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Cardiovascular Research 2006 69(4):816-824; doi:10.1016/j.cardiores.2005.10.006
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Copyright © 2005, European Society of Cardiology

Caveolae modulate excitation–contraction coupling and β2-adrenergic signalling in adult rat ventricular myocytes

Sarah Calaghan* and Ed White

Research Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9NQ, UK

* Corresponding author. Tel.: +44 113 343 4309; fax: +44 113 343 4228. Email address: s.c.calaghan{at}leeds.ac.uk

Received 8 September 2005; revised 14 October 2005; accepted 18 October 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Caveolae, flask shaped invaginations of the cell membrane, influence signalling cascades in many cell types. We have tested the hypothesis that caveolae modulate excitation–contraction coupling (ECC) and β-adrenergic stimulation in the adult cardiac myocyte.

Methods: Shortening, [Ca2+]i and L-type Ca2+ current (ICa,L) were recorded in rat ventricular myocytes. Caveolae were disrupted with methyl-β-cyclodextrin (MβC).

Results: Shortening and [Ca2+]i transient amplitude were reduced in myocytes treated with MβC. MβC did not alter the density or characteristics of ICa,L or the sarcoplasmic reticulum (SR) Ca2+ load, but significantly reduced fractional SR Ca2+ release. The inotropic response of myocytes to β1-adrenoceptor stimulation was insensitive to caveolae disruption. By contrast, the increase in shortening, [Ca2+]i transient and ICa,L seen following β2 stimulation was markedly enhanced (3–5 fold) following MβC treatment, and the effect on ICa,L could be mimicked by dialyzing cells with an antibody to caveolin 3. When the G{alpha}i pathway was disabled with pertussis toxin (PTX), control cells showed a similar response to β2 stimulation as seen in MβC-treated myocytes, whereas MβC-treated cells were insensitive to PTX.

Conclusions: Caveolae modulate ECC via the efficiency of the Ca2+-induced Ca2+ release process, rather than Ca2+ influx. Our data are also consistent with the hypothesis that interaction of Gi protein cascade components with caveolin in the caveolae is necessary for effective signalling by this pathway. This suggests that changes in caveolin expression in the adult heart seen during aging and in disease will have consequences for baseline cardiac function and β-adrenergic responsiveness.

KEYWORDS Caveolae; Calcium; Adrenergic agonists; Ca-channel; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Caveolae (‘little caves’) are specialized areas of the cell membrane, enriched in cholesterol and sphingolipids, and coated on the internal surface with the protein caveolin which gives them their characteristic flask shape. Caveolae are important in a variety of cellular functions including signal transduction (see Refs. [1–4] for reviews). Caveolin targets signalling molecules to caveolae and regulates their activity [2,5]. That many signalling molecules and cascades are regulated by interaction with caveolin suggests the importance of this microdomain in orchestrating cell signalling pathways. We are interested in the role of caveolae in excitation–contraction coupling (ECC) and β-adrenergic signalling in the adult heart.

The idea that caveolae may influence cardiac ECC is suggested by findings that many molecules critical for Ca2+ regulation are enriched in caveolae [6–8]. There is little functional data defining the role of caveolae in Ca2+ signalling in the adult heart, but in neonatal cardiac myocytes and vascular smooth muscle cells, depletion of caveolae modulates unitary Ca2+ release events from the sarcoplasmic reticulum (SR) without affecting ICa,L [9]. However, these cells do not contain t-tubules. The adult ventricular myocyte has a well defined t-tubular structure which plays a vital role in co-ordinating ECC [10]. Caveolae are present in these t-tubules [11] but their role in ECC has not been studied functionally.

One of the major mechanisms for regulating contractility of the heart is via β-adrenergic stimulation. Both β1 and β2 adrenoceptors are expressed in the ventricle, and are functionally relevant [12,13]. In the adult heart, β2 receptors, G{alpha}s, and adenyl cyclase V/VI are concentrated in caveolae [14–16]. In neonatal myocytes disruption of caveolae has been shown to potentiate the increase in spontaneous beating frequency in response to β2 but not β1 stimulation [17], although others have reported that it enhances cyclic AMP accumulation in response to both β1 and β2 stimulation [5]. However, there are developmental changes in the β2 signalling pathway and the global increase in cAMP in neonatal ventricular myocytes in response to β2 stimulation is not seen in the adult myocyte [13,15]. In the adult myocyte, β2 stimulation increases ICa,L through the action of a local pool of cAMP [18,19].

