Role of sonic hedgehog signaling pathway in the regulation of ion channels: focus on its association with cardio-cerebrovascular diseases

Sonic hedgehog (SHH) signaling is vital for cell differentiation and proliferation during embryonic development, yet its role in cardiac, cerebral, and vascular pathophysiology is under debate. Recent studies have demonstrated that several compounds of SHH signaling regulate ion channels, which in turn affect the behavior of target cells. Some of these ion channels are involved in the cardio-cerebrovascular system. Here, we first reviewed the SHH signaling cascades, then its interaction with ion channels, and their impact on cardio-cerebrovascular diseases. Considering the complex cross talk of SHH signaling with other pathways that also affect ion channels and their potential impact on the cardio-cerebrovascular system, we highlight the necessity of thoroughly studying the effect of SHH signaling on ion homeostasis, which could serve as a novel mechanism for cardio-cerebrovascular diseases. Activation of SHH signaling influence ion channels activity, which in turn influence ion homeostasis, membrane potential, and electrophysiology, could serve as a novel strategy for cardio-cerebrovascular diseases


Introduction
Sonic hedgehog (SHH) protein, one of the three hedgehog ligands, encoded by the shh gene, belongs to the hedgehog family.It functions as an endogenous ligand of SHH signaling and plays a crucial role in cell differentiation and proliferation during embryonic development [41].Most injuries, including ischemia and trauma, stimulate SHH signaling activation, which is vital for tissue repair, angiogenesis, and neurogenesis [8,81].SHH signaling can be divided into canonical and non-canonical pathways.Canonical SHH signaling activates several anti-inflammatory and anti-apoptosis gene expression that promote tissue repair [12,82].From this perspective, most studies announced that SHH signaling activation by recombinant SHH protein or SHH signaling agonist, smoothened agonist (SAG), protect against cardio-cerebrovascular disease (CCVDs) during the pathological stimulus.In contrast, noncanonical SHH signaling that does not participate in the downstream target gene expression is activated under pathological conditions [67].Recent studies indicate that the non-canonical SHH signaling participates in regulating ion channels, which impact ion homeostasis and the target cells.
The human genome encodes more than 400 ion channels [5].Disorders of ion channels affect multiple aspects of cytobiological functions, including bioelectrical conduction, proliferation and differentiation, and metabolism [10].Most ion channels are sensitive to guanine-nucleotide-binding regulatory protein (G protein) coupled receptors (GPCRs) and their associated kinases.Smoothened (SMO) is a well-studied coreceptor of the SHH signaling pathway.SMO is coupled with several GPCR-sensitive protein kinases (PKs) like PKA and PKC that regulate ion channels [37].
Evidence of SHH signaling with CCVDs is insufficient and full of controversy.Considering that the ion channels heavily affect the cardio-cerebrovascular system, the relationship between SHH signaling and ion channels could provide novel mechanisms for CCVDs.In this review, we summarized the current research on SHH signaling with ion channels that relate to CCVDs.We hope to shed light on the interactions between SHH signaling and ion channels, which would be helpful for further understanding the pathogenesis of CCVDs.

