Cholecystokinin-A signaling regulates automaticity of pacemaker cardiomyocytes

Abstract

Aims: The behavior of pacemaker cardiomyocytes (PCs) in the sinoatrial node (SAN) is modulated by neurohormonal and paracrine factors, many of which signal through G-protein coupled receptors (GPCRs). The aims of the present study are to catalog GPCRs that are differentially expressed in the mammalian SAN and to define the acute physiological consequences of activating the cholecystokinin-A signaling system in isolated PCs. Methods and results: Using bulk and single cell RNA sequencing datasets, we identify a set of GPCRs that are differentially expressed between SAN and right atrial tissue, including several whose roles in PCs and in the SAN have not been thoroughly characterized. Focusing on one such GPCR, Cholecystokinin-A receptor (CCK_AR), we demonstrate expression of Cckar mRNA specifically in mouse PCs, and further demonstrate that subsets of SAN fibroblasts and neurons within the cardiac intrinsic nervous system express cholecystokinin, the ligand for CCK_AR. Using mouse models, we find that while baseline SAN function is not dramatically affected by loss of CCK_AR, the firing rate of individual PCs is slowed by exposure to sulfated cholecystokinin-8 (sCCK-8), the high affinity ligand for CCK_AR. The effect of sCCK-8 on firing rate is mediated by reduction in the rate of spontaneous phase 4 depolarization of PCs and is mitigated by activation of beta-adrenergic signaling. Conclusion: (1) PCs express many GPCRs whose specific roles in SAN function have not been characterized, (2) Activation of the cholecystokinin-A signaling pathway regulates PC automaticity.

Keywords: GPCR (G protein coupled receptor); cardiac nervous system; cholecystokinin; pacemaker cell automaticity; sinoatrial node.

FIGURE 1

Differential Expression of GPCRs in Mammalian Sinoatrial Node. Heatmap demonstrates differentially expressed GPCRs from a previously published dataset 1 with 3 biological replicates of right atrial (RA) cardiomyocytes and sinoatrial node (SAN) pacemaker cardiomyocytes. Color scale indicates z-score. Cckar is highlighted.

FIGURE 2

Cckar is Expressed in Mouse Cardiac Pacemaker Cells. (A) Read counts for Cckar from Bulk RNA sequencing of sinoatrial node (SAN) tissue and right atrial tissue isolated from 3 embryonic day 14.5 Hcn4-GFP BAC transgenic mouse embryos using laser capture microdissection (original dataset from Ref. [9]) A two-tailed t-test was used to assess for significance. ‘*’ denotes p < 0.05 (B) Read counts for Cckar from Bulk RNA sequencing of sorted Hcn4-GFP+ pacemaker cells and atrial cardiomyocytes from neonatal mouse hearts shown in the heatmap. A two-tailed t-test was used to assess for significance. ‘***’ denotes p < 0.001. (C) Quantitative PCR for Cckar mRNA in samples manually dissected from adult mouse SAN and right atrium, using GAPDH as an endogenous control (n = 3 biological replicates). A one-sample t-test was used to assess for significance since Cckar was not detected in the RA samples. “**” denotes p < 0.01 (D) Uniform Manifold Approximation Projection (UMAP) plot of leiden clustering analysis of single cell RNA sequencing data derived from 6 E16.5 mouse embryonic heart tissue samples taken from the SAN region demonstrated a pacemaker cell population (red circle, cluster 11), marked by co-expression of Hcn4 (E) and Isl1 (F), as well as several other identifiable cell types (original dataset from ref [12]). Cckar was differentially expressed in the pacemaker cluster (C11), as seen in the UMAP plot (G) and in the violin plot (H).

FIGURE 3

Expression of Cck in Neurons and Cardiac Fibroblasts. (A) Uniform Manifold Approximation and Projection (UMAP) plot of a single cell RNA sequencing dataset from neonatal sinoatrial node (SAN) (Ref [12]) demonstrates expression of Cck in a population of fibroblasts and in a cardiac neuron, as seen in the magnified images in panel (B). Adjacent serial sections from a neonatal Cck-Cre;ROSA ^mTmG mouse heart stained with acetylcholinesterase to mark the right atrial cardiac ganglion (C) or imaged with epifluorescence to visualize Td-tomato and GFP expression (D, E) demonstrate that GFP+ cells, representing Cck expressing cells, are detectable in neuronal cell bodies within the ganglia. Sectioning and epifluorescence visualization of the SAN Cck-Cre;ROSA ^mTmG showed few GFP+ cells (F) but as shown in (G) and (H), GFP+ axons could be observed coursing alongside Hcn4+ pacemaker cells (white arrowheads). Notably, GFP expression did not overlap with Hcn4 expression. A nearby section from the SAN (I) also revealed GFP+ cells with fibroblast morphologies in the SAN that did not express Hcn4 (J, K) – yellow arrowheads). “*” denotes the sinoatrial node artery.

