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Keywords:

  • cardiac morphogenesis;
  • shear stress;
  • myocardial function

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. BRIEF REVIEW OF HEART MORPHOGENESIS
  5. BIOMECHANICAL THEORIES OF HEART DEVELOPMENT
  6. INSTANCES OF ASSOCIATED DEFECTS IN BOTH HEART FUNCTION AND MORPHOGENESIS
  7. POTENTIAL MECHANISMS FOR THE FUNCTION–FORM LINK
  8. FUTURE DIRECTIONS
  9. REFERENCES

For years, biomechanical engineers have studied the physical forces involved in morphogenesis of the heart. In a parallel stream of research, molecular and developmental biologists have sought to identify the molecular pathways responsible for embryonic heart development. Recently, several studies have shown that these two avenues of research should be integrated to explain how genes expressed in the heart regulate early heart function and affect physical morphogenetic steps, as well as to conversely show how early heart function affects the expression of genes required for morphogenesis. This review combines the perspectives of biomechanical engineering and developmental biology to lay out an integrated view of the role of mechanical forces in heart development. Developmental Dynamics 233:373–381, 2005. © 2005 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. BRIEF REVIEW OF HEART MORPHOGENESIS
  5. BIOMECHANICAL THEORIES OF HEART DEVELOPMENT
  6. INSTANCES OF ASSOCIATED DEFECTS IN BOTH HEART FUNCTION AND MORPHOGENESIS
  7. POTENTIAL MECHANISMS FOR THE FUNCTION–FORM LINK
  8. FUTURE DIRECTIONS
  9. REFERENCES

During embryogenesis, the heart is the first organ to begin mechanical function and that function is initiated well before structural organogenesis is complete. Whereas it would seem obvious that organs with structural defects will also have functional defects, the fact that the heart begins to function before final structure is achieved raises the possibility that its early mechanical function affects its own morphogenesis. In the embryo, proper structure and function of many organs is not required for survival, but defects in heart function or structure are often embryonic-lethal. Therefore, the heart's ability to support the needs of the embryo while undergoing very significant changes in morphology is fascinating and makes this the premier organ for study if one wishes to examine structure–function or function–structure relationships during organogenesis.

The relationship between function and form of the heart has been an area of vigorous research among those interested in late-onset or acquired cardiac diseases, such as the cardiomyopathies. Studies in this literature have shown that changes in myocardial function due to either intrinsic or extrinsic factors cause a remodeling of the heart in an attempt to compensate for the functional change, and ongoing research is concentrated on identifying the genes that mediate remodeling events and determining how changes in function lead to the activation of these genes. Recent data that will be discussed in the following pages indicate that this phenomenon—one for which function affects form—exists during the initial modeling of the heart as well. As research in each of these fields continues, it will be interesting to see if the genes and processes involved in connecting early heart function with morphogenesis are the same as those involved in connecting late heart function with remodeling.

BRIEF REVIEW OF HEART MORPHOGENESIS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. BRIEF REVIEW OF HEART MORPHOGENESIS
  5. BIOMECHANICAL THEORIES OF HEART DEVELOPMENT
  6. INSTANCES OF ASSOCIATED DEFECTS IN BOTH HEART FUNCTION AND MORPHOGENESIS
  7. POTENTIAL MECHANISMS FOR THE FUNCTION–FORM LINK
  8. FUTURE DIRECTIONS
  9. REFERENCES

Heart morphogenesis varies somewhat between species but can generally be broken down into the steps of cardiomyocyte determination and specification, formation of the heart tube, looping, chamber development and growth, and endocardial cushion, valve, and septal morphogenesis (Fig. 1). Many excellent reviews discuss the molecular biology of these steps in detail (e.g., Srivastava and Olson, 2000; Srivastava, 2001), so the reader is directed elsewhere for further information. Pertaining to structure–function relationships, the heartbeat appears to initiate during the late period of heart tube formation and early looping; therefore, all steps subsequent to this potentially may be affected by myocardial function.

