, implying an association between Cav-3 and DOR (Fig. 2C) in the same structural complex. The localization of DOR to caveolae and their association with Cav-3 was further confirmed by immunohistochemistry. Cav-3 and DOR localized to the plasma membrane and in a transverse pattern along the length of the myocyte (78.5 ± 6.3% colocalization), a pattern consistent with T tubules; the proteins colocalized predominantly at the membrane, as seen most readily in the overlap and mask images, which assess localization of overlapping pixels (Fig. 2D).


Figure 2
View larger version (46K):
[in this window]
[in a new window]
  		Fig. 2. Biochemical and immunohistochemical evaluation of caveolin distribution and interaction with {delta}-opioid receptor (DOR). A: fractionated cardiac myocytes (CM) demonstrate a buoyant [fractions 4 and 5, buoyant fractions (BF)] localization of caveolin-3 (Cav-3) and heavier membrane fractions [fractions 9–12, heavy fractions (HF)] with less Cav-3 enrichment. IB, immunoblot. B: DOR are found in BF where Cav-3 is enriched. WCL, whole cell lysate. C: Cav-3 immunoprecipitates or detergent-solubilized WCL show that Cav-3 and DOR specifically interact. IP, immunoprecipitate. D: whole cells were costained with antibodies for Cav-3 and DOR. Cav-3 stains predominantly the cell membrane and also inside the cell in a pattern consistent with the distribution of T tubules. DOR stains predominantly the sarcolemmal membrane with Cav-3 (78.5 ± 6.3% colocalization, n = 12 myocytes from 4 rats).

 
Effect of MbetaCD treatment on the expression of caveolae of cardiac myocytes. We treated cardiac myocytes with MbetaCD, an agent that disrupts caveolae by depletion of cholesterol (5, 14, 30). Electron microscopy revealed disruption of cardiac myocyte caveolae with increasing concentrations of MbetaCD but absence of such disruption by treatment with cholesterol-loaded MbetaCD, which is unable to deplete membrane cholesterol (Fig. 3A; see Refs. 5 and 33). Quantification revealed that MbetaCD treatment reduced the number of caveolae (per µm of membrane), an effect not observed with cholesterol-loaded MbetaCD (Fig. 3B).


Figure 3
View larger version (35K):
[in this window]
[in a new window]
  		Fig. 3. Disruption of cardiac myocyte caveolae by methyl-beta-cyclodextrin (MbetaCD). Control cardiac myocytes or cells treated with MbetaCD (1 and 2 mM, 1 h) were scraped and fixed for electron microscopic (EM) analysis. Random sections were taken for each treatment group and quantified as the no. of caveolae/µm membrane. A: representative EM membrane images of control and MbetaCD-treated (1 and 2 mM) cells. There are fewer caveolae in the MbetaCD-treated groups compared with control. Cardiac myocytes treated with cholesterol-loaded MbetaCD expressed a similar number of caveolae as did the control group. Cho, cholesterol loaded. B: bar graph representing caveolae/membrane length in control and MbetaCD-treated cells (1 and 2 mM). *P < 0.05 vs. control group.

 
Role of caveolae in IPC- and opioid-induced cardiac protection in isolated myocytes. Cytoprotection by IPC to blunt simulated ischemia was attenuated in cardiac myocytes pretreated with MbetaCD for 1 h (Fig. 4A) and was not altered by cholesterol-loaded MbetaCD, which is not able to deplete cholesterol. Similar results were observed in experiments performed on freshly isolated myocytes (data not shown). Cytoprotection produced by the DOR agonist SNC-121 was also attenuated by MbetaCD, but treatment with cholesterol-loaded MbetaCD did not blunt SNC-121-induced cytoprotection (Fig. 4B).


