生理学报　Acta Physiologica Sinica, April 25, 2006, 58 (2): 95-103

Brief Review

Developmental regulation of intracellular calcium homeostasis in early cardiac myocytes

FU Ji-Dong, YANG Huang-Tian^*

Key Laboratory of Stem Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200025, China

Abstract: The proper intracellular Ca²⁺ signaling is essential for normal cell functions and organ development, and the maintaining Ca²⁺ homeostasis in cardiac myocytes is of functional importance for the intact heart. As the first functional organ in the vertebrate embryo, the heart is continuously remodeled and maintains its physiologic pumping function in response to increasing circulatory demands. The expressions of Ca²⁺ handing proteins in the embryonic heart, however, are different from those in neonatal and adult hearts, which means that the regulation of Ca²⁺ transients in embryonic cardiomyocytes is different from that in adult cardiac myocytes. Recent advances in molecular and cellular biology, as well as the application of embryonic stem cell differentiation system, have made progress in uncovering the regulation of Ca²⁺ homeostasis during cardiomyogenesis. This paper briefly summarizes the Ca²⁺ homeostasis during early development of cardiomyocytes and reviews current knowledge of the regulatory mechanisms controlling Ca²⁺ homeostasis during cardiomyocyte development.

Key words: Ca²⁺ homeostasis; cardiomyocytes; development

心肌细胞发育过程中胞浆内钙稳态的调控

傅继东，杨黄恬^*

中国科学院干细胞生物学重点实验室，上海 200025

摘 要：Ca²⁺信号是细胞和各器官生长发育、行使其生理功能的基础，维持心肌细胞的钙稳态是保持正常心脏功能的先决条件。作为在胚胎发育过程中最早出现并行使功能的器官，胚胎期心脏的形态结构发生了明显的变化，泵血功能不断增强，以适应不断增强的机体的生理需求。从胚胎到成年，心肌细胞的功能有非常大的改变，各钙离子通道的表达也发生明显变化。因此，发育早期心肌细胞的钙稳态调控与成熟心肌细胞有明显的不同，在发育过程中引起细胞收缩的Ca ²⁺来源也有明显的变化。随着分子和细胞生物学研究的发展，以及胚胎干细胞体外分化模型的应用，人们对心肌细胞发育过程中钙稳态的调控有了进一步的认识。本文综述了早期心肌细胞发育过程中胞浆内钙稳态的变化，总结了早期心肌细胞钙稳态调控机制的最新研究进展。

关键词：钙稳态；心肌细胞；分化发育

中图分类号：Q463; R331.3⁺1 Received 2005-10-20 Accepted 2006-01-11

This work was supported in part by the National Natural Science Foundation of China (No.30270656), Programs from Science and Technology Committee of Shanghai Municipality (No.03DJ14019, 01JC14048).

^*Corresponding author.
Tel: +86-21-63854240;
Fax: +86-21-63852593;
E-mail: htyang@sibs.ac.cn

Ca²⁺ ions are major players in an intracellular signaling system that translate extracellular stimuli into the regulation of a bewildering number of phenomena such as muscle contraction, neurotransmitter release and other secretion processes, cell proliferation, gene expression and cell death. Therefore, the proper intracellular Ca²⁺ signaling is essential for normal cell functions and organ development^[1]. Ca²⁺ homeostasis in cardiac myocytes is of functional importance for at least three reasons^[2]. First, cardiac myocytes must achieve a resting cytosolic free calcium ion concentration ([Ca²⁺]_i) of <200 nmol/L if the contractile elements are to relax. With extracellular Ca²⁺ concentration at 1 mmol/L, this low [Ca²⁺]_i must be maintained in the presence of a 5 000-fold gradient for Ca²⁺ across the sarcolemma. Second, contraction in cardiomyocytes arises by the Ca²⁺ induced Ca²⁺ release (CICR) mechanism, i.e. Ca²⁺ entering through sarcolemmal specific ion channels activates Ca²⁺ release channels (RyRs) in the sarcoplasmic reticulum (SR). This is the base of cardiac excitation-contraction (E-C) coupling. Upon the extrusion of cytosolic Ca²⁺ back to the SR and out of the sarcolemma, relaxation occurs. The fundamental principle of this process is to maintain a steady Ca²⁺ homeostasis, the amount of Ca²⁺ entering the cell with each contraction must be extruded before the subsequent contraction. Third, the contractile force of cardiac myocytes is modulated by variations in the magnitude and rise or decay of the Ca²⁺ transients. Therefore, any alteration of Ca²⁺ homeostasis would cause abnormal contractions and drugs that modify Ca²⁺ homeostasis may significantly alter the contractile force of an individual cardiomyocyte and thus of the intact heart.

