Lack of evidence for longitudinal dissociation of the atrioventricular conduction axis.

Longitudinal dissociation of the aggregated specialized cardiomyocytes within the non-branching portion of atrioventricular conduction axis has proved a controversial topic for both morphologists and electrophysiologists. We have now used morphological methods, including three-dimensional assessment, to revisit, in human, canine, and bovine hearts, the presence or absence of interconnections between the aggregated cardiomyocytes making up the non-branching bundle. We analyzed three datasets from human and canine hearts, and two from bovine hearts, using longitudinal and orthogonal serial histological sections. In addition, we assessed three hearts using translucent India ink injected specimens, permitting assessment of the three-dimensional arrangement of the cardiomyocytes. Using the longitudinal sections, we found numerous oblique interconnections between the groups of specialized cardiomyocytes. When assessing orthogonal sections, we noted marked variation in the grouping of the cardiomyocytes. We interpreted this finding as evidence of bifurcation and convergence of the groups seen in the longitudinal sections. The three-dimensional assessment of the bovine material confirmed the presence of the numerous interconnections. The presence of multiple connections between the cardiomyocytes in the non-branching bundle rules out the potential for longitudinal dissociation.

[The antagonistic function of the heart muscle sustains the autoregulation according to Frank and Starling : Part I: Structure and function of heart muscle]. / Die antagonistische Funktion des Herzmuskels unterstützt die Autoregulation nach Frank-Starling : Teil I: Struktur und Funktion des Herzmuskels.

In the tradition of Harvey and according to Otto Frank the heart muscle structure is arranged in a strictly tangential fashion hence all contractile forces act in the direction of ventricular ejection. In contrast, morphology confirms that the heart consists of a 3-dimensional network of muscle fibers with up to two fifths of the chains of aggregated myocytes deviating from a tangential alignment at variable angles. Accordingly, the myocardial systolic forces contain, in addition to a constrictive also a (albeit smaller) radially acting component. Using needle force probes we have correspondingly measured an unloading type of force in a tangential direction and an auxotonic type in dilatative transversal direction of the ventricular walls to show that the myocardial body contracts actively in a 3-dimensional pattern. This antagonism supports the autoregulation of heart muscle function according to Frank and Starling, preserving ventricular shape, enhances late systolic fast dilation and attenuates systolic constriction of the ventricle wall. Auxotonic dilating forces are particularly sensitive to inotropic medication. Low dose beta-blocker is able to attenuate the antagonistic activity. All myocardial components act against four components of afterload, the hemodynamic, the myostructural, the stromatogenic and the hydraulic component. This complex interplay critically complicates clinical diagnostics. Clinical implications are far-reaching (see Part II, https://doi.org/10.1007/s00059-018-4735-x).

The E3 ubiquitin ligase SMURF1 regulates cell-fate specification and outflow tract septation during mammalian heart development.

Smad ubiquitin regulatory factor 1 (SMURF1) is a HECT-type E3 ubiquitin ligase that plays a critical role in vertebrate development by regulating planar cell polarity (PCP) signaling and convergent extension (CE). Here we show that SMURF1 is involved in mammalian heart development. We find that SMURF1 is highly expressed in outflow tract cushion mesenchyme and Smurf1-/- mouse embryos show delayed outflow tract septation. SMURF1 is expressed in smooth muscle cells of the coronary arteries and great vessels. Thickness of the aortic smooth muscle cell layer is reduced in Smurf1-/- mouse embryos. We show that SMURF1 is a negative regulator of cardiomyogenesis and a positive regulator of smooth muscle cell and cardiac fibroblast differentiation, indicating that SMURF1 is important for cell-type specification during heart development. Finally, we provide evidence that SMURF1 localizes at the primary cilium where it may regulate bone morphogenetic protein (BMP) signaling, which controls the initial phase of cardiomyocyte differentiation. In summary, our results demonstrate that SMURF1 is a critical regulator of outflow tract septation and cell-type specification during heart development, and that these effects may in part be mediated via control of cilium-associated BMP signaling.