Figure 4
View larger version (33K):
[in this window]
[in a new window]
  		Fig. 4. Effect of caveolar disruption on ischemic preconditioning (IPC) and opioid-induced cytoprotection. A: rat cardiac myocytes were subjected to simulated ischemia (mineral oil layering) or to preconditioning (IPC, 10 min ischemia with 30 min recovery) in the presence and absence of MbetaCD (2 mM for 1 h). Cells treated with MbetaCD could no longer be preconditioned. *P < 0.05 vs. respective ischemia group. B: isolated cells were subjected to ischemia or pretreated with SNC-121 (1 mM) to induce protection in the presence and absence of MbetaCD and cholesterol-loaded MbetaCD. SNC-121 did not provide protection in MbetaCD-treated cells but did in cholesterol-loaded MbetaCD-treated cells. *P < 0.05 vs. respective ischemia group.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we show that compartmentation of opioid receptors contributes to cardiac protection; DOR, the opioid receptor subtype thought to mediate cardiac protection, localizes to caveolae, and this localization appears to be critical for such protection. The results imply the necessity of having intact caveolar microdomains to achieve cardiac protection by either preconditioning or a receptor agonist. Our combined use of subcellular fractionation with immunoblot analysis, immunofluorescence, and immunoprecipitation provides strong complementary evidence that Cav-3 organizes opioid receptors in caveolae of adult cardiac myocytes. The ability of MbetaCD treatment to attenuate cytoprotection in response to agonist treatment in cardiac myocytes implies a role for intact caveolae in IPC. Importantly, MbetaCD preloaded with cholesterol, which is unable to deplete membrane cholesterol and therefore does not disrupt caveolae, did not hinder the ability of SNC-121 to protect cardiac myocytes from ischemia-induced cell death.

Caveolins function as scaffolds to compartmentalize signaling molecules in caveolae, providing a mechanism for temporal and spatial regulation of signal transduction and cross-talk among signaling molecules (40, 50). Such interactions may contribute to regulation of cell death. For example, infusion of a peptide comprising the scaffolding domain of Cav-1 in isolated-perfused rat hearts produces cardiac protection (53). In prostate cancer, the cav-1 gene can be downregulated by c-myc, and maintaining high levels of cav-1 results in suppression of c-myc-induced apoptosis and sustains active Akt (48). Such data suggest multiple mechanisms whereby caveolins may have a role in apoptosis and perhaps necrosis.

In the heart, overexpression of Cav-3 in neonatal rat cardiac myocytes inhibits myocyte hypertrophy (19), which contributes to adverse cardiac remodeling and the progression of cardiomyopathy (10). Moreover, transgenic mice that overexpress L-type Ca2+ channels show decreased inotropic response to {alpha}-adrenergic stimulation linked to a deficiency in protein kinase C signaling secondary to suppression of Cav-3 (31). In contrast, Cav-1 and Cav-3 double knockout mice show a reduction in caveolae and develop severe cardiomyopathy (29). Dissociation of caveolin from caveolae has been associated with aging and myocardial ischemia-induced heart failure (32). Collectively, these data, as well as the current findings, suggest that increased expression of caveolins may be beneficial in the ischemic heart, and interventions that decrease their expression may be detrimental.

We show that cholesterol depletion can disrupt caveolae formation and impair cardiac protection; this finding would appear to be contradictory to cholesterol reduction produced by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (e.g., statins), which inhibit the rate-limiting enzyme in cholesterol synthesis and produce cardiac protection. We believe this is likely because of the inability of statins to alter membrane caveolae in cardiac myocytes but more importantly because of "pleiotropic" actions of statins that are not necessarily related to plasma cholesterol reduction (8, 20, 51). In preliminary studies (data not shown), we have found that statins do not appear to alter the distribution of Cav-3 in cardiac myocyte sucrose density fractions, suggesting that statins may not alter caveolae in cardiac myocytes. Statins have been shown to have other effects that would influence cardiac protection, such as attenuation of oxidant stress (18), activation of protein kinase C (46), and inactivation of glycogen synthase kinase-3beta (4). In addition, another response to statins, prevention of cardiac hypertrophy, is thought to occur via cholesterol-independent mechanisms (22).

Limitations of the study include the use of a treatment protocol whereby SNC-121 was not washed out; such a protocol differs from IPC. We used this protocol to mimic in vivo pharmacological studies in which an opioid is given as a preconditioning stimulus and ischemia-reperfusion is induced shortly thereafter (13). In addition, one of the original protocols using simulated ischemia in isolated cardiac myocytes used isotonic or hypotonic media for reperfusion (15), whereas more recent studies do not incorporate a reperfusion protocol (34, 35). In most of our studies, we used cells that were allowed to stabilize for 24 h after isolation with induction of ischemia by mineral oil layering and performed reperfusion using normal media that resulted in a sufficient "death response" in the simulated ischemia group. We undertook key confirmatory studies with freshly isolated myocytes and obtained similar results to those of the 24-h cultures (data not shown).

