Nicotinic Acid Adenine Dinucleotide Phosphate Induces Intracellular Ca2+ Signalling and Stimulates Proliferation in Human Cardiac Mesenchymal Stromal Cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a newly discovered second messenger that gates two pore channels 1 (TPC1) and 2 (TPC2) to elicit endo-lysosomal (EL) Ca²⁺ release. NAADP-induced lysosomal Ca²⁺ release may be amplified by the endoplasmic reticulum (ER) through the Ca²⁺-induced Ca²⁺ release (CICR) mechanism. NAADP-induced intracellular Ca²⁺ signals were shown to modulate a growing number of functions in the cardiovascular system, but their occurrence and role in cardiac mesenchymal stromal cells (C-MSCs) is still unknown. Herein, we found that exogenous delivery of NAADP-AM induced a robust Ca²⁺ signal that was abolished by disrupting the lysosomal Ca²⁺ store with Gly-Phe β-naphthylamide, nigericin, and bafilomycin A1, and blocking TPC1 and TPC2, that are both expressed at protein level in C-MSCs. Furthermore, NAADP-induced EL Ca²⁺ release resulted in the Ca²⁺-dependent recruitment of ER-embedded InsP₃Rs and SOCE activation. Transmission electron microscopy revealed clearly visible membrane contact sites between lysosome and ER membranes, which are predicted to provide the sub-cellular framework for lysosomal Ca²⁺ to recruit ER-embedded InsP₃Rs through CICR. NAADP-induced EL Ca²⁺ mobilization via EL TPC was found to trigger the intracellular Ca²⁺ signals whereby Fetal Bovine Serum (FBS) induces C-MSC proliferation. Furthermore, NAADP-evoked Ca²⁺ release was required to mediate FBS-induced extracellular signal-regulated kinase (ERK), but not Akt, phosphorylation in C-MSCs. These finding support the notion that NAADP-induced TPC activation could be targeted to boost proliferation in C-MSCs and pave the way for future studies assessing whether aberrant NAADP signaling in C-MSCs could be involved in cardiac disorders.

1 Introduction

Nicotinic acid adenine dinucleotide phosphate (NAADP) has emerged as a the most powerful (already in the nanomolar concentration range) Ca²⁺-releasing second messenger in mammalian cells (Galione, 2015; Patel, 2015). NAADP elicits an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) by gating a novel family of intracellular Ca²⁺-releasing channels, known as two-pore channels (TPCs), which present two isoforms in mammals (i.e., TPC1 and TPC2) and mobilize endo-lysosomal (EL) Ca²⁺ into the cytosol (Patel, 2015; Galione, 2019; Jin et al., 2020). Jupiter microtubule-associated homolog 2 (JPT2) (Gunaratne et al., 2021) and the RNA-binding protein, Lsm2 (Zhang et al., 2021), serve as auxiliary protein to bind NAADP and thereby contribute to mediate TPC-mediated EL Ca²⁺ release. The Ca²⁺ response to NAADP may remain spatially confined in proximity of EL vesicles (Ruas et al., 2010; Vassileva et al., 2020) or it can be amplified into a regenerative Ca²⁺ wave through the Ca²⁺-dependent recruitment of juxtaposed ryanodine and inositol-1,4,5-trisphosphate (InsP₃) receptors at membrane contact sites (MCSs) between lysosomes and endoplasmic reticulum (ER) (Kinnear et al., 2004; Davis et al., 2012; Kilpatrick et al., 2013; Penny et al., 2014). Lysosomal Ca²⁺ refilling is impaired by alkalinization of the EL lumen (Ronco et al., 2015), although the mechanisms whereby intraluminal pH recharges EL vesicles with Ca²⁺ remains a controversial issue (Morgan et al., 2011; Garrity et al., 2016; Faris et al., 2018).

NAADP has been recognized as the trigger of the cellular Ca²⁺ response to extracellular stimuli in multiple tissues (Galione, 2015; Patel, 2015), including the cardiovascular system (Fameli et al., 2017; Moccia et al., 2021a; Negri et al., 2021b). NAADP-induced Ca²⁺ release through TPC2 increases the Ca²⁺ content within the sarcoendoplasmic reticulum in ventricular (Macgregor et al., 2007) and atrial myocytes (Collins et al., 2011), both at rest (Macgregor et al., 2007; Collins et al., 2011) and during β-adrenergic receptor stimulation (Macgregor et al., 2007; Collins et al., 2011; Lewis et al., 2012; Capel et al., 2015). Likewise, a flurry of reports showed that NAADP-induced intracellular Ca²⁺ signals elicit contraction in multiple types of vascular smooth muscle cells (VSMCs) (Kinnear et al., 2004; Jiang et al., 2013; Fameli et al., 2014; Trufanov et al., 2019). For instance, NAADP gates TPC2 to promote the Ca²⁺-dependent recruitment of RyR3 and global cytosolic Ca²⁺ waves in pulmonary artery VSMCs stimulated with either endothelin-1 (Kinnear et al., 2004; Jiang et al., 2013) or angiotensin II (Lee et al., 2015). Finally, NAADP may serve as a trigger of the Ca²⁺ response to extracellular stimuli also in vascular endothelial cells (Favia et al., 2014; Zuccolo et al., 2019; Negri et al., 2021a) and circulating endothelial colony forming cells (ECFCs) (Balducci et al., 2021; Moccia et al., 2021b). Aberrant NAADP signalling in cardiac myocytes may result in arrhythmia (Nebel et al., 2013) and ischemia-reperfusion injury (Davidson et al., 2015), whereas it could lead to pulmonary artery hypertension in VSMCs (Jiang et al., 2018; Hu et al., 2021).

Once regarded as mere bystanders of the contractile function effected by neighbouring cardiac myocytes, cardiac mesenchymal stromal cells (C-MSCs) are required to maintain myocardial structure and function and, therefore, to ensure effective cardiac contraction (Brown et al., 2005; Camelliti et al., 2005). C-MSCs contribute to wound healing and fibrotic remodelling after ischemic injury (Jugdutt, 2003; Camelliti et al., 2005) and they have been put forward as a promising cellular substrate to induce cardiac repair (Bagno et al., 2018; Braunwald, 2018). Furthermore, C-MSCs could stimulate cardiac myocytes to undergo proliferation or hypertrophy depending on whether this interaction takes place during embryonic development or in the adult heart (Kakkar and Lee, 2010). Finally, C-MSCs exhibit significant immunomodulatory potential by attenuating the inflammatory response in the infarcted myocardium (Czapla et al., 2016; Diedrichs et al., 2019). In agreement with their contribution to the structural, biochemical and electro-chemical features of the myocardium, C-MSCs are involved in the pathogenic mechanisms of multiple cardiac diseases (Brown et al., 2005; Camelliti et al., 2005). For instance, C-MSCs provide a source of adipocytes (Sommariva et al., 2016; Stadiotti et al., 2017) and support fibrotic remodelling (Maione et al., 2021) in arrhythmogenic cardiomyopathy (ACM), a rare genetic disorder that is featured by fibro-fatty myocardium substitution, malignant arrhythmias, and heart failure and that can lead to sudden death in young individuals (Moccia et al., 2019). It has long been known that an increase in [Ca²⁺]_i regulates multiple functions in human MSCs (Moccia et al., 2015; Forostyak et al., 2016; Jiang et al., 2017), including proliferation (Foreman et al., 2006), migration (Peng et al., 2016), gene expression (Kawano et al., 2006), and differentiation (Kawano et al., 2006; Tao et al., 2011). However, it is still unclear whether and how NAADP evokes intracellular Ca²⁺ signals and whether lysosomal-ER MCSs do exist in C-MSCs. This information could be extremely helpful to boost the design of alternative strategies to effectively target C-MSCs in a variety of life-threatening cardiac disorders. In the present investigation, we first provided the evidence that NAADP evokes robust lysosomal Ca²⁺ mobilization, which is amplified into a global increase in [Ca²⁺]_i by InsP₃ receptors (InsP₃Rs). Transmitted electron microscopy (TEM) then revealed clearly discernible MCSs between lysosomes and ER membrane in C-MSCs. Finally, we found that NAADP-induced Ca²⁺-dependent crosstalk between lysosomes and ER triggers the intracellular Ca²⁺ signals whereby Fetal Bovine Serum (FBS) induces cell proliferation. The role of Ca²⁺ signalling in regulating proliferation and differentiation in MSCs confer these findings the potential to provide the molecular framework for further studies aiming at manipulating C-MSCs for therapeutic purposes.

2 Materials and Methods

2.1 Ethical Statement

This study complies with the WMA Declaration of Helsinki. The use of human cells from biopsy samples of healthy subjects (cardiomyopathies ruled out) was approved by IEO-CCM IRCCS Ethic Committee (project CCM1072). Written informed consent was obtained from all participants.

2.2 C-MSC Isolation and Culture

Cells were obtained from endomyocardial specimens and characterized as previously described (Pilato et al., 2018) and cultured with Iscove’s Modified Dulbecco’s Medium (Thermo Fisher Scientific, MA, United States) supplemented with 20% Fetal Bovine Serum (FBS), 10 ng/ml basic fibroblast growth factor, 10,000 U/ml Penicillin, 10,000 μg/ml Streptomycin, and 0.02 M L-Glutamine.

2.3 Solutions

Physiological salt solution (PSS) had the following composition (in mM): 150 NaCl, 6 KCl, 1.5 CaCl₂, 1 MgCl₂, 10 Glucose, 10 Hepes. In Ca²⁺-free solution (0Ca²⁺), Ca²⁺ was substituted with 2 mM NaCl, and 0.5 mM EGTA was added. Solutions were titrated to pH 7.4 with NaOH. The osmolality of the extracellular solution, as measured with an osmometer (Wescor 5500, Logan, UT, United States), was 300–310 mmol/kg.

2.4 [Ca²⁺]_i Measurements and Statistics of Ca²⁺ Signals

C-MSCs were loaded with 2 µM fura-2 acetoxymethyl ester (fura-2/AM; 1 mM stock in dimethyl sulfoxide) in PSS for 30 min at room temperature (RT). The details of the Ca²⁺ recording set-up have been described in Moccia et al. (2021b) and are reported in the Supplementary Material. All the experiments were performed at RT. The amplitude of intracellular Ca²⁺ release in response to each agonist (NAADP or FBS) or drug [Gly-Phe β-naphthylamide (GPN), nigericin, bafilomycin A1, and cyclopiazonic acid (CPA)] was measured as the difference between the ratio at the peak of intracellular Ca²⁺ mobilization and the mean ratio of 1 min baseline before the peak. Pooled data are given as mean ± SE and statistical significance (p < 0.05) was evaluated by the Student’s t-test for unpaired observations or one-way Anova analysis followed by the post-hoc Dunnett’s test as appropriate (Negri et al., 2021a; Remigante et al., 2021). Data relative to Ca²⁺ signals are presented as mean ± SE, while the number of cells analysed is indicated in the corresponding bar histograms.

2.5 mRNA Extraction and qRT-PCR Assay

Cell cultures were lysed in RL lysis buffer (Norgen Biotek Corp., Thorold, ON, Canada). RNA was isolated from cells by using a Total RNA Purification kit (Norgen Biotek Corp., Thorold, ON, Canada). The quantification of the isolated RNA was determined by NanoDrop spectrophotometer (ND-1000, EuroClone, Milan, Italy). Reverse transcription was conducted with SuperScript III (Invitrogen, Carlsbad, CA, United States) following the manufacturer’s instructions. qRT-PCR was performed with the use of the iQTM SYBR Green Super Mix (Bio-Rad Laboratories, Hercules, CA, United States) and specific primers (reported in Table 1). All reactions were performed in a 96-well format with the 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific, MA, United States). The relative quantities of specific mRNA were obtained with the use of the comparative Ct method and were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Maione et al., 2021; Zuccolini et al., 2022). The expression of each target gene was assessed in triplicate (Ferrera et al., 2021; Maione et al., 2021).

TABLE 1

TABLE 1. Primer sequences 5′-3′.

2.6 Protein Extraction and Western Blot Analysis

C-MSCs were lysed in cell lysis buffer (Cell Signalling Technology, Danvers, MA, United States) supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich, Saint Louis, MO, United States). Total protein extracts were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad, CA, United States). The membranes were blocked for 1 h at room temperature in 5% non-fat dry milk in Wash Buffer (Tris Buffer Sulfate, 0.1% Tween-20) and then incubated O/N at 4°C with the appropriate primary antibodies (reported in Table 2). The membranes were incubated with peroxidase-conjugated secondary antibodies (GE Healthcare, Chicago, IL, United States) for 1 h. Signals were visualized using the LiteUP Western Blot Chemiluminescent Substrate (EuroClone, Milan, Italy). Images were acquired with the ChemiDocTM MP Imaging System (Bio-Rad, CA, United States), and densitometric analysis of membranes was performed using the ImageJ software (National Institutes of Health, Bethesda, MD, United States). C-MSC proteins were normalized according to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) signal.

TABLE 2

TABLE 2. Primary antibodies.

