Role of protein kinase A and A kinase anchoring proteins in buffering and compartmentation of cAMP signalling in human airway smooth muscle cells.

BACKGROUND AND PURPOSE: In human airway smooth muscle (hASM) cells, not all receptors stimulating cAMP production elicit the same effects. This can only be explained if cAMP movement throughout the cell is restricted, yet the mechanisms involved are not fully understood. Phosphodiesterases (PDEs) contribute to compartmentation of many cAMP responses, but PDE activity alone is predicted to be insufficient if cAMP is otherwise freely diffusible. We tested the hypothesis that buffering of cAMP by protein kinase A (PKA) associated with A kinase anchoring proteins (AKAPs) slows cAMP diffusion and that this contributes to receptor-mediated, compartmentalized responses. EXPERIMENTAL APPROACH: Raster image correlation spectroscopy (RICS) was used to measure intracellular cAMP diffusion coefficients and evaluate the contribution of PKA-AKAP interactions. Western blotting and immunocytochemistry were used to identify the AKAPs involved. RNA interference was used to down-regulate AKAP expression and determine its effects on cAMP diffusion. Compartmentalized cAMP responses were measured using fluorescence resonance energy transfer (FRET) based biosensors. KEY RESULTS: Cyclic AMP movement was significantly slower than that of free-diffusion in hASM cells, and disrupting PKA-AKAP interactions significantly increased the diffusion coefficient. PKA associated with the outer mitochondrial membrane appears to play a prominent role in this effect. Consistent with this idea, knocking down expression of D-AKAP2, the primary mitochondrial AKAP, increased cAMP diffusion and disrupted compartmentation of receptor-mediated responses. CONCLUSION AND IMPLICATIONS: Our results confirm that AKAP-anchored PKA contributes to the buffering of cAMP and is consequential in the compartmentation of cAMP responses in hASM cells.

A multiscale predictive digital twin for neurocardiac modulation.

Cardiac function is tightly regulated by the autonomic nervous system (ANS). Activation of the sympathetic nervous system increases cardiac output by increasing heart rate and stroke volume, while parasympathetic nerve stimulation instantly slows heart rate. Importantly, imbalance in autonomic control of the heart has been implicated in the development of arrhythmias and heart failure. Understanding of the mechanisms and effects of autonomic stimulation is a major challenge because synapses in different regions of the heart result in multiple changes to heart function. For example, nerve synapses on the sinoatrial node (SAN) impact pacemaking, while synapses on contractile cells alter contraction and arrhythmia vulnerability. Here, we present a multiscale neurocardiac modelling and simulator tool that predicts the effect of efferent stimulation of the sympathetic and parasympathetic branches of the ANS on the cardiac SAN and ventricular myocardium. The model includes a layered representation of the ANS and reproduces firing properties measured experimentally. Model parameters are derived from experiments and atomistic simulations. The model is a first prototype of a digital twin that is applied to make predictions across all system scales, from subcellular signalling to pacemaker frequency to tissue level responses. We predict conditions under which autonomic imbalance induces proarrhythmia and can be modified to prevent or inhibit arrhythmia. In summary, the multiscale model constitutes a predictive digital twin framework to test and guide high-throughput prediction of novel neuromodulatory therapy. KEY POINTS: A multi-layered model representation of the autonomic nervous system that includes sympathetic and parasympathetic branches, each with sparse random intralayer connectivity, synaptic dynamics and conductance based integrate-and-fire neurons generates firing patterns in close agreement with experiment. A key feature of the neurocardiac computational model is the connection between the autonomic nervous system and both pacemaker and contractile cells, where modification to pacemaker frequency drives initiation of electrical signals in the contractile cells. We utilized atomic-scale molecular dynamics simulations to predict the association and dissociation rates of noradrenaline with the ß-adrenergic receptor. Multiscale predictions demonstrate how autonomic imbalance may increase proclivity to arrhythmias or be used to terminate arrhythmias. The model serves as a first step towards a digital twin for predicting neuromodulation to prevent or reduce disease.

