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Cardiovascular Research 2001 52(1):65-75; doi:10.1016/S0008-6363(01)00349-2
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Copyright © 2001, European Society of Cardiology

Divergent expression of delayed rectifier K+ channel subunits during mouse heart development

Diego Francoa,b,*, Sophie Demolombea,c, Sabina Kupershmidtd, Robert Dumainee, Jorge N Dominguezb, Dan Rodend, Charles Antzelevitche, Denis Escanded and Antoon F.M Moormana

aExperimental Molecular Cardiology Group, AMC, University of Amsterdam, Amsterdam, The Netherlands
bDepartment of Experimental Biology, Faculty of Health and Experimental Sciences, University of Jaén, 23071 Jaén, Spain
cINSERM U533 Hôpital Hotel-Dieu, Nantes, France
dDepartments of Medicine and Pharmacology, Vanderbilt University, Nashville, TN, USA
eDepartment of Molecular Biology and Pharmacology, Utica, NY, USA

dfranco{at}ujaen.es

* Corresponding author. Tel.: +34-953-002-604; fax: +34-953-012-141

Received 21 February 2001; accepted 8 May 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
The repolarization phase of the cardiac action potential is dependent on transmembrane K+ currents. The slow (IKs) and fast (IKr) components of the delayed-rectifier cardiac K+ current are generated by pore-forming {alpha} subunits KCNQ1 and KCNH2, respectively, in association with regulatory β-subunit KCNE1, KCNE2 and perphaps KCNE3. In the present study we have investigated the distribution of transcripts encoding these five potassium channel-forming subunits during mouse heart development as well as the protein distribution of KCNQ1 and KCNH2. KCNQ1 and KCNH2 mRNAs (and protein) are first expressed at embryonic day (E) 9.5, showing comparable levels of expression within the atrial and ventricular myocardium during the embryonic and fetal stages. In contrast, the β-subunits display a more dynamic pattern of expression during development. KCNE1 expression is first observed at E9.5 throughout the entire myocardium and progressively is confined to the ventricular myocardium. With further development (E16.5), KCNE1 expression is mainly confined to the compact ventricular myocardium. KCNE2 is first expressed at E9.5 and it is restricted already to the atrial myocardium. KCNE3 is first expressed at E8.5 throughout the myocardium and with further development, it becomes restricted to the atrial myocardium. The fact that {alpha} subunits are homogeneously distributed within the myocardium, whereas the β subunits display a regionalized expression profile during cardiac development, suggest that differences in the slow and fast component of the delayed-rectifier cardiac K+ currents between the atrial and the ventricular cardiomyocytes are mainly determined by differential β-subunit distribution.

KEYWORDS K-Channels; Gene expression; Embryology; Developmental biology; Ion channels


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The heart develops from a peristaltoid-contracting tube without valves into a synchronously contracting four-chambered heart with valves [1]. During the subsequent processes of looping and septation many changes in the patterns of gene expression are observed (for a review, see Ref. [2]). Soon after the formation of the tubular heart, five different segments can be distinguished based on morphological and functional characteristics, namely, inflow tract, atrium, atrioventricular canal, ventricle and outflow tract [1,3]. The atrial segment contracts and relaxes quickly, allowing it to function as the drainage pool of the embryo. The ventricular segment contracts quickly but shows a longer-lasting relaxation [4,5]. These two myocardial regions are flanked by remnants of the primary myocardium [1,6], i.e., the inflow tract, the atrioventricular junction and the outflow tract that act as ‘embryonic valves’. The functional differences between the distinct regions are correlated with distinct molecular phenotypes (for a review, see Refs. [1,2]).