2.7 Transmission Electron Microscopy

For transmission electron microscopy (TEM) analysis, following trypsinization cells were centrifuged at 800 rpm for 5 min and then fixed with 2.5% glutaraldehyde in culture medium, for 2 h at RT (Carriero et al., 2021). The cell pellet was then rinsed in PBS overnight, post-fixed in 1% aqueous OsO4 for 3 h at room temperature and rinsed in H2O. Cells were pre-embedded in 2% agarose in water, dehydrated in acetone and then embedded in epoxy resin (Electron Microscopy Sciences, EM-bed812). Ultrathin sections (60–80 nm) were cut on a Reichert OM-U3 ultramicrotome, collected on nickel grids and then stained with uranyl acetate and lead citrate. The specimens were observed with a JEM 1200 EX II (JEOL, Peabody, MA, United States) electron microscope operating at 100 kV and equipped with a MegaView G2 CCD camera (Olympus OSIS, Tokyo, Japan).

2.8 Cell Proliferation

C-MSCs were plated in 6-well plates (100,000 cells/well) and serum starved for 4 h. Cells were then stimulated with 20% FBS in the absence (Ctrl) or presence of 100 µM of NED-19, a selective TPC blocker (Galione, 2015; Jin et al., 2020). 24 and 48 h after stimulation with FBS, the medium was removed, cells detached from the plates, and counted.

2.9 Flow Cytometry

To evaluate whether blocking TPCs with NED-19 was able to induce apoptosis in C-MSCs, Annexin V Alexa Fluor™ 488 Dye (Thermo Fisher Scientific, MA, United States) has been used, according to the manufacturer’s instructions. Briefly, cells were detached using TrypLE™ Select Enzyme (Thermo Fisher Scientific, MA, United States) and incubated with Annexin V Alexa Fluor™ 488 Dye for 15 min at RT. The fluorescence emission at 530 nm corresponding to apoptotic cells has been measured using flow cytometry (Gallios, Beckman Coulter, Brea, CA, United States).

3 Results

3.1 Nicotinic Acid Adenine Dinucleotide Phosphate Induces Intracellular Ca²⁺ Signals by Mobilizing Lysosomal Ca²⁺ in Cardiac Mesenchymal Stromal Cells

In order to assess whether they are endowed with a NAADP-sensitive Ca²⁺ store, C-MSCs were loaded with Fura-2/AM (2 µM), a Ca²⁺ sensitive fluorophore, as shown elsewhere (Maione et al., 2020a). Human MSCs may exhibit spontaneous oscillations in [Ca²⁺]_i (Kawano et al., 2002; Kawano et al., 2003; Kawano et al., 2006). Consistently, a fraction of C-MSCs (≈56.4%) exhibited a few (1-4) Ca²⁺ spikes in the absence of extracellular stimulation (Supplementary Figure S1). These cells were, therefore, discarded from subsequent analysis as shown elsewhere (Zuccolo et al., 2017; Zuccolo et al., 2019), since the spontaneous, unpredictable Ca²⁺ activity could mask or even prevent (in case of transient depletion of endogenous target organelle) the Ca²⁺ response to NAADP. We then assessed whether NAADP-AM, a membrane-permeable analogue of NAADP (Macgregor et al., 2007; Brailoiu et al., 2010), was able to increase the [Ca²⁺]_i in C-MSCs. NAADP-AM (1 µM) evoked a short train of intracellular Ca²⁺ oscillations that declined ≈25 min after their onset in the presence of extracellular Ca²⁺ in 88 out of 164 cells (53.6%) (Figure 1A). In 58 out of 164 cells (35.4%), NAADP-AM (1 µM) induced a transient increase in [Ca²⁺]_i that lasted ≈800 s returned to the baseline in the continuous presence of the agonist (Figure 1A). Eighteen cells (11%) were not responsive to NAADP-AM (1 µM). Under 0Ca²⁺ conditions, NAADP-AM (1 µM) induced only a transient increase in [Ca²⁺]_i that was not followed by additional Ca²⁺ spikes (Figure 1B). Intriguingly, the duration of the elevation in [Ca²⁺]_i was significantly shorter, i.e., ≈280 s, while the peak amplitude was higher (Figure 1C), as compared to the Ca²⁺ transient recorded in the presence of extracellular Ca²⁺. The subsequent restitution of extracellular Ca²⁺ after the full recovery of [Ca²⁺]_i to the baseline resulted in a second Ca²⁺ signal that was due to extracellular Ca²⁺ entry (Figure 1B). NAADP-AM was removed from the perfusate 100 s before re-addition of extracellular Ca²⁺ (Figure 1B), which suggests that the Ca²⁺ entry pathway recruited downstream of NAADP-AM-induced Ca²⁺ release is provided by store-operated Ca²⁺ entry (SOCE), as more widely discussed below (Yamazaki et al., 2007; Sanchez-Hernandez et al., 2010; Negri et al., 2020). The statistical analysis of the two distinct components of the Ca²⁺ response to NAADP-AM (i.e., endogenous Ca²⁺ release and SOCE) is presented in Figure 1D. NAADP is recognized as a mobilizer of the lysosomal Ca²⁺ pool (Galione, 2015; Patel, 2015). Control experiments confirmed that adding back extracellular Ca²⁺ after 700 s exposure to 0Ca²⁺ conditions did not increase the in C-MSCs (Supplementary Figure S2). In accord, NAADP-AM-evoked intracellular Ca²⁺ release was significantly (p < 0.001) reduced by discharging the lysosomal Ca²⁺ store with the lysosomotropic compound, dipeptide glycyl-l-phenylalanine 2-naphthylamide (GPN; 200 μM, 30 min) (Kilpatrick et al., 2013; Yuan et al., 2021) (Figures 1E,F). Of note, GPN has recently been reaffirmed as a reliable pharmacological tool to mobilize lysosomal Ca²⁺ (Yuan et al., 2021). Furthermore, NAADP-AM-evoked endogenous Ca²⁺ mobilization was abolished by collapsing the lysosomal H⁺ gradient that maintains lysosomal Ca²⁺ refilling with the H⁺/K⁺ ionophore, nigericin (50 μM, 30 min) (Figures 1E,F), or with the v-ATPase inhibitor, bafilomycin A1 (1 μM, 30 min) (Figures 1E,F) (Morgan et al., 2011; Ronco et al., 2015; Faris et al., 2019; Yuan et al., 2021). Supplementary Figure S3 shows that GPN (200 µM), nigericin (50 µM), and bafilomycin A1 (1 µM) induced a remarkable reduction in Lysotracker Red fluorescence, thereby confirming that all of these drugs target lysosomal Ca²⁺ (Pandey et al., 2009; Faris et al., 2019; Yuan et al., 2021). In accord with these observations, ammonium chloride (NH₄Cl), which disrupts the lysosomal Ca²⁺ pool by inducing intraluminal alkalinization (Christensen et al., 2002), also reduced Lysotracker Red Fluorescence and impaired NAADP-AM-evoked intracellular Ca²⁺ mobilization (Supplementary Figure S4).

FIGURE 1

FIGURE 1. NAADP induces intracellular Ca²⁺ signals by mobilizing lysosomal Ca²⁺ in C-MSCs. (A) Exogenous administration of NAADP-AM (1 µM) indues either intracellular Ca²⁺ oscillations or a transient increase in [Ca²⁺]_i. (B) In absence of external Ca²⁺ (0Ca²⁺), NAADP-AM (1 µM) induced only a transient increase in [Ca²⁺]_i, whereas subsequent restitution of extracellular Ca²⁺ after the full recovery of [Ca²⁺]_i to the baseline resulted in a second Ca²⁺ signal that was due to extracellular Ca²⁺ entry. (C) Mean ± SE of the amplitude of the peak Ca²⁺ response to NAADP in the presence and absence of extracellular Ca²⁺. Student’s t-test: ***p < 0.001. (D) Mean ± SE of the amplitude of NAADP-induced intracellular Ca²⁺ release and SOCE. (E) Disrupting the lysosomal Ca²⁺ store with GPN (200 μM, 30 min), nigericin (50 μM, 30 min) or bafilomycin A1 (1 μM, 30 min) severely affected the intracellular Ca²⁺ response to NAADP-AM. (F) Mean ± SE of the amplitude of the peak Ca²⁺ response to NAADP-AM in the absence and in the presence of GPN, nigericin (Nig), or bafilomycin A1 (Baf). One-Way Anova followed by the post-hoc Dunnett’s test: ***p < 0.001.

Overall, these findings provide the first evidence that NAADP may induce lysosomal Ca²⁺ release followed by extracellular Ca²⁺ entry in C-MSCs.

3.2 Nicotinic Acid Adenine Dinucleotide Phosphate-Induced Intracellular Ca²⁺ Release is Mediated by TPCs in Cardiac Mesenchymal Stromal Cells

TPCs mediate NAADP-induced intracellular Ca²⁺ release throughout the phylogenetic tree (Patel, 2015; Galione, 2019; Jin et al., 2020), including the cardiovascular system (Moccia et al., 2021a; Negri et al., 2021b). In accord, qRT-PCR analysis showed that both TPC1 and TPC2 transcripts are expressed in C-MSCs, although TPC1 mRNA is slightly more abundant (Figure 2A). Negative controls were performed by omitting reverse transcriptase from the reaction (not shown) (Faris et al., 2019). Immunoblotting confirmed that TPC1 and TPC2 are also expressed at protein level. Two single bands of, respectively, 94 and 85 kDa were found for TPC1 and TPC2 proteins (Figure 2B). C-MSCs are not amenable for lipofectamine-mediated transfection of selective small interfering RNAs (Maione, Sommariva, and Pompilio, unpublished results), which is the strategy we have recently employed to downregulate TPC1 expression in different cellular models (Faris et al., 2019; Moccia et al., 2021b). Therefore, we probed the effect of NED-19, a selective TPC inhibitor (Galione, 2015; Jin et al., 2020), which has been widely employed to inhibit NAADP-dependent TPC activation throughout the cardiovascular system (Macgregor et al., 2007; Jiang et al., 2013; Hu et al., 2021; Moccia et al., 2021a; Negri et al., 2021a). As predicted, NED-19 (100 μM, 30 min) fully suppressed NAADP-AM-evoked intracellular Ca²⁺ mobilization (Figures 2C,D). Likewise, NED-K (10 μM, 30 min), a chemically modified analogue of NED-19 that has recently been shown to selectively inhibit TPC1 (Davidson et al., 2015), and tetrandrine (10 μM, 30 min), a traditional Chinese herbal remedy that block both TPC1 and TPC2 (Sakurai et al., 2015; Moccia et al., 2021a), respectively, inhibited (p < 0.001) and abrogated NAADP-AM-evoked intracellular Ca²⁺ release (Figures 2C,D). In aggregate, these data demonstrate that NAADP stimulates TPCs to mobilize lysosomal Ca²⁺ in c-MSCs.

FIGURE 2

FIGURE 2. Two-pore channels (TPCs) mediate NAADP-induced lysosomal Ca²⁺ release in C-MSCs. (A) TPC1 and TPC2 gene expression in total RNA extracts of C-MSCs. qRT-PCR data are shown as transcript abundance (genes threshold cycles [Ct] with respect to the house-keeping gene GAPDH). n = 4/group. Student’s t-test: ***p < 0.001. (B) Western Blot analysis of TPC1 and TPC2 proteins in total cellular extracts. n = 3/group. (C) The Ca²⁺ response to NAADP-AM was suppressed by incubating the cells with the following TPC inhibitors: NED-19 (100 μM, 30 min), NED-K (10 μM, 30 min), and tetrandrine (10 μM, 30 min). (D) Mean ± SE of the amplitude of the peak Ca²⁺ response to NAADP in the absence (Ctrl) and in the presence of NED-19, NED-K and tetrandrine (Tetra). Student’s t-test: ***p < 0.001.