Compartmentalized cAMP signaling in cardiac ventricular myocytes.

Activation of different receptors that act by generating the common second messenger cyclic adenosine monophosphate (cAMP) can elicit distinct functional responses in cardiac myocytes. Selectively sequestering cAMP activity to discrete intracellular microdomains is considered essential for generating receptor-specific responses. The processes that control this aspect of compartmentalized cAMP signaling, however, are not completely clear. Over the years, technological innovations have provided critical breakthroughs in advancing our understanding of the mechanisms underlying cAMP compartmentation. Some of the factors identified include localized production of cAMP by differential distribution of receptors, localized breakdown of this second messenger by targeted distribution of phosphodiesterase enzymes, and limited diffusion of cAMP by protein kinase A (PKA)-dependent buffering or physically restricted barriers. The aim of this review is to provide a discussion of our current knowledge and highlight some of the gaps that still exist in the field of cAMP compartmentation in cardiac myocytes.

Mitochondrial A-kinase anchoring proteins in cardiac ventricular myocytes.

Compartmentation of cAMP signaling is a critical factor for maintaining the integrity of receptor-specific responses in cardiac myocytes. This phenomenon relies on various factors limiting cAMP diffusion. Our previous work in adult rat ventricular myocytes (ARVMs) indicates that PKA regulatory subunits anchored to the outer membrane of mitochondria play a key role in buffering the movement of cytosolic cAMP. PKA can be targeted to discrete subcellular locations through the interaction of both type I and type II regulatory subunits with A-kinase anchoring proteins (AKAPs). The purpose of this study is to identify which AKAPs and PKA regulatory subunit isoforms are associated with mitochondria in ARVMs. Quantitative PCR data demonstrate that mRNA for dual specific AKAP1 and 2 (D-AKAP1 & D-AKAP2), acyl-CoA-binding domain-containing 3 (ACBD3), optic atrophy 1 (OPA1) are most abundant, while Rab32, WAVE-1, and sphingosine kinase type 1 interacting protein (SPHKAP) were barely detectable. Biochemical and immunocytochemical analysis suggests that D-AKAP1, D-AKAP2, and ACBD3 are the predominant mitochondrial AKAPs exposed to the cytosolic compartment in these cells. Furthermore, we show that both type I and type II regulatory subunits of PKA are associated with mitochondria. Taken together, these data suggest that D-AKAP1, D-AKAP2, and ACBD3 may be responsible for tethering both type I and type II PKA regulatory subunits to the outer mitochondrial membrane in ARVMs. In addition to regulating PKA-dependent mitochondrial function, these AKAPs may play an important role by buffering the movement of cAMP necessary for compartmentation.

Mechanisms of cAMP compartmentation in cardiac myocytes: experimental and computational approaches to understanding.

The small diffusible second messenger 3',5'-cyclic adenosine monophosphate (cAMP) is found in virtually every cell in our bodies, where it mediates responses to a variety of different G protein coupled receptors (GPCRs). In the heart, cAMP plays a critical role in regulating many different aspects of cardiac myocyte function, including gene transcription, cell metabolism, and excitation-contraction coupling. Yet, not all GPCRs that stimulate cAMP production elicit the same responses. Subcellular compartmentation of cAMP is essential to explain how different receptors can utilize the same diffusible second messenger to elicit unique functional responses. However, the mechanisms contributing to this behaviour and its significance in producing physiological and pathological responses are incompletely understood. Mathematical modelling has played an essential role in gaining insight into these questions. This review discusses what we currently know about cAMP compartmentation in cardiac myocytes and questions that are yet to be answered.

Compartmentation of ß2 -adrenoceptor stimulated cAMP responses by phosphodiesterase types 2 and 3 in cardiac ventricular myocytes.

