Cardiac Caveolae Research Articles

Functional interaction of the two-pore domain potassium channel TASK-1 and caveolin-3

The two-pore domain potassium channel TASK-1 is strongly expressed in the heart and has been shown to modulate the resting membrane potential and action potential. However, little is known about the regulation of TASK-1 channels. The present study was designed to determine whether TASK-1 is modulated by caveolin-3, a primary structural protein of cardiac caveolae. Functional studies with the whole-cell voltage clamp technique showed that the expression of caveolin-3 decreased recombinant TASK-1 currents significantly in HEK293T cells, and this effect was prevented by co-expressing the dominant negative mutant caveolin-3 P104L. Immunofluorescence imaging revealed the colocalization of TASK-1 and caveolin-3. Co-immunoprecipitation analysis indicated that caveolin-3 associated with TASK-1. When co-expressed with caveolin-3 P104L, the fluorescence intensity of caveolin-3 on the cell periphery was reduced. This agrees with the functional evidence that caveolin-3 P104L prevented the inhibitory effect of caveolin-3 on TASK-1 currents, possibly via reducing the plasma membrane targeting of caveolin-3. Further, our data from cardiomyocytes suggest that TASK-1 is associated with caveolin-3. In summary, our study indicates that TASK-1 is functionally regulated by caveolin-3, possibly via association with each other on the cell surface. These results point out a novel mechanism in the regulation of TASK-1.

Identification of Caveolar Resident Proteins in Ventricular Myocytes Using a Quantitative Proteomic Approach: Dynamic Changes in Caveolar Composition Following Adrenoceptor Activation

The lipid raft concept proposes that membrane environments enriched in cholesterol and sphingolipids cluster certain proteins and form platforms to integrate cell signaling. In cardiac muscle, caveolae concentrate signaling molecules and ion transporters, and play a vital role in adrenergic regulation of excitation–contraction coupling, and consequently cardiac contractility. Proteomic analysis of cardiac caveolae is hampered by the presence of contaminants that have sometimes, erroneously, been proposed to be resident in these domains. Here we present the first unbiased analysis of the proteome of cardiac caveolae, and investigate dynamic changes in their protein constituents following adrenoreceptor (AR) stimulation.Rat ventricular myocytes were treated with methyl-β-cyclodextrin (MβCD) to deplete cholesterol and disrupt caveolae. Buoyant caveolin-enriched microdomains (BCEMs) were prepared from MβCD-treated and control cell lysates using a standard discontinuous sucrose gradient. BCEMs were harvested, pelleted, and resolubilized, then alkylated, digested, and labeled with iTRAQ reagents, and proteins identified by LC-MS/MS on a LTQ Orbitrap Velos Pro. Proteins were defined as BCEM resident if they were consistently depleted from the BCEM fraction following MβCD treatment. Selective activation of α-, β1-, and β2-AR prior to preparation of BCEMs was achieved by application of agonist/antagonist pairs for 10 min in populations of field-stimulated myocytes.We typically identified 600–850 proteins per experiment, of which, 249 were defined as high-confidence BCEM residents. Functional annotation clustering indicates cardiac BCEMs are enriched in integrin signaling, guanine nucleotide binding, ion transport, and insulin signaling clusters. Proteins possessing a caveolin binding motif were poorly enriched in BCEMs, suggesting this is not the only mechanism that targets proteins to caveolae. With the notable exception of the cavin family, very few proteins show altered abundance in BCEMs following AR activation, suggesting signaling complexes are preformed in BCEMs to ensure a rapid and high fidelity response to adrenergic stimulation in cardiac muscle.

The Role of CAV3 in Long–QT Syndrome

Background— Mutations in CAV3 , coding for caveolin-3, the major constituent scaffolding protein of cardiac caveolae, have been associated with skeletal muscle disease, cardiomyopathy, and most recently long–QT syndrome (LQTS) and sudden infant death syndrome. We examined the occurrence of CAV3 mutations in a large cohort of patients with LQTS. Methods and Results— Probands with LQTS (n=167) were screened for mutations in CAV3 using direct DNA sequencing. A single proband (0.6%) was found to be a heterozygous carrier of a previously described missense mutation, caveolin-3:p.T78M. The proband was also a heterozygous carrier of the trafficking-deficient Kv11.1:p.I400N mutation. The caveolin-3:p.T78M mutation was found isolated in 3 family members, none of whom had a prolonged QT c interval. Coimmunoprecipitations of caveolin-3 and the voltage-gated potassium channel subunit (Kv11.1) were performed, and the electrophysiological classification of the Kv11.1 mutant was carried out by patch-clamp technique in human embryonic kidney 293 cells. Furthermore, the T-wave morphology was assessed in mutation carriers, double mutation carriers, and nonmutation carriers by applying a morphology combination score. The morphology combination score was normal for isolated caveolin-3:p.T78M carriers and of LQT2 type in double heterozygotes. Conclusions— Mutations in CAV3 are rare in LQTS. Furthermore, caveolin-3:p.T78M did not exhibit a LQTS phenotype. Because no association has ever been found between LQTS and isolated CAV3 mutations, we suggest that LQTS9 is considered a provisional entity.