3.3 InsP₃Rs at MCSs are Activated Downstream of NAADP-AM-Induced Intracellular Ca²⁺ Release in Cardiac Mesenchymal Stromal Cells

The local release of lysosomal Ca²⁺ evoked by NAADP has long been known to be amplified into a global increase in [Ca²⁺]_i by the recruitment of juxtaposed InsP₃Rs on the ER membrane (Churchill and Galione, 2001; Kinnear et al., 2004; Davis et al., 2012; Faris et al., 2019; Moccia et al., 2021b). To assess whether the ER Ca²⁺ store is required to maintain lysosomal Ca²⁺ release, we first exploited cyclopiazonic acid (CPA), an established inhibitor of Sarco-Endoplasmic reticulum Ca²⁺-ATPase activity, as recently shown elsewhere (Kilpatrick et al., 2013; Faris et al., 2019; Moccia et al., 2021b). In the absence of extracellular Ca²⁺ (0Ca²⁺), CPA (30 µM) induced a transient elevation in [Ca²⁺]_i due to Ca²⁺ efflux into the cytosol through ER leakage channels followed by Ca²⁺ extrusion across the plasma membrane (Figure 3A). While NAADP-AM (1 µM) was able to induce robust Ca²⁺ release in not-treated cells (Figure 3B), it failed to evoke endogenous Ca²⁺ mobilization upon CPA-induced depletion of the ER Ca²⁺ store (Figure 3A). A preliminary characterization of the Ca²⁺ handling machinery revealed that C-MSCs express InsP₃Rs, but not RyRs, and that InsP₃-induced ER Ca²⁺ discharge activates SOCE (Maione et al., 2020a). To assess the contribution of InsP₃Rs to NAADP-induced intracellular Ca²⁺ mobilization, we adopted a similar strategy to that described in (Kilpatrick et al., 2013; Kilpatrick et al., 2016; Faris et al., 2019; Moccia et al., 2021b). The transient increase in [Ca²⁺]_i evoked by NAADP-AM (1 µM) was significantly (p < 0.001) reduced by blocking InsP₃Rs with 2-Aminoethoxydiphenyl borate (2-APB) (50 μM, 30 min) (Figure 3C) (Kilpatrick et al., 2013; Kilpatrick et al., 2016) and was suppressed by inhibiting the basal production of InsP₃ with U73122 (10 μM, 10 min) (Figure 3C), which selectively interferes with phospholipase C (PLC) activity (Moccia et al., 2006; Negri et al., 2021a). The statistical analysis of these data has been presented in Figure 3D. The lack of full inhibition of NAADP-AM-evoked intracellular Ca²⁺ mobilization could be due to the incomplete inhibition of InsP₃Rs, as also reported in ECFCs (Moccia et al., 2021b), rat gastric smooth muscle cells (Pereira et al., 2014), and MDA-MB-231 breast cancer cells (Vismara et al., 2021). Therefore, InsP₃Rs provide a robust source of Ca²⁺ during lysosomal Ca²⁺ mobilization and, based upon previous observations (Davis et al., 2012; Kilpatrick et al., 2013; Ronco et al., 2015; Kilpatrick et al., 2016; Faris et al., 2019; Moccia et al., 2021b), it can be concluded that they can be recruited by CICR upon NAADP-induced lysosomal Ca²⁺ release. TEM was then exploited to assess whether MCSs between lysosomal vesicles and ER cisternae can also be detected and thereby sustain the Ca²⁺-dependent cross-talk between the two organelles also in C-MSCs (Kilpatrick et al., 2013). For this purpose, after glutaraldehyde fixation cells have been post-fixed in aqueous OsO₄ in order to darkly stain lipids and membranes, as described in Section 2. TEM micrographs indicated extensive ER-lysosome MCSs (<20 nm, 14.3 ± 1.13, n = 27 from five cells) with ultrastructural resolution (Figure 4). As also reported in human fibroblasts, in the regions of close appositions (e.g., Figure 4A), fibres that appear to tether lysosomes and ER membranes were clearly discernible. In addition, we detected regions where the apposing membranes appeared to be physically coupled with no visible space between them (e.g., Figures 4B,C). Quantification in random sections showed that 60.5% of lysosomes established contact sites with the ER. As also discussed in Kilpatrick et al. (2013), this is likely to be an underestimate as lysosomal diameter spans between 200 and 500 nm and is, therefore, predicted to extend over several sections above and below the selected plane, where additional contact sites might have been established. Of note, lysosomes could establish extensive contact sites both with the smooth (Figure 4D) and the rough (Figure 4E) ER. We also found that ER cisternae could come in direct contacts with more than one lysosome (Figure 4E). Overall, these findings provide the ultrastructural evidence that the architecture of lysosomes and ER MCSs is fully consistent with the recruitment of ER-embedded InsP₃Rs by NAADP-induced lysosomal Ca²⁺ release through TPCs.

FIGURE 3

FIGURE 3. InsP₃Rs support the Ca²⁺ response to NAADP. (A) Administration of NAADP-AM (1 µM) after the pharmacological depletion of the ER Ca²⁺ pool with CPA (30 μm, 30 min) failed to induce intracellular Ca²⁺ release. CPA induced a transient increase in [Ca²⁺]_i that reflects passive ER Ca²⁺ leakage from the ER. (B) The intracellular Ca²⁺ transient evoked by NAADP-AM (1 µM) in the absence of extracellular Ca²⁺ (0Ca²⁺) under control conditions. (C) The intracellular Ca²⁺ release evoked by NAADP-AM (1 µM) under control (Ctrl) conditions was severely affected by blocking InsP₃Rs with 2-APB (50 μm, 30 min) or inhibiting PLC activity with U73122 (10 μm, 10 min). (D) Mean ± SE of the amplitude of the peak intracellular Ca²⁺ response to NAADP-AM under the designated treatments. Student’s t-test: ***p < 0.001.

FIGURE 4

FIGURE 4. Ultrastructural analysis of C-MSCs. Several examples of membrane contacts between lysosomes (Ly) and ER are shown. (A) Note the closeness of the lysosomes with the cell nucleus and ER. Scale bar: 1 µm. (B) The lysosome-ER contact site is indicated (arrow and inset). Scale bar: 1 μm; inset scale bar: 100 nm. (C) The arrow indicates the membrane contact site. Scale bar: 500 nm. (D) Extensive contact (arrows) between the lysosomal and the ER membranes. Scale bar: 500 nm. (E) Note the contact (arrows) between the ribosomes-rich ER and the lysosome. Scale bar: 200 nm. (F) Two close lysosomes; the one on the right is in tight contact with the ER (arrows and inset). Scale bar: 1 μm; inset scale bar: 100 nm.

3.4 SOCE Maintains NAADP-AM-Evoked Intracellular Ca²⁺ Signals in Cardiac Mesenchymal Stromal Cells

Figure 1B clearly shows that NAADP-AM-induced mobilization of intercellularly stored Ca²⁺ resulted in extracellular Ca²⁺ entry even after the agonist washout from the perfusate. This feature clearly hints at SOCE as the Ca²⁺ entry pathway sustaining the long-lasting increase in [Ca²⁺]_i evoked by NAADP in the presence of extracellular Ca²⁺. Indeed, InsP₃-dependent ER Ca²⁺ mobilization results in SOCE activation virtually in all mammalian cells (Prakriya and Lewis, 2015; Emrich et al., 2021), including C-MSCs (Maione et al., 2020a). In order to assess whether NAADP-AM-induced lysosomal Ca²⁺ release can lead to SOCE via intermediate ER Ca²⁺ depletion, we repeated the “Ca²⁺ add-back” protocol described in Figure 1 in the absence and presence of BTP-2 or Pyr6, two selective blockers of SOCE (Schleifer et al., 2012; Moccia et al., 2016). This strategy has long been exploited to selectively evaluate the blocking effect of SOCE-targeting drugs on agonist-evoked extracellular Ca²⁺ entry rather than on the previous phase of endogenous Ca²⁺ mobilization (Sanchez-Hernandez et al., 2010; Jairaman et al., 2015; Rahman and Rahman, 2017; Scarpellino et al., 2019; Negri et al., 2020; Schach et al., 2020). The influx of Ca²⁺ secondary to Ca²⁺ restitution to the perfusate after removal of NAADP-AM (Figure 5A) from the perfusate was significantly (p < 0.001) attenuated by BTP-2 (20 μM, 20 min) and abrogated by Pyr6 (10 μM, 10 min) (Figure 5A). The statistical analysis of these data has been presented in Figure 5B. These observations demonstrate that NAADP-induced lysosomal Ca²⁺ mobilization in C-MSCs is functionally coupled to SOCE via InsP₃-dependent ER Ca²⁺ release. Therefore, lysosomal Ca²⁺ release must induce depletion of ER Ca²⁺ via InsP₃Rs, thereby leading to SOCE recruitment on the plasma membrane. To further support this conclusion, Supplementary Figure S5A shows that also the pharmacological depletion of the lysosomal Ca²⁺ store with nigericin (50 µM) induced both intracellular Ca²⁺ release and extracellular Ca²⁺ entry. Furthermore, the intracellular Ca²⁺ response to nigericin (50 µM) was significantly (p < 0.001) reduced by blocking InsP₃Rs with 2-APB (50 μM, 30 min) (Supplementary Figures S5B,C) and by interfering with basal InsP₃ production with U73122 (10 μM, 10 min) (Supplementary Figures S5B,C), as recently shown in primary cultures of colorectal cancer cells (Faris et al., 2019) and in circulating ECFCs (Moccia et al., 2021b). Finally, nigericin-evoked extracellular Ca²⁺ entry was significantly (p < 0.001) attenuated by blocking SOCE with either BTP-2 (20 μM, 20 min) or Pyr6 (10 μM, 10 min) (Figures 5C,D). This result is, therefore, consistent with the evidence reported above that NAADP-induced Ca²⁺ release through TPCs is able to induce ER Ca²⁺ depletion followed by SOCE activation.

FIGURE 5

FIGURE 5. NAADP-AM-induced lysosomal Ca²⁺ mobilization is functionally coupled to SOCE in C-MSCs. (A) The influx of extracellular Ca²⁺ evoked by NAADP-AM (1 µM) upon depletion of intracellular Ca²⁺ stores (Ctrl) was severely affected by inhibiting SOCE with Pyr6 (10 μM, 10 min) or BTP-2 (20 μm, 20 min). The previous NAADP-AM-evoked endogenous Ca²⁺ release has not been shown. (B) Mean ± SE of the amplitude of NAADP-AM-evoked Ca²⁺ entry evoked by nigericin in the absence (Ctrl) and presence of Pyr-6 and BTP-2. Student’s t-test: ***p < 0.001. (C) Nigericin-evoked extracellular Ca²⁺ entry was attenuated or inhibited by, respectively, blocking SOCE with BTP-2 (20 μM, 20 min) or Pyr6 (10 μM, 10 min). (D) Mean ± SE of the amplitude of Ca²⁺ entry evoked by nigericin in the absence (Ctrl) and presence of Pyr-6 and BTP-2. Student’s t-test: ***p < 0.001.

3.5 Nicotinic Acid Adenine Dinucleotide Phosphate-Induced Lysosomal Ca²⁺ Release via TPCs Supports FBS-Induced Intracellular Ca²⁺ Oscillations in C-MSCs

FBS has been shown to induce intracellular Ca²⁺ signals to stimulate proliferation in primary MSCs harvested from rat bone marrow (Foreman et al., 2006). 20% FBS induced intracellular Ca²⁺ oscillations also in ≈26% of C-MSCs, whereas it promoted a transient increase elevation in [Ca²⁺]_i in the remaining 74% cells (Figure 6A). Intracellular Ca²⁺ oscillations lasted for at least 30 min, while the transient Ca²⁺ signal took approximately 13 min to decline to pre-stimulation levels (Figure 6A). In the absence of extracellular Ca²⁺ (0Ca²⁺), 20% FBS induced a rapid (≈3 min) increase in [Ca²⁺]_i that reflected endogenous Ca²⁺ mobilization. The subsequent re-addition of extracellular Ca²⁺, 100 s after FBS removal from the bath, resulted in a second bump in [Ca²⁺]_i, which was due to extracellular Ca²⁺ entry and was likely to be mediated by SOCE (Figure 6B). FBS-induced intracellular Ca²⁺ signals are known to be triggered by InsP₃-induced ER Ca²⁺ mobilization and maintained over time by SOCE (Foreman et al., 2006; Hu et al., 2009; Zuccolo et al., 2018b). Preliminary experiments confirmed that 20% FBS-induced intracellular Ca²⁺ release was abrogated by depleting the ER Ca²⁺ store with CPA (30 μM, 30 min) (Figures 6C,D), inhibiting InsP₃Rs with 2-APB (50 μM, 30 min) (Figures 6C,D), and blocking PLC with U73122 (10 μM, 10 min) (Figures 6C,D). Furthermore, 20% FBS-induced extracellular Ca²⁺ entry was significantly (p < 0.001) reduced by inhibiting SOCE with BTP-2 (20 μM, 20 min) or Pyr6 (10 μM, 10 min) (Figures 6E,F).

FIGURE 6

FIGURE 6. Fetal bovine serum (FBS)-induces intracellular Ca²⁺ release from endogenous stores and enhance SOCE. (A) 20% FBS induced oscillatory or transient increases in [Ca²⁺]_i. (B) Under 0Ca²⁺ conditions, 20% FBS induced a transient elevation in [Ca²⁺]_i. Subsequent re-addition of extracellular Ca²⁺, 100 s after agonist removal from the bath, resulted in a second bump in [Ca²⁺]_i that was indicative of SOCE. (C) The intracellular Ca²⁺ release evoked by 20% FBS (Ctrl) was inhibited by depleting the ER Ca²⁺ pool with CPA (30 μM, 30 min), by blocking InsP₃Rs with 2-APB (50 μM, 30 min), or inhibiting PLC with U73122 (10 μM, 10 min). (D) Mean ± SE of the amplitude of the peak intracellular Ca²⁺ response to 20% FBS under the designated treatments. (E) Subsequent to store depletion by 20% of FBS application (data are not shown her(E), FBS were washed out from bath, then extracellular Ca²⁺ added to the bath to bath in the presence and absence of SOCE inhibitors, Pyr6 (10 μm, 10 min) or BTP-2 (20 μm, 20 min). (F) Mean ± SE of the amplitude of Ca²⁺ entry evoked by 20% FBS in the absence (Ctrl) and presence of Pyr6 and BTP-2. The asterisk indicates ***p < 0.001.

