Hypoxia Alters Sympathetic Terminal Function and Innervation Patterns in the Rat Left Atrium
Authors: Saleeb-Mousa James , Ahmed Shadman , Nathanael Demitris , Kalla Manish , Herring Neil , Coney Andrew , Brain Keith 
Published: May 2024

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia (Hindricks et al., 2021). Emerging evidence suggests that the association of AF with conditions such as obstructive sleep apnoea, COPD and heart failure may be partly attributable to atrial autonomic remodelling in the setting of chronic hypoxia (CH) and chronic-intermittent hypoxia (CIH) (Saleeb-Mousa et al. 2023). In particular, hypoxic remodelling of the atrial autonomic system is associated with a shift towards sympathetic predominance. We hypothesised that sympathetic remodelling in CH and CIH is characterised by altered regulation of prejunctional terminal function. Procedures were performed in accordance with the UK Animals (Scientific Procedures) Act 1986. A novel confocal fluorescence assay was employed to dynamically monitor single-terminal noradrenaline handling in vitro (Cao et al. 2020). This technique utilises the neurotransmitter transporter uptake assay (NTUA, Molecular Devices), a fluorescent monoamine mimetic which accumulates in sympathetic terminals by entering via the noradrenaline transporter (NAT). Experiments were performed in atrial tissues derived from male adult Wistar rats (200-300g) kept in normoxia (N; n=24), CH (FIO2=0.1-0.12, 9-10 days; n=9) and CIH (FIO2=0.06-0.21, 15 cycles hour−1, 8 hours day−1, 21-24 days; n=4) using a hypoxia chamber. Hearts were excised under non-recovery terminal inhalation isoflurane (3-5% in O2, flow rate 1.5L min−1) with death by cervical dislocation. Tissues were dissected into left atrial appendage (LAA), left atrial posterior wall (LAPW), right atrium (RA), right atrial appendage (RAA) and pulmonary veins. Sections were pinned flat and transferred to a confocal microscope. Tissues were superfused with NTUA and images were captured over 15 minutes to allow for quantification of fluorescence uptake. To determine innervation density, additional imaging of NTUA-loaded tissues was performed at low magnification in each region. Further structural images were captured in an additional group of rats (nN=2, nCH=6) using an anterograde tracing technique. Tissues were excised with sympathetic chains intact, and a dextran-conjugated fluorophore (Dextran Texas Red, Invitrogen) was loaded over 21 hours into either the left or right stellate ganglion. Tissues were subsequently dissected and confocal imaging was performed to visualise atrial sympathetic fibres. Analysis of fluorescence uptake in all tissues revealed a two-phase association characterized by an initial 6-minute linear uptake followed by a plateau phase. In preliminary CIH experiments, no significant changes in NTUA uptake were observed (p>0.05). In CH tissues, linear uptake was not significantly different compared with N (p=0.09). CH was associated with an increase in maximum fluorescence (p=0.0004); however, a significant effect was only observed in the LAA (p=0.008). CH tissues also demonstrated a sympathetic hyperinnervation (p<0.0001), with post-hoc testing revealing a significant effect in the LAPW (p=0.0007). Stellate tracing in all animals revealed diffuse innervation of the LAPW, but an absence of fluorescence in the RA, LAA and RAA. Overall, our data demonstrate regionally dependent functional and structural adaptations of the cardiac sympathetic system to hypoxia. Further work should aim to determine the contribution of atrial autonomic heterogeneity to arrhythmogenesis in conditions associated with CH. This research was funded by the Kennedy Trust, grant number KENN 20 21 04-Saleeb-Mousa, and the British Heart Foundation, grant number FS/PhD/20/29093. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.