Candidate genes for the hereditary component of cardiac hypertrophy

Cardiac hypertrophy is an independent risk factor for morbidity and mortality [1]. Specifically, cardiac hypertrophy often is a precursor to congestive heart failure. The aetiology of cardiac hypertrophy is multifactorial and can involve systemic factors such as volume or pressure overload, with the latter related to an increased peripheral vasoconstriction [2]. It can also involve local factors, such as neurotransmitters and hormones directly acting on the cardiomyocytes [3–7]. Moreover, genetic factors also appear to play a role, and some patients are more likely to develop cardiac hypertrophy than others [8]. The interest in the subject is underscored by the fact that a Medline search with the Key words: ‘heart’, ‘hypertrophy’ and ‘genetics’ yields more than 3000 published articles. The identification of the genes involved in the heritability of cardiac hypertrophy uses two major approaches. First, a genome-wide scan can be used to test for associations of markers with risk for cardiac hypertrophy [9]. The advantage of this approach is that it does not involve an untested hypothesis, but such shot-gun approaches are labour intensive, requiring rather large research teams, and may not directly identify the responsible gene or genes. An alternative approach is the use of candidate genes for which physiological data plausibly suggest that they may be involved in the control of cardiomyocyte growth. Although this may miss genes that do not fit current hypotheses, it allows a more focused approach, which is also feasible for smaller groups of researchers. The search for candidate genes originally focused on genes related to the factors causing cardiomyocyte growth in cultured cardiomyocytes, such as the genes encoding receptors for noradrenaline, angiotensin II or endothelin [8,10,11]. An extension of this idea is the search for candidate genes involved in the formation of such transmitters [12] or in the signal transduction of their receptors (e.g. G-proteins and subsequently activated protein kinases) [13]. However, the above factors are not only involved in the development of cardiac hypertrophy, but also in the control of vascular tone. This implies that it is difficult, or even impossible, to determine whether a given factor is involved in the development of cardiac hypertrophy by direct effects on the heart or indirectly via an increased after load due to alterations of vascular tone [14,15]. Although it can be argued that, from a clinical point of view, it is of little relevance whether the association between a gene and cardiac hypertrophy is direct or indirect via vasoconstriction, such reasoning is relevant for the identification of the molecular chain of events linking a given genetic factor to the hypertrophic phenotype. In this issue of the journal, Sharma et al. [16] importantly demonstrate the heritability of human cardiac hypertrophy based upon a large cohort of normotensive twins [16]. This implies that at least a major fraction of the genetic factors involved in cardiac hypertrophy is unrelated to the genes that also are involved in vasoconstriction. Hence, many of the previous studies investigating candidate genes with regard to cardiac hypertrophy are pathophysiologically interesting but may be barking up the wrong tree with regard to the genetics of cardiac hypertrophy. Therefore, we would like to draw attention to another group of candidate genes. These are the genes that are involved in embryonic ventricular development and might be linked to hyper- or hypoplastic ventricular development. They are also of interest because the hypertrophic and failing heart reactivates a foetal gene expression programme. Cardiac chamber formation Very early in development, a straight heart tube is formed from cardiac progenitor cells. This heart tube comprises of two layers: the endocardium (inner layer) and the myocardium (outer layer). During subsequent development, the heart tube bends to the right and, at the major outer curvature of the tube, the forming ventricle becomes evident. Ventricular development becomes evident by the formation of trabeculae at embryonic day (E) 8 of mouse development. Subsequently, the morphology of the ventricular myocardium starts to change with the enveloping of the heart tube by epicardium at E9.5. Initially, the ventricular myocardium consists of a one-to-two cell thick outer layer with long thin trabeculae that project into the lumen. With the formation of the epicardium, the outer myocardial layer starts to expand and proliferate, forming the compact myocardium, whereas the trabeculae remodel and become thicker and (relatively) shorter. As a result, two compartments become evident within the ventricle: the compact myocardium and the trabecular myocardium. In principle, the ventricle has now acquired its adult form and only needs to grow to reach its adult size [17]. Although the formation of the ventricle has been studied extensively, the molecular mechanisms underlying the induction of chamber formation in the linear heart tube and the subsequent development of the compact and trabecular myocardium are poorly understood. Nevertheless, a large body of literature describes different ventricular phenotypes in mice in which individual genes have been disrupted. Among those not considered here are knockouts showing cardiac abnormalities together with general oedema and/or haemorrhages because such phenotypes imply an aberrant vasculature formation, suggesting that the cardiac abnormalities most probably occur secondarily. Rather, we propose to look at genes that, upon functional impairment in mice, lead to an absence of chamber