Cardiac myocyte‐specific caveolin‐3 overexpression alters β‐adrenergic receptor activity

Caveolins are scaffolding proteins essential to the formation of caveolae, plasma membrane invaginations that compartmentalize signaling molecules. Mice with cardiac myocyte‐specific overexpression of caveolin‐3 (Cav‐3 OE) have increased caveolae number in cardiac myocytes and are protected from ischemia‐reperfusion injury, while mice lacking Cav‐3 (Cav‐3 KO) lack cardiac caveolae and are more sensitive to cardiac injury. β‐adrenergic receptors (β‐ARs) localize to caveolae and when stimulated by agonists alter cardiac inotropy and chronotropy but whether increased Cav‐3 expression alters β‐AR agonist response in the heart is unknown. We treated Langendorff‐perfused hearts from transgene‐negative (TGneg), Cav‐3 KO, and Cav‐3 OE mice with the β‐AR agonist isoproterenol (Iso). Cav‐3 OE mice had augmented Iso‐stimulated inotropy (+dP/dt) and lusitropy (−dP/dt) relative to TGneg mice but response was reduced in Cav‐3 KO mice. Cardiac myocytes (CM) isolated from Cav‐3 OE mice had enhanced Iso‐stimulated production of cAMP while Cav‐3 KO CM had reduced cAMP production. These data indicate that the expression of Cav‐3 is a key modifier of β‐AR‐promoted cAMP formation and contractile function. Physiological or pathological alteration in Cav‐3 expression thus has the potential to modulate cardiac response to sympathetic activation. Research supported by VMIRF and NIH research grants.

Serotonin 5-HT2A receptor-mediated hypertrophy is negatively regulated by caveolin-3 in cardiomyoblasts and neonatal cardiomyocytes

The serotonin 5-HT2A receptor belongs to the G-protein-coupled receptors (GPCRs) superfamily and mediates the hypertrophic response to serotonin (5-HT) in cardiac myocytes. At present the regulatory mechanisms of 5-HT2A receptor-induced myocyte hypertrophy are not fully understood. The localization and the compartmentation of GPCRs within specialized membrane microdomains are known to modulate their signalling pathway. Therefore, we hypothesized that caveolae microdomains and caveolin-3, the predominant isoform of cardiac caveolae, might be regulators of 5-HT2A receptor signalling. We demonstrate that 5-HT2A receptors interact with caveolin-3 upon 5-HT stimulation and traffic into caveolae membrane microdomains. We provide evidence that caveolin-3 knockdown abolishes the redistribution of 5-HT2A receptors into caveolae and enhances 5-HT2A receptor-induced myocyte hypertrophic markers such as cell size, protein synthesis and ANF gene expression. Importantly, we demonstrate that caveolin-3 and caveolae structures are negative regulators of 5-HT2A receptor-induced nuclear factor of activated T cells (NFAT) transcriptional activation. Taken together, our data demonstrate that caveolin-3 and caveolae microdomains are important regulators of the hypertrophic response induced by 5-HT2A receptors. These findings thus open new insights to target heart hypertrophy under the enhanced serotonin system. This article is part of a Special Issue entitled “Local Signaling in Myocytes”.

A computational investigation of cardiac caveolae as a source of persistent sodium current

Recent studies of cholesterol-rich membrane microdomains, called caveolae, reveal that caveolae are reservoirs of “recruitable” sodium ion channels. Caveolar channels constitute a substantial and previously unrecognized source of sodium current in cardiac cells. In this paper we model for the first time caveolar sodium currents and their contributions to cardiac action potential morphology. We show that the β-agonist-induced opening of caveolae may have substantial impacts on peak overshoot, maximum upstroke velocity, and ultimately conduction velocity. Additionally, we show that prolonged action potentials and the formation of potentially arrhythmogenic afterdepolarizations, can arise if caveolae open intermittently throughout the action potential. Our simulations suggest that caveolar sodium current may constitute a route, which is independent of channelopathies, to delayed repolarization and the arrhythmias associated with such delays.

