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Research Highlights from the Literature

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2004; Volume 14(6) from Clinical Autonomic Research    
                                      
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Identification of the muscarinic receptor responsible for sinus bradycardia
   
The presence of multiple muscarinic acetylcholine receptor (mAChR) subtypes in the heart and lung, combined with the lack of mAChR subtype-selective ligands, have complicated the task of identifying the mAChR subtypes mediating cardiac slowing (bradycardia) and airway narrowing (bronchoconstriction) due to vagal innervation. To determine which of the five mAChRs are responsible for the cholinergic control of heart rate and airway caliber in vivo, Fisher et al studied mutant mice lacking the two prime candidates for such control, the M2 or M3 mAChR. Bradycardia caused by direct vagal stimulation or administration of the muscarinic agonist methacholine (MCh) was abolished in mice lacking functional M2 mAChRs (M2-/- mice). In contrast, heart rate responses remained unchanged in M3 receptor-deficient mice (M3-/- mice). A reduced hypotensive response of M3-/- mice to MCh suggests M3 mAChRs contribute to peripheral vasodilation. The M2-/- mice showed significantly enhanced in vivo bronchoconstrictor responses to vagal stimulation or MCh administration. In contrast, bronchoconstrictor responses were totally abolished in M3-/- mice. Thus, M2 muscarinic receptors mediate vagally-mediated bradycardia.

Fisher,JT, Vincent,SG, Gomeza,J et al (2004)  Loss of vagally mediated bradycardia and bronchoconstriction in mice lacking M2 or M3 muscarinic acetylcholine receptors.  FASEB J 18:711-713.

 

Hirschsprung aganglionosis: A multigenic disease
   
Hirschsprung disease is a multigenic, congenital disorder that affects 1 in 5,000 newborns and is characterized by the absence or reduction of neural crest-derived enteric ganglia in a variable portion of the distal gastrointestinal tract. Most familial cases are related to mutations in the receptor-type tyrosine kinase (RET), but mutations in the endothelin receptor-B gene (EdnrB) have also been described.  Progenitor cells from the neural crest have to express EDNRB during development to complete their migration to the hindgut.  Zhu et al reported that a partial deletion of a 1 kb region in the promoter region of EdnrB results in fatal megacolon in mice.  This region also contains binding sites for Sox10, an SRY-related transcription factor that is also associated with familial Hirschsprung disease.  Others have shown that RET also modulates EdnrB function.  The interaction of these genes within the neural crest stem cells may explain why seemingly unrelated mutations give rise to a common phenotype, Hirschsprung disease.
    In a parallel study focusing specifically on the potential effects of gene interactions in enteric neural crest, Cantrell et al explored the interactions between genes in the endothelin pathway and Sox10 using Sox10Dom mutant mice.  Sox10Dom mice carry a single-base mutation in the Sox10 locus that produces a dominant negative form of the protein.  These mice suffer from aganglionosis of variable penetrance and severity, analogous to the variable aganglionosis seen in human HSCR families.  Single locus association analysis of an extended pedigree of Sox10Dom mice identified a highly significant effect of EdnrB alleles on the Sox10Dom aganglionosis phenotype.  Crosses between EdnrB and Sox10 mutants corroborate this gene interaction, with double mutant progeny exhibiting significantly more severe aganglionosis. The background strain of the EdnrB mutant was found to further influence the phenotype of Sox10/EdnrB double mutant progeny, implying the action of additional modifiers on this phenotype.  These results further support the hypothesis that Hirschsprung disease is the consequence of multiple gene interactions that modulate the ability of enteric neural crest cells to populate the developing gut.  Interactions between these and other genes can account for the variability in disease expression observed in patients.

Zhu,L, Lee,HO, Jordan,CS et al (2004)  Spatiotemporal regulation of endothelin receptor-B by SOX10 in neural crest-derived enteric neuron precursors.  Nat Genet 36:732-737.

Cantrell,VA, Owens,SE, Chandler,RL et al (2004)  Interactions between Sox10 and EdnrB modulate penetrance and severity of aganglionosis in the Sox10Dom mouse model of Hirschsprung disease.  Hum Mol Genet  (Published on line ahead of print).

 

Autonomic nervous system and obesity
   
Sympathetic nervous system activity is increased in many forms of human obesity.  Indeed, muscle sympathetic nerve activity (MSNA) is linearly related to body mass index (BMI), and this correlation is tighter than with most other biological markers.  Circulating leptin, originating from fat cells, is increased in obesity and is a potential mediator of this sympathetic activation.  BMI, however, is an imperfect indicator of obesity, and measurements of fat mass and fat distribution are more informative.  E.g., visceral fat is a better predictor of poor cardiovascular outcomes than subcutaneous fat.  Alvarez et al examined the relationship between MSNA, plasma leptin, and fat mass in 9 subcutaneously obese (SUBOB) and 15 nonobese (NO) men with similar levels of abdominal visceral fat (determined by computer tomography).  As expected, body mass (94±4 vs. 71±2 kg), total fat mass (25±2 vs. 12±1 kg), and abdominal subcutaneous fat (307±36 vs. 132±12 cm2) were significantly higher in the SUBOB group compared with NO peers, but the level of abdominal visceral fat did not differ significantly in the two groups (69±7 vs. 55±5 cm2). MSNA was not different between SUBOB and NO men (23±3 vs. 24±2 bursts/min; P > 0.05, respectively) despite approximately 2.6-fold higher (P < 0.05) plasma leptin concentration in the SUBOB men. Furthermore, abdominal visceral fat was the only body composition phenotype that correlated with MSNA (r = 0.45; P < 0.05). In addition, abdominal visceral fat was related to MSNA in NO (r = 0.58; P = 0.0239) but not SUBOB (r = 0.39; P = 0.3027) men. With the caveat that this study involved a small number of subjects and type II errors cannot be excluded, these findings suggest that abdominal visceral fat is more important than subcutaneous fat or leptin levels in increasing sympathetic tone in obesity. 
   