In conclusion, the present study provides evidence for a mechanism utilized by DOR to protect cardiac myocytes from ischemic damage. The findings emphasize the importance of the cellular distribution and spatial organization of signaling components in caveolae in cardiac myocytes and suggest that cardiac pathophysiology can be impacted by subcellular organization of GPCRs in general, and perhaps DOR in particular. Based on evidence that disruption of caveolae blunts cardiac protection produced by DOR, the results provide a mechanistic link between receptor compartmentation in caveolae, opioid receptor stimulation, and cardiac protection.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by National Institutes of Health Grants HL-74625 (to H. H. Patel), HL-66941 (to P. A. Insel), and HL-66941–01A1 (to D. M. Roth) and by a Merit Award from the Department of Veterans Affairs (to D. M. Roth).


   ACKNOWLEDGMENTS
 
We thank Marilyn G. Farquhar and James R. Feramisco for the use of their laboratories and their guidance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. H. Patel, Univ. of California, San Diego, VA San Diego Healthcare System, #125, 3350 La Jolla Village Dr., San Diego, CA 92161-5085 (e-mail: hepatel{at}ucsd.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* P. A. Insel and D. M. Roth share equal senior authorship; they contributed equally to the preparation of the manuscript. Back


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Agard DA, Hiraoka Y, Shaw P, and Sedat JW. Fluorescence microscopy in three dimensions. Methods Cell Biol 30: 353–377, 1989.[ISI][Medline]
  2. Agard DA and Sedat JW. Three-dimensional architecture of a polytene nucleus. Nature 302: 676–681, 1983.[CrossRef][Medline]
  3. Benedict PE, Benedict MB, Su TP, and Bolling SF. Opiate drugs and {delta}-receptor-mediated myocardial protection. Circulation 100, Suppl II: II-357–II-360, 1999.
  4. Bergmann MW, Rechner C, Freund C, Baurand A, Jamali AE, and Dietz R. Statins inhibit reoxygenation-induced cardiomyocyte apoptosis: role for glycogen synthase kinase 3beta and transcription factor beta-catenin. J Mol Cell Cardiol 37: 681–690, 2004.[CrossRef][ISI][Medline]
  5. Christian AE, Haynes MP, Phillips MC, and Rothblat GH. Use of cyclodextrins for manipulating cellular cholesterol content. J Lipid Res 38: 2264–2272, 1997.[Abstract]
  6. Chun M, Liyanage UK, Lisanti MP, and Lodish HF. Signal transduction of a G protein-coupled receptor in caveolae: colocalization of endothelin and its receptor with caveolin. Proc Natl Acad Sci USA 91: 11728–11732, 1994.[Abstract/Free Full Text]
  7. De Vries L, Elenko E, McCaffery JM, Fischer T, Hubler L, McQuistan T, Watson N, and Farquhar MG. RGS-GAIP, a GTPase-activating protein for Galphai heterotrimeric G proteins, is located on clathrin-coated vesicles. Mol Biol Cell 9: 1123–1134, 1998.[Abstract/Free Full Text]
  8. Elrod JW and Lefer DJ. The effects of statins on endothelium, inflammation and cardioprotection (Abstract). Timely Top Med Cardiovasc Dis 9: E20, 2005.[Medline]
  9. Feron O, Smith TW, Michel T, and Kelly RA. Dynamic targeting of the agonist-stimulated m2 muscarinic acetylcholine receptor to caveolae in cardiac myocytes. J Biol Chem 272: 17744–17748, 1997.[Abstract/Free Full Text]
  10. Frey N and Olson EN. Cardiac hypertrophy: the good, the bad, and the ugly. Annu Rev Physiol 65: 45–79, 2003.[CrossRef][ISI][Medline]
  11. Fryer RM, Auchampach JA, and Gross GJ. Therapeutic receptor targets of ischemic preconditioning. Cardiovasc Res 55: 520–525, 2002.[Abstract/Free Full Text]
  12. Fryer RM, Hsu AK, Eells JT, Nagase H, and Gross GJ. Opioid induced second window of cardioprotection: potential role of mitochondrial KATP channels. Circ Res 84: 846–851, 1999.[Abstract/Free Full Text]
