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β2-Adrenergic Receptor Redistribution in Heart Failure Changes cAMP Compartmentation

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Science  26 Mar 2010:
Vol. 327, Issue 5973, pp. 1653-1657
DOI: 10.1126/science.1185988

Heart Cell Signaling in 3D

A healthy heart relies on the proper transduction of cellular signals through the β1- and β2-adrenergic receptors (βARs), which are located on the surface of the heart's muscle cells (cardiomyocytes). The surface of these cells resembles a highly organized series of hills and valleys and it has been unclear whether this topography plays a role in the βAR signaling events that are critical to cell function. Nikolaev et al. (p. 1653, published online 25 February; see Perspective by Dorn) monitored the cyclic adenosine monophosphate (cAMP) signals generated by the βARs in living cardiomyocytes. In cells from healthy rats and from rats with heart failure, the β1ARs were localized across the entire cell surface. In contrast, the spatial localization of the β2ARs differed in healthy and failing cells. In healthy cardiomyocytes, the β2ARs resided exclusively within surface invaginations called transverse tubules, thereby producing spatially confined cAMP signals, whereas in failing cardiomyocytes, the β2ARs redistributed to other cell surface areas, thereby producing diffuse cAMP signals. Thus, changes in the spatial localization of β2AR-induced cAMP signaling may contribute to heart failure.

Abstract

The β1- and β2-adrenergic receptors (βARs) on the surface of cardiomyocytes mediate distinct effects on cardiac function and the development of heart failure by regulating production of the second messenger cyclic adenosine monophosphate (cAMP). The spatial localization in cardiomyocytes of these βARs, which are coupled to heterotrimeric guanine nucleotide–binding proteins (G proteins), and the functional implications of their localization have been unclear. We combined nanoscale live-cell scanning ion conductance and fluorescence resonance energy transfer microscopy techniques and found that, in cardiomyocytes from healthy adult rats and mice, spatially confined β2AR-induced cAMP signals are localized exclusively to the deep transverse tubules, whereas functional β1ARs are distributed across the entire cell surface. In cardiomyocytes derived from a rat model of chronic heart failure, β2ARs were redistributed from the transverse tubules to the cell crest, which led to diffuse receptor-mediated cAMP signaling. Thus, the redistribution of β2ARs in heart failure changes compartmentation of cAMP and might contribute to the failing myocardial phenotype.

The β1 and β2 adrenergic receptors (βARs) are heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors found on the surface of cardiac muscle cells (cardiomyocytes). They mediate cardiac responses to catecholamine hormones through their coupling to Gs proteins and to the production of the common second messenger cyclic adenosine monophosphate (cAMP) (1). Selective stimulation of these two receptor subtypes leads to distinct physiological and pathophysiological responses. β1ARs but not β2ARs stimulate the cAMP-dependent protein kinase (PKA)–mediated phosphorylation of phospholamban and cardiac contractile proteins (2), induce hypertrophy upon moderate overexpression (3, 4), and promote cardiomyocyte apoptosis (5, 6). In ventricular cardiomyocytes isolated from transgenic mice expressing a fluorescence resonance energy transfer (FRET)–based cAMP sensor, we recently showed that, upon local receptor stimulation, β1AR-induced receptor-mediated cAMP signaling propagated throughout the entire cell, whereas β2AR-cAMP responses were locally confined by an unknown mechanism (7). The spatial localization of the receptors and the compartmentation of their signaling is thought to play a critical role in cardiac physiology and the development of cardiac disease, such as heart failure (812). However, the precise distribution of the β1 and β2ARs in cardiomyocytes with respect to the highly organized sarcomeric structure of these cells and its potential functional implications are still elusive. As for most G protein–coupled receptors, immunocytochemical or electron microscopy detection of native βARs receptors with antibodies is limited by the low expression level of these proteins and by insufficient antibody specificity.

Here, we present an alternative approach to determine both position and function of endogenous βAR subtypes, by combining scanning ion conductance microscopy (SICM) with measurements of cAMP production by FRET after local receptor stimulation (13). SICM is a nonoptical method (14) in which a nanopipette is used as a scanning probe for noncontact visualization of the three-dimensional surface topography of living cells (15, 16). SICM is based on measuring the changes in ion flow through the pipette positioned at the cell surface and allows resolution of the structural features of cardiomyocytes, such as Z-grooves, cell crests located between them, and transverse (T)-tubules (Fig. 1), with a resolution equal to the pipette’s inner diameter (17), typically ~50 to 100 nm, as confirmed by electron microscopy. The SICM pattern of T-tubule distribution colocalized well with the T-tubular marker di-8-ANEPPS, a voltage-sensitive dye (fig. S1A).

