Robert G. Gourdie

Epicardium-Derived Cells Contribute a Novel Population to the Myocardial Wall and the Atrioventricular Cushions

The epicardium and dorsal mesocardium are known to be the source of structures that form the wall of the coronary vessels. Because mouse knockout studies have shown that proper epicardial formation is also essential for myocardial development, we have studied in detail the migration and differentiation of epicardium-derived cells (EPDCs) within the developing heart. We constructed chicken-quail chimeras by grafting the quail epicardial organ, including a piece of primordial liver, at essentially stages 16 and 17. The embryos were studied at stages 25 to 43. To detect quail-derived EPDCs, an anti-quail nucleus antibody was used in combination with several differentiation markers, eg, for muscle actin, for vascular smooth muscle cells, for procollagen-I, for quail endothelium, and for Purkinje fibers. At stages 25 to 31, EPDCs are encountered in the myocardial wall and the subendocardial region. The latter deposition is spatially facilitated as the endocardium protrudes through transient discontinuities in the myocardium to contact the subepicardial layer. Later on, at stages 32 to 43, EPDCs invaded, by way of the atrioventricular sulcus, the atrioventricular cushion tissue. The localization is apparent at the interface with the myocardium, as well as subendocardially, but never within the endocardial lining. The origin of endothelium, smooth muscle cells, and fibroblasts of the coronary vessel wall from the epicardial graft were confirmed in accordance with already published data. The functional role of the novel EPDCs in the subendocardium, myocardium, and atrioventricular cushions remains to be investigated. A close positional relationship is found with the differentiating Purkinje fibers. Furthermore, a regulatory role is postulated in the process of endocardial-mesenchymal transformation. The ultimate fate of EPDCs seems to be a cardiac fibroblast cell line involved in the formation of the fibrous heart skeleton.

Dissociated Spatial Patterning of Gap Junctions and Cell Adhesion Junctions During Postnatal Differentiation of Ventricular Myocardium

Nonuniformity in the spatial patterning of gap junctions between heart muscle cells is now recognized as an important determinant of electromechanical function in working myocardium. Breakdown of the normal geometry of electrical intercellular connectivity in diseased myocardium correlates with reentry, arrhythmia, and conduction disturbance. The developmental mechanism(s) that determines this precise spatial order in gap junction organization in normal myocardium is at present unknown. To examine this question, we have used immunoelectron and immunoconfocal microscopy to analyze the spatial distributions of gap junctional (connexin43), desmosomal (desmoplakin), and adherens junctional (N-cadherin) components during maturation of rodent and canine left ventricular myocardium. In rats, a striking divergence in the distribution of gap junctions and cell adhesion junctions emerged within the first 20 days of postnatal life. It was found that although gap junctions initially demonstrated dispersed distributions across myocyte cell membranes, desmosomes and adherens junctions showed more rapid polarization toward cell termini (ie, nascent intercalated disks) after birth. Over subsequent postnatal development (20 to 90 postnatal days), gap junctions became progressively concentrated in these cell adhesion junction-rich zones of membrane. Quantitative analyses of this process in a series of rats aged 15 embryonic and 1, 5, 10, 20, 40, 70, and 90 postnatal days indicated that significantly higher levels (P < .01) of N-cadherin and desmoplakin than of connexin43 were immunolocalized to cell termini by as early as postnatal day 5. Although all three junctions types showed increasing polarization to myocyte termini with development, variation between junctions remained significant (P < .05) at all times points between 5 and 70 postnatal days. Only at 90 postnatal days, when the animals were nearly full grown, did the proportions of gap junction, desmosome, and adherens junction at intercalated disks become statistically similar (P > .05). Examination of myocardium from 1- and 3-month-old canines revealed that related differential changes to the spatiotemporal distribution of intercellular junctions occurred during postnatal maturation of the dog heart, suggesting that the process was not rodent specific. It is concluded that this progressive change in the organization and pattern of association between gap junctions and cell adhesion junctions is likely to be an important factor in maturation of electromechanical function within the mammalian heart.