  13. Fryer RM, Hsu AK, Nagase H, and Gross GJ. Opioid-induced cardioprotection against myocardial infarction and arrhythmias: mitochondrial versus sarcolemmal ATP-sensitive potassium channels. J Pharmacol Exp Ther 294: 451–457, 2000.[Abstract/Free Full Text]
  14. Furuchi T and Anderson RG. Cholesterol depletion of caveolae causes hyperactivation of extracellular signal-related kinase (ERK). J Biol Chem 273: 21099–21104, 1998.[Abstract/Free Full Text]
  15. Ganote CE, Armstrong S, and Downey JM. Adenosine and A1 selective agonists offer minimal protection against ischaemic injury in isolated rat cardaomyocytes. Cardiovasc Res 27: 1670–1676, 1993.[Abstract/Free Full Text]
  16. Howells RD, Kilpatrick DL, Bailey LC, Noe M, and Udenfriend S. Proenkephalin mRNA in rat heart. Proc Natl Acad Sci USA 83: 1960–1963, 1986.[Abstract/Free Full Text]
  17. Insel PA, Head BP, Ostrom RS, Patel HH, Swaney JS, Tang CM, and Roth DM. Caveolae and lipid rafts: G protein-coupled signaling microdomains in cardiac myocytes. Ann NY Acad Sci 1047: 166–172, 2005.[CrossRef][ISI][Medline]
  18. Jones SP, Teshima Y, Akao M, and Marban E. Simvastain attenuates oxidant-induced mytochondrial dysfunction in cardiac myocytes. Circ Res 93: 697–699, 2003.[Abstract/Free Full Text]
  19. Koga A, Oka N, Kikuchi T, Miyazaki H, Kato S, and Imaizumi T. Adenovirus-mediated overexpression of caveolin-3 inhibits rat cardiomyocyte hypertrophy. Hypertension 42: 213–219, 2003.[Abstract/Free Full Text]
  20. Liao JK and Laufs U. Pleiotropic effects of statins. Annu Rev Pharmacol Toxicol 45: 89–118, 2005.[CrossRef][ISI][Medline]
  21. Lisanti MP, Scherer PE, Tang Z, and Sargiacomo M. Caveolae, caveolin and caveolin-rich membrane domains: a signaling hypothesis. Trends Cell Biol 4: 231–235, 1994.[CrossRef][Medline]
  22. Nakagami H, Jensen KS, and Liao JK. A novel pleiotropic effect of statins: prevention of cardiac hypertrophy by cholesterol-independent mechanisms. Ann Med 35: 398–403, 2003.[CrossRef][ISI][Medline]
  23. Ostrom RS, Gregorian C, Drenan RM, Xiang Y, Regan JW, and Insel PA. Receptor number and caveolar co-localization determine receptor coupling efficiency to adenylyl cyclase. J Biol Chem 276: 42063–42069, 2001.[Abstract/Free Full Text]
  24. Ostrom RS and Insel PA. The evolving role of lipid rafts and caveolae in G protein-coupled receptor signaling: implications for molecular pharmacology. Br J Pharmacol 143: 235–245, 2004.[CrossRef][ISI][Medline]
  25. Ostrom RS, Liu X, Head BP, Gregorian C, Seasholtz TM, and Insel PA. Localization of adenylyl cyclase isoforms and G protein-coupled receptors in vascular smooth muscle cells: expression in caveolin-rich and non-caveolin domains. Mol Pharmacol 62: 983–992, 2002.[Abstract/Free Full Text]
  26. Ostrom RS, Post SR, and Insel PA. Stoichiometry and compartmentation in G protein-coupled receptor signaling: implications for therapeutic interventions involving Gs. J Pharmacol Exp Ther 294: 407–412, 2000.[Abstract/Free Full Text]
  27. Ostrom RS, Violin JD, Coleman S, and Insel PA. Selective enhancement of beta-adrenergic receptor signaling by overexpression of adenylyl cyclase type 6: colocalization of receptor and adenylyl cyclase in caveolae of cardiac myocytes. Mol Pharmacol 57: 1075–1079, 2000.[Abstract/Free Full Text]
  28. Palade G. Fine structure of blood capilaries. J Appl Physiol 24: 1424–1436, 1953.
  29. Park DS, Woodman SE, Schubert W, Cohen AW, Frank PG, Chandra M, Shirani J, Razani B, Tang B, Jelicks LA, Factor SM, Weiss LM, Tanowitz HB, and Lisanti MP. Caveolin-1/3 double-knockout mice are viable, but lack both muscle and non-muscle caveolae, and develop a severe cardiomyopathic phenotype. Am J Pathol 160: 2207–2217, 2002.[Abstract/Free Full Text]