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Figure 1. Steps of heart morphogenesis. Illustrations depict cardiac development with conserved shading of morphologically related regions, seen from a ventral view. Cardiogenic precursors form a crescent (far-left panel) that is specified to form specific segments of the linear heart tube, which is patterned along the anteroposterior axis to form the various regions and chambers of the looped and mature heart. Each cardiac chamber balloons out from the outer curvature of the looped heart tube in a segmental manner. Neural crest cells populate the bilaterally symmetric aortic arch arteries (III, IV, and VI) and aortic sac (AS) that together contribute to specific segments of the mature aortic arch. Mesenchymal cells form the cardiac valves from the contruncal (CT) and atrioventricular valve (AVV) segments. Corresponding days of human embryonic development are indicated. A, atrium; V, ventricle; RV, right ventricle; LV, left ventricle; RA, right atrium; LA, left atrium; PA, pulmonary artery; Ao, aorta; DA, ductus arteriosus; RSCA, right subclavian artery; LSCA, left subclavian artery; RCC, right common carotid; LCC, left common carotid. Used, with permission, from Nature Publishing Group (Srivastava and Olson, 2000).

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Despite our increasing knowledge of the genes expressed during heart development, we know little about how these genes cause mechanical rearrangements during morphogenesis or how expression of these genes may in turn be affected by heart mechanics. For example, Shh, nodal, lefty, fibroblast growth factor (FGF), and Pitx2 (Linask et al., 2002), among others, are involved in heart looping (all reviewed in Yost, 2001), yet we do not know how asymmetric expression and action of these molecules leads to a mechanical looping and structural asymmetry of the heart. During chamber development, pressure and volume loading of the heart should create wall stress, and later on, the developing endocardial cushions and valves are subject to significant shear stress, yet we have limited understanding of how mechanical forces affect localized gene expression and, therefore, influence morphogenesis in these tissues. As we later discuss, there is mounting evidence of associations between function and form during heart development, but models that unify biomechanics and molecular/cellular biology still need to be further developed by those working on both sides of this field.

BIOMECHANICAL THEORIES OF HEART DEVELOPMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. BRIEF REVIEW OF HEART MORPHOGENESIS
  5. BIOMECHANICAL THEORIES OF HEART DEVELOPMENT
  6. INSTANCES OF ASSOCIATED DEFECTS IN BOTH HEART FUNCTION AND MORPHOGENESIS
  7. POTENTIAL MECHANISMS FOR THE FUNCTION–FORM LINK
  8. FUTURE DIRECTIONS
  9. REFERENCES

The extraordinary transformation of the vertebrate heart during cardiac morphogenesis is an excellent example of the precise interplay between form and function during development. Cardiogenesis involves changes in organ mass (growth), physical properties (remodeling), and shape (morphogenesis). Before birth, the developing heart grows by increasing the number (hyperplasia) of cardiac myocytes, whereas postparturition growth is characterized by increasing the size (hypertrophy) of those cells (Clark et al., 1989). Despite the dramatic morphological changes occurring during its genesis, the heart continues to serve its primary function, pumping blood. As a result, the heart is under a constantly changing barrage of biophysical stimulation induced by forcing blood through a closed circulatory system. These forces include wall shear stress (friction caused by moving blood next to the heart wall), transmural pressure (fluid pressure exerted against the walls due to contraction and relaxation of the heart muscle), and circumferential stretch (cyclical distention of the cardiac tissues resulting from pulsatile blood flow). While the fundamental information dictating how the heart develops is clearly defined with an organism's genetic programming, there is little doubt that these biophysical forces play a part in modifying the hardwired plan.

Heart Tube Formation

The early heart tube forms as a result of the migration and eventual fusion of two primordial epithelial tubes (Li et al., 2004). This critical initial process continues after the heart begins to beat and loop, as the atrium and future left ventricle form. The specific forces modulating heart tube formation are not well-understood. One interesting hypothesis posits that the cylindrical bending of a sheet of epithelial cells could result in the formation of a hollow tube (Taber, 1998). The key to this theoretical model is the contraction of actin microfilaments surrounding the apices of the epithelial cells. Such an asymmetric contraction might cause the normally cylindrical cells to become more wedge-shaped, forcing the apical surface of the sheet into the inner curvature of the developing tube. The ability of individual cells to resist apical–basal elongation during this contraction will result in more a substantive bending of the sheet (Keller et al., 2003). Confocal laser scanning microscopic studies have revealed that the early cardiac epithelial cells do in fact have such a microfilament arrangement (Shiraishi et al., 1992), and pharmacological inhibition of the microfilaments results in failed heart tube formation (Ettensohn, 1985). Although no definitive experimental proof has shown that actin-induced cell deformation is the cause of tube formation, the work done to date is suggestive enough to spur interest in this biomechanical theory as a potential mechanism of tube formation not only for the early heart but for gastrulation and neurulation as well (Odell et al., 1981).