During embryonic development, similar to adulthood, the atrial and ventricular cardiomyocytes display differences in action potential configuration [3,7] (for a review, see Ref. [8,9]). Several voltage-dependent types of potassium currents are involved in the repolarization of the action potential, which have been thoroughly characterised electrophysiologically, and more recently molecularly (for a review, see Ref. [9]). The slow component of the delayed-rectifier cardiac K+ current is generated by the pore-forming K+ channel subunit termed KCNQ1 (KvLQT1), in association with a regulatory β-subunit KCNE1 (minK/IsK) (for a review, see Ref. [10]). Mutations in either KCNQ1 or KCNE1 are associated with long QT syndrome, cardiac arrhythmia and sudden death in humans [11]. The fast component of the delayed-rectifier cardiac K+ current is generated by a pore-forming subunit KCNH2 (HERG). Recently, a new ancillary subunit has been described, named KCNE3 (also called MiRP2 [12]). Co-transfection experiments of KCNE3 and KCNQ1 lead to a constitutive open conformation of the KCNQ1 channel [13], although its putative role within cardiomyocytes remains to be explored. It has been suggested that KCNE1 and KCNE2 (also called MiRP1) interact with KCNH2 to modulate the IKr potassium current in cardiomyocytes [14,15]. Similarly to KCNE3, KCNE2 has also been proposed to interact with KCNQ1 leading to permanently open KCNQ1 channels [16]. Mutations in KCNH2 or KCNE2 are also associated with cardiac arrhythmias [14,15,17,18].

In the adult, expression of KCNQ1, KCNE1 and KCNE3 is not confined to the heart, but it is present in other organs in different species [13,19–23] (for a review, see Ref. [10]). Similarly, KCNE2 expression is not only observed in the heart but also in skeletal muscle [14]. In recent years, mice have become an increasing source of information concerning cardiac physiology because they are amenable to genetic manipulation (see, e.g., Refs. [24,25]), although specific differences in the electrophysiology of mice and humans are clearly documented [10,11]. At present, the functional role of the ancillary subunits of the delayed-rectifier K+ channels is still debated. Scant information is available about the distribution pattern of delayed-rectifier K+ channel subunits during mouse heart development. Therefore, the aim of this study is to investigate the expression profiles of KCNQ1, KCNH2, KCNE1, KCNE2, KCNE3 mRNAs, and KCNQ1 and KCNH2 protein during development.

We show that KCNQ1 and KCNH2 display a homogeneous expression within the different myocardial chambers of the developing heart whereas their ancillary subunits, KCNE1, KCNE2 and KCNE3 display a more heterogeneous and dynamic expression pattern. Our observations suggest a predominant role of the β-subunits in the determination of regional heterogeneity of the delayed rectifier cardiac K+ current during cardiac development.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Embryos
Control FVB embryos (Charles Rivers Benelux) ranging from E7.5 to E18.5 were used to detect the endogenous expression of KCNQ1 (KvLQT1), KCNH2 (HERG), KCNE1 (IsK/minK) KCNE2 (MiRP1) and KCNE3 (MiRP2) mRNAs by whole-mount in situ hybridization and KCNQ1 and KCNH2 protein distribution by immunohistochemistry. Embryos were excised from the uterus and the thoracic wall was removed (E12.5–E18.5) exposing the heart to allow maximal penetrance of fixatives and reagents. Specimens were fixed in 4% freshly prepared formaldehyde overnight and rinsed twice in phosphate-buffered saline (PBS). Subsequently the embryos were dehydrated in increasing concentrations of ethanol and stored at –20°C for whole-mount in situ hybridization or alternatively embedded in paraplast for immunohistochemistry. The investigation conforms with principles outlined in the Declaration of Helsinki.

2.2 Immunohistochemistry
Sections were deparaffinised, hydrated in graded ethanol steps, briefly rinsed in PBS and incubated in TENG-T (10 mM Tris, 5 mM EDTA, 150 mM NaCl, 0.25% gelatine, 0.05% Tween-20, pH 8.0; 30 min) to diminish non-specific binding. Immunohistochemical detection was essentially as described by Franco et al. [26]. The sections were incubated overnight with specific primary polyclonal antibodies against KCNQ1 (R. Dumaine, unpublished data), KCNH2 (Alamone Labs, Irsael) and desmin (Monosan). Visualization of the first antibody binding was performed using either alkaline phosphatase-conjugated secondary antibody (KCNH2) or a peroxidase-conjugated secondary antibody (KCNQ1).