BACKGROUND AND PURPOSE: In cardiac myocytes, cyclic AMP (cAMP) produced by both ß1 - and ß2 -adrenoceptors increases L-type Ca2+ channel activity and myocyte contraction. However, only cAMP produced by ß1 -adrenoceptors enhances myocyte relaxation through phospholamban-dependent regulation of the sarco/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2). Here we have tested the hypothesis that stimulation of ß2 -adrenoceptors produces a cAMP signal that is unable to reach SERCA2 and determine what role, if any, phosphodiesterase (PDE) activity plays in this compartmentation. EXPERIMENTAL APPROACH: The cAMP responses produced by ß1 -and ß2 -adrenoceptor stimulation were studied in adult rat ventricular myocytes using two different fluorescence resonance energy transfer (FRET)-based biosensors, the Epac2-camps, which is expressed uniformly throughout the cytoplasm of the entire cell and the Epac2-αKAP, which is targeted to the SERCA2 signalling complex. KEY RESULTS: Selective activation of ß1 - or ß2 -adrenoceptors produced cAMP responses detected by Epac2-camps. However, only stimulation of ß1 -adrenoceptors produced a cAMP response detected by Epac2-αKAP. Yet, stimulation of ß2 -adrenoceptors was able to produce a cAMP signal detected by Epac2-αKAP in the presence of selective inhibitors of PDE2 or PDE3, but not PDE4. CONCLUSION AND IMPLICATIONS: These results support the conclusion that cAMP produced by ß2 -adrenoceptor stimulation was not able to reach subcellular locations where the SERCA2 pump is located. Furthermore, this compartmentalized response is due at least in part to PDE2 and PDE3 activity. This discovery could lead to novel PDE-based therapeutic treatments aimed at correcting cardiac relaxation defects associated with certain forms of heart failure.

Effect of Adenylyl Cyclase Type 6 on Localized Production of cAMP by ß-2 Adrenoceptors in Human Airway Smooth-Muscle Cells.

ß 2-Adrenoceptors (ß 2ARs) are concentrated in caveolar lipid raft domains of the plasma membrane in airway smooth-muscle (ASM) cells, along with adenylyl cyclase type 6 (AC6). This is believed to contribute to how these receptors can selectively regulate certain types of cAMP-dependent responses in these cells. The goal of the present study was to test the hypothesis that ß 2AR production of cAMP is localized to specific subcellular compartments using fluorescence resonance energy transfer-based cAMP biosensors targeted to different microdomains in human ASM cells. Epac2-MyrPalm and Epac2-CAAX biosensors were used to measure responses associated with lipid raft and nonraft regions of the plasma membrane, respectively. Activation of ß 2ARs with isoproterenol produced cAMP responses that are most readily detected in lipid raft domains. Furthermore, overexpression of AC6 somewhat paradoxically inhibited ß 2AR production of cAMP in lipid raft domains without affecting ß 2AR responses detected in other subcellular locations or cAMP responses to EP2 prostaglandin receptor activation, which were confined primarily to nonraft domains of the plasma membrane. The inhibitory effect of overexpressing AC6 was blocked by inhibition of phosphodiesterase type 4 (PDE4) activity with rolipram, inhibition of protein kinase A (PKA) activity with H89, and inhibition of A kinase anchoring protein (AKAP) interactions with the peptide inhibitor Ht31. These results support the idea that overexpression of AC6 leads to enhanced feedback activation of PDE4 via phosphorylation by PKA that is part of an AKAP-dependent signaling complex. This provides insight into the molecular basis for localized regulation of cAMP signaling in human ASM cells.

Compartmentalized cAMP Signaling Associated With Lipid Raft and Non-raft Membrane Domains in Adult Ventricular Myocytes.