The evidence reported above clearly showed that NAADP-induced lysosomal Ca²⁺ release via TPCs was able to promote InsP₃-induced Ca²⁺ release from the ER, thereby resulting in SOCE activation on the plasma membrane. Therefore, we sought to assess the role of NAADP-induced lysosomal Ca²⁺ release in the Ca²⁺ response to 20% FBS (Figure 7A). The depletion of the lysosomal Ca²⁺ store with either GPN (200 μM, 30 min) (Figures 7A,B) or nigericin (50 μM, 30 min) (Figures 7A,B) abrogated FBS-induced intracellular Ca²⁺ mobilization. The same effect was achieved upon pharmacological blockade of TPCs with NED-19 (100 μM, 30 min) (Figures 7C,D), NED-K (10 μM, 30 min) (Figures 7C,D), and tetrandrine (Figures 7C,D). Therefore, NAADP plays a crucial role in igniting the Ca²⁺ response to 20% FBS in C-MSCs.

FIGURE 7

FIGURE 7. TPCs mediate 20% FBS-induced intracellular Ca²⁺ release. (A) Intracellular Ca²⁺ release induced by 20% FBS was abolished upon depletion of the lysosomal Ca²⁺ pool with either GPN (200 μM, 30 min) or nigericin (50 μM, 30 min). (B) Mean ± SE of the amplitude of the intracellular Ca²⁺ peak evoked by 20% FBS under the designated treatments. (C) 20% FBS induced an intracellular Ca²⁺ transient that was significantly reduced or inhibited by blocking TPCs with, respectively, NED-K (10 μM, 30 min), tetrandrine (10 μM, 30 min) or NED-19 (100 μM, 30 min). (D) Mean ± SE of the amplitude of the intracellular Ca²⁺ peak evoked by 20% FBS in the absence (Ctrl) or presence of NED-19, NED-K or tetrandrine (Tetra). Student’s t-test: ***p < 0.001.

3.6 TPCs Mediate 20% FBS-Induced Proliferation and ERK Phosphorylation in Cardiac Mesenchymal Stromal Cells

In order to assess the physiological role of NAADP-induced lysosomal Ca²⁺ release through TPCs, 20% FBS-induced C-MSC proliferation was evaluated in the absence (Ctrl) and presence of NED-19 (100 μM, 30 min). Figure 8A shows that the pharmacological blockade of TPCs significantly (p < 0.05) reduced the total cell number at 24 and 48 h, thereby showing the crucial role of TPCs in supporting C-MSC proliferation. Flow cytometric analysis of Annexin V fluorescence confirmed that pre-treating C-MSCs with NED-19 did not induce apoptosis (Supplementary Figure S6). In order to determine whether TPCs recruit mitogen-associated protein kinases (MAPKs), we evaluated the phosphorylated levels of the Ca²⁺-dependent extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and of the survival kinase, Akt (Zuccolo et al., 2018a; Faris et al., 2019; Negri et al., 2021b). Figures 8B,C illustrate that 20% FBS-induced ERK1/2, but not Akt, phosphorylation was significantly (p < 0.05) inhibited by blocking TPCs with NED-19 (100 μM, 30 min). Overall, these findings demonstrate that NAADP-induced lysosomal Ca²⁺ release through TPCs stimulates C-MSC proliferation by engaging ERK1/2.

FIGURE 8

FIGURE 8. TPCs mediate 20% FBS-induced proliferation and ERK phosphorylation in C-MSCs. Following 4 h of growth without serum, cells were treated with NED-19 (100 μM, 30 min) and subsequently stimulated with 20% FBS. (A) Following 24 and 48 h of FBS stimulation, the medium was removed, cells detached from the plates, and counted by hemocytometer (n = 3/group). Student’s t-test: *p < 0.05. (B) The cells were lysate after 60 min of FBS stimulation. Total protein extract from treated cells was subjected to Western blot analysis to visualize active phosphorylated form and total of ERK and AKT using specific antibodies. Phospho-ERK1/2 and Phospho-AKT levels were corrected by total ERK1/2 and AKT densitometry respectively. (C) Western blot data are presented as the fold change of target protein expression. The results are expressed as mean ± SEM (n = 3/group). Student’s t-test: *p < 0.05.

4 Discussion

NAADP is emerging as a crucial regulator of intracellular Ca²⁺ signalling and Ca²⁺-dependent processes in the cardiovascular system (Macgregor et al., 2007; Collins et al., 2011; Fameli et al., 2017; Moccia et al., 2021a). C-MSCs represent the large majority of supportive cells in the heart, are critical to normal cardiac function and contribute to maladaptive cardiac remodelling under multiple pathological conditions. Herein, we showed for the first time that NAADP mobilizes EL Ca²⁺ via TPCs also in C-MSCs. NAADP-evoked intracellular Ca²⁺ signals are amplified by InsP₃-sensitive ER Ca²⁺ release at lysosomes-ER C-MCSs followed by SOCE activation. The functional crosstalk between NAADP-evoked lysosomal Ca²⁺ release, InsP₃-induced ER Ca²⁺ mobilization and SOCE sustains FBS-induced intracellular Ca²⁺ signals and proliferation by promoting ERK phosphorylation.

4.1 Nicotinic Acid Adenine Dinucleotide Phosphate Evokes Complex Ca²⁺ Signals in Cardiac Mesenchymal Stromal Cells

Intracellular Ca²⁺ signals tightly control a plethora of crucial functions in human MSCs from multiple sources, as reviewed in Moccia et al. (2015), Forostyak et al. (2016), and Jiang et al. (2017). According to the canonical model, the Ca²⁺ response evoked by chemical stimulation in human MSCs is triggered by InsP₃-dependent ER Ca²⁺ and may be maintained over time by SOCE (Kawano et al., 2002; Peng et al., 2016; Kotova et al., 2018). Herein, we expanded our knowledge of the molecular mechanisms shaping intracellular Ca²⁺ signalling in these cells by focusing on the role played by NAADP in C-MSCs. The intracellular delivery of NAADP mobilizes acidic Ca²⁺ stores throughout the cardiovascular system (Moccia et al., 2021a), e.g., in guinea pig ventricular (Macgregor et al., 2007) and atrial (Collins et al., 2011) cardiomyocytes, rat pulmonary artery VSMCs (Kinnear et al., 2004), human aortic endothelial cells (Brailoiu et al., 2010), mouse brain endothelial cells (Zuccolo et al., 2019), and circulating ECFCs (Di Nezza et al., 2017; Moccia et al., 2021b). Likewise, NAADP-AM, a membrane-permeable analogue of NAADP, could induce either a transient elevation in [Ca²⁺]_i or a burst of intracellular Ca²⁺ oscillations. This latter observation is in accord with the evidence that: 1) intracellular delivery of NAADP may induce oscillatory Ca²⁺ signals in human Jurkat T-lymphocytes (Berg et al., 2000), cytotoxic T lymphocytes (Davis et al., 2012), and human pancreatic β-cells (Johnson and Misler, 2002); 2) NAADP contributes to agonist-induced repetitive Ca²⁺ spikes in several types of endothelial cells (Zuccolo et al., 2019; Berra-Romani et al., 2020; Balducci et al., 2021), and that 3) NAADP induces intracellular Ca²⁺ oscillations in mouse cardiomyocytes during reperfusion injury (Davidson et al., 2015). Early work conducted on echinoderms first suggested that NAADP was able to elicit repetitive Ca²⁺ oscillations by promoting a Ca²⁺-dependent crosstalk between two different Ca²⁺ pools (Churchill and Galione, 2001), which were later shown to be located in acidic vesicles and ER (Churchill et al., 2002; Moccia et al., 2006).

4.2 Nicotinic Acid Adenine Dinucleotide Phosphate-Induced Intracellular Ca²⁺ Signals Are Triggered by Lysosomal Ca²⁺ Release via TPCs, Amplified by InsP₃-Evoked ER Ca²⁺ Release and Maintained by SOCE

The Ca²⁺ response to NAADP in C-MSCs comprised an early phase of intracellular Ca²⁺ mobilization followed by a later phase of extracellular Ca²⁺ entry, which required the previous depletion of the endogenous Ca²⁺ pool but not the NAADP-AM presence in the perfusate. First, we found that GPN, nigericin, and bafilomycin A1, which provide three established pharmacological tools to mobilize acidic Ca²⁺ stores (Kilpatrick et al., 2013; Ronco et al., 2015; Morgan and Galione, 2021; Yuan et al., 2021), prevent NAADP-induced intracellular Ca²⁺ mobilization. In agreement with the hypothesis that the lysosomal compartment represents the primary source of this increase in [Ca²⁺]_i, all of these drugs, as well as NH₄Cl, induced a rapid reduction in Lysotracker Red fluorescence. Although a recent investigation questioned the documented GPN ability to release lysosomal Ca²⁺ (Atakpa et al., 2019), Patel’s group provided the clear-cut evidence that this compound mobilizes Ca²⁺ from acidic organelles and may, therefore, be safely exploited to probe the endogenous store primarily targeted by NAADP (Morgan et al., 2020; Yuan et al., 2021). We further showed that C-MSCs express both TPC1 and TPC2 transcripts and proteins, and that the Ca²⁺ response to NAADP was inhibited by blocking TPCs with two selective antagonists, such as NED-19 (Macgregor et al., 2007; Di Nezza et al., 2017; Jin et al., 2020; Moccia et al., 2021a) and NED-K (Davidson et al., 2015), and the traditional Chinese herbal remedy, tetrandrine, which can target both TPC1 and TPC2 (Sakurai et al., 2015; Moccia et al., 2021a). As recently reviewed in Moccia et al. (2021a) and Negri et al. (2021b), TPC1 and TPC2 are both present in mouse ventricular cardiomyocytes, but this is the first time that they were reported in any other cellular component of the human heart. As reviewed in Pitt et al. (2016), TPC1 presents a limited Ca²⁺ permeability, while TPC2 is predicted to release more Ca²⁺ upon activation. Nevertheless, it has been shown that even a small Ca²⁺ flux through TPC1 can generate a global increase in [Ca²⁺]_i when lysosomal vesicles are juxtaposed to ER cisternae (Galione, 2019). For instance, TPC1 alone supports NAADP-induced intracellular Ca²⁺ oscillations in circulating ECFCs (Di Nezza et al., 2017; Moccia et al., 2021b) and in mouse cardiac myocytes undergoing the ischemia-reperfusion injury (Davidson et al., 2015). Furthermore, TPC1 was sufficient to maintain the intracellular Ca²⁺ response to nutrients or incretins in mouse pancreatic β cells deficient for TPC2 (Cane et al., 2016). Three pieces of evidence suggest that InsP₃Rs in ER cisternae contribute to amplify NAADP-induced lysosomal Ca²⁺ release. First, depletion of the ER Ca²⁺ pool with CPA suppressed or attenuated the intracellular Ca²⁺ release evoked by both NAADP and the H⁺/K⁺ antiporter, nigericin. Second, NAADP-induced endogenous Ca²⁺ mobilization was impaired by inhibiting InsP₃Rs with 2-APB and by blocking basal InsP₃ production with U73122. Conversely, functional RyRs are absent in C-MSCs (Maione et al., 2020a). The requirement for InsP₃Rs to sustain the increase in [Ca²⁺]_i resulting from NAADP-AM-evoked Ca²⁺ release from lysosomal vesicles is in full agreement with previous work carried out on human fibroblasts (Kilpatrick et al., 2013), human ECFCs (Moccia et al., 2021b), COS-7 cells (Morgan and Galione, 2021), HeLa cells (Ronco et al., 2015), and human metastatic colorectal cancer cells (Faris et al., 2019). Third, TEM revealed clearly discernible ER-lysosomes MCSs, which closely resemble those previously described in human fibroblasts (Kilpatrick et al., 2013) and could provide the sub-cellular framework to enable InsP₃R recruitment by local Ca²⁺ release through TPCs (Penny et al., 2014). Likewise, the MCSs between lysosomal vesicles and ER cisternae in C-MSCs are similar to the cytoplasmic nanojunctions between lysosomes and sarcoplasmic reticulum (SR) recently reported in rat aortic VSMCs (Fameli et al., 2014).

The different extent of coupling between lysosomal TPCs and ER-embedded InsP₃Rs (due to changes in either their distribution or density at MCSs) could explain the onset of a long-lasting elevation in [Ca²⁺]_i that replaces the intracellular Ca²⁺ oscillations in a fraction of C-MSCs. For instance, computational modelling indicated that TPC clustering within the microdomain could accelerate the frequency of InsP₃Rs-driven Ca²⁺ oscillations (Penny et al., 2014), which could ultimately lead to the fusion of the Ca²⁺ spikes and the occurrence of a single, broader increase in [Ca²⁺]_i (Bartlett et al., 2020).