2.3 Whole-mount in situ hybridization
Complementary RNA probes against mouse KCNQ1 (2054–2843 nt [23]), human KCNH2 (1755–2880 nt [28]), mouse KCNE1 (full length [25,27,29]), KCNE2 (full length [15]) and KCNE3 (full length [13]) mRNAs were labeled with digoxigenin-UTP by in vitro transcription according to standard protocols [30,31]. Sense probes for KCNQ1, KCNH2, KCNE1, KCNE2 and KCNE3 were used as negative controls, yielding no hybridization signal as previously described by Delomombe et al. [23]. Hybridization conditions were as described by Henrique et al. [31] with slight modifications [6].


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Expression profile of {alpha} subunits in the developing heart
3.1.1 KCNQ1 (KvLQT1) mRNA and protein expression
We have analysed the expression pattern of KCNQ1 transcripts from the early tubular heart stage (E8.5) to the fully septated heart stage (E18.5). Weak homogeneous expression is first observed at E9.5 throughout the entire myocardial tube (Fig. 1A,B). No signs of antero-posterior polarity were observed.


Figure 1
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  		Fig. 1 KCNQ1 mRNA expression during heart development. Ventral (A,C,E) and dorsal (B,D,F) views of whole-mount in situ hybridization against KCNQ1 mRNA in mouse embryos of E9.5 (A,B), E12.5 (C,D) and E16.5 (E,F). KCNQ1 is weakly expressed along the entire cardiac tube at E9.5. At E12.5, expression is homogeneous along the atria and ventricles. KCNQ1 transcripts remain evenly expressed at E16.5 within the atria and the ventricles. No that the atrioventricular junction shows similar staining as the myocardium elsewhere in the fetal heart (F). RV, right ventricle; LV, left ventricle; RA, right atrium, LA, left atrium.

 
In the prototypical embryonic heart stage, E10.5, KCNQ1 mRNA expression levels are similar in all cardiac compartments, i.e., the atrial and ventricular myocardium as well as in the remnants of the primary heart tube, the atrioventricular canal and the outflow tract. The expression of KCNQ1 transcripts remains unchanged during the subsequent stages of cardiac development (E12.5–E18.5) (Fig. 1C–F). Within the ventricular compartment, the trabeculated layer and the compact layer, including the ventricular septum, show a homogeneous pattern of expression of KCNQ1 transcripts.

The expression profile of KCNQ1 protein during development is similar to that observed at the mRNA level during early stages of cardiac development. KCNQ1 protein is first observed at E9.5 evenly expressed throughout the entire myocardium (Fig. 2A,B). During the embryonic (E10.5–E14.5) and fetal (E15.5–E18.5) stages, KCNQ1 protein remains evenly expressed within the atrial and ventricular myocardium (Fig. 2C–E). At late fetal stages (E18.5), enhanced expression of KCNQ1 protein is observed in the ventricular conduction system, i.e., atrioventricular node, bundle of His and bundle branches (Fig. 2F).


Figure 2
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  		Fig. 2 KCNQ1 protein expression during cardiac development. Immunohistochemical detection of desmin (A,C,E) and KCNQ1 (B,D–F) protein in transverse sections of mouse embryos of E9.5 (A,B), E12.5 (C,D), E16.5 (E) and E18.5 (F). KCNQ1 protein expression in the embryonic (B,D) and fetal heart (E,F) is observed homogeneously distributed in the atrial and ventricular myocardium. At late fetal stages, enhanced expression is observed preferentially in the ventricular conduction system, i.e., bundle of His (His). Desmin immunostaining illustrates the myocardial boundaries (A,C). RA, right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle; OFT, outflow tract.