The aim of this study was to test the hypotheses that caveolae modulates ECC and the response to β-adrenergic stimulation in the adult ventricle by determining the functional consequences of disrupting two of the main components of caveolae – cholesterol and caveolin 3.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
This investigation conforms with the UK Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act of 1986 and with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). Ventricular myocytes were isolated enzymatically from the hearts of male Wistar rats (250–280 g) (see Ref. [20]).

2.1 Disruption of caveolae
Caveolae were disrupted using methyl-β-cyclodextrin (MβC). MβC depletes cholesterol from the cell membrane and this targets caveolae which are highly enriched in cholesterol. MβC treatment has been shown to flatten caveolae and cause dissociation of the marker protein caveolin [21]. This technique is very well validated in cells from many different tissues including cardiac muscle and vascular smooth muscle [5,9,22]. Cells were treated with methyl-β-cyclodextrin (MβC) either 1 mmol/l for 1 h at 37 °C or 5 mmol/l for 2 h at room temperature (see Refs. [9,22]). For each incubation condition, the concentration of MβC represents the highest concentration that gave viable cells (rod-shaped with striations visible). In a population of cells in which the 2 MβC treatment protocols were directly compared, both had identical effects on shortening. Data from cells receiving both treatments were therefore pooled.

To verify that effects of MβC are due to cholesterol depletion which targets caveolae, rather than a non-specific effect of MβC, some myocytes were treated with MβC conjugated with cholesterol (molar ratio 8:1) [23]. The other major constituent of caveolae in the heart is caveolin 3. Some cells were dialyzed with an antibody to caveolin 3 (25 µg/ml) through the patch pipette to selectively disrupt caveolin-dependent signalling (see Ref. [16]).

2.2 Measurement of shortening and [Ca2+]i
Shortening and intracellular Ca2+ ([Ca2+]i) were measured simultaneously in cells loaded with fura-2, AM (Molecular Probes) as described previously [20]. Shortening was also recorded in a population of cells that were not fura-loaded. Myocytes were superfused with solution (22–25 °C) containing (mmol/l): NaCl 137; KCl 5.4; NaH2PO4 0.33; MgCl2 0.5; HEPES 5; glucose 5.5; CaCl2 1 (pH 7.4), and field-stimulated at 0.5 Hz.

2.3 Ruptured patch–clamp studies
For measurement of ICa,L the ruptured patch configuration was used (in conjunction with an Axopatch 1D amplifier). Pipette solution contained (mmol/l): CsCl 125; NaCl 8; MgCl2 0.5; MgATP 5; Na2GTP 0.4; Glucose 5; HEPES 10; EGTA 5 (pH 7.2). Cells were superfused with solution as described above, but with K+ replaced by Cs+. Pipette resistance was 1.5–4.0 M{Omega}, access resistance was 7.0 ± 0.3 M{Omega}, and series resistance was electronically compensated by 72 ± 1% (n=45). Peak ICa,L was elicited from a holding potential of –80 mV by a 50 ms step depolarization to –40 mV (to inactivate INa), followed by a 500 ms step to 0 mV (0.5 Hz). Current–voltage relationships, steady-state activation and inactivation of ICa,L were measured using conventional double-pulse protocols. After stepping to –40 mV, myocytes were held for 500 ms at potentials from –60 to +60 mV, stepped back to –40 mV for 5 ms, then to 0 mV for 500 ms. There was no difference in rundown at 2 min between control (to 89 ± 4%, n=13) and MβC-treated (to 90 ± 5%, n=9) myocytes.

