Localization of caveolae and mitochondria in adult cardiac myocytes: implications for reductive signaling

Abstract

Caveolae, membrane invaginations composed of cholesterol and the protein caveolin (Cav), are involved in trafficking and compartmentation of signaling molecules, including endothelial nitric oxide synthase (eNOS) in cardiovascular cells. Recently, heme oxygenase (HO), the rate-limiting enzyme in the oxidative degradation of heme to carbon monoxide (CO), biliverdin and iron, was detected in caveolae in endothelial cells. Here, we used cardiac myocytes (CM) isolated from adult rats and sought to determine whether HO localizes with other enzymes that modulate reactive species in caveolae. Immunoblotting of sucrose density gradient fractions of CM membranes detected enrichment of HO-1, eNOS, cytochrome P450 reductase, and Cu-Zn and Mn superoxide dismutase in buoyant fractions (caveolae); biliverdin reductase was not found in buoyant fractions. Immunoprecipitation studies revealed co-immunoprecipitation of all Cav isoforms and HO-1. Using immunohistochemistry, we found co-localization between Cav-3, HO-1 and cytochrome C, a mitochondrial protein marker, along the sarcolemma. HO-1 and HO-2 co-localized with Cav-3 at both the sarcolemma and intercalated discs. Electron microscopy revealed close apposition of sub-sarcolemmal mitochondria and sarcolemmal caveolae. The localization with caveolin of HO and modulatory enzymes for other reactive species and the close apposition of caveolae and mitochondria suggest that caveolae are microdomains that generate both CO and NO, and may serve as “reductive antioxidant sinks” for free radicals produced by adjacent mitochondria in CM.