Sonic hedgehog signaling
The essential hedgehog axis contained signaling molecules (ligands), receptors and co-receptors, and effectors (functional proteins and transcriptional factors) (Fig. 1).In mammals, three ligands have been identified, including SHH, Indian hedgehog (IHH), and Desert hedgehog (DHH).SHH is the most well-studied and most relevant to cardiovascular system [9].The SHH precursor (molecular weight ~ 45 kDa) is autocleaved to produce the SHH N-terminus (SHH-N, ~ 19 kDa) that mediated by SHH C-terminus (SHH-C, ~ 25 kDa), and the C-terminus of SHH-N is modified with cholesterol.Then, the N-terminus of SHH-N is modified with palmitic acid by skinny acyltransferase (SKI).The matured SHH-N is carried outside through extracellular vesicular particles (termed exovesicles) and released from the cell surface mediated by Dispatched (Disp) and transferred across cells with the help of Scube2 (signal peptide, CUB and EGF-like domaincontaining protein 2), then the Scube2-SHH complex binds with Cdon (cell adhesion molecule downregulated by oncogenes) and Boc (brother of Cdon) of the target cell surface, and growth arrest-specific-1 (Gas1) mediates SHH release from the tetramer (Scube2-SHH-Cdon-Boc) and mediates the combination of SHH to the patched receptors [37].
Two receptors of SHH pathways have been identified: patched 1 (PTCH1) and patched 2 (PTCH2).PTCH1 mainly locates in mesenchymal cells, and PTCH2 is primarily expressed in testicular and skin epithelial cells.Several coreceptors involve in SHH signaling, and SMO is the most well-studied.At the inactivated status of SHH signaling, PTCHs combine and inhibit SMO.When SHH signaling is activated, the matured SHH protein combines and leading to the degradation of PTCHs, followed with the release of SMO and subsequent activation of the downstream pathway [35].The other co-receptors, including Boc and Cdon, mediate negative feedback in the SHH signaling pathway [70].
The SHH pathway is divided into canonical and noncanonical pathways.The canonical pathway mediates the gene transcription via transcriptional factors, zinc finger protein GLIs, while non-canonical pathway does not.In the canonical pathway, when SMO is activated, GLIs translocate into the nucleus, triggering the transcription of SHH target genes [67].GLI1 contains one, and GLI2/3 contains two activation domains in the C-terminus; the GLI2/3 contains a repression domain in N-terminus, but GLI1 does not.GLI2 often exists in a full-length form or completely degraded, while GLI3 often appears as a cleaved form of GLI3 N-terminus, which functions as a transcriptional repressor.Hence, GLI1/2 is often seen as the transcriptional activators, and GLI3 as a transcriptional repressor in the SHH pathway [67,58].PKA, casein kinase 1 (CK1), and glycogen synthase kinase 3β (GSK3β) can phosphorylate GLI2/3 and promote their full or partial degradation, while the degradation of GLI1 is facilitated after phosphorylation by PKA and GSK3β [45,53,56,79].Intriguingly, activation of SMO relies on the phosphorylation by PKA, while SMO activation lowers the cAMP level and inhibits PKA activation, exerting negative feedback of the SHH signaling pathway.This negative feedback loop could help to fine tune the signaling response and potentially prevent excessive activation because the collapse negative feedback of SHH signaling is carcinogenic [4,46].Missing in metastasis (MIM) and mammalian target of rapamycin (mTOR) are two positive regulators that interact and release GLIs from the SUFU-GLI complex.Atypical PKCs (aPKCs) can bind with the GLI-MIM complex and further phosphorylate GLI1, which magnifies the affinity of GLI1 to DNA [23].The canonical SHH signaling pathway's targeting genes mainly regulate cell development, differentiation, and death [67].Several other regulators also participate in the regulation of GLIs [37].
The non-canonical pathways do not involve gene transcription and classify as two types.For type I, or SMO-independent mechanism, SHH binds and inhibits PTCH1, which inhibits the recruitment of caspases and suppresses apoptosis.For type II, or SMO-dependent mechanism, SHH binds with PTCH1, and SMO is released without the subsequent activation of GLI1-3 [3].The type I non-canonical pathway, or the SMO-independent pathway, mainly involves tumorigenesis, and the type II non-canonical pathway, or the SMO-dependent pathway, could contribute to cardio-cerebrovascular diseases.SMO possesses the characteristics of GPCR such as the sevenpass transmembrane structure, the extracellular N-terminal tail and intracellular C-terminal tail, and it can form heterotrimeric with other proteins [4].SMO interacts with G i proteins following with recruitment of several GPCR-sensitive protein kinases.Up to now, SMO is found to activate G αi subunit and inhibit adenyl cyclase, which then inhibit cAMP production and downregulate PKA activity; yet G βγi subunits activate PKC and PI3K [4].The inhibition of PKA, or the activation of PKC and PI3K, is related to the changes in the physiological and pathological state of the body.For example, most ion channels are sensitive to PKA and PKC [16], which establish the link between SHH signaling and ion channels.

