Alterations in autonomic function are known to occur in cardiac conditions including sudden cardiac death. Cardiac stimulation via sympathetic neurons can potentially trigger arrhythmias. Dissecting direct neural-cardiac interactions at the cellular level is technically challenging and understudied due to the lack of experimental model systems and methodologies. Here we demonstrate the utility of optical interrogation of sympathetic neurons and their effects on macroscopic cardiac monolayer dynamics to address research targets such as the effects of adrenergic stimulation via the release of neurotransmitters, the effect of neuronal numbers on cardiac wave behaviour and the applicability of optogenetics in mechanistic in vitro studies. We combine photo-uncaging or optogenetic neural stimulation with imaging of cardiac monolayers to measure electrical activity in an automated fashion, illustrating the power and high throughput capability of such interrogations. The methods described highlight the challenges and benefits of co-cultures as experimental model systems.

Aims: Macroscopic cultures of cardiac myocytes are a useful model to study arrhythmogenic mechanisms, but the absence of sympathetic stimulation limits our ability to study one of the main triggers of arrhythmia. In this study, we grow myocytes and cardiac stellate sympathetic neurons (CSSN) in confluent monolayer's to investigate how neurons modulate propagating wave patterns. Methods: Cardiac monolayer's were prepared by plating petri-dishes with neonatal myocytes from Sprague Dawley rat ventricle cells. After 24 hours, co-cultures were produced by adding CSSN isolated from litter mates. Co-cultures were imaged using state-of-the-art dye free optical mapping techniques. Results: SCCN form structural (evidenced by scanning electron microscopy and conventional microscopy) and functional connections with myocytes. We demonstrate that co-cultures are able to support target, spiral and wavelet patterns. Basal unstimulated wave speed of co-cultures was significantly faster than myocyte monocultures (+30%), and the number of co-cultures displaying multiple wave fronts was lower than that found in monocultures (P < 0.05). 10 μM nicotine stimulation of co-cultures showed an immediate increase in beat rate and in cases where the cardiac cultures are in target (analogous to sinus rhythm) acute addition of nicotine may initiate a re-entrant activity or can cause the breakup of the target wave. Conclusion: CSSN affect cardiac conduction velocity and wave patterns in in-vitro co-cultures. This model system may be used for the quantitative investigation of the influence of neural activation on cardiac activity during arrhythmias.

Hypertension is associated with over-activity of cardiac sympathetic neurons resulting in greater transmitter release that is coupled to abnormally high intracellular Ca2+ transients in the spontaneously hypertensive rat (SHR). These neurons also express lower levels of nNOS, the beta1 subunit of soluble guanylate cyclase (sGC), and cGMP compared to normotensive control WKY rats. We tested the hypothesis that dysregulation of the voltage gated Ca2+ channel could provide the trigger for the calcium phenotype seen in pro-hypertensive SHRs that is linked impaired cyclic nucleotide signalling. Cardiac sympathetic stellate ganglions were acutely isolated from 4 week, pro-hypertensive SHR and WKY rats. Whole cell voltage clamp (10 mV steps from -50 to +50) was performed at 37°. The whole cell maximum Ca2+ conductance (in nS/pF) in the SHR was significantly larger when compared to the normotensive WKY (2.48 ± 0.11 SEM, n = 10 vs 2.09 ± 0.13 SEM, n = 10, p = 0.037). Application of Conotoxin GVIA reduced the max conductance by approximately 83% and 78% (0.40 ± 0.05 SEM, n = 6 and 0.46 ± 0.04 SEM, n = 3, p < 0.0001), suggesting a major role for the N-type Ca2+ channel. Increasing the cGMP levels (8-bromo-cGMP) did not affect the WKY (1.96 ± 0.17 SEM, n = 7, p = 0.54), but normalised the SHR conductance to levels seen in WKYs (2.02 ± 0.13 SEM, n = 7, p = 0.0184, all unpaired t-test). There was no change in voltage dependency of the conductances or the half maximum activation (mV) between the groups. In conclusion, an enhanced N-Type Ca2+ channel conductance suggests a channelopathy may be an early hallmark of sympathetic impairment in the SHR that is coupled to impaired cyclic nucleotide regulation.