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Opioid-Induced Preconditioning Is Dependent on Caveolin-3 Expression

Tsutsumi, Yasuo M. MD, PhD*; Kawaraguchi, Yoshitaka MD; Niesman, Ingrid R. MS; Patel, Hemal H. PhD; Roth, David M. MD, PhD

doi: 10.1213/ANE.0b013e3181f3351a
Cardiovascular Anesthesiology: Brief Report
Chinese Language Editions

We tested the hypothesis that caveolin-3 (Cav-3) is essential for opioid-induced preconditioning in vivo. Cav-3 overexpressing mice, Cav-3 knockout mice, and controls were exposed to myocardial ischemia/reperfusion (I/R) in the presence of SNC-121 (SNC), a δ-selective opioid agonist, or naloxone, a nonselective opioid antagonist. Controls were protected from I/R injury by SNC. No protection was produced by SNC in Cav-3 knockout mice. Cav-3 overexpressing mice showed innate protection from I/R compared with controls that was abolished by naloxone. Our results show that opioid-induced preconditioning is dependent on Cav-3 expression and that endogenous protection in Cav-3 overexpressing mice is opioid dependent.

Published ahead of print August 24, 2010 Supplemental Digital Content is available in the text.

From the *Department of Anesthesiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan; and Department of Anesthesiology, University of California, San Diego, and VAMC San Diego, San Diego, California.

Study funding: Funding information is provided at the end of the article.

Disclosure: The authors report no conflicts of interest.

Reprints will not be available from the author.

Address correspondence to Yasuo M. Tsutsumi, MD, PhD, Department of Anesthesiology, Institute of Health Biosciences, the University of Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima, Japan 770-8503. Address e-mail to

Accepted July 13, 2010

Published ahead of print August 24, 2010

Myocardial ischemia/reperfusion (I/R) injury is a major cause of morbidity and mortality in the operative and nonoperative settings. Experimentally, myocardial I/R injury can be attenuated using a variety of interventions (termed “preconditioning”) including brief episodes of ischemia, opioids, and volatile anesthetics.1,2 Caveolae are flask-like invaginations (approximately100 mm in diameter) of the sarcolemmal membrane that are enriched in lipids (e.g., cholesterol and glycosphingolipids), structural proteins (caveolins), and signaling molecules.3 Recently, we have shown that caveolins are essential in myocardial preconditioning and that cardiac-specific overexpression of caveolin-3 (Cav-3) results in innate cardiac protection.4,5 Additionally, we identified that cardiac protection produced by opioid-induced preconditioning is absent when caveolae are disrupted in vitro.6 The impact of Cav-3 and caveolae on opioid-induced preconditioning in vivo has not been investigated. In addition, the role of opioid receptors in the innate cardiac protection observed in Cav-3 overexpressing mice is unknown. Therefore, we tested the hypothesis that expression of Cav-3 is a critical component of opioid-induced preconditioning in vivo and that the innate cardiac protection observed in Cav-3 overexpressing mice is opioid dependent.

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All animals were treated in compliance with the Guide for the Care and Use of Laboratory Animals, and with animal use protocols approved by the VA San Diego Healthcare System Institutional Animal Care and Use Committee (San Diego, CA). Male C57BL/6 Cav-3 knockout (KO) mice and C57BL/6 transgenic mice with cardiac myocyte–specific overexpression of Cav-3 were generated as reported previously.4,7 Male C57BL/6 mice (Jackson Laboratory, Sacramento, CA) served as controls.

In untreated hearts from all 3 experimental groups, left ventricular homogenates were used for immunoblotting for Cav-3 expression as described previously.4 Homogenates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis with 10% polyacrylamide precast gels and transferred by electroelution. Membranes were visualized using primary (Cav-3 and GAPDH [glyceraldehyde 3-phosphate dehydrogenase]) and secondary antibodies (antimouse). Additionally, electron microscopy was performed on myocardium as described previously4 to assess morphologic caveolae in all experimental groups.

Myocardial I/R was induced in vivo as previously described.4 Briefly, mice were anesthetized with pentobarbital and the lungs mechanically ventilated. Cardiac catheterization via the right carotid artery was performed with a microtip pressure transducer for the determination of hemodynamics. Ischemia was produced by occluding the left coronary artery with a snare occluder for 30 minutes. Hearts were reperfused for 2 hours.

Cav-3 KO and control mice were randomly assigned to receive the δ-opioid receptor agonist, SNC-121 (10 mg/kg),8 15 minutes before I/R to initiate opioid-induced preconditioning (Fig. 1). A subset of Cav-3 overexpressing mice were randomly treated with naloxone (a nonselective opioid receptor antagonist; 3.0 mg/kg IV)9 10 minutes before ischemia (Fig. 1).

Figure 1

Figure 1

After reperfusion, the area at risk (AAR) and the myocardial infarct size were determined as described before.4 Cardiac troponin I in serum was measured with a high-sensitivity mouse cardiac troponin I enzyme-linked immunosorbent assay kit.

