Mutation of weak atrium/atrial myosin heavy chain disrupts atrial function and influences ventricular morphogenesis in zebrafish

The embryonic vertebrate heart is composed of two major chambers, a ventricle and an atrium, each of which has a characteristic size, shape and functional capacity that contributes to efficient circulation. Chamber-specific gene expression programs are likely to regulate key aspects of chamber formation. Here, we demonstrate that epigenetic factors also have a significant influence on chamber morphogenesis. Specifically, we show that an atrium-specific contractility defect has a profound impact on ventricular development. We find that the zebrafish locus weak atrium encodes an atrium-specific myosin heavy chain that is required for atrial myofibrillar organization and contraction. Despite their atrial defects, weak atrium mutants can maintain circulation through ventricular contraction. However, the weak atrium mutant ventricle becomes unusually compact, exhibiting a thickened myocardial wall, a narrow lumen and changes in myocardial gene expression. As weak atrium/atrial myosin heavy chain is expressed only in the atrium, the ventricular phenotypes in weak atrium mutants represent a secondary response to atrial dysfunction. Thus, not only is cardiac form essential for cardiac function, but there also exists a reciprocal relationship in which function can influence form. These findings are relevant to our understanding of congenital defects in cardiac chamber morphogenesis.     

Organization of cardiac chamber progenitors in the zebrafish blastula

Organogenesis requires the specification of a variety of cell types and the organization of these cells into a particular three-dimensional configuration. The embryonic vertebrate heart is organized into two major chambers, the ventricle and atrium, each consisting of two tissue layers, the myocardium and endocardium. The cellular and molecular mechanisms responsible for the separation of ventricular and atrial lineages are not well understood. To test models of cardiac chamber specification, we generated a high-resolution fate map of cardiac chamber progenitors in the zebrafish embryo at 40% epiboly, a stage prior to the initiation of gastrulation. Our map reveals a distinct spatial organization of myocardial progenitors: ventricular myocardial progenitors are positioned closer to the margin and to the dorsal midline than are atrial myocardial progenitors. By contrast, ventricular and atrial endocardial progenitors are not spatially organized at this stage. The relative orientations of ventricular and atrial myocardial progenitors before and after gastrulation suggest orderly movements of these populations. Furthermore, the initial positions of myocardial progenitors at 40% epiboly indicate that signals residing at the embryonic margin could influence chamber fate assignment. Indeed, via fate mapping, we demonstrate that Nodal signaling promotes ventricular fate specification near the margin, thereby playing an important early role during myocardial patterning.     

Patterning the zebrafish heart tube: acquisition of anteroposterior polarity.

The patterning of an internal organ, like the heart, is little understood. Central to this patterning is the formation, or the acquisition, of an anteroposterior (A-P) axis. We have approached the question of how the heart tube acquires polarity in the zebrafish, Brachydanio rerio, which offers numerous advantages for studying cardiac morphogenesis. During the early stages of organogenesis in the fish, the heart tube lies in an A-P orientation with the venous end lying anteriorly and the arterial end lying posteriorly. High doses (10(-6)-10(-5)M) of retinoic acid (RA) cause truncation of the body axis, as they do in Xenopus. Low doses of retinoic acid (10(-8)-10(-7) M), which do not appear to affect the rest of the embryo, have pronounced effects upon heart tube morphogenesis, causing it to shrink progressively along the A-P axis. To investigate this further, we identified monoclonal antibodies that distinguish between the zebrafish cardiac chambers and used them to show that the RA-induced cardiac truncation always begins at the arterial end of the heart tube. There is a continuous gradient of sensitivity from the arterial to the venous end, such that increasing RA exposure causes the progressive and sequential deletion first of the bulbus arteriosus and then, in order, of the ventricle, the atrium, and the sinus venosus. As exposure increases, parts of chambers are deleted before entire chambers; thus, the sensitivity to RA appears to be independent of chamber boundaries. The analysis of the heart tube's sensitivity to RA and its timing suggest that polarity is established during or shortly after initial commitment to the cardiac lineage.     