Fig. 1

Functional localization of βAR-induced cAMP signaling and the principle of the combined nanoscale SICM-FRET approach. Typical SICM image of a rat cardiomyocyte shows defined morphological structures that are used to position the nanopipette and to stimulate receptors in a highly localized fashion. Receptor activity is measured by monitoring the production of cAMP by Epac2-camps, a FRET-based cAMP sensor that changes its conformation and fluorescence properties upon activation—i.e., cAMP binding. YFP and CFP, yellow and cyan fluorescent proteins, respectively.

We isolated ventricular cardiomyocytes from healthy adult rats, introduced into the cells an adenovirus vector encoding the cytosolic cAMP sensor Epac2-camps (18), and acquired an image of the cell surface topography by SICM. Next, we positioned the pipette onto various membrane regions of defined morphology and applied receptor ligands in a highly localized fashion. On selective local stimulation of β1 and β2ARs, we measured cAMP synthesis and its accumulation in the cell cytosol by FRET microscopy (Fig. 1). Local stimulation was achieved by applying pressure to the pipette while constantly superfusing the cells with buffer solution (7). Pressure application resulted in the delivery of ~1.6 pl/s of the agonist solution onto the cell surface (fig. S1B) and did not cause any membrane deformations, as measured by SICM (fig. S1C). Local ligand application ensured activation of a small (≤500 nm) sarcolemmal area, as confirmed by labeling of the membrane with the fluorescent βAR ligand BODIPY-TMR-CGP12177 (fig. S1D). This area was in the range of the size of a T-tubule opening and well below the size of a sarcomere.

To study the localization of receptors and cAMP signals in cardiomyocytes isolated from healthy adult rat hearts, we locally stimulated β1 and β2ARs either in the T-tubule or on the cell crest. Selective stimulation of β1ARs in both regions resulted in a robust decrease of FRET, reflecting the activation of cAMP synthesis by the receptors localized in different parts of the membrane (Fig. 2, A to C). In sharp contrast, β2-cAMP signals were observed only after local T-tubular stimulation, but not after stimulation at the cell crest (Fig. 2, D to F). We quantified changes in FRET (Fig. 2G) after local receptor stimulations and investigated whether the absence of the β2AR signal on the cell crest results from higher rates of local cAMP degradation by phosphodiesterases (PDEs) (8) or from coupling of the β2AR to Gi proteins (19, 20) in this particular compartment. Inhibition of PDEs with 3-isobutyl-1-methylxanthine (IBMX) led to slightly higher cAMP levels after stimulation in the T-tubules but did not produce any signals after stimulation in the cell crest. Likewise, Gi-protein inactivation with pertussis toxin (PTX) did not change the localized pattern of the β2AR signals (Fig. 2H). These data indicate that, in normal cardiomyocytes, β2ARs are exclusively localized to the T-tubules, whereas β1ARs are present in both the cell crests and the T-tubules. To exclude the possibility that the absence of the β2AR signal on the cell crest was due to the lower expression level of the β2 versus β1ARs in cardiomyocytes, we partially blocked β1ARs by applying 40 nM of the β1AR antagonist CGP20712A, so that the β2 and β1 stimuli were of equal strength (7). Even in this case, β1AR signals were detected equally well in the T-tubules and on the cell crest (fig. S2). We also tested the possibility that β3ARs might blunt cAMP production, as reported for neonatal cardiomyocytes (21). Addition of the selective β3AR antagonist SR59230A did not cause any change in FRET signals induced by local selective β1 and β2AR stimulations (fig. S3).

Fig. 2

Distribution of βAR-induced cAMP signaling in cardiomyocytes from healthy rats. (A to C) SICM image and corresponding FRET YFP/CFP ratio traces recorded from whole cardiomyocytes after local β1AR stimulation in the cell crest (B) and in the T-tubule (C), as indicated by arrows in (A). Decrease in the FRET ratio indicates an increase in cAMP. The cell was superfused with 50 nM of the β2AR antagonist ICI118551, and β1ARs were then locally stimulated from the scanning nanopipette filled with isoproterenol (ISO, 10 μM) and ICI118551 (ICI, 5 μM) by applying pressure (414 kPa) for 50 s. (D to F) SICM image and corresponding ratio traces after local β2AR stimulation in the cell crest (E) and in the T-tubule (F). Cells were superfused with 100 nM of the β1AR antagonist CGP20712A, and then β2ARs were selectively stimulated from the pipette with ISO (10 μM) and CGP20712A (CGP, 10 μM). (G) Quantification of the changes in FRET YFP/CFP ratios from experiments shown in (A) to (F). Data are plotted as means ± SEM (n ≥ 9). (H) Effect of 300 μM IBMX and of pretreatment with 1.5 μg/ml PTX on the localized β2AR signaling, means ± SEM (n ≥ 6). *P < 0.01, by analysis of variance (ANOVA).