2.4 Immunofluorescence confocal microscopy
Myocytes were fixed and immunolabelled according to the method described by Howarth et al. [24] with an antibody to the muscle-specific isoform of caveolin, caveolin 3 (BD Transduction Laboratories). To visualize the t-tubular system, cells were loaded with di-8-ANNEPS (Molecular Probes) as described by Brette et al. [25]. Sections of approximately 2 µm thickness were imaged using confocal laser microscopy (Zeiss LSM 5 Pascal).

2.5 β-adrenergic stimulation
Selective stimulation of β1 adrenoceptors was achieved using isoprenaline in the presence of the β2 antagonist ICI 118,551 (10–7 mol/l). Selective stimulation of β2 adrenoceptors was achieved using salbutamol in the presence of the β1 antagonist atenolol (10–6 mol/l). Cells were pre-exposed to antagonist before being exposed to agonist in the continued presence of antagonist. Neither ICI 118,551 nor atenolol had any significant effect on the magnitude of shortening or the amplitude of the [Ca2+]i transient in control or MβC-treated cells (P>0.05; data not shown). Parameters were measured under basal conditions and at steady state following stimulation. To inhibit Gi protein function, cells were treated with 1.5 µg/ml pertussis toxin (PTX) for 3 h at 37 °C [26].

2.6 Statistics
Results are expressed as mean ± S.E.M of n observations. Statistical analysis was performed using the Student's t-test (paired and unpaired) or ANOVA (and post hoc analysis by the Tukey test) as appropriate.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 Effect of MβC on caveolin 3 and t-tubular structure
The distribution of caveolae in adult rat ventricular myocytes, was revealed by immunolabelling caveolin 3. Caveolin 3 staining was confined to sarcolemmal membranes, and was localized to both surface and t-tubular sarcolemma (Fig. 1A,B). The signal was stronger at the surface membrane, consistent with other work showing that the density of caveolae is higher at the cell surface than in the t-tubules (see Ref. [11]). Following MβC treatment, caveolin 3 moves from the light membrane fraction (which contains caveolae) to the heavy fraction [5], although resolution of confocal microscopy is not sufficient to detect this (see Ref. [27]).


Figure 1
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  		Fig. 1 Distribution of caveolin 3 and effect of methyl-β-cyclodextrin (MβC) on t-tubule integrity in the adult ventricular mycoyte. A. Representative confocal image of the mid-plane of a myocyte immunolabelled with an antibody to caveolin 3 showing both surface (short arrow) and t-tubular (long arrow) staining. B. Surface and t-tubular membranes from area defined in A, at a higher magnification. C, D. Representative confocal images taken through the mid-plane of control (C) and MβC-treated (D) myocytes labelled with di-8-ANNEPS. T-tubular structure is preserved after treatment with MβC. The control myocyte was maintained at 37 °C for 1 h; the MβC-treated myoycte was exposed to 1 mmol/l MβC for 1 h at 37 °C. Scale bar represents 20 µm for cell images and 5 µm for enlargement (B).

 
In fetal skeletal muscle, MβC has been shown to affect t-tubular structures. Therefore we used the lipophilic voltage sensitive dye di-8-ANNEPS to visualize the sarcolemma following treatment of cells with MβC. In the adult cardiac myocyte, gross t-tubular structure was not disrupted following MβC treatment (Fig. 1C,D).

3.2 Effect of MβC on basal shortening and [Ca2+]i transient
We began by looking at the effect of disrupting caveolae using MβC on the magnitude of the [Ca2+]i transient. Myocytes treated with MβC had significantly smaller (P<0.05) [Ca2+]i transients than control cells. This was reflected in a reduction in the degree of cell shortening (Fig. 2). To assess the contribution from a decrease in myofilament Ca2+ sensitivity to reduced shortening, we plotted the slope of the phase–plane relationship of cell length and fura-2 ratio units during re-lengthening (relaxation). Cytosolic [Ca2+] is in quasi-equilibrium with the myofilaments during this phase (see Ref. [28]). There was no difference (P>0.05) in the slope of the phase–plane relationship between control (48.4 ± 8.4 µm/ratio unit; n=35) and MβC-treated myocytes (51.8 ± 6.4 µm/ratio unit; n=35). These data suggest that the effect of MβC on shortening is due to the reduction in [Ca2+]i transient amplitude.


