The expression pattern of the TASK-1 protein was analyzed during early heart development in chicken, using a polyclonal antibody to human TASK-1. This antibody was made to detect an epitope in human TASK-1 that is 100% conserved in chicken TASK-1 (Fig. 1). TASK-1 protein is detected in the myocardium (M) of the fusing cardiac primordium (CP) (Fig. 2A) and the primitive heart tube (HT) (Fig. 2B) as early as Hamburger and Hamilton stage 10. At stage 18, the heart is a looped, two-chambered, tube with one primitive atrium (PA) and one primitive ventricle (PV). TASK-1 is seen throughout the myocardium of the heart tube, in the outflow tract (OF) (Fig. 2C), as well as the primitive atrium and ventricles (Fig. 2D). By embryonic day (E) 5 or stage 27, the four chambers of the heart are beginning to form, TASK-1 continues to be expressed throughout the myocardium of the outflow, ventricles (V), and the atria (A) (Fig. 2E,F). Interestingly, at stage 18 and E5, TASK-1 staining is also detected in regions of the endocardium (E), where cells are undergoing a transformation into mesenchymal cells as the endocardial cushions (EC) form (Fig. 2C-F). To demonstrate staining is specific to TASK-1, we performed controls at all stages. Sections were labeled with the TASK-1 antibody, which had been pre-absorbed with the TASK-1 peptide, followed by the same secondary antibody used for all TASK-1 staining. No signal was observed in these controls (Fig. 2G and data not shown). Consistent with protein expression, mRNA encoding cTASK-1 is seen throughout the heart tube at stage 18 (Fig. 2H) and in all four chambers of the heart at E5 (Fig. 2I).
TASK-1 Expression Is Restricted to the Developing Ventricular Conduction System in the Chicken Embryo
As embryogenesis proceeds, a heterogeneous distribution of TASK-1 develops. In the ventricles, TASK-1 expression is gradually down-regulated in the compact myocardium (CM) and is maintained in the trabeculated myocardium (TM); this population of cells is thought to differentiate into ventricular conduction system (reviewed in Gourdie et al.,[1999]; Moorman and Christoffels,[2003]). In the mature heart, the ventricular conduction system consists of the bundle of His and the bundle branches as well as a peripheral conduction network of Purkinje fibers. In avians, the Purkinje network is well developed and, unlike mammals, extends into periarterial regions. By E7 (stage 31) in chicken, TASK-1 staining is strongest at the crest edge of the interventricular septum (IVS) as well as in the trabeculated myocardium. In comparison, expression in the compact myocardium is weaker (Fig. 3A). This expression pattern suggests that TASK-1 may become restricted to components of the developing ventricular conduction system as the heart develops.
Рис.3. | Expression of two-pore domain potassium channel (TASK-1) in the maturing ventricular conduction system of chicken. TASK-1 expression, as detected by an antibody to human TASK-1, in frozen frontal sections of excised embryonic day (E) 7-E11 hearts. A-C: A heterogeneous distribution of TASK-1 expression becomes apparent at E7 (s31), cells with high levels of TASK-1 expression (green) also express polysialic acid-nerve cell adhesion molecule (PSA-NCAM, red). D-F: TASK-1 (green) and PSA-NCAM (red) expression at E8 (s34) in the bundle branches of the interventricular septum. G-I: TASK-1 (green) and HNK-1 (red) in the interventricular septum at E8, G is a serial section of D-F, and H and G are at 50-m intervals anteriorly. Arrows highlight a region of TASK-1 staining that is negative for PSA-NCAM and HNK-1 (see text). J: TASK-1 expression in the left bundle branch and bundle of His in the interventricular septum at E11. IVS, interventricular septum; TM, trabeculated myocardium; CM, compact myocardium; LBB, left bundle branch; BH, bundle of His. Scale bars = 50 m.
One of the earliest molecular markers of the developing conduction system in chickens is polysialic acid-nerve cell adhesion molecule (PSA-NCAM). This cell surface carbohydrate is seen in the trabeculated myocardium as early as stage 20 and transiently marks cells that go on to form the Purkinje fibers and bundle branches, but not the bundle of His, in the mature conduction system (Watanabe et al.,[1992]; Chuck and Watanabe,[1997]). At E7, strong TASK-1 staining colocalizes with PSA-NCAM expression in the subendocardial cells of the trabeculated myocardium and the septum (Fig. 3A-C). During heart development, the bundle of His and bundle branches develop relatively early (mostly before E8), whereas the peripheral Purkinje fibers develop later and continue to differentiate until after hatching (Gourdie et al.,[1995]; Cheng et al.,[1999]; Thompson et al.,[2000]). At E8 (stage 34), in the ventricular septum, bundle branch precursors appear to be defined by PSA-NCAM staining (Fig. 3E; Watanabe et al.,[1992]; Chuck and Watanabe,[1997]). At this stage, TASK-1 is expressed in PSA-NCAM positive bundle branch precursors as well as a population of cells superior to this (see arrows in Fig. 3D-F). It is possible that these TASK-1-positive cells are part of the bundle branch that is not marked by PSA-NCAM. Another explanation is that this staining represents the developing bundle of His. It has previously been proposed that HNK-1 marks the bundle of His (Chuck and Watanabe,[1997]). However, others suggest that HNK-1 is expressed in the myocardium surrounding the bundle of His and that the bundle itself does not express HNK-1 (Verberne et al.,[2000]). Despite these conflicting observations we have characterized the expression of TASK-1 in relation to HNK-1 for completeness (Fig. 3G is a serial section of Fig. 3D-F; Fig. 3H,G are at 50-m intervals anteriorly). Our results demonstrate that, at E8, TASK-1 (green) is expressed robustly in a population of HNK-1-negative cells that are surrounded by HNK-1 (red)-positive myocardium at the top of the interventricular septum (see arrows). By E11, it is clear that TASK-1 expression is mostly restricted to the bundle of His and the bundle branches of the interventricular septum (Fig. 5J; this section plane shows only the left bundle branch).