To analyze individual effects of β1 and β2ARs, we studied adult cardiomyocytes isolated from either β2 or β1AR knockout (KO) mice transgenically expressing the FRET-based cAMP sensor. We first confirmed that cardiomyocytes from wild-type mice exhibited a pattern of β1 and β2AR distribution similar to the pattern we noted for healthy rat cardiomyocytes (fig. S4, A to D). We then showed that cardiomyocytes from wild-type and KO mice had a similar surface morphology (fig. S4, B, F, and I). Consistent with the rat data, we found that β1AR-cAMP signals in the β2AR-KO myocytes were detectable on β1AR stimulation in both cell crests and T-tubular regions, whereas β2ARs in β1AR-KO cells showed locally confined cAMP signals in the T-tubules (fig. S4, G, K, and L).

We next studied whether βAR localization was altered in a rat model of chronic heart failure induced by myocardial infarction. We have shown severe functional deficits in this model both at the whole-organ and cellular levels, with cardiomyocytes demonstrating marked structural abnormalities, including an extensive loss of the T-tubules (17) (Fig. 3, A and D), and βAR desensitization, a hallmark of cardiac failure (fig. S5). We analyzed the distribution of the β1AR- and β2AR-mediated cAMP signaling in experiments similar to those performed with the healthy cells (Fig. 2). Selective local stimulation of β1ARs led to cAMP signals equally detectable in T-tubules and in the nontubular regions (Fig. 3, A to C). Note that selective local β2AR-stimulation of failing cells revealed cAMP signals originating not only from the T-tubules, but also from the receptors locally activated in the nontubular areas (Fig. 3, D to F). The amplitudes of these β2AR-cAMP signals were smaller than the β2AR signals from the T-tubules of healthy cells [P < 0.01, by analysis of variance (ANOVA), compare Fig. 3G and Fig. 2G], but the cAMP production was clearly detectable after local stimulation of different sarcolemmal regions (Fig. 3, E to G), which suggested that, in heart failure, β2ARs redistribute from the T-tubules to the cell crest. Once again, modulation of PDE activity and Gi coupling had only minor effects (Fig. 3H). We observed a similar redistribution of β2ARs in rat and in β1AR-KO mouse cardiomyocytes after artificial formamide-induced detubulation, which suggested that it is the loss of T-tubules in disease that drives the selective redistribution of the β2AR subtype (fig. S6).

Fig. 3

βAR-induced cAMP signals in cardiomyocytes from rats with chronic heart failure. (A to C) SICM image and corresponding FRET ratio traces after local β1AR stimulation in the detubulated area (B) and in the T-tubule (C). The cells were superfused and stimulated from the nanopipette as described in Fig. 2. (D to F) SICM image and corresponding ratio traces after local β2AR stimulation in the detubulated area (E) and in the T-tubule (F). (G) Quantification of the changes in cAMP-FRET ratios from experiments shown in (A) to (F). Data are means ± SEM (n ≥ 8). (H) Effect of 300 μM IBMX and of pretreatment with 1.5 μg/ml PTX on the localized β2AR signaling, means ± SEM (n ≥ 5).

We explored the spatial organization of cAMP signaling from differentially localized βARs in normal and failing cardiomyocytes by analyzing the distribution of the FRET signals in different parts of the cell cytosol after local β2AR stimulation. To validate the measurements of cAMP diffusion using a soluble sensor protein, we confirmed that the diffusion of the sensor itself did not change upon receptor stimulation (fig. S7, A to C). β2AR-induced cAMP signals were highly locally confined when this receptor was activated in the T-tubules of healthy cells (Fig. 4, A and E). Note that stimulation of β2ARs in detubulated areas of failing cardiomyocytes produced diffuse cAMP signaling that propagated throughout the entire cytosol (Fig. 4, B and E), similar in behavior to the β1AR-generated signal (fig. S7D).

Fig. 4

Differences in spatial organization of cAMP signaling in cardiomyocyte from healthy rats and rats with heart failure. (A) Local stimulation of β2ARs in the T-tubule of a healthy cardiomyocyte, as described in Fig. 2F, demonstrates a locally confined cAMP response detectable only at the site of stimulation (arrow, n ≥ 7). The graph shows FRET ratio changes measured in five different regions marked with colored ovals (left). (B) β2AR stimulation in a detubulated area of a failing cell induces a global cAMP signal propagating throughout the cytosol (n ≥ 7). Note the loss of striated PKA localization pattern in failing cells (right). (C and D) Treatment of healthy cells with 50 μM Ht31 (C) but not with control Ht31P peptide (D) for 40 to 60 min induces the disruption of the striated PKA localization (right) and propagating cAMP signals following local β2AR stimulation (n = 5). Scale bars in (A) to (D), 20 μM. (E) Quantification of cAMP signal propagation for experiments presented in (A) to (D). Data are means ± SEM.

