Iroquois Homeodomain transcription factors in ventricular conduction system and arrhythmia.

Iroquois homeobox genes, Irx, encode cardiac transcription factors, Irx1-6 in most mammals. These six transcription factors are expressed in different patterns mainly in the ventricular part of the heart. Existing researches show that Irx genes play key roles in the differentiation and development of ventricular conduction system and the establishment and maintenance of gradient expression of potassium channels, Kv4.2. Our main focus of this review is on the recent advances in the discovery of above-mentioned genes and the function of the encoding products, how Irx genes establish ventricular conduction system and regulate ventricular repolarization, how the individual and complementary functions can be verified to complement our cognition and leads to novel therapeutic approaches.

Introduction

The conduction of electrocardiosignals and the synchronous contraction between ventricles with rhythmicity are essentially important for forming effective cardiac beating cycles and thus ensuring unidirectional blood flow circulation in whole bodies. As an indispensable part of the heart, ventricular conduction system (VCS) plays a vital role in coordinating the heartbeat to activate contraction from the apex and maximize efficiency of expulsion of the blood through aorta (systemic circulation) and pulmonary artery (pulmonary circulation) at the base of the heart. VCS is majorly composed of the following parts: atrioventricular bundle (AVB), left and right bundle branches (BB), and Purkinje fiber (PF) network. The deficiency or damage of this system and genetic loci involved in the pathogenesis will lead to slowed conduction velocities, abnormal activation patterns of the ventricles, different kinds of ventricular arrhythmia and even sudden cardiac death 1-3.

Homeobox genes encode various kinds of transcription factors containing homeodomains, and mutations of these genes or transcription factors can lead to dysembryoplasia, or even early embryonic death. Among which, recent studies have revealed Iroquois homeobox (Irx) family of cardiac transcription factors are important for the development of VCS4 and heterogeneity of the ventricular repolarization of rodents and humans5, 6. This review focuses on the regulatory effects of these Irx transcription factors in VCS and grants a summarization of some unanswered questions about their developmental and molecular functions.

Iroquois Homeodomain transcription factors

Three homeobox genes of the Irx family, caupolican, araucan and mirror were first discovered in Drosophila which play a critical role in establishing an organizing center that is essential for formation of follicle7, head8, eyes9, 10, muscle11, mesothorax12, lateral notum13, bristles on the thorax14 and heart development15 and specification of the veins in the wing and associated sensory organs16-19. Irx gene family consists of Irx1, Irx2 and Irx4 in the IrxA cluster, and Irx3, Irx5 and Irx6 in the IrxB cluster in mouse and human20 (Figure 1), 11 genes, Ziro1a, 2a, 4a of IrxAa, Ziro1b, 4b of IrxAb, Ziro3a, 5a, 6a of IrxBa, Ziro3b, 5b of IrxBb and Ziro7 in Zebrafish. In vertebrates, Irx gene family members encode highly conserved homeodomain-containing transcription factors of a 13 amino-acid domain (the Iro box) and the 3-amino-acid-loop-extension (TALE) family25 (Figure 2), which are required for the opportune patterning and formation of the nervous system26-31 and the heart32, specification of the membranous labyrinth33 and nephron segment fate during kidney development 34-36, lung development and maturation37, pancreatic, retinal and microvascular endothelial cell development and function38-40, female gonad41 and early limb development42 and also give an advantage to morbid obesity, type 2 diabetes mellitus 43, fused toes44 and tumor cells45. In the heart, all the Irx transcription factors are expressed in unique and overlapping patterns, abnormality or absence of encoding Irx may cause congenital structural heart disease46, cardiomyopathy47, 48 and a variety of arrhythmia49. The present article focuses on their roles in VCS development, ionic channels maintenance and thus involvement in arrhythmia as below (Table 1).

Figure 1

Schematic illustration of Irx and Irxl1 genes. Location and organization of Irx and Irxl1 genes in mouse (Mus) and human (Hs).

Figure 2

Schematic representation of Irx and Irxl1 proteins. The Irx, Irxl1 proteins and homeobox domains are depicted according to the size. TALE, 3-amino acid-loop-extension.

Table 1

Expression, function and associated phenotype of Irx genes in conduction system.