Aim: Confining cAMP production to discrete subcellular locations makes it possible for this ubiquitous second messenger to elicit unique functional responses. Yet, factors that determine how and where the production of this diffusible signaling molecule occurs are incompletely understood. The fluid mosaic model originally proposed that signal transduction occurs through random interactions between proteins diffusing freely throughout the plasma membrane. However, it is now known that the movement of membrane proteins is restricted, suggesting that the plasma membrane is segregated into distinct microdomains where different signaling proteins can be concentrated. In this study, we examined what role lipid raft and non-raft membrane domains play in compartmentation of cAMP signaling in adult ventricular myocytes. Methods and Results: The freely diffusible fluorescence resonance energy transfer-based biosensor Epac2-camps was used to measure global cytosolic cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. We found that ß-adrenergic receptors, which are expressed in lipid raft and non-raft domains, produce cAMP responses near the plasma membrane that are distinctly different from those produced by E-type prostaglandin receptors, which are expressed exclusively in non-raft domains. We also found that there are differences in basal cAMP levels associated with lipid raft and non-raft domains, and that this can be explained by differences in basal adenylyl cyclase activity associated with each of these membrane environments. In addition, we found evidence that phosphodiesterases 2, 3, and 4 work together in regulating cAMP activity associated with both lipid raft and non-raft domains, while phosphodiesterase 3 plays a more prominent role in the bulk cytoplasmic compartment. Conclusion: These results suggest that different membrane domains contribute to the formation of distinct pools of cAMP under basal conditions as well as following receptor stimulation in adult ventricular myocytes.

cAMP Signaling Compartmentation: Adenylyl Cyclases as Anchors of Dynamic Signaling Complexes.

It is widely accepted that cAMP signaling is compartmentalized within cells. However, our knowledge of how receptors, cAMP signaling enzymes, effectors, and other key proteins form specific signaling complexes to regulate specific cell responses is limited. The multicomponent nature of these systems and the spatiotemporal dynamics involved as proteins interact and move within a cell make cAMP responses highly complex. Adenylyl cyclases, the enzymatic source of cAMP production, are key starting points for understanding cAMP compartments and defining the functional signaling complexes. Three basic elements are required to form a signaling compartment. First, a localized signal is generated by a G protein-coupled receptor paired to one or more of the nine different transmembrane adenylyl cyclase isoforms that generate the cAMP signal in the cytosol. The diffusion of cAMP is subsequently limited by several factors, including expression of any number of phosphodiesterases (of which there are 24 genes plus spice variants). Finally, signal response elements are differentially localized to respond to cAMP produced within each locale. A-kinase-anchoring proteins, of which there are 43 different isoforms, facilitate this by targeting protein kinase A to specific substrates. Thousands of potential combinations of these three elements are possible in any given cell type, making the characterization of cAMP signaling compartments daunting. This review will focus on what is known about how cells organize cAMP signaling components as well as identify the unknowns. We make an argument for adenylyl cyclases being central to the formation and maintenance of these signaling complexes.

Compartmentalized cAMP responses to prostaglandin EP2 receptor activation in human airway smooth muscle cells.

BACKGROUND AND PURPOSE: Previous studies indicate that prostaglandin EP2 receptors selectively couple to AC2 in non-lipid raft domains of airway smooth muscle (ASM) cells, where they regulate specific cAMP-dependent responses. The goal of the present study was to identify the cellular microdomains where EP2 receptors stimulate cAMP production. EXPERIMENTAL APPROACH: FRET-based cAMP biosensors were targeted to different subcellular locations of primary human ASM cells. The Epac2-camps biosensor, which expresses throughout the cell, was used to measure bulk cytoplasmic responses. Epac2-MyrPalm and Epac2-CAAX were used to measure responses associated with lipid raft and non-raft regions of the plasma membrane respectively. Epac2-NLS was used to monitor responses at the nucleus. KEY RESULTS: Activation of AC with forskolin or ß2 -adrenoceptors with isoprenaline increased cAMP in all subcellular locations. Activation of EP2 receptors with butaprost produced cAMP responses that were most readily detected by the non-raft and nuclear sensors, but only weakly detected by the cytosolic sensor and not detected at all by the lipid raft sensor. Exposure to rolipram, a PDE4 inhibitor, unmasked the ability of EP2 receptors to increase cAMP levels associated with lipid raft domains. Overexpression of AC2 selectively increased EP2 receptor-stimulated production of cAMP in non-raft membrane domains. CONCLUSIONS AND IMPLICATIONS: EP2 receptor activation of AC2 leads to cAMP production in non-raft and nuclear compartments of human ASMs, while ß2 adrenoceptor signalling is broadly detected across microdomains. The activity of PDE4 appears to play a role in maintaining the integrity of compartmentalized EP2 receptor responses in these cells.

