Background:

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome caused by mutations in RYR2 (encoding ryanodine receptor 2, RyR2). Investigation into the pro-arrhythmic effects of these mutations have focused exclusively on cardiomyocytes. However, RyR2 is also present in neuronal tissue. Moreover, patients often present with clinical signs of autonomic nervous system (ANS) dysfunction, and treatment strategies frequently target the ANS.

Objective:

To assess whether RyR2 mutations affect the neurons that modulate cardiac function and/or impact on cardiac innervation, using an established CPVT mouse model (RyR2-R2474S).

Methods:

RNA sequencing, immunocytochemistry, live-cell fluorescence imaging, immunohistochemistry and liquid chromatography–mass spectrometry.

Results:

Analysis of published RNA sequencing datasets revealed that RyR2 is abundantly expressed in mouse stellate ganglia (SG; crucial autonomic modulators of cardiac function). Using immunocytochemistry, we confirm the presence of RyR2 protein in these peripheral, cardiac-modulating neurons. Functional investigation revealed a reduction of caffeine-induced calcium release in isolated CPVT SG neurons (Fura Red AM F/F0 CPVT 0.53±0.01 vs wild type (WT) 0.45±0.01; p<0.01), indicating the occurrence of intracellular calcium leakage. Moreover, cultured CPVT SG neurons showed increased neurite outgrowth (βIII tubulin/DAPI-positive area CPVT 6.9±0.6 vs WT 4.1±0.6 µm²; p<0.01). Immunohistochemical staining of mouse heart cryosections with tyrosine hydroxylase (TH) demonstrated a significant increase in the density of sympathetic neuron innervation in ventricular tissue of CPVT mice (TH-positive area CPVT 3.8±0.5 vs WT 1.8±0.4 µm²; p<0.01), and increased innervation heterogeneity (CPVT 5.4±1.3 vs WT 2.3±0.5 Δ µm²; p<0.05). Neurotransmitter quantification in ventricular tissue revealed reduced levels of epinephrine in CPVT hearts (CPVT 0.023±0.002 vs WT 0.041±0.005 nmol/g; p<0.01), elevated levels of the norepinephrine (NE) metabolite normetanephrine (NMN), and a consequent increase in the NMN/NE ratio, which is an index of NE turnover (CPVT 0.26±0.02 vs WT 0.16±0.01 nmol/g; p<0.05). Quantification of blood plasma neurotransmitter metabolites revealed an increase in the dopamine metabolite 3-MT (CPVT 2.4±0.3 vs WT 1.3±0.2 nmol/g; p<0.05).

Conclusion:

The neurons that modulate cardiac function abundantly express RyR2. The RyR2-R2474S mutation leads to significant alterations of stellate ganglia neuron calcium homeostasis, increased neurite outgrowth, sympathetic hyperinnervation and increased innervation heterogeneity of ventricular myocardium, and alterations of cardiac neurotransmitters and their metabolites. These functional and structural alterations of the ANS would have important consequences for cardiac function and arrhythmogenesis and may provide a novel blood biomarker for improved risk stratification and treatment design.