Nkx2-5 mutation causes anatomic hypoplasia of the cardiac conduction system

Heterozygous mutations of the cardiac transcription factor Nkx2-5 cause atrioventricular conduction defects in humans by unknown mechanisms. We show in KO mice that the number of cells in the cardiac conduction system is directly related to Nkx2-5 gene dosage. Null mutant embryos appear to lack the primordium of the atrioventricular node. In Nkx2-5 haploinsufficiency, the conduction system has half the normal number of cells. In addition, an entire population of connexin40(-)/connexin45(+) cells is missing in the atrioventricular node of Nkx2-5 heterozygous KO mice. Specific functional defects associated with Nkx2-5 loss of function can be attributed to hypoplastic development of the relevant structures in the conduction system. Surprisingly, the cellular expression of connexin40, the major gap junction isoform of Purkinje fibers and a putative Nkx2-5 target, is unaffected, consistent with normal conduction times through the His-Purkinje system measured in vivo. Postnatal conduction defects in Nkx2-5 mutation may result at least in part from a defect in the genetic program that governs the recruitment or retention of embryonic cardiac myocytes in the conduction system.

Connexin45 (alpha 6) expression delineates an extended conduction system in the embryonic and mature rodent heart.

We previously demonstrated that alpha 6 (Cx45), one of the three connexins of the mammalian myocardium, is preferentially expressed in the peripheral portion of the ventricular conduction system in rats and mice. Here we report that alpha 6 is also prominently immunolocalized in the atrioventricular node and His bundle of these species. The distribution of immunolocalized alpha 6 reveals that the node and bundle form part of an extended central conductive network circumscribing the AV and outflow junctional regions of the fetal, and less continuously, the adult heart. Of the three cardiac connexins, alpha 6 is the isoform most continuously expressed by conduction tissues, and may thus account for the recently reported viability of the alpha 5 (Cx40) knockout mouse. It is concluded that alpha 6 expression is a defining feature of the heterogenous tissues comprising the atrioventricular conduction system of the rodent heart.

Immunolabelling patterns of gap junction connexins in the developing and mature rat heart

SummaryThe distribution of gap junctions in prenatal, postnatal, and adult rat hearts was studied by laser scanning confocal microscopy, using antiserum raised to a peptide (HJ) matching part of the sequence of connexin43 (a cardiac gap junction protein). Using digital reconstruction of optically-sectioned tissue volumes, a highly sensitive detection of immunolabelled gap junctions was achieved. The distribution of positive anti-HJ immunolabelling was regionalised in the prenatal heart from its first detection at 10 days post-coitus. High levels of immunopositive staining occurred in the trabeculae of the embryonic ventricles. Other zones of the early myocardium including early central conduction tissues had no detectable signal. The prenatal outflow tract, interventricular septum and a narrow zone of myocardium subjacent to the epicardial free wall also had low levels of immunopositive signal. During postnatal growth and in the adult rat heart, a marked distinction emerged between the central conducting tissues of the atria and ventricles. Whilst small immunostained gap junctions became detectable within the atrioventricular node on the atrial side of the junction, between the interatrial and interventricular septa, no immunolabelling was found within the ventricular branching bundle. This difference between the atrioventricular node and branching bundle is consistent with potential functional distinctions between these two structures, and is not consistent with the recent proposal that the His bundle and its branches act as an extended atrioventricular node in smaller mammals such as the rat. Ventricular Purkinje fibres, distal to the branching bundle, showed high levels of anti-HJ immunostaining. Organisation of gap junctions into intercalated disks within the ventricle proceeded late into the adolescent stages of heart growth. The distribution of a second connexin protein, MP70, not previously characterised in the heart, was studied using monoclonal antibodies. MP70 was transiently immunolabelled in the heart during the postnatal period, but only within valves. Previously, this protein has been reported only in the eye lens. MP70-containing gap junctions may represent a specialisation in avascular tissues, since blood vessels are not present in either the eye lens or the cusps of heart valves.

Connexin 43 connexon to gap junction transition is regulated by zonula occludens-1

Cx43 gap junctions (GJs) are integral to the function of the mammalian heart. It is shown that ZO-1 dynamically regulates the transition between Cx43 connexons and GJ intercellular channels, determining the balance of connexon-mediated cell permeability to GJ communication. Importantly, a novel domain proximal to GJs is identified—the perinexus.

