Constitutive Overexpression of Muscarinic Receptors Leads to Vagal Hyperreactivity

BACKGROUND: Alterations in muscarinic receptor expression and acetylcholinesterase (AchE) activity have been observed in tissues from Sudden Infant Death Syndrome (SIDS). Vagal overactivity has been proposed as a possible cause of SIDS as well as of vasovagal syncopes. The aim of the present study was to seek whether muscarinic receptor overexpression may be the underlying mechanism of vagal hyperreactivity. Rabbits with marked vagal pauses following injection of phenylephrine were selected and crossed to obtain a vagal hyperreactive strain. The density of cardiac muscarinic receptors and acetylcholinesterase (AchE) gene expression were assessed. Blood markers of the observed cardiac abnormalities were also sought. METHODOLOGY/PRINCIPAL FINDINGS: Cardiac muscarinic M(2) and M(3) receptors were overexpressed in hyperreactive rabbits compared to control animals (2.3-fold and 2.5-fold, respectively) and the severity of the phenylephrine-induced bradycardia was correlated with their densities. A similar overexpression of M(2) receptors was observed in peripheral mononuclear white blood cells, suggesting that cardiac M(2) receptor expression can be inferred with high confidence from measurements in blood cells. Sequencing of the coding fragment of the M(2) receptor gene revealed a single nucleotide mutation in 83% of hyperreactive animals, possibly contributing for the transcript overexpression. Significant increases in AchE expression and activity were also assessed (AchE mRNA amplification ratio of 3.6 versus normal rabbits). This phenomenon might represent a compensatory consequence of muscarinic receptors overexpression. Alterations in M(2) receptor and AchE expression occurred between the 5th and the 7th week of age, a critical period also characterized by a higher mortality rate of hyperreactive rabbits (52% in H rabbits versus 13% in normal rabbits) and preceeded the appearance of functional disorders. CONCLUSIONS/SIGNIFICANCE: The results suggest that cardiac muscarinic receptor overexpression plays a critical role in the development of vagal hyperreactivity, whereas AchE hyperactivity appears as a compensatory consequence of it. Since similar vagal disorders were observed recently by us in SIDS, muscarinic receptor overexpression could become a marker of risk of vasovagal syncopes and SIDS

L'hypertension artérielle chez l'enfant après réparation chirurgicale d'une coarctation de l'aorte

STRASBOURG-Medecine (674822101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

M₂ muscarinic receptor gene expression in peripheral mononuclear white blood cells from normal (N) and vagal hyperreactive (H) rabbits.

<p>R-R intervals were measured in conscious rabbits challenged with PNE 500 µg kg⁻¹ following the procedure described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015618#s2" target="_blank">Material and Methods</a>. M<b>₂</b> muscarinic gene expression was assessed in peripheral mononuclear white blood cells by quantitative RT-PCR and normalized to the rabbit 18S housekeeping gene. Values in (<b>a</b>) show amplification ratio calculated according to the 2^{−ΔΔ<i>C</i>t} method of 9 (N) and 16 (H) experiments. In (<b>b</b>), each symbol represents one animal; ΔCt M<b>₂</b>-18S corresponds to the number of amplification cycles needed to detect M<b>₂</b> fluorescence standardized to 18S; thus, the lower the ΔCt M₂-18S, the greater M<b>₂</b> mRNA initial quantity. *: <i>P</i><0.0001 <i>versus</i> N.</p

Polymorphism of the M₂ cholinergic muscarinic receptor gene of normal (N) and vagal hyperreactive (H) rabbits.

<p>(<b>a</b>) DNA sequence analysis of the coding fragment of the M₂ muscarinic receptor gene (Chmr2) showing the normal CCT codon and the single nucleotide substitution at position 1311 (G instead of T). (<b>b</b>) The severity of vagal pauses was evaluated in conscious animals by measuring the duration of R-R interval on the ECG recording after challenge with PNE 500 µg kg⁻¹ following the procedure described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015618#s2" target="_blank">Material and Methods</a>. DNA sequence analysis of the coding fragment of the M₂ muscarinic receptor gene was carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015618#s2" target="_blank">Material and Methods</a>. Each symbol represents one animal.</p

Effect of AchE enzyme blockade in normal (N) and vagal hyperreactive (H) rabbits.