Sonic hedgehog signaling and ion channels
Na + and K + are crucial for almost all the physiological processes that mediate the switch of action potential and resting potential.Besides an essential role in action and resting potential, Ca 2+ also plays a key role in multiple physiological functions such as muscle contraction, neurotransmitter release, signaling transduction, and cell proliferation.However, in the central nervous system (CNS), Na + , K + , and Ca 2+ induce an excitatory effect leading to an action potential, whereas chloride ions act to inhibit this effect.Dysregulation of ion channels contributes to multiple cardiocerebrovascular diseases (CCVDs), especially arrhythmia and epilepsy.Since SMO possesses GPCR characteristics and recruits several kinases that regulate ion channels, including PKA and PKC [44], during the activation of the non-canonical SHH pathway, it is likely that SHH signaling participates in the regulation of ion channels.Here, we summarized the direct and indirect interactions of SHH signaling with ion channels (Table 1).

SHH signaling and voltage-gated ion channels
The voltage-gated ion channels (VGICs) are activated by the changes in membrane potentials and form selective pores that mediate the rapid transmembrane flux to switch the membrane depolarization and repolarization.VGICs are the key channels that regulate electrophysiological functions, including cardiac pacemaking, autonomy, systolic and diastolic functions, and neuron electrical conduction.They are also coupled with calcium release channels on the sarcoplasmic reticulum (SR) to regulate the intracellular calcium concentration.Dysregulation of these channels causes cellular abnormalities, including electrophysiological disturbance, ion overload, or insufficient intracellular ion concentration, eventually leading to severe incidences, including arrhythmia and epilepsy.Evidence has shown that SHH signaling regulates several VGICs.
There is direct evidence that SHH signaling impacts the heart's voltage-gated potassium channel 4.3 (K v 4.3).Before the SHH combines with PTCHs, the latter combine and inhibit smoothened (SMO), pro-tein kinase A (PKA), casein kinase 1 (CK1), and glycogen synthase kinase 3β (GSK3β) can phosphorylate GLI2/3 and promote their full or partial degradation, following with the generation of GLI3R, the transcriptional repression form of GLI3, and GLI1 degradation is facilitated after phosphorylation by PKA and GSK3β, then the SHH targeting gene transcription is inhibited.When SHH combine with PTCHs, the PTCH-SHH complex transfers into the cell and degraded.Then, the SMO is released and activates the downstream canonical pathways that mediated by transcriptional factor GLIs. (Right panel) Noncanonical SHH signaling pathway.The SMO independent noncanonical pathway (or type I noncanonical pathway) is activated after the combination of SHH and PTCH and broke the PTCH-caspase-3 axis [67], and apoptosis is inhibited; the SMO dependent noncanonical pathway (or type II noncanonical pathway) mainly relied on the GPCR characteristics of SMO, which will recruit G i and regulate the downstream protein kinases purmorphamine inhibited K v 4.3 in cardiomyocytes and ex vivo mice hearts in an inhibitory G protein (G i )-dependent manner, while SMO antagonist KAAD-cyclopamine reversed this effect.Besides, cardiomyocytes or hearts isolated from G i CT/TTA mice (which disrupt the interactions of SMO with G i ) have no such effect [13].As we mentioned above, G i regulates several protein kinases including PKA and PKC; however, Cheng's study did not mention the mediator between G i and K v 4.3 [13].

Cheng et al. reported that SMO was activated by SHH or
SHH signaling triggers Ca 2+ oscillation patterns during chicken feather development and elongation that are mediated by T-type Ca 2+ currents, and cyclopamine inhibits feather bud polarization and elongation.This effect relies on the effect of SHH signaling on the expression of connexin-43 and STIM1 (sensor of Ca 2+ release-activated Ca 2+ channels), but not directly on Ca 2+ channels [51].Nevertheless, the L-type Ca 2+ currents could not exclude the impact of SHH signaling on the model above.The role of T-type Ca 2+ currents have not been well elucidated, and the T-type Ca v knockout or overexpress did not show abnormal phenotypes in adult mice [40].Specific to the heart, T-type Ca 2+ currents were decreased during development but awake under pathological conditions, especially in ventricular remodeling of hypertrophic or infarcted hearts [75].Although the T-type Ca 2+ current played in cardiomyopathy is not clear, it has not attracted enough interest in the cardiac system in recent years.However, the T-type Ca 2+ channels could be targets for multiple CNS diseases.Ca v 3.2 mediates the neuropathic pain [52], an adverse effect of vincristine.Ca v 3.1 knockout in mice inhibits trigeminal neuropathic pain [15].T-type Ca 2+ channel antagonists also inhibit the absence epilepsy [73].Interestingly, Ca 2+ channel antagonist nifedipine and diltiazem alleviated abdominal aortic aneurysms related to their anti-inflammatory effect rather than their effect on blood pressure [61,62], which showed the diverse role of Ca 2+ .Given by the information above, illuminating the relationship between SHH signaling and T-type Ca 2+ channels in adult individuals could identify the key signaling pathway in the pathogenesis of CNS diseases.However, it is difficult to distinguish the T-type from L-type Ca 2+ channels.Whether the SHH signaling affects L-type Ca 2+ channels, it also needs to be clarified.
For voltage-gated sodium channels (Na v s), we have not found any direct evidence that can unveil their relationship with SHH signaling.Indirect evidence from Iqbal SM et al.'s study [39] shows that the PKA plays a key role in regulating Na v s activity.Both Na v 1.2 and Na v 1.5 can be phosphorylated by PKA, while Na v 1.2 is inhibited, and Na v 1.5 is activated under phosphorylation modification [39] (Fig. 2).Although PKA is regulated by multiple GPCRs, it is still unknown whether the effect of SHH signaling on PKA is sufficient enough to regulate Na v 1.2 and Na v 1.5.