The Purkinje fibers of the ventricular conduction system develop from the trabeculated myocardium of the ventricular walls (reviewed in Moorman et al.,[1998]). To determine whether TASK-1 expression is maintained as the Purkinje network differentiates, its expression was closely examined between E5 and E19 in the trabeculated myocardium of the ventricular walls. At E5, TASK-1 is expressed throughout the ventricular myocardium (Fig. 4A). At E11, TASK-1 expression is strongest at the outer edge of the trabeculated myocardium but is not yet completely absent in rest of the ventricular wall (Fig. 4B). By E19, the trabeculated myocardium has become more compact and TASK-1 expression is restricted to a subpopulation located close to the ventricular lumen (L) (Fig. 4C). Thus, a temporal restriction of the TASK-1 protein is occurring in the trabeculated myocardium as embryogenesis proceeds. Further analysis of TASK-1 staining in E19 chickens revealed periarterial expression of TASK-1, in addition to that seen at the edge of ventricular walls (Fig. 4D-F). This expression pattern is characteristic of the Purkinje network in chickens and suggests that TASK-1 is restricted to cells of the ventricular conduction system by E19. To demonstrate that a population of the TASK-1-positive cells are Purkinje fibers, embryos were double labeled with TASK-1 (green) and ALD-58 (red; a marker specific for slow skeletal muscle-type myosin heavy chain that labels the mature Purkinje system in chickens; Gonzales-Sanchez and Bader,[1985]). Coexpression of TASK-1 and ALD-58 is seen in periarterial Purkinje cells (Fig. 4D-F) as well as subendocardial Purkinje cells of the ventricular walls (Fig. 4G-I) of E19 chickens. Taken together, these results demonstrate that, in chicken, TASK-1 is initially expressed throughout the myocardium of the developing heart tube but in the ventricles is restricted to components of the conduction system as the heart differentiates.
Рис.4. | Restriction of two-pore domain potassium channel (TASK-1) expression to the mature conduction system in the developing chicken embryo. TASK-1 protein expression in transverse (A and B) and frontal (C-I) frozen sections of the ventricular wall as detected by an antibody to human TASK-1. A: Embryonic day (E) 5 (s27). B: E11. C-I: At E19, TASK-1 expression (green) is seen subendocardial (E,F) and periarterial Purkinje fibers (G-I) of the ventricular walls marked by ALD-58.
Рис.5. | TASK-1 expression in the developing mouse heart. TASK-1 protein expression as detected by an antibody to human TASK-1. TASK-1 is expressed throughout the myocardium at embryonic day (E) 8.5 (A), E9.5 (B), and E10.5 (C). In serial sections of E13.5 and E18.5 hearts, TASK-1 expression (green) becomes restricted to the developing conduction system marked by Cx40 (red). D: Cx40 expression at E13.5. E: TASK-1 expression at E13.5. F: Cx40 in the trabeculated myocardium at E18.5. G: TASK-1 in the trabeculated myocardium of E18.5 embryos. Cx40 (H) and TASK-1 (I) in the right bundle branch of the interventricular septum at E18.5. M, myocardium; E, endocardium; IVS, interventricular septum; BB, bundle branch; TM, trabeculated myocardium. Scale bars = 50 m.