Heart is the first organ that becomes functional in the vertebrate embryo. Approximately embryonic day (E) 7.25 in mouse, the precardiac mesoderm forms a primitive tubular heart, which starts beating at E8. Heart is continuously remodeled until the four-chambered organ is formed, and maintains its physiologic pumping function in response to increasing circulatory demands^[3]. The ensuing development of E-C coupling is fundamental to the embryonic cardiac function during embryogenesis. The expression of Ca²⁺ handing proteins in embryonic heart, however, is different from that in the neonatal and adult heart^[4,5], which means that the process of Ca²⁺ transients in embryonic cardiomyocytes may be different from that in adult cardiac myocytes. Because of the known difficulties to obtain cardiomyocytes from the very early mammalian embryos (e.g., before day 12 to 13 of gestation in mouse), there is only limited knowledge on the developmental aspects of cardiomyocyte functions. Recent studies have established the embryonic stem (ES) cell in vitro differentiation system as a tool for the investigation of early cardiomyogene-sis^[6-11]. The ES cell-derived cardiomyocytes (ESCMs) represent specialized cell types of the heart, such as atrial-like, ventricular-like, sinus nodal-like, and Purkinje-like cells^[6]. During differentiation, the ultrastructural^[7], molecular biological^[9] and electrophysiological^[6,12] studies have demonstrated that within ES cell-formed embryoid bodies (EBs), the various stages of cardiomyogenesis parallel the murine heart development. This in vitro model system is especially useful to identify gene function during cardiac development when "loss of function" studies result in early embryonic lethality^[6,9,13]. Furthermore, the development of the new technology allows observing intracellular Ca²⁺ movement and, thus, provides the new knowledge on the regulation of early cardiac Ca²⁺ homeostasis. The present paper reviews the new interpretation on the developmental regulation of the Ca²⁺ homeostasis in early cardiomyocytes.

Developmental changes of Ca²⁺ homeostasis

During the heart remodeling process, regular heart beating is already observed in the very early period of heart development, which indicates that an intracellular system of contraction, as well as cardiac automaticity, establishes in the early period. With regard to the diastolic [Ca²⁺]_i of (102±1) nmol/L in 12-day fetal and (151±4) nmol/L in adult cardiomyocytes^[14], it is reasonable that there is an increasing change in the rest [Ca²⁺]_i during the cardiac maturation. During the cardiac differentiation from ES cells in vitro, the basal [Ca²⁺]_i rises significantly from (94±7) nmol/L at early developed stage to (135±11) nmol/L at late developed stage^[15]. Ca²⁺ sparks, which reflect the Ca²⁺-dependent activation of RyRs, are rarely detected in the early fetal cardiac myocytes, and Ca²⁺ transients in the fetal myocytes are characterized by a slower upstroke and decay of the [Ca²⁺]_i compared to those in adult myocytes^[14]. In early to late differentiated cardiomyocytes derived from ES cells, the more rapid and higher spontaneous Ca²⁺ transients are developed gradually^[15,16].

Control of resting [Ca²⁺]_i

In the adult cardiomyocytes, the resting [Ca²⁺]_i is determined by a Ca²⁺ leak that is compensated for by the ATP-dependent sarcolemmal Ca²⁺ pump (sarcolemmal Ca²⁺-ATPase) and the sarcolemmal Na⁺-Ca²⁺ exchanger (NCX). The cardiac NCX contains 970 amino acids and has a molecular mass of 108 kDa^[17]. The NCX is reversible and carries out ion transport in a consecutive or "ping-pong"reaction mechanism. NCX transports 3 Na⁺ inwardly and releases an intracellular Ca²⁺ ion to the extracellular space after completing a cycle. This would result in movement of one net charge inward per cycle and thus Ca²⁺ extrusion constitutes an inward current of NCX (I_Na/Ca). This electrogenicity also indicates that NCX is sensitive to membrane potential as well as intracellular and extracellular [Ca²⁺] and [Na⁺]^[18]. It is identical to the reversal potential of NCX (around -40 mV) in resting cardiomyocytes. If membrane potential depolarizes to more positive than the reversal potential, the NCX will function as the "reverse" (Ca²⁺ in/Na⁺ out) mode. The membrane potential in the normal resting myocytes is known to be more negative than the reversal potential, therefore, the NCX functions in a "forward" (Na⁺ in/Ca²⁺ out) mode and extrudes Ca²⁺ from the cells.