Screening mosaic F1 females for mutations affecting zebrafish heart induction and patterning

The genetic pathways underlying the induction and anterior-posterior patterning of the heart are poorly understood. The recent emergence of the zebrafish model system now allows a classical genetic approach to such challenging problems in vertebrate development. Two large-scale screens for mutations affecting zebrafish embryonic development have recently been completed; among the hundreds of mutations identified were several that affect specific aspects of cardiac morphogenesis, differentiation, and function. However, very few mutations affecting induction and/or anterior-posterior patterning of the heart were identified. We hypothesize that a directed approach utilizing molecular markers to examine these particular steps of heart development will uncover additional such mutations. To test this hypothesis, we are conducting two parallel screens for mutations that affect either the induction or the anterior-posterior patterning of the zebrafish heart. As an indicator of cardiac induction, we examine expression of nkx2.5, the earliest known marker of precardiac mesoderm; to assess anterior-posterior patterning, we distinguish ventricle from atrium with antibodies that recognize different myosin heavy chain isoforms. In order to expedite the examination of a large number of mutations, we are screening the haploid progeny of mosaic F1 females. In these ongoing screens, we have identified four mutations that affect nkx2.5 expression as well as 21 that disrupt either ventricular or atrial development and thus far have recovered several of these mutations, demonstrating the value of our approach. Future analysis of these and other cardiac mutations will provide further insight into the processes of induction and anterior-posterior patterning of the heart. Dev. Genet. 22:288–299, 1998. © 1998 Wiley-Liss, Inc.     

心脏特异表达绿色荧光斑马鱼模型的建立与评估

本课题组前期研究中, 利用斑马鱼cmlc2 (Cardiac myosin light chain 2)基因启动子构建了一个用于斑马鱼心脏组织特异表达外源基因的转基因表达载体pTol2-cmlc2-IRES-EGFP。文章利用该载体构建了一个稳定表达EGFP的转基因斑马鱼品系, 并初步分析了EGFP的表达对该转基因斑马鱼品系的心脏发育和功能的影响。结果表明, 在建立的转基因斑马鱼品系早期胚胎发育过程中, 绿色荧光信号在心脏中特异表达, 该表达模式与原位杂交分析的cmlc2的表达模式结果相同; 该转基因斑马鱼品系的心脏形态及发育生长正常; 进一步通过M-Mode分析心脏生理学功能的结果表明：该转基因品系心动周期、心率、收缩与舒张表面积及表面积缩短率等重要生理指标与正常野生型的斑马鱼对照组相比没有显著差别。以上结果表明该转基因品系中绿色荧光蛋白的表达对斑马鱼心脏的发育和功能没有影响。研究结果为进一步利用该载体建立外源目的基因转基因表达模型, 研究心脏表达基因的功能奠定了重要基础。     

Connexin43基因抑制对斑马鱼心血管系统发育的影响

为了研究cx43基因抑制对斑马鱼胚胎心血管系统发育的影响,针对cx43的翻译起始位点设计两个吗啉修饰的反义寡核苷酸抑制其表达,在斑弓鱼受精卵一到两细胞期混合注射并且验证其有效性.注射后用原位杂交和原位免疫荧光检测心脏标志基因的表达以及心脏的表型,同时利用显微荧光造影和原位杂交检测血管的发育情况.用心室心房的标志基因vmhc和amhc反义RNA探针进行的原位杂交结果显示,vmhc表达抑制,而amhc表达上调.原位免疫荧光显示与原位杂交一致的结果表明:心房扩张心室缩小,并且心脏环化不全.用血管标志基凶flk-1的RNA探针原位杂交和显微荧光造影表明,cx43基因抑制的斑马鱼胚胎血管无明显缺陷.此外,cx43基因抑制的斑马鱼胚胎心脏功能也有明显改变,包括心脏搏动无力,有血液回流现象.抑制cx43的表达可能通过影响两个细胞群的迁移导致斑马鱼胚胎心脏的发育缺陷,从而影响了心脏的功能,但是未发现胚胎血管系统发育的明显缺陷.     

斑马鱼心肌特异性启动子的克隆及其功能鉴定*

目的:探讨心室肌球蛋白重链(vmhc)基因启动子的心肌组织特异性.方法:利用PCR技术从斑马鱼基因组中克隆了vmhc编码区5’上游大小为1952bp的调控区域,应用酶切连接方法将vmhc启动子插入pGEFP-N1质粒,成功构建pEGFP-vmhc重组载体.再应用高保真DNA聚合酶PCR扩增包含vmhc启动子序列,增强型绿色荧光蛋白(EGFP)基因序列及3'UTR序列的基因片段,经过纯化后通过显微注射将vmhc-EGFP基因片段导入斑马鱼受精卵中.结果:注射后的斑马鱼心脏中出现绿色荧光,而其他部位无荧光出现.结论:vmhc启动子能够正确有效地驱动外源基因在斑马鱼心脏中特异表达,适合应用于心血管疾病的基因功能研究,基因靶向治疗等.     