<p>R-R intervals were measured in conscious rabbits challenged with PNE 250 µg kg^-1 before (control) and after administration of neostigmine 25 µg kg⁻¹. Values are means ± SD of 4 N and 4 H experiments. *: <i>P</i> = 0.02 <i>versus</i> N control; **: <i>P</i> = 0.03 <i>versus</i> H control.</p

AchE cardiac gene expression and enzyme activity in erythrocytes from normal (N) and vagal hyperreactive (H) rabbits.

<p>R-R intervals were measured in conscious rabbits challenged with PNE 500 µg kg⁻¹ following the procedure described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015618#s2" target="_blank">Material and Methods</a>. (<b>a,b</b>) Cardiac AchE gene expression was assessed by quantitative RT-PCR and normalized to the rabbit 18S housekeeping gene. Values in (<b>a</b>) show amplification ratio calculated according to the 2^{−ΔΔ<i>C</i>t} method of 7 (N) and 10 (H) experiments. In (<b>b</b>), each symbol represents one animal; ΔCt AchE-18S corresponds to the number of amplification cycles needed to detect AchE fluorescence standardized to 18S; thus, the lower the ΔCt AchE-18S, the greater AchE mRNA initial quantity. (<b>c</b>) AchE enzyme activity was assayed colorimetrically in erythrocyte hemolysates from 7 (N) and 13 (H) animals. *: <i>P</i> = 0.008 <i>versus</i> N. **: <i>P</i> = 0.02 <i>versus</i> N.</p

Age-dependent changes in R-R interval and M₂ muscarinic receptor and AchE gene expression in peripheral mononuclear white blood cells from normal (N) and vagal hyperreactive (H) rabbits.

<p>(<b>a</b>) M₂ muscarinic receptor and AchE gene expression were assessed in peripheral mononuclear white blood cells by quantitative RT-PCR and normalized to the rabbit 18S housekeeping gene. Values show amplification ratio calculated according to the 2^{−ΔΔ<i>C</i>t} method of 8–9 (N) and 7–11 (H) experiments. *: <i>P</i> = 0.0262 and **: <i>P</i> = 0.0122 <i>versus</i> 5 weeks-old rabbits within the N and the H group, respectively. (<b>b</b>) R-R intervals were measured in conscious rabbits challenged with PNE 500 µg kg⁻¹ following the procedure described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015618#s2" target="_blank">Material and Methods</a>. Values are means ± SD of 11 N and 15 H rabbits. *: <i>P</i> = 0.0179 and **: <i>P</i> = 0.0110 <i>versus</i> 7 weeks-old rabbits within the H group.</p

Correlations between R-R interval and total, M₂ and M₃ muscarinic receptor density in hearts from normal (N) and vagal hyperreactive (H) rabbits.

<p>R-R intervals were measured in conscious rabbits challenged with PNE 500 µg kg⁻¹ following the procedure described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015618#s2" target="_blank">Material and Methods</a>. Total, M₂ and M₃ muscarinic receptor densities in hearts (B_max; fmol mg⁻¹ protein) were determined from Scatchard analysis of the saturation data using [³H]NMS, [³H]AF-DX 384 and [³H]4-DAMP, respectively, as radioligands. Binding conditions were as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015618#s2" target="_blank">Material and Methods</a>. Each symbol represents one animal. (<b>a</b>) Total muscarinic receptors; <i>n</i> = 9 H (full symbols) and 6 N (open symbols). (<b>b</b>) M₂ muscarinic receptors; <i>n</i> = 10 H (full symbols) and 7 N (open symbols). (<b>c</b>) M₃ muscarinic receptors; <i>n</i> = 10 H (full symbols) and 6 N (open symbols).</p