  30. Parpal S, Karlsson M, Thorn H, and Stralfors P. Cholesterol depletion disrupts caveolae and insulin receptor signaling for metabolic control via insulin receptor substrate-1, but not for mitogen-activated protein kinase control. J Biol Chem 276: 9670–9678, 2001.[Abstract/Free Full Text]
  31. Petrashevskaya NN, Bodi I, Koch SE, Akhter SA, and Schwartz A. Effects of {alpha}1-adrenergic stimulation on normal and hypertrophied mouse hearts. Relation to caveolin-3 expression. Cardiovasc Res 63: 561–572, 2004.[Abstract/Free Full Text]
  32. Ratajczak P, Damy T, Heymes C, Oliviero P, Marotte F, Robidel E, Sercombe R, Boczkowski J, Rappaport L, and Samuel JL. Caveolin-1 and -3 dissociations from caveolae to cytosol in the heart during aging and after myocardial infarction in rat. Cardiovasc Res 57: 358–369, 2003.[Abstract/Free Full Text]
  33. Rong JX, Shapiro M, Trogan E, and Fisher EA. Transdifferentiation of mouse aortic smooth muscle cells to a macrophage-like state after cholesterol loading. Proc Natl Acad Sci USA 100: 13531–13536, 2003.[Abstract/Free Full Text]
  34. Sato T, Sasaki N, O'Rourke B, and Marban E. Adenosine primes the opening of mitochondrial ATP-sensitive potassium channels: a key step in ischemic preconditioning? Circulation 102: 800–805, 2000.[Abstract/Free Full Text]
  35. Sato T, Sasaki N, Seharaseyon J, O'Rourke B, and Marban E. Selective pharmacological agents implicate mitochondrial but not sarcolemmal K(ATP) channels in ischemic cardioprotection. Circulation 101: 2418–2423, 2000.[Abstract/Free Full Text]
  36. Schultz JEJ, Hsu AK, and Gross GJ. Ischemic preconditioning in the intact rat heart is mediated by {delta}1- but not µ- or {kappa}-opioid receptors. Circulation 97: 1282–1289, 1998.[Abstract/Free Full Text]
  37. Schultz JEJ, Hsu AK, Nagase H, and Gross GJ. TAN-67, a {delta}1-opioid receptor agonist, reduces infarct size via activation of Gi/o proteins and KATP channels. Am J Physiol Heart Circ Physiol 274: H909–H914, 1998.[Abstract/Free Full Text]
  38. Schulz R, Gres P, and Heusch G. Role of endogenous opioids in ischemic preconditioning but not in short-term hibernation in pigs. Am J Physiol Heart Circ Physiol 280: H2175–H2181, 2001.[Abstract/Free Full Text]
  39. Sedarat F, Xu L, Moore ED, and Tibbits GF. Colocalization of dihydropyridine and ryanodine receptors in neonate rabbit heart using confocal microscopy. Am J Physiol Heart Circ Physiol 279: H202–H209, 2000.[Abstract/Free Full Text]
  40. Shaul PW and Anderson RG. Role of plasmalemmal caveolae in signal transduction. Am J Physiol Lung Cell Mol Physiol 275: L843–L851, 1998.[Abstract/Free Full Text]
  41. Smart EJ, Ying YS, Mineo C, and Anderson RG. A detergent-free method for purifying caveolae membrane from tissue culture cells. Proc Natl Acad Sci USA 92: 10104–10108, 1995.[Abstract/Free Full Text]
  42. Song KS, Li S, Okamoto T, Quilliam LA, Sargiacomo M, and Lisanti MP. Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains. Detergent-free purification of caveolae microdomains. J Biol Chem 271: 9690–9697, 1996.[Abstract/Free Full Text]
  43. Song KS, Scherer PE, Tang Z, Okamoto T, Li S, Chafel M, Chu C, Kohtz DS, and 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 271: 15160–15165, 1996.[Abstract/Free Full Text]
  44. Steinberg SF and Brunton LL. Compartmentation of g protein-coupled signaling pathways in cardiac myocytes. Annu Rev Pharmacol Toxicol 41: 751–773, 2001.[CrossRef][ISI][Medline]
  45. Taggart MJ. Smooth muscle excitation-contraction coupling: a role for caveolae and caveolins? News Physiol Sci 16: 61–65, 2001.[Abstract/Free Full Text]
  46. Tanaka K, Honda M, and Takabatake T. Anti-apoptotic effect of atorvastatin, a 3-hydroxy-3-methylglutaryl coexzyme a reductase inhibitor, on cardiac myocytes through protein kinase C activation. Clin Exp Pharmacol Physiol 31: 360–364, 2004.[Medline]
