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L‐type voltage‐gated calcium channels in stem cells and tissue engineering

Abstract L‐type voltage‐gated calcium ion channels (L‐VGCCs) have been demonstrated to be the mediator of several significant intracellular activities in excitable cells, such as neurons, chromaffin cells and myocytes. Recently, an increasing number of studies have investigated the function of L‐VGCCs in non‐excitable cells, particularly stem cells. However, there appear to be no systematic reviews of the relationship between L‐VGCCs and stem cells, and filling this gap is prescient considering the contribution of L‐VGCCs to the proliferation and differentiation of several types of stem cells. This review will discuss the possible involvement of L‐VGCCs in stem cells, mainly focusing on osteogenesis mediated by mesenchymal stem cells (MSCs) from different tissues and neurogenesis mediated by neural stem/progenitor cells (NSCs). Additionally, advanced applications that use these channels as the target for tissue engineering, which may offer the hope of tissue regeneration in the future, will also be explored.

of L-VGCCs and the transduction of L-VGCCs-mediated signals into the nucleus. 4,5 Structurally, L-VGCCs are composed of several different subunits, encompassing the main pore-forming α1 subunit, auxiliary subunits α2/δ, β and γ. 6 The α1 subunit, which contains four homologous repeats, determines the main biophysical and pharmacological properties of the channels including voltage sensing, ion permeability and drug binding. Each of the repeats is composed of six membrane-spanning helices. 7 Specifically, The S4 helix in each repeat can serve as voltage sensor and the S5-S6 helices in Repeat III are binding sites for L-VGCCs blockers, especially for dihydropyridines (DHPs). The α2δ subunit protrudes far into the extracellular space and influences the voltage-dependent and kinetic properties of the calcium currents. 8 It also takes part in cell attachment of skeletal myocytes and synaptogenesis in neurons. 5 The β subunit is a cytoplasmic protein containing a conserved Src homology 3 (SH3) domain and a guanylate kinase-like domain. It binds to the cytoplasmic linker between domains I and II of the α1 subunit via the guanylate kinase domain and facilitates trafficking of calcium channel complexes by preventing E3 ubiquitin ligase-induced proteasomal degradation of the α1 subunit. 6,9 The γ subunit was purified from skeletal muscle, but it can be absent, especially in heart tissue. Compared with other auxiliary subunits, its role is limited and defined as a transmembrane AMPA-glutamate receptor modifying protein. 6 All these subunits can undergo alternative splicing, a process that enables a single gene to code for multiple proteins by rearranging the pattern of introns and exons. This process is a key mechanism for the regulation of pharmacological properties of L-VGCCs, generating a variety of unique splice isoforms with distinct cell and tissue distribution, with significance for specific physiological and pathological processes. 10,11 According to differences of the α1 subunits, L-VGCCs have four subtypes, ranging from Ca v 1.1 to Ca v 1.4, which are distributed widely among different tissues and cells, with distinct pharmacological and biophysical properties to support specific cellular tasks. 6,12 Calcium influx through these channels can serve as a second messenger of electrical signalling, initiating intracellular events such as membrane depolarization, secretion, synaptic transmission and gene expression. 6,13 Concretely, Ca v 1.1, which is mainly present in skeletal muscle and expressed at low levels in other tissues, can serve as the voltage sensor mediating the process of excitation-contraction coupling. 14 Ca v 1.2 is widely expressed in various tissues including smooth muscle, 15,16 bone, 17 brain 18,19 and heart. 20 In cardiac tissues, Ca v 1.2 is indispensable for calcium ions to enter the cardiac cells and initiate cardiac excitation-contraction coupling during the plateau phase of the action potential. Additionally, Ca v 1.2 is also expressed in most types of neurons, where it activates calcium-dependent enzymes and initiates calcium-dependent gene transcription. 7 The distribution pattern of Ca v 1.3 is similar to that of Ca v 1.2. 7 Recently, Ca v 1.3 has been shown to initiate pace-making in dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) 21 and the sinoatrial node (SAN). 22 The calcium currents associated with Ca v 1.3 regulate the secretion of catecholamine from chromaffin cells 23 and are also essential for the development of normal immature inner hair cells (IHC). 24  While there is ample evidence supporting the vital function of L-VGCCs in excitable cells including neurons, 29 chromaffin cells 30 and myocytes, 31,32 this mediation in non-excitable cells such as stem cells has not been described firmly. However, recent studies have found that L-VGCCs also contribute to the proliferation F I G U R E 1 Topology of a voltage-gated calcium channel subunit. L-VGCCs are formed of subunits encompassing the pore-forming α1 subunit, auxiliary subunits α2/δ, β subunit and γ subunit (not shown). The α1 subunit consists of four homologous repeats, each with six membrane-spanning helices. The S4 helix in each repeat can serve as voltage sensor and the S5-S6 helices in Repeat III are binding sites for L-VGCCs blockers, especially for dihydropyridines (DHPs). The α2/δ subunit comprises an α2 subunit with an extracellular protrusion and a δ subunit buried within the cell membrane. The β subunit is the cytoplasmic protein containing an SH3 domain and a guanylate kinase-like domain. The γ subunit can be absent, especially in heart tissue, which is not shown in Figure 1. L-VGCCs, L-type voltage-gated calcium ion channels and differentiation of several kinds of stem cells. This review will discuss the possible involvement of L-VGCCs in the function of stem cells isolated from different accessible sources (Figure 2), especially as it pertains to the osteogenic differentiation of MSCs and neurogenic differentiation of NSCs. Moreover, considering that the effects of L-VGCCs on stem cells offer the hope of tissue regeneration in the future, we will also discuss the potential applications of L-VGCCs in tissue engineering.
F I G U R E 2 Functional regulation of various stem cell types by L-VGCCs. L-VGCCs are involved in the functional regulation of various stem cell types, including MSCs from bone marrow, oral cavity, adipose and skin tissues, as well as other stem cells, such as ESCs, NSCs, CPCs and HFSCs. The corresponding regulatory mechanisms influence cell proliferation and multipotent differentiation, such as osteogenic, neurogenic and myogenic differentiation. The γ subunit can be absent in cardiac and neuronal calcium channels. AMSCs, adipose-derived mesenchymal stem cells; CPCs, cardiac progenitor cells; ESCs, embryonic stem cells; HFSCs, hair follicle stem cells; L-VGCCs, L-type voltage-gated calcium ion channels; NSCs, neural stem/ progenitor cells TA B L E 1 Functional regulation of MSCs from different sources by L-type voltage-gated calcium ion channels (L-VGCCs)   Table 1.

