Rad-GTPase contributes to heart rate via L-type calcium channel regulation

https://doi.org/10.1016/j.yjmcc.2021.01.005Get rights and content

Highlights

  • SAN cardiomyocytes express Rad.

  • Rad deletion in SAN cardiomyocytes increases ICa,L consistent with Rad's role as an endogenous LTCC governor.

  • Rad regulates intrinsic HR consistent with βAR modulated ICa,L regulation.

  • HR during the sleep cycle is selectively elevated by Rad deletion.

  • Rad – LTCC association may be a useful target for future therapeutics to treat symptomatic bradycardia.

Abstract

Sinoatrial node cardiomyocytes (SANcm) possess automatic, rhythmic electrical activity. SAN rate is influenced by autonomic nervous system input, including sympathetic nerve increases of heart rate (HR) via activation of β-adrenergic receptor signaling cascade (β-AR). L-type calcium channel (LTCC) activity contributes to membrane depolarization and is a central target of β-AR signaling. Recent studies revealed that the small G-protein Rad plays a central role in β-adrenergic receptor directed modulation of LTCC. These studies have identified a conserved mechanism in which β-AR stimulation results in PKA-dependent Rad phosphorylation: depletion of Rad from the LTCC complex, which is proposed to relieve the constitutive inhibition of CaV1.2 imposed by Rad association. Here, using a transgenic mouse model permitting conditional cardiomyocyte selective Rad ablation, we examine the contribution of Rad to the control of SANcm LTCC current (ICa,L) and sinus rhythm. Single cell analysis from a recent published database indicates that Rad is expressed in SANcm, and we show that SANcm ICa,L was significantly increased in dispersed SANcm following Rad silencing compared to those from CTRL hearts. Moreover, cRadKO SANcm ICa,L was not further increased with β-AR agonists. We also evaluated heart rhythm in vivo using radiotelemetered ECG recordings in ambulating mice. In vivo, intrinsic HR is significantly elevated in cRadKO. During the sleep phase cRadKO also show elevated HR, and during the active phase there is no significant difference. Rad-deletion had no significant effect on heart rate variability. These results are consistent with Rad governing LTCC function under relatively low sympathetic drive conditions to contribute to slower HR during the diurnal sleep phase HR. In the absence of Rad, the tonic modulated SANcm ICa,L promotes elevated sinus HR. Future novel therapeutics for bradycardia targeting Rad – LTCC can thus elevate HR while retaining βAR responsiveness.

Introduction

Spontaneous electrical activity of the sinoatrial node (SAN) initiates cardiac rhythm and is a critical determinant of heart rate (HR). Cardiomyocytes of the SAN (SANcm) are highly specialized cells that generate automatic activity and form the cellular basis for cardiac pacemaking. SANcm exhibit a spontaneous diastolic depolarization. The early phase of diastolic depolarization is dominated by inward cationic funny current (If [1] carried by HCN4 [2]). The latter phase of diastolic depolarization is driven by voltage-gated L-type calcium channels (LTCC, with the main pore forming subunit, CaV1.2 or CaV1.3) [3]. To a large extent, If and the LTCC complex determine the ‘membrane clock’ that interacts with the calcium clock in the SAN [4,5]. The inter-relation of the membrane clock and calcium clock is underscored by studies of the genetic model of loss of CaV1.3 impairing sarcoplasmic reticulum Ca2+-release [6], and by computational studies linking feedback loops among sarcoplasmic Ca2+-release, NCX, and ICa,L [7]. The ability of ICa,L to simultaneously contribute to membrane- and calcium-clocks suggests that targeted regulation of the LTCC is a potentially powerful approach to control HR. Although cellular and molecular mechanisms that contribute to pacemaker activity are well established [8], bradyarrhythmia treatment options are limited mainly to electronic devices that are insensitive to autonomic nervous system regulation. Experimentally, nifedipine (an LTCC antagonist) has a negative chronotropic effect on the leading pacemaker site of the SAN [9]; similarly, genetic knockout of CaV1.3 induces bradycardia [10]. A fundamental premise of this study is that modulation of LTCC activity conferred by Rad provides a central nodal mechanism for sympathetic nervous system (SNS) control of SAN rhythm.