A multiscale computational modelling approach predicts mechanisms of female sex risk in the setting of arousal-induced arrhythmias.

KEY POINTS: This study represents a first step toward predicting mechanisms of sex-based arrhythmias that may lead to important developments in risk stratification and may inform future drug design and screening. We undertook simulations to reveal the conditions (i.e. pacing, drugs, sympathetic stimulation) required for triggering and sustaining reentrant arrhythmias. Using the recently solved cryo-EM structure for the Eag-family channel as a template, we revealed potential interactions of oestrogen with the pore loop hERG mutation (G604S). Molecular models suggest that oestrogen and dofetilide blockade can concur simultaneously in the hERG channel pore. ABSTRACT: Female sex is a risk factor for inherited and acquired long-QT associated torsade de pointes (TdP) arrhythmias, and sympathetic discharge is a major factor in triggering TdP in female long-QT syndrome patients. We used a combined experimental and computational approach to predict 'the perfect storm' of hormone concentration, IKr block and sympathetic stimulation that induces arrhythmia in females with inherited and acquired long-QT. More specifically, we developed mathematical models of acquired and inherited long-QT syndrome in male and female ventricular human myocytes by combining effects of a hormone and a hERG blocker, dofetilide, or hERG mutations. These 'male' and 'female' model myocytes and tissues then were used to predict how various sex-based differences underlie arrhythmia risk in the setting of acute sympathetic nervous system discharge. The model predicted increased risk for arrhythmia in females when acute sympathetic nervous system discharge was applied in the settings of both inherited and acquired long-QT syndrome. Females were predicted to have protection from arrhythmia induction when progesterone is high. Males were protected by the presence of testosterone. Structural modelling points towards two plausible and distinct mechanisms of oestrogen action enhancing torsadogenic effects: oestradiol interaction with hERG mutations in the pore loop containing G604 or with common TdP-related blockers in the intra-cavity binding site. Our study presents findings that constitute the first evidence linking structure to function mechanisms underlying female dominance of arousal-induced arrhythmias.

A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation.

Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and temporal distribution of cAMP at the subcellular level could be important for developing new strategies for the prevention or treatment of unfavorable responses associated with different disease states.

Mechanisms Restricting Diffusion of Intracellular cAMP.

Although numerous receptors stimulate cAMP production in a wide array of cells, many elicit distinct, highly localized responses, implying that the subcellular distribution of cAMP is not uniform. One often used explanation is that phosphodiesterases, which breakdown cAMP, act as functional barriers limiting diffusion. However, several studies refute the notion that this is sufficient, suggesting that phosphodiesterase-independent movement of cAMP must occur at rates slower than free diffusion. But, until now this has never been demonstrated. Using Raster Image Correlation Spectroscopy (RICS), we measured the diffusion coefficient of a fluorescently-labeled cAMP derivative (φ450-cAMP) as well as other fluorescent molecules in order to investigate the role that molecular size, cell morphology, and buffering by protein kinase A (PKA) play in restricting cAMP mobility in different cell types. Our results demonstrate that cytosolic movement of cAMP is indeed much slower than the rate of free diffusion and that interactions with PKA, especially type II PKA associated with mitochondria, play a significant role. These findings have important implications with respect to cAMP signaling in all cells.

Caveolin contributes to the modulation of basal and ß-adrenoceptor stimulated function of the adult rat ventricular myocyte by simvastatin: a novel pleiotropic effect.

