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

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

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 -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--cyclodextrin (MCD, 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 MCD and MCD 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). MCD 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 MCD, 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

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