Increased GlcNacation and decreased caveolin‐3 in cardiac myocyte caveolae during diabetes mellitus

Cardiomyopathy, independent of the vascular effects of hypertension or coronary artery disease, occurs in patients with diabetes mellitus (DM), thus suggesting that factors within cardiac myocytes contribute to DM‐associated cardiac remodeling. Caveolin‐3 (Cav‐3), a myocyte‐specific protein, localizes numerous molecules within caveolae (flask‐like invaginations of the membrane) and can modulate cardiac remodeling; Knockout of Cav‐3 in mice results in a cardiomyopathic phenotype. To test the hypothesis that post‐translational modification of caveolar proteins occur in DM, we assessed hearts from control or streptozotocin‐induced DM mice using fractionation/immunoblot analysis and electron microscopy (EM). We found that diabetic hearts have reduced Cav‐3 expression in caveolar fractions and increased O‐GlcNacation (a post‐translational modification) of caveolar‐resident proteins. EM revealed that diabetic hearts had disruption of cytoarchitecture with disorganized, rounded mitochondria and fewer caveolar invaginations. DM is thus associated with enhanced Glc‐Nacation of caveolar proteins and decreased expression of Cav‐3 in caveolae. Thus, therapies designed to enhance Cav‐3/caveolae expression are potentially novel approaches to limit DM‐associated cardiac remodeling.

Long Qt Syndrome Mutations In Caveolin-3 Cause Loss Of The Kir2.1-mediated Inward Rectifier Potassium Current (iK1)

Caveolin-3 (CAV3) is a key structural protein in cardiac caveolae that serves as an anchoring protein and a regulatory element for protein signaling in human cardiac myocytes. We have demonstrated previously that mutations in CAV3-encoded caveolin-3 are associated with long QT syndrome (LQT9) and increase late sodium current (INa).

The function of α‐ and β‐adrenoceptors of the saphenous artery in caveolin‐1 knockout and wild‐type mice

Adrenoceptors can associate with cardiac caveolae. To investigate the function of vascular caveolae, adrenoceptor-mediated effects were compared in the saphenous artery of caveolin-1 knockout (cav-1KO) and wild-type (WT) mice. Electronmicroscopy was used to detect caveolae. Real-Time quantitative PCR was used for adrenoceptor subtypes. Catecholamine-evoked contractions and relaxations were studied in arterial segments. Caveolae were found in arterial smooth muscle from WT but not from cav-1KO mice. Arterial mRNA levels for the adrenoceptors alpha1A, alpha1B, alpha1D, beta1, beta2 and beta3 were similar in cav-1KO and WT. (-)-Noradrenaline contracted cav-1KO (-log EC50M=7.1) and WT (-log EC50M=7.3) arteries through prazosin-sensitive receptors. Maximum (-)-noradrenaline-evoked contractions were greater in cav-1KO than WT arteries. (-)-Isoprenaline relaxed WT arteries (-log EC50M=7.3) more potently than cav-1KO arteries (-log EC50M=6.8); the effects were antagonized partially and similarly by the beta2-selective antagonist ICI118551 (50 nM). The (-)-isoprenaline-evoked relaxation was partially antagonized by the beta1-adrenoceptor-selective antagonist CGP20712 (300 nM) in WT but not cav-1KO arteries. The beta3-adrenoceptor-selective antagonist L748337 (100 nM) partially antagonized the relaxant effects of (-)-isoprenaline in cav-1KO but not in WT arteries. BRL37344 partially relaxed arteries through beta3-adrenoceptors in cav-1KO but not WT. The relaxant effects of BRL37344 were decreased by the NO synthase inhibitor OmegaL-nitroarginine. The function of arterial alpha1- and beta2-adrenoceptors is similar in cav-1KO and WT mice. beta1-adrenoceptor-mediated relaxation in WT is lost in cav-1KO and replaced by the appearance of beta3-adrenoceptors.