FIGURE 4

Heart Rhythm in Cckar ^−/− Mice. (A) Electrocardiographic (ECG) tracings from awake unrestrained WT (top, black) and Cckar ^−/− (bottom, red) adult mice. (B) Diurnal heart rate variation among 7 WT and 11 Cckar ^−/− adult mice implanted with ECG transmitters. Each point shows mean±SEM. (C) Activity counts for 7 WT and 11 Cckar ^−/− adult mice implanted with transmitters. Each point shows mean±SEM. (D) Heart rate before (baseline) and after atropine and propranolol injection (A/P) in 4 WT (black) and 7 Cckar ^−/− mice (red) under anesthesia. Error bars denote standard deviation. (E) Heart rate before (baseline) and after isoproterenol injection (Max) in 5 WT (black) and 6 Cckar ^−/− mice (red). Error bars denote standard deviation. A Mann-Whitney test was used to test for significance with p < 0.05 deemed significant. ‘ns’ denotes non-significant. (F) Averaged histograms of heart rate during low activity period (left) and high activity period (right) in 7 WT (black) and 11 Cckar ^−/− mice (red) with 10 beat per minute bins.

FIGURE 5

sCCK-8 Reduces Spontaneous Firing Rate of Pacemaker Cells. (A) Spontaneous firing rate for an isolated adult WT pacemaker cell (PC) before, during perfusion of 50 pM sCCK-8, and after washout. (B) Superimposed spontaneous action potentials from an adult WT pacemaker cell firing spontaneously at baseline (solid line) and after perfusion with 5 nM sCCK-8 (dashed line). (C) Spontaneous firing rate for an isolated adult Cckar ^−/− pacemaker cell before and during perfusion with 5 nM sCCK-8. (D) Superimposed spontaneous action potentials from an adult Cckar ^−/− pacemaker cell at baseline (solid line) and during perfusion with 5 nM sCCK-8 (dashed line). (E) Change in spontaneous firing rate of WT PCs at baseline (BL) and after perfusion of 50 nM sCCK-8 (n = 5, left panel) or 5 nM sCCK-8 (n = 8, middle panel) compared with Cckar ^−/− PCs exposed to 5 nM sCCK-8 (n = 5, right panel). Statistical comparison was made with a paired t-test with the indicated p values (“NS” denotes non-significant). (F) Percent changein spontaneous firing rate for isolated WT pacemaker cells 1 min after perfusion of 10 pM (n = 3), 25 pM (n = 4), 50 pM (n = 5), 1 nM (n = 7), or 5 nM (n = 8) sCCK-8. Error bars denote standard deviation. Percent change was calculated as 100 x [(R _BL–R _sCCK-8)/R _BL], where ‘R _BL’ denotes rate at baseline and ‘R _sCCK-8’ denotes rate after perfusion of sCCK-8.

FIGURE 6

sCCK-8 Reduces Early Spontaneous Depolarization in Pacemaker Cardiomyocytes. (A). Definition of action potential parameters. The top tracing, V _m (t), shows spontaneous action potentials (APs) from an isolated adult pacemaker cell and the bottom tracing shows the first derivative (dV/dt). The minimum diastolic potential (MDP) was defined as the minimum potential recorded between action potentials; Take off potential (TOP) was defined as the membrane potential at which dV/dt reach 10% of its maximum value; and early diastolic depolarization rate (EDDR) was defined as the average value of dV/dt over the shaded area in the bottom panel (from 10% to 50% of the MDP-to-TOP interval). (B) AP parameters including Firing Rate, Amplitude, APD₅₀, MDP, TOP, and EDDR were measured under baseline conditions in WT and Cckar ^−/− PCs (n = 8 and n = 5, respectively). Measurements of these parameters were also compared before and 3 min after perfusion with 5 nM sCCK-8 for (C) WT and (D) Cckar ^−/− PCs. Statistical comparisons between baseline values and between pre- and post-sCCK-8 values were made with a Mann-Whitney test (“ns” denotes non-significant, “*” denotes p < 0.05).