Gene	Cluster	Chromosomal position	Expression ranges	Physiological function	Associated phenotype
Irx1	A	13 C1(Mus)5p15.33(Hs)	E10.5, interventricular septum trabecularE11.5, ventricular septumE14.5, regions of the developing VCS, including AVB and BB	Potentially involved the development of VCS	Irx1b-MO in zebrafish, bradycardia
Irx2	A	13 C1(Mus)5p15.33(Hs)	identical to Irx1	Not been verified	None
Irx3	B	8 C5(Mus)16q12.2(Hs)	From E9.5, trabeculated component of the ventricles	Establish fast conduction in the VCS	Irx3-KO mice, prolonged QRS, notched R waves and RBBB on ECG
Irx4	A	13 C1(Mus)5p15.33(Hs)	E7.5, anterior domain of the linear heart;E9.5, atrioventricular canal, inner curvature, and proximal outflow tract;E11.5, ventricular myocardium.	Regulate chamber-specific gene expression, act as a multifunctional co-factor	No defect in cardiacelectricalactivity
Irx5	B	8 C5(Mus)16q12.2(Hs)	E9, heart tube ventricle;E9.5, endocardial chamber myocardium (atrioventricular canal, inner curvature, and outflow track);E14.5, ventricular trabeculae, AVB and BB.	Establish and maintain the gradients of Ito,f in ventricles	Irx5-/-mice, T-wave alterations
Irx6	B	8 C5(Mus)16q12.2(Hs)	E10.5, endocardial lining of atrial and ventricular chambers	Not been verified	None
Irxl1		18 A1(Mus)10p12.1(Hs)	No expression found in the heart	Not been verified	No defect in cardiacelectricalactivity

Formation of ventricular conduction system during cardiac development

Before heart becomes developmentally mature, the electric signals generated from pacemaker tissue can be slowly transmitted through the ventral and dorsal parts of atrioventricular canal (AVC) between embryonic atrium and ventricle50. After the cardiac chambers have been divided, the fibrotic nonconductors consist mainly of atrioventricular cushion and extraepicardial mesenchymal tissues shield electric conduction between atrium and ventricle, with the exclusive atrioventricular electric channel, AVB. AVB is part of the VCS, deriving from interventricular septum crista and physically connecting with AVC and atrioventricular node (AVN). With the further development of interventricular septum, both left and right BB develop inch by inch and form from AVB branches under endocardium51. The cells forming AVB and BB continue to feature in primitive cardiomyocytes and are more like the genetic and electrophysiological phenotypes of AVN. This specific lineage of cardiomyocytes can substitute for the upper positioned pacemaker tissues to discharge rhythmic electric impulses, junctional rhythms, during atrioventricular block. Different than AVN, Cx40 is enriched between the cells from these tissues, leading to relatively fast conduction velocity.

Within the maturely developed heart, the distal BB and PF network are the tissues containing only several layers of cells, and they constitute the rapid intraventricular conduction system with the maturation of endocardial trabecula52. Even in the lower vertebrates, similar conduction characteristics can also be recorded53. During the developmental process of embryonic ventricles, the regions specifically expressing Cx40, Nppa and TASK-1 in the heart are gradually restricted within the trabecula tissues, and form peripheral ventricular conduction system simultaneously with the cardiac pericardium linking layer not expressing the above-mentioned ventricular chamber and conduction system specific molecular markers. With the Nkx2.5 regulating the cardiac developmental maturation, the trabecular region of myocardium finally composes the peripheral ventricular conduction system in terms of structure and function.

Expression of Irx and roles in the VCS

During the development process of heart, Irx1 expression could be detected as early as E10.5 in the ventricular septal trabecular myocardium, which is restricted to the ventricular septum at E11.5, and expands to the developing VCS regions from E14.5 onward, including the AVB and BB 54 (Figure 3), which implies that Irx1 may be involved in the development of VCS. Zebrafish, after injection of a morpholino oligonucleotide designed to knockdown Ziro1b, a gene homologous to Irx1, exhibits profound dose-dependent bradycardia, with slower heart rate reduced by 35% of the normal control55. Further studies should be carried out to evaluate the functions of Irx1 knockout (KO) in conduction system.