Looping

Perhaps no aspect of cardiac morphogenesis better characterizes the biomechanical complexity of heart development than does the sequence of bending and twisting actions that transform the linear heart tube into an asymmetrical heart loop. Over time, several attempts to describe precisely which morphogenetic events are encompassed by looping have been put forth. These descriptions range from only considering the bending of the linear heart tube into a C-shape (so-called “dextral looping”) through the completion of events leading to complete cardiac septation (Manner, 2000). Detailed descriptions of the events comprising cardiac looping have been made in several species (e.g., Ramos and Macias, 1998; Manner, 2000; Gormley and Nascone-Yoder, 2003).

Numerous attempts to quantify the stresses and strains within the developing heart have been made (e.g., Clark et al., 1989; Chabert and Taber, 2002; Ling et al., 2002; Zamir et al., 2003), and most experimental studies have focused on dextral looping. Potential mechanisms underlying looping include expansion of the cardiac jelly (Manasek et al., 1984), differential growth of the heart tube (Stalsberg, 1970), and shortening of the dorsal mesocardium due to residual stress (Taber, 1995). Despite the considerable effort put forth, none of these hypotheses are entirely consistent with the available experimental data. A potential explanation may be that normal looping is affected by a host of partially redundant mechanisms, any of which could be compensated for by the others (Taber, 1995). Most studies done to date indicate that the bending portion of dextral looping is under control of forces intrinsic to the heart tube itself. However, recent evidence (Voronov and Taber, 2002; Voronov et al., 2004) suggests that the rotational component of c-looping may be externally influenced by forces imposed on the heart tube from motions of adjacent tissues.

To address this possibility, researchers are turning to computational modeling techniques. Models of volumetric heart growth (Rodriguez et al., 1994) and tissue morphogenesis (Odell et al., 1981; Oster et al., 1983) recently have been combined into a more unified theory of heart development (Taber and Perucchio, 2000; Taber, 2001). Of course, such models still depend on experimental data to provide them with realistic boundary conditions, and as we have seen, these are often contradictory. The results of such models will have to be considered speculative until better empirical data become available.

Trabeculation

Myocardial trabeculae have been implicated in enhancing contractility (Challice and Viragh, 1973), ventricular septation (Ben-Shachar et al., 1985), intraventricular conduction (de Jong et al., 1992), and helping to direct blood flow before septation (Hogers et al., 1995). Early trabecular formation is often described as ridge-like incursions of endocardium (Icardo and Fernandez-Teran, 1987) running circumferentially along the primitive heart tube. Forces produced by the peristaltic contractile pattern characterizing the early heart may cause a “buckling” of the endocardial surface and stimulate patterned trabecular morphogenesis (Thompson et al., 2000). The primary mechanical consequences of ventricular trabeculation are more uniform transmural stress distribution and increased intramyocardial blood flow (Taber, 1998). Mathematical models describing trabeculation as mesenchymal morphogenesis are based on fundamental balance laws of continuum mechanics (Oster et al., 1983; Taber, 1998).

The process of trabecular morphogenesis is described by the emergence and lengthening of the primary trabeculae, followed by the emergence of copious secondary offshoots from the primary projections and their spatial orientation within the heart chambers. Regardless of the native fiber orientation, which appears to vary across species, trabecular patterning has been shown experimentally to respond to hemodynamic changes (Clark et al., 1984; Sedmera et al., 1997, 2000). Unfortunately, the geometric irregularity in ventricle trabeculation makes computational modeling (e.g., finite element methods) of the developing heart exceedingly difficult (Taber and Perucchio, 2000; Ling et al., 2002).

Valvulogenesis

Cardiac valves are multileaflet structures that help to ensure unidirectional blood flow within the heart. Abnormal development of the heart valves and associated structures can lead to regurgitation (backward flow of blood) and/or stenosis (narrowing of the valve orifice) and accounts for more than 25% of all cardiovascular defects (Armstrong and Bischoff, 2004). Although little is known about the precise mechanisms driving valve formation, we know that the atrioventricular and conotruncal valves in vertebrates arise from endocardial cushions that form between the atrium and ventricle and between the ventricle and outflow tract. It has been suggested that these cells are prepatterned for this activity and await later myocardial signaling (Walsh and Stainier, 2001). The cardiac cushions begin as endocardial bulges caused by regional swellings of the cardiac jelly sandwiched between the inner endocardium and the outer myocardium of the heart tube. Some time later these primordia are “seeded” by migrating cardiac endothelial cells that metamorphose into cardiac mesenchymal cells and begin proliferating, eventually forming the endocardial cushions proper (Markwald et al., 1977). The mesenchymal cells then presumably proliferate into the fibrous valvular tissues. The process by which these cushions emerge from the myocardium to form leaflets is also poorly understood.

