Spontaneous Calcium Oscillations Regulate Human Cardiac Progenitor Cell Growth

Abstract

Rationale

The adult heart possesses a pool of progenitor cells stored in myocardial niches but the mechanisms involved in the activation of this cell compartment are currently unknown.

Objective

Ca²⁺ promotes cell growth raising the possibility that changes in intracellular Ca²⁺ initiate division of c-kit-positive human cardiac progenitor cells (hCPCs) and determine their fate.

Methods and Results

Ca²⁺ oscillations were identified in hCPCs and these events occurred independently from coupling with cardiomyocytes or the presence of extracellular Ca²⁺. These findings were confirmed in the heart of transgenic mice in which EGFP was under the control of the c-kit-promoter. Ca²⁺ oscillations in hCPCs were regulated by the release of Ca²⁺ from the ER through activation of inositol 1,4,5-triphosphate receptors (IP3Rs) and the re-uptake of Ca²⁺ by the sarco/endoplasmic reticulum Ca²⁺ pump (SERCA). IP3Rs and SERCA were highly expressed in hCPCs while ryanodine receptors were not detected. Although Na⁺-Ca²⁺ exchanger, store-operated Ca²⁺-channels and plasma membrane Ca²⁺-pump were present and functional in hCPCs, they had no direct effects on Ca²⁺ oscillations. Conversely, Ca²⁺ oscillations and their frequency markedly increased with ATP and histamine which activated purinoceptors and histamine-1 receptors highly expressed in hCPCs. Importantly, Ca²⁺ oscillations in hCPCs were coupled with the entry of cells into the cell cycle and BrdUrd incorporation. Induction of Ca²⁺ oscillations in hCPCs prior to their intramyocardial delivery to infarcted hearts was associated with enhanced engraftment and expansion of these cells promoting the generation of a large myocyte progeny.

Conclusion

IP3R-mediated Ca²⁺ mobilization control hCPC growth and their regenerative potential.

The recognition that the adult heart in animals and humans possesses a pool of stem/progenitor cells^1-3 has raised the critical question concerning the mechanisms involved in the activation of this cell compartment and the modulation of cardiac homeostasis and repair. During the course of life before the manifestations of myocardial aging become apparent,⁴ dying parenchymal cells are continuously replaced by newly formed myocytes^5,6 through activation and commitment of quiescent cardiac progenitor cells (CPCs) stored in myocardial niches.^7,8 However, the signals responsible for the initiation of the cell cycle in CPCs, cardiomyocyte generation and preservation of the steady state of the organ are currently unknown. Calcium has two fundamental functions in the heart: it activates growth processes^9,10 and modulates the mechanical behavior of cardiomyocytes.^11,12 Critical for understanding physiological cell turnover and myocardial regeneration following injury is the identification of the mechanisms by which CPCs divide and acquire the myocyte phenotype. Changes of calcium levels in CPCs may occur and trigger a cascade of events that dictate their ultimate fate. Therefore, the objectives of the current study were: 1. to determine the pathways that regulate intracellular Ca²⁺ in human CPCs (hCPCs); 2. to establish whether Ca²⁺ oscillations in hCPCs condition cell replication; and 3. to assess whether Ca²⁺ oscillations are intrinsic to the cells or are triggered by interaction of hCPCs with cardiomyocytes. This cell-to-cell communication may favor the translocation of Ca²⁺ from myocytes to quiescent hCPCs initiating a cellular growth response. Moreover, transmembrane Ca²⁺ fluxes may contribute to rapid and transient rise in cytosolic Ca²⁺ promoting the entry of hCPCs into the cell cycle. These variables include a functional endoplasmic reticulum (ER) where Ca²⁺ is stored, the activity of ER channels that promote Ca²⁺ release, and the membrane systems modulating the exchange of Ca²⁺ with the extracellular compartment.

Methods

hCPCs were isolated from myocardial specimens obtained from patients who underwent cardiac surgery. Cytosolic Ca²⁺ levels in cultured hCPCs were measured utilizing the Ca²⁺ indicator Fluo-3 and two-photon microscopy. Cell proliferation in vivo and in vitro was evaluated by BrdUrd incorporation. Results are shown as mean±SEM. An expanded Materials and Methods section can be found in the online data supplement available at http://circres.ahajournals.org.

