Glucagon-like peptide-1 increases heart rate by a direct action on the sinus node

Abstract

Aims: Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) are increasingly used to treat type 2 diabetes and obesity. Albeit cardiovascular outcomes generally improve, treatment with GLP-1 RAs is associated with increased heart rate, the mechanism of which is unclear.

Methods and results: We employed a large animal model, the female landrace pig, and used multiple in vivo and ex vivo approaches including pharmacological challenges, electrophysiology, and high-resolution mass spectrometry to explore how GLP-1 elicits an increase in heart rate. In anaesthetized pigs, neither cervical vagotomy, adrenergic blockers (alpha, beta, or combined alpha-beta blockade), ganglionic blockade (hexamethonium), nor inhibition of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (ivabradine) abolished the marked chronotropic effect of GLP-1. GLP-1 administration to isolated perfused pig hearts also increased heart rate, which was abolished by GLP-1 receptor blockade. Electrophysiological characterization of GLP-1 effects in vivo and in isolated perfused hearts localized electrical modulation to the atria and conduction system. In isolated sinus nodes, GLP-1 administration shortened the action potential cycle length of pacemaker cells and shifted the site of earliest activation. The effect was independent of HCN blockade. Collectively, these data support a direct effect of GLP-1 on GLP-1 receptors within the heart. Consistently, single nucleus RNA sequencing showed GLP-1 receptor expression in porcine pacemaker cells. Quantitative phosphoproteomics analyses of sinus node samples revealed that GLP-1 administration leads to phosphorylation changes of calcium cycling proteins of the sarcoplasmic reticulum, known to regulate heart rate.

Conclusion: GLP-1 has direct chronotropic effects on the heart mediated by GLP-1 receptors in pacemaker cells of the sinus node, inducing changes in action potential morphology and the leading pacemaker site through a calcium signalling response characterized by PKA-dependent phosphorylation of Ca2+ cycling proteins involved in pacemaking. Targeting the pacemaker calcium clock may be a strategy to lower heart rate in people treated with GLP-1 RAs.

Keywords: Calcium clock; Calcium signalling; Chronotropic; GLP-1; Glucagon-like peptide 1; Heart rate; Pacemaker; Sinus node.

Graphical abstract

Figure 1

GLP-1 increases heart rate in the anaesthetized pig, but its metabolite GLP-1 (9-36) does not. (A–C) GLP-1 was infused at 10 pmol/kg/min for two consecutive 45 min periods with two 45 min washout periods (n = 8). Heart rate increased similarly in both infusion periods and was followed by a return to near-baseline during washout. (D–F) GLP-1 was infused at 15 pmol/kg/min for 1 h as a control, followed by a bolus injection of the DPP-4 inhibitor Val-Pyr (300 µmol/kg). Subsequently, an hour infusion of GLP-1 at 15 pmol/kg/min either preceded or followed by an hour infusion of the metabolite GLP-1 (9-36) at 10 pmol/kg/min was performed (n = 4). GLP-1 increased heart rate consistently, whereas its metabolite GLP-1 (9-36) did not. Tested with two-way repeated measures ANOVA with Bonferroni multiple comparisons testing (B and E), paired t-test (C), and repeated measures one-way ANOVA (F); *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 2

The increase in heart rate induced by GLP-1 is independent of alpha- and beta-adrenergic stimulation. GLP-1 was infused at 15 pmol/kg/min for two 60 min periods. Before the second infusion period, bolus injections of either propranolol (1 mg/kg; A and B), phentolamine (1 + 0.5 mg/kg; C and D), or a combination of propranolol and phentolamine (E and F) were given to block the beta-adrenergic receptors (n = 5), alpha-adrenergic receptors (n = 6), or both (n = 5), respectively. Heart rate increased with subsequent GLP-1 infusion regardless of the pre-treatment with any of the adrenergic blockers. The GLP-1-induced increase in heart rate after the interventions was comparable between interventions (G). Tested with two-way repeated measures ANOVA with Bonferroni multiple comparisons testing (B, D, and F) and one-way ANOVA with Dunnett’s multiple comparisons testing (G); **P < 0.01, ****P < 0.0001.

Figure 3

The increase in heart rate induced by GLP-1 is independent of the autonomic nervous system or the HCN channel. (A–C) GLP-1 was infused at 10 pmol/kg/min for 45 min as a control. Subsequently cervical vagotomy was performed, followed by a second infusion of GLP-1. This was followed by a bolus injection of the HCN channel blocker ivabradine (0.35 mg/kg) and a third infusion of GLP-1 (n = 6). All infusions of GLP-1 increased heart rate. (D–F) GLP-1 was infused at 12.5 pmol/kg/min for 45 min as a control, followed by two bolus injections of the ganglionic blocker hexamethonium (8 mg/kg, administered twice), and another 45 min infusion of GLP-1 (n = 5). Even after ganglionic block, heart rate increased during the GLP-1 infusion. Tested with two-way repeated measures ANOVA with Bonferroni multiple comparisons testing (B and E), repeated measures one-way ANOVA with Dunnett’s multiple comparisons testing (C), and paired t-test (F); *P < 0.05, ****P < 0.0001.