The other Ca²⁺ transport system that contributes to the maintenance of low cytosolic Ca²⁺ concentration in ventricular myocytes is the sarcolemmal Ca²⁺ ATPase. The purified protein was cloned with a 108 kDa molecular mass, and a central stretch of ～80 kDa is all required for Ca²⁺ transport^[19] that contains the regulatory calmodulin binding domain and has regulatory sites phosphorylated by PKA and PKC^[20]. One Ca²⁺ ion seems to be transported per ATP hydrolyzed and Ca²⁺ extrusion by this pump appears to be coupled to proton influx (1Ca²⁺:1H⁺). Although the sarcolemmal Ca²⁺-ATPase probably does contribute to the maintenance of [Ca²⁺]_i in resting myocytes, its importance seems to be minor in comparision with the NCX. In cultured guinea-pig adult ventricular myocytes, the rate at which the sarcolemmal Ca²⁺-ATPase extrudes the Ca²⁺ from the myocytes appears to be about 1/10 that of the NCX over the range of physiological cytosolic Ca²⁺concentrations^[21].

It is still unclear whether the mechanisms to modulate the rest [Ca²⁺]_i in embryonic cardiomyocytes is different from that in adult cardiac myocytes. However, NCX is homogenously expressed in early embryonic and fetal heart, and the expression level is high in fetal heart but decreases postnatally^[22]. The NCX current density is also declined rapidly after birth^[23]. The highly expression of NCX in cardiomyocytes is thought to be required to maintain a low [Ca²⁺]_i. NCX1-null mouse embryos lack a heartbeat and have abnormal myofibrillar organization, and die before E10.5^[24-26]. Therefore, NCX is more important in developing hearts and may play a pronounced role in maintaining a low [Ca²⁺]_i during the development of cardiomyocytes. Recently, we observed that both ryanodine, a specific inhibitor of RyRs, and thapsigargin, a specific SR Ca²⁺-pump ATPase inhibitor, significantly elevate resting [Ca²⁺]_i in cardiomyocytes derived from ES cells at early, intermittent and late differentiation stages, demonstrating that functional SR also takes part in maintaining basal [Ca²⁺]_i^[15]. With regard to the increasing rest [Ca²⁺]_i, both SR and the sarcolemmal Ca²⁺ transport proteins may contribute to the maintenance of the resting [Ca²⁺]_i in early developing cardiomyocytes.

Regulation of Ca²⁺ influx in the E-C coupling

In adult cardiac myocytes, the increase of [Ca²⁺]_i from the 100 nmol/L at diastole to a peak of ～10 mmol/L at maximal contraction occurs because a small quantity of Ca²⁺ enters the sarcolemma and triggers a much larger release of Ca²⁺ from the SR Ca²⁺ release channel RyRs by the process of CICR, which leads to a dramatic increase of [Ca²⁺]_i^[2]. For the contraction of the hearts, a Ca²⁺-influx from extracellular space is required because the removal of Ca²⁺ from extracellular solution abolishes cardiac contraction. This phenomenon is also observed in embryonic cardiomyocytes. Therefore, the developmental expressions of those sarcolemmal Ca²⁺ channels have a direct influence on the modulatory mechanisms of E-C coupling.

