Identification of Mineralocorticoid Receptors, Aldosterone, and Its Processing Enzyme CYP11B2 on Parasympathetic and Sympathetic Neurons in Rat Intracardiac Ganglia.

Lukas Dehe,Noureddin Aboryag,Shaaban A Mousa,Sascha Treskatsch,Antje Beyer,Mohammed Shaqura,Michael Schäfer

doi:10.3389/fnana.2021.802359

Abstract

Recent interest has focused on the mineralocorticoid receptor (MR) and its impact on the myocardium and the performance of the heart. However, there is a lack of evidence about MR expression and its endogenous ligand aldosterone synthesis with specific regard to the intrinsic cardiac nervous system. Therefore, we looked for evidence of MR and aldosterone in sympathetic and parasympathetic neurons of intracardiac ganglia. Tissue samples from rat heart atria were subjected to conventional reverse-transcriptase polymerase chain reaction (PCR), Western blot, and double immunofluorescence confocal analysis of MR, corticosterone-inactivating enzyme 11β-hydroxysteroid-dehydrogenase-2 (11β-HSD2), aldosterone, and its processing enzyme CYP11B2 together with the neuronal markers vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH). Our results demonstrated MR, 11β-HSD2, and CYP11B2 specific mRNA and protein bands in rat heart atria. Double immunofluorescence labeling revealed coexpression of MR immunoreactivity with VAChT in large diameter parasympathetic principal neurons. In addition, MR immunoreactivity was identified in TH-immunoreactive small intensely fluorescent (SIF) cells and in nearby VAChT- and TH-immunoreactive nerve terminals. Interestingly, the aldosterone and its synthesizing enzyme CYP11B2 and 11β-HSD2 colocalized in MR– immunoreactive neurons of intracardiac ganglia. Overall, this study provides first evidence for the existence of not only local expression of MR, but also of 11β-HSD2 and aldosterone with its processing enzyme CYP11B2 in the neurons of the cardiac autonomic nervous system, suggesting a possible modulatory role of the mineralocorticoid system on the endogenous neuronal activity on heart performance.

Highlights

Corticosteroids are steroid hormones produced in the adrenal cortex and bind to two types of receptors, the glucocorticoid and mineralocorticoid receptor (Gray et al, 2017)
A thick cardiac nerve consisting of non-varicose or varicose vesicular acetylcholine transporter (VAChT)-positive nerve fibers coexpressing mineralocorticoid receptor (MR) immunoreactivity was identified in rat atria (Figures 3A–H)
Our experiments identified mRNA and proteins specific for MR, its protecting enzyme 11β-HSD2, as well as the aldosterone synthesizing enzyme CYP11B2 in the right and left atria of the rat

Summary

INTRODUCTION

Corticosteroids are steroid hormones produced in the adrenal cortex and bind to two types of receptors, the glucocorticoid and mineralocorticoid receptor (Gray et al, 2017). Until now, there is no conclusive evidence of the expression and anatomical localization of the mineralocorticoid receptors, its endogenous ligand aldosterone, the enzyme aldosterone synthase CYP11β2, and the MR protecting enzyme 11β-HSD2 with specific regard to the parasympathetic and sympathetic innervations of the heart. Due to the recent increasing interest of the modulating effects of local mineralocorticoid receptors on the autonomic cardiac nervous system, we set out to systematically examine the expression of MR in rat heart atria containing intracardiac ganglia using conventional PCR and Western blot. We aimed at the systematic investigation of the colocalization of MR, 11β-HSD2, and CYP11β2 with specific markers for parasympathetic neurons (vesicular acetylcholine transporter— VAChT) and catecholaminergic (including sympathetic) neurons (tyrosine hydroxylase (TH) by double immunofluorescence confocal microscopy The results of these findings may provide anatomical evidence of the local mineralocorticoid receptor and its corresponding endogenous ligand for the autonomic regulation of cardiac function

MATERIALS AND METHODS

RESULTS

DISCUSSION

ETHICS STATEMENT