Previous epidemiological studies have suggested an associated between systolic blood pressure (SBP) responses to orthostatic stress and BMI.  To study if these phenotypes are genetically linked, North et al conducted a bivariate linkage analysis of postural SBP change and BMI. Sibships with hypertension, their parents, and selected nonmedicated offspring were recruited at five centers (1636 whites and 1747 blacks).  They then performed a maximum likelihood bivariate genome scan for quantitative trait loci influencing postural SBP change and BMI, adjusted for race, study center, sex and. The maximum genome-wide logarithm of odds (LOD) score of 3.2 was detected on chromosome 13 at 24 cM. This marker (D13S493) lies within 20 cM of a marker previously linked to BMI and is substantially higher than the univariate linkage for each trait (LOD scores for BMI and postural SBP change were 2.4 and 0.9, respectively). These findings suggest that a gene(s) on chromosome 13q jointly regulates the SBP response to postural change and BMI.  Previous studies have suggested an association between orthostatic hypotension and low BMI and, conversely, an increase in orthostatic SBP with higher BMI.  Epidemiological studies are no longer limited by our ability to genotype populations, but by difficulties in precise phenotyping.  E.g., in the present study 75% of participants were taking antihypertensive medications which could have modulated orthostatic SBP changes.  Nonetheless, LOD scores remained significant after correcting for this variable.

Alvarez,GE, Ballard,TP, Beske,SD et al (2004)  Subcutaneous obesity is not associated with sympathetic neural activation.  Am J Physiol Heart Circ Physiol 287:H414-H418.

North,KE, Rose,KM, Borecki,IB et al (2004)  Evidence for a gene on chromosome 13 influencing postural systolic blood pressure change and body mass index.  Hypertension 43:780-784.

 

Mechanisms of orthostatic hypocapnea
   
End-tidal PCO2 decreases on standing.  This otherwise normal response is exaggerated in POTS patients, and this may contribute to cerebral vasoconstriction and symptoms of orthostatic intolerance (Low et al Am J Med Sci. 1999;317:124.  This orthostatic hypocapnea can be explained by the increase in ventilation observed on standing, but other factors that are modulated during standing may contribute as well; such as a decrease in stroke volume (SV), a ventilation-perfusion (V/Q) gradient and an increase in functional residual capacity (FRC).  .  Gisolf et al use a mathematical model to explore the individual contribution of these to orthostatic hypocapnia, and validated the model with experimental data obtained in normal subjects.  On average, the end-tidal PCO2 decreased from 40 mmHg to 36 mmHg after standing. Both the model and experimental data demonstrated that this decrease is due primarily to an increase in tidal volume, and transiently to decreased SV and increased FRC.  Gravity-induced V/Q mismatch also contributes to orthostatic hypocapnia, but only slightly.

Gisolf,J, Wilders,R, Immink,RV et al (2004)  Tidal volume, cardiac output and functional residual capacity determine end-tidal CO(2) transient during standing up in humans.  J Physiol 554:579-590.

 

Advances in analysis of autonomic cardiovascular rhythms
   
Spectral analysis of high frequency variability of heart rate has proven useful in the evaluation of vagal cardiac modulation and as a predictor of poor cardiovascular outcomes.  Evaluation of sympathetic cardiac modulation is more challenging.  Studies using autonomic withdrawal with ganglionic blockade and autonomic failure patients have shown that the low/high frequency ratio of heart rate variability does indeed contain information about sympathetic cardiac modulation (see Autonomic News, Clin Autonom Res 2003;13:302).   A clear separation of the two autonomic nervous activities, however, is not possible with a standard spectral analysis approach. Zhong et al used a modified “Principal Dynamic Modes” (PDM) method to separate the dynamics of sympathetic and parasympathetic nervous activities. They found that the first two dominant PDMs have similar frequency characteristics for parasympathetic and sympathetic activities in healthy human subjects. Applications of atropine or propranolol significantly decreased the amplitude of the waveforms that correspond to each nervous activity. Furthermore, near complete elimination of these dynamics was observed when both drugs were given to the subjects. This approach may provide a more accurate assessment of the cardiac autonomic function than traditional heart rate variability.
    Even though spectral analysis of heart rate provides limited information about sympathetic function, low frequency oscillations of blood pressure reflect sympathetic modulation of vascular tone.  Tsai et al used b
iorthogonal wavelets analysis to quantify the relationship between blood pressure fluctuations (BP) and sympathetic nerve activity (SNA). They controlled SNA fluctuations using electrical stimulation of the medulla in anesthetized, paralyzed, vagotomized, cardiac sympathetic-blocked, baroreceptor-denervated, and angiotensin II-converting enzyme-inhibited rats. Spectral analysis showed a close coupling between fluctuations of BP and SNA at frequencies between 0.25 and ~0.4 Hz.  These results corroborate that neurovascular transmission, which entails a complex function of neural activation, neurotransmitter release, postsynaptic activation and presynaptic modulation, can be modeled using low-frequency variabilities of BP to quantitatively estimate fluctuations of SNA in the time domain.

Zhong,Y, Wang,H, Ju,KH et al (2004)  Nonlinear analysis of the separate contributions of autonomic nervous systems to heart rate variability using principal dynamic modes.  IEEE Trans Biomed Eng 51:255-262.

Tsai,ML, Shann,WC, Luo,WR et al (2004)  Wavelet-based analysis of low-frequency fluctuations of blood pressure and sympathetic nerve activity in rats.  Neurosci Lett 358:165-168.