Re-evaluation of hypoplastic left heart syndrome from a developmental and morphological perspective.

BACKGROUND: Hypoplastic left heart syndrome (HLHS) covers a spectrum of rare congenital anomalies characterised by a non-apex forming left ventricle and stenosis/atresia of the mitral and aortic valves. Despite many studies, the causes of HLHS remain unclear and there are conflicting views regarding the role of flow, valvar or myocardial abnormalities in its pathogenesis, all of which were proposed prior to the description of the second heart field. Our aim was to re-evaluate the patterns of malformation in HLHS in relation to recognised cardiac progenitor populations, with a view to providing aetiologically useful sub-groupings for genomic studies. RESULTS: We examined 78 hearts previously classified as HLHS, with subtypes based on valve patency, and re-categorised them based on their objective ventricular phenotype. Three distinct subgroups could be identified: slit-like left ventricle (24%); miniaturised left ventricle (6%); and thickened left ventricle with endocardial fibroelastosis (EFE; 70%). Slit-like ventricles were always found in combination with aortic atresia and mitral atresia. Miniaturised left ventricles all had normally formed, though smaller aortic and mitral valves. The remaining group were found to have a range of aortic valve malformations associated with thickened left ventricular walls despite being described as either atresia or stenosis. The degree of myocardial thickening was not correlated to the degree of valvar stenosis. Lineage tracing in mice to investigate the progenitor populations that form the parts of the heart disrupted by HLHS showed that whereas Nkx2-5-Cre labelled myocardial and endothelial cells within the left and right ventricles, Mef2c-AHF-Cre, which labels second heart field-derived cells only, was largely restricted to the endocardium and myocardium of the right ventricle. However, like Nkx2-5-Cre, Mef2c-AHF-Cre lineage cells made a significant contribution to the aortic and mitral valves. In contrast, Wnt1-Cre made a major contribution only to the aortic valve. This suggests that discrete cardiac progenitors might be responsible for the patterns of defects observed in the distinct ventricular sub-groups. CONCLUSIONS: Only the slit-like ventricle grouping was found to map to the current nomenclature: the combination of mitral atresia with aortic atresia. It appears that slit-like and miniature ventricles also form discrete sub-groups. Thus, reclassification of HLHS into subgroups based on ventricular phenotype, might be useful in genetic and developmental studies in investigating the aetiology of this severe malformation syndrome.

Pneumatic Distension of Ventricular Mural Architecture Validated Histologically.

Purpose: There are ongoing arguments as to how cardiomyocytes are aggregated together within the ventricular walls. We used pneumatic distension through the coronary arteries to exaggerate the gaps between the aggregated cardiomyocytes, analyzing the pattern revealed using computed tomography, and validating our findings by histology. Methods: We distended 10 porcine hearts, arresting 4 in diastole by infusion of cardioplegic solutions, and 4 in systole by injection of barium chloride. Mural architecture was revealed by computed tomography, measuring also the angulations of the long chains of cardiomyocytes. We prepared the remaining 2 hearts for histology by perfusion with formaldehyde. Results: Increasing pressures of pneumatic distension elongated the ventricular walls, but produced insignificant changes in mural thickness. The distension exaggerated the spaces between the aggregated cardiomyocytes, compartmenting the walls into epicardial, central, and endocardial regions, with a feathered arrangement of transitions between them. Marked variation was noted in the thicknesses of the parts in the different ventricular segments, with no visible anatomical boundaries between them. Measurements of angulations revealed intruding and extruding populations of cardiomyocytes that deviated from a surface-parallel alignment. Scrolling through the stacks of tomographic images revealed marked spiraling of the aggregated cardiomyocytes when traced from base to apex. Conclusion: Our findings call into question the current assumption that cardiomyocytes are uniformly aggregated together in a tangential fashion. There is marked heterogeneity in the architecture of the different ventricular segments, with the aggregated units never extending in a fully transmural fashion. Key Points: • Pneumographic computed tomography reveals an organized structure of the ventricular walls.• Aggregated cardiomyocytes form a structured continuum, with marked regional heterogeneity.• Global ventricular function results from antagonistic forces generated by aggregated cardiomyocytes. Citation Format: • Burg MC, Lunkenheimer P, Niederer P et al. Pneumatic Distension of Ventricular Mural Architecture Validated Histologically. Fortschr Röntgenstr 2016; 188: 1045 - 1053.