 
3.1.2 KCNH2 (HERG) mRNA and protein expression
We have analysed the expression pattern of KCNH2 mRNA from the early tubular heart stage (E8.5) to the fully septated heart stage (E18.5). Similar to KCNQ1 mRNA, first expression of KCNH2 can be detected first at E9.5 throughout the entire heart. Similar expression levels are observed within the distinct transcriptional domains of the developing heart, atria, atrioventricular canal, ventricles and outflow tract (Fig. 3A). This expression profile remains unaltered with further development (Fig. 3B). No differences in expression levels have been observed within the ventricular compartment; compact and trabeculated layers have similar expression profiles. The expression profile of KCNH2 protein fully recapitulated that observed at the mRNA level during cardiac development (Fig. 3C,D).


Figure 3
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  		Fig. 3 KCNH2 mRNA and protein expression. Left lateral view (A) and ventral view (B) of whole-mount in situ hybridization against KCNH2 mRNA in mouse embryos of E10.5 (A) and E12.5 (B). Observe that expression of KCNH2 is homogeneously distributed along the entire myocardium at both stages (A,B). Immunohistochemical detection of KCNH2 protein in transverse sections of E16.5 mouse hearts. Observe that KCNH2 protein is evenly expressed in the atrial and ventricular myocardium, including the pulmonary vein (PV) and the caval vein myocardium (LSCV; C). Note that expression in the developing ventricular conduction system, i.e., bundle of His (arrow; D) is similar to that of the surrounding working myocardium. RV, right ventricle; LV, left ventricle; RA, right atrium; LA, left atrium; OFT, outflow tract; LSCV, left superior caval vein.

 
3.2 Expression profile of β subunits in the developing heart
3.2.1 KCNE1 (IsK/minK) mRNA expression
We have analysed the expression pattern of KCNE1 mRNA from the early tubular heart stage (E8.5) to the fully septated heart stage (E18.5). In contrast to KCNQ1, KCNE1 displays a dynamic profile of expression during heart development.

KCNE1 mRNA is first expressed at E9.5 through the entire myocardial tube. Atrial and ventricular myocardial chambers display similar KCNE1 expression levels (Fig. 4A,B). At this stage, high levels of expression of KCNE1 transcripts are observed in the most distal region of the outflow tract, just at the boundary between outflow tract myocardium and the mesenchymal aortic sac, as compared to the atrial and ventricular chambers. This profile of expression is maintained in the prototypical embryonic stage of heart development, at E10.5.


Figure 4
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  		Fig. 4 KCNE1 mRNA expression during heart development. Right lateral (A), ventral (B,C) and transverse section (D) views of whole-mount in situ hybridization against KCNE1 mRNA in mouse embryos of E9.5 (A,B) and E12.5 (C,D). Ventral (E) and dorsal (F) views of a mouse E12.5 heart sectioned along the atrioventricular axis and hybridized against KCNE1 mRNA. Left lateral (G) and right lateral (H) views of a E16.5 mouse heart sectioned along the atrioventricular axis and hybridized against KCNE1 mRNA. Observe that KCNE1 mRNA is first expressed within the atrial and the ventricular myocardium, having higher expression levels in the distal outflow tract (A,B). With further development, KCNE1 transcripts become confined to the ventricular myocardium (C,D). Expression is very high along the ventricular septum, whereas the ventricular trabeculations are almost devoid of expression. At E16.5, expression of KCNE1 transcripts is mainly confined to the outer compact myocardial layers, with no detectable levels in the ventricular septum (IVS). RV, right ventricle; LV, left ventricle; RA, right atrium, LA, left atrium.

 

With further development (E12.5), KCNE1 expression becomes confined to the ventricular and outflow tract myocardium, whereas no expression can be observed in the atrioventricular canal, atrial and inflow tract myocardium (Fig. 4C,D). Enhanced expression of KCNE1 is no longer observed in the outflow tract derived myocardium. Within the ventricular myocardium, robust expression is observed in the compact myocardial layer, whereas the trabeculations show a slightly lower level of expression. No differences between right and left cardiac ventricular compartments have been observed. Strikingly, KCNE1 transcripts are highly expressed within the entire ventricular septum (Fig. 4E,F). Short exposure hybridization experiments show enhanced expression in the myocardium forming the interventricular sulcus (groove), at both the dorsal and the ventral side (data not shown). This profile of KCNE1 expression in the early septating heart is maintained at E13.5 of gestation.