Removal of extracellular Ca²⁺ shortened the duration of the Ca²⁺ response to NAADP-AM. Therefore, NAADP is predicted to gate a Ca²⁺-permeable pathway on the plasma membrane. This observation is supported by the evidence that restitution of extracellular Ca²⁺ following exposure to NAADP (or nigericin) under 0Ca²⁺ conditions, results in a second bump in [Ca²⁺]_i that reflects extracellular Ca²⁺ entry. This influx of Ca²⁺ occurs after washout of the agonist from the bath and, therefore, it is exclusively coupled to the previous depletion of endogenous Ca²⁺ stores. As discussed elsewhere (Yamazaki et al., 2007; Sanchez-Hernandez et al., 2010; Negri et al., 2020), this feature hints at SOCE as being responsible for NAADP-induced extracellular Ca²⁺ entry. In agreement with this hypothesis, NAADP-evoked Ca²⁺ influx was remarkably reduced in the presence of either BTP-2 or Pyr6, two different inhibitors of Orai1 channels, which provide the pore-forming subunit of store-operated Ca²⁺ channels in non-excitable cells (Prakriya and Lewis, 2015; Emrich et al., 2021) and MSCs (Lee et al., 2016; Peng et al., 2016). SOCE activation ultimately results from the reduction of ER Ca²⁺ concentration (Emrich et al., 2021). As discussed elsewhere (Davis et al., 2012; Brailoiu and Brailoiu, 2016), the engagement of SOCE by NAADP (and nigericin) hints at the depletion of the ER Ca²⁺ content as the intermediate step between lysosomal Ca²⁺ release and extracellular Ca²⁺ entry. However, extracellular Ca²⁺ entry directly evoked by NAADP delivery was not always engaged during acidic Ca²⁺ signalling in the cell types where this functional interplay has been investigated (Faris et al., 2019; Moccia et al., 2021b). Therefore, it is conceivable that lysosomal Ca²⁺ release recruits ER sub-domains that are functionally coupled to the SOCE machinery in C-MSCs, but not in other cell types, as widely discussed in Parekh and Putney (2005). These observations hint at NAADP as a Ca²⁺-releasing second messenger that can trigger a functional crosstalk among multiple Ca²⁺ sources (lysosomes, ER, and plasma membrane) in C-MSCs. In these cells, NAADP may serve as a provider of the “trigger” Ca²⁺ response to extracellular stimulation that is subsequently amplified by InsP₃Rs on the ER and maintained over time by SOCE activation on the plasma membrane, as previously reported in many mammalian cells, including human fibroblasts (Kilpatrick et al., 2013), human ECFCs (Moccia et al., 2021b) and brain microvascular endothelial cells (Zuccolo et al., 2019), human metastatic colorectal cancer cells (Faris et al., 2019), human primary CTL cells (Davis et al., 2012), and rat pulmonary artery VSMCs (Kinnear et al., 2004).

4.3 Lysosomal Ca²⁺ Release via TPCs is Crucial to FBS-Induced Intracellular Ca²⁺ Signalling and Proliferation in Cardiac Mesenchymal Stromal Cells

It has long been known that FBS stimulates proliferation through an increase in [Ca²⁺]_i that can adopt either a biphasic (Faris et al., 2019) or an oscillatory pattern (Tao et al., 2011) in a variety of cell types, including rat bone marrow MSCs (Foreman et al., 2006). FBS-induced intracellular Ca²⁺ signals are known to impinge on the interplay between InsP₃-induced Ca²⁺ release from the ER and SOCE (Foreman et al., 2006; Hu et al., 2009). Intriguingly, a recent investigation reported the first evidence that NAADP-evoked lysosomal Ca²⁺ release via TPC1 interacts with InsP₃-dependent ER Ca²⁺ mobilization and SOCE to promote FBS-induced proliferation in human metastatic colorectal cancer cells (Faris et al., 2019). Unveiling the molecular mechanisms that drive C-MSC proliferation is crucial to improve the therapeutic outcome of regenerative strategies aiming at utilizing these cells to promote cardiac repair (Bagno et al., 2018; Braunwald, 2018). Preliminary analysis showed that FBS evoked a complex increase in [Ca²⁺]_i also in C-MSCs, which displayed either an oscillatory or a biphasic Ca²⁺ signal. Pharmacological manipulation confirmed that the Ca²⁺ response to FBS comprised InsP₃-induced ER Ca²⁺ mobilization followed by SOCE activation. Indeed, FBS-induced intracellular Ca²⁺ release was suppressed by inhibiting InsP₃Rs with 2-APB, by blocking basal InsP₃ production with U73122 and by depleting the ER Ca²⁺ store with CPA, whereas FBS-induced extracellular Ca²⁺ entry was remarkably attenuated by blocking SOCE with BTP-2 and Pyr6. Next, we provided the evidence that the NAADP-sensitive acidic Ca²⁺ store is crucial to FBS-induced intracellular Ca²⁺ signals and proliferation in C-MSCs. Indeed, FBS-induced intracellular Ca²⁺ release was abrogated by depleting the lysosomal Ca²⁺ store with either GPN or nigericin, as previously shown in human metastatic colorectal cancer cells (Faris et al., 2019). In agreement with these observations, the selective blockade of TPCs with NED-19, NED-K or tetrandrine also abolished the intracellular Ca²⁺ response to FBS. Therefore, NAADP-induced lysosomal Ca²⁺ release is indispensable to trigger the cytosolic Ca²⁺ response to FBS and this requires the functional recruitment of InsP₃Rs on the ER via CICR at lysosomal-ER MCSs. That the ER is depleted via InsP₃Rs-mediated ER Ca²⁺ release following NAADP-induced lysosomal Ca²⁺ mobilization in response to FBS is also suggested by FBS-induced SOCE activation, which requires a reduction in ER Ca²⁺ concentration (Brailoiu et al., 2009; Davis et al., 2012). The mechanism whereby FBS stimulation results to intracellular NAADP generation in C-MSCs, as well as in human metastatic cancer cells (Faris et al., 2019), remains to be elucidated. Nevertheless, FBS is likely to engage the multifunctional enzyme CD38, which catalyses the “base exchange” of the nicotinamide moiety of NADP with nicotinic acid, thereby resulting in NAADP production in most cell types (Galione, 2015), including cardiomyocytes (Negri et al., 2021b). A recent paper suggested that the dual NADPH oxidases, DUOX1 and DUOX2, contribute to NAADP biosynthesis in murine T lymphocytes (Gu et al., 2021), but their role in NAADP-dependent Ca²⁺ response to FBS is yet to be investigated.

The physiological role of NAADP-induced intracellular Ca²⁺ signals were further assessed by evaluating the effect of NED-19 on C-MSC proliferation. The pharmacological blockade of TPCs with NED-19 strongly reduced FBS-induced C-MSC proliferation at 24 and 48 h. Preliminary experiments indicated that the massive release of Ca²⁺ induced by nigericin per se resulted in C-MSC cell death already at 24 h from exposure to this lysosomotropic compound. While this observation is in accord with the reported effects of nigericin on various cell models (Murakami et al., 2012), it prevented us from probing its ability to interfere with FBS-induced proliferation. Previous work showed that NAADP-induced Ca²⁺ release may stimulate proliferation by recruiting the Ca²⁺-dependent ERK1/2 and Akt signalling pathways (Faris et al., 2019; Negri et al., 2021b). Consistently, FBS-induced ERK1/2 phosphorylation was impaired by the pharmacological blockade of TPCs with NED-19, whereas Akt engagement was unaffected. Interestingly, ERK1/2, but not Akt, was harnessed by intracellular Ca²⁺ oscillations to drive FBS-induced proliferation also in human bone marrow MSCs (Tao et al., 2011). Additionally, NAADP-induced intracellular Ca²⁺ oscillations could underpin another crucial function of C-MSCs, i.e., the regulation of extracellular matrix (ECM) composition (Maione et al., 2020b). For instance, bone marrow-derived human MSCs exhibited repetitive Ca²⁺ spikes during aligned collagen matrix formation (Gilchrist et al., 2019), whereas extracellular Ca²⁺ entry in human airway epithelial cells drives the expression and secretion of matrix-degrading enzymes, such as matrix metalloprotease 1 (Li et al., 2011). Interestingly, an increase in [Ca²⁺]_i in cardiac fibroblasts may also regulate collagen remodelling in mouse hearts (Adapala et al., 2020). Therefore, future studies will have to assess the role of NAADP-induced Ca²⁺ signalling in the modulation of ECM composition by C-MSCs.

In conclusion, this study demonstrated that NAADP induces intracellular Ca²⁺ signals in C-MSCs by promoting lysosomal Ca²⁺ release via TPCs that is in turn amplified by ER-embedded InsP₃Rs at lysosomal-ER MCSs. The following depletion of the ER Ca²⁺ pool activates SOCE, which prolongs the Ca²⁺ response to NAADP. FBS impinges on the NAADP-induced Ca²⁺-dependent crosstalk between lysosomes and ER to stimulate proliferation through the Ca²⁺-dependent ERK1/2 signalling pathway. These findings pave the way for future studies assessing whether NAADP signalling in C-MSCs could be targeted to favour cardiac repair upon an ischemic insult or to other pathologies associated to maladaptive cardiac remodelling, such as ACM, heart failure and cardiac fibrosis.

Data Availability Statement

The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.

Ethics Statement

The studies involving human participants were reviewed and approved by the IEO-CCM IRCCS Ethic Committee (project CCM1072). The patients/participants provided their written informed consent to participate in this study.

Author Contributions

FM conceived and directed the project in collaboration with AM. FM, MB, and AM conceived the experiments. PF, CC, AM, SN, and LI performed the experiments and analysed the data. All authors contributed to the article and approved the submitted version.

Funding

The authors gratefully acknowledge financial support from: Italian Ministry of Education, University and Research (MIUR): Dipartimenti di Eccellenza Program (2018–2022)—Department of Biology and Biotechnology “L. Spallanzani,” University of Pavia (MB and FM); Fondo Ricerca Giovani from the University of Pavia (MB and FM); EU Horizon 2020 FETOPEN-2018-2020 Programme “LION-HEARTED,” grant agreement No. 828984 (FM); Italian Ministry of Health—RCXXXXXX—(AM); “Fondazione di Comunità di Milano” and “Fondo Giacomo Ponzone” (AM).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Acknowledgments

The authors gratefully acknowledge the Laboratory of Electron Transmission Microscopy, Centro Grandi Strumenti of the University Pavia, for excellent technical and scientific support.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2022.874043/full#supplementary-material

References

Adapala, R. K., Kanugula, A. K., Paruchuri, S., Chilian, W. M., and Thodeti, C. K. (2020). TRPV4 Deletion Protects Heart from Myocardial Infarction-Induced Adverse Remodeling via Modulation of Cardiac Fibroblast Differentiation. Basic Res. Cardiol. 115, 14. doi:10.1007/s00395-020-0775-5

PubMed Abstract | CrossRef Full Text | Google Scholar

Atakpa, P., Van Marrewijk, L. M., Apta-Smith, M., Chakraborty, S., and Taylor, C. W. (2019). GPN Does Not Release Lysosomal Ca2+ but Evokes Ca2+ Release from the ER by Increasing the Cytosolic pH Independently of Cathepsin C. J. Cel Sci 132. doi:10.1242/jcs.223883

PubMed Abstract | CrossRef Full Text | Google Scholar

Bagno, L., Hatzistergos, K. E., Balkan, W., and Hare, J. M. (2018). Mesenchymal Stem Cell-Based Therapy for Cardiovascular Disease: Progress and Challenges. Mol. Ther. 26, 1610–1623. doi:10.1016/j.ymthe.2018.05.009

PubMed Abstract | CrossRef Full Text | Google Scholar

Balducci, V., Faris, P., Balbi, C., Costa, A., Negri, S., Rosti, V., et al. (2021). The Human Amniotic Fluid Stem Cell Secretome Triggers Intracellular Ca2+ Oscillations, NF‐κB Nuclear Translocation and Tube Formation in Human Endothelial colony‐forming Cells. J. Cel Mol Med 25, 8074–8086. doi:10.1111/jcmm.16739

CrossRef Full Text | Google Scholar

Bartlett, P. J., Cloete, I., Sneyd, J., and Thomas, A. P. (2020). IP3-Dependent Ca2+ Oscillations Switch into a Dual Oscillator Mechanism in the Presence of PLC-Linked Hormones. iScience 23, 101062. doi:10.1016/j.isci.2020.101062

PubMed Abstract | CrossRef Full Text | Google Scholar

Berg, I., Potter, B. V. L., Mayr, G. W., and Guse, A. H. (2000). Nicotinic Acid Adenine Dinucleotide Phosphate (Naadp+) Is an Essential Regulator of T-Lymphocyte Ca2+-Signaling. J. Cel Biol 150, 581–588. doi:10.1083/jcb.150.3.581