A mathematical model of the mechanical link between shortening of the cardiomyocytes and systolic deformation of the left ventricular myocardium.

BACKGROUND: Left ventricular myocytes are arranged in a complex three-dimensional mesh. Since all myocytes contract approximately to the same degree, mechanisms must exist to enable force transfer from each of these onto the framework as a whole, despite the transmural differences in deformation strain. This process has hitherto not been clarified in detail. OBJECTIVE: To present a geometrical model that establishes a mechanical link between the three-dimensional architecture and the function of the left ventricular myocardium. METHODS: The left ventricular equator was modeled as a cylindrical tube of deformable but incompressible material, composed of virtual cardiomyocytes with known diastolic helical and transmural angles. By imposing reference circumferential, longitudinal, and torsional strains onto the model, we created a three-dimensional deformation field to calculate passive shortening of the myocyte surrogates. We tested two diastolic architectures: 1) a simple model with longitudinal myocyte surrogates in the endo- and epicardium, and circular ones in the midwall, and 2) a more accurate architecture, with progressive helical angle distribution varying from -60° in the epicardium to 60° in the endocardium, with or without torsion and transmural cardiomyocyte angulation. RESULTS: The simple model caused great transmural unevenness in cardiomyocyte shortening; longitudinal surrogates shortened by 15% at all depths equal to the imposed longitudinal strain, whereas circular surrogates exhibited a maximum shortening of 23.0%. The accurate model exhibited a smooth transmural distribution of cardiomyocyte shortening, with a mean (range) of 17.0 (13.2-20.8)%. Torsion caused a shortening of 17.0 (15.2-18.9)% and transmural angulation caused a shortening of 15.2 (12.4-18.2)%. Combining the effects of transmural angulation and torsion caused a change of 15.2 (13.2-16.5)%. CONCLUSION: A continuous transmural distribution of the helical angle is obligatory for smooth shortening of the cardiomyocytes, but a combination of torsional and transmural angulation changes is necessary to execute systolic mural thickening whilst keeping shortening of the cardiomyocytes within its physiological range.

Molecular architecture of the human specialised atrioventricular conduction axis.

The atrioventricular conduction axis, located in the septal component of the atrioventricular junctions, is arguably the most complex structure in the heart. It fulfils a multitude of functions, including the introduction of a delay between atrial and ventricular systole and backup pacemaking. Like any other multifunctional tissue, complexity is a key feature of this specialised tissue in the heart, and this complexity is both anatomical and electrophysiological, with the two being inextricably linked. We used quantitative PCR, histology and immunohistochemistry to analyse the axis from six human subjects. mRNAs for ~50 ion and gap junction channels, Ca(2+)-handling proteins and markers were measured in the atrial muscle (AM), a transitional area (TA), inferior nodal extension (INE), compact node (CN), penetrating bundle (PB) and ventricular muscle (VM). When compared to the AM, we found a lower expression of Na(v)1.5, K(ir)2.1, Cx43 and ANP mRNAs in the CN for example, but a higher expression of HCN1, HCN4, Ca(v)1.3, Ca(v)3.1, K(ir)3.4, Cx40 and Tbx3 mRNAs. Expression of some related proteins was in agreement with the expression of the corresponding mRNAs. There is a complex and heterogeneous pattern of expression of ion and gap junction channels and Ca(2+)-handling proteins in the human atrioventricular conduction axis that explains the function of this crucial pathway.

Application of discriminant analysis with clustered data to determine anthropogenic metals contamination.

Environmental site assessments involve, among other things, characterization of the nature and extent of contamination. In general, environmental assessors are interested in empirical methodologies that can be applied to a broad range of environmental media (e.g., soils, sediments, etc.) and situations. To date, no unified guidance has been adopted, and site investigations usually involve a tiered process with multiple analyses. We propose a multivariate analysis methodology utilizing discriminant analysis with clustered chemical concentrations as a novel application to environmental site assessments that determine, in relative order of magnitude, contaminated chemicals. Finite mixture models are presented as a means to assess latent chemical clusters with some basis in statistical inference. The methodology is illustrated with a typical localized data set containing total metal and metalloid (i.e., chemical) concentrations, extracted from bulk soil collected from reference and site-related locations, obtained from a former military installation in the southeast United States. The illustration is particularly applicable because site-related soils inherently possessed higher background chemical levels than reference soils, which biased conventional analyses. However, contrasting chemical compositions were inferred within site-related samples illustrating the versatility of the proposed methodology. Using these results along with known information regarding the history of contamination at the site, a qualitative and quantitative assessment of contaminated chemicals was made. Results are intended for illustration purposes only and are discussed within the context of environmental site assessment.

Partitioning species variability from soil property effects on phytotoxicity: ECx normalization using a plant contaminant sensitivity index.

Soil properties mitigate hazardous effects of contaminants through soil chemical sequestration and should be considered when evaluating ecological risk from terrestrial contamination. Empirical models that quantify relationships between soil properties and toxicity to ecological receptors are necessary for site-specific adjustments to ecological risk assessments. However, differential sensitivities of test organisms in dose-response studies may limit the utility of such models. We present a novel approach to toxicity estimation that partitions the effect of differential sensitivities of test organisms from that of soil chemical/physical properties. Five soils that ranged in selected properties were spiked with five concentrations of sodium arsenate. Bioassays were conducted where above ground dry matter growth and the corresponding tissue arsenic concentrations were evaluated for three terrestrial plants (Alfalfa, Medicago sativa L.; Perennial ryegrass, Lolium perrene L.; and Japanese millet, Echinochloa crusgalli L.). Estimates were combined into a plant contaminant sensitivity index (PCSI) and used to normalize phytotoxicity parameters to the most sensitive species (i.e., alfalfa) where necessary. Simple linear regression and ANCOVA indicated a 36.5% increase in the explanatory power of the modifying effects of soil properties on phytotoxicity when differential arsenate sensitivities were accounted for by PCSI (r(2) = 0.477-0.833). Normalization of ecotoxicity parameters by PCSI is a seemingly effective approach to quantify the modifying effects of soil properties on phytotoxicity endpoints when it is of interest to consider multiple plant species (or varieties within a species) with differential sensitivities to experimental contaminants.

The anatomy of the tendon of the infundibulum revisited.

The heart is a muscular organ supported by collagenous tissue. The collagenous tissue is condensed in certain areas to form a supporting framework, often called the fibrous skeleton. The so-called tendon of the infundibulum has previously been described as part of this skeleton, but its structure and incidence remain ill defined. The tendon was initially described as a strip of fibrous tissue running between the aortic root and the pulmonary trunk. Since information on its structure is vague, we sought to evaluate its existence in 100 formalin-fixed adult human hearts obtained from subjects ranging in age from 22 to 86 years, in 20 hearts from infants and children aged from 2 months to 6 years at the time of their death and in 10 cattle hearts. We used classical macroscopic anatomical techniques to demonstrate all the possible connections between the sinuses of the aorta and the pulmonary trunk. We then supplemented the macroscopic techniques with serial transverse histological sections taken through the vascular roots, staining the sections with the haematoxylin-eosin, van Gieson, Masson trichrome and orcein staining methods. Fascial bands surrounded by connective tissue were observed in all hearts. In 80 adult hearts and in 16 neonatal hearts we found fascial bands or strips, which connected the aortic and pulmonary roots. Only in two hearts, however, were we able to identify tendon-like structures, and histology revealed that these were formed by tightly packed collagen fibres intermingled with fat, most likely due to advanced age. Thus in those cases where a "tendon" was present it was no more than condensed fascial bands joining together the apposing sinuses of the arterial trunks. In our opinion, therefore, accounts in the literature describing the "tendon of the infundibulum" as a tendinous structure connecting the aortic and pulmonary roots do not accurately represent this anatomical structure.

Hearts with concordant ventriculoarterial connections but parallel arterial trunks.

OBJECTIVES: To determine the characteristic morphological features of hearts with concordant ventriculoarterial connections and parallel arterial trunks, and to provide unequivocally a method to describe their anatomy. DESIGN, METHODS AND PATIENTS: The entire cardiac database and cardiac pathological archive at the Hospital for Sick Children, Toronto, Ontario, Canada, was interrogated to identify all patients with concordant ventriculoarterial connections and parallel arterial trunks. The clinical records, autopsy reports and actual cardiac specimens of those who underwent autopsy, were reviewed. RESULTS: 8 cases meeting our criteria were identified. The infundibular anatomy was variable, including four hearts with bilateral infundibulums, three with subpulmonary infundibulums and one with bilaterally absent infundibulums. Considerable variability was also found in the type of atrial arrangement, along with the morphology of the atrioventricular junctions. The most common findings were the usual atrial arrangement (n = 5), left juxtaposition of the right atrial appendages (n = 3), an atrial septal defect (n = 6), univentricular atrioventricular connection (n = 5), ventricular septal defect (n = 8) and pulmonary obstruction (n = 4). In addition, five specimens had either a single coronary artery or two coronary arteries arising from the anticipated right coronary aortic sinus. CONCLUSIONS: Concordant ventriculoarterial connections with parallel arterial trunks can be found in a variety of segmental combinations. An accurate diagnosis of these rare hearts can be achieved by detailed analysis of not only the ventriculoarterial connections but also the infundibular anatomy and the spatial relationship of the arterial trunks. Particular attention to the coronary arteries is warranted.

Pulmonary atresia with intact ventricular septum: predictors of early and medium-term outcome in a population-based study.

OBJECTIVES: Pulmonary atresia with intact ventricular septum is a form of congenital heart disease usually associated with right-heart hypoplasia, with considerable morphologic heterogeneity and often poor outlook. Ascertainment of risk factors for poor outcome is an important step if an improvement in outcome is to be achieved. METHODS: The UK and Ireland Collaborative study of Pulmonary Atresia with Intact Ventricular Septum is an ongoing population-based study of all patients born with this disease from 1991 through 1995. All available clinical, morphologic, and investigative variables were directly reviewed, and risk factor analysis was performed for poor outcome. RESULTS: One hundred eighty-three patients presented with pulmonary atresia with intact ventricular septum. Fifteen underwent no procedure, and all died. Of the remainder, 67 underwent a right ventricular outflow tract procedure (catheter or surgical), 18 underwent an outflow tract procedure with shunt, and 81 underwent a systemic-to-pulmonary shunt alone. One- and 5-year survival was 70.8% and 63.8%, respectively. Results from Cox proportional hazards model analysis showed that low birth weight (P = .024), unipartite right ventricular morphology (P = .001), and the presence of a dilated right ventricle (P < .001) were independent risk factors for death. The presence of coronary artery fistulae, right ventricular dependence, or the tricuspid valvar z score did not prove to be risk factors for death. After up to 9 years of follow-up, 29% have achieved a biventricular repair, 3% a so-called one-and-a-half ventricular repair, and 10.5% a univentricular repair, with 16.5% still having a mixed circulation (41% died). CONCLUSIONS: This population-based study has shown which features at presentation place an infant in a high-risk group. This is important information for counseling in fetal life and for surgical strategy after birth.

Sinus node revisited in the era of electroanatomical mapping and catheter ablation.