Further changes in the pattern of expression of KCNE1 transcripts are observed at E14.5. Expression of KCNE1 remains confined to the ventricular chambers, mainly within the outermost compact myocardial layers. However, the ventricular septum, at this stages, does not show expression of KCNE1. This expression profile is maintained in the subsequent stages of cardiac development (E15.5–E18.5) (Fig. 4G,H).

3.2.2 KCNE2 (MiRP1) mRNA expression
In contrast to the ubiquitous expression of the pore-forming subunits of the IKs and IKr mediating K+ channels in the myocardium, the expression of KCNE2 mRNA is restricted to the atrial myocardium starting at E9.5, when it is first observed. Expression of KCNE2 transcripts during more advanced embryonic (E10.5) and fetal (E13.5) developmental stages remains confined to the atrial myocardium as illustrated in Fig. 5A,B.


Figure 5
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  		Fig. 5 KCNE2 and KCNE3 mRNA expression during cardiac development. Left lateral view (A) and ventral view (B) of whole-mount in situ hybridization against KCNE2 mRNA in mouse embryos of E10.5 (A) and E12.5 (B). Observe that expression of KCNE2 is already confined to the developing atrial chambers at early embryonic stage (A) and remains a such during further development (B). Ventral view (C,E,F) and right lateral view (D) of whole-mount in situ hybridization against KCNE3 mRNA in mouse embryos of E8.5 (C), E9.5 (D), E12.5 (E). and E14.5 (F). Expression of KCNE3 mRNA is first observed evenly within the entire heart (C–E), whereas in fetal stages, expression of KCNE3 mRNA becomes mainly confined to the atrial chambers (F). Note that expression of KCNE3 mRNA is also detected in the developing branchial arches (D).

 

3.2.3 KCNE3 (Mirp2) mRNA expression
The expression profile of KCNE3 transcripts during cardiac development is dynamic. First evidence of KCNE3 expression is observed at the tubular heart stage (E8.5) throughout the entire heart (Fig. 5C) and it remains similar during the embryonic stages (E9.5–E12.5; Fig. 5D,E). In addition to cardiac expression, KCNE3 is also expressed in the branchial arches and the developing limb buds (data not shown). With further development, the expression of KCNE3 becomes progressively down-regulated in the ventricular chambers, being the highest expression confined to the atrial chambers (Fig. 5F). Expression of KCNE3 is down-regulated during late fetal stages (E16.5), when it is expressed only at basal levels (data not shown).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The heart is the first embryonic organ to be formed during embryogenesis and first beats are already observed at the linear heart tube stage [32–34]. At this stage, several components of the excitation–contraction coupling system are already differentially expressed along the antero-posterior axis [2,4,5]. We have observed that the initial expression of the pore-forming subunits of the slow and fast delayed-rectifier cardiac K+ currents can be detected around E8.5–E9.0, shortly after the cardiac tube starts to acquire distinct atrial and ventricular chamber identities and a synchronous pattern of contraction [34]. At this time, expression of KCNQ1, KCNH2, KCNE2 and KCNE3 is observed within all cardiac compartments, including the forming atrial and ventricular working myocardium, as well as the myocardial areas reminiscent of the primary cardiac tube (‘primary myocardium’; outflow tract, atrioventricular canal and inflow tract). This extensive myocardial expression profile seems to correlate with the developmental window at which a peristaltoid-like contraction wave can be recorded in the embryonic heart. Whereas KCNQ1 and KCNH2 display homogeneous expression along the antero-posterior cardiac axis, both at the mRNA and protein levels, the enhanced expression of KCNE1 transcripts in the distal myocardial portion of the outflow tract at E9.5–E10.5 might be reminiscent of the contraction gradient established by dominant pacemaker activity observed invariably at the venous pole of the cardiac tube at this stage [34]. Unfortunately, the analysis of the pattern of expression of KCNE1 protein is hampered by the lack of specific antibodies against such potassium channel subunit. On the other hand, KCNE2 transcripts are already restricted to the atrial myocardium at this early stage, contrasting with the patterns often seen during development of the mammalian heart (for a review, see Ref. [2]). Recently, several transcription factors have been reported to be confined to discrete transcriptional domains as early as in the tubular heart stage [35,36]. In chicken, the restricted expression of AMHC1 mRNA to the prospective atrial myocardial cells at the linear heart stage, is in contrast to the more broad expression of VMHC1 mRNA at similar stages [37,38], underscoring the hypothesis that confinement of ‘atrial-specific’ genes may occur earlier during ontogenesis than ‘ventricular-specific’ genes. It may be postulated that a similar mechanism applies to the ion channel subunits expression during mammalian cardiogenesis.