SHH signaling and ligand-gated ion channels
Ligand-gated ion channels (LGICs, also named ionotropic receptors) participate in multiple physiological processes, such as nerve conduction, muscle contraction, and hormone secretion [36].They are activated by neurotransmitters or ligands like ATP and glutamate and allow ion transport across the membrane.In the central nervous system (CNS), LGICs mainly mediate excitotoxicity, a kind of specific neurotoxicity that is caused by high extracellular glutamate concentration and result in neuron death [49].Elevated extracellular glutamate levels have long been considered as a predisposing factor for multiple neuron disorders, including epilepsy and stroke [59].Extracellular glutamate activates multiple glutamate-gated ion channels (also termed ionotropic glutamate receptors, iGluRs), including AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors (AMPARs) and NMDA (N-methyl-D-aspartate) receptors (NMDARs), and, in turn, increases intracellular Ca 2+ concentration [20].In addition to acute injury, anti-NMDAR therapeutics have gained promising results in the clinical trial of treating Alzheimer's disease [66].
Glutamate transporters could be a key component of SHH-LGIC signaling [28,78].In a mouse model of epilepsy, SHH signaling was activated, and then the glutamate transporter EAAC1 (excitatory amino acid transporter proteins, also named EAAT3, SLC1A1) in hippocampal neurons was inhibited, leading to the increase of extracellular glutamate levels through the interaction with SMO-G αi/o proteins [28].The inhibition of SHH signaling by cyclopamine or SHH neutralizing antibody 5E1 suppressed the epilepsy progress.It should be noted that SHH signaling did not affect NMDA-or AMPA-induced currents in hippocampal neurons.Wang et al. found that the SMO-dependent noncanonical SHH signaling pathway was activated in a mouse model of ischemic stroke, then GLT-1 was phosphorylated by PKCα, leading to the decrease in the site of membrane, and glutamate was detained in extracellular space [78].
There were reports that SHH-SMO signaling promoted the membrane stability of potassium-chloride co-transporter 2 (KCC2) in the postnatural neurons, which hyperpolarized GABAergic nerve and lowered the central excitability [31].Many studies have shown that KCC2 activation has a protective effect against epilepsy, and mutations that impair KCC2 activity are an underlying mechanism of congenital epilepsy [21].That revealed the multiple effect of SHH signaling on the same disease.