OBJECTIVE: To study the architecture of the human sinus node to facilitate understanding of mapping and ablative procedures in its vicinity. METHODS: The sinoatrial region was examined in 47 randomly selected adult human hearts by histological analysis and scanning electron microscopy. RESULTS: The sinus node, crescent-like in shape, and 13.5 (2.5) mm long, was not insulated by a sheath of fibrous tissue. Its margins were irregular, with multiple radiations interdigitating with ordinary atrial myocardium. The distances from the node to endocardium and epicardium were variable. In 72% of the hearts, the whole nodal body was subepicardial and in 13 specimens (28%) the inner aspect of the nodal body was subendocardial. The nodal body cranial to the sinus nodal artery was more subendocardial than the remaining nodal portion, which was separated from the endocardium by the terminal crest. In 50% of hearts, the most caudal boundaries of the body of the node were at least 3.5 mm from the endocardium. When the terminal crest was > 7 mm thick (13 hearts, 28%), the tail was subepicardial or intramyocardial and at least 3 mm from the endocardium. CONCLUSIONS: The length of the node, the absence of an insulating sheath, the presence of nodal radiations, and caudal fragments offer a potential for multiple breakthroughs of the nodal wavefront. The very extensive location of the nodal tissue, the cooling effect of the nodal artery, and the interposing thick terminal crest caudal to this artery have implications for nodal ablation or modification with endocardial catheter techniques.

The forces generated within the musculature of the left ventricular wall.

OBJECTIVES: To test the hypothesis that two populations of myocardial fibres-fibres aligned parallel to the surfaces of the wall and an additional population of fibres that extend obliquely through the wall-when working in concert produce a dualistic, self stabilising arrangement. METHODS: Assessment of tensile forces in the walls of seven porcine hearts by using needle probes. Ventricular diameter was measured with microsonometry and the intracavitary pressure through a fluid filled catheter. Positive inotropism was induced by dopamine, and negative inotropism by thiopental. The preload was raised by volume load and lowered by withdrawal of blood. Afterload was increased by inflation of a balloon in the aortic root. The anatomical orientation of the fibres was established subsequently in histological sections. RESULTS: The forces in the fibres parallel to the surface decreased 20-35% during systolic shrinkage of the ventricle, during negative inotropism, and during ventricular unloading. They increased 10-30% on positive inotropic stimulation and with augmentation in preload and afterload. The forces in the oblique transmural fibres increased 8-65% during systole, on positive inotropic medication, with an increase in afterload and during ventricular shrinkage, and decreased 36% on negative inotropic medication. There was a delay of up to 147 ms in the drop in activity during relaxation in the oblique transmural fibres. CONCLUSION: Although the two populations of myocardial fibres are densely interwoven, it is possible to distinguish their functions with force probes. The delayed drop in force during relaxation in obliquely oriented fibres indicates that they are hindered in their shortening to an extent that parallels any increase in mural thickness. The transmural fibres, therefore, contribute to stiffening of the ventricular wall and hence to confining ventricular compliance.

Respiratory effects of sulphur dioxide: a hierarchical multicity analysis in the APHEA 2 study.

BACKGROUND: Sulphur dioxide (SO(2)) was associated with hospital admissions for asthma in children in the original APHEA study, but not with other respiratory admissions. AIMS: To assess the association between daily levels of SO(2) and daily levels of respiratory admissions in a larger and more recent study. METHODS: Time series of daily counts of hospital emergency admissions were constructed for asthma at ages 0-14 years and 15-64 years, COPD and asthma, and all respiratory admissions at ages 65+ years in the cities of Birmingham, London, Milan, Paris, Rome, Stockholm, and in the Netherlands for periods of varying duration between the years 1988 and 1997. A two stage hierarchical modelling approach was used. In the first stage generalised additive Poisson regression models were fitted in each city controlling for weather and season. These results were then combined across cities in a second stage ecological regression that looked at potential effect modifiers. RESULTS: For an increase of 10 micro g/m(3) of SO(2) the daily number of admissions for asthma in children increased 1.3% (95% CI 0.4% to 2.2%). Effect modification among cities by levels of other air pollutants or temperature was not found. The SO(2) effect disappeared after controlling for PM(10) or CO, but correlation among these pollutants was very high. Other respiratory admissions were not associated with SO(2). CONCLUSION: SO(2) is associated with asthma admissions in children, indicating that reduction in current air pollution levels could lead to a decrease in the number of asthma admissions in children in Europe.