Original article
Localization of f-channels to caveolae mediates specific β2-adrenergic receptor modulation of rate in sinoatrial myocytes

https://doi.org/10.1016/j.yjmcc.2006.09.018Get rights and content

Abstract

β1- and β2-adrenergic receptors (ARs) coexist in different regions of the heart. The β21 expression ratio is higher in the sinoatrial node (SAN) than in atria and ventricles, but the specific contribution of either type of receptor to rate modulation is still not well established. We have recently demonstrated that pacemaker (“funny”) f-channels are located in lipid rafts of the rabbit SAN. Since in ventricular myocytes β2-, but not β1-ARs, localize to caveolae, we hypothesized that modulation of f-channels and of pacemaker activity in SAN myocytes is controlled mainly by β2-AR activation. To address this point, we investigated the caveolar localization of proteins by co-immunoprecipitation and immunocytochemistry, and found that f-channels interact with caveolin 3. We also recorded If current and spontaneous activity from SAN myocytes, and found that β-AR activation by the non-selective agonists isoproterenol and fenoterol shifted the If activation curve similarly (by 6.3 and 5.3 mV) and increased similarly spontaneous rate (by 23.1% and 21.6%, respectively). Specific β2 stimulation had similar effects (4.9 mV shift of the activation curve and 16.9% rate increase), but specific β1 stimulation was less effective (1.7 mV shift and 7.2% rate increase). However, after caveolar disorganization by MβCD (2%), stimulation of β1-ARs was as effective as non-specific β-AR stimulation. These data show that specific stimulation of β2-ARs is the main mechanism by which heart rate is modulated through a positive shift of the If activation curve and that this mechanism requires specific membrane compartmentation.

Introduction

In the mammalian heart, spontaneous activity is an intrinsic property of pacemaker cells of the SAN. Although generation of rhythmic activity does not require nervous inputs, autonomic innervation of the SAN region is responsible for the modulation of heart rate.

Fine tuning of cardiac rhythm is achieved by the opposing actions of the neurotransmitters norepinephrine and acetylcholine released by sympathetic and parasympathetic branches, respectively, of the autonomic nervous system. Activated adrenergic and muscarinic receptors, acting through different G-proteins, stimulate and inhibit adenylate cyclase causing an increase and a decrease in cytoplasmic cAMP concentration, respectively.

To date, three different subtypes of β-ARs are known (i.e., β1, β2, and β3). Only β1- and β2-ARs are abundantly expressed in cardiac tissue, with a predominance of the β1-ARs subtype. In mammals, the β12 ratio varies among different cardiac regions: it is very high in the ventricles, lower in the atria and lower still in the SAN. The distribution pattern varies somewhat in different species but the trend is the same, with the SAN being the region with the highest level of expression of β2-ARs [1], [2], [3], [4].

In the heart, functional coupling to adenylate cyclase is reported to be more efficient for β2- than for β1-adrenergic receptors [4], [5], [6]. Although this difference could be due simply to the intrinsic properties of receptors, it could also result from differences in the subcellular distribution of receptors and the specific signal transduction proteins to which each type of receptor is coupled.

There are indeed indications that different β-adrenergic receptors have different subcellular localizations. In rat ventricular myocytes, for example, β2-ARs are confined to caveolar spaces along with isoforms V/VI of adenylate cyclase, while β1-ARs are for the most part excluded from the same membrane microdomains [7], [8], [9], [10].

We have recently shown that pacemaker f-channels localize to membrane lipid rafts both in rabbit SAN myocytes and in HEK293 cells expressing HCN4, the major HCN channel isoform contributing to native f-channels [11]. The If current plays a crucial role in the generation and modulation of the slow diastolic depolarization and heart rate [12], [13], [14]. f-channels mediate autonomic modulation of rate by their sensitivity to cAMP, which binds directly to channels and increases the probability of channel opening via a depolarizing shift of the open probability curve [15]; the consequent current increase steepens the diastolic depolarization rate of action potentials and is thus responsible for rate acceleration [12]. We have recently shown that a form of familial sinus bradycardia is associated with a mutation of the HCN4 gene. The mutation causes the channels to activate at more negative voltages than wild-type channels; this effect, similar to the physiologic effect of vagal stimulation, is able to slow heart rate by decreasing the pacemaker current during diastolic depolarization [16].