SHH signaling and intracellular Ca 2+ release channels
Normal physiological functions depend on a certain level of intracellular Ca 2+ in most cells.The intracellular Ca 2+ level is controlled by the endoplasmic reticulum (ER)/SR [25].RyRs are believed to mediate the transduction of SHH signaling during neural tube development [48].Knockout of each RyR family member, including RyR1a, RyR1b, RyR2a, and RyR3, have all diminished the SHH signaling in corresponding responder cells in zebrafish.The RyR inhibitor azumolene inhibited the SHH signaling in a dose-dependent manner.Azumolene could also affect the functions of SHHdependent cells and had little effect on SHH-independent cells.RyRs are dispensable for SHH secretion but indispensable for SHH signaling transduction.Besides, azumolene failed to inhibit the overexpression of the dominant-negative form of PKA (dnPKA)-induced SHH signaling activation, while the overexpression of constitutively active form of SMO-induced SHH signaling activation was inhibited by azumolene, which showed that the RyRs involving in SHH signaling resided the downstream of SMO and upstream of PKA.
IP3Rs are another group of Ca 2+ release channels on SR and also related with SHH signaling.Chihiro et al. identified a direct interaction between SHH signaling and IP3Rs in astrocytes because they found that SHH and SAG mediated Ca 2+ oscillation in the cultured and brain slice astrocytes [1].In the cultured astrocytes, SAG mediated Ca 2+ oscillation even in Ca 2+ free medium.During this process, the extracellular ATP was needed, because carbenoxolone (CBX) and 1-octanol (inhibitors of connexin hemichannels) and Gd 3+ (inhibitor of maxianion) all could inhibit the SAG-induced the enhancement of Ca 2+ oscillations (both connexin hemichannels and maxianion are ATP-permeable).In contrast, the P2X7 inhibitor brilliant blue G (BBG) showed little effect on Ca 2+ oscillations, consistent with the SAG-induced Ca 2+ oscillation in a Ca 2+ medium, where Ca 2+ oscillation is independent of Ca 2+ influx.Further results showed that the Ca 2+ oscillation was mediated by the release of Ca 2+ from SR/ER through IP3R channels, because the IP3R inhibitor 2-APB and ER Ca-ATPase inhibitor thapsigargin (Tg) inhibited the Ca 2+ oscillations, while RyR inhibitor dantrolene does not.The Ca 2+ oscillation in astrocytes relies on the SMO-dependent non-canonical SHH pathways, because partial activation of SMO, which did not initiate SHH target genes transcription, also induce Ca 2+ oscillation [71], and the couple disruption of SMO-G i by pertussis toxin (PTX) partially suppressed Ca 2+ oscillation.Because of the wide distribution of RyRs and IP3Rs, the effect of the SHH pathway on them should be considered during the interference of SHH signaling as a therapeutic goal.

SHH signaling and mechanically activated ion channels
Cardiac pumping functions to ensure blood supply is circulated to the whole body.During this process, mechanical stress constantly exists along with myocardial and vessel contraction and dilatation.Pathological conditions, particularly in the cardiovascular system, like hypertension, arteriosclerosis, and cardiac hypertrophy, change the mechanical stress and aggravate the pathophysiological process [38].Mechanically gated ion channels that are sensitized by the changes of mechanical stress termed mechanically activated ion channels, convert mechanical stimuli into electrical signals [47].Several transient receptor potential (TRP) channels (including TRPC1, 5, 6; TRPV1, 2, 4; TRPM3, 7; TRPA1; TRPP2) and Piezo channels are sensed by mechanical changes [38].Most of the TRP channels are coupled with the GPCRs [76]; PKA and PKC serve as the second messengers to reduce the activation thresholds of TRP channels via phosphorylating them.Dysregulation of TRP channels is related to several diseases, including arrhythmia, neuropathic and inflammatory pain, hypertension, atherosclerosis, and aneurysm [38,76].
TRPV1 is a cation channel that is related to multiple pathophysiological responses, including chronic pain, cardiac remodeling, and epilepsy [2,85].It has been shown Fig. 2 Smoothened-dependent noncanonical sonic hedgehog signaling pathway regulating ion channels.The smoothened (SMO) recruits G i proteins without initializing gene transcription.The α-subunit of G i (G αi ) activation by SMO inhibits PKA activation, while the βγ-subunit of G i (G βγi ) activation activates PKC and PI3K.Na v 1.2 phosphorylation on N1466 residue by PKA and PKC inhibits its slow inactivation in rat [11]; Na v 1.5 phosphorylation on S525 and S528 (or S526 and S529 in rat) by PKA increases I Na , while S1503 (or S1505 in rat) phosphorylation by PKC inhibits I Na in human [39].TRPP2 phosphorylation on S829 by PKA upregulates its activation, and S807 phosphorylation by PKC is essential for its normal function in human [19].Blue fonts represent activation, and orange fonts represent inhibition that the SHH signaling is involved in the TRPV1 regulation in pancreatic cancer pain [32].The high level of SHH elevated the TRPV1 expression in pancreatic stellate cells and TRPV1 current, and inhibition of SHH signaling with SHH siRNA or cyclopamine decreased the TRPV1 level along with the downregulated levels of pain-related peptides, substance P (SP), and calcitonin gene-related peptide (CGRP).
Mechanically activated TRP channels are not only regulated by SHH signaling, their activity also affects the SHH ligand expression during the primary cilia development.For examples, TRPM7 can regulate Indian hedgehog (IHH) expression in rat chondrocyte [55], and TRPP family members always formed transmembrane (TM) proteins, including 11-TM proteins consist of polycystin (PKD) 1, PKD1-L1, and PKD1-L2 (PKD1 also named TRPP1) and 6-TM proteins consist of PKD2, PKD2-L1, PKD2-L2 (also named TRPP2, TRPP3, TRPP5, respectively) take part in the regulation of SHH signaling during primary cilia development [18].Furthermore, TRPP2 and TRPP3 activity are heavily relied on PKA phosphorylation [19, 64] (Fig. 2).Although no direct evidence supports the regulation of SHH signaling by the mechanically activated TRP channels, the GPCRs have been reported to serve as the mechanical sensors [76], and the resident organelle of SMO, primary cilia, is also sensitive to mechanical stimuli [42].Whether SMO affects TRP channels, it needs further study.