CrossRef Full Text | Google Scholar

Berra-Romani, R., Faris, P., Pellavio, G., Orgiu, M., Negri, S., Forcaia, G., et al. (2020). Histamine Induces Intracellular Ca2+ Oscillations and Nitric Oxide Release in Endothelial Cells from Brain Microvascular Circulation. J. Cel Physiol 235, 1515–1530. doi:10.1002/jcp.29071

CrossRef Full Text | Google Scholar

Brailoiu, G. C., Brailoiu, E., Parkesh, R., Galione, A., Churchill, G. C., Patel, S., et al. (2009). NAADP-mediated Channel 'chatter' in Neurons of the Rat Medulla Oblongata. Biochem. J. 419 (91-97), 91–97. doi:10.1042/BJ20081138

PubMed Abstract | CrossRef Full Text | Google Scholar

Brailoiu, G. C., and Brailoiu, E. (2016). Modulation of Calcium Entry by the Endo-Lysosomal System. Adv. Exp. Med. Biol. 898, 423–447. doi:10.1007/978-3-319-26974-0_18

PubMed Abstract | CrossRef Full Text | Google Scholar

Brailoiu, G. C., Gurzu, B., Gao, X., Parkesh, R., Aley, P. K., Trifa, D. I., et al. (2010). Acidic NAADP-Sensitive Calcium Stores in the Endothelium. J. Biol. Chem. 285, 37133–37137. doi:10.1074/jbc.c110.169763

CrossRef Full Text | Google Scholar

Braunwald, E. (2018). Cell-Based Therapy in Cardiac Regeneration. Circ. Res. 123, 132–137. doi:10.1161/circresaha.118.313484

PubMed Abstract | CrossRef Full Text | Google Scholar

Brown, R. D., Ambler, S. K., Mitchell, M. D., and Long, C. S. (2005). The Cardiac Fibroblast: Therapeutic Target in Myocardial Remodeling and Failure. Annu. Rev. Pharmacol. Toxicol. 45, 657–687. doi:10.1146/annurev.pharmtox.45.120403.095802

PubMed Abstract | CrossRef Full Text | Google Scholar

Camelliti, P., Borg, T., and Kohl, P. (2005). Structural and Functional Characterisation of Cardiac Fibroblasts. Cardiovasc. Res. 65, 40–51. doi:10.1016/j.cardiores.2004.08.020

PubMed Abstract | CrossRef Full Text | Google Scholar

Cane, M. C., Parrington, J., Rorsman, P., Galione, A., and Rutter, G. A. (2016). The Two Pore Channel TPC2 Is Dispensable in Pancreatic β-cells for normal Ca2+ Dynamics and Insulin Secretion. Cell Calcium 59, 32–40. doi:10.1016/j.ceca.2015.12.004

PubMed Abstract | CrossRef Full Text | Google Scholar

Capel, R. A., Bolton, E. L., Lin, W. K., Aston, D., Wang, Y., Liu, W., et al. (2015). Two-pore Channels (TPC2s) and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) at Lysosomal-Sarcoplasmic Reticular Junctions Contribute to Acute and Chronic β-Adrenoceptor Signaling in the Heart. J. Biol. Chem. 290, 30087–30098. doi:10.1074/jbc.m115.684076

PubMed Abstract | CrossRef Full Text | Google Scholar

Carriero, F., Martinelli, C., Gabriele, F., Barbieri, G., Zanoletti, L., Milanesi, G., et al. (2021). Berberine Photo-Activation Potentiates Cytotoxicity in Human Astrocytoma Cells through Apoptosis Induction. J. Pers Med. 11. doi:10.3390/jpm11100942

CrossRef Full Text | Google Scholar

Christensen, K. A., Myers, J. T., and Swanson, J. A. (2002). pH-dependent Regulation of Lysosomal Calcium in Macrophages. J. Cel Sci 115, 599–607. doi:10.1242/jcs.115.3.599

CrossRef Full Text | Google Scholar

Churchill, G. C., and Galione, A. (2001). NAADP Induces Ca2+ Oscillations via a Two-Pool Mechanism by Priming IP3- and cADPR-Sensitive Ca2+ Stores. EMBO J. 20, 2666–2671. doi:10.1093/emboj/20.11.2666

PubMed Abstract | CrossRef Full Text | Google Scholar

Churchill, G. C., Okada, Y., Thomas, J. M., Genazzani, A. A., Patel, S., and Galione, A. (2002). NAADP Mobilizes Ca2+ from Reserve Granules, Lysosome-Related Organelles, in Sea Urchin Eggs. Cell 111, 703–708. doi:10.1016/s0092-8674(02)01082-6

PubMed Abstract | CrossRef Full Text | Google Scholar

Collins, T. P., Bayliss, R., Churchill, G. C., Galione, A., and Terrar, D. A. (2011). NAADP Influences Excitation-Contraction Coupling by Releasing Calcium from Lysosomes in Atrial Myocytes. Cell Calcium 50, 449–458. doi:10.1016/j.ceca.2011.07.007

PubMed Abstract | CrossRef Full Text | Google Scholar

Czapla, J., Matuszczak, S., Wiśniewska, E., Jarosz-Biej, M., Smolarczyk, R., Cichoń, T., et al. (2016). Human Cardiac Mesenchymal Stromal Cells with CD105+CD34- Phenotype Enhance the Function of Post-Infarction Heart in Mice. PLoS One 11, e0158745. doi:10.1371/journal.pone.0158745

PubMed Abstract | CrossRef Full Text | Google Scholar

Davidson, S. M., Foote, K., Kunuthur, S., Gosain, R., Tan, N., Tyser, R., et al. (2015). Inhibition of NAADP Signalling on Reperfusion Protects the Heart by Preventing Lethal Calcium Oscillations via Two-Pore Channel 1 and Opening of the Mitochondrial Permeability Transition Pore. Cardiovasc. Res. 108, 357–366. doi:10.1093/cvr/cvv226

PubMed Abstract | CrossRef Full Text | Google Scholar

Davis, L. C., Morgan, A. J., Chen, J.-L., Snead, C. M., Bloor-Young, D., Shenderov, E., et al. (2012). NAADP Activates Two-Pore Channels on T Cell Cytolytic Granules to Stimulate Exocytosis and Killing. Curr. Biol. 22, 2331–2337. doi:10.1016/j.cub.2012.10.035

PubMed Abstract | CrossRef Full Text | Google Scholar

Di Nezza, F., Zuccolo, E., Poletto, V., Rosti, V., De Luca, A., Moccia, F., et al. (2017). Liposomes as a Putative Tool to Investigate NAADP Signaling in Vasculogenesis. J. Cel. Biochem. 118, 3722–3729. doi:10.1002/jcb.26019

CrossRef Full Text | Google Scholar

Diedrichs, F., Stolk, M., Jürchott, K., Haag, M., Sittinger, M., and Seifert, M. (2019). Enhanced Immunomodulation in Inflammatory Environments Favors Human Cardiac Mesenchymal Stromal-like Cells for Allogeneic Cell Therapies. Front. Immunol. 10, 1716. doi:10.3389/fimmu.2019.01716

PubMed Abstract | CrossRef Full Text | Google Scholar

Emrich, S. M., Yoast, R. E., and Trebak, M. (2021). Physiological Functions of CRAC Channels. Annu. Rev. Physiol. 84:355. doi:10.1146/annurev-physiol-052521-013426

PubMed Abstract | CrossRef Full Text | Google Scholar

Fameli, N., Evans, A. M., and Van Breemen, C. (2017). Tissue Specificity: The Role of Organellar Membrane Nanojunctions in Smooth Muscle Ca2+ Signaling. Adv. Exp. Med. Biol. 993, 321–342. doi:10.1007/978-3-319-57732-6_17

PubMed Abstract | CrossRef Full Text | Google Scholar

Fameli, N., Ogunbayo, O. A., Van Breemen, C., and Evans, A. M. (2014). Cytoplasmic Nanojunctions between Lysosomes and Sarcoplasmic Reticulum Are Required for Specific Calcium Signaling. F1000Res 3, 93. doi:10.12688/f1000research.3720.1

PubMed Abstract | CrossRef Full Text | Google Scholar

Faris, P., Pellavio, G., Ferulli, F., Di Nezza, F., Shekha, M., Lim, D., et al. (2019). Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Induces Intracellular Ca2+ Release through the Two-Pore Channel TPC1 in Metastatic Colorectal Cancer Cells. Cancers (Basel) 11. E542. doi:10.3390/cancers11040542

PubMed Abstract | CrossRef Full Text | Google Scholar

Faris, P., Shekha, M., Montagna, D., Guerra, G., and Moccia, F. (2018). Endolysosomal Ca(2+) Signalling and Cancer Hallmarks: Two-Pore Channels on the Move. TRPML1 Lags behind!. Cancers (Base1) 11, 27. doi:10.3390/cancers11010027

PubMed Abstract | CrossRef Full Text | Google Scholar

Favia, A., Desideri, M., Gambara, G., D'alessio, A., Ruas, M., Esposito, B., et al. (2014). VEGF-induced Neoangiogenesis Is Mediated by NAADP and Two-Pore Channel-2-dependent Ca2+ Signaling. Proc. Natl. Acad. Sci. 111, E4706–E4715. doi:10.1073/pnas.1406029111

PubMed Abstract | CrossRef Full Text | Google Scholar

Ferrera, L., Barbieri, R., Picco, C., Zuccolini, P., Remigante, A., Bertelli, S., et al. (2021). TRPM2 Oxidation Activates Two Distinct Potassium Channels in Melanoma Cells through Intracellular Calcium Increase. Int. J. Mol. Sci. 22. doi:10.3390/ijms22168359

CrossRef Full Text | Google Scholar

Foreman, M. A., Smith, J., and Publicover, S. J. (2006). Characterisation of Serum-Induced Intracellular Ca2+ Oscillations in Primary Bone Marrow Stromal Cells. J. Cel. Physiol. 206, 664–671. doi:10.1002/jcp.20521

CrossRef Full Text | Google Scholar

Forostyak, O., Forostyak, S., Kortus, S., Sykova, E., Verkhratsky, A., and Dayanithi, G. (2016). Physiology of Ca2+ Signalling in Stem Cells of Different Origins and Differentiation Stages. Cell Calcium 59, 57–66. doi:10.1016/j.ceca.2016.02.001

PubMed Abstract | CrossRef Full Text | Google Scholar

Galione, A. (2019). NAADP Receptors. Cold Spring Harb Perspect. Biol. 11. doi:10.1101/cshperspect.a035071

PubMed Abstract | CrossRef Full Text | Google Scholar

Galione, A. (2015). A Primer of NAADP-Mediated Ca2+ Signalling: From Sea Urchin Eggs to Mammalian Cells. Cell Calcium 58, 27–47. doi:10.1016/j.ceca.2014.09.010

PubMed Abstract | CrossRef Full Text | Google Scholar

Garrity, A. G., Wang, W., Collier, C. M., Levey, S. A., Gao, Q., and Xu, H. (2016). The Endoplasmic Reticulum, Not the pH Gradient, Drives Calcium Refilling of Lysosomes. Elife 5. doi:10.7554/eLife.15887

CrossRef Full Text | Google Scholar

Gilchrist, C. L., Leddy, H. A., Kaye, L., Case, N. D., Rothenberg, K. E., Little, D., et al. (2019). TRPV4-mediated Calcium Signaling in Mesenchymal Stem Cells Regulates Aligned Collagen Matrix Formation and Vinculin Tension. Proc. Natl. Acad. Sci. USA 116, 1992–1997. doi:10.1073/pnas.1811095116

CrossRef Full Text | Google Scholar

Gu, F., Krüger, A., Roggenkamp, H. G., Alpers, R., Lodygin, D., Jaquet, V., et al. (2021). Dual NADPH Oxidases DUOX1 and DUOX2 Synthesize NAADP and Are Necessary for Ca2+ Signaling during T Cell Activation. Sci. Signal. 14, eabe3800. doi:10.1126/scisignal.abe3800

PubMed Abstract | CrossRef Full Text | Google Scholar

Gunaratne, G. S., Brailoiu, E., He, S., Unterwald, E. M., Patel, S., Slama, J. T., et al. (2021). Essential Requirement for JPT2 in NAADP-Evoked Ca2+ Signaling. Sci. Signal. 14. doi:10.1126/scisignal.abd5605

CrossRef Full Text | Google Scholar

Hu, R., He, M.-l., Hu, H., Yuan, B.-X., Zang, W.-J., Lau, C.-P., et al. (2009). Characterization of Calcium Signaling Pathways in Human Preadipocytes. J. Cel. Physiol. 220, 765–770. doi:10.1002/jcp.21823

CrossRef Full Text | Google Scholar

Hu, W., Zhao, F., Chen, L., Ni, J., and Jiang, Y. (2021). NAADP‐induced Intracellular Calcium Ion Is Mediated by the TPCs (Two‐pore Channels) in Hypoxia‐induced Pulmonary Arterial Hypertension. J. Cel Mol Med 25, 7485–7499. doi:10.1111/jcmm.16783