Revived interest in the effects of blood flow-induced forces on endothelial cell behavior (Sidi and Rosa, 2004) suggests that hemodynamics may play a role in valve formation. Significant reduction of wall sheer stress in the developing fish heart has been shown to result in gross cardiac dysmorphogenesis including failed valvulogenesis (Hove et al., 2003). Bartman et al. (2004) demonstrated that myocardial function in fact may be the key factor regulating endocardial cushion development with hemodynamic shear stress playing a secondary role. These studies will be described in more detail later in the review, where it will be pointed out that the difficulty in completely separating heart muscle mechanics from blood flow necessitate further study if we are to discover how biomechanical forces influence valve development.

Septation

The role of blood flow-induced biophysical forces in the regulation of cardiovascular septation dates back to the initial description of two spiral streams within the developing heart (Bremer, 1932) and the suggestion that septae could form between these streams due to intracardiac pressure gradients (Jaffee, 1963). This so called “flow molding” hypothesis is a source of controversy even today. Proponents suggest that the endothelial cells lining the entire vertebrate cardiovascular system are able to discriminate between subtle variations in flow velocity and direction and result in repeatable cell morphologies and orientations (Dewey et al., 1981; Davies, 1995; Takahashi et al., 1997) and substantial arterial remodeling (Kamiya and Togawa, 1980; Langille and O'Donnell, 1986). It follows, they argue, that these same forces also help to shape cardiac jelly and trabeculae within the developing heart into septa that separate the cardiac chambers. Experimental alteration of intracardiac flow has been shown to induce a variety of cardiac malformations (Clark et al., 1989; Sedmera et al., 1999; Tobita and Keller, 2000) supporting this contention. Others dispute the cause and effect relationship between flow and septal formation. Using imaging techniques superior to those available to Bremer, Yoshida et al. (1983) have convincingly shown that the blood flowing from the early vitelline veins do not form the classically described twin, spiraling streams at all, leaving the question unanswered of whether blood flow guides septal formation or septa guide blood flow.

INSTANCES OF ASSOCIATED DEFECTS IN BOTH HEART FUNCTION AND MORPHOGENESIS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. BRIEF REVIEW OF HEART MORPHOGENESIS
  5. BIOMECHANICAL THEORIES OF HEART DEVELOPMENT
  6. INSTANCES OF ASSOCIATED DEFECTS IN BOTH HEART FUNCTION AND MORPHOGENESIS
  7. POTENTIAL MECHANISMS FOR THE FUNCTION–FORM LINK
  8. FUTURE DIRECTIONS
  9. REFERENCES

Recently, several studies have shown that embryos with abnormal heart function (e.g., due to mutation of genes involved in mechanical function), frequently have associated defects in cardiac morphogenesis. Many of these studies were focused on describing functional changes, and, therefore, mentioned only peripherally the presence of structural changes in these embryos. Some of these authors may also have assumed that morphogenetic defects were nonspecific, instead of specific examples of the function–form link. More recently, especially in the zebrafish system where heart function and structural form can be visualized simultaneously and can be followed over time in one embryo, there has been a greater awareness of the role of heart function in controlling late heart morphogenesis. In fact, the association between early heart function and late morphogenesis may have been presaged in the original forward genetic screens in zebrafish, where mutants classified as having poor myocardial function often had structural defects as well (Chen et al., 1996; Stainier et al., 1996). The following are some recently published examples of situations in which changes in function and form have been coordinately affected.