β-Subunit of cardiac Na+-K+-ATPase dictates the concentration of the functional enzyme in caveolae

Previous studies showed the presence of a significant fraction of Na(+)-K(+)-ATPase alpha-subunits in cardiac myocyte caveolae, suggesting the caveolar interactions of Na(+)-K(+)-ATPase with its signaling partners. Because both alpha- and beta-subunits are required for ATPase activity, to clarify the status of the pumping function of caveolar Na(+)-K(+)-ATPase, we have examined the relative distribution of two major subunit isoforms (alpha(1) and beta(1)) in caveolar and noncaveolar membranes of adult rat cardiac myocytes. When cell lysates treated with high salt (Na(2)CO(3) or KCl) concentrations were fractionated by a standard density gradient procedure, the resulting light caveolar membranes contained 30-40% of alpha(1)-subunits and 80-90% of beta(1)-subunits. Use of Na(2)CO(3) was shown to inactivate Na(+)-K(+)-ATPase; however, caveolar membranes obtained by the KCl procedure were not denatured and contained approximately 75% of total myocyte Na(+)-K(+)-ATPase activity. Sealed isolated caveolae exhibited active Na(+) transport. Confocal microscopy supported the presence of alpha,beta-subunits in caveolae, and immunoprecipitation showed the association of the subunits with caveolin oligomers. The findings indicate that cardiac caveolar inpocketings are the primary portals for active Na(+)-K(+) fluxes, and the sites where the pumping and signaling functions of Na(+)-K(+)-ATPase are integrated. Preferential concentration of beta(1)-subunit in caveolae was cell specific; it was also noted in neonatal cardiac myocytes but not in fibroblasts and A7r5 cells. Uneven distributions of alpha(1) and beta(1) in early and late endosomes of myocytes suggested different internalization routes of two subunits as a source of selective localization of active Na(+)-K(+)-ATPase in cardiac caveolae.

Behavior of Caveolae and Caveolin-3 During the Development of Myocyte Hypertrophy

Recent studies have indicated that caveolae are enriched in a variety of signaling molecules, some of which are associated with cardiomyocyte hypertrophy. Caveolin-3, a major constituent of cardiac caveolae, has been suggested to interact with several signaling molecules. We investigated the morphologic changes of caveolae and caveolin-3 expression in hypertrophied cardiomyocytes induced by an alpha1-adrenergic agonist. Cultured rat neonatal cardiomyocytes were used for the experiments. Phenylephrine induced cellular hypertrophy associated with an increase of the number of caveolae and an up-regulation of caveolin-3. Although PMA increased the number of caveolae and the caveolin-3 expression, the extent of these up-regulations was less than that by phenylephrine. Moreover, ionomycin increased the number of caveolae and up-regulated caveolin-3 as much as phenylephrine. Phenylephrine-induced up-regulations of caveolae and caveolin-3 expression were inhibited by BAPTA, suggesting that the intracellular Ca2+ is involved in those regulations. Inhibitors of calcineurin and Ca2+calmodulin-dependent kinase II attenuated the phenylephrine-induced up-regulation of caveolin-3. In pressure-overloaded rat hearts, caveolin-3 protein levels were increased compared with sham-operated rats. In conclusion, the number of caveolae and the expression of caveolin-3 were up-regulated in rat hypertrophied cardiomyocytes, possibly via the alterations of intracellular Ca2+ and protein kinase C.

Adenovirus-mediated overexpression of caveolin-3 inhibits rat cardiomyocyte hypertrophy.

Caveolae are omega-shaped organelles of the cell surface. The protein caveolin-3, a structural component of cardiac caveolae, is associated with cellular signaling. To investigate the effect of adenovirus-mediated overexpression of caveolin-3 on hypertrophic responses in cardiomyocytes, we constructed an adenovirus that encoded human wild-type caveolin-3 (Ad.Cav-3), mutant caveolin-3 (Ad.Cav-3Delta), or bacterial beta-galactosidase (Ad.LacZ). This mutant has been reported to cause human limb-girdle muscular dystrophy. It lacks 9 nucleotides in the caveolin scaffolding domain and behaves in a dominant-negative fashion. Rat neonatal cardiomyocytes were infected with the virus and then harvested 36 hours after infection. In noninfected cells, phenylephrine (PE) and endothelin-1 (ET) increased cell size and [3H]leucine incorporation, along with the induction of sarcomeric reorganization and the reexpression of beta-myosin heavy chain, indicating myocyte hypertrophy. Infection with Ad.LacZ had no effect on those parameters. Ad.Cav-3 prevented the PE- and ET-induced increases in cell size, leucine incorporation, sarcomeric reorganization, and reexpression of beta-myosin heavy chain. Ad.Cav-3 also blocked the PE- and ET-induced phosphorylations of extracellular signal-regulated kinases (ERKs) but did not affect c-Jun amino-terminal kinase and p38 mitogen-activated protein kinase activities. In contrast, Ad.Cav-3Delta significantly augmented hypertrophic responses to ET, which were associated with increased ET-induced phosphorylation of ERK1/2. These results suggest that caveolin-3 behaves as a negative regulator of hypertrophic responses, probably through suppression of ERK1/2 activity.

Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase.