  47. Tang Z, Scherer PE, Okamoto T, Song K, Chu C, Kohtz DS, Nishimoto I, Lodish HF, and Lisanti MP. Molecular cloning of caveolin-3, a novel member of the caveolin gene family expressed predominantly in muscle. J Biol Chem 271: 2255–2261, 1996.[Abstract/Free Full Text]
  48. Timme TL, Goltsov A, Tahir S, Li L, Wang J, Ren C, Johnston RN, and Thompson TC. Caveolin-1 is regulated by c-myc and suppresses c-myc-induced apoptosis. Oncogene 19: 3256–3265, 2000.[CrossRef][ISI][Medline]
  49. Weil J, Eschenhagen T, Fleige G, Mittmann C, Orthey E, and Scholz H. Localization of preproenkephalin mRNA in rat heart: selective gene expression in left ventricular myocardium. Am J Physiol Heart Circ Physiol 275: H378–H384, 1998.[Abstract/Free Full Text]
  50. Williams TM and Lisanti MP. The caveolin proteins (Abstract). Gene 5: 214, 2004.
  51. Wright DG and Lefer DJ. Statin mediated protection of the ischemic myocardium. Vascul Pharmacol 42: 265–270, 2005.[CrossRef][ISI][Medline]
  52. Yamada E. The fine structure of the gall bladder epithelium of the mouse. J Biophys Biochem Cytol 1: 445–458, 1955.[Medline]
  53. Young LH, Ikeda Y, and Lefer AM. Caveolin-1 peptide exerts cardioprotective effects in myocardial ischemia-reperfusion via nitric oxide mechanism. Am J Physiol Heart Circ Physiol 280: H2489–H2495, 2001.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 CirculationHome page
Y. M. Tsutsumi, Y. T. Horikawa, M. M. Jennings, M. W. Kidd, I. R. Niesman, U. Yokoyama, B. P. Head, Y. Hagiwara, Y. Ishikawa, A. Miyanohara, et al.
Cardiac-Specific Overexpression of Caveolin-3 Induces Endogenous Cardiac Protection by Mimicking Ischemic Preconditioning
Circulation, November 4, 2008; 118(19): 1979 - 1988.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
C. L. Quinlan, A. D. T. Costa, C. L. Costa, S. V. Pierre, P. Dos Santos, and K. D. Garlid
Conditioning the heart induces formation of signalosomes that interact with mitochondria to open mitoKATP channels
Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H953 - H961.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S. H. Deo, M. A. Barlow, L. Gonzalez, D. Yoshishige, and J. L. Caffrey
Cholinergic location of {delta}-opioid receptors in canine atria and SA node
Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H829 - H838.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
M.-H. Huang, H.-Q. Wang, W. R. Roeske, Y. Birnbaum, Y. Wu, N.-P. Yang, Y. Lin, Y. Ye, D. J. McAdoo, M. G. Hughes, et al.
Mediating {delta}-opioid-initiated heart protection via the beta2-adrenergic receptor: role of the intrinsic cardiac adrenergic cell
Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H376 - H384.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
H. H. Patel, Y. M. Tsutsumi, B. P. Head, I. R. Niesman, M. Jennings, Y. Horikawa, D. Huang, A. L. Moreno, P. M. Patel, P. A. Insel, et al.
Mechanisms of cardiac protection from ischemia/reperfusion injury: a role for caveolae and caveolin-1
FASEB J, May 1, 2007; 21(7): 1565 - 1574.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
J.-F. Jasmin, S. Malhotra, M. Singh Dhallu, I. Mercier, D. M. Rosenbaum, and M. P. Lisanti
Caveolin-1 Deficiency Increases Cerebral Ischemic Injury
Circ. Res., March 16, 2007; 100(5): 721 - 729.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/1/H344    most recent
01100.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (14)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Patel, H. H.
Right arrow Articles by Roth, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Patel, H. H.
Right arrow Articles by Roth, D. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.