Cardiomyocyte Specification/Fate

Function of the myocardial cells may affect their specification from a very early stage. For example, Iijima et al. (2003) have shown that skeletal muscle-derived cells can transdifferentiate into cardiomyocytes in coculture with cardiac cells. This transdifferentiation depended on cell-to-cell contact between the skeletal and cardiac muscle cells. Addition of nifedipine to the culture to stop beating suppressed the transdifferentiation of skeletal muscle, although passive stretch of the culture could overcome the nifedipine treatment. This study suggests that mechanical forces applied to muscle cells, completely unrelated to any flow phenomena, can program these cells to accept a cardiac fate. An in vivo example of this explanation seems to come from the silent-heart zebrafish, which carries a mutation in cardiac troponin T (tnnt2). The myocardial cells of these embryos do not beat but also exhibit a reduction in the amount of Tmpa and an absence of Tnni3, indicating that the lack of Tnnt2 function leads to a down-regulation of these other sarcomeric proteins (Sehnert et al., 2002). This result was recapitulated with the use of the tnnt2 morpholino. These two studies, therefore, suggest that rhythmic stretch or beating of muscle cells may be necessary and sufficient for determining a cardiomyocyte fate.

The silent heart embryos show that there are alterations in embryonic myocardial gene expression when the heart does not beat. Other studies have demonstrated alterations in myocardial gene expression as a function of when in the cardiac cycle mechanical forces are applied. For example, activation of p44/42 mitogen-activated protein kinase (MAPK) and phosphorylation of MEK1/2 can be significantly increased by applying strain to the myocardium solely during the systolic phase, but this effect is greatly diminished if the strain is applied during diastole. In contrast, activation of p38 MAPK was similar, regardless of the timing of strain on the heart (Yamamoto et al., 2001). Therefore, cardiomyocyte function may affect heart development from a very early stage.

Heart-Tube Formation and Looping

Other examples exist in which a heartbeat is lacking, yet heart development is not perturbed until a later stage. For example, Ncx-1−/− (sodium–calcium exchanger) embryos lack a heartbeat yet specification of cardiomyocytes appears normal. Furthermore, looping and chamber formation appeared normal as well, and these embryos have appropriate expression of markers such as MLC2a, MLC2v, dHand, and eHand (Koushik et al., 2001). These mice die at E11–E11.5, presumably due to a lack of oxygen transport in the embryos. The aforementioned silent-heart embryos also have normal looping and chamber development, despite their changes in myocardial gene expression. It therefore seems possible that, as long as initial cardiomyocyte specification occurs, later steps of organogenesis may proceed for a while even as specification is being lost. However, both of these mutants have defects in key aspects of later morphogenesis such as endocardial cushion formation, which will be mentioned later.

Another zebrafish mutant that shows morphogenetic aberrations with changes in function is heart and mind, which encodes the α1B1 isoform of Na,K-ATPase (Shu et al., 2003). The mutant embryos have normal specification of cardiomyocytes as assayed with vmhc, nkx2.5, and cmlc2 expression, but abnormal extension of the fused heart tube at approximately 24 hours postfertilization (hpf). At 48 hpf, the heart is not beating and chamber-specific markers are aberrantly expressed, but these phenotypes may be secondary to defects in extension of the heart tube or may be causally related to each other. Of interest, embryos injected with a morpholino directed against the α2 subunit of Na,K-ATPase have normal contractility and chamber development but have a cardiac laterality phenotype. Therefore, the two different α-subunits of this ATPase appear to play different roles in early cardiac morphogenesis.

Poor heart function due to pharmacologic treatment of zebrafish embryos has been shown to phenocopy mutations that affect function as well. For example, treatment of embryos with polycyclic aromatic hydrocarbons can mimic both the functional and subsequent structural abnormalities seen in silent-heart (Incardona et al., 2004). Taken together, these studies indicate that lack of myocardial beating can have variable effects on maintenance of myocardial fate, tube formation, and looping, but the reason for the phenotypic variability seen between these studies remains to be explained.

Chamber Development

Defects in heart function have also been associated with morphogenetic defects of chamber formation. For example, a knockout of the Mlc2a gene also leads to early embryonic lethality (E10.5–E11.5), with significant defects in chamber morphogenesis. At E8.5 (during the linear heart tube stage), mutant hearts were enlarged and amorphous compared with their wild-type littermates, and although the mutant hearts did proceed to loop, there were aberrations in the development of the chambers and in the architecture of looping itself (Huang et al., 2003). Chamber development was disturbed both in the atrium and the ventricle, even though Mlc2a is only required for function of the atrium. Notably, mutant embryos also lacked endocardial cushion development, reminiscent of the Ncx1−/− and sih−/− embryos. The Mlc2a study demonstrates that development of intracardiac structures, including the ventricle, is dependent on the function of the atrium. The mechanism for this effect is not proven in the study, but the suggestion is that alterations in intracardiac fluid forces are responsible for the effect, a hypothesis to be discussed later.