formation or affect the development of compact and/or trabecular myocardium and hence are candidates potentially involved in hypertrophic phenotypes. Early inducers of chamber formation The first sets of genes affecting left ventricular mass are the genes that induce chamber formation. If ventricular formation is not induced, trabecular formation is absent from the straight heart tube, and the mice die early during development (approximately E9–10.5). Such a phenotype is observed in mice in which the myocardially expressed growth factor bone morphogenetic protein 10 [18], the endocardially expressed Tie-2 receptor ligand angiopoietin-1 [19], or the endocardially expressed Tie-2 receptor are functionally disrupted [20]. Furthermore, the heart tube retains its primary phenotype in mice in which the serum response factor, a transcription factor, is deleted specifically from the cardiomyocytes [21], or if the T-box transcription factor 2 is ectopically overexpressed throughout the forming primary heart tube [22]. It is therefore conceivable that deregulation of early inducers of chamber formation might lead to an over- or underdevelopment of the ventricle. Role of epicardium in chamber formation Concomitant with the formation of the epicardium, the ventricular myocardium differentiates into an outer compact layer and trabeculae at the luminal side. If the formation of the epicardium is aberrant, the compact myocardium is not formed and the trabeculae retain their early embryonic phenotype, being long and slender. Mice in which the epicardium forms but immediately disintegrates and falls off the heart die at midgestation (approximately E13–15). Such a phenotype is observed in mice in which the vascular cell adhesion molecule-1 [23], β4-integrin [24], Wilms’ tumour-1 [25], erythropoietin [26], or the erythropoietin-receptor [26] genes are disrupted. These findings point to an essential interaction between the epicardium and myocardium that leads to the further development and growth of the compact myocardium. Although the epicardium is necessary for chamber formation, it is not sufficient because it has long been known that chamber development also depends on the presence of coronary vessels. Similar to the epicardium, these are derived from the pro-epicardium [27]. The finding that mice in which the transcription factor Friend-of-GATA-2 (FOG-2) was knocked out had no coronary development and thin ventricular walls [28] underscored this idea. Moreover, a genetically engineered transcription factor GATA 4 that is unable to interact with FOG-2 also shows an absence of coronary vessel development, similar to the knockout of FOG-2, whereas functional disruption of GATA 4 results in early embryonic lethality due to aberrant folding of the embryo [29]. However, although ventricles depend on coronary vessels for growth, we do not anticipate that an excess of coronary vessels leads to increased left ventricular mass. Therefore, we believe that genes involved in coronary vessel formation are secondary and therefore not prime candidate genes for ventricular mass. Interaction between epicardium and myocardium in chamber formation Although the epicardium and coronaries are essential for normal chamber formation, they are not sufficient as such. For example, non-compaction in combination with long slender trabeculae is observed in mice in which an epicardium and coronaries are formed. This phenotype was reported in mice in which the retinoic acid signalling was altered by disruption of either the Retinoic-X Receptor α [30] gene or the rate-limiting enzyme retinaldehyde-dehydrogenase 2 (Raldh2) gene [31]. Although their expression patterns suggest a direct role in ventricular formation, detailed analysis revealed a non-cell autonomous effect. This indicates that direct signalling between the epicardium and the underlying myocardium is essential in the induction of the formation of the adult ventricular myocardium. Potential candidates are epicardially expressed growth factors and myocardially expressed receptors. The finding that disruption of the myocardially expressed gp130 receptor leads to non-compaction with grossly normal trabeculae [32] suggests an involvement of the interleukin growth factor family. However, G-protein-coupled receptors are also possible candidates because disruption of the myocardially expressed G-protein-coupled β-adrenergic receptor kinase 1 [33] results in a similar cardiac phenotype. The fact that growth factor signalling appears to regulate the formation of compact and trabecular myocardium indicates the possible involvement of myocardially expressed factors, which are regulated by the respective growth factor signalling pathways. Unexpectedly, rather general transcription factors, such as N-myc [34], transcriptional enhancer factor 1 [35], myocardin related transcription factor B [36], FoxP1 [37] and K-Ras [38], result in aberrant formation of the compact outer myocardial layer of the heart. Based on these observations, we infer that rather broadly expressed transcription factors might qualify as potential candidates in the regulation of ventricular mass. Interaction between endocardium and myocardium in chamber formation The regulation of the formation of the compact myocardium is even further complicated by the finding that altering the expression of the endocardially expressed receptors neuropilin [39] and PlexinD1 [40] results in thin ventricular myocardium. Similarly, disruption of the myocardial receptor Semaphorin6D, which is able to interact with PlexinA1, causes a similar phenotype. An elegant study evaluating the role of PlexinA1 and Semaphorin6D in chicken embryos found that expression of both genes in the