Hearts and Hands: the good, the bad, and the ugly

This editorial refers to ‘HAND1 loss-of-function within the embryonic myocardium reveals survivable congenital cardiac defects and adult heart failure’, by B.A. Firulli et al., pp. 605–618. Congenital heart defects (CHD) are common and deadly as they constitute the most common birth defects and affect up to 1% of live births.1 These defects are highly variable and include: valvular, septal, muscular, and vascular abnormalities. The aetiology of CHD is complex and includes both environmental as well as genetic causes.2 Elegant studies using gene disruption or transgenic technologies in mouse, zebrafish, fly, or other genetic models have deciphered key regulatory nodes or pathways that govern cardiac morphogenesis and perturbation of these pathways contributes to CHD. Estimates suggest that mutations involving one of more than 400 different genes may contribute to CHD. Furthermore, emerging technologies such as genetic screens are enhancing our understanding of cardiogenesis and genetic contributions to CHD.3 These screens and genetic studies have emphasized the importance of transcription factors as regulators of molecular programs during cardiogenesis and CHD.4 The Hand proteins are core transcription factors that have been shown to be essential regulators of cardiac development and CHD.5 Hand1 and Hand2 are basic helix–loop–helix (bHLH) transcription factors that are expressed in the precardiogenic mesoderm, where they heterodimerize and govern essential gene regulatory networks to promote early cardiac development. The discovery of the bHLH Hand proteins was undertaken in the Olson lab by Cserjesi, Srivastava, and Firulli.6–8 The global genetic deletion of Hand1 in mice by Firulli et al.,5 resulted in early embryonic lethality (E8.5) and had severe defects in cardiac looping as well as extra-embryonic mesodermal defects. The global knockout in the mouse died so early, that a full study of the cardiac consequences of Hand1 was not possible. While Hand1 was expressed in the left ventricle of the developing heart, Hand2 had complementary expression and was localized in the right ventricle and outflow tract in the developing mouse heart.7 Gene disruption of the Hand2 gene resulted in embryonic lethality by E10.5 with complete absence of the right ventricle and an abrupt connection between the conotruncus and the left ventricle.8 Studies have emphasized the functional role(s) of these Hand transcription factors was mediated via their context-dependent interactions with other transcriptional regulators, such as Mef2c, Gata4, Nfatc1, P300, and others. Prior to chamber specification, both Hand1 and Hand2 were expressed in the precardiogenic mesoderm and later were restricted to their respective left- and right-ventricular domains. Moreover, dimerization of Hand1 and Hand2 has been described, suggesting that overall gene dosage could be a critical contributor to the phenotype during cardiac development.9 Furthermore, the regulation of chamber specific expression has been shown to be dependent on other transcription factors, such as Gata4 to specific enhancer regions. The importance of Hand transcription factors has been amplified by the identification of mutations in human patients that lead to congenital heart defects, such as septal defects and hypoplastic left heart syndrome.10–12 Due to the important role of Hand transcription factors in cardiac development and in congenital heart disease, there remains intense interest in the role of Hand transcription factors during embryogenesis and their role in the postnatal heart. In the article by Firulli et al.,13 the authors pursued a conditional gene knockout strategy to delete the Hand1 gene specifically in the developing heart. The authors used Cre recombinase dependent gene deletion mouse models that were under control of either the early cardiac transcription factor Nkx2-5 or the cardiac-specific gene myosin heavy chain 6. Using these genetic tools, the authors demonstrated that embryos with Hand1 deletion, specifically in the myocardial lineage, displayed congenital heart defects including ventricular septal defects (see Figure 1). To define the genetic regulatory pathways that were controlled by the transcription factor Hand1, the authors performed RNA sequencing of RNA from right and left ventricles of control and conditionally deleted Hand1 (CKO) embryos. Analysis of the differences in gene expression revealed that Hand1 was critically important for essential gene networks that include the regulation of cardiac genes such as Nppa, Cited1, and other genes. Surprisingly, a number of CKO embryos survived to adulthood despite the presence of CHD, in contrast to a previously published study using similar genetic approaches.14 Whether the differences between these two studies were due to the genetic background of the mouse lines, the precise genetic strategy utilized, or due to some extracardiac role of Hand1 in the previous study is unclear. Nevertheless, the current study enforces the notion that precise dosing of Hand transcription factors was critical for early foetal development and proper cardiac morphogenesis. Hand1 is an essential transcriptional regulator that governs cardiac morphogenesis. Left panel: Is a scanning electron micrograph of a developing murine heart at E9.5 pseudocoloured red for the expression domain of Hand1. Right panel: Hand1 expression results in normal cardiac morphogenesis. However, if cardiac expression of Hand1 during development is perturbed, the study by Firulli et al. demonstrated a phenotype that results in ventricular septal defects, increased trabeculation, and an absence of chordae tendineae associated with the mitral valve. A second important finding from the current study was that the authors identified valvular abnormalities downstream of Hand1 deletion. These findings suggested that there could be an important interplay between the myocardium and the cardiac neural crest-derived structures, such as the outflow tract and the valves that required proper functioning of Hand transcription factors. In the current study, the authors describe a feature of the Hand1 CKO adult heart having an absence of chordae tendineae associated with the mitral valve that is similar to the previously described CHD, mitral valve arcade (see Figure 1).15 Finally, the notion that relatively mild CHDs that do not appear to impact early pre- or post-natal development can result in cardiac dysfunction in adulthood was a novel result in the Firulli et al., study. This finding was emphasized by the presence of cardiac dysfunction in the animals that did survive to adulthood, such as perturbed diastolic performance. This finding further suggests that there could likely be a whole spectrum of subclinical variations in cardiovascular developmental differences that warrants future investigation. As an example, one subclinical phenotype could be the number of cardiomyocytes that populate the newborn heart as Hand1 is known to regulate cardiomyocyte proliferation during development, and Hand1 mutation can lead to hypoplastic left heart syndrome. It is possible that mild gene dosage variations could lead to individual differences in overall cardiomyocyte content of the heart, which predisposes individuals to the development of heart failure in the presence of other risk factors, such as hypertension, previous myocardial infarction or valvular dysfunction. These CHDs, the subclinical variations, and the important insights learned from the RNA sequencing experiments are new information and principles highlighted in the manuscript by Firulli et al.13 As outlined in this manuscript,13 the essential role of Hand1 during cardiogenesis represents The Good, the subclinical variations of the adult heart that lacks Hand1 is The Bad, and the embryonic lethality associated with Hand1 deletion represents The Ugly. Collectively, this study and others using emerging technologies such as bulk RNA-seq, single-cell RNA-seq, and reprogramming strategies will provide a platform that will enhance our understanding of CHD and hopefully result in future therapies for this patient population. Conflict of interest: none declared. J.H.v.B. is supported by a Biomedical Research Scholar award from The Hartwell Foundation and the NIH. D.J.G. is supported by grants from RMM, AHA, the NIH, and the DoD. The opinions expressed in this article are not necessarily those of the Editors of Cardiovascular Research or of the European Society of Cardiology.