The L-type Ca²⁺ channel is an oligomeric complex of five subunits (a1, a2/d, b and g) and can be phosphorylated by cyclic AMP (cAMP)-dependent protein kinase (PKA)^[27]. L-type Ca²⁺ channels are thought to be the main transporter for trans-sarcolemmal Ca²⁺ influx in adult cardiomyocytes^[28]. In the early (3-day) stage of development, the L-type Ca²⁺ channels appear in embryonic chick heart cells, and the L-type Ca²⁺ current density in 3-day is higher than that in 17-day cells^[29]. In mouse and rat hearts, the density of Ca²⁺ currents increases during fetal development and reaches its maximal level at about the time of birth^[30,31]. In the 9.5-day postcoitum mouse heart^[32] and in early-stage ESCMs^[33], b-adrenergic receptor (b-AR) stimulation already modulates L-type Ca²⁺ channel currents. In brief, L-type Ca²⁺ channels are considered to be of great importance in E-C coupling in early embryonic cardiomyocytes.

Ca²⁺ currents in the cardiomyocytes generally consist of two components: a high threshold slow (L-type) Ca²⁺ current and a low threshold early transient (T-type) Ca²⁺ current. The T-type Ca²⁺ current is also a trigger for the Ca²⁺ release from the SR, although its function is much less compared to that of the L-type current^[34,35]. The expression pattern of T-type Ca²⁺ current depends on species, tissues, and developmental stages. The T-type Ca²⁺ currents is relatively abundant in the conduction system, such as sinoatrial nodal cells and Purkinje cells, whereas it is negligible in some ventricular cells (e.g., rat) ^[28]. It has been documented that T-type Ca²⁺ currents present in early fetal and ES cell-derived early-stage cardiomyocytes^[36,37] and that the mRNA expression of T-type channel increases significantly following a decline during the cardiomyocyte differentiation from ES cells^[37]. T-type Ca²⁺ channels also present in embryonic human cardiomyocytes and the mRNA expression decreases with development^[38]. In chick-ventricular development, a Ca²⁺ entry via the T-type channels plays a significant role in E-C coupling in the developing heart through the stimulation of CICR^[39]. Pharmacological approaches also suggest that the Ca²⁺ entered via the T-type Ca²⁺ channels contributes to excitability in midgestational fetal mouse myocardium^[36].

In developing cardiomyocytes, the Ca²⁺ influx can also be mediated via NCX "reverse" mode (Ca²⁺ in/Na⁺ out). In theory, when the membrane depolarizes to the reversal potential of the NCX, a Ca²⁺ influx should occur as the exchange reverses. This process may be further stimulated by subsarcolemmal rises in sodium concentration caused by the sodium influx via the cardiac sodium channels^[40]. The reverse NCX Ca²⁺ current can trigger SR Ca²⁺ release in adult rabbit ventricular myocytes^[41]. The expressions of NCX mRNA and protein are maximal near the time of birth and decline postnatally in chick, rat and rabbit hearts^[22,42,43]. Similarly, the density of the outward NCX current (the reverse mode) is highest at birth and decreases postnatally^[23,44]. The high expression of NCX in developing cardiomyocytes is thought to be required to maintain a low intracellular Ca²⁺ concentration, and may play a role in E-C coupling^[26,28]. Furthermore, such a Ca²⁺ influx appears to be sufficient to produce contraction in neonate cells, but not in adult cells^[44]. It is quite likely that the Ca²⁺ efflux mediated by NCX1 might compensate for the low Ca²⁺ uptake by the SR in immature cells and its significance in the regulation of the contraction and relaxation becomes less upon the maturation of the SR.

SR regulation and CICR

The RyR, as the major Ca²⁺ release channel in the SR, is a very large protein (565 kDa) and it exists as a homotetramer (2.2 MDa). The large size of this homotetramer has helped to identify it ultrastructurally as the junctional "foot"process, and the majority of the RyR molecular mass is located between the SR and sarcolemmal membranes where it forms a 28 nm×28 nm structure that extends ～12 nm above the SR membrane. This is the region where numerous ligands interact with RyRs, such as calmodulin, Ca²⁺, FK-506-binding proteins, PKA, phosphatases 1 and 2A, imperatoxin A^[45]. There are three RyR isoforms in mam-mals, and the main isoform in cardiac cells is RyR2^[46]. The contraction of the adult myocardium is highly dependent on SR function and less dependent on trans-sarcolemmal Ca²⁺ influx when compared with the neonatal myocardium^[47]. Some studies have shown that in fetal heart cells, the SR is scarce when observed with electron-microscope^[48], and isolated SR vesicles from the fetal heart have lower volume, lower density, and a decreased capability to load Ca²⁺ compared to those isolated from the mature heart^[49-51]. The expression levels of RyR2 and cardiac sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2) mRNAs and proteins in fetal cardiomyocytes are obviously lower than those in adult cells^[4]. Ryanodine, a specific inhibitor of RyR, has little to no effect on Ca²⁺ transients in fetal cells^[4,48,52]; and RyR2 has been postulated to act as a Ca²⁺ leak channel and not as a functional Ca²⁺ release channel in embryonic cardiomyocytes. Therefore, it is proposed that the contraction of fetal cardiomyocyte is regulated predominantly by sarcolemmal Ca²⁺ influx rather than Ca²⁺ release from SR.