CrossRef Full Text | Google Scholar

Jairaman, A., Yamashita, M., Schleimer, R. P., and Prakriya, M. (2015). Store-Operated Ca2+ Release-Activated Ca2+ Channels Regulate PAR2-Activated Ca2+ Signaling and Cytokine Production in Airway Epithelial Cells. J.I. 195, 2122–2133. doi:10.4049/jimmunol.1500396

PubMed Abstract | CrossRef Full Text | Google Scholar

Jiang, L.-H., Mousawi, F., Yang, X., and Roger, S. (2017). ATP-induced Ca2+-Signalling Mechanisms in the Regulation of Mesenchymal Stem Cell Migration. Cell. Mol. Life Sci. 74, 3697–3710. doi:10.1007/s00018-017-2545-6

PubMed Abstract | CrossRef Full Text | Google Scholar

Jiang, Y.-L., Lin, A. H. Y., Xia, Y., Lee, S., Paudel, O., Sun, H., et al. (2013). Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Activates Global and Heterogeneous Local Ca2+ Signals from NAADP- and Ryanodine Receptor-Gated Ca2+ Stores in Pulmonary Arterial Myocytes. J. Biol. Chem. 288, 10381–10394. doi:10.1074/jbc.m112.423053

CrossRef Full Text | Google Scholar

Jiang, Y., Zhou, Y., Peng, G., Tian, H., Pan, D., Liu, L., et al. (2018). Two-pore Channels Mediated Receptor-Operated Ca2+ Entry in Pulmonary Artery Smooth Muscle Cells in Response to Hypoxia. Int. J. Biochem. Cel Biol. 97, 28–35. doi:10.1016/j.biocel.2018.01.012

CrossRef Full Text | Google Scholar

Jin, X., Zhang, Y., Alharbi, A., Hanbashi, A., Alhoshani, A., and Parrington, J. (2020). Targeting Two-Pore Channels: Current Progress and Future Challenges. Trends Pharmacol. Sci. 41, 582–594. doi:10.1016/j.tips.2020.06.002

PubMed Abstract | CrossRef Full Text | Google Scholar

Johnson, J. D., and Misler, S. (2002). Nicotinic Acid-Adenine Dinucleotide Phosphate-Sensitive Calcium Stores Initiate Insulin Signaling in Human Beta Cells. Proc. Natl. Acad. Sci. 99, 14566–14571. doi:10.1073/pnas.222099799

PubMed Abstract | CrossRef Full Text | Google Scholar

Jugdutt, B. I. (2003). Ventricular Remodeling after Infarction and the Extracellular Collagen Matrix. Circulation 108, 1395–1403. doi:10.1161/01.cir.0000085658.98621.49

PubMed Abstract | CrossRef Full Text | Google Scholar

Kakkar, R., and Lee, R. T. (2010). Intramyocardial Fibroblast Myocyte Communication. Circ. Res. 106, 47–57. doi:10.1161/circresaha.109.207456

PubMed Abstract | CrossRef Full Text | Google Scholar

Kawano, S., Otsu, K., Kuruma, A., Shoji, S., Yanagida, E., Muto, Y., et al. (2006). ATP Autocrine/paracrine Signaling Induces Calcium Oscillations and NFAT Activation in Human Mesenchymal Stem Cells. Cell Calcium 39, 313–324. doi:10.1016/j.ceca.2005.11.008

PubMed Abstract | CrossRef Full Text | Google Scholar

Kawano, S., Otsu, K., Shoji, S., Yamagata, K., and Hiraoka, M. (2003). Ca2+ Oscillations Regulated by Na+ -Ca2+ Exchanger and Plasma Membrane Ca2+ Pump Induce Fluctuations of Membrane Currents and Potentials in Human Mesenchymal Stem Cells. Cell Calcium 34, 145–156. doi:10.1016/s0143-4160(03)00069-1

PubMed Abstract | CrossRef Full Text | Google Scholar

Kawano, S., Shoji, S., Ichinose, S., Yamagata, K., Tagami, M., and Hiraoka, M. (2002). Characterization of Ca2+ Signaling Pathways in Human Mesenchymal Stem Cells. Cell Calcium 32, 165–174. doi:10.1016/s0143416002001240

PubMed Abstract | CrossRef Full Text | Google Scholar

Kilpatrick, B. S., Yates, E., Grimm, C., Schapira, A. H., and Patel, S. (2016). Endo-lysosomal TRP Mucolipin-1 Channels Trigger Global ER Ca2+ Release and Ca2+ Influx. J. Cel Sci 129, 3859–3867. doi:10.1242/jcs.190322

PubMed Abstract | CrossRef Full Text | Google Scholar

Kilpatrick, B. S., Eden, E. R., Schapira, A. H., Futter, C. E., and Patel, S. (2013). Direct Mobilisation of Lysosomal Ca2+ Triggers Complex Ca2+ Signals. J. Cel Sci 126, 60–66. doi:10.1242/jcs.118836

CrossRef Full Text | Google Scholar

Kinnear, N. P., Boittin, F.-X., Thomas, J. M., Galione, A., and Evans, A. M. (2004). Lysosome-Sarcoplasmic Reticulum Junctions. J. Biol. Chem. 279, 54319–54326. doi:10.1074/jbc.m406132200

CrossRef Full Text | Google Scholar

Kotova, P. D., Bystrova, M. F., Rogachevskaja, O. A., Khokhlov, A. A., Sysoeva, V. Y., Tkachuk, V. A., et al. (2018). Coupling of P2Y Receptors to Ca2+ Mobilization in Mesenchymal Stromal Cells from the Human Adipose Tissue. Cell Calcium 71, 1–14. doi:10.1016/j.ceca.2017.11.001

PubMed Abstract | CrossRef Full Text | Google Scholar

Lee, S. H., Park, Y., Song, M., Srikanth, S., Kim, S., Kang, M. K., et al. (2016). Orai1 Mediates Osteogenic Differentiation via BMP Signaling Pathway in Bone Marrow Mesenchymal Stem Cells. Biochem. Biophysical Res. Commun. 473, 1309–1314. doi:10.1016/j.bbrc.2016.04.068

CrossRef Full Text | Google Scholar

Lee, S., Paudel, O., Jiang, Y., Yang, X.-R., and Sham, J. S. K. (2015). CD38 Mediates Angiotensin II-Induced Intracellular Ca2+Release in Rat Pulmonary Arterial Smooth Muscle Cells. Am. J. Respir. Cel Mol Biol 52, 332–341. doi:10.1165/rcmb.2014-0141oc

CrossRef Full Text | Google Scholar

Lewis, A. M., Aley, P. K., Roomi, A., Thomas, J. M., Masgrau, R., Garnham, C., et al. (2012). SS-Adrenergic Receptor Signaling Increases NAADP and cADPR Levels in the Heart. Biochem. Biophysical Res. Commun. 427, 326–329. doi:10.1016/j.bbrc.2012.09.054

CrossRef Full Text | Google Scholar

Li, J., Kanju, P., Patterson, M., Chew, W.-L., Cho, S.-H., Gilmour, I., et al. (2011). TRPV4-mediated Calcium Influx into Human Bronchial Epithelia upon Exposure to Diesel Exhaust Particles. Environ. Health Perspect. 119, 784–793. doi:10.1289/ehp.1002807

PubMed Abstract | CrossRef Full Text | Google Scholar

Macgregor, A., Yamasaki, M., Rakovic, S., Sanders, L., Parkesh, R., Churchill, G. C., et al. (2007). NAADP Controls Cross-Talk between Distinct Ca2+ Stores in the Heart. J. Biol. Chem. 282, 15302–15311. doi:10.1074/jbc.m611167200

CrossRef Full Text | Google Scholar

Maione, A. S., Faris, P. S., Bisonni, L., Lodola, F., Casella, M., Catto, V., et al. (2020a). Imbalance of Calcium-dependent Mechanisms in Cardiac Mesenchymal Stromal Cells from Arrhythmogenic Cardiomyopathy Patients. Vasc. Pharmacol. 132, 106732. doi:10.1016/j.vph.2020.106732

CrossRef Full Text | Google Scholar

Maione, A. S., Pilato, C. A., Casella, M., Gasperetti, A., Stadiotti, I., Pompilio, G., et al. (2020b). Fibrosis in Arrhythmogenic Cardiomyopathy: The Phantom Thread in the Fibro-Adipose Tissue. Front. Physiol. 11, 279. doi:10.3389/fphys.2020.00279

PubMed Abstract | CrossRef Full Text | Google Scholar

Maione, A. S., Stadiotti, I., Pilato, C. A., Perrucci, G. L., Saverio, V., Catto, V., et al. (2021). Excess TGF-Beta1 Drives Cardiac Mesenchymal Stromal Cells to a Pro-fibrotic Commitment in Arrhythmogenic Cardiomyopathy. Int. J. Mol. Sci. 22. doi:10.3390/ijms22052673

CrossRef Full Text | Google Scholar

Moccia, F., Lodola, F., Stadiotti, I., Pilato, C. A., Bellin, M., Carugo, S., et al. (2019). Calcium as a Key Player in Arrhythmogenic Cardiomyopathy: Adhesion Disorder or Intracellular Alteration? Int. J. Mol. Sci. 20. doi:10.3390/ijms20163986

PubMed Abstract | CrossRef Full Text | Google Scholar

Moccia, F., Negri, S., Faris, P., Perna, A., De Luca, A., Soda, T., et al. (2021a). Targeting Endolysosomal Two-Pore Channels to Treat Cardiovascular Disorders in the Novel COronaVIrus Disease 2019. Front. Physiol. 12, 629119. doi:10.3389/fphys.2021.629119

CrossRef Full Text | Google Scholar

Moccia, F., Nusco, G. A., Lim, D., Kyozuka, K., and Santella, L. (2006). NAADP and InsP3 Play Distinct Roles at Fertilization in Starfish Oocytes. Develop. Biol. 294, 24–38. doi:10.1016/j.ydbio.2006.02.011

PubMed Abstract | CrossRef Full Text | Google Scholar

Moccia, F., Ruffinatti, F., and Zuccolo, E. (2015). Intracellular Ca2+ Signals to Reconstruct A Broken Heart: Still A Theoretical Approach? Cdt 16, 793–815. doi:10.2174/1389450116666141219121723

PubMed Abstract | CrossRef Full Text | Google Scholar

Moccia, F., Zuccolo, E., Di Nezza, F., Pellavio, G., Faris, P. S., Negri, S., et al. (2021b). Nicotinic Acid Adenine Dinucleotide Phosphate Activates Two‐pore Channel TPC1 to Mediate Lysosomal Ca2+ Release in Endothelial colony‐forming Cells. J. Cel Physiol 236, 688–705. doi:10.1002/jcp.29896

CrossRef Full Text | Google Scholar

Moccia, F., Zuccolo, E., Poletto, V., Turin, I., Guerra, G., Pedrazzoli, P., et al. (2016). Targeting Stim and Orai Proteins as an Alternative Approach in Anticancer Therapy. Cmc 23, 3450–3480. doi:10.2174/0929867323666160607111220

PubMed Abstract | CrossRef Full Text | Google Scholar

Morgan, A. J., and Galione, A. (2021). Lysosomal Agents Inhibit Store-Operated Ca2+ Entry. J. Cel Sci 134. doi:10.1242/jcs.248658

CrossRef Full Text | Google Scholar

Morgan, A. J., Platt, F. M., Lloyd-Evans, E., and Galione, A. (2011). Molecular Mechanisms of Endolysosomal Ca2+ Signalling in Health and Disease. Biochem. J. 439, 349–378. doi:10.1042/bj20110949

PubMed Abstract | CrossRef Full Text | Google Scholar

Morgan, A. J., Yuan, Y., Patel, S., and Galione, A. (2020). Does Lysosomal Rupture Evoke Ca2+ Release? A Question of Pores and Stores. Cell Calcium 86, 102139. doi:10.1016/j.ceca.2019.102139

PubMed Abstract | CrossRef Full Text | Google Scholar

Murakami, T., Ockinger, J., Yu, J., Byles, V., Mccoll, A., Hofer, A. M., et al. (2012). Critical Role for Calcium Mobilization in Activation of the NLRP3 Inflammasome. Proc. Natl. Acad. Sci. 109, 11282–11287. doi:10.1073/pnas.1117765109

PubMed Abstract | CrossRef Full Text | Google Scholar

Nebel, M., Schwoerer, A. P., Warszta, D., Siebrands, C. C., Limbrock, A.-C., Swarbrick, J. M., et al. (2013). Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-mediated Calcium Signaling and Arrhythmias in the Heart Evoked by β-Adrenergic Stimulation. J. Biol. Chem. 288, 16017–16030. doi:10.1074/jbc.m112.441246

CrossRef Full Text | Google Scholar

Negri, S., Faris, P., Maniezzi, C., Pellavio, G., Spaiardi, P., Botta, L., et al. (2021a). NMDA Receptors Elicit Flux-independent Intracellular Ca2+ Signals via Metabotropic Glutamate Receptors and Flux-dependent Nitric Oxide Release in Human Brain Microvascular Endothelial Cells. Cell Calcium 99, 102454. doi:10.1016/j.ceca.2021.102454

