Dysregulation of Neuronal Ca ²⁺ Channel Linked to Heightened Sympathetic Phenotype in Prohypertensive States

Abstract

Hypertension is associated with impaired nitric oxide (NO)-cyclic nucleotide (CN)-coupled intracellular calcium (Ca 2ϩ ) homeostasis that enhances cardiac sympathetic neurotransmission. Because neuronal membrane Ca 2ϩ currents are reduced by NO-activated S-nitrosylation, we tested whether CNs affect membrane channel conductance directly in neurons isolated from the stellate ganglia of spontaneously hypertensive rats (SHRs) and their normotensive controls. Using voltage-clamp and cAMP-protein kinase A (PKA) FRET sensors, we hypothesized that impaired CN regulation provides a direct link to abnormal signaling of neuronal calcium channels in the SHR and that targeting cGMP can restore the channel phenotype. We found significantly larger whole-cell Ca 2ϩ currents from diseased neurons that were largely mediated by the N-type Ca 2ϩ channel (Cav 2.2 ). Elevating cGMP restored the SHR Ca 2ϩ current to levels seen in normal neurons that were not affected by cGMP. cGMP also decreased cAMP levels and PKA activity in diseased neurons. In contrast, cAMP-PKA activity was increased in normal neurons, suggesting differential switching in phosphodiesterase (PDE) activity. PDE2A inhibition enhanced the Ca 2ϩ current in normal neurons to a conductance similar to that seen in SHR neurons, whereas the inhibitor slightly decreased the current in diseased neurons. Pharmacological evidence supported a switching from cGMP acting via PDE3 in control neurons to PDE2A in SHR neurons in the modulation of the Ca 2ϩ current. Our data suggest that a disturbance in the regulation of PDE-coupled CNs linked to N-type Ca 2ϩ channels is an early hallmark of the prohypertensive phenotype associated with intracellular Ca 2ϩ impairment underpinning sympathetic dysautonomia.

Sympathetic neurons are a powerful driver of myocyte function in cardiovascular disease

“…These neurons show impaired nitric oxide (NO) - cGMP signalling resulting in lower cGMP levels and disinhibition of neurotransmission51525354. Moreover we recently reported that the neuronal Ca 2+ channel phenotype in the SHR is linked to a phosphodiesterase – cyclic nucleotide impairment and failure of cGMP signalling to inhibit I CaN 16.…”

“…Nevertheless cardiac sympathetic hyperactivity is a well established early hallmark of heart failure45, post myocardial infarction6 and hypertension, both in humans7891011 and in the spontaneously hypertensive rat (SHR)1213. In the pro-hypertensive SHR, the sympathetic stellate neurons that predominantly innervate the heart14 show increased membrane Ca 2+ currents1516, intracellular Ca 2+ transients17 and significant impairment of the noradrenaline reuptake transporter (NET)18 that all contribute to enhanced noradrenaline (NA) release131920. This heightened sympathetic activity at the end organ results in ß-adrenergic hyper-responsiveness of the myocyte212223.…”

Novel target identification using single cell sequencing and electrophysiology of cardiac sympathetic neurons in disease

“…The main difference between these two sympathetic neuronal populations is that one also expressed the co-transmitter neuropeptide-Y. Differential expression analysis between the 6 Wistar and SHR sympathetic neuron populations identified in Figure 2C highlight a significant decrease in 5 ion channel subunit encoding genes that may contribute to firing rate of stellate ganglia sympathetic neurons (Table 1) (Full list in supplementary 2D).…”

Impaired cAMP-cGMP cross-talk during cardiac sympathetic dysautonomia

“…3 Moreover, it is widely accepted that autonomic imbalance contributes to the pathogenesis of hypertension itself, where emerging research is beginning to shed light on the key cellular and molecular changes that occur in diseased neurons. 3,4 The postganglionic sympathetic stellate neurons (PGSNs) of the SHR that predominantly innervate the heart, display increased intracellular calcium [Ca 2C ] i transients linked to impaired neuronal nitric oxide synthase (nNOS) activity. 3 Together, with downstream reductions in nitric oxide (NO)-cyclic guanosine monophosphate (cGMP), this results in enhanced end-organ neurotransmission.…”

“…Recently, we published data suggesting that sympathetic hyperactivity in the stellate neurons from the pro-SHR may be triggered by dysregulated Ca 2C channel activity, resulting in greater Ca 2C influx. 4 N-type Ca channels (Ca v 2.2) were identified as the major contributor to Ca 2C entry in PGSNs, that in turn facilitate sympathetic neurotransmission. Inhibition of the N-type Ca 2C channel also reduces the propensity for fatal ventricular arrhythmias and ameliorates autonomic dysfunction in a heart failure mouse model.…”