Results

Intracellular Ca²⁺ in hCPCs

Changes in [Ca²⁺]_i occur in excitable and non-excitable cells raising the possibility that a similar phenomenon is present in hCPCs and may have a functional role. Thus, hCPCs were loaded with the Ca²⁺ sensitive dye Fluo-3 and the intensity of the fluorescent signal was monitored over a period of ~30 minutes. During this interval, 79% hCPCs maintained stable levels of [Ca²⁺]_i while 21% displayed one or more consecutive Ca²⁺ oscillations. Repetitive events were restricted to a small percentage of cells and were comparable in amplitude and duration (Figure 1A through 1C). The fraction of hCPCs displaying Ca²⁺ oscillations increased with time up to 2 hours although the frequency of these episodes remained low (Figure 1D). These cells were all positive for the stem cell antigen c-kit (Supplemental Figure I). Ca²⁺ oscillations increased in hCPCs at the G₁-S phase transition but decreased at G₂-M (Figure 1 E and Supplemental Figure II).

Figure 1.

Intracellular Ca²⁺ in hCPCs. A, Cytosolic Ca²⁺ levels in a quiescent hCPC (upper trace) and in two hCPCs showing single (middle trace) and multiple (lower trace) Ca²⁺ oscillations. B, Distribution of Ca²⁺ oscillations, from 1 to more than 8, in hCPCs over a period of 33 min. C, Amplitude and duration of Ca²⁺ events in hCPCs. D, Ca²⁺ oscillations in hCPCs (Active Cells) analyzed for a period of 132 minutes. E, Ca²⁺ oscillations in hCPCs in control condition (Ctrl) and at the G₁-S and G₂-M transition.

Cell-to-Cell Interaction and Ca²⁺ Oscillations in hCPCs

The next objective was to establish whether Ca²⁺ oscillations in hCPCs are modulated at the single cell level or are mediated by adjacent cells. hCPCs are nested in myocardial niches and connexins are found between hCPCs and myocytes which operate as supporting cells.^3,7 These intercellular communications may account for the generation of Ca²⁺ oscillations in hCPCs, a process that is commonly observed in cardiomyocytes (Supplemental Figure III and Movie 1 in the online data supplement). Therefore, to identify the origin of Ca²⁺ oscillations in hCPCs, these cells were cultured alone or together with neonatal cardiomyocytes. Initially, dye transfer assays were performed^3,7,13 to document the formation of functional gap junctions between hCPCs. The fluorescent dye cascade blue was microinjected in individual hCPCs and found to rapidly migrate to neighboring cells through gap junction channels expressing connexin 43 (Figure 2A through 2E). However, the high molecular weight rhodamine-labeled dextran, injected simultaneously with cascade blue, failed to translocate to adjacent hCPCs. Additionally, DiI labeled hCPCs loaded with calcein were cultured with untreated cells. After ~12 hours, calcein was detected in unlabeled hCPCs structurally connected to DiI-calcein-positive cells (Supplemental Figure IV). Intracellular Ca²⁺ was then measured before and after exposure of hCPCs to the connexin hemi-gap-junction channel blocker octanol. Octanol did not affect the frequency and properties of Ca²⁺ oscillations in hCPCs (Figure 2F and 2G).

Figure 2.

Cell-to-cell interaction and Ca²⁺ oscillations. A-C, Cascade blue (blue) microinjected in a single hCPC (A) translocated spontaneously to adjacent cells (B and C). D, Rhodamine-labeled dextran (red), delivered simultaneously with cascade blue, remained confined to the injected cell. Scale bar: 20 μm. Cascade blue translocation was detected in 6 experiments. E, Connexin 43 (Cx43, white) is present between hCPCs (c-kit, green). Nuclei are stained by DAPI (blue). Scale bar: 20 μm. A group of cells is shown at higher magnification on the right panel. The inset shows Cx43 labeling in a myocyte [α-sarcomeric actin, (α-SA) red]. Scale bar: 10 μm. F, Intracellular Ca²⁺ in hCPCs before (left trace) and after exposure to octanol (right trace). G, Effects of uncoupling on Ca²⁺ oscillations.