We hypothesized that, as well as in ventricular myocytes, β2-ARs might be preferentially located in caveolae also in rabbit SAN myocytes, and might therefore colocalize with f-channels in pacemaker cells. If this were the case, adrenergic modulation of If current and of cardiac chronotropism would be expected to depend mostly on stimulation of β2-ARs. We therefore investigated the subcellular distribution of β-ARs and the efficiency of f-channel and rate modulation by β1-AR vs. β2-AR stimulation in rabbit SAN myocytes.

Section snippets

Isolation of rabbit SAN myocyctes

The procedures employed in this work conformed to guidelines for the care and use of laboratory animals as established by State (D.L. 116/1992) and European directives (86/609/CEE). Rabbit SAN myocytes were isolated as previously reported [17], stored at 4 °C in Tyrode solution (in mM: NaCl, 140; KCl, 5.4; CaCl2, 1.8; MgCl2, 1; d-glucose, 5.5; HEPES–NaOH, 5; pH 7.4) and used for the day.

Cholesterol depletion

Membrane cholesterol depletion in SAN myocytes was achieved by 2% methyl-β-cyclodextrin treatment (MβCD,

Results

Previous evidence has indicated that pacemaker f-channels of SAN cells localize to lipid raft-enriched fractions of the plasma membrane [11]. The available data however cannot distinguish between caveolar and non-caveolar lipid rafts. In order to discriminate caveolar from non-caveolar localization of f-channels, we immunoprecipitated proteins from a SAN extract (see Materials and methods) using either anti-caveolin 3 or anti-HCN4 primary antibodies and checked in the precipitated proteins for

Discussion

The role played by funny channels in the generation and autonomic modulation of heart rate is well established [12]. f-channels are directly activated by cAMP [15], whose free cytoplasmic concentration is set at any time by the opposing actions of adenylate cyclase and cAMP phosphodiesterase. Since the activity of adenylate cyclase, and hence the cAMP concentration, are increased by β-adrenergic and decreased by muscarinic stimulation, the cAMP/f-channel-dependent mechanism is well suited to

Acknowledgments

This work was supported by grants from the Ministero dell'Istruzione, Università e Ricerca (FIRB 2003) to DD.

References (35)

  • F.O. Levy et al.

    Efficacy of β1-adrenergic receptors is lower than that of β2-adrenergic receptors

    Proc. Natl. Acad. Sci. U. S. A.

    (1993)
  • A. Barbuti et al.

    Localization of pacemaker channels in lipid rafts regulates channel kinetics

    Circ. Res.

    (2004)
  • D. DiFrancesco

    Pacemaker mechanisms in cardiac tissue

    Annu. Rev. Physiol.

    (1993)
  • E.A. Accili et al.

    From funny current to HCN channels: 20 years of excitation

    News Physiol. Sci.

    (2002)
  • R.B. Robinson et al.

    Hyperpolarization-activated cation currents: from molecules to physiological function

    Annu. Rev. Physiol.

    (2003)
  • D. DiFrancesco et al.

    Direct activation of cardiac pacemaker channels by intracellular cyclic AMP

    Nature

    (1991)
  • R. Milanesi et al.

    Familial sinus bradycardia associated with a mutation in the cardiac pacemaker channel

    N. Engl. J. Med.

    (2006)
  • Cited by (87)

    • The funny current: Even funnier than 40 years ago. Uncanonical expression and roles of HCN/f channels all over the body

      2021, Progress in Biophysics and Molecular Biology
      Citation Excerpt :

      Of note, this decrease in HCN expression has been associated with a DCP-dependent decrease in the number of caveolae (and of caveolin-3 levels) and knockdown of caveolin-3 in control ICCs elicited a phenotype similar to that of DCP since it decreased both the HCN protein expression and the If current (Dong et al., 2016). This finding is in agreement with previous works demonstrating that caveolin-3 interacts with and modulates HCN function, and impairment of such interaction decreases the expression of channels (Barbuti et al, 2007, 2012). Several studies have elegantly shown that the If blocker ivabradine, a drug used in the clinic to reduce heart rate, also displays relevant effects on endothelial functions, oxidative stress, and lymphocytes migration (Bonadei et al., 2018; Dallapellegrina et al., 2020; Li et al., 2016; Walcher et al., 2010).

    • The funny current in genetically modified mice

      2021, Progress in Biophysics and Molecular Biology
    View all citing articles on Scopus
    View full text