SHH signaling in cardio-cerebrovascular disease
Many studies have demonstrated an essential role of SHH signaling in the angiogenesis and development of the heart and brain.In contrast, its role in cardio-cerebrovascular disease (CCVDs) is limited.Most studies focused on the protective role of exogenous SHH protein in chronic CCVDs, particularly in the ischemic models.In acute CCVD models, the role of SHH signaling is controversial.Since many of the pathological changes in CCVDs are related to the dysregulation of ion channels, here we focus on the role of SHH signaling in CCVDs via regulation of ion channels (Table 2).

Cardiac disease
The myocardium is not only sensitive to hypoxia but also to ion homeostasis.As we mentioned in the section "SHH signaling and voltage-gated ion channels," activation of SHH signaling coupled the SMO with inhibitory G proteins (G i ) and inhibited voltage-gated potassium channel 4.3 (K v 4.3), which prolonged the ventricular repolarization time (QT interval) and induced ventricular arrhythmias [13].In addition, K v 4.3 also showed a protective effect on cardiac remodeling and hypertrophy [69,77].These results indicated that the inhibition of SHH signaling relevant to K v 4.3 negatively affects the heart.Interestingly, Ghaleh et al. reported that the injection of recombinant SHH protein protects the pig heart from arrhythmia in a myocardial ischemia/reperfusion model [29].However, no specific ion channel had been identified in this study.These two studies suggest the difference in the effect on arrhythmia and ion homeostasis between exogenous and endogenous SHH protein.
As we mentioned in the section "SHH signaling and intracellular Ca2 + 241 release channels," SHH signaling positively regulated RyR2 activity.Under normal condition, the opening of RyR2 is mainly regulated by I Ca,L , which is mainly mediated by Ca v 1.2.Under multiple pathological conditions, RyR2 opened uncoupling of I Ca,L and caused Ca 2+ efflux out of SR in resting state, termed "Ca 2+ leakage," leading to the decrease of the Ca 2+ storage in SR [6].Besides, the continuous Ca 2+ leakage caused cytoplasm Ca 2+ overload, a critical toxic effector in cardiomyocytes, to cause cell death [72,74].In the ischemic heart, posttranslational modifications of RyR2 usually made it open during the phase that caused Ca 2+ leakage from SR, which led to cytosolic Ca 2+ overload.In the early stage of reperfusion, RyR2 open-induced transit Ca 2+ oscillations cause reperfusion arrhythmias [27].
To date, direct or indirect evidence about the relationship between SHH signaling and heart failure is not available.The relationship between SHH signaling and ion channels could give some clues.Na v 1.5 is essential for the zero phase of cardiac action potential, and it is downregulated in heart failure [74,34].Inhibition of Na v 1.5 reduced cardiac contractility and caused slow conduction and ventricular arrhythmias [26,57,83].It should be noted that Na v 1.5 overactivation enhanced the I NaL and impacted calcium handling, prolonging the QT interval and leading to lethal arrhythmia [84].As we mentioned above, Na v 1.5 is activated by PKA phosphorylation, and considering the effect of SHH signaling on K v 4.3 through G i , it is reasonable to speculate that the effect of SHH signaling on PKA and PKC is sufficient enough to drive the downstream ion channels, whether the activation of SHH signaling inhibits Na v 1.5 via its inhibition of PKA may be worth studying.