In vitro motility assay of atrial and ventricular myosin from pig

The role of myosin isoforms in determining contractile filament velocity in the atrium and ventricle of the pig heart was studied by measuring the motion of fluorescently labeled actin over myosin (in vitro motility assay). A rapid and relatively simple method for purification of myosin from small tissue samples was used. The relative extent of light chain-2 phosphorylation was about 30% in both atrial and ventricular myosin extracts. Although the extracted myosin was not free from contaminating proteins, mainly actin, the mean velocity at optimal pH and 32°C of both atrial (3.3 μm/s) and ventricular (2.3 μm/s) myosin were similar to those obtained using extensively purified myosin. The filament sliding velocities using isolated myosin and actin are lower than those estimated from previously published experiments on skinned fiber preparations, which might reflect an influence on sliding velocity by the filament organization or regulatory proteins in the muscle fiber. However, the ratio between velocities of atrial and ventricular myosin was similar in the motility assay (1.5) and muscle fiber experiments (1.6), which might suggest that these two methods reflect the same fundamental processes in cardiac contraction and that the difference in filament sliding velocity between the atrium and ventricle of the pig heart is determined my their myosin isoforms. J. Cell. Biochem. 67:241–247, 1997. © 1997 Wiley-Liss, Inc.     

Semaphorin3D regulates invasion of cardiac neural crest cells into the primary heart field

The primary heart field in all vertebrates is thought to be derived exclusively from lateral plate mesoderm (LPM), which gives rise to a cardiac tube shortly after gastrulation. The heart tube then begins looping and additional cells are added from other embryonic regions, including the secondary heart field, cardiac neural crest and the proepicardial organ. Here we show in zebrafish that neural crest cells invade and contribute cardiac myosin light chain2 (cmlc2)-positive cardiomyocytes to the primary heart field. Knockdown of semaphorin3D, which is expressed in the neural crest but apparently not in LPM, reduces the size of the primary heart field and the number of cardiomyocytes in the primary heart field by 20% before formation of the primary heart tube. Sema3D morphants have subsequent complex congenital heart defects, including hypertrophic cardiomyocytes, decreased ventricular size and defects in trabeculation and in atrioventricular (AV) valve development. Neuropilin1A, a semaphorin receptor, is expressed in LPM but apparently not in the neural crest, and nrp1A morphants have cardiac development defects. We propose that a population of sema3D-dependent neural crest cells follow a novel migratory pathway, perhaps toward nrp1A-expressing LPM, and serve as an important early source of cardiomyocytes in the primary heart field.     

The slow force response to stretch in atrial and ventricular myocardium from human heart: functional relevance and subcellular mechanisms

Mechanical load is an important regulator of cardiac force. Stretching human atrial and ventricular trabeculae elicited a biphasic force increase: an immediate increase (Frank-Starling mechanism) followed by a further slow increase (slow force response, SFR). In ventricle, the SFR was unaffected by AT- and ET-receptor antagonism, by inhibition of protein-kinase-C, PI-3-kinase, and NO-synthase, but attenuated by inhibition of Na+/H+- (NHE) and Na+/Ca2+ exchange (NCX). In atrium, however, neither NHE- nor NCX-inhibition affected the SFR. Stretch elicited a large NHE-dependent [Na+]i increase in ventricle but only a small, NHE-independent [Na+]i increase in atrium. Stretch-activated non-selective cation channels contributed to basal force development in atrium but not ventricle and were not involved in the SFR in either tissue. Interestingly, inhibition of AT receptors or pre-application of angiotensin II or endothelin-1 reduced the atrial SFR. Furthermore, stretch increased phosphorylation of atrial myosin light chain 2 (MLC2) and inhibition of myosin light chain kinase (MLCK) attenuated the SFR in atrium and ventricle. Thus, in human heart both atrial and ventricular myocardium exhibit a stretch-dependent SFR that might serve to adjust cardiac output to increased workload. In ventricle, there is a robust NHE-dependent (but angiotensin II- and endothelin-1-independent) [Na+]i increase that is translated into a [Ca2+]i and force increase via NCX. In atrium, on the other hand, there is an angiotensin II- and endothelin-dependent (but NHE- and NCX-independent) force increase. Increased myofilament Ca2+ sensitivity through MLCK-induced phosphorylation of MLC2 is a novel mechanism contributing to the SFR in both atrium and ventricle.     