Subsequently, the effect of Ca²⁺ cycling in myocytes on hCPC function was assessed by plating together DiI-labeled hCPCs and unlabeled cardiomyocytes. In spite of the presence of functional gap junctions between these two cell populations (Figure 3A and Supplemental Figure V), spontaneous or electrically stimulated Ca²⁺ transients in myocytes had no detectable consequence on [Ca²⁺]_i of hCPCs (Figure 3B and 3C). In fact, Ca²⁺ oscillations in hCPCs persisted in the presence of cadmium which abolished Ca²⁺ transients in myocytes.

Figure 3.

Ca²⁺ cycling in myocytes and CPCs. A, Cx43 (white) between DiI-labeled hCPCs (DiI, red) and myocytes (α-SA, green). Nuclei are identified by DAPI. Scale bars: 10 μm. B, Intracellular Ca²⁺ in hCPCs (blue traces) and adjacent co-cultured neonatal myocytes (black traces). The effects of electrical stimulation and cadmium chloride are also shown. Different cells were used in the three conditions. C, Intracellular Ca²⁺ in hCPCs and neonatal myocytes in line scan mode. Red identifies hCPCs loaded with DiI and green corresponds to Fluo-3; scale bar: 20 μm. D, Cytosolic Ca²⁺ in a quiescent mCPC (upper trace) and two mCPCs showing a single (middle trace) and multiple (lower trace) Ca²⁺ oscillations. E, Properties of Ca²⁺ oscillations in hCPCs and mCPCs. E, c-kit-EGFP mouse heart loaded with Rhod-2 (red), stimulated at 1 Hz and analyzed in line-scan mode (arrows). CPCs were identified by EGFP (green). G, Ca²⁺ transients in myocytes (red trace) did not affect Ca²⁺ levels in the neighboring EGFP-positive CPC (green trace). Identical results were obtained in 7 other experiments. Scale bars: 20 μm.

Cell-to-Cell Interaction and Ca²⁺ Oscillations in Mouse CPCs

To strengthen these in vitro results, a transgenic mouse model in which EGFP was under the control of the c-kit promoter¹⁴ was employed to test whether Ca²⁺ cycling in myocytes triggers Ca²⁺ oscillations in CPCs in situ within the myocardium. Preliminary studies were conducted to evaluate whether EGFP-positive mouse CPCs (mCPCs) in vitro showed spontaneous Ca²⁺ oscillations, mimicking the behavior of hCPCs. The percentage of mCPCs exhibiting Ca²⁺ oscillation was comparable to that measured in human cells. Similarly, the rate of these events and their duration did not differ in these two cell classes while the amplitude was larger in mCPCs (Figure 3D and 3E).

The heart of these transgenic mice was then examined ex vivo by two-photon microscopy^3,13 following perfusion of the coronary circulation with the Ca²⁺ indicator Rhod-2. The possibility that EGFP may interfere with the detection of Rhod-2 was excluded in preliminary studies conducted in EGFP-positive mouse myocytes (Supplemental Figure VI). Based on these observations, the mouse heart was stimulated at 1 Hz and the Ca²⁺ levels in EGFP-positive mCPCs were found not to be affected by the changes in Ca²⁺ transients in neighboring cardiomyocytes (Figure 3F and 3G). Thus, human and mouse CPCs appear to possess an intracellular Ca²⁺ regulatory system which is independent from that of terminally-differentiated parenchymal cells.

Intracellular Ca²⁺ Control in hCPCs

We then determined whether activation of inositol 1,4,5-triphosphate receptors (IP3Rs) and/or ryanodine receptors (RyRs) resulted in the release of Ca²⁺ from the ER and Ca²⁺ oscillatory events. Moreover, sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) is responsible for the re-uptake of Ca²⁺ into the ER and restoration of Ca²⁺ stores.^10,11 Before conducting the functional studies, quantitative RT-PCR and immunolabeling were employed to document in hCPCs the presence of transcripts and proteins for IP3Rs and SERCA. Both IP3Rs and SERCA were highly expressed in hCPCs. However, RyRs were not identified in these cells (Figure 4A through 4D).

Figure 4.