compact myocardium was necessary for its expansion, whereas the expression of only Semaphorin6D in the trabecular myocardium was required for trabecular formation [41]. Additional evidence for interactions between endocardium and myocardium in chamber formation is provided by studies investigating the neuregulin-1 ErbB2/ErbB4 pathway. ErbB2, a receptor tyrosine kinase, heteromerizes with ErbB4 and together form a high-affinity receptor for neuregulin-1. During normal development, neuregulin-1 is produced in the endocardium and signals in a paracrine manner to the Erbb2/ErbB4 receptor heteromers that are present in the directly opposed myocardium. Both the ErbB2/4 receptors and neuregulin-1 ligand are essential for heart development because mutations in these genes result in impaired ventricular trabeculation, causing midgestation lethality [42]. Interestingly, disruption of the tumour necrosis factor-β-converting enzyme (TACE or ADAM17), which is expressed in a transmural gradient that is highest at the endocardial side of the myocardium, results in hyper-trabeculation [43]. Although TACE was initially only indicated in tumour necrosis factor-β activation, it is now clear that TACE is a membrane-anchored, Zn-dependent metalloprotease that can release an extensive array of membrane-bound or -anchored growth regulators by ectodomain shedding. Additional analyses are required to evaluate whether the observed effect of TACE on trabeculation is a direct or an indirect effect due to interference with endocardial–myocardial signalling. Thus, signalling from the endocardium towards the myocardium plays a key role in chamber formation. Autocrine regulation of ventricular formation Interestingly, embryos can survive up to weaning in the absence of compaction when the trabeculae overdevelop, referred to as hypertrabeculation. The finding of many long and thick trabeculations in mice in which the ubiquitously expressed intracellular signalling protein FKBP12 is disrupted [44] documents this possibility. Although the intracellular role of FKBP12 is not yet firmly established, a role in the regulation of tumour growth factor (TGF) β-superfamily signalling via interaction with the ALK receptors is indicated. In this respect, it is of interest that: (i) mice in which ALK3 (BMPRIA) is specifically deleted from the heart muscle cells show hypo-trabeculation and (ii) knockouts of the myocardially expressed TGFβ receptor III (also referred to as betaglycan) [45] show a reduction of the formation of compact myocardium. Because TGFβRIII is required for TGFβ2 signalling, it is not surprising that TGFβ2 null embryos display aberrant ventricle formation [46]. Because TGFβ2 is expressed in the myocardium, this suggests that growth factor signalling in an autocrine manner is also regulating ventricular development. Cell cycle and apoptosis in ventricular formation Obviously, regulators of the cell-cycle and apoptosis are potential candidates of regulating ventricular mass. The crucial role of cell-cycle regulation is illustrated by triple cyclin D1–3 deficient mice displaying severely thinned ventricular walls mainly affecting the compact zone [47]. Apoptosis or programmed cell death was found to play an important role in trabecular formation because mice homozygously deficient in the apoptosis signalling pathway, comprising caspase 8 [48], FLIP (associated with FADD and caspase-8) [49] and FADD (Fas-associated death domain) [48], die in mid-gestation with thin disorganized trabeculae. Interestingly, in mice in which both the transcription factors Hey-1 and Hey-2 are disrupted, the compact as well as the trabecular layers start to form but, subsequently, the trabecular component disappears as a result of apoptosis [50]. Taken together, these findings suggest that disruption of the balance between cell-cycle and apoptosis regulation leads to alterations in ventricular mass. Heritability of left ventricular mass Given the unexpected large heritable component of the left ventricular mass even in normotensive subjects, as reported by Sharma et al. [16], and the fact that it is highly unlikely that a single gene in a normally distributed population can account for this level of genetic variance [51], it is likely that multiple genes determine left ventricular mass in any population. Moreover, work from monogenic familial hypertrophy studies has failed to demonstrate that such genes have a significant influence on common sporadic left ventricular hypertrophy. Consequently, it is likely that combinations of single nucleotide polymorphisms (SNPs), which alter the function and/or expression of genes, in some of the above-mentioned genes (and others) will affect left ventricular mass. Although a candidate gene approach in SNP association studies would be successful, it is now possible to test such associations between SNPs and left ventricular mass in a genome-wide setting with the use of SNP-chips containing 100 000 or more SNPs. Genome-wide methods have the advantage of being assumption-free and therefore likely would yield all associations, and not just the most obvious ones. However, it is of the utmost importance to investigate genomic associations in well-defined and described populations, such as the one reported by Sharma et al. [16], because this will safeguard against spurious associations. Ultimately, genome-wide association studies will provide clues with respect to the influence of genetic components that underlie the normal range of left ventricular mass, and perhaps even to the susceptibility to cardiac hypertrophy in the general population. Acknowledgement M.J.B.vdH. and A.V.P. are financially supported by The Netherlands Heart Foundation (M96.002).