Hemodynamics in Cardiac Development

Abstract

The beating heart is subject to intrinsic mechanical factors, exerted by contraction of the myocardium (stretch and strain) and fluid forces of the enclosed blood (wall shear stress). The earliest contractions of the heart occur already in the 10-somite stage in the tubular as yet unsegmented heart. With development, the looping heart becomes asymmetric providing varying diameters and curvatures resulting in unequal flow profiles. These flow profiles exert various wall shear stresses and as a consequence different expression patterns of shear responsive genes. In this paper we investigate the morphological alterations of the heart after changing the blood flow by ligation of the right vitelline vein in a model chicken embryo and analyze the extended expression in the endocardial cushions of the shear responsive gene Tgfbeta receptor III. A major phenomenon is the diminished endocardial-mesenchymal transition resulting in hypoplastic (even absence of) atrioventricular and outflow tract endocardial cushions, which might be lethal in early phases. The surviving embryos exhibit several cardiac malformations including ventricular septal defects and malformed semilunar valves related to abnormal development of the aortopulmonary septal complex and the enclosed neural crest cells. We discuss the results in the light of the interactions between several shear stress responsive signaling pathways including an extended review of the involved Vegf, Notch, Pdgf, Klf2, eNos, Endothelin and Tgfβ/Bmp/Smad networks.

Keywords: Klf2; TGF beta; cardiogenesis; endocardial cushions; growth factors; hemodynamics; neural crest; outflow tract septum; semilunar valve; shear stress.

Figure 1

Cardiac looping in normal and ligated embryos HH20. Scanning electron micrograph (SEM) of ventral views. (a) Normal embryo with cardiac segments indicated. (b) Ligated embryo. The retarded looping resembles that of a HH17 embryo with an open inner curvature (*). The AV canal is relatively long. (c,d) Interior view of dorsal heart halves. (c) The inferior AV cushion and the OFT cushions are well developed, ventricular trabeculations have formed. (d) AV and OFT cushions are non-existent, spongy trabeculations and the compact myocardium is thin (arrowheads). AV: Atrioventricular groove, DOT: distal OFT, IAV inferior AV cushion, LA: left part of atrium, M: compact myocardium, OTC: OFT cushions, POT: proximal OFT, VI ventricular inlet, VO: ventricular outlet, * inner curvature, arrowheads: thin compact myocardium.

Figure 2

Expression of TGFβ type III receptor. (a) Normal left and right atrium of HH20. No TBRIII expression in atrial endocardium. (b) Ligated embryo with ectopic TBRIII expression along the atrial septum and atrial floor (arrows). (c) AV cushions of normal HH22 embryo, TBRIII expression is downregulated. (d) Ligated embryo with prolonged endocardial TBRIII expression (arrows). (e) Normal HH20 embryo, ventricular trabeculations lack TBRIII. (f) Ectopic TBRIII expression of the endocardium lining ventricular trabeculations after ligation (arrows). (g) Higher magnification of the boxed area of (e). (h) Higher magnification of the boxed area of (f). IAV: inferior AV cushion, LA: left atrium, RA: right atrium, SA: superior AV cushion.

Figure 3

SEM of HH34 postseptation embryos viewed from apex to base. (a) Normal heart. The subpulmonary infundibulum (*) is continuous with the interventricular septum (IVS). (b) Ligated embryo with a subaortic ventricular septal defect (VSD). The line of the fused OFT cushions is indicated (arrow). Both arterial orifices (SA and P) are situated above the right ventricle (double outlet right ventricle). The aorta is stenotic (SA) and the mitral valve leaflets (MV) are abnormal. The myocardium is severely affected, as seen by a thin IVS and compact layer (M) and spongy trabeculations (TR). IVS: interventricular septum, LVO: left ventricular OFT, M: compact myocardium, MV: mitral valve, P: pulmonary orifice, RVO: right ventricular OFT, SA: stenotic aorta, TR: trabeculations, TV: tricuspid valve.

Figure 4

LacZ tracing and apoptosis of neural crest (NC) cells immunostained for actin. (a) Proximal OFT region of a retrovirus infected, non-ligated HH31 embryo. Myocardialization of the OFT is nearly complete (*) as the opposing parts of the myocardium (M) almost touch (arrows). NC cells (blue) are present in the mesenchyme and myocardium (arrowheads). (b) Adjacent section with magnified AP septal complex, subjected to Tdt-mediated dUTP nick end labeling (TUNEL) for apoptotic cells (brown). Most of the apoptotic (brown) cells are also blue, indicating NC cells. After apoptosis, X-gal granules give way the position of the original NC cell (arrows). (c) HH37 ligated embryo with a subarterial VSD, showing confluence of the semilunar valve leaflets without myocardialization (arrows far apart). (d) Retrovirus infected, ligated HH37 embryo. Numerous blue NC cells in the ventral prong of the AP-septal complex. Adjacent TUNEL stained sections (not shown) presented no apoptotic cells. (e) Retrovirus infected, non-ligated HH37 embryo with full myocardialization (arrows meet each other) only a single blue NC cell (arrowhead) has been registered. (f) Ligated HH37 embryo showing a subaortic VSD with substantial myocardialization of the AP septal complex (compare with (c)). Ao: aorta, LVO: left ventricular OFT, M myocardium, P: pulmonary trunk, RA: right atrium, RV: right ventricle, RVO: right ventricular OFT.

Figure 5

Alteration in laminar flow invokes changes in mechanoreceptors such as monocilia and integrins, followed by activation of e.g., the Mek/Erk-Mef2 cascade. Mef2 activates nuclear expression of Klf2 thereby influencing Et-1, eNos, Vegf, Smads (particularly P-Smad2, and Smad4/7) and probably Pdgf-B. Upregulation of Klf2 by changes in laminar flow (but also by Angiopoietin-activation of the Tie2 receptor and by statins) involves down-regulation of most of the latter genes. It is interesting to note that oxidative stress and cytokine but also oscillatory flow not sensed by monocilia downregulate Klf2 expression. Data mainly based on [3,5,63,105].