Ca²⁺ regulatory proteins in hCPCs. A-D, Expression at the mRNA (A) and protein (B-D) levels of the components of the ER that are implicated in Ca²⁺ homeostasis. Myocytes were used as positive control for RyRs. Human heart (hHeart) was used as positive control. Scale bars: 20 μm. E, Repetitive Ca²⁺ oscillations in hCPCs in the presence of IP3R agonist. F-H, Ca²⁺ oscillations in hCPCs at baseline (Tyrode) and in the presence of activation (F) and inhibition (G and H) of IP3R function. Xestopsongin-C, XeC. I-N, Ca²⁺ in hCPCs in the presence of modulators of SERCA (I and J) and RyRs (K-N). *P<0.05 vs. Tyrode.

Subsequently, IP3 binding to IP3Rs was enhanced by thimerosal and this intervention markedly increased the number of hCPCs displaying Ca²⁺ oscillations and the frequency of Ca²⁺ oscillatory episodes per cell (Figure 4E and 4F). An opposite response was observed by inhibition of IP3R function with 2-amino-ethoxydiphenyl borate (2-APB) or xestospongin-C or attenuation of IP3 formation by phospholipase-C (PLC) blockade with U-73122 (Figure 4G and 4H). Similarly, inhibition of SERCA with cyclopiazonic acid (CPA) reduced the fraction of activated hCPCs (Figure 4I and 4J). Additionally, Ca²⁺ oscillations were smaller in amplitude and prolonged in duration, pointing to SERCA as a relevant component of hCPC function. Consistent with the lack of identify RyRs in hCPCs, their agonists ryanodine and caffeine had no effects on Ca²⁺ oscillations; the number of active cells and the frequency, amplitude and duration of Ca²⁺ events did not differ from baseline (Figure 4K through 4N). These data point to the IP3-IP3R system and SERCA as the predominant modulators of Ca²⁺ in hCPCs.

ATP-specific P2-purinoceptors (P2Y2) and histamine (H1) receptors are G_q-protein coupled receptors that are implicated in the release of intracellular Ca²⁺ from the ER by activation of PLC, IP3 synthesis and, ultimately, IP3R binding.^15,16 P2Y2 and H1 receptors were expressed at the mRNA and protein level in hCPCs (Figure 5A through 5C), suggesting that they may be implicated in Ca²⁺ cycling of these progenitor cells. In the presence of ATP or histamine, a more than 3-fold increase in the number of hCPCs exhibiting oscillations was detected. Similarly, the number of events per cell markedly increased while the amplitude and duration of Ca²⁺ elevations did not change (Figure 5D through 5G). Inhibition of IP3Rs or IP3 formation prevented the effects of ATP and histamine on Ca²⁺ mobilization (Supplemental Figure VII).

Figure 5.

G_q-protein coupled receptors and intracellular Ca²⁺ in hCPCs. A-C, Expression at the mRNA (A) and protein (B and C) levels of P2Y2 and H1 receptors in hCPCs. Human heart (hHeart) was used as positive control. Scale bars: 10 μm. D-G, Ca²⁺ oscillations in hCPCs in the presence of ATP (D and E) or histamine (F and G). *P<0.05 vs. Tyrode.

Since PLC-β3 may be modulated by P2Y2 receptor activation,¹⁷ the expression of PLC-β subunits was evaluated in hCPCs. Additionally, the functional role of PLC-β3 in mediating intracellular Ca²⁺ mobilization upon ATP stimulation was established. PLC-β3 mRNA in hCPCs was significantly higher than that of the other subunits (Supplemental Figure VIII, A through C). Thus, siRNA strategy was employed to downregulate PLC-β3 in hCPCs prior to their stimulation with ATP. This intervention markedly attenuated the ability of ATP to dramatically increase the pool of hCPCs displaying Ca²⁺ oscillations (Supplemental Figure VIII, D and E).