| L-VGCCs and dental MSCs
Dental tissues are accessible resources that provide an abundant reservoir of MSCs, such as dental pulp stem cells (DPSCs), stem cells from exfoliated deciduous teeth (SHED), stem cells from the apical papilla (SCAPs) and periodontal ligament stem cells

| L-VGCCs and MSCs from other tissues
In addition to the bone marrow, MSCs also exist in many other tissues, including adipose and skin tissues. The patch-clamp analysis demonstrated that human adipose-derived mesenchymal stem cells (AMSCs) possess functional VGCCs, although they account for <1%. 48 However, analysis of cytoplasmic Ca 2+ concentration evoked by ATP, high K + solution, GABA and caffeine demonstrated that functional VGCCs are absent in AMSCs isolated from rats. 49 The previous work on AMSCs from chronic kidney disease pa- Ca v 1.3 knock-down rats produced using adeno-associated virus (AAV) or retrovirus-based technologies showed impaired dendritic or axonal formation in vivo. 63 To further study the mechanism of the regulation of neurogenic activities by L-VGCCs, researchers investigated the involvement of Ca 2+ -dependent regulatory genes in differentiation, such as BETA2/NeuroD1, 64 which are essential to neuronal survival and/or maturation in many neuronal tissues, including the dentate hippocampus, the cerebellum and the olfactory bulb, 67,68 and the research showed that BETA2/NeuroD1 were involved in L-VGCCs-mediated regulation. Moreover, it has been reported that activation of the GABA B receptor can increase Ca v 1.3 expression in NSCs, 69 suggesting that GABA receptors may be a part of a signalling pathway for stem cell maintenance. The depolarization mediated by glutamate and GABA A receptors would facilitate the Ca 2+ influx through L-type Ca 2+ channels. 64 However, the role of GABA receptors in the regulation of L-VGCCs is controversial, since the evidence indicating that GABA receptors may be involved in the maintenance and activation of hippocampal NSCs is inconsistent 70 .