Nkx genes regulate heart tube extension and exert differential effects on ventricular and atrial cell number

Heart formation is a complex morphogenetic process, and perturbations in cardiac morphogenesis lead to congenital heart disease. NKX2-5 is a key causative gene associated with cardiac birth defects, presumably because of its essential roles during the early steps of cardiogenesis. Previous studies in model organisms implicate NKX2-5 homologs in numerous processes, including cardiac progenitor specification, progenitor proliferation, and chamber morphogenesis. By inhibiting function of the zebrafish NKX2-5 homologs, nkx2.5 and nkx2.7, we show that nkx genes are essential to establish the original dimensions of the linear heart tube. The nkx-deficient heart tube fails to elongate normally: its ventricular portion is atypically short and wide, and its atrial portion is disorganized and sprawling. This atrial phenotype is associated with a surplus of atrial cardiomyocytes, whereas ventricular cell number is normal at this stage. However, ventricular cell number is decreased in nkx-deficient embryos later in development, when cardiac chambers are emerging. Thus, we conclude that nkx genes regulate heart tube extension and exert differential effects on ventricular and atrial cell number. Our data suggest that morphogenetic errors could originate during early stages of heart tube assembly in patients with NKX2-5 mutations.     

The heart-forming fields: one or multiple?

The recent identification of a second mesodermal region as a source of cardiomyocytes has challenged the views on the formation of the heart. This second source of cardiomyocytes is localized centrally on the embryonic disc relative to the remainder of the classic cardiac crescent, a region also called the pharyngeal mesoderm. In this review, we discuss the concept of the primary and secondary cardiogenic fields in the context of folding of the embryo, and the subsequent temporal events involved in formation of the heart. We suggest that, during evolution, the heart developed initially only with the components required for a systemic circulation, namely a sinus venosus, a common atrium, a 'left' ventricle and an arterial cone, the latter being the myocardial outflow tract as seen in the heart of primitive fishes. These components developed in their entirety from the classic cardiac crescent. Only later in the course of evolution did the appearance of novel signalling pathways permit the central part of the cardiac crescent, and possibly the contiguous pharyngeal mesoderm, to develop into the cardiac components required for the pulmonary circulation. These latter components comprise the right ventricle, and that part of the left atrium that derives from the mediastinal myocardium, namely the dorsal atrial wall and the atrial septum. It is these elements which are now recognized as developing from the second field of pharyngeal mesoderm. We suggest that, rather than representing development from separate fields, the cardiac components required for both the systemic and pulmonary circulations are derived by patterning from a single cardiac field, albeit with temporal delay in the process of formation.     

Myosin heavy chain isoforms in the myocardium of the atrioventricular junction of Scyliorhinus canicula (Chondrichthyes,Carcharhiniformes)

The atrioventricular junction of the fish heart, namely the segment interposed between the single atrium and the single ventricle, has been studied anatomically and histologically in several chondrichthyan and teleost species. Nonetheless, knowledge about myosin heavy chain (MyHC) in the atrioventricular myocardium remains scarce. The present report is the first one to provide data on the MyHC isoform distribution in the myocardium of the atrioventricular junction in chondrichthyans, specifically in the lesser spotted dogfish, Scyliorhinus canicula, a shark species whose heart reflects the primitive cardiac anatomical design in gnathostomes. Hearts from five dogfish were examined using histochemical and immunohistochemical techniques. The anti-MyHC A4.1025 antibody was used to detect differences in the occurrence of MyHC isoforms in the dogfish, as the fast-twitch isoforms MYH2 and MYH6 have a higher affinity for this antibody than the slow-twitch isoforms MYH7 and MYH7B. The histochemical findings show that myocardium of the atrioventricular junction connects the trabeculated myocardium of the atrium with the trabeculated layer of the ventricular myocardium. The immunohistochemical results indicate that the distribution of MyHC isoforms in the atrioventricular junction is not homogeneous. The atrial portion of the atrioventricular myocardium shows a positive reactivity against the A4.1025 antibody similar to that of the atrial myocardium. In contrast, the ventricular portion of the atrioventricular junction is not labelled, as is the case with the ventricular myocardium. This dual condition suggests that the myocardium of the atrioventricular junction has two contraction patterns: the myocardium of the atrial portion contracts in line with the atrial myocardium, whereas that of the ventricular portion follows the contraction pattern of the ventricular myocardium. Thus, the transition of the contraction wave from the atrium to the ventricle may be established in the atrioventricular segment because of its heterogeneous MyHC isoform distribution. The findings support the hypothesis that a distinct MyHC isoform distribution in the atrioventricular myocardium enables a synchronous contraction of inflow and outflow cardiac segments in vertebrates lacking a specialized cardiac conduction system.     