The number of people taking statins is increasing across the globe, highlighting the importance of fully understanding statins' effects on the cardiovascular system. The beneficial impact of statins extends well beyond regression of atherosclerosis to include direct effects on tissues of the cardiovascular system ('pleiotropic effects'). Pleiotropic effects on the cardiac myocyte are often overlooked. Here we consider the contribution of the caveolin protein, whose expression and cellular distribution is dependent on cholesterol, to statin effects on the cardiac myocyte. Caveolin is a structural and regulatory component of caveolae, and is a key regulator of cardiac contractile function and adrenergic responsiveness. We employed an experimental model in which inhibition of myocyte HMG CoA reductase could be studied in the absence of paracrine influences from non-myocyte cells. Adult rat ventricular myocytes were treated with 10 µM simvastatin for 2 days. Simvastatin treatment reduced myocyte cholesterol, caveolin 3 and caveolar density. Negative inotropic and positive lusitropic effects (with corresponding changes in [Ca2+]i) were seen in statin-treated cells. Simvastatin significantly potentiated the inotropic response to ß2-, but not ß1-, adrenoceptor stimulation. Under conditions of ß2-adrenoceptor stimulation, phosphorylation of phospholamban at Ser16 and troponin I at Ser23/24 was enhanced with statin treatment. Simvastatin increased NO production without significant effects on eNOS expression or phosphorylation (Ser1177), consistent with the reduced expression of caveolin 3, its constitutive inhibitor. In conclusion, statin treatment can reduce caveolin 3 expression, with functional consequences consistent with the known role of caveolae in the cardiac cell. These data are likely to be of significance, particularly during the early phases of statin treatment, and in patients with heart failure who have altered ß-adrenoceptor signalling. In addition, as caveolin is ubiquitously expressed and has myriad tissue-specific functions, the impact of statin-dependent changes in caveolin is likely to have many other functional sequelae.

Role of membrane microdomains in compartmentation of cAMP signaling.

Spatially restricting cAMP production to discrete subcellular locations permits selective regulation of specific functional responses. But exactly where and how cAMP signaling is confined is not fully understood. Different receptors and adenylyl cyclase isoforms responsible for cAMP production are not uniformly distributed between lipid raft and non-lipid raft domains of the plasma membrane. We sought to determine the role that these membrane domains play in organizing cAMP responses in HEK293 cells. The freely diffusible FRET-based biosensor Epac2-camps was used to measure global cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. Disruption of lipid rafts by cholesterol depletion selectively altered cAMP responses produced by raft-associated receptors. The results indicate that receptors associated with lipid raft as well as non-lipid raft domains can contribute to global cAMP responses. In addition, basal cAMP activity was found to be significantly higher in non-raft domains. This was supported by the fact that pharmacologic inhibition of adenylyl cyclase activity reduced basal cAMP activity detected by Epac2-CAAX but not Epac2-MyrPalm or Epac2-camps. Responses detected by Epac2-CAAX were also more sensitive to direct stimulation of adenylyl cyclase activity, but less sensitive to inhibition of phosphodiesterase activity. Quantitative modeling was used to demonstrate that differences in adenylyl cyclase and phosphodiesterase activities are necessary but not sufficient to explain compartmentation of cAMP associated with different microdomains of the plasma membrane.

CaV1.2 signaling complexes in the heart.

L-type Ca(2+) channels (LTCCs) are essential for generation of the electrical and mechanical properties of cardiac muscle. Furthermore, regulation of LTCC activity plays a central role in mediating the effects of sympathetic stimulation on the heart. The primary mechanism responsible for this regulation involves ß-adrenergic receptor (ßAR) stimulation of cAMP production and subsequent activation of protein kinase A (PKA). Although it is well established that PKA-dependent phosphorylation regulates LTCC function, there is still much we do not understand. However, it has recently become clear that the interaction of the various signaling proteins involved is not left to completely stochastic events due to random diffusion. The primary LTCC expressed in cardiac muscle, CaV1.2, forms a supramolecular signaling complex that includes the ß2AR, G proteins, adenylyl cyclases, phosphodiesterases, PKA, and protein phosphatases. In some cases, the protein interactions with CaV1.2 appear to be direct, in other cases they involve scaffolding proteins such as A kinase anchoring proteins and caveolin-3. Functional evidence also suggests that the targeting of these signaling proteins to specific membrane domains plays a critical role in maintaining the fidelity of receptor mediated LTCC regulation. This information helps explain the phenomenon of compartmentation, whereby different receptors, all linked to the production of a common diffusible second messenger, can vary in their ability to regulate LTCC activity. The purpose of this review is to examine our current understanding of the signaling complexes involved in cardiac LTCC regulation.