At the same time, Berdougo et al. (2003) described the zebrafish mutant weak-atrium, which, as its name suggests, is defective in atrial function. This mutant was positionally cloned and shown to be the amhc gene (Berdougo et al., 2003). The amhc mutant embryos have defective atrial contraction from the first heartbeat, but normal ventricular heart rate. Because the functional defect is limited to the atrium, the embryos continue to have circulation due to ventricular contraction, and extracardiac development appears normal (with some embryos surviving to adulthood). However, isolated loss of atrial function does have an effect on ventricular development. Weak-atrium embryos have normal ventricular form and function until 36 hpf, but by 48 hpf, the ventricle becomes more compact with thickening of the ventricular wall, a phenotype reminiscent of hypertrophic cardiomyopathy. Furthermore, amhc−/− embryos show up-regulation of atrial natriuretic factor and cmlc2 in both cardiac chambers, furthering the analogy with cardiomyopathies, because these changes in gene expression are seen in that disease state as well (Aronow et al., 2001).

Two other zebrafish mutants demonstrate that changes in function can lead to defective modeling of the cardiac chambers—pickwik, which encodes Titin (Xu et al., 2002), and island beat, which encodes an L-type calcium channel subunit (Rottbauer et al., 2001). Although pickwik encodes a sarcomeric protein and, therefore, might be expected to have a direct role in both cardiomyocyte function and shape, island beat should primarily affect cardiac conduction; therefore, it would appear that the effect on ventricular growth seen in these embryos is secondary to the change in heart function.

Endocardial Cushion, Septum, and Valve Development

As noted above, several functional mutants in mouse and zebrafish exhibit defects specifically in endocardial cushion, septum, and valve morphogenesis. For example, both the mouse Ncx-1 knockout and the zebrafish silent heart mutation have relatively specific effects on cushion development. In mouse, loss of connexin40 and/or connexin43 (either as nulls of one gene or double heterozygous loss) leads to expected conduction problems, but frequently also causes an associated common atrioventricular junction or ventricular septal defect (Kirchhoff et al., 2000). Mutations in nkx2.5 cause a wide range of cardiac phenotypes in mouse and man (Schott et al., 1998; Biben et al., 2000; Jay et al., 2004; Kasahara and Benson, 2004; Pashmforoush et al., 2004), including both functional errors (conduction delays) as well as structural errors, primarily of the cushions and septum (atrial septal defects, outflow tract misalignments). With nkx2.5, it is not entirely clear if the mutations lead to conductive and structural problems independently by affecting different downstream targets of nkx2.5 signaling or if the conduction deficits leads to abnormal embryonic heart function, which subsequently is responsible for the structural defects. However, these studies together indicate that cushion, septum, and valve formation may be particularly sensitive to changes in heart function.

One model to explain how change in heart function leads to defects in endocardial morphogenesis is that altered myocardial function leads to decreased blood flow. This change decreases shear stress on the endocardial cells, thereby altering their genetic program or the signals that they send to the myocardium. Although this mechanism has not been extensively tested in vivo, two recent studies have attempted to examine the role of mechanics in cushion/valve development specifically.

One study that directly addressed this issue involved preventing intracardiac blood flow in zebrafish embryos through microdissection and implantation of beads in the inflow or outflow tract of the zebrafish heart (Hove et al., 2003). Flow was interrupted at 37 hpf at the primitive heart stage, and embryos were assayed at 4.5 days postfertilization (dpf) for errors in morphogenesis. Embryos with impaired flow (by blockage at either location) showed phenotypes of abnormal development of the bulbus venosus, impaired looping, and lack of endocardial cushion and/or valve formation. The lack of cushion/valve formation was demonstrated by a lack of up-regulation of a tie2::GFP transgene in the regions of the developing valves. An exact measurement of the changes in shear stress was not made on surgically manipulated embryos but was estimated to be approximately 10-fold lower than baseline, possibly in the range of 5 to 10 dyn cm−2 as opposed to approximately 76 dyn cm−2 in wild-type 4.5 dpf embryos. This finding would suggest that alterations in the shear forces within the heart do have a profound effect on morphogenesis, especially with development of the endocardial cushions and valves.