Although IP3R-mediated Ca²⁺ mobilization from the ER is responsible for the generation of Ca²⁺oscillations in hCPCs, transmembrane Ca²⁺ fluxes maybe operative contributing to intracellular calcium cycling. Exposure of hCPCs to Ca²⁺ free medium did not alter the frequency and characteristics of Ca²⁺ oscillations (Supplemental Figure IX), strengthening the role of intracellular stores as the source of Ca²⁺ for oscillatory events. Immunolabeling and PCR data, together with patch clamp and cytosolic Ca²⁺ imaging (Supplemental Figures X and XI), revealed that store operated channels (SOC), Na⁺-Ca²⁺ exchanger (NCX) and plasma membrane Ca²⁺ pump (PMCA) were functional in hCPCs. However, these systems did not appear to participate in the generation of Ca²⁺ oscillations in these primitive cells.

Ca²⁺ Oscillations and hCPC Growth

To evaluate the functional import of Ca²⁺ oscillations in hCPC replication, these cells were cultured in serum free medium for 24 hours and were exposed to either ATP or histamine to induce Ca²⁺ release from the ER and oscillatory events. BrdUrd was added to the medium and its incorporation in hCPCs was measured 24 hours later. To exclude the potential confounding effect of cell death on the evaluation of cell proliferation, apoptosis was also determined. With either ATP or histamine, a nearly 2-fold increase in BrdUrd labeling of hCPCs was detected (Figure 6A and 6B). Conversely, inhibition of Ca²⁺ release from the ER led to a decrease in BrdUrd incorporation in hCPCs to values lower than those seen at baseline (Figure 6A through 6C). Moreover, purinergic stimulation failed to promote proliferation of hCPCs when PLC-β3 was downregulated and the P2Y2-IP3R axis was disrupted (Supplemental Figure XII). ATP and histamine had no influence on hCPC apoptosis (Figure 6D and 6E), suggesting that increases in Ca²⁺ oscillations were not coupled with the activation of the cell death program.

Figure 6.

hCPC growth and apoptosis. A-C, ATP and histamine increase Ca²⁺ oscillations and proliferation of hCPCs. Inhibitors of Ca²⁺ oscillations prevent the effects of ATP and histamine. Control, Ctrl; histamine, His. *P<0.05 vs. Ctrl, **P<0.05 vs. agonist. D and E, Apoptosis of hCPCs measured by Annexin V labeling and FACS. PI, propidium iodide. Q2, late apoptotic or necrotic cells; Q3, alive cells; Q4, cells undergoing apoptosis. F and G, IGF-1R transcript and protein in hCPCs and hHeart. Scale bar: 20 μm. Right panel in G illustrates selected cells at higher magnification. H and I, Intracellular Ca²⁺ in hCPCs exposed to IGF-1. *P<0.05 vs. Tyrode. J, Proliferation of hCPCs in the presence of IGF-1 alone or in combination with inhibitors of Ca²⁺ oscillations. *P<0.05 vs. Ctrl, ** P<0.05 vs. IGF-1.

To strengthen the possibility of a cause and effect relationship between Ca²⁺ oscillations and cell cycle activation in hCPCs, the impact of a well-established activator of progenitor cell division, insulin-like growth factor-1 (IGF-1) was determined. Resident cardiac progenitors express IGF-1 receptors (Figure 6F and 6G) and synthesize and secrete the ligand.⁶ IGF-1 increased the percentage of hCPCs showing Ca²⁺ oscillations by nearly 3-fold (Figure 6H and 6I). Similarly, there was an increase in the number of Ca²⁺ oscillations per cell while the amplitude and duration of Ca²⁺ events remained essentially constant. When Ca²⁺ oscillations were blocked, the growth promoting effects of IGF-1 on hCPCs were completely prevented. IGF-1 increased hCPC proliferation by ~2-fold and blockade of Ca²⁺ release from the ER decreased cell replication to levels below baseline values (Figure 6J). Similarly, Ca²⁺ oscillations mediated by IGF-1 were abrogated by PLC and IP3R antagonists (supplemental Figure XIII).

To establish whether Ca²⁺ oscillations in hCPCs favor the acquisition of the myocyte lineage, these cells were cultured in differentiating medium³ in the absence or presence of ATP or histamine. After one week, the fraction of primitive cells expressing α-sarcomeric actin was comparable in stimulated and non-stimulated hCPCs (Supplemental Figure XIV), suggesting that these agents did not impact on the differentiation of hCPCs into the myocyte phenotype.