Cerebral disease
SHH signaling is involved in the pathophysiological process of multiple cerebral diseases, particularly in cerebral ischemic diseases.Although its role in the acute ischemic brain injury is still under debate, the ion channel-mediated SHH signaling is believed to play deleterious role in acute cerebral ischemic disease.
In an acute ischemic stroke mice model, Wang et al. revealed that SHH signaling was activated, accompanied by the glutamate transporter GLT1 inhibition, leading to extracellular glutamate accumulation and neuronal excitotoxicity.In contrast, suppression of SHH signaling by SMO inhibitor (cyclopamine) or SMO knockout reduced the cerebral ischemic injury [78].Since extracellular glutamate disorder is related to multiple cerebral diseases and ion channel disturbance [17], it is likely that the ionotropic glutamate receptors (iGluRs) play a bridge role between SHH signaling and cerebral diseases.
IP3R2 contributes to the detrimental effect of SHH signaling on acute ischemic brain injury, too.As we mentioned earlier, SHH signaling can mediate Ca 2+ oscillation via activating IP3Rs in hippocampal astrocytes [1], and disruption of Ca 2+ signaling in astrocytes of IP3R2 knockout mice inhibits ischemia-induced cerebral injury [50].Moreover, global knockout of IP3R2 did not significantly change brain cytoarchitecture compared with wild-type mice, indicating that astrocyte IP3R2 could be a promising target of ischemic brain disease.
Different from its detrimental effect on acute ischemic brain injury, SHH signaling may benefit ischemic brain recovery in a long run due to its function in stimulating angiogenesis and neurogenesis [43].Wang J et al. reported that stimulation of SHH signaling post-stroke leads to increased neurogenesis and improved behavioral functions after stroke [80].Using conditional knockout of SHH signaling receptor Smo gene in neural stem cells, they have further demonstrated that SHH signaling abolished stroke-induced neurogenesis in the knockout mice.Compared to control mice, Smo knockout mice also showed delayed motor function recovery and increased anxiety level after stroke.In Zhou et al.' s study, Dl-3-N-butylphthalide (NBP) has been reported to attenuate the brain infarct volume and promote angiogenesis via upregulation of SHH in 14 days after ischemic stroke [87], while in Sims et al.'s work, inhibition of SHH signaling with cyclopamine has been shown to reduce neuron progenitor proliferation in 10 days after cerebral ischemia in mice [68].For more information regarding the beneficial effects of SHH signaling on cerebral ischemia and other neurological disorders, please refer to an excellent review contributed by Patel et al. [65].
Besides neurons, SHH signaling also possesses a key function in astrocytes.Hill et al. revealed that SHH signaling in mature astrocytes is required for establishing structural organization and remodeling of cortical synapses in a cell type-specific manner.Selective disruption of SHH signaling in astrocytes produced a dramatic increase in synapse number specifically on layer V apical dendrites that emerged during adolescence and persists into adulthood.In conditional knockout mice of Smo gene, SHH signaling in astrocytes was impaired, concomitant with the reduced glial-specific K ir 4.1 and enhanced neuronal excitability, while impairment of SHH signaling in neurons did not show any differences in spine density [33], supporting a specific role of SHH signaling in astrocyte-mediated modulation of neuronal activity.
Furthermore, SHH signaling-ion channels also have a role in psychiatric disorders, including epilepsy.As previously mentioned, SHH signaling activation in cerebral ischemia led to extracellular glutamate accumulation and aggravated ischemic injury [78], which is one of the mechanisms for SHH signaling in promoting epilepsy [28].Furthermore, Na v 1.2, which is inhibited by PKA but activated by PKC, exerts a proepilepsy effect because Na v 1.2 antagonists have showed an anti-epilepsy effect [60].Clarifying whether SHH signaling regulates Na v 1.2 via PKA or PKC could be helpful for the study regarding epilepsy.