Figure 3

Expression distribution of Irx transcription factors in the heart. Schematic illustration of Irx transcription factors expression in the embryonic and mature hearts.

Expression pattern of Irx2 is nearly identical to that of Irx1 54 (Figure 3). Despite the high expression in the developing heart, Irx2 KO mice are fertile and well-grown, and show no phenotype. And Electrocardiographic analysis of 8- to 10-week-old mice revealed no difference between Irx2-/- mice and wildtype controls56, implying that Irx2 is unessential to the cardiac conduction system, probably as a result of functional redundancy of other Irx genes.

In embryonic hearts, Irx3 can be detected in regions contributing to the VCS, such as the ventricular septum and trabeculae at E9.5 54, 57. Irx3 expression is overlapped in the mature heart with Cx40, a major functional gap junction protein of the AVB, BB, and PF of the VCS51, 58-60, and accurately labels the mature VCS4 (Figure 3). Irx3-null mice show QRS complex duration prolongation, notched R waves (R') on an electrocardiogram and an increase of HV interval (the conduction time between the AVB and ventricles) in electrophysiological examination, all indicative of prolonged conduction times in the VCS, by 2 weeks of age4. And telemetry ECG displays right axis deviation in 70% of Irx3 KO mice, consistent with right bundle-branch block (RBBB) or abnormal impulse conduction in the right ventricular free wall as published in dog and mouse studies previously59-61. The impaired conduction in the VCS is identified with decreased expression of Cx40, which forms gap junction channels with a high conductance and is expressed in all compartments of the fast conduction system, from the AVB, left and right BB, to the whole PF network60, 62. Meanwhile, ectopic expression of Cx43, characterize the nodal conduction structures with slow velocity63, was also verified in the proximal VCS with a direct coupling to the septal myocardium. In accordance with the results that Irx3 activates Cx40 expression while inhibits Cx43. These results suggest that transcription of Cx40 is activated by Irx3 repressing an unidentified repressor indirectly, nevertheless, expression of Cx43 is likely to be suppressed by Irx3 directly through binding to the Cx43 promoter and antagonizing Nkx2.5 dependent activation, which is required for the postnatal differentiation of VCS64, 65. Thus, Irx3 plays a key role for maintaining rapid electric conduction through the VCS as well as proper ventricular activation. It is still unclear that whether deficiency in the VCS lacking Irx3 would induce the changes of the expression of gap junction expression located in the VCS. Additionally, optical mapping utilizing intact Zebrafish expressing the in vivo cardiac-specific fluorescent calcium indicator Tg(cmlc2:gCaMP) showed that knockdown of Ziro3a led to low conduction velocity and asynchronization66, 67. Aforementioned results illuminate that Irx3 is required for normal ventricular activation and impulse conduction.

In early stage of heart development at E7.5, Irx4 starts to be found in the anterior cardiac primordia, and in atrioventricular canal, inner curvature, ventricles, and proximal outflow tract at E9.5. Irx4 does not show any expression until E11.5 in the myocardial but not in non-myocardial cushion cells located in the atrioventricular canal. After E11.5, expression of Irx4 is restricted to the ventricular myocardium while absent from both atria and the outflow tract 54 (Figure 3). Unlike its involvement in establishing chamber-specific gene expression and differentiation of atrial and ventricular myocytes in the developmental heart 68-70, there is no direct evidence that Irx4 participates in the differentiation and development of VCS.

Irx5 expression was first detected at E9 at the ventral side of the looping heart tube. At E9.5, Irx5 started to be observed in the endocardial lining of the atrial and ventricular chamber myocardium, conversely, it is absent from the endocardium of the atrioventricular canal, inflow tract, inner curvature, outflow tract, and endothelium of the aortic arch arteries 54. By E14.5, expression region of Irx5 in ventricles is confined to the ventricular trabeculae, AVB and BB57 (Figure 3). The pattern of expression remained unchanged during expansion of the chambers 54. Irx5 expresses in the region of immature AVB and BB, which opens the possibility of the sealed regulatory effect of the developing VCS.