The restricted expression of KCNE1 to the ventricular chambers and of KCNE3 to the atrial chambers coincides with the onset of more synchronous contractions of the atria and ventricles [38], as the cardiac chambers obtain distinct functional properties [5]. On the other hand, KCNQ1 and KCNH2 (mRNA and protein) remain expressed at similar levels throughout the entire heart. A summary of the expression domains of the different K channel subunits is provided in Fig. 6. These data suggest that β-subunit expression plays a major role in establishing the regional heterogeneity (e.g., atrial versus ventricular) of the delayed rectifier K+ currents during cardiac development. Our data are in line with previous electrophysiological measurements of IKs and IKr currents in atrial and ventricular myocytes. Davies et al. [39] reported that IKr is predominantly recorded in atrial cells at early embryonic stages, coinciding with the restricted expression of KCNE2 to the atrial myocardium. Similarly, the IKs current is increasingly more prominent in the ventricular myocardial cells during cardiac development but failed to be recorded in atrial cells at any stage of development. These data are in agreement with the ventricular myocardium-restricted distribution profile of KCNE1 transcripts reported in this study. Although the functional implications of the dynamic expression of KCNE3 transcripts in the developing heart remains to be elucidated, its expression profile underscores the hypothesis of a major role of the β-subunits in establishing K+ current heterogeneity during cardiogenesis.


Figure 6
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  		Fig. 6 K channel subunits expression domains. Schematic representation of the distribution pattern of KCNQ1, KCNH2, KCNE1, KCNE2 and KCNE3 transcripts in the different cardiac regions at embryonic and fetal stages. IFT, inflow tract; A, atria; AVC, atrioventricular canal; V, ventricle; OFT, outflow tract.

 
Surprisingly, the expression profiles of KCNQ1 mRNA and protein diverge within the working and specialized conduction system myocardium in fetal stages. KCNQ1 mRNA is expressed at similar levels in both myocardial tissues [22], whereas an enhanced expression of KCNQ1 protein is observed in the fetal and adult ventricular conduction system. These data suggest that a tissue-specific post-transcriptional mechanism is controlling the distribution of functional KCNQ1 channels during fetal development and adulthood.

In summary, the present data show that differential expression of KCNE1 and KCNE3 is turned on concomitantly with the onset of a co-ordinated atrial/ventricular contraction wave in the developing heart, whereas restriction of KCNE2 is observed at the earliest time points. Additionally, we show that the regional variation in the physiological characteristics of the delayed-rectifier outward K+ current would be mainly dependent on the expression domains of the β-subunits (KCNE1, KCNE2 and KCNE3), since the pore-forming subunits (KCNQ1 and KCNH2) are evenly expressed, both at mRNA and protein level, in atrial and ventricular myocardial compartments.

Time for primary review 19 days.


   Acknowledgements
 
We are indebted to Corrie de Gier-de Vries for technical support and to Connie Bezzina and Andrew Munk for critical reading of the manuscript. D. Franco is supported by NWO (902-16-219) and Dutch Heart Foundation (97206). Denis Escande and A.F.M. Moorman laboratories are supported by an NWO/INSERM grant.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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