Figure 2
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  		Fig. 2 Disruption of caveolae reduces shortening and [Ca2+]i transient amplitude in the adult ventricular myocyte. Panels show representative traces (A) and mean data (B) for shortening (expressed as a percent of resting cell length) in control (Con) and MβC-treated (MβC) myocytes measured in the absence of fura-2 loading (n=74–76 cells). There was no difference in resting cell length between control (111 ± 1 µm) and MβC-treated (110 ± 2 µm) myocytes. Representative [Ca2+]i transients (C) and mean [Ca2+]i transient data (D) in myocytes loaded with fura-2 are shown below (n=35). There was no difference in diastolic [Ca2+]i between control (0.67 ± 0.01 ratio units, RU) and MβC-treated (0.67 ± 0.01 RU) myocytes. * P<0.05; ** P<0.01 compared with control group (Student's unpaired t-test).

 
The effects of MβC on shortening and [Ca2+]i were not seen when MβC was conjugated with cholesterol. Shortening and [Ca2+]i transient amplitude were significantly greater (P<0.05) in cells treated with MβC-cholesterol compared with cells treated with MβC alone, and not different (P>0.05) to these parameters in control cells (n=30 cells in each group).

3.3 Effect of MβC on ICa,L
ICa,L is the main source of Ca2+ entry and the major trigger for Ca2+ release from the SR. One possible reason for the reduction in [Ca2+]i transient amplitude following depletion of caveolae with MβC is that ICa,L is affected. Fig. 3 shows the characteristics of ICa,L in control and MβC-treated cells. Cell capacitance was not different (P>0.05) in MβC-treated cells compared with controls (151 ± 8 vs. 172 ± 9 pF, n=1620). There was no difference (P>0.05) in peak ICa,L or peak ICa,L density (measured when stepping from –40 to 0 mV) between the two groups of cells. Indeed, the current–voltage relationship was identical in control and MβC-treated myocytes (Fig. 3B). Likewise, steady-state activation and inactivation were similar in control and MβC-treated cells (Fig. 3C); voltages for half maximal activation were –16.2 ± 0.4 vs. –15.9 ± 0.5 mV, and for inactivation –35.3 ± 0.5 vs. –35.2 ± 0.6 mV.


Figure 3
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  		Fig. 3 Disruption of caveolae does not modulate ICa,L in the adult ventricular myocyte. A. Representative traces for peak ICa,L recorded during a step from –40 to 0 mV in a control (Con) and an MβC-treated myocyte. B. Current voltage relationships. C. Steady-state activation and inactivation measured during a conventional double-pulse protocol. Measurements of ICa,L were made in the presence of atenolol which had no effect on basal ICa,L amplitude in control (n=7) or MβC-treated cells (n=7; data not shown). Open circles are control cells (n=12–20), closed circles are MβC-treated cells (n=11–16).

 
3.4 Effect of MβC on sarcoplasmic reticulum (SR) Ca2+ release
Next, we looked at the effect of MβC on SR Ca2+ load and fractional Ca2+ release to see if a change in SR Ca2+ release could account for the smaller [Ca2+]i transient recorded in the absence of a change in ICa,L following MβC treatment. The size of the [Ca2+]i transient recorded in response to rapid perfusion with 10 mmol/l caffeine (an index of SR Ca2+ load) was not different (P>0.05) in MβC-treated myoyctes compared with controls (Fig. 4A). However, fractional SR Ca2+ release (the amplitude of electrically stimulated [Ca2+]i transients at steady state/the amplitude of the caffeine-induced transient) was significantly lower (P<0.0001) after MβC treatment (Fig. 4B).


Figure 4
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  		Fig. 4 Disruption of caveolae reduces fractional Ca2+ release without affecting sarcoplasmic reticulum Ca2+ load. A. Amplitude of Ca2+ transients recorded in response to 10 mmol/l caffeine (an index of SR Ca2+ load, expressed as fura-2 ratio units (RU). B. Fractional SR Ca2+ release (the amplitude of electrically induced [Ca2+]i transient/amplitude of caffeine stimulated [Ca2+]i transient). n=24 cells in each group. *** P<0.001 vs. control group.

