Cardiac development in the chick embryo with reference to conduction and structure using a myosin heavy chain knock down model and global RNA sequencing in an outflow tract banded model

In the developing embryo the heart is the first organ to develop and thus supply the rest of the developing embryo with a good blood supply. Regulation of cardiogenesis in these early stages of development is key as any dysregulation will result in defects in the heart. Dysregulation of sarcomeric proteins has been associated with a range of cardiomyopathies and septal defects. This demonstrates the importance of structure on development. However, structural genes have not been linked to conduction disorders in the heart. Myosin heavy chain genes (MYH) encode sarcomeric structural proteins (MHC). Previous work by Rutland et al. (2011) showed that alpha myosin heavy chain (αMHC), beta myosin heavy chain (βMHC) and embryonic myosin heavy chain (eMHC) are necessary for correct Ca2+ transients, with eMHC also required for a normal action potential and normal intracellular K+. The thesis uses a chick model to analyse the effect of structure on the conduction system. The first part of the thesis utilises antisense oligonucleotide morpholino technology for gene knockdown (KD) of the mRNA of αMCH, βMHC and eMHC proteins to analyse the effect on structure and conduction in the heart. Cultures of atrial and ventricular KD HH29 cells showed no differences in beating rate, though 2 out of 9 samples of αMYH and eMYH culture failed to form beating syncytiums, compared to all controls that did. The structural maturity of KD cultures was assessed through Z-disc integrity by immunocytochemistry. Decreased maturity of both the atria and ventricular culture was found KDs. Expression of selected conduction genes was also assed with the pace maker cell potassium channel HCN4 showing decreased levels at the sinus venosus region by in situ hybridisation and significant decreases by RT-qPCR in whole chick heart embryos at HH20. The decrease in expression of such genes could be caused by disruption to internal cell architecture that organises expression of proteins at the cell membrane. Structure at the Z-disc, which show immaturity in KD hearts, is key for this process. Altered haemodynamics and cardiomyopathy is known to effect internal heart structure and the next phase of the project utilised an out flow tract banding (OFT-banding) technique that has been shown previously to alter haemodynamics, effect heart structure and show features of cardiomyopathy. Global sequencing was carried out in order to assess the effect that OFT-banding may have on the conduction system in HH29 chick embryos. In order to carry out global gene expression on OFT-banded hearts, a library preparation method was optimised that removed excess haemoglobin from the hearts and an >99% reduction in all embryonic globin genes was seen, this then allowed detailed gene analysis even of low read genes by RNA sequencing. Sequencing revealed differential expression of calcium sequestering genes in what appears to be a conductive cardioprotective mechanism to maintain coordinated contraction. Interestingly, sequencing also revealed a gene profile that would be expected to alter AMPK signalling that could lead to a multitude of disorders or affects such glycogen storage cardiomyopathy or increased inhibition of myosin expression

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

Cardiogenesis is influenced by both environmental and genetic factors, with blood flow playing a critical role in cardiac remodelling. Perturbation of any of these factors could lead to abnormal heart development and hence the formation of congenital heart defects. Although abnormal blood flow has been associated with a number of heart defects, the effects of abnormal pressure load on the developing heart gene expression profile have to date not clearly been defined. To determine the heart transcriptional response to haemodynamic alteration during development, outflow tract (OFT) banding was employed in the chick embryo at Hamburger and Hamilton stage (HH) 21. Stereological and expression studies, including the use of global expression analysis by RNA sequencing with an optimised procedure for effective globin depletion, were subsequently performed on HH29 OFT‐banded hearts and compared with sham control hearts, with further targeted expression investigations at HH35. The OFT‐banded hearts were found to have an abnormal morphology with a rounded appearance and left‐sided dilation in comparison with controls. Internal analysis showed they typically had a ventricular septal defect and reductions in the myocardial wall and trabeculae, with an increase in the lumen on the left side of the heart. There was also a significant reduction in apoptosis. The differentially expressed genes were found to be predominately involved in contraction, metabolism, apoptosis and neural development, suggesting a cardioprotective mechanism had been induced. Therefore, altered haemodynamics during development leads to left‐sided dilation and differential expression of genes that may be associated with stress and maintaining cardiac output

Cardiac troponin T is necessary for normal development in the embryonic chick heart

The heart is the first functioning organ to develop during embryogenesis. The formation of the heart is a tightly regulated and complex process, and alterations to its development can result in congenital heart defects. Mutations in sarcomeric proteins, such as alpha myosin heavy chain and cardiac alpha actin, have now been associated with congenital heart defects in humans, often with atrial septal defects. However, cardiac troponin T (cTNT encoded by gene TNNT2) has not. Using gene-specific antisense oligonucleotides, we have investigated the role of cTNT in chick cardiogenesis. TNNT2 is expressed throughout heart development and in the postnatal heart. TNNT2-morpholino treatment resulted in abnormal atrial septal growth and a reduction in the number of trabeculae in the developing primitive ventricular chamber. External analysis revealed the development of diverticula from the ventricular myocardial wall which showed no evidence of fibrosis and still retained a myocardial phenotype. Sarcomeric assembly appeared normal in these treated hearts. In humans, congenital ventricular diverticulum is a rare condition, which has not yet been genetically associated. However, abnormal haemodynamics is known to cause structural defects in the heart. Further, structural defects, including atrial septal defects and congenital diverticula, have previously been associated with conduction anomalies. Therefore, to provide mechanistic insights into the effect that cTNT knockdown has on the developing heart, quantitative PCR was performed to determine the expression of the shear stress responsive gene NOS3 and the conduction gene TBX3. Both genes were differentially expressed compared to controls. Therefore, a reduction in cTNT in the developing heart results in abnormal atrial septal formation and aberrant ventricular morphogenesis. We hypothesize that alterations to the haemodynamics, indicated by differential NOS3 expression, causes these abnormalities in growth in cTNT knockdown hearts. In addition, the muscular diverticula reported here suggest a novel role for mutations of structural sarcomeric proteins in the pathogenesis of congenital cardiac diverticula. From these studies, we suggest TNNT2 is a gene worthy of screening for those with a congenital heart defect, particularly atrial septal defects and ventricular diverticula