A subsequent study has shown that a genetic mutation that affects early myocardial performance can also, relatively specifically, affect cushion and valve formation (Bartman et al., 2004). In this study, the cardiofunk mutation was described as having a lack of cushion/valve formation, also demonstrated by lack of tie2::GFP up-regulation at the atrioventricular boundary at 48 hpf. These mutant embryos go on to develop significant atrioventricular regurgitation, also indicative of a lack of cushion/valve development. Positional cloning of the mutation showed that cfk encoded a sarcomeric actin, which was likely redundant to cardiac actin. The mutant form of cfk had a missense mutation that would be expected to have a dominant-negative affect on the cardiac actin, a theory supported by in vitro studies (Wen and Rubenstein, 2003). Closer examination of cfk embryos showed that the initial defect in the heart is poor contractility and blood flow at 36 hpf, before cushion formation is initiated, suggesting that the cushion phenotype was secondary. Pharmacologic impairment of heart function through use of 2,3-butane dione monoxime (2,3-BDM, an inhibitor of myofibrillar ATPase; Herrmann et al., 1992) from 24–48 hpf showed that the ability to form endocardial cushions decreased in a dose-dependent manner as the dose of 2,3-BDM was increased. It was noted that cushion formation could be prevented even if blood flow was present, and inversely, cushion formation could occur even if blood flow was absent, suggesting that it is the level of myocardial function, not blood flow, that is responsible for the effect seen with 2,3-BDM. However, because measurements of shear stress on the endothelial cells were not taken in this study and because decreasing myocardial function is likely to lead to a respective decrease in shear stress (as opposed to a all-or-none phenomenon), it remains debatable what the force is that lies upstream of cushion development.

POTENTIAL MECHANISMS FOR THE FUNCTION–FORM LINK

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. BRIEF REVIEW OF HEART MORPHOGENESIS
  5. BIOMECHANICAL THEORIES OF HEART DEVELOPMENT
  6. INSTANCES OF ASSOCIATED DEFECTS IN BOTH HEART FUNCTION AND MORPHOGENESIS
  7. POTENTIAL MECHANISMS FOR THE FUNCTION–FORM LINK
  8. FUTURE DIRECTIONS
  9. REFERENCES

The above biomechanical studies and in vivo examples strongly suggest that heart function affects development of form. Possible mechanisms for this affect include (1) mechanical motion of the heart directly pushes cells and tissues into their locations, and/or (2) mechanical function affects signaling pathways and, therefore, the cellular program of cells and their developmental fate. The later could occur through at least two different mechanisms.

First, shear stress on endocardial cells might induce local signaling changes, as has been demonstrated with endothelial cells in both in vitro and in vivo studies (e.g., Icardo, 1989; Helmlinger et al., 1991, 1995). Some of the molecules that may sense this shear stress include the integrins (Ingber, 1999), platelet endothelial cell adhesion molecule-1 (PECAM-1; Fujiwara et al., 2001), VE-cadherin (Shay-Salit et al., 2002), ion channels (Barakat, 1999), vascular endothelial growth factor (VEGF) receptor 2 (Shay-Salit et al., 2002), and G proteins (Gudi et al., 1996). Several studies, including some using microarray technology, have identified some of those genes downstream of the stress signals (Resnick et al., 1997; Chien et al., 1998; Malek et al., 1999; Gimbrone et al., 2000; McCormick et al., 2003; Black, 2000), and it is possible that some of these genes may affect cardiac morphogenesis. Development of the cushions and valves seems particularly sensitive to changes in heart function, and molecules known to be expressed in the endocardium and responsible for endocardial cushion/valve morphogenesis include ES/130 (Ramsdell et al., 1998), transforming growth factor (TGF) -β3 (Nakajima et al., 1994, 1998; Brown et al., 1996; Ramsdell and Markwald, 1997; Boyer et al., 1999), VEGF (Dor et al., 2003), nuclear factor of activated T cell (NFAT) c1 (de la Pompa et al., 1998; Ranger et al., 1998), Notch and hesr2 (Kokubo et al., 2004), Wnt/β-catenin (Hurlstone et al., 2003; Liebner, 2004), bone morphogenetic protein/TGF-β (Nakajima et al., 1998), ErbB, and NF1/Ras (all nicely reviewed in Armstrong and Bischoff, 2004). It remains to be seen which, if any, of these endothelial/endocardial signaling pathways are perturbed in instances of poor heart function.