 
ECC can be indexed by measuring the relationship between SR Ca2+ release and the magnitude of the Ca2+ current that produces that release, often referred to as the ‘gain’ of the system [29,30]. The maximum rate of rise of the [Ca2+]i transient can be used as an indicator of SR Ca2+ release [30]. This parameter was significantly lower (P<0.05) in MβC-treated cells (17.7 ± 1.2 ratio unit/s, n=35) compared with controls (22.5 ± 1.7 ratio unit/s, n=35). Gain, indexed as the mean maximum rate of rise of the [Ca2+]i transient/mean ICa,L amplitude, was lower when caveolae were depleted with MβC (3.9 x 10–3 ratio unit/s/pA/pF) than in controls (5.0 x 10–3 ratio unit/s/pA/pF).

3.5 Effect of MβC on the response to selective β-adrenergic stimulation
To look at the effect of MβC treatment on the response to β-adrenergic stimulation, we recorded shortening and [Ca2+]i in response to a low concentration of agonist and a maximal concentration of agonist. This equated to 5 x 10–9 and 10–7 mol/l isoprenaline [20] and 5 x 10–6 and 5 x 10–5 mol/l salbutamol [12]. There was no significant difference in the response of shortening or the [Ca2+]i transient to sub-maximal or maximal β1 stimulation between control and MβC-treated cells (Fig. 5A,B). By contrast, there was a marked difference in the response of MβC-treated cells to selective β2 stimulation. The response of [Ca2+]i to sub-maximal and maximal β2 stimulation was significantly enhanced following MβC treatment, and this was reflected in an enhanced inotropic response (P<0.05; Fig. 5 C,D).


Figure 5
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  		Fig. 5 Disruption of caveolae potentiates the contractile and [Ca2+]i response to β2, but not β1, adrenoceptor stimulation in the adult ventricular myocyte. Shortening (A) and [Ca2+]i transient (B) response to selective β1 stimulation with a low and high concentration of isoprenaline (Iso) in the presence of ICI 118,551 (ICI). Shortening (C) and [Ca2+]i transient (D) response to selective β2 stimulation with a low and high concentration of salbutamol (Salb) in the presence of atenolol (At). Open circles are controls, closed circles are MβC-treated cells.* P<0.05; *** P<0.01 vs. control group (n=12–19 cells).

 
One of the main factors influencing the magnitude of the Ca2+ transient following β2 stimulation is Ca2+ entry via ICa,L. The increase in ICa,L in response to maximal β2 stimulation was much greater in MβC-treated cells compared with controls (P<0.001) (Fig. 6A).


Figure 6
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  		Fig. 6 Effect of MβC and intracellular dialysis with caveolin 3 antibody on the response of ICa,L to β2-stimulation in the adult ventricular myocyte. A. Percent change in peak ICa,L recorded during a step from –40 to 0 mV in response to 5 x 10–5 mol/l salbutamol (+10–6 mol/l atenolol) in control (n=10) and MβC-treated myocytes (n=10). B. The effect of inclusion of caveolin 3 antibody (25 µg/ml; cav 3 ab) in the pipette on peak response to 5 x 10–5 mol/l salbutamol (+10–6 mol/l atenolol) in control myocytes (n=5–6 myocytes). * P<0.05; *** P<0.001 vs. control group.

 
To ascertain whether the effect of MβC could be mimicked by disrupting caveolin binding specifically, we looked at the effect of dialyzing the cell interior with an antibody to caveolin 3 (see Ref. [16]). The response to salbutamol was recorded at ~10 min after achieving the whole-cell configuration. We used pipettes with lower resistance for these studies to increase cellular dialysis, and corrected for rundown which was slightly higher under these conditions (to 85% at 2 min). Including caveolin 3 antibody in the pipette significantly potentiated (P<0.05) the response of ICa,L to salbutamol when compared with controls (Fig. 6B).