Altered haemodynamics cause aberrations in the epicardium

During embryo development, the heart is the first functioning organ. Although quiescent in the adult, the epicardium is essential during development to form a normal four‐chambered heart. Epicardial‐derived cells contribute to the heart as it develops with fibroblasts and vascular smooth muscle cells. Previous studies have shown that a heartbeat is required for epicardium formation, but no study to our knowledge has shown the effects of haemodynamic changes on the epicardium. Since the aetiologies of many congenital heart defects are unknown, we suggest that an alteration in the heart's haemodynamics might provide an explanatory basis for some of them. To change the heart's haemodynamics, outflow tract (OFT) banding using a double overhang knot was performed on HH21 chick embryos, with harvesting at different developmental stages. The epicardium of the heart was phenotypically and functionally characterised using a range of techniques. Upon alteration of haemodynamics, the epicardium exhibited abnormal morphology at HH29, even though migration of epicardial cells along the surface of the heart was found to be normal between HH24 and HH28. The abnormal epicardial phenotype was exacerbated at HH35 with severe changes in the structure of the extracellular matrix (ECM). A number of genes tied to ECM production were also differentially expressed in HH29 OFT‐banded hearts, including DDR2 and collagen XII. At HH35, the differential expression of these genes was even greater with additional downregulation of collagen I and TCF21. In this study, the epicardium was found to be severely impacted by altered haemodynamics upon OFT banding. The increased volume of the epicardium at HH29, upon OFT‐banding, and the expression changes of ECM markers were the first indicative signs of aberrations in epicardial architecture; by HH35, the phenotype had progressed. The decrease in epicardial thickness at HH35 suggests an increase in tension, with a force acting perpendicular to the surface of the epicardium. Although the developing epicardium and the blood flowing through the heart are separated by the endocardium and myocardium, the data presented here demonstrate that altering the blood flow affects the structure and molecular expression of the epicardial layer. Due to the intrinsic role the epicardium in cardiogenesis, defects in epicardial formation could have a role in the formation of a wide range of congenital heart defects

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

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

Cardiac development in the chick embryo with reference to conduction and structure using a myosin heavy chain knock down model and global RNA sequencing in an outflow tract banded model

In the developing embryo the heart is the first organ to develop and thus supply the rest of the developing embryo with a good blood supply. Regulation of cardiogenesis in these early stages of development is key as any dysregulation will result in defects in the heart. Dysregulation of sarcomeric proteins has been associated with a range of cardiomyopathies and septal defects. This demonstrates the importance of structure on development. However, structural genes have not been linked to conduction disorders in the heart. Myosin heavy chain genes (MYH) encode sarcomeric structural proteins (MHC). Previous work by Rutland et al. (2011) showed that alpha myosin heavy chain (αMHC), beta myosin heavy chain (βMHC) and embryonic myosin heavy chain (eMHC) are necessary for correct Ca2+ transients, with eMHC also required for a normal action potential and normal intracellular K+. The thesis uses a chick model to analyse the effect of structure on the conduction system. The first part of the thesis utilises antisense oligonucleotide morpholino technology for gene knockdown (KD) of the mRNA of αMCH, βMHC and eMHC proteins to analyse the effect on structure and conduction in the heart. Cultures of atrial and ventricular KD HH29 cells showed no differences in beating rate, though 2 out of 9 samples of αMYH and eMYH culture failed to form beating syncytiums, compared to all controls that did. The structural maturity of KD cultures was assessed through Z-disc integrity by immunocytochemistry. Decreased maturity of both the atria and ventricular culture was found KDs. Expression of selected conduction genes was also assed with the pace maker cell potassium channel HCN4 showing decreased levels at the sinus venosus region by in situ hybridisation and significant decreases by RT-qPCR in whole chick heart embryos at HH20. The decrease in expression of such genes could be caused by disruption to internal cell architecture that organises expression of proteins at the cell membrane. Structure at the Z-disc, which show immaturity in KD hearts, is key for this process. Altered haemodynamics and cardiomyopathy is known to effect internal heart structure and the next phase of the project utilised an out flow tract banding (OFT-banding) technique that has been shown previously to alter haemodynamics, effect heart structure and show features of cardiomyopathy. Global sequencing was carried out in order to assess the effect that OFT-banding may have on the conduction system in HH29 chick embryos. In order to carry out global gene expression on OFT-banded hearts, a library preparation method was optimised that removed excess haemoglobin from the hearts and an >99% reduction in all embryonic globin genes was seen, this then allowed detailed gene analysis even of low read genes by RNA sequencing. Sequencing revealed differential expression of calcium sequestering genes in what appears to be a conductive cardioprotective mechanism to maintain coordinated contraction. Interestingly, sequencing also revealed a gene profile that would be expected to alter AMPK signalling that could lead to a multitude of disorders or affects such glycogen storage cardiomyopathy or increased inhibition of myosin expression