Muscarinic receptor agonists and antagonists: effects on cardiovascular function.

Muscarinic receptor activation plays an essential role in parasympathetic regulation of cardiovascular function. The primary effect of parasympathetic stimulation is to decrease cardiac output by inhibiting heart rate. However, pharmacologically, muscarinic agonists are actually capable of producing both inhibitory and stimulatory effects on the heart as well as vasculature. This reflects the fact that muscarinic receptors are expressed throughout the cardiovascular system, even though they are not always involved in mediating parasympathetic responses. In the heart, in addition to regulating heart rate by altering the electrical activity of the sinoatrial node, activation of M2 receptors can affect conduction of electrical impulses through the atrioventricular node. These same receptors can also regulate the electrical and mechanical activity of the atria and ventricles. In the vasculature, activation of M3 and M5 receptors in epithelial cells can cause vasorelaxation, while activation of M1 or M3 receptors in vascular smooth muscle cells can cause vasoconstriction in the absence of endothelium. This review focuses on our current understanding of the signaling mechanisms involved in mediating these responses.

Caveolae compartmentalise ß2-adrenoceptor signals by curtailing cAMP production and maintaining phosphatase activity in the sarcoplasmic reticulum of the adult ventricular myocyte.

Inotropy and lusitropy in the ventricular myocyte can be efficiently induced by activation of ß1-, but not ß2-, adrenoceptors (ARs). Compartmentation of ß2-AR-derived cAMP-dependent signalling underlies this functional discrepancy. Here we investigate the mechanism by which caveolae (specialised sarcolemmal invaginations rich in cholesterol and caveolin-3) contribute to compartmentation in the adult rat ventricular myocyte. Selective activation of ß2-ARs (with zinterol/CGP20712A) produced little contractile response in control cells but pronounced inotropic and lusitropic responses in cells treated with the cholesterol-depleting agent methyl-ß-cyclodextrin (MBCD). This was not linked to modulation of L-type Ca(2+) current, but instead to a discrete PKA-mediated phosphorylation of phospholamban at Ser(16). Application of a cell-permeable inhibitor of caveolin-3 scaffolding interactions mimicked the effect of MBCD on phosphorylated phospholamban (pPLB) during ß2-AR stimulation, consistent with MBCD acting via caveolae. Biosensor experiments revealed ß2-AR mobilisation of cAMP in PKA II signalling domains of intact cells only after MBCD treatment, providing a real-time demonstration of cAMP freed from caveolar constraint. Other proteins have roles in compartmentation, so the effects of phosphodiesterase (PDE), protein phosphatase (PP) and phosphoinositide-3-kinase (PI3K) inhibitors on pPLB and contraction were compared in control and MBCD treated cells. PP inhibition alone was conspicuous in showing robust de-compartmentation of ß2-AR-derived signalling in control cells and a comparatively diminutive effect after cholesterol depletion. Collating all evidence, we promote the novel concept that caveolae limit ß2-AR-cAMP signalling by providing a platform that not only attenuates production of cAMP but also prevents inhibitory modulation of PPs at the sarcoplasmic reticulum. This article is part of a Special Issue entitled "Local Signaling in Myocytes".

Caveolae create local signalling domains through their distinct protein content, lipid profile and morphology.

Compartmentation of signalling allows multiple stimuli to achieve diverse cellular responses with only a limited pool of second messengers. This spatial control of signalling is achieved, in part, by cellular structures which bring together elements of a particular cascade. One such structure is the caveola, a flask-shaped lipid raft. Caveolae are well-recognised as signalosomes, platforms for assembly of signalling complexes of receptors, effectors and their targets, which can facilitate efficient and specific cellular responses. Here we extend this simple model and present evidence to show how the protein and lipid profiles of caveolae, as well as their characteristic morphology, define their roles in creating local signalling domains in the cardiac myocyte. This article is part of a Special Issue entitled "Local Signaling in Myocytes."