3.6 Effect of disrupting Gi signalling on the response to β2 stimulation
β1 adrenoceptors couple to Gs proteins while β2 adrenoceptors couple to both Gs and Gi [31]. In the adult myocyte, it has been proposed by some (but not all) workers that Gi coupling to the β2 receptor negates Gs-dependent global cAMP signalling and compartmentalizes this to a subsarcolemmal microdomain [15,26,31,32]. In the present study, the effect of MβC on the response to β2 stimulation is consistent with an uncoupling of the Gi pathway. Therefore, we determined whether the effects of caveolar disruption could be mimicked by inhibiting Gi-dependent signalling with PTX (Fig. 7). PTX significantly potentiated (P<0.05) the contractile response of control cells to β2-selective stimulation indicating that Gi signalling normally counters the effect of Gs activation [26,32]. There was no difference (P>0.05) between control cells treated with PTX and MβC-treated cells in the response to β2 stimulation. Furthermore, PTX had no effect (P>0.05) on the response of MβC-treated cells to β2 stimulation. These data are consistent with an effect of caveolar disruption on Gi coupling.


Figure 7
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  		Fig. 7 Effect of inhibition of Gi signalling on the response to β2 stimulation in control and MβC-treated myocytes. Bars show percent increase in shortening in response to selective β2 stimulation (5 x 10–5 mol/l salbutamol+10–6 mol/l atenolol) in control (Con) and MβC-treated cells with or without a 3 h incubation in pertussis toxin (PTX). n=26 myocytes in each group. * P<0.05 vs. control group (ANOVA).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
Several studies have addressed the role of caveolae in ECC and signal transduction in the cardiac cell by characterizing the location of components within caveolae, and their interaction with caveolin 3 (e.g. Ref. [11]). This important information does not tell us about the functional relevance of caveolae. Previous functional studies have generally used neonatal cardiac myocytes which differ from the adult contractile phenotype [5,9,17], or caveolin-null animals [33] which show histological abnormalities in muscle and defects in the t-tubular network [34,35]. We have used functional assays to assess the role of caveolae in ECC and β-adrenergic signalling in the normal adult ventricular myocyte.

We used the cholesterol-sequestering compound MβC to disrupt caveolae. Although the specificity of this process has been questioned [36], it is commonly used to study caveolae (e.g. Refs. [5,9,22,37]) and electron microscopy has shown that it removes caveolae from neonatal cardiac myocytes [9] and vascular smooth muscle [9,38]. We know that MβC does not cause major disruption to the t-tubules in the adult cardiac myocyte because, in MβC-treated myocytes, t-tubular membranes were clearly visible and cell capacitance was not different to that in control cells. We were able to prevent the effects of MβC by conjugating MβC with cholesterol, and to mimic them with by dialyzing cells with an antibody to caveolin 3. Furthermore, our finding that MβC influences ECC via the SR rather than ICa,L, and adrenergic signalling via the β2 adrenoceptor pathway, is consistent with the cellular location of these specific signalling proteins and caveolins (see below) [11,14–16].

4.1 Effect of caveolar disruption on excitation–contraction coupling
We report that disruption of caveolae in the adult ventricular cardiac myocyte modulates basal cell function, decreasing the amplitude of the [Ca2+]i transient and thereby reducing contraction. The effect of caveolar disruption on basal performance reported in the present study is consistent with reduced fractional shortening measured in vivo in the intact heart of caveolin 3-null mice [33]. The reduction in [Ca2+]i transient amplitude with MβC is not due to a change in ICa,L. Because we see a reduction in the maximal rate of rise of the Ca2+ transient, in fractional SR Ca2+ release, and in gain, we conclude that disruption of caveolae reduces the efficiency of coupling between Ca2+ entry and Ca2+ release from the SR.