Figure 4

GLP-1 increases heart rate in the isolated perfused pig heart, which is dependent on the GLP-1 receptor, and increases AV nodal conduction. (A and B) Isolated perfused hearts were exposed to consecutive boluses of GLP-1 (2.2 nmol), GLP-1 (4.4 nmol), the GLP-1 receptor blocker exendin 9-39 (100 nmol), and GLP-1 (4.4 nmol) (n = 6). Heart rate increased with both doses of GLP-1, but GLP-1 no longer affected heart rate after the bolus of the GLP-1 receptor blocker. (C) To evaluate other electrophysiological effects of GLP-1, ECG was measured in vivo in the anaesthetized pig during atrial pacing (400 ms basic cycle length) before and after 30 min infusion of GLP-1 (10 pmol/kg/min) (n = 6). PQ interval shortened, but the other parameters were not affected, indicating increased AV nodal conduction. Tested with two-way repeated measures ANOVA with Bonferroni multiple comparisons testing (B) and with paired t-test (C); *P < 0.05.

Figure 5

GLP-1 decreases cycle length and shifts the site of earliest activation in the isolated superfused pig sinus node independent of HCN channel block. (A–H) Isolated superfused sinus node tissues were exposed to GLP-1 (1 nM), ivabradine (1 µM), or both compounds combined. (A) Effect of GLP-1 on action potential cycle length over time (n = 7). (B) Quantification of the effect of GLP-1 on sinus node action potential cycle length (n = 7). (C) Representative monophasic action potential traces recorded at baseline (black) or after GLP-1 superfusion (orange). Note differences in rate and diastolic depolarization. (D) Diastolic depolarization (dV/dt) at baseline and after GLP-1 superfusion (n = 4), identifying a steeper diastolic depolarization during GLP-1 exposure (for method of analysis, see Supplementary material online, Figure S7B). (E) GLP-1 shortened sinus node cycle length in the presence of HCN channel blocker ivabradine (n = 7). (F) Illustration showing mapping electrode array placement on the sinus node. (G) High-density maps of activation times were measured across the array (n = 4). Representative maps at baseline (left) and after GLP-1 superfusion (right) are shown. Note the shift in site of earliest activation towards the crista terminalis. (H) Representative high-resolution maps of activation times measured at baseline (left) and in the presence of ivabradine (middle) and ivabradine + GLP-1 (right). Ivabradine did not affect the shift in site of earliest activation by GLP-1. Statistical tests: paired t-test (B and D) and a Friedman test with Dunn’s multiple comparison testing (E); **P < 0.01 and ***P < 0.001. CT, crista terminalis; ICV, intercaval vein; IVC, inferior vena cava; LAT, local activation time; RA, right atrium; SAN, sinoatrial node; SEA, site of earliest activation; SVC, superior vena cava.

Figure 6

GLP-1 receptor localizes to pacemaker cells in the pig sinus node. (A–D) Neighbour embedding of snRNAseq of isolated nuclei from porcine sinoatrial tissue (n = 2), which predominantly contains cardiomyocytes and fibroblasts. Expression of GLP-1 receptor (B), HCN4 (C), and CACNA1D (D) in cardiomyocytes. snRNAseq, single nucleus RNA sequencing.

Figure 7

Phosphoproteomics analysis suggests modulation of cAMP/Ca²⁺ signalling pathways in pig sinus node upon GLP-1 infusion. (A) Protocol of infusion of GLP-1 or vehicle prior to tissue collection (n = 4). (B) Workflow for phosphoproteomic experiments of pig sinus node tissues. Proteins were extracted from anatomical sections of sinus node tissue, tryptic digests were multiplexed with TMTpro reagents, and phosphopeptides were enriched using TiO₂ beads. The enriched peptides were fractionated at high pH and analysed by LC-MS/MS. (C) Heatmap of Pearson correlation coefficients for all measured phosphorylated peptide intensities across all samples. (D) Principal component analysis (PCA) of phosphorylated peptide intensities displaying separation of the samples according to treatment along the third component. (E) Gene set enrichment analysis (GSEA) using phosphorylation fold changes as input identified enriched pathways consequent to GLP-1 infusion. For multi-phosphorylated proteins, the greatest phosphorylation fold change was used. Enriched pathways are indicated in the plot; for all pathways, the adjusted P-value was below 1e⁻⁵. The gene set size refers to the number of genes covered in our data set of the respective pathways, and the colour indicates the normalized enrichment score (NES). All proteins measured in our dataset for the pathways highlighted in bold are visualized in the network in panel F. (F) Gene–KEGG pathway network (cnetplot) displaying the proteins contributing to the overrepresentation of the four pathways shown. Nodes are coloured by the greatest phosphorylation event fold change measured for the protein. Proteins known to be involved in heart rate regulation of the sinus node are encircled by a dashed line. (G) Western blot analysis of sinus node samples from pigs infused with GLP1 (n = 4) or vehicle (n = 4). Representative data of phospholamban phosphorylated at amino acid serine 16 [p-PLN(S16)] and loading control (GAPDH). Upper panel: Comparison of ratios of p-PLN(S16)/total PLN intensities. Lower panel: one-way Mann–Whitney U test. AU, arbitrary units.