Introduction

While extrinsic autonomic input regulates atrioventricular (AV) conduction, the functional role of intrinsic non-neuronal catecholaminergic sources is incompletely understood. Dopamine β-hydroxylase-expressing cardiomyocytes (Dbh-CMs) were abundantly identified within the AV node however, their contribution to nodal excitability remains untested (Sun et al., 2023). Here, we use a tamoxifen (TAM)-inducible cardiomyocyte-specific Dbh conditional knockout line (Dbh-cKO) to bypass developmental compensation and assess whether intrinsic Dbh-CMs influence AV nodal conduction in the adult heart—bridging structural discovery with functional insight.

Aims

To functionally characterise intrinsic catecholaminergic cardiomyocyte influence on AV nodal conduction reserve.

Methodology

Dbh-cKO mice (C57BL/6J mixed-sex) were generated by crossing Dbh^+/Flox mice with MHC-CreERT2 (A1cf^{Tg(Myh6-cre/Esr1*)1Jmk}) mice. Four experimental groups were defined: (1) Dbh-cKO (Cre⁺Flox⁺TAM⁺); (2) Cre⁺ vehicle control (Cre⁺Flox⁺TAM^–); (3) floxed TAM⁺ control (Cre^–Flox⁺TAM⁺); and (4) floxed baseline control (Cre^–Flox⁺TAM^–). Six- to nine-week-old mice received TAM administration via oral gavage (100 mg/kg) or intraperitoneal (50 mg/kg or 35 mg/kg) for five consecutive days. Mice were monitored using echocardiography under general anaesthesia and permitted one-week recovery. Mice treated with 100 mg/kg experienced complete lethality and were excluded from downstream analysis. Electrophysiological experiments were performed during sixteen- to eighteen-weeks of age. Mice were intraperitoneally anaesthetised with 0.3 mL of 1.2% 2,2,2-tribromoethanol and 0.3 mL of 2500 U/mL heparin to prevent clot formation. After five minutes, mice were humanely sacrificed by cervical dislocation in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. Hearts were excised and retrogradely perfused at physiological temperature via Langendorff apparatus with oxygenated Krebs-Henseleit buffer. Two pacing protocols were employed: (1) S1S1—twenty consecutive pulses at 100 ms cycle length with 5 ms decrements until 2:1 AV conduction block (Wenckebach point, WBP-AV); and (2) S1S2—a train of eight S1 stimuli followed by a single premature S2 stimulus with progressively shortened S1S2 intervals to determine the AV nodal effective refractory period (AVNERP).

Results

Statistical analyses were conducted in GraphPad Prism v10.5.0 using Shapiro-Wilk tests for distributional normality and Kruskal-Wallis tests for non-parametric comparisons. Data presented as median ± interquartile range. Median RR-intervals were broadly comparable: Dbh-cKO (n = 3) 213.4 ± 34.0 ms; Cre⁺Flox⁺TAM^– (n = 2) 206.0 ± 16.0 ms; Cre^–Flox⁺TAM⁺ (n = 3) 179.5 ± 45.5 ms; and Cre^–Flox⁺TAM^– (n = 2) 243.3 ± 66.8 ms (P = 0.5783). AVNERP demonstrated no significant differences: Dbh-cKO (n = 3) 80.0 ± 10.5 ms; Cre⁺Flox⁺TAM^– (n = 1); Cre^–Flox⁺TAM⁺ (n = 3) 86.0 ± 11.5 ms; Cre^–Flox⁺TAM^– (n = 2) 79.0 ± 7.0 ms (P = 0.8163). WBP-AV were comparable: Dbh-cKO (n = 2) 100.0 ± 5.0 ms; Cre^–Flox⁺TAM⁺ (n = 3) 100.0 ± 0.0 ms; and Cre^–Flox⁺TAM^– (n = 2) 97.5 ± 2.5 ms (P = 0.4286). One Dbh-cKO mouse exhibited non-sustained ventricular tachycardia following S1S2 pacing at a 77 ms coupling interval, suggestive of provocation-induced conduction instability.

Conclusions

These preliminary findings provide the first functional assessment of AV nodal conduction following adult-onset cardiomyocyte-specific deletion of Dbh. While no statistically significant intergroup differences in basal conduction metrics were observed, the emergence of provocation-induced arrhythmia in a Dbh-cKO heart suggests a potential role for Dbh-CMs in preserving AV nodal function.