Ca²⁺ Oscillations in hCPCs and Myocardial Regeneration

In both animals and humans, shortly after ischemic myocardial injury, there is an increase of resident progenitors mostly restricted to the border zone of the infarcted heart.¹ These cells rapidly acquire the myocyte lineage and result in small foci of cardiac repair.¹⁸ The possibility that factors naturally released in the damaged area facilitate this process was confirmed here in our transgenic mouse model in which EGFP-labeling made rather easy the identification of c-kit-positive CPCs. Myocardial infarction at 2 days led to a 15-fold and 5-fold increase in CPCs in the region bordering and remote from the infarction, respectively (Supplemental Figure XV).

However, spontaneous regeneration is severely limited and only a minimal fraction of a variety of progenitor cells delivered to the infarcted myocardium survives and integrates in the unfavorable environment of the necrotic tissue.^13,19 Activation of hCPCs with ATP and histamine may enhance their engraftment, growth and formation of a myocyte progeny. EGFP-labeled hCPCs were exposed to ATP or histamine 30 min prior to their injection in the area bordering an acute infarct in immunosuppressed mice. Untreated hCPCs were used as control. All cell preparations were serum-starved for 24 hours before ATP or histamine exposure. Animals were examined 2 days later when cell engraftment is completed, cell death is markedly attenuated and the number of cells available for cardiac repair is established.^13,20 Mice were exposed to BrdUrd to obtain cumulative values of cell regeneration. Additional groups of mice were sacrificed at 7 days to asses the impact of this protocol on myocardial regeneration and cardiac function.

Of the 60 × 10³ hCPCs injected in each heart, 7,000 EGFP-positive cells were found in control hearts while nearly 19,000 cells (P<0.002) were detected in hearts in which hCPCs were treated with ATP or histamine. Cell engraftment was confirmed by the detection of connexin 43 and N-cadherin at the interface of hCPCs and spared myocytes (Supplemental Figure XVI). Moreover, the number of EGFP-positive cells labeled by BrdUrd was 10-12-fold higher in infarcts injected with ATP or histamine activated hCPCs (Figure 7A through 7C). Most importantly, the aggregate number of EGFP-positive cells expressing the myocyte transcription factors Nkx2.5 was ~4-fold larger in hearts treated with cells exposed to ATP or histamine.

Figure 7.

Ca²⁺ oscillations and growth of hCPCs in vivo. A, EGFP-positive hCPCs 48 hours after implantation in the infarcted mouse heart under control conditions and following activation of hCPCs with ATP or histamine. Proliferation of EGFP-positive cells (green) is documented by BrdU labeling (magenta, arrows). B, Nkx2.5 (white) is present in several EGFP-positive cells (arrows). Scale bars: 20 μm. C, Results are shown as mean±SEM. *P<0.05 vs. Ctrl.

Seven days after coronary artery occlusion and cell implantation, the extent of myocardial regeneration associated with the delivery of activated hCPCs was markedly superior to that obtained with untreated cells. The number of newly formed cardiomyocytes with ATP-histamine treatment was ~3-fold greater than in controls. However, the volume of cardiomyocytes was similar in the two groups (Figure 8A and 8B). Importantly, LV hemodynamics revealed that pre-treatment of hCPCs with ATP or histamine enhanced the effects of cell transplantation and positively interfered with the deterioration of cardiac function following myocardial injury (Figure 8 C).

Figure 8.

Myocardial regeneration by activated hCPC. A, Mouse heart treated with histamine-stimulated hCPCs. The mid-portion of the infarct is replaced by EGFP-positive (upper panel, green) α-SA-positive cardiomyocytes (lower panel, red). The area in the rectangle is shown at higher magnification in the inset. B, Extent of regeneration mediated by hCPCs non activated (Ctrl hCPCs) or exposed to ATP or histamine (ATP-His hCPCs). C, LV function in sham operated (SO), infarcted untreated (MI + PBS) and hCPC-treated (MI + hSCPs) mice 7 days after coronary ligation. Ctrl, ATP and His identify non-stimulated, ATP-stimulated and histamine-stimulated hCPCs, respectively. LVEDP, LV end-diastolic pressure; LVDevP and +dP/dt. *P<0.05 vs. SO, **P<0.05 vs. MI + PBS, † P<0.05 vs. MI injected with untreated hCPCs.