Expression of TASK-1 in Mouse Is Restricted to the Developing Conduction System in Mouse
To determine whether a similar profile of TASK-1 expression occurs during mammalian cardiogenesis, we analyzed the distribution of this protein in the developing mouse heart. TASK-1 expression is detected in the M of the fusing cardiac primordium as early as E8.5 (Fig. 5A). In the early heart tube, at E9.5, TASK-1 is present throughout the myocardium (Fig. 5B). At E10.5, TASK-1 continues to be expressed in the myocardium of both the atria and ventricles. However, even at this early stage, expression is strongest in the TM of the ventricles (Fig. 5C). In addition, TASK-1 staining is also seen in regions of endocardium (E) undergoing endocardial to mesenchymal transition as the endocardial cushions develop (Fig. 5C). To compare the expression profile of TASK-1 in relation to the development of the murine conduction system, its distribution was analyzed in association with the ventricular conduction system marker, connexin40 (cx40) (Miquerol et al.,[2003]). At 13.5, cx40 is prominent in the TM and at the outer edge of the IVS; regions that will eventually differentiate into the mature ventricular conduction system (Fig. 5D). Likewise, TASK-1 is expressed predominantly in the trabeculated myocardium of the ventricles and at the outer edge of the interventricular septum with only low levels in the CM (Fig. 5E). By E18.5 TASK-1 is largely confined to the ventricular conduction tissues that are defined by cx40, expression is seen in what remains of the trabeculated myocardium (Fig. 5F,G) and the bundle branches of the interventricular septum (Fig. 5H,I). These data demonstrate that, in the mouse ventricles, TASK-1 expression is restricted to the developing conduction system in a similar manner to that seen in the chicken and are consistent with a mechanism of conduction system development common to chicken and mouse.
DISCUSSION
В этом исследовании мы описали белковые последовательности куриного TASK-1 во время развития вентрикулярной проводящей системы у эмбрионов кур и мышей. У обоих организмов TASK-1 первоначально экспрессируется по всему миокарду на ст. ранней сердечной трубки. Однако, по ходу кардиогенеза экспрессия в желудочках становится ограниченной сначала трабекулярным миокардом и в конце концов пучками Гиса, веточками пучков и волокнами Пуркинье (Figs. 2-5). Этот паттерн экспрессии особенно интересен, т.к. он коррелирует с рекрутированием клеток в проводящую вентрикулярную систему в ходе эмбриогенеза. Пространственно-временное распределение TASK-1 сходно с некоторыми др. маркерами ткани проводящей системы. Напр., у кур с нейральным белком EAP 300, экспрессируемым сначала по всему миокарду во время раннего эмбриогенеза, а затем ограничивающегося вентрикулярной проводящей системой по мере развития сердца (McCabe et al.,[1995]). Сходным образом, у кур, мышей и людей транскрипционный фактор Nkx2.5 обнаруживает стадио-зависимое ограничение компонентами вентрикулярной проводящей системы (Takebayashi-Suzuki et al., 2001; Thomas et al.,[2001]; Harris et al.,[2004]). Исследования по отслеживанию клонов, а также пространственное и временное картирование клеточной пролиферации элегантно продемонстрировали, что вентрикулярная проводящая система происходит из миоцитов трабекулярного миокарда, который клонально родственен миоцитам первичной сердечной трубки (Gourdie et al.,[1995]; Thompson et al.,[1995]; Cheng et al.,[1999]; Thompson et al.,[2000]; Sedmera et al.,[2003]; rev. Moorman et al.,[1998]; Moorman and Christoffels,[2003]). Наши данные демонстрируют, что пучок Гиса, веточки пучка и волокна Пуркинье дифференцируются из TASK-1-позитивных миоцитов ранней сердечной трубки, которые продолжают экспрессировать TASK-1 в ходе всего кардиогенеза. Эти результаты согласуются с общепринятым механизмом дифференцировки вентрикулярной проводящей системы у кур и мышей, несмотря на анатомические различия их зрелых проводящих систем.
Хотя физиологическая роль TASK-1 в развивающемся сердце пока неизвестна, но наши результаты согласуются с предположением, что экспрессия TASK-1 может быть связана с проводящей способностью тканей сердца во время развития. Морфологические, так как и физиологические исследования подтверждают, что весь миокард ранней сердечной трубки существенен для проводящей системы, также как и для работающего миокарда (Viragh and Challice,[1977]; Kamino et al.,[1991]). Позднее в развитии, перед тем как вентрикулярная проводящая система будет полностью развита, трабекулярный миокард, по-видимому, функционирует как проводящий компонент желудочков (De Jong et al.,[1992]). Истечение калия через фоновые каналы является важным для восстановления потенциала покоящихся мембран и TASK-1 каналы, как сообщалось ранее, участвуют в генерации и модуляции resting membrane potential (Lesage and Lazdunski,[1998]). Возможно, что в быстро проводящей ткани зрелой проводящей системы функция каналов TASK-1 помогает реполяризовать клетки при подготовке последующих потенциалов действия. Чувствительность к колебаниям в O2 и pH ещё больше подтверждает, что TASK-1 может затрагивать вентрикулярное проведение в условиях гипоксии и/или ацидоза и alkalosis и может представлять механизм для поддержания кардиального гомеостаза в этих условиях.
Итак, мы идентифицировали TASK-1 как маркер развивающейся вентрикулярной проводящей системы у птиц и млекопитающих. Полученные результаты подтверждают механизм развития у этих двух видов, у которых вентрикулярная проводящая система дифференцируется из пред-существующих миоцитов, которые продолжают экспрессировать TASK-1.
Сайт создан в системе
uCoz