Effect of altered haemodynamics on the developing mitral valve in chick embryonic heart

Abstract

Intracardiac haemodynamics is crucial for normal cardiogenesis, with recent evidence showing valvulogenesis is haemodynamically dependent and inextricably linked with shear stress. Although valve anomalies have been associated with genetic mutations, often the cause is unknown. However, altered haemodynamics have been suggested as a pathogenic contributor to bicuspid aortic valve disease. Conversely, how abnormal haemodynamics impacts mitral valve development is still poorly understood. In order to analyse altered blood flow, the outflow tract of the chick heart was constricted using a ligature to increase cardiac pressure overload. Outflow tract-banding was performed at HH21, with harvesting at crucial valve development stages (HH26, HH29 and HH35). Although normal valve morphology was found in HH26 outflow tract banded hearts, smaller and dysmorphic mitral valve primordia were seen upon altered haemodynamics in histological and stereological analysis at HH29 and HH35. A decrease in apoptosis, and aberrant expression of a shear stress responsive gene and extracellular matrix markers in the endocardial cushions were seen in the chick HH29 outflow tract banded hearts. In addition, dysregulation of extracellular matrix (ECM) proteins fibrillin-2, type III collagen and tenascin were further demonstrated in more mature primordial mitral valve leaflets at HH35, with a concomitant decrease of ECM cross-linking enzyme, transglutaminase-2. These data provide compelling evidence that normal haemodynamics are a prerequisite for normal mitral valve morphogenesis, and abnormal blood flow could be a contributing factor in mitral valve defects, with differentiation as a possible underlying mechanism.

Characterisation of the developing heart in a pressure overloaded model utilising RNA sequencing to direct functional analysis

“…These features are in line with previously reported characteristics of pressure overloaded embryonic hearts (Sedmera et al ; Tobita et al ; Hall et al ). Consistent with the literature (Pang et al ), phenotypic analysis revealed a VSD at HH29 (n = 26/31) (Fig. Bb,b’; denoted by an asterisk in Fig.…”

“…In addition, previous data from our group show changes in the ECM of the epicardium at a molecular and morphological level with a progressive aberrant phenotype from HH29 to HH35 (Perdios et al ). However, overall embryonic growth was not affected upon banding, as the cardiac output and overall embryo size are the similar in control and banded embryos (Clark et al ; Keller et al ; Pang et al ). In this study, evidence is provided of molecular mechanisms involved in OFT‐banded hearts, highlighting potential cardioprotective responses, with further analyses characterising the levels of apoptosis and glycogen accumulation.…”

“…The suture was passed through and removed immediately on sham controls. Untreated (UT) control embryos were opened and staged but no further procedures performed (Sedmera et al ; Pang et al ). All eggs were sealed and incubated for an additional 3–5 days until HH29‐35.…”

Development and evolution of the metazoan heart

“…Heterospecific interactions between a small number of the transcription factors Tbx5 and Nkx2.5, and GATA4, also part of the cardiogenic tool kit, underlying gene expression patterns in specific tissues, have been demonstrated in mammalian cardiogenesis . However, this shows much complexity and requires fine‐tuning involving additional partners, including histone modifications, epigenetic regulation, involvement of hemodynamics, regulation of cell movement, and participation of microRNAs to provide for expansion of cardiac progenitors, compartments, and cell differentiation.…”

“…Gene regulatory networks in the cardiac tool kits have been studied in mammalian embryos. These include but are not limited to Notch1, Slit‐Robo, Krox 20, Wnt signaling, TGFβ signaling, NFATc1, ADAM17, Msx, and matrix proteins such as laminin, versican, periostin, hyaluronan, tenascin and fibrillin, elastin, collagens, and others (see Pucéat).…”