However, this view of the embryonic cardiomyocytes without functional SR has been challenged by recent repor-ts. The recent observations demonstrate that SR RyR2^[9,53] and SERCA2a^[54,55] mRNAs/proteins are already abundantly expressed from the earliest stages of beating myocardium and functional SR Ca²⁺ stores are present in E13 rat ventricle. Early stage ESCMs continuously contract by the spontaneous intracellular [Ca²⁺]_i oscillations, which are from RyR sensitive Ca²⁺ stores^[56]. Moreover, the amplitude of Ca²⁺ transients in fetal cardiomyocytes is inhibited by both ryanodine and thapsigargin, a specific SERCA2 inhibitor^[14,15]. Takeshima et al.^[57-59] demonstrated that RyR2 deficient and the junction protein (JP-2) null cardiomyocytes have slow, weak, and irregular Ca²⁺ transients with early embryo lethality, indicating that RyR2 released Ca²⁺ is critical for Ca²⁺ homeostasis in early embryonic cardiomyocytes. The SR is functional in the embryonic chick heart before hatching, although most of the Ca²⁺ associated with the transients comes through the sarcolemmal Ca²⁺ channels^[60]. To specifically test whether SR Ca²⁺ release directly contributes to or regulates Ca²⁺ homeostasis and contraction in early developing cardiomyocytes, we have compared the dynamics of [Ca²⁺]_i transients and its relationship to cardiomyocyte contraction in cells differentiated from wild-type (RyR2^+/+) and RyR2 deficient (RyR2^-/-) ES cells both with time of in vitro differentiation and following pharmacological intervention. With time of RyR2^+/+ ESCM differentiation, SR function develops progressively with characteristics of an enhanced frequency and amplitude and a progressive decrease in the duration of Ca²⁺ transients that could be inhibited by ryanodine and thapsigargin. RyR2^-/- ESCMs, comparatively, demonstrate a significantly prolonged time-to-peak and reduced frequency of Ca²⁺ transients and contractions. b-adrenergic stimulation of RyR2^+/+ ESCMs increases the frequency and amplitude of Ca²⁺ transients in a differentiation-dependent manner, but b-adrenergic responses are much weaker in RyR2^-/- ESCMs. We also demonstrate that SR load and RyR2 are essential for upstroke and frequency of Ca²⁺ transients, and consequently contraction and beating rate even at an early developmental stage and that RyR2 and SR are important for b-AR stimulation in these cells^[13,15]. These results firmly establish that SR and RyR2 are not only essential for determining the beating rate and the rate of diastolic depolarization^[13], but also for regulating the contraction through more rapid and robust Ca²⁺ transients in early cardiac development^[15]. Moreover, the SR function in regulation of Ca²⁺ homeostasis increases with cardiomyocyte developme-nt^[13,15]. With the SR structure and function maturation, the [Ca²⁺]_i fluctuations in cardiac precursor cells at very early developmental stage develops gradually into the Ca²⁺ sparks and the global [Ca²⁺]_i transients with a marked increase in the frequency and the amplitude in late maturation cardiomyocytes^[16].