Irx6's level of expression is much lower compared to the other five Irx genes and is detected first at E10.5 in the heart71. Expression pattern of Irx6 is restricted to the endocardial lining of atrial and ventricular chambers, similar to Irx5 (Figure 3). The function of Irx6 in VCS has not been verified.

Actually, there is another divergent Irx family transcription factor gene Irxl1, Iroquois homeobox-like 1, or Mkx, Mohawk, which also belongs to the TALE (three-amino-acid loop extension) superclass of the atypical homeobox gene family72 (Figure 1). Irxl1/Mkx encodes transcription factor Irxl1 (Figure 2) that regulates the expression of matrix molecule genes and differentiation in developing tendons73-76, including type 1 collagen and Tenomodulin, volar plates77, homeostasis of the periodontal ligament 78, MyoD expression and myoblast differentiation 79, brain and pharyngeal arch morphogenesis80 and participates in some pathological process, such as, diabetic tendinopathy81, anterior cruciate ligament degradation82, serous ovarian cancer83, and Rett Syndrome84. Mouse Mkx shares 56% homology with Irx2 over the entire homeodomain, though, no expression found in the heart.

Irx in cardiacelectricalactivity and ionic channel

In mammalian mature heart, both Irx3 and Irx5 are expressed in the ventricles with transmural, endocardium to epicardium gradients 5, 85-87. Irx5mice exhibit abnormal ECG characterized by T-wave alterations, whereas heart rate, atrioventricular (PR interval) and intraventricular conduction (QRS duration) are not affected, consistent with accelerated repolarization and reduced ventricular refractoriness 5. Depression of T-wave originated from ventricular repolarization abnormity which in mice is attributed mainly to gradients of the fast transient outward K+ currents, Ito,f88-91. Ito,f in the rodent heart is initiated mainly by two voltage-gated potassium (Kv) channels, Kv4.2 and Kv4.392, along with KChIP2, an accessory subunit 93-95, and cardiovascular disease-related96, 97. In Irx5 KO mice, the Ito,f gradient in ventricular walls flattens as a result of selective increases in Ito,f and Kv4.2 along with shorter action potential duration in endocardium5, 87. The inverse relationship between Irx5 and Kv4.2 expression suggests that an Irx5 repressor gradient negatively regulates Kv4.2 gene expression, forms an opposite gradient of Ito,f and coordinates repolarization in the heart. Also, we should note that Irx5 can act as either an activator or repressor in a cell-type-dependent manner. For instance, the C-terminal portion of Irx5 physically binds to the N-terminal region of Irx4, through which Irx4 could suppress Kv4.2 gene promoter activation mediated by Irx5 in non-cardiomyocytes, like 10T1/2 fibroblastic and PC12 cells. Further tests demonstrate endogenous Irx4 inhibits the function of transfected Irx5 to increase Kv4.2 promoter activity which degree may be determined by the expression ratio of Irx5/Irx4 6. This contradictorily activated or repressive impact of Irx5 in cardiomyocytes or non-cardiomyocytes is gained from recruitment of mBop (Smyd1), which has been verified as a cardiac corepressor essential for cardiomyocyte differentiation and cardiac morphogenesis98, 99. However, physical interaction between Irx5 and mBop might not exist, nor affect Irx5-mediated Kcnd2 promoter activation 6, this is just contrary to previous conclusion that Irx5 represses Kv4.2 expression through mBop recruitment 5. Therefore, further studies will be necessary to examine whether and how Irx5, Irx4 and mBop are interacting to regulate Kv4.2 expression.

Recent studies have revealed crucial roles of miRNAs as regulators of the development and function of the heart 100-103. Irx gene expression is also regulated by miRNAs in the heart, including miR-1,which is highly conserved and enriched in heart and encoded by two separate genes, miR-1-1 and miR-1-2 and has important pathophysiological functions (regulates cardiac arrhythmogenic potential) in the heart106. There is a miR-1 binding site in the 3`UTR of Irx5 , and in miR-1-2 mice, Irx4 and Irx5 expression increase in the heart, which exhibit lower heart rate, prolonged PR interval and QRS complex, consistent with the observations in Irx5 KO mice, a decrease in Kcnd2 along with increased Irx5 expression107.

