Dysregulation of Cardiac cAMP in Nicotine Stimulated Sympathetic Neuronal‐Myocyte Co‐Cultures from Hypertensive Rats: Are Sympathetic Neurons the Primary Driver of Autonomic Hypertension?
Authors: Larsen Hege E , Lefkimmiatis Konstantinos , Paterson David J 
Published: November 2023

Hypertension is associated with marked sympathetic over‐activity leading to end organ hyper responsiveness. The autonomic dysfunction manifests itself in significantly larger neuronal N‐type Ca2+ currents and greater intracellular Ca2+ transients, resulting in greater noradrenaline (NA) release into the cardiac synaptic cleft (2). There is also a reduced reuptake of NA due to reduced functionality and expression of the NA transporter (NET). At the end organ, the sinoatrial node cells show beta adrenergic hyper responsiveness associated with up regulation of the L‐type Ca2+ current (1) leading to significantly increased heart rate responses to adrenergic stimulation (2).

We tested whether activation of coupled cardiac sympathetic neurons onto beating myocytes resulted in a distinctive cellular phenotype in co‐cultures from normotensive and pro‐hypertensive rats. The neurons were physiologically stimulated using nicotine to induce native NA release, before the resulting beta adrenergic response was measured in the myocytes using a FRET cAMP sensor. Cardiac sympathetic neurons and neonatal beating ventricular myocytes from pre‐hypertensive, spontaneously hypertensive rats (SHR) and their normotensive controls (WKY) were isolated using enzymatic digestion before and plated together to form co‐cultures. Cells were grown for 5 days to allow synapse formation before the myocytes were selectively infected with the FRET cAMP sensor.

Application of Nicotine rapidly increased cAMP levels in the myocytes that returned to baseline following NA removal from the synaptic cleft. A significantly larger beta adrenergic response was seen in the SHR co‐cultures when compared to the WKY at 1 μM (21.82 ± 2.76, n=29 vs 2.12 ± 0.63, n=24) and 10 μM Nicotine (and 16.26 ± 2.40, n=28 vs 7.17 ± 1.53, n=29; all % FRET change from baseline, p<0.0001). The SHR myocyte‐alone cultures were found to be over‐responsive to Isoprenaline concentrations > 3nM when compared to WKY myocytes (40–60%). Attempts were made to mimic the sympathetic dysfunction in the healthy myocytes by cross culturing hypertensive SHR neurons onto normotensive WKY myocytes. These cross cultures increased the cAMP levels in the WKY myocytes to that seen in the SHR myocytes suggesting the sympathetic impairment seen in hypertension is driven primarily, but not solely by the cardiac sympathetic neurons during nicotinic activation. The action of nicotine was blocked by the acetylcholine receptor antagonist Mechamylamine. Nicotine had no effects on myocytes cultured alone (n=33) and all co‐culture responses were blocked by the beta blocker metoprolol (10 μM, n=27) in all cells.

Taken together these results suggest that the cardiac sympathetic nerves have a more dominant role in hypertension than has previously been described. The ability of the SHR neurons to induce a hypertensive phenotype on the otherwise healthy WKY myocytes, suggest an increased amount of NA released per unit of Nicotine in the SHR neurons.

Support or Funding Information

Wellcome Trust OXION initiative, British Heart Foundation