In adult cardiomyocytes, Ca²⁺ influx through L-type Ca²⁺ channels activates RyR2 and triggers SR Ca²⁺ release at T- tubule-SR junctions. This CICR process is the predominant mechanism of E-C coupling and it is structurally required that the sarcolemmal Ca²⁺ channels and SR Ca²⁺ release channels are located in close proximity. The T-tubule network is absent in the early fetal myocytes^[14], which is one of the reasons that have led to the assumption that CICR is not critical to E-C coupling in embryonic cardiomyocytes. However, Lohn et al.^[61] reported a distance of 20～50 nm between the abundant caveolae and SR, and this close proximity could be responsible for the localized Ca²⁺ sparks observed in neonatal myocytes. Two types of junctional membrane complexes between the cell-surface membrane and the SR with gap size of ～12 and ～30 nm in cardiomyocytes were observed from E9.5 mouse embryos^[58]. Because of the ～12 nm junctional gap in mature mouse myocytes, fetal cardiomyocytes might also possess functional peripheral couplings as do in adult cells. This is supported by that JP-2 is expressed at time coincident with the expression pattern of RyR2 in RyR2^+/+ ESCMs but decreased in RyR2^-/- cells^[15], and that JP-2 null E9.5 cardiomyocytes have slow, weak, and irregular Ca²⁺ transients^[58]. In addition, the recent study in cat atrial myocytes also demonstrate that subsarcolemmal junctional SR sites are activated in a stochastic fashion by the opening of voltage-dependent sarcolemmal Ca²⁺ channels, and subsequently, central nonjunctional SR sites are activated by Ca²⁺-induced Ca²⁺ release propagating from the periphery^[c]. During the development of cardiac myocytes, the gradual development of the T-tubule network, the maturation of the SR system, and the spatial colocalization of L-type Ca²⁺ channel and RyR2 lead to more and more Ca²⁺ sparks triggered simultaneously. Therefore, the SR is becoming more important in the regulation of [Ca²⁺]_i homeostasis during the cardiomyocyte development^[14].

Decay of [Ca²⁺]_i during the relaxation

The amount of Ca²⁺ which enters the cytosol of myocytes at each steady state twitch must also be extruded from the cytosol; otherwise the [Ca²⁺]_i homeostasis will alter. The decay of the Ca²⁺ transients occurs in adult cardiomyocytes because of the reuptake of Ca²⁺ into the SR through the Ca²⁺-pump ATPase and the extrusion of Ca²⁺ from the myocyte by the NCX^[28], as discussed above.

The cardiac SERCA2, a 100～115 kDa protein, is concentrated in the longitudinal component of the SR^[63]. The SERCA transports two Ca²⁺ ions for each ATP molecule consumed in cardiac SR. Phospholamban (PLB) is an endogenous inhibitor of SERCA in cardiac myocytes. While the level of SERCA2a mRNA remains unchanged, its protein level increases with human heart development^[38]. It has also been proven that the abundance of SERCA2 gene products is regulated primarily though post-transcriptional mechanisms during the rat perinatal period^[64].

A Ca²⁺ extrusion from the cells via the NCX1 also occurs during the decline phase of the Ca²⁺ transients. The two fundamental principles to maintain steady-state contraction of cardiac physiological function are^[2], (i) a balance exists between the amount of Ca²⁺ entering the cell via the Ca²⁺ currents and the amount of Ca²⁺ extruded by the NCX; and (ii) the amount of Ca²⁺ released from the SR equals that sequestered by the SR Ca²⁺-pump ATPase. If an abrupt change in this balance of fluxes takes place, the Ca²⁺ content of the SR will be affected. There is a dynamic competition between NCX1 and SERCA2 during relaxation, and both of them contribute to a variable amount toward [Ca²⁺]_i decline depending on species, developmental stages and physiologic conditions. The expression of SERCA2 and PLB increase gradually during the cardiac development, and the NCX expression level in early developed cells is twice as in adult myocytes^[4,522,43]. These complementary temporal gradients in SERCA and NCX expression suggest a role for NCX activity to compensate for less SERCA expression in the embryonic/early postnatal state of development. Pharmacological researches also demonstrated that in rat cardiomyocytes the contributions of the SERCA2 uptake to the twitch relaxation associated with [Ca²⁺]_i decline increase from ～75% to 92%, and the contributions of NCX decrease from ～24% to 5% from birth to adulthood^[65].