Vascular disease
Certain vascular diseases including atherosclerosis and aortic aneurysms are closely related to the disorder of differentiation, proliferation, and secretion of vascular resident cells, such as endothelial cells, smooth muscle cells, and macrophages, which contribute to vascular dysfunction.Atherosclerosis is a chronic inflammatory disease involved in multiple pathological processes, and the underlying mechanisms are not fully elucidated.It is well known that the switch of vascular smooth muscle cell (VSMC) to macrophage-like cells and foam cell in plaques is the leading factors for atherosclerosis [7,22,63].A variety of ion channels have been reported to be involved in atherosclerosis.As we mentioned before, T-type Ca 2+ current was affected by SHH signaling, and the cross talk of the T-type Ca 2+ channel (mainly inferred to Ca v 3.2)-RyR axis with BKCa could be related to the reactions of endothelium in atherosclerosis [14].Both the Ca v 3.2 and RyRs have been shown to be regulated by SHH signaling, which might be novel pathophysiological mechanism of atherosclerosis.The TRP channels that are sensitive to PKA have shown contradictory roles in atherosclerosis.The TRPV4 aggravated atherosclerosis by mediating foam cell formation [24,30], and TRPA1 showed a protective role in atherosclerosis [86].TRPV1 could be directly regulated by SHH signaling, and it also showed a protective effect on atherosclerosis [32,54].The relationship between ion channels that are regulated by SHH signaling and atherosclerosis is much more complex and even the pathological mechanism of atherosclerosis has not been well elucidated.Thus, the regulatory role of SHH signaling in ion channels provides us with a novel approach to unravel atherosclerosis pathogenesis.Aortic aneurysms (AA) are a vascular disease that appears in the abnormal dilation of the artery with damage to vascular structures, resulting in vascular rupture with high mortality.Growing evidence has identified a causal link between SHH signaling and aneurysms, with most studies demonstrating the effective effects of SHH in AA.However, the effect of SHH signaling on ion channels also needs to be considered.AA is also an ion-sensitive disease; deficiency of TRPP2 has been reported to be related to aneurysms [38].Effect of SHH signaling on ion channels, particularly the inhibition of TRPP2 along with PKA inhibition by SHH signaling should be noticed.

Summary
Ion homeostasis has been reported to affect almost all organs and is the pathogenesis of multiple diseases.Ion channels provide a bridge that links the SHH signaling under multiple conditions, not limited to cardio-cerebrovascular diseases (CCVDs).However, based on the existing data, we cannot tell whether the interaction between SHH signaling and ion channels is occasional or wellregulated.According to the available evidence, we propose that the impact of SHH signaling on ion channels, at least those involved in CCVDs, should be considered in SHH-targeted therapeutics.Unfortunately, there is a paucity of research regarding the ion channel drug targets and how they implicate SHH signaling to date.Apart from the given ion channels affected by SHH signaling, more studies are needed to be done to unravel the exact mechanism between SHH signaling and ion channels and broaden to the newly discovered channels.With the development of novel molecular biology techniques, like the applications of cryo-electron microscopy and single-cell sequencing, biomolecular interactions and locations will be clarified.Finally, we agreed with Cheng that the agonist of SHH signaling for the therapeutic aim should be carefully reconsidered [13].

Fig. 1
Fig. 1 Sonic hedgehog signaling.(Left panel) Matured sonic hedgehog (SHH) ligand release from SHH donor cells.The SHH precursor auto-cleaved mediate by SHH-C terminal and SHH-N undergoes cholesterol modification, then the N-terminal of SHH-N undergoes palmitoylation by skinny acyltransferase (SKI).The modified SHH-N is transferred to the cell membrane and then released from the surface of SHH donor cells that mediated by Dispatched (Disp) and transferred across cells with the help of Scube2 (signal peptide, CUB and EGF-like domain-containing protein 2).(Middle panel) SHH combine with membrane receptors on SHH receiving cells and canonical SHH signaling pathways.Scube2-SHH complex transfers to the SHH receiving cells and binds with Cdon (cell adhesion molecule downregulated by oncogenes) and Boc (Brother of Cdon) on the target cell surface, then the growth arrest-specific-1 (Gas1) mediates SHH release from the tetramer (Scube2-SHH-Cdon-Boc) and combines with the patched receptors (PTCHs).Before the SHH combines with PTCHs, the latter combine and inhibit smoothened (SMO), pro-

Table 1
Sonic hedgehog signaling-related ion channels