Ouabain binding to Na(+)/K(+)-ATPase activates Src/epidermal growth factor receptor (EGFR) to initiate multiple signal pathways that regulate growth. In cardiac myocytes and the intact heart, the early ouabain-induced pathways that cause rapid activations of ERK1/2 also regulate intracellular Ca(2+) concentration ([Ca(2+)](i)) and contractility. The goal of this study was to explore the role of caveolae in these early signaling events. Subunits of Na(+)/K(+)-ATPase were detected by immunoblot analysis in caveolae isolated from cardiac myocytes, cardiac ventricles, kidney cell lines, and kidney outer medulla by established detergent-free procedures. Isolated rat cardiac caveolae contained Src, EGFR, ERK1/2, and 20-30% of cellular contents of alpha(1)- and alpha(2)-isoforms of Na(+)/K(+)-ATPase, along with nearly all of cellular caveolin-3. Immunofluorescence microscopy of adult cardiac myocytes showed the presence of caveolin-3 and alpha-isoforms in peripheral sarcolemma and T tubules and suggested their partial colocalization. Exposure of contracting isolated rat hearts to a positive inotropic dose of ouabain and analysis of isolated cardiac caveolae showed that ouabain caused 1) no change in total caveolar ERK1/2, but a two- to threefold increase in caveolar phosphorylated/activated ERK1/2; 2) no change in caveolar alpha(1)-isoform and caveolin-3; and 3) 50-60% increases in caveolar Src and alpha(2)-isoform. These findings, in conjunction with previous observations, show that components of the pathways that link Na(+)/K(+)-ATPase to ERK1/2 and [Ca(2+)](i) are organized within cardiac caveolae microdomains. They also suggest that ouabain-induced recruitments of Src and alpha(2)-isoform to caveolae are involved in the manifestation of the positive inotropic effect of ouabain.

Characterization of caveolae from rat heart: Localization of postreceptor signal transduction molecules and their rearrangement after norepinephrine stimulation

Caveolae are plasma membrane subcompartments that have been implicated in signal transduction. In many cellular systems, caveolae are rich in signal transduction molecules such as G proteins and receptor-associated tyrosine kinases. An important structural component of the caveolae is caveolin. Recent evidence show that among the caveolin gene family, caveolin-3 is expressed in skeletal and cardiac muscle and caveolae are present in cardiac myocyte cells. Both the ANP receptor as well as the muscarinic receptor have been localized to the caveolae of cardiac myocytes in culture. These findings prompted us to conduct a further analysis of cardiac caveolae. In order to improve our understanding of the mechanisms of signal transduction regulation in cardiac myocytes, we isolated cardiac caveolae by discontinuous sucrose density gradient centrifugation from rat ventricles and rat neonatal cardiocytes. Our analysis of caveolar content demonstrates that heterotrimeric G proteins, p21ras and receptor-associated tyrosine kinases are concentrated within these structures. We also show that adrenergic stimulation induces an increase in the amount of diverse a- and b-subunits of G proteins, as well as p21ras, in both in vivo and in vitro experimental settings. Our data show that cardiac caveolae are an important site of signal transduction regulation. This finding suggests a potential role for these structures in physiological and pathological states. J. Cell. Biochem. 77:529-539, 2000. © 2000 Wiley-Liss, Inc.

Characterization of caveolae from rat heart: Localization of postreceptor signal transduction molecules and their rearrangement after norepinephrine stimulation

Caveolae are plasma membrane subcompartments that have been implicated in signal transduction. In many cellular systems, caveolae are rich in signal transduction molecules such as G proteins and receptor-associated tyrosine kinases. An important structural component of the caveolae is caveolin. Recent evidence show that among the caveolin gene family, caveolin-3 is expressed in skeletal and cardiac muscle and caveolae are present in cardiac myocyte cells. Both the ANP receptor as well as the muscarinic receptor have been localized to the caveolae of cardiac myocytes in culture. These findings prompted us to conduct a further analysis of cardiac caveolae. In order to improve our understanding of the mechanisms of signal transduction regulation in cardiac myocytes, we isolated cardiac caveolae by discontinuous sucrose density gradient centrifugation from rat ventricles and rat neonatal cardiocytes. Our analysis of caveolar content demonstrates that heterotrimeric G proteins, p21ras and receptor-associated tyrosine kinases are concentrated within these structures. We also show that adrenergic stimulation induces an increase in the amount of diverse alpha- and beta-subunits of G proteins, as well as p21ras, in both in vivo and in vitro experimental settings. Our data show that cardiac caveolae are an important site of signal transduction regulation. This finding suggests a potential role for these structures in physiological and pathological states.