| L-VG CC S AND OTHER S TEM CELL S
Cardiac progenitor cells (CPCs), which are mainly located in the myocardium, express the cardiogenic genes. 71

| P OTENTIAL APPLI C ATI ON S OF L-VG CC S IN TISSUE ENG INEERING
Tissue engineering is an emerging interdisciplinary field involving the use of an interactive triad of scaffolds, signalling molecules, engineering and cells, focusing on producing functional replacement tissues and creating favourable conditions for tissue regeneration. 75 The latest developments in tissue engineering have provided new perspectives for the regeneration and replacement of defective tissues, such as bone and heart tissue. 76 Signalling molecules, which are vital for all tissue engineering strategies, can promote spatiotemporal signalling cascades to maximize the functionality of engineered tissues. 77 Given that L-VGCCs are of significance for the osteogenic, myogenic and neural differentiation of several stem cell types, we will focus on their potential applications in the tissue engineering of bone, heart and neurons for organ repair and regeneration ( Figure 3). that L-VGCCs may be a potential target for periodontal tissue engineering. 82 Other researchers also elucidated that L-VGCCs are involved in in vitro osteogenic differentiation induced by calcium phosphate (CaP)-bearing biomineralized scaffolds, as well as in in vivo bone tissue regeneration of human ESCs and MSCs. 83 Thus, the application of L-VGCCs agonists is a promising approach for bone tissue engineering.

| L-VGCCs and cardiomyogenesis
Heart failure, especially myocardial infarction, is one of the leading global health challenges that threaten the well-being of more than thirty million people annually. 84 Resident cardiomyocytes of patients with heart disease have a compromised proliferation and differentiation potential. Current strategies for treating heart dysfunction, such as organ transplantation, are limited due to immunological rejection and insufficient availability of donor organs. 85 Tissue engineering has emerged as a promising therapeutic strategy for cardiac regeneration. To date, diverse cell sources have been adopted for repair and regeneration of the impaired myocardium in animal models, with a subset of them undergoing clinical assessment. 86 Stem cell-based therapy has been considered as an attractive option for treating defective myocardium. 87,88 Several stem cell types have been used in clinical trials for treating functional deficiency of the heart, such as CPCs. A modest improvement of cardiac performance was observed in several clinical trials, but the methodology is still inadequate, partly due to functional immaturity and electrical insufficiency. 83 The addition of small-molecule compounds in tissue engineering is favourable to structural and functional recovery of the treated hearts. 89 However, this approach is complicated by the fact that heart regeneration is a process involving cell proliferation, differentiation and maturation. 90 Adequate knowledge about the regulatory processes that are active during cardiomyogenesis is indispensable for using biomolecules with unique activity to target stem cell function.
Researchers have confirmed that calcium influx is an integral second messenger for the early steps of cardiomyocyte specification and commitment, leading to the differentiation of stem cells by modulating cardiac gene expression. 71 Recent data also showed that Ca 2+ regulates excitation-contraction coupling, as well as the localization of cardiogenic transcription factors in CPCs. 72

| L-VGCCs and neurogenesis
Since adult NSCs are limited in differentiation and distribution, neurons are refractory to replication. The reprogramming or regeneration of human neural tissue 96 is considered to be difficult to achieve.
According to several studies, the predominant isoforms of L-VGCCs

| CON CLUS IONS
In this review, we discussed not only the important roles of L-VGCCs in the functional regulation of several stem cell types, but also the potential application of drugs targeting L-VGCCs in tissue engineering. As discussed above, L-VGCCs partake in the regulation of osteogenic, myogenic and neural differentiation, which makes their agonists or antagonists highly promising candidates for the repair and regeneration of bone, heart and neural tissue.
Notwithstanding, there are still considerable challenges within this field. First, functional L-VGCCs are the precondition for the efficacy of the drugs targeting them, but they are present only in a portion of stem cells. Secondly, the role of L-VGCCs in osteogenic differentiation still remains controversial, partly due to the varying characteristics of different stem cell types. Thus, more basic research is needed before attempting the therapy targeting L-VGCCs for bone regeneration. Thirdly, it has been reported that L-VGCCs can be detrimental to possibly cause severe neurodegenerative diseases. For example, researchers found that increasing Ca 2+ influx by the overexpression of L-VGCCs modulated the processing of the beta amyloid (Aβ) precursor protein, leading to a positive feedback loop and ultimately the development of Alzheimer's disease. 102,103 Despite this, it is clear that selective application of L-VGCCs activators or inhibitors based on their effect to distinct stem cell types provides a new avenue for tissue regeneration.

CO N FLI C T O F I NTE R E S T
The authors have declared that no competing interest exists.

AUTH O R CO NTR I B UTI O N S
Yi-zhou Tan