Sample size was determined for the primary end point of myocardial infarct size. The standard deviation in measurement of infarct size was determined from historic control mice of similar strain undergoing a similar I/R protocol (SD = 6%). We determined that the sample size needed would be at least 6 mice per experimental group assuming 2-tailed α of 0.05 at 90% power with a hypothetical difference of 15%. Statistical analyses were performed by 1-way analysis of variance, followed by Bonferroni post hoc test or unpaired Student t test. All data are expressed as mean ± SD. Statistical significance was defined as P < 0.05.

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We first assessed Cav-3 expression in the 3 treatment groups by immunoblot and caveolae by electron microscopy. Immunoblots of left ventricular homogenates revealed expression of Cav-3 in both control mice and Cav-3 overexpressing mice, and the absence of Cav-3 protein in Cav-3 KO mice (Fig. 2A). The relative expression of Cav-3 protein was greater in Cav-3 overexpressing mice compared with control mice. Electron microscopy revealed caveolae formation in control and Cav-3 overexpressing mice; however, no caveolae were observed in Cav-3 KO mice (Fig. 2, B–D).

Figure 2

Figure 2

We next assessed hemodynamics and myocardial infarction in the experimental groups with opioid receptor agonism or antagonism. There were no differences in preocclusion baseline hemodynamics (Table 1) or in the cardiac AAR among groups (Fig. 3A). Mice preadministered SNC-121 had a reduction in myocardial infarct size compared with control mice (30% ± 2% vs 41% ± 2%, P < 0.05, respectively; Fig. 3B). This cardiac-protective effect of opioid-induced preconditioning was abolished in Cav-3 KO mice (40% ± 4%). Additionally, we confirmed the previous findings of an innate preconditioning-like cardiac protection in Cav-3 overexpressing mice. The innate protective effect of Cav-3 overexpression was eliminated by pretreatment with naloxone (25% ± 4% vs 41% ± 2% of AAR, P < 0.05, respectively; Fig. 3B). The myocardial infarct-sparing effects of opioid-induced preconditioning and Cav-3 overexpression were confirmed by cardiac troponin I measurements (Fig. 3C).

Table 1

Table 1

Figure 3

Figure 3

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The signal transduction pathways described for myocardial preconditioning are complex. It is thought that preconditioning involves activation of G protein– coupled receptors. Specific receptors that have been identified in producing preconditioning include A1 and A3 adenosine,1012 adrenergic,13 M2 muscarinic,14,15 B2 bradykinin,16,17 δ-opioid,18 and cannabinoid19 receptors. Opioid receptor activation produces preconditioning and cardiac protection from myocardial I/R injury in experimental animals and in patients.20,21 Opioid peptides are expressed in the heart.22 Furthermore, it has been demonstrated that the δ-opioid receptor, the dominant opioid receptor in the heart, facilitates preconditioning and cardiac protection.23

Caveolae are cholesterol and sphingolipid-enriched invaginations of the plasma membrane, and caveolins, especially Cav-3, are the structural proteins essential for caveolae formation in myocytes. Caveolins function as chaperones and scaffolds recruiting signaling molecules to caveolae to provide direct temporal and spatial regulation of signal transduction.3 Specifically, many G protein– coupled receptors including opioid receptors localize to caveolae and coimmunoprecipitate with caveolins.24 Previous work in isolated-adult cardiac myocytes from our laboratory showed that caveolae were essential for protection from hypoxia/reoxygenation in vitro and immunohistochemistry showed Cav-3 organized opioid receptors in caveolae.6,24 Our previous work suggested that caveolae and the expression of Cav-3 might be essential for opioid-induced preconditioning in vivo. The current study confirmed this notion by showing that Cav-3–deficient mice, in which no caveolae were observed, were resistant to opioid-induced preconditioning. We also showed that the endogenous cardiac protection in Cav-3 overexpressing mice could be abolished by pretreatment with naloxone, a nonspecific opioid receptor antagonist. The mechanism involving opioid dependence of the innate cardiac protection afforded by Cav-3 overexpression is under investigation. A limitation of the current study is the lack of use of selective opioid receptor antagonists.

In conclusion, the expression of Cav-3 seems to be essential for δ-opioid receptor–induced cardiac protection from myocardial I/R injury. Our results suggest that the caveolae and caveolins within the heart are critical for opioid-induced preconditioning and that caveolins may be novel therapeutic targets for preconditioning the heart to myocardial I/R injury.

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Supported by a Grant-in-Aid for Young Scientists (A) 20591833 (to Dr. Tsutsumi) from the Japan Society for the Promotion of Science, Tokyo; Takeda Science Foundation, Tokyo (to Dr. Tsutsumi); Scientist Development Grant 060039N (to Dr. Patel) from the American Heart Association, Burlingame, CA; a VA Merit grant (to Dr. Roth) from the Department of Veterans Affairs, Washington, DC; and National Institutes of Health grants HL081400 (to Dr. Roth), HL066941 (to Dr. Roth), and HL091071 (to Dr. Patel) from the United States Public Health Service, Bethesda, MD.

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