Discussion

The results of the current study indicate that hCPCs display spontaneous elevations in intracellular Ca²⁺ due to IP3R-mediated Ca²⁺ release from the ER. Re-uptake of Ca²⁺ into the ER is accomplished by SERCA which replenishes the Ca²⁺ stores allowing repetitive oscillations with preserved amplitude and duration. The Ca²⁺ handling molecules NCX, PMCA and SOC are functional and contribute to Ca²⁺ homeostasis in hCPCs, but are not implicated in the initiation and incidence of Ca²⁺ oscillations in these undifferentiated cells. Agonists of G_q-protein coupled receptors and histamine and ATP stimulate PLC and IP3 formation leading to an increase in the number of activated hCPCs and frequency of Ca²⁺ oscillatory episodes per cell in vitro. These Ca²⁺ oscillations promote hCPC proliferation, documenting that cytosolic Ca²⁺ plays a primary role in hCPC growth. Induction of Ca²⁺ oscillatory events in hCPCs prior to their intramyocardial delivery in vivo was coupled with enhanced the engraftment of these cells within the infarcted heart, their expansion in the unfavorable environment of the necrotic tissue and the generation of a myocyte progeny.

Origin of Ca²⁺ Oscillations in hCPCs

In the present study, a fundamental issue in need of resolution involved the recognition whether Ca²⁺ oscillations in hCPCs represent an intrinsic property of these primitive cells or the consequence of Ca²⁺ entry from cardiomyocytes and/or the extracellular compartment. Collectively, our results on the regulation of Ca²⁺ in hCPCs in vitro and in mouse CPCs within the myocardium in ex vivo preparations, suggest that cell-to-cell communication and the interstitial milieu are not implicated in the rapid and transient elevations of Ca²⁺ in CPCs. Under our experimental conditions, Ca²⁺ cycling in myocytes appears to have no influence on Ca²⁺ oscillations in CPCs. However, changes in the rate and amplitude of Ca²⁺ transients in myocytes may affect Ca²⁺ loading in CPCs. Cardiomyocytes function as supporting cells in myocardial niches⁷ and are connected by gap and adherens junctions to CPCs making them the ideal candidate for the translocation of Ca²⁺ and the initiation of oscillatory processes in CPCs. Although this was not found to be the case, this phenomenon may occur later during differentiation; the intercellular passage of Ca²⁺ may activate in lineage committed CPCs the release of Ca²⁺ from the ER conditioning the acquisition of the adult cardiomyocyte phenotype and contractile function.

The most significant regulator of IP3R-mediated Ca²⁺ release from the ER is Ca²⁺ itself. IP3R open channel probability is stimulated at low [Ca²⁺]_i while high [Ca²⁺]_i exerts an inhibitor effect.¹⁶ Accordingly, changes in [Ca²⁺]_i may initiate and end Ca²⁺ oscillations in hCPCs. IP3 regulates IP3R channels mainly by enhancing their sensitivity to Ca²⁺. Two receptor ligand systems, P2Y2-ATP and H1-histamine, were identified in hCPCs and their importance in the modulation of intracellular Ca²⁺ and progenitor cell growth was defined in vitro and in vivo to characterize their potential function in cardiac homeostasis and regeneration. Importantly, the doses of ATP and histamine employed here have previously been shown to exert a powerful effect on Ca²⁺ mobilization in other cell systems.^21,22

In physiological conditions, cell loss by normal wear and tear may result in an increase in the local level of ATP¹⁵ leading to Ca²⁺ oscillations in neighboring hCPCs which in turn activate cell replication and expansion. Additionally, the release of ATP from synaptic vesicles in terminal nerves¹⁵ may produce a comparable role in the hCPC compartment. These two mechanisms of ATP accumulation in the interstitial space are enhanced by prolonged myocardial ischemia and myocyte death.^15,23 Cardiac pathology potentiates the load on the spared myocardium and mechanical stretch further enhances exocytosis of ATP from cardiomyocytes.¹⁵ In humans and animals, myocardial infarction is associated with CPC division and the generation of functionally competent cardiomyocytes,¹ supporting the notion that ATP-mediated CPC growth may be critical for cardiac repair.