PubMed Abstract | CrossRef Full Text | Google Scholar

Negri, S., Faris, P., and Moccia, F. (2021b). Endolysosomal Ca2+ Signaling in Cardiovascular Health and Disease. Int. Rev. Cel Mol Biol 363, 203–269. doi:10.1016/bs.ircmb.2021.03.001

CrossRef Full Text | Google Scholar

Negri, S., Faris, P., Pellavio, G., Botta, L., Orgiu, M., Forcaia, G., et al. (2020). Group 1 Metabotropic Glutamate Receptors Trigger Glutamate-Induced Intracellular Ca2+ Signals and Nitric Oxide Release in Human Brain Microvascular Endothelial Cells. Cel. Mol. Life Sci. 77, 2235–2253. doi:10.1007/s00018-019-03284-1

CrossRef Full Text | Google Scholar

Pandey, V., Chuang, C.-C., Lewis, A. M., Aley, P. K., Brailoiu, E., Dun, N. J., et al. (2009). Recruitment of NAADP-Sensitive Acidic Ca2+ Stores by Glutamate. Biochem. J. 422, 503–512. doi:10.1042/bj20090194

PubMed Abstract | CrossRef Full Text | Google Scholar

Parekh, A. B., and Putney, J. W. (2005). Store-operated Calcium Channels. Physiol. Rev. 85, 757–810. doi:10.1152/physrev.00057.2003

PubMed Abstract | CrossRef Full Text | Google Scholar

Patel, S. (2015). Function and Dysfunction of Two-Pore Channels. Sci. Signal. 8, re7. doi:10.1126/scisignal.aab3314

PubMed Abstract | CrossRef Full Text | Google Scholar

Peng, H., Hao, Y., Mousawi, F., Roger, S., Li, J., Sim, J. A., et al. (2016). Purinergic and Store-Operated Ca2+ Signaling Mechanisms in Mesenchymal Stem Cells and Their Roles in ATP-Induced Stimulation of Cell Migration. Stem Cells 34, 2102–2114. doi:10.1002/stem.2370

PubMed Abstract | CrossRef Full Text | Google Scholar

Penny, C. J., Kilpatrick, B. S., Han, J. M., Sneyd, J., and Patel, S. (2014). A Computational Model of Lysosome-ER Ca2+ Microdomains. J. Cel Sci 127, 2934–2943. doi:10.1242/jcs.149047

CrossRef Full Text | Google Scholar

Pereira, G. J. S., Hirata, H., Do Carmo, L. G., Stilhano, R. S., Ureshino, R. P., Medaglia, N. C., et al. (2014). NAADP-sensitive Two-Pore Channels Are Present and Functional in Gastric Smooth Muscle Cells. Cell Calcium 56, 51–58. doi:10.1016/j.ceca.2014.04.005

PubMed Abstract | CrossRef Full Text | Google Scholar

Pilato, C. A., Stadiotti, I., Maione, A. S., Saverio, V., Catto, V., Tundo, F., et al. (2018). Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients. J. Vis. Exp. 132, e57263. doi:10.3791/57263

PubMed Abstract | CrossRef Full Text | Google Scholar

Pitt, S. J., Reilly-O'donnell, B., and Sitsapesan, R. (2016). Exploring the Biophysical Evidence that Mammalian Two-Pore Channels Are NAADP-Activated Calcium-Permeable Channels. J. Physiol. 594, 4171–4179. doi:10.1113/jp270936

CrossRef Full Text | Google Scholar

Prakriya, M., and Lewis, R. S. (2015). Store-Operated Calcium Channels. Physiol. Rev. 95, 1383–1436. doi:10.1152/physrev.00020.2014

PubMed Abstract | CrossRef Full Text | Google Scholar

Rahman, S., and Rahman, T. (2017). Unveiling Some FDA-Approved Drugs as Inhibitors of the Store-Operated Ca2+ Entry Pathway. Sci. Rep. 7, 12881. doi:10.1038/s41598-017-13343-x

PubMed Abstract | CrossRef Full Text | Google Scholar

Remigante, A., Zuccolini, P., Barbieri, R., Ferrera, L., Morabito, R., Gavazzo, P., et al. (2021). NS-11021 Modulates Cancer-Associated Processes Independently of BK Channels in Melanoma and Pancreatic Duct Adenocarcinoma Cell Lines. Cancers (Basel) 13. doi:10.3390/cancers13236144

CrossRef Full Text | Google Scholar

Ronco, V., Potenza, D. M., Denti, F., Vullo, S., Gagliano, G., Tognolina, M., et al. (2015). A Novel Ca2+-Mediated Cross-Talk between Endoplasmic Reticulum and Acidic Organelles: Implications for NAADP-dependent Ca2+ Signalling. Cell Calcium 57, 89–100. doi:10.1016/j.ceca.2015.01.001

PubMed Abstract | CrossRef Full Text | Google Scholar

Ruas, M., Rietdorf, K., Arredouani, A., Davis, L. C., Lloyd-Evans, E., Koegel, H., et al. (2010). Purified TPC Isoforms Form NAADP Receptors with Distinct Roles for Ca2+ Signaling and Endolysosomal Trafficking. Curr. Biol. 20, 703–709. doi:10.1016/j.cub.2010.02.049

PubMed Abstract | CrossRef Full Text | Google Scholar

Sakurai, Y., Kolokoltsov, A. A., Chen, C.-C., Tidwell, M. W., Bauta, W. E., Klugbauer, N., et al. (2015). Two-pore Channels Control Ebola Virus Host Cell Entry and Are Drug Targets for Disease Treatment. Science 347, 995–998. doi:10.1126/science.1258758

PubMed Abstract | CrossRef Full Text | Google Scholar

Sánchez-Hernández, Y., Laforenza, U., Bonetti, E., Fontana, J., Dragoni, S., Russo, M., et al. (2010). Store-Operated Ca2+ Entry Is Expressed in Human Endothelial Progenitor Cells. Stem Cell Develop. 19, 1967–1981. doi:10.1089/scd.2010.0047

CrossRef Full Text | Google Scholar

Scarpellino, G., Genova, T., Avanzato, D., Bernardini, M., Bianco, S., Petrillo, S., et al. (2019). Purinergic Calcium Signals in Tumor-Derived Endothelium. Cancers (Basel). 11, 766. doi:10.3390/cancers11060766

PubMed Abstract | CrossRef Full Text | Google Scholar

Schach, C., Wester, M., Leibl, F., Redel, A., Gruber, M., Maier, L. S., et al. (2020). Reduced Store‐operated Ca2+ Entry Impairs Mesenteric Artery Function in Response to High External Glucose in Type 2 Diabetic ZDF Rats. Clin. Exp. Pharmacol. Physiol. 47, 1145–1157. doi:10.1111/1440-1681.13300

PubMed Abstract | CrossRef Full Text | Google Scholar

Schleifer, H., Doleschal, B., Lichtenegger, M., Oppenrieder, R., Derler, I., Frischauf, I., et al. (2012). Novel Pyrazole Compounds for Pharmacological Discrimination between Receptor‐operated and Store‐operated Ca2+ Entry Pathways. Br. J. Pharmacol. 167, 1712–1722. doi:10.1111/j.1476-5381.2012.02126.x

CrossRef Full Text | Google Scholar

Sommariva, E., Brambilla, S., Carbucicchio, C., Gambini, E., Meraviglia, V., Dello Russo, A., et al. (2016). Cardiac Mesenchymal Stromal Cells Are a Source of Adipocytes in Arrhythmogenic Cardiomyopathy. Eur. Heart J. 37, 1835–1846. doi:10.1093/eurheartj/ehv579

PubMed Abstract | CrossRef Full Text | Google Scholar

Stadiotti, I., Catto, V., Casella, M., Tondo, C., Pompilio, G., and Sommariva, E. (2017). Arrhythmogenic Cardiomyopathy: the Guilty Party in Adipogenesis. J. Cardiovasc. Trans. Res. 10, 446–454. doi:10.1007/s12265-017-9767-8

PubMed Abstract | CrossRef Full Text | Google Scholar

Tao, R., Sun, H.-Y., Lau, C.-P., Tse, H.-F., Lee, H.-C., and Li, G.-R. (2011). Cyclic ADP Ribose Is a Novel Regulator of Intracellular Ca2+ Oscillations in Human Bone Marrow Mesenchymal Stem Cells. J. Cel Mol Med 15, 2684–2696. doi:10.1111/j.1582-4934.2011.01263.x

CrossRef Full Text | Google Scholar

Trufanov, S. K., Rybakova, E. Y., Avdonin, P. P., Tsitrina, A. A., Zharkikh, I. L., Goncharov, N. V., et al. (2019). The Role of Two-Pore Channels in Norepinephrine-Induced [Ca(2+)]i Rise in Rat Aortic Smooth Muscle Cells and Aorta Contraction. Cells 8. 1144. doi:10.3390/cells8101144

PubMed Abstract | CrossRef Full Text | Google Scholar

Vassileva, K., Marsh, M., and Patel, S. (2020). Two-pore Channels as Master Regulators of Membrane Trafficking and Endocytic Well-Being. Curr. Opin. Physiol. 17, 163–168. doi:10.1016/j.cophys.2020.08.002

PubMed Abstract | CrossRef Full Text | Google Scholar

Vismara, M., Zarà, M., Negri, S., Canino, J., Canobbio, I., Barbieri, S. S., et al. (2021). Platelet-derived Extracellular Vesicles Regulate Cell Cycle Progression and Cell Migration in Breast Cancer Cells. Biochim. Biophys. Acta (Bba) - Mol. Cel Res. 1868, 118886. doi:10.1016/j.bbamcr.2020.118886

CrossRef Full Text | Google Scholar

Yamazaki, D., Ohya, S., Asai, K., and Imaizumi, Y. (2007). Characteristics of the ATP-Induced Ca2+-Entry Pathway in the T-BBEC 117 Cell Line Derived from Bovine Brain Endothelial Cells. J. Pharmacol. Sci. 104, 103–107. doi:10.1254/jphs.sc0070080

PubMed Abstract | CrossRef Full Text | Google Scholar

Yuan, Y., Kilpatrick, B. S., Gerndt, S., Bracher, F., Grimm, C., Schapira, A. H., et al. (2021). The Lysosomotrope GPN Mobilises Ca2+ from Acidic Organelles. J. Cel Sci 134. doi:10.1242/jcs.256578

CrossRef Full Text | Google Scholar

Zhang, J., Guan, X., Shah, K., and Yan, J. (2021). Lsm12 Is an NAADP Receptor and a Two-Pore Channel Regulatory Protein Required for Calcium Mobilization from Acidic Organelles. Nat. Commun. 12, 4739. doi:10.1038/s41467-021-24735-z

PubMed Abstract | CrossRef Full Text | Google Scholar

Zuccolini, P., Ferrera, L., Remigante, A., Picco, C., Barbieri, R., Bertelli, S., et al. (2022). The VRAC Blocker DCPIB Directly gates the BK Channels and Increases Intracellular Ca(2+) in Melanoma and Pancreatic Duct Adenocarcinoma (PDAC) Cell Lines. Br. J. Pharmacol., 1–18. doi:10.1111/bph.15810

CrossRef Full Text | Google Scholar

Zuccolo, E., Di Buduo, C., Lodola, F., Orecchioni, S., Scarpellino, G., Kheder, D. A., et al. (2018a). Stromal Cell-Derived Factor-1α Promotes Endothelial Colony-Forming Cell Migration through the Ca2+-dependent Activation of the Extracellular Signal-Regulated Kinase 1/2 and Phosphoinositide 3-Kinase/AKT Pathways. Stem Cell Develop. 27, 23–34. doi:10.1089/scd.2017.0114

PubMed Abstract | CrossRef Full Text | Google Scholar

Zuccolo, E., Kheder, D. A., Lim, D., Perna, A., Nezza, F. D., Botta, L., et al. (2019). Glutamate Triggers Intracellular Ca2+ Oscillations and Nitric Oxide Release by Inducing NAADP‐ and InsP 3 ‐dependent Ca2+ Release in Mouse Brain Endothelial Cells. J. Cel Physiol 234, 3538–3554. doi:10.1002/jcp.26953

CrossRef Full Text | Google Scholar

Zuccolo, E., Laforenza, U., Ferulli, F., Pellavio, G., Scarpellino, G., Tanzi, M., et al. (2018b). Stim and Orai Mediate Constitutive Ca2+ Entry and Control Endoplasmic Reticulum Ca2+ Refilling in Primary Cultures of Colorectal Carcinoma Cells. Oncotarget 9, 31098–31119. doi:10.18632/oncotarget.25785

PubMed Abstract | CrossRef Full Text | Google Scholar

Zuccolo, E., Lim, D., Kheder, D. A., Perna, A., Catarsi, P., Botta, L., et al. (2017). Acetylcholine Induces Intracellular Ca2+ Oscillations and Nitric Oxide Release in Mouse Brain Endothelial Cells. Cell Calcium 66, 33–47. doi:10.1016/j.ceca.2017.06.003

PubMed Abstract | CrossRef Full Text | Google Scholar