Second, changes in myocardial function may affect gene expression within that layer, and these molecular changes may affect both myocardial and endocardial cell development. An abundant literature describes muscle genes whose expression is affected by muscle function (reviewed in Seidman and Seidman, 2001), although this literature focuses on the remodeling events that take place in adult hearts. However, some of the genes expressed in these altered conditions are those used for heart morphogenesis in the embryo. For example, eHand and dHand, two prominent players in chamber formation, experience dynamic changes in their level of expression in cardiomyopathies (Ritter et al., 1999; Natarajan et al., 2001). Other molecules that regulate morphogenesis and demonstrate changes in expression or posttranslational regulation in response to mechanical forces include β-catenin (Masuelli et al., 2003) and the calcineurin/NFAT pathway (Molkentin et al., 1998). Because the calcineurin/NFATc pathway is affected by changes in heart function in adult animals, is also involved in endocardial cushion and valve morphogenesis (Uhing et al., 1993; de la Pompa et al., 1998; Ranger et al., 1998; Graef et al., 2001; Chang et al., 2004), and because changes in heart function affects development of the valves (Bartman et al., 2004), this pathway becomes an extremely attractive candidate for linking embryonic heart function with cushion and valve formation. Studies that have used cyclosporin A (CsA) to suppress NFAT signaling have demonstrated similar cushion/valve defects to the NFATc1 null mice (Uhing et al., 1993; Graef et al., 2001). However, cyclosporin treatment is known to affect contractile activity (Abbott et al., 1998; Janssen et al., 2000), which may indicate that the defects seen with CsA treatment of mouse embryos may be secondary to changes in heart function. In chick embryos treated with CsA, defects were seen in ventricular wall morphology, heart looping, and formation of the endocardial cushions (Liberatore and Yutzey, 2004). Presumably, heart function was poor in these embryos due to the changes in ventricular wall morphology, but this was not clearly demonstrated.

One recent study that attempted to tease apart the issues of NFAT's role in function and form in utero ultrasound biomicroscopy to study NFATc1−/− embryos and control littermates sequentially from E10.5 to E14.5 (Phoon et al., 2004). These authors showed that null mice developed defects in diastolic dysfunction and valvular regurgitation, despite maintaining normal contractile (systolic) function. Further studies such as these will be required to prove or disprove the relationship between NFAT's role in heart function and its role in heart morphogenesis.

FUTURE DIRECTIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. BRIEF REVIEW OF HEART MORPHOGENESIS
  5. BIOMECHANICAL THEORIES OF HEART DEVELOPMENT
  6. INSTANCES OF ASSOCIATED DEFECTS IN BOTH HEART FUNCTION AND MORPHOGENESIS
  7. POTENTIAL MECHANISMS FOR THE FUNCTION–FORM LINK
  8. FUTURE DIRECTIONS
  9. REFERENCES

The literature cited in this review is only a subset of that which shows, quite convincingly, that (1) morphogenesis of the heart requires mechanical events to take place (such as looping), (2) some of the signaling pathways required for heart development are affected by local mechanical or physical factors, (3) multiple mutations and knock-outs have demonstrated defects in heart function that result in secondary structural phenotypes, and (4) remodeling of the heart in adults due to mechanical disturbances uses some of the pathways known to be involved in primary heart morphogenesis.

To date, many investigators have focused on studying one of these four areas. However, it appears that we are on the verge of understanding of how mechanical forces in the developing heart are translated into alterations in gene expression or function, and how activation of the molecular pathways that are involved in heart morphogenesis can create a physical change in the structure of the heart. Hopefully, as physicists and biomechanical engineers collaborate with molecular and developmental biologists, we will greatly expand our integrated study of these processes. The implications for such work are that we may come to a better understanding of the causes of human congenital heart diseases and may identify previously unsuspected genes whose primary function is to regulate embryonic heart function but in which mutations can lead to structural heart disease. Furthermore, because development of the cushions and valve seems to be particularly affected by mechanical forces and because acquired valvular disease is a significant problem in adults, this integrated understanding may lead to novel therapies for heart disease in adults or provide insight into the best way to create artificial biological valves for transplant.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. BRIEF REVIEW OF HEART MORPHOGENESIS
  5. BIOMECHANICAL THEORIES OF HEART DEVELOPMENT
  6. INSTANCES OF ASSOCIATED DEFECTS IN BOTH HEART FUNCTION AND MORPHOGENESIS
  7. POTENTIAL MECHANISMS FOR THE FUNCTION–FORM LINK
  8. FUTURE DIRECTIONS
  9. REFERENCES