Inositol 1, 4, 5-Triphosphate Receptors and Human Left Ventricular Myocytes

Abstract

Background

Little is known concerning the function of inositol 1,4,5-triphosphate receptors (IP3Rs) in the adult heart experimentally. Moreover, whether these Ca²⁺ release channels are present and play a critical role in human cardiomyocytes remains to be defined. IP3Rs may be activated following Gαq-protein-coupled receptors (GPCR) stimulation affecting Ca²⁺ cycling, enhancing myocyte performance and, potentially, favoring an increase in the incidence of arrhythmias.

Methods and Results

IP3R function was determined in human left ventricular (LV) myocytes and this analysis was integrated with assays in mouse myocytes to identify the mechanisms by which IP3Rs influence the electrical and mechanical properties of the myocardium. We report that IP3Rs are expressed and operative in human LV myocytes. Following GPCR activation, Ca²⁺ mobilized from the sarcoplasmic reticulum via IP3Rs contributes to the decrease in resting membrane potential, prolongation of the action-potential, and occurrence of early after-depolarizations. Ca²⁺ transient amplitude and cell shortening are enhanced, and extra-systolic and dysregulated Ca²⁺ elevations and contractions become apparent. These alterations in the electromechanical behavior of human cardiomyocytes are coupled with increased isometric twitch of the myocardium and arrhythmic events, suggesting that GPCR activation provide inotropic reserve, which is hampered by electrical instability and contractile abnormalities. Additionally, our findings support the notion that increases in Ca²⁺ load by IP3Rs promote Ca²⁺ extrusion by forward mode Na⁺/Ca²⁺ exchange, an important mechanism of arrhythmic events.

Conclusions

Thus, the GPCR/IP3R axis modulates the electromechanical properties of the human myocardium and its propensity to develop arrhythmias.

Myocyte function relies on Ca²⁺ entry from the extracellular space and its release from the sites of storage in the sarcoplasmic reticulum (SR), the latter being mediated by activation of ryanodine receptors (RyRs).¹ Following membrane depolarization, opening of the RyRs is triggered by Ca²⁺ influx through voltage-activated L-type channels, allowing translocation of Ca²⁺ to the cytoplasm where myofilament cross-bridge formation initiates cell shortening.^1,2 Relaxation is promoted by a reduction in cytosolic Ca²⁺ through its re-uptake in the SR by the sarco-endoplasmic Ca²⁺ pump (SERCA), and Ca²⁺ extrusion via the Na⁺/Ca²⁺ exchanger (NCX). Dysfunctional RyRs result in spontaneous Ca²⁺ release or enhanced Ca²⁺ leak, pathologies found in inherited and acquired arrhythmogenic diseases,^3–7 strengthening the notion that defects in Ca²⁺ homeostasis impact on the electrical properties of the heart.

The release of Ca²⁺ from the endoplasmic reticulum via inositol 1,4,5-triphosphate receptors (IP3Rs) promotes spontaneous Ca²⁺ oscillations in human cardiac stem cells (hCSCs), inducing cell cycle reentry and modulating the fate of these cells, following their injection in the infarcted heart of immunosuppressed mice.⁸ Additionally, modulation of Ca²⁺ signals via IP3Rs controls apoptosis and lineage commitment of differentiating embryonic stem cells,⁹ a mechanism which may be operative in cardiomyogenesis. Although limited data in rodents and rabbits suggest that IP3Rs are expressed in left ventricular (LV) myocytes,^10,11 whether the presence of IP3Rs in resident hCSCs is preserved in the derived human myocyte progeny,^12,13 playing a role in Ca²⁺ homeostasis remains to be defined.

Impaired IP3R-Ca²⁺ signaling and alterations of the calcineurin-NFAT pathway lead to developmental defects and abrogate cardiac hypertrophy induced by isoproterenol or angiotensin II.^14,15 Moreover, initial observations raise the possibility that enhanced IP3R activity may increase Ca²⁺ transient amplitude, the incidence of extra-systolic Ca²⁺ elevations and the propensity for arrhythmias.¹¹ Thus, the hypothesis may be raised that abnormalities in Ca²⁺ transients in the presence of Gαq-protein-coupled receptors (GPCR) agonists may involve IP3R-mediated Ca²⁺ mobilization, cytoplasmic Ca²⁺ overload, perturbations in transmembrane potential, and/or sensitization of RyRs, leading to elementary release of Ca²⁺ and electrical instability.

Ca²⁺ mobilized by IP3Rs relies on intracellular levels of IP3, which is produced by phospholipase C (PLC). GPCR agonists promote PