Wouter H. Lamers

Development of the Cardiac Conduction System

In the formed heart, it is convention to distinguish working myocardium (the primary function of which is contraction) from the conduction system (the primary function of which is the generation and conduction of the electrical impulse). The conduction system comprises separate components with distinct functions. The SAN, which contains the leading pacemaker, generates the impulse. The impulse is subsequently conducted, via the atrial myocardium, which in this sense is part of the conduction pathway as well, toward the AVN. With a delay, the impulse is then rapidly transmitted from the AVN via the bundle branches and PPN to ensure a coordinated activation of the ventricular myocardium from apex to base. Classic reports cover the anatomy,1 pathology,1 and histology2 of the adult and developing conduction system. The myocytes of the conduction system share with those of the ordinary working myocardium four basic elements: (1) contraction, (2) autorhythmicity, (3) intercellular conduction, and (4) electromechanical coupling. In the early embryonic heart tube, an ECG, similar to an adult ECG, can be recorded, indicating the presence of sequentially activated chambers.3 Given this observation, it is as confusing to accept the presence of a conduction system because it is functionally present as it is to deny its existence because it is not morphologically recognizable. Rather, it is of paramount importance to appreciate that the arrangement of myocyte populations, with distinct contractile, conductive, and pacemaking properties, establishes the coordinated activation of the heart. Departures from these tenets have led to a confusing and fruitless search for so-called “cardiac specialized tissues” during development. The obvious key question is how this arrangement is being achieved. Early cardiac development starts with the formation of a primary heart tube from the cardiogenic mesoderm (Fig 1⇓); this topic has been reviewed recently.4 The primary heart …

Formation of the Tricuspid Valve in the Human Heart

BACKGROUND Some of the problems concerning the origin of the inlet component of the definitive right ventricle were resolved in a previous study in which we showed it to be derived exclusively from the embryonic right ventricle. Questions remain, however, concerning the relative contributions of endocardial cushion tissue and myocardium to the definitive valvar apparatus guarding the right atrioventricular orifice and the origin of the valvar leaflets. METHODS AND RESULTS The formation of the tricuspid valve was studied by scanning electron microscopic and immunohistochemical techniques. Concurrent with the development of the right atrioventricular connection, a myocardial ridge forms at the boundary between the atrioventricular canal and the embryonic right ventricle. It grows to become a myocardial gully that funnels atrial blood beneath the lesser curvature of the initial heart tube toward the middle of the right ventricle. Fenestrations in the floor of the gully create an additional inferior opening in the funnel, transforming its initial anterior rim into the septomarginal trabeculation. The septum formed by the fusion of the endocardial ridges of the outflow tract becomes myocardialized in its inferior portion to form, in part, the outlet septum and, in part, the supraventricular crest. The smooth atrial surface of the tricuspid valvar leaflets develops from endocardial cushion tissue. The leaflets become freely movable, however, only after delamination of the tension apparatus within the myocardium. The inferior and septal leaflets derive from the gully and the ventricular septum, their delamination being a single, continuous process. The antero-superior leaflet forms by delamination from the developing supraventricular crest. CONCLUSIONS The leaflets of the tricuspid valve develop equally from the endocardial cushion tissues and the myocardium. The myocardium contributing to the valve comes from two sources, the tricuspid gully complex and the developing supraventricular crest. These findings facilitate the understanding of several congenital malformations.

Development of the heart: (3) Formation of the ventricular outflow tracts, arterial valves, and intrapericardial arterial trunks

In the first part of our review of cardiac development,1 we explained the changes occurring during the transformation of the solitary primary heart tube into the primordiums of the definitive heart, describing how this involved the processes of looping, and subsequent formation from the primary tube of the components of the atriums and ventricles. In the second part of our review,2 we then accounted for the steps involved in separation of the atrial and ventricular chambers, emphasising that the processes were more complicated than the simple formation of partitions within the respective atrial and ventricular primordiums. The subject of this, our third review, is the transformation of the initially solitary outflow portion of the heart tube into the intrapericardial parts of the aorta and the pulmonary trunk, their arterial valves and sinuses, and the subarterial ventricular outflow tracts. In our first review, we summarised some of the problems that continue to plague the understanding of the development of these outflow structures. Thus, initially the entirety of the primary heart tube contained within the confines of the pericardial cavity possesses a myocardial phenotype. Yet, in the definitive heart, the walls of the intrapericardial arterial trunks, along with the sinuses of the arterial valves, and small parts of the subarterial ventricular outlets, have an arterial or fibrous phenotype. The steps involved in the changes of the walls from the myocardial to the arterial and fibrous phenotypes have yet to be clarified. And then, cushions, or ridges, of endocardial tissue initially fuse to divide the entirety of the solitary outflow segment into the presumptive systemic and pulmonary outlets. With subsequent development, these cushions lose their septal function, as the arterial valves and trunks, along with the subpulmonary muscular infundibulum, develop as free-standing structures with their own discrete walls within the pericardial …

DEVELOPMENT OF THE HEART: (1) FORMATION OF THE CARDIAC CHAMBERS AND ARTERIAL TRUNKS

Through the 20th century, knowledge of the events occurring during cardiac development was clouded by conflicting descriptions, coupled with use of notably different terminologies. Furthermore, not all accounts were based on direct study of embryonic material, instead being constructed on the basis of interpretations of previous reports, supported by inferences made from the structure of the congenitally malformed heart. Such processes, in themselves, are understandable, since it is axiomatic that proper appreciation of the events occurring during formation of the heart will aid in the analysis of the morphogenesis of cardiac malformations, this being a desirable prerequisite in the search for optimal treatment. Over the past decade, this has all changed. There has been an explosion of work, both anatomical and molecular, devoted to cardiac development. Advances in technology, coupled with the use of suitable animal models, now enable us to provide a more accurate account of the steps involved in formation and septation of the cardiac chambers. Not all of this new information is concordant with the “classical” accounts. In these reviews, therefore, we will describe, first, the steps involved in formation of the primary heart tube, and its conversion to the four cardiac chambers and the paired arterial trunks. We will then look in greater detail at the events occurring during the separation of the initial solitary heart tube into discrete systemic and pulmonary circulations. The mesodermal tissues that give rise to the heart first become evident when the embryo is undergoing the process known as gastrulation. In the human, this occurs during the third week of development, while for the mouse, at a comparable stage of development, around seven days will have elapsed from fertilisation, and the embryo will be in the presomitic stage. The embryonic plate in humans, initially possessing two layers, is ovoid, and is …

Downregulation of Connexin 45 Gene Products During Mouse Heart Development

The electrical activity in heart is generated in the sinoatrial node and then propagates to the atrial and ventricular tissues. The gap junction channels that couple the myocytes are responsible for this propagation process. The gap junction channels are dodecamers of transmembrane proteins of the connexin (Cx) family. Three members of this family have been demonstrated to be synthesized in the cardiomyocytes: Cx40, Cx43, and Cx45. In addition, each of them has been shown to form channels with unique and specific electrophysiological properties. Understanding the conduction phenomenon requires detailed knowledge of the spatiotemporal expression pattern of these Cxs in heart. The expression patterns of Cx40 and Cx43 have been previously described in the adult heart and during its development. Here we report the expression of Cx45 gene products in mouse heart from the stage of the first contractions (8.5 days postcoitum [dpc]) to the adult stage. The Cx45 gene transcript was demonstrated by reverse transcriptase-polymerase chain reaction experiments to be present in heart at all stages investigated. Between 8.5 and 10.5 dpc it was shown by in situ hybridization to be expressed in low amounts in all cardiac compartments (including the inflow and outflow tracts and the atrioventricular canal) and then to be downregulated from 11 to 12 dpc onward. At subsequent fetal stages, the transcript was weakly detected in the ventricles, with the most distinct expression in the outflow tract. Cx45 protein was demonstrated by immunofluorescence microscopy to be expressed in the myocytes of young embryonic hearts (8.5 to 9.5 dpc). However, beyond 10.5 dpc the protein was no longer detected with this technique in the embryonic, fetal, or neonatal working myocardium, although it could be shown by immunoblotting that the protein was still synthesized in neonatal heart. In the major part of adult heart, Cx45 was undetectable. It was, however, clearly seen in the anterior regions of the interventricular septum and in trace amounts in some small foci dispersed in the ventricular free walls. Cx45 gene is the first Cx gene so far demonstrated to be activated in heart at the stage of the first contractions. The coordination of myocytes during the slow peristaltic contractions that occur at this stage would thus appear to be controlled by the Cx45 channels.

Abnormal Cardiac Conduction and Morphogenesis in Connexin40 and Connexin43 Double-Deficient Mice

Connexin40-deficient (Cx40−/−/Cx43+/+) and connexin43-heterozygous knockout mice (Cx40+/+/Cx43+/−) are viable but show cardiac conduction abnormalities. The ECGs of adult double heterozygous animals (Cx40+/−/Cx43+/−) suggest additive effects of Cx40 and Cx43 haploinsufficiency on ventricular, but not on atrial, conduction. We also observed additive effects of both connexins on cardiac morphogenesis. Approximately half of the Cx40−/−/Cx43+/+ embryos died during the septation period, and an additional 16% died after birth. The majority of the latter mice had cardiac hypertrophy in conjunction with common atrioventricular junction or a ventricular septal defect. All Cx40−/−/Cx43+/− progeny exhibited cardiac malformations and died neonatally. The most frequent defect was common atrioventricular junction with abnormal atrioventricular connection, which was more severe than that seen in Cx40−/−/Cx43+/+ mice. Furthermore, muscular ventricular septal defects, premature closure of the ductus arteriosus, and subcutaneous edema were noticed in these embryos. Cx40+/−/Cx43−/− embryos showed the same phenotype (ie, obstructed right ventricular outflow tract) as reported for Cx40+/+/Cx43−/− mice. These findings demonstrate that Cx43 haploinsufficiency aggravates the abnormalities observed in the Cx40−/− phenotype, whereas Cx40 haploinsufficiency does not worsen the Cx43−/− phenotype. We conclude that the gap-junctional proteins Cx40 and Cx43 contribute to morphogenesis of the heart in an isotype-specific manner.

Development of the heart: (2) Septation of the atriums and ventricles

In the first part of our review of cardiac development,1 we discussed the initial changes involved in transformation of the heart forming regions of the embryo into the great veins, the atrial and ventricular chambers, and the arterial trunks. We showed that this first part of cardiac development could be divided into phases of formation of the primary myocardial tube, looping of the tube, during which additional parts are added that give the future compartments their definitive topography, and the assembly of these components into the cardiac chambers and arterial trunks. In this second review, we discuss the processes that complete the separation of the two sides of the definitive heart, for the most part involving septation of the parts of the primary tube not themselves directly involved in formation of the chamber-specific compartments of the atriums and ventricles. In this respect, when concluding our first review, we explained how the arterial trunks were also formed by septation within the solitary outflow tract of the primary heart tube. We also showed, however, that subsequent to formation of the two arterial trunks, there was disappearance of the cushions that initially divided them. Thus, in the definitive heart, the proximal parts of the aorta and pulmonary trunk, along with the sinuses of the arterial roots and the subpulmonary infundibulum, possess their own discrete walls, separated by extra-cardiac space. Although septation by fusion of endocardial cushions will be a topic included in this second review, septation and separation of the outflow tracts is sufficiently complicated to require special treatment. Because of this, we will devote a third review specifically to the mechanisms underscoring the remodelling of the outflow tracts. In this review, therefore, we will confine our considerations to the formation of the atrial, atrioventricular, and ventricular septal structures. As will become …

Lack of specificity of commercially available antisera against muscarinergic and adrenergic receptors

Commercially available antisera against five subtypes of muscarinic receptors and nine subtypes of adrenoceptors showed highly distinct immunohistochemical staining patterns in rat ureter and stomach. However, using the M1–4 muscarinic receptor subtypes and α2B-, β2-, and β3-adrenoceptors as examples, Western blots with membranes prepared from cell lines stably expressing various subtypes of muscarinic receptors or adrenoceptors revealed that each of the antisera recognized a set of proteins that differed between the cell lines used but lacked specificity for the claimed target receptor. We propose that receptor antibodies need better validation before they can reliably be used.

Causes of fecal and urinary incontinence after total mesorectal excision for rectal cancer based on cadaveric surgery: a study from the Cooperative Clinical Investigators of the Dutch total mesorectal excision trial

PURPOSE Total mesorectal excision (TME) for rectal cancer may result in anorectal and urogenital dysfunction. We aimed to study possible nerve disruption during TME and its consequences for functional outcome. Because the levator ani muscle plays an important role in both urinary and fecal continence, an explanation could be peroperative damage of the nerve supply to the levator ani muscle. METHODS TME was performed on cadaver pelves. Subsequently, the anatomy of the pelvic floor innervation and its relation to the pelvic autonomic innervation and the mesorectum were studied. Additionally, data from the Dutch TME trial were analyzed to relate anorectal and urinary dysfunction to possible nerve damage during TME procedure. RESULTS Cadaver TME surgery demonstrated that, especially in low tumors, the pelvic floor innervation can be damaged. Furthermore, the origin of the levator ani nerve was located in close proximity of the origin of the pelvic splanchnic nerves. Analysis of the TME trial data showed that newly developed urinary and fecal incontinence was present in 33.7% and 38.8% of patients, respectively. Both types of incontinence were significantly associated with each other (P = .027). Low anastomosis was significantly associated with urinary incontinence (P = .049). One third of the patients with newly developed urinary and fecal incontinence also reported difficulty in bladder emptying, for which excessive perioperative blood loss was a significant risk factor. CONCLUSION Perioperative damage to the pelvic floor innervation could contribute to fecal and urinary incontinence after TME, especially in case of a low anastomosis or damage to the pelvic splanchnic nerves.

Patterns of expression in the developing myocardium: towards a morphologically integrated transcriptional model

The heart is the first embryonic organ to function. Early in development, the heart shows autorhythmycity and peristaltoid contraction waves [1, 2]. Contraction requires the expression of a specific set of proteins that form the contractile apparatus, i.e. the sarcomere. The contraction–relaxation cycle of the sarcomeric apparatus is mediated by changing local concentrations of free calcium. This function is achieved by another set of specific proteins, located in the sarcoplasmic reticulum and in the sarcolemma. Fascinating questions that are still poorly understood are how the cardiogenic lineage becomes established to form the peristaltoid contracting tube without valves and how this tube becomes transformed into the synchronous-contracting four-chambered heart with unidirectional valves. It is well documented that the expression of the different isoforms of contractile proteins changes considerably during these stages (for a review see [3]). However, a detailed analysis of the changes in the patterns of gene expression in relation to cardiac morphogenesis is lacking. In the present review we try to fill this gap. We have centred our attention on gene products (mRNA and protein) expressed in the working myocardium of mammals and birds. No distinction has been made when mRNA and protein display the same pattern of expression, however we have highlighted those cases where the pattern of expression differs between mRNA and protein. The development and expression pattern of genes of the conduction system of the heart merits an independent review [4](Moorman et al., Circ. Res., in press). Data referring to other experimental models as Drosophila , Xenopus or zebrafish ( Danio rerio ) are included only if they are helpful for our general understanding. Often only gene expression in the presumptive atria and/or presumptive ventricles is mentioned, whereas understanding the functional significance of the patterns of gene expression requires knowledge of the entire pattern including the … * Corresponding author. Tel.: +31 (20) 5664928; fax: +31 (20) 6976177; e-mail: a.f.moorman@amc.uva.nl