Mast cells are the predominant source of histamine in the myocardium.²⁴ The number of mast cells and CPCs is comparable in the rodent and human heart.^3,7,25,26 Additionally, tissue damage and inflammation recruit mast cells and CPCs, suggesting that histamine released from mast cells may be implicated in the activation of cardiac progenitors and the creation of myocytes. ATP is formed largely by cardiomyocytes that represent ~85% of the myocardium while histamine is synthesized by a very small number of mast cells, ~2-3/mm² of tissue.^15,25,26 However, extracellular ATP is rapidly degraded to inactive ADP. Conversely, histamine has a longer half-life time,^15,27 indicating that these two molecules may have complementary function in the modulation of hCPC function.

As shown here for hCPCs, IP3R-mediated Ca²⁺ mobilization from the ER has been reported in human mesenchymal stem cells²⁸ and mouse embryonic stem cells.^29,30 In both cases, the increases in intracellular Ca²⁺ have been linked to cell growth and lineage specification. Embryonic stem cells and hCPCs differentiate into cardiomyocytes and the characteristics of Ca²⁺ cycling in these cells is regulated by the Ca²⁺-induced Ca²⁺-release mechanism that controls myocyte mechanics and ventricular function in vivo.¹¹ Whether mesenchymal stem cells have a similar capacity is currently debatable.

IGF-1 and Ca²⁺ Oscillations in hCPCs

The function of IGF-1 is largely mediated by binding to the receptor tyrosine kinase IGF-1R. Phosphorylation of IGF-1R leads to recruitment of the insulin receptor substrate protein 1 (IRS-1) that modulates the effects of IGF-1R on cellular responses in the heart. The recruitment of IRS-1 upregulates PI3 kinase which phosphorylates Akt; Akt activation favors cell differentiation, hypertrophy or proliferation.^31,32 IRS-1 also promotes the interaction of Ras, Raf and ERK which may lead to cellular hypertrophy or division.³³ Surprisingly, in the current study, IGF-1 induced Ca²⁺ oscillatory episodes in hCPCs through the activation of IP3Rs and the release of Ca²⁺ from the ER. Whether this was a direct effect or was mediated by the generation of IP3 is currently unclear. However, the mitogenic properties of IGF-1 appear to be mediated, at least in part, by the release of Ca²⁺ from the ER, strengthening the notion that Ca²⁺ mobilization via IP3R is involved in cell cycle progression and growth of hCPCs.

Ca²⁺ Oscillations and hCPC In Vivo

A critical component of cell therapy is related to the recognition of the variables implicated in the engraftment and expansion of the delivered cells within the damaged myocardium.³⁴ The function of progenitor cells is determined by causes inherent to the cells and risk factors for cardiovascular diseases. The former includes the telomere-telomerase axis, DNA damage and the expression of genes implicated in the forced entry of cells into in an irreversible quiescent state and/or activation of the endogenous cell death program.^6,35 The latter involves several pathologic states such as diabetes, hypertension, coronary artery disease, valvular defects, dilated cardiomyopathy and myocardial aging.³⁴

Non-standard Abbreviations and Acronyms

CPA

cyclopiazonic acid

CPCs

cardiac progenitor cells

H1

histamine receptor-1

hCPCs

human cardiac progenitor cells

His

histamine

IP3

inositol 1,4,5-triphosphate

IP3Rs

inositol 1,4,5-triphosphate receptors

NCX

Na⁺-Ca²⁺ exchanger

PMCA

plasma membrane Ca²⁺ pump

P2Y2

P2-purinoceptors

RyRs

ryanodine receptors

SERCA

sarco/endoplasmic reticulum Ca²⁺ pump

SOC

store operated channels

XeC

xestospongin-C

2-APB

2-aminoethyl diphenylborinate

BrdUrd

5-bromodeoxyuridine

CPC

cardiac progenitor cell

Dil

1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine

EGFP

enhanced green fluorescent protein

ER

endoplasmic reticulum

IGF

insulin-like growth factor

IGF-1R

insulin-like growth factor-1 receptor

IRS

insulin receptor substrate protein

LV

left ventricular

mCPC

mouse cardiac progenitor cell

PLC

phosphohpase-C