Although caveolae were morphologically identified by electron microscopy more than fifty years ago as defined invaginations of the surface membrane, it is only in the past 15 years that their composition and functional significance have begun to be understood. Caveolae are distinct in their membrane lipid composition, being highly enriched in cholesterol and sphingolipids, and caveolins are signature proteins essential for the formation of caveolae. Specifically Cav-3 is expressed in muscle cells including the cardiomyocytes. A subset of cardiac ion channels have been proposed to reside in caveolae in cardiomyocytes including HCN4, Cav1.2 Kv1.5 channels, Kir6.2/Sur2a channels, Nav1.5, and NCX. These caveolar ion channels are regulated by a variety of signaling pathways in a highly localized fashion in part due to the localization of multiple components of specific signaling pathways to the caveolar microdomain. Dysfunction of caveolar ion channels or their regulation have been implicated in the genesis of inherited arrhythmias such as LQT3 and LQT9 and potentially in acquired arrhythmias in conditions such as heart failure. However, understanding the composition and functional roles of caveolae in the heart as well as their contribution to arrhythmia syndromes is only beginning. Many critical questions remain to be answered.
Limitations in the techniques used to determine whether channel proteins are present in caveolae remain and need to be carefully considered. Early studies oftentimes identified proteins as localized to caveolae based on biochemical membrane fractionation studies revealing a co-distribution of the protein with Cav-3 in low density fraction sucrose gradients. Although this is supportive evidence, it is possible that these proteins reside in noncaveolar lipid rafts or are nonspecifically present in these membrane fractions. Co-immunoprecipitation studies demonstrating that proteins associate with Cav-3 are commonly employed, but these studies share the limitations of all immunoprecipitation studies of possible nonspecific associations and require careful control studies. Imaging approaches are useful, but the spatial resolution of standard light microscopy including confocal microscopy cannot definitively localize proteins to the small 50–100 nm caveolae. Electron microscopy studies can provide spatially defined localization of proteins to caveolae, but these studies are technically challenging and limited by the available specific antibodies that can be used. Approaches to disrupt caveolae using MβCD and evaluating the impact on channel localization can be helpful, but as a nonspecific chelator of cholesterol, MβCD can exert a wide range of effects in addition to disrupting caveolae which can confuse data interpretation. Reducing or eliminating Cav-3 expression is another technique using transgenic technology or siRNA approaches which may offer a more specific manner to disrupt caveolae. The most definitive studies have used a variety of techniques to demonstrate caveolar localization of the ion channels or signaling molecules to caveolae. It is likely that as additional research is performed, the list of caveolar proteins will continue to be refined.
Even more challenging than localizing a protein to caveolae, is isolating the functional properties of that protein in the caveolar microdomain in the intact cell. In the case of ion channels, determining the ionic current properties specifically of caveolar-localized channels is difficult. The loss of function studies using MβCD have the limitations previously described in specificity for caveolae. Disrupting caveolae by knocking down Cav-3 expression can be accomplished, but whether this will disrupt the function of the ion channel previously present in caveolae is not a certainty, as the channel may simply redistribute to other membrane domains. The interpretation of these results can be challenging. The most common supportive finding in caveolar channel functional studies is a dysregulation of the channels by signaling pathways following disruption of caveolae. Future efforts are needed to more specifically define the functional roles of caveolar ion channels to cardiac electrophysiology and pathology.
There are many unanswered fundamental questions regarding ion channels in caveolae and cardiac arrhythmias. How are ion channels targeted to caveolae, and how are these channels trafficked distinctly to the surface membrane? Are there multiple types of caveolae in a single cell type which may have different compositions and functional properties? For any given ion channel, what is the distribution of the channel in caveolar membranes vs. noncaveolar membranes? Is the caveolar distribution of a given ion channel dynamic and how is it regulated? How do various disease processes impact the function and regulation of caveolae that may alter the risk of arrhythmias? These are among the many interesting and important questions that will be addressed by future research.