Differential regulation of the atrial isoforms of the myosin light chains during striated muscle development.

We have isolated a cDNA that encodes the human regulatory myosin light chain isoform predominant in adult atrial muscle. The cDNA contains an open reading frame of 175 amino acids and encodes a hydrophilic protein of a largely helical structure with two potential phosphorylation sites. The protein is different from any other regulatory myosin light chain so far described and is the product of a previously uncharacterized single copy gene. An isoform-specific probe was used to analyze the expression of this isoform in adult muscle and in cardiac and skeletal muscle development in vivo and in vitro. Parallel analysis of the corresponding human alkali myosin light chain (predominant in adult atrium) showed that both isoforms are expressed in early heart development, in both atrium and ventricle. Although the atrial alkali light chain is expressed throughout embryonic striated muscle development, the regulatory myosin light chain was not detected in skeletal myogenesis in vivo or in vitro. Thus the atrial isoforms are not universally or exclusively "paired" and can be independently regulated. We propose that the manner in which these particular isoforms fulfill the functional requirements of the muscle at different developmental times may have direct impact on their regulation.     

Increased cardiac alpha-myosin heavy chain in left atria and decreased myocardial insulin-like growth factor (Igf-I) expression accompany low heart rate in hibernating grizzly bears

Grizzly bears (Ursus arctos horribilis) tolerate extended periods of extremely low heart rate during hibernation without developing congestive heart failure or cardiac chamber dilation. Left ventricular atrophy and decreased left ventricular compliance have been reported in this species during hibernation. We evaluated the myocardial response to significantly reduced heart rate during hibernation by measuring relative myosin heavy-chain (MyHC) isoform expression and expression of a set of genes important to muscle plasticity and mass regulation in the left atria and left ventricles of active and hibernating bears. We supplemented these data with measurements of systolic and diastolic function via echocardiography in unanesthetized grizzly bears. Atrial strain imaging revealed decreased atrial contractility, decreased expansion/reservoir function (increased atrial stiffness), and decreased passive-filling function (increased ventricular stiffness) in hibernating bears. Relative MyHC-α protein expression increased significantly in the atrium during hibernation. The left ventricle expressed 100% MyHC-β protein in both groups. Insulin-like growth factor (IGF-I) mRNA expression was reduced by ～50% in both chambers during hibernation, consistent with the ventricular atrophy observed in these bears. Interestingly, mRNA expression of the atrophy-related ubiquitin ligases Muscle Atrophy F-box (MAFBx) and Muscle Ring Finger 1 did not increase, nor did expression of myostatin or hypoxia-inducible factor 1α (HIF-1α). We report atrium-specific decreases of 40% and 50%, respectively, in MAFBx and creatine kinase mRNA expression during hibernation. Decreased creatine kinase expression is consistent with lowered energy requirements and could relate to reduced atrial emptying function during hibernation. Taken together with our hemodynamic assessment, these data suggest a potential downregulation of atrial chamber function during hibernation to prevent fatigue and dilation due to excessive work against an optimally filled ventricle, a response unpredicted by the Frank-Starling mechanism.     

The PAF1 complex differentially regulates cardiomyocyte specification

The specification of an appropriate number of cardiomyocytes from the lateral plate mesoderm requires a careful balance of both positive and negative regulatory signals. To identify new regulators of cardiac specification, we performed a phenotype-driven ENU mutagenesis forward genetic screen in zebrafish. In our genetic screen we identified a zebrafish ctr9 mutant with a dramatic reduction in myocardial cell number as well as later defects in primitive heart tube elongation and atrioventricular boundary patterning. Ctr9, together with Paf1, Cdc73, Rtf1 and Leo1, constitute the RNA polymerase II associated protein complex, PAF1. We demonstrate that the PAF1 complex (PAF1C) is structurally conserved among zebrafish and other metazoans and that loss of any one of the components of the PAF1C results in abnormal development of the atrioventricular boundary of the heart. However, Ctr9, Cdc73, Paf1 and Rtf1, but not Leo1, are required for the specification of an appropriate number of cardiomyocytes and elongation of the heart tube. Interestingly, loss of Rtf1 function produced the most severe defects, resulting in a nearly complete absence of cardiac precursors. Based on gene expression analyses and transplantation studies, we found that the PAF1C regulates the developmental potential of the lateral plate mesoderm and is required cell autonomously for the specification of cardiac precursors. Our findings demonstrate critical but differential requirements for PAF1C components in zebrafish cardiac specification and heart morphogenesis.