We hypothesized that cAMP buffering by the cAMP-dependent protein kinase (PKA) (22) might contribute to the localized β2AR-cAMP signaling. It was noteworthy that immunostaining of the kinase’s RII subunit revealed a loss of the striated PKA localization pattern in cells from rats with heart failure (Fig. 4, A and B), although the pattern of potential PKA targeting structures, such as sarcoplasmic reticulum (SR), was not distorted (fig. S7F). Treatment of healthy cells with the Ht31 peptide, which blocks the interaction between RII subunits and A-kinase–anchoring proteins (10, 23), led to disruption of the T-tubular localization of the PKA and to propagation of β2AR-cAMP signals throughout larger parts of the cell (Fig. 4, C to E). This finding suggests that the cAMP produced on stimulation of β2ARs in the T-tubules may be locally confined by the interaction with localized PKA molecules enriched in this compartment. In addition, activation of PKA by cAMP may lead to a local increase in PDE4 activity, which provides a negative-feedback control of the cAMP levels (8). To test this possibility, we blocked PDE4 by rolipram and observed that β2AR-cAMP signals now propagated from locally activated T-tubules (fig. S7E). Thus, local buffering of cAMP by PKA and activation of PDE4 by this kinase contribute to the confinement of β2AR-cAMP signaling.

One possible mechanism that might be responsible for T-tubule–selective β2AR localization is the interaction of this receptor with lipid rafts (11). To investigate the role of these structures in the β2AR localization and signaling, we performed SICM-FRET experiments in rat cardiomyocytes after membrane cholesterol depletion by methyl-β-cyclodextrin (MβCD). MβCD treatment did not cause any loss of T-tubules but induced β2AR redistribution and propagation of β2AR-cAMP signals from the crest of the cell (fig. S8), which suggested that the interaction of β2ARs with cholesterol-rich membrane domains is important for normal β2AR localization and signal compartmentation.

On the basis of the observed distribution of β1 and β2AR signals in failing versus healthy cardiomyocytes, we propose a model in which the compartmentation of the β2AR-cAMP signaling changes in heart failure (fig. S9). Redistribution of the β2AR from the T-tubules to the cell crest in failing cardiomyocytes and the loss of proper PKA localization, observed also in human heart failure (24), results in uncoupling of the β2ARs from the localized pools of PKA that are responsible for the compartmentation of the β2AR-cAMP signaling. Thus, in failing cells, activation of β2ARs leads to cell-wide cAMP signal propagation patterns, similar to the patterns observed for β1ARs (compare Fig. 4B and fig. S7D). Upon redistribution of the receptor, β2AR signaling may lose its normally cardioprotective properties and may acquire the characteristics of the β1AR response, thus contributing to the heart failure phenotype. It has been previously noted that β2AR signals in ventricular myocytes from failing human hearts and animal heart failure models had functional effects more characteristic of the β1AR (25, 26). The propagating β2AR-cAMP gradients that we observed in failing cardiomyocytes and in normal cardiomyocytes treated with rolipram or MβCD are compatible with the ability of β2ARs to induce phospholamban and troponin I phosphorylation that was reported in these experimental settings and that is associated with arrhythmogenic effects of the β2AR in heart failure (2730).

In summary, using the SICM-FRET technique, we were able to functionally localize β1ARs and β2ARs to the surface structures of adult ventricular cardiomyocytes and to uncover the mechanisms leading to the abnormal cAMP compartmentation in heart failure. These findings should provide a deeper understanding of this cardiac disease and facilitate the development of new therapeutic strategies.

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1185988/DC1

Materials and Methods

Figs. S1 to S9

References

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. We thank P. O’Gara for cardiomyocyte isolation; C. Dees for receptor binding assays; and L. Jaffe, F. Klauschen, and S. Gambaryan for critical reading of the manuscript. This study was supported by the Wellcome Trust (WTN 084064 to J.G.), Action Medical Research Grant (to J.G.), a U.K. Biotechnology and Biological Sciences Research Council grant (to Y.E.K.), a U.K. Medical Research Council grant (to J.G. and Y.E.K.) and a grant from Leducq Foundation (to S.E.H. and M.J.L.). Y.E.K. is a shareholder of, and P.N. has a consultancy agreement with, Ionscope Ltd., a company that sells scanning ion conductance microscopes. The cAMP sensor Epac2-camps used in this study is covered by a patent belonging to the University of Würzburg. It is being provided at no cost, but with a material transfer agreement, to nonprofit research institutes and universities.
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