The LTCC is a heteromultimeric protein complex [11]. The pore-forming subunit of the LTCC in SANcm is carried by CaV1.3 [10,12] and CaV1.2 [3] channels. Key auxiliary subunits of the LTCC complex include CaVβ2, α2δ, and CaM [11]. Recent work (13)highlights the importance of RGK proteins [14] as regulators of ICa,L via association with the β subunits [[15], [16], [17], [18]]. Rad is a constituent of the LTCC complex in the myocardium [18,19]. Sympathetic nervous system drive acts via β-AR signaling cascades, resulting in the activation of PKA which in turn modulates LTCC activity [20]. β-AR modulation is maintained in transgenic mouse models expressing mutant CaV1.2 and CaVβ that cannot be phosphorylated by PKA [13,21], suggesting that a non-channel PKA target is central to channel modulation. Using proximity biotinylation to analyze β-adrenergic-dependent changes within the LTCC complex, Rad was recently shown to be depleted from the LTCC complex following acute β-AR stimulation. Rad depletion from the LTCC relieves the constitutive inhibition imposed by Rad association [15,17,18,22]. In keeping with its presumptive role as an endogenous inhibitor of LTCC function, we showed that LTCC activity is increased In Rad-null mice (Rad−/−) with properties mirroring β-AR modulation of ICa,L [17]. ECG telemetry showed that Rad−/− mice have a complex phenotype that includes differential vascular and inflammatory properties [16]. However, this model has multi-organ involvement [16,23] and possible development-related effects that potentially confound analysis of heart rhythm modification originating from channel modulation in cardiomyocytes. To circumvent these effects, we recently developed an inducible myocardium-restricted Rad knockout (cRadKO) mouse [15]. Cardiomyocyte selective Rad deletion was shown to phenocopy β-AR modulated LTCC properties, increasing basal ventricular contractile function [15]. Using this same conditional KO model, we now test the hypothesis that myocardial Rad-knockout will modulate sinus heart rate. Here we show that cRadKO mice exhibit an elevated intrinsic heart rate, and elevated sleep phase heart rate. Mechanistically, Rad regulation of HR appears to be driven by modulated ICa,L following Rad loss in SANcm. This work reveals Rad – LTCC interactions as a novel target for future therapeutics for symptomatic bradycardia.

Section snippets

Materials and methods

All experimental procedures and protocols were approved by the Animal Care and Use Committee of the University of Kentucky and conformed to the National Institute of Health “Guide for the Care and Use of Laboratory Animals.”

SANcm express Rad

In the cRadKO mouse model cre-recombinase is driven by a myosin heavy chain 6 promoter limiting expression to all cardiomyocytes (reference [24], and personal communication Jeff Molkentin). Following tamoxifen-mediated cre-recombination Rad protein levels are not detected from the whole hearts of cRadKO [15] mice, suggesting cardiomyocyte-restricted expression of Rad in the heart. Similarly, single nucleus RNAseq (snRNAseq) analysis of the mouse SAN showed that RRAD transcript is enriched

Discussion

We showed previously that Rad-deficiency confers a β-AR –modulated phenotype on basal ICa,L without structural or functional remodeling of the heart [37]. The main findings of this study are that in SANcm, the deletion of Rad results in elevated ICa,L with properties approximating SNS modulated ICa,L. Second, the absence of Rad elevates intrinsic HR. Third, HR in ambulatory mice is unchanged during the active phase, but is significantly elevated during the sleep/resting phase. Early studies

Funding

This research was funded by National Institutes of Health (NIH), NHLBI R01 HL131782 (DAA, JS), Department of Defense, United States W81XWH-20-1-0418 (DAA, JS), AHA pre-doctoral fellowship to BMA (19PRE34380909), and Institutional Development Award (IDeA) from NIH NIGMS P30 GM127211.

Declaration of competing interest

We, the authors have no competing interests.

Acknowledgements

The authors would like to thank Andrea Sebastian, Wendy Katz, and Tanya Seward for outstanding technical support including heart dispersal, histology, and ECG telemetry surgical implantations. We are grateful for help from Matthew Hazzard, University of Kentucky Medical Illustrations support team.

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