Irx and arrhythmia

As the Irx genes are critical for efficient conduction in the VCS development, defects of Irx genes may be associated with arrhythmia. Loss of Irx3 leads to demolition of the rapid concerted spread of excitation in ventricles, prolonged QRS, notched R waves (R') and an increase of HV interval, right bundle branch block to be specific, which is considered to be associated with increase of mortality in patients with acute myocardial infarction108, and a reasonable candidate of cardiac resynchronization therapy for heart failure patients with evidence of either electrical or mechanical left-sided delay109.

Among the many factors can impact cardiac rhythms and cause arrhythmias, ion channel abnormality and defect in repolarization could contribute 2. Establishment and maintenance of electric transmural gradients play a key role in ventricular repolarization. Irregular myocardium repolarization is associated with many kinds of malignant arrhythmia, including torsades de pointes (in long-QT, short-QT and Brugada syndromes), ventricular tachycardia and fibrillation. The Ito,f gradient in ventricular walls reduces and ventricular tachyarrhythmia could be reproducibly induced by intracardiac programmed stimulation in Irx5 KO mice, consistent with alterations in ventricular repolarization to induce arrhythmia, so there is a fair speculation that defects of IRX5 may be related to cardiac arrhythmia in human. 5 These findings suggest that loss of the ventricular transmural gradient of Ito,f in Irx5-/- hearts is the major cause of enhanced arrhythmia susceptibility, or simply the shorten of ventricular refractory period. It will clearly be of interest to further explore the increased pathogenesis of arrhythmias in the Irx5 KO mice.

Irx as new therapy for clinical applications

Recently, Ban et al. developed a method of profound significance to isolate pure ventricular cardiomyocytes by targeting the mRNA of Irx4, with high specificity using molecular beacons from differentiating human and mouse pluripotent stem cells110. This approach of producing a pure population of working cardiomyocytes offers a new option for cell therapy of heart failure and arrhythmia caused by it, and can be used to model the abnormal functional phenotype of genetic or idiopathic cardiac diseases, such as long QT syndrome, and to identify potential new therapeutic agents. For ischemic heart disease, the proangiogenic function of Irx3 in human microvascular endothelial cells opens new avenues for basic research and clinical applications40.

Discussion

The six Irx genes of mammals are mainly distributed in the ventricles during embryonic development, including the parts that develop into the conduction system. Although only Irx3 has been confirmed to participate in the establishment of the ventricular conduction system, other Irx genes have not been shown any abnormal phenotypes in the conduction system after the individual knockout (normal ECG characteristics), given the overlap and functional redundancy of these six genes, more often function as the form of two clusters, A and B111, wherein Irx genes are closely related to each other, and regulate certain physiological and pathological processes in contradictory ways, so it's very likely that all these six genes play certain roles in the development of the VCS. Even if it is difficult to identify individual genes alone, it is still necessary to conduct further experiments through multiple gene editing methods to find specific effects of some or certain cluster of Irx genes.

In the same way, like the previous experiment, Irx3 indirectly promotes Cx40 expression by combining with an unknown factor in the development of conduction system, Irx4 plays an important role in the process of cardiac compartmentation by combination with Vitamin D, the interaction between Irx4, Irx5 and mBop has yet to be clarified, and in addition, in recent years, the understanding of the three-dimensional structure of Irx gene clusters has been further deepened. In the process of verifying the function of individual Irx genes, it also needs to verify the function of the complexes and their structures.

In an interesting study, the gender-dependent effects of Irx genes on ionic channels have been verified86, it may be possible to explain that female gender is associated with a higher risk of torsades de pointes than the opposite sex112. And further research in such gender difference could blaze new trails on the basis of that Irx3 gene was confirmed to be involved in the development of the female sex gland41 and several sex hormones, including progesterone, estradiol and testosterone, rained influence on ionic currents, ventricular action potential and QTc interval duration112, 113.

Irx genes play critical roles in the differentiation development of VCS and maintenance of normal cardiacelectricalactivity. With the in depth development of genome-editing and stem cell technologies, people will gain in-depth understanding of the development process of VCS, the mechanisms of various cardiac arrhythmia and regulation pathways involved, the clinical application of Irx gene family as a genomic target of arrhythmia will develop ideas for initiating new therapeutic methods.