Hemodynamics Is a Key Epigenetic Factor in Development of the Cardiac Conduction System

Abstract— The His-Purkinje system (HPS) is a network of conduction cells responsible for coordinating the contraction of the ventricles. Earlier studies using bipolar electrodes indicated that the functional maturation of the HPS in the chick embryo is marked by a topological shift in the sequence of activation of the ventricle. Namely, at around the completion of septation, an immature base-to-apex sequence of ventricular activation was reported to convert to the apex-to-base pattern characteristic of the mature heart. Previously, we have proposed that hemodynamics and/or mechanical conditioning may be key epigenetic factors in development of the HPS. We thus hypothesized that the timing of the topological shift marking maturation of the conduction system is sensitive to variation in hemodynamic load. Spatiotemporal patterns of ventricular activation (as revealed by high-speed imaging of fluorescent voltage-sensitive dye) were mapped in chick hearts over normal development, and following procedures previously characterized as causing increased (conotruncal banding, CTB) or reduced (left atrial ligation, LAL) hemodynamic loading of the embryonic heart. The results revealed that the timing of the shift to mature activation displays striking plasticity. CTB led to precocious emergence of mature HPS function relative to controls whereas LAL was associated with delayed conversion to apical initiation. The results from our study indicate a critical role for biophysical factors in differentiation of specialized cardiac tissues and provide the basis of a new model for studies of the molecular mechanisms involved in induction and patterning of the HPS in vivo.

Novel therapies for scar reduction and regenerative healing of skin wounds

Fibrotic scars deposited during skin wound healing can cause disfiguration and loss of dermal function. Scar differentiation involves inputs from multiple cell types in a predictable and overlapping sequence of cellular events that includes inflammation, migration/proliferation and extracellular matrix deposition. Research into the molecular mechanisms underpinning these processes in embryonic and adult wounds has contributed to the development of a growing number of novel therapeutic approaches for improving scar appearance. This review discusses some of these emerging strategies for shifting the balance of healing from scarring to regeneration in the context of non-pathological wounds. Particular focus is given to potential therapies based on transforming growth factor (TGF)-beta signaling and recent unexpected findings involving targeting of gap junctional connexins. Lessons learned in promoting scarless healing of cutaneous injuries might provide a basis for regenerative healing in other scenarios, such as spinal cord rupture or myocardial infarction.

Altered patterns of cardiac intercellular junction distribution in hypertrophic cardiomyopathy.

OBJECTIVE: To examine the distribution pattern of intercellular junctions (the mechanically coupling desmosomes and the electrically coupling gap junctions) in hypertrophic cardiomyopathy (HCM) hearts showing myofibre disarray. DESIGN: Samples from six necropsied hearts were studied, representing the interventricular septum and the free walls of the left and right ventricles. Immunohistochemical labelling of desmoplakin was used as a marker for desmosomes, and of connexin43 as a marker for gap junctions, in single and double stainings. The slides were examined by confocal laser scanning microscopy. RESULTS: Marked disorganisation of intercalated discs was observed in areas featuring myofibre disarray. Besides overall derangement, localised abnormalities in desmosome organisation were evident, which included: (1) the formation of abnormally enlarged megadiscs; (2) the presence of intersecting disc structures; and (3) aberrant side to side desmosomal connections. Gap junctional abnormalities included: (1) random distribution of gap junctions over the surface of myocytes, rather than localisation to intercalated discs; (2) abundant side to side gap junction connections between adjacent myocytes; and (3) formation of abnormally shaped gap junctions. Circles of myocytes continuously interconnected by gap junctions were also observed. Regions of the diseased hearts lacking myofibre disarray, and control hearts of normal patients and patients with other cardiac diseases, did not show these alterations. CONCLUSIONS: The disorganisation of the intercellular junctions associated with myofibre disarray in HCM may play an important role in the pathophysiological manifestations of the disease. The remodelling of gap junction distribution may underlie the formation of an arrhythmogenic substrate, thereby contributing to the generation and maintenance of cardiac arrhythmias associated with HCM.

A Novel Genetic Pathway for Sudden Cardiac Death via Defects in the Transition between Ventricular and Conduction System Cell Lineages

HF-1 b, an SP1 -related transcription factor, is preferentially expressed in the cardiac conduction system and ventricular myocytes in the heart. Mice deficient for HF-1 b survive to term and exhibit normal cardiac structure and function but display sudden cardiac death and a complete penetrance of conduction system defects, including spontaneous ventricular tachycardia and a high incidence of AV block. Continuous electrocardiographic recordings clearly documented cardiac arrhythmogenesis as the cause of death. Single-cell analysis revealed an anatomic substrate for arrhythmogenesis, including a decrease and mislocalization of connexins and a marked increase in action potential heterogeneity. Two independent markers reveal defects in the formation of ventricular Purkinje fibers. These studies identify a novel genetic pathway for sudden cardiac death via defects in the transition between ventricular and conduction system cell lineages.