In neonatal cardiac myocytes and arterial smooth muscle cells, which lack defined t-tubular structure, MβC does not modulate ICa,L, but reduces the frequency and width of Ca2+ sparks, without affecting SR Ca2+ load [9]. A model to account for this places Ca2+ channels in caveolae, where the invagination of the sarcolemma ensures a closer proximity between the channel and adjacent RyRs. This reduction in the diffusion distance means that a greater number of RyRs will be recruited for a given Ca2+ influx [9]. This explanation may not be fully applicable to the adult ventricular myocyte where the majority of L-type Ca2+ channels are in the t-tubules (see Ref. [10] for a review) where they form close juxtapositions (dyads) with ryanodine receptors in the junctional SR membrane [39,40]. There are several reasons why the presence of ‘caveolemmal’ Ca2+ channels may not explain our data: electron microscopy has shown that there are no caveolae within the dyad [40]; the distance between Ca2+ channel and RyR in the dyad (10–12 nm) [41] is significantly less than the depth of a caveolae (50–100 nm) [1,2]; Scriven et al. [11] report little co-localisation of t-tubular caveolin 3 with the {alpha}-subunit of the L-type Ca2+ channel.

However, 40% of RyR in the myocyte has been shown to be outside the dyad, situated in the corbular SR [42] and Scriven et al. [11] observed caveolin 3 adjacent to a group of extra-dyadic ryanodine receptors. This raises the question as to whether Ca2+ release from non-dyadic RyR contributes to the global Ca2+ transient, and whether caveolae play a modulatory role in this process. In this regard, it is interesting that Head et al. [14] have shown that RyR co-immunoprecipitates with caveolin 3, implying some interaction between the 2 proteins.

4.2 Effect of caveolar disruption on β-adrenergic signalling
Disruption of caveolae markedly potentiated the response of ICa,L, [Ca2+]i and contraction to β2 adrenoceptor stimulation, without affecting the response to β1 stimulation. We can mimic the effect of MβC on the response to β2 stimulation with PTX suggesting that MβC disables Gi protein dependent signalling.

In adult cardiac myocytes, it has been shown that β2 adrenoceptors, G{alpha}s, and adenyl cyclase V/VI are enriched in the caveolar microdomain, whereas β1 receptors are distributed between caveolar and non-caveolar membrane fractions [14–16]. Rybin et al. [15] report that G{alpha}i2 is located predominantly in the caveolar fraction, whereas Head et al. [14] report that both G{alpha}i2 and G{alpha}i3 are excluded from caveolae. Because we can mimic the effects of caveolar disruption by dialyzing the cell with an antibody to caveolin 3, the implication is that interaction of one or more Gi-dependent signalling components with caveolin protein acts to enhance signalling. Although there is no consensus as to the location of Gi proteins in the adult myocyte, we are cautious in suggesting that it is the Gi protein itself that is modulated by caveolin because in vitro studies have shown that interaction between caveolin and G proteins negatively regulates G protein activity [43].

Our data suggest that caveolae contribute to the efficiency of the Ca2+-induced Ca2+ release process and tonically enhance the Gi-dependent signalling pathway. Is it possible that there is a common caveolae-based mediator that modulates both these processes? Endothelial nitric oxide (eNOS) interacts with caveolin and this targets eNOS to caveolae [44]. NO can enhance SR Ca2+ release via s-nitrosylation of RyR [45], attenuates the response to β stimulation [46], and is a component of the Gi signalling pathway [19]. However, although location of eNOS in caveolae is required for eNOS activation, caveolin binding to eNOS maintains the enzyme in an inactive state [47] which is not consistent with the effect of caveolae disruption on cardiac cell function.

The fact that ECC and β-adrenergic signalling molecules are concentrated in caveolae would suggest that caveolae play an important part in these signalling pathways; our observations support this hypothesis by showing functional consequences of caveolae disruption consistent with the structural data [11,14–16]. The role of caveolae in the heart may be relevant to cardiac disease states. Caveolin 3 expression decreases in the heart in models of cardiac hypertrophy [48,49] and dissociation of caveolin from caveolae is associated with aging and heart failure [50]. The inotropic response to β1 stimulation is relatively robust compared with that to β2 stimulation in the healthy heart [51], but the balance of β1:β2 signalling changes in the failing heart changes so that the response to β2 stimulation becomes more important [52]. Given that caveolin expression changes in cardiac disease, and with age, our data suggest that this will have consequences for both baseline cardiac function and for β-adrenergic responsiveness.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was sponsored by the British Heart Foundation.


   Notes
 
Time for primary review 34 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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