The role of inositol-1,4,5-trisphosphate receptor (IP₃R) in Ca²⁺ homeostasis

Except RyRs, there is an inositol-1,4,5-trisphosphate (IP₃) sensitive Ca²⁺ release channel on the SR membrane. Three IP₃R subtypes are expressed in a wide variety of tissues, and only the type-2 IP₃R is expressed in isolated ventricular myocytes^[66]. The IP₃R released Ca²⁺ plays an important regulatory role in the cellular proliferation and apoptosis, whereas RyR-released Ca²⁺ is required for muscle contraction^[66]. The IP₃R mRNA expression and the IP₃-induced intracellular Ca²⁺ release are detected as early as 5.5-day postcoitum in the mouse embryo, which is earlier than the expression of RyR2 mRNA^67]. In adult guinea pig ventricular myocytes, the low concentrations of IP₃ (1～10 µmol/L) transiently increase isotonic contractions which accord with the receptor-initiated SR Ca²⁺ release^[68]. The complementary temporal changes in RyR2 and IP₃R function in the regulation of Ca²⁺ transients indicate that IP₃R has a critical role in the regulation of Ca²⁺ homeostasis in early developed cardiomyocytes. A recent study showed that IP₃-dependent shuttle of free Ca²⁺ in and out of the SR is essential for a proper generation of pacemaker activity during early cardiomyogenesis and fetal life^[69]. Therefore, the possible contribution of IP₃R to Ca²⁺ homeostasis in early developed cardiomyocytes should be considered and explored further.

Summary

Dynamic changes in the regulation of Ca²⁺ homeostasis occur during the development of the heart, and the source of Ca²⁺ for producing contraction is especially altered during development (Fig.1).

In embryonic developing cardiac cells, most Ca²⁺ required for contraction is derived from the Ca²⁺ influx through the voltage-dependent sarcolemmal Ca²⁺ channels (L-type and T-type), and RyR2-mediated SR Ca²⁺ release is critical and irreplaceable in Ca²⁺ homeostasis. Ca²⁺ entering through the sarcolemmal Ca²⁺ channels and then being released from Ca²⁺-release channels in peripheral SR may diffuse into the intracellular space, which in turn stimulates Ca²⁺-release channels in the deep space. However, as the absence of the T-tubule and the immature SR Ca²⁺ store, the whole linkage of CICR produces a slow kinetics of Ca²⁺ transients in the fetal myocytes.

In neonatal developing cardiac cells, the relative mature SR plays a role as the main source of Ca²⁺ required for contraction compared to that in the fetus. Especially, the

SR has been well organized and the T-tubules start to develop in the neonatal period. These morphological changes produce a short distance between the sarcolemmal Ca²⁺ channels and SR Ca²⁺ release channel, and Ca²⁺ influx through the sarcolemmal can activate Ca²⁺ release from the SR by CICR. Thus the kinetics of Ca²⁺ transient becomes faster.

During the process of the cardiac maturation, the SR develops gradually into a major mechanism to regulate cardiac Ca²⁺ homeostasis, which becomes the physiologic basis for the cardiac E-C coupling, such as, the proteins expression of RyR2 and SERCA2 and their functions increase, the T-tubule network develops, the junctophilin-2 links the SR membrane with the T-tubule wall membrane, and the Ca²⁺-binding protein calsequestrin expresses in the SR lumen.

As discussed above, the source of Ca²⁺ for producing contraction is altered during cardiac development, and the function of SR is critical for cardiac Ca²⁺ homeostasis. Hence, functional abnormalities or gene mutation of RyR2^[57] and SERCA2^[70] during development may be involved in the genesis of cardiac arrhythmias and sudden deaths. Furthermore, the failing heart has an altered program of gene expression that with embryonic characteristics^71,72], and the expression of RyRs and SR Ca²⁺-ATPase are decreased^[73,74] with defective RyR2 channel function^[72]. Moreover, transplantation of exogenous cells into injured myocardium, such as fetal cardiomyocytes^[75], bone marrow cells^[76] and ESCMs^[77], has emerged for the regeneration of damaged myocardium and for the improvement of cardiac function in post infarcted hearts in recent years. Therefore, it is significant to further investigate the detailed knowledge about the establishment of the E-C coupling and the altered regulation of Ca²⁺ homeostasis during development, which is also important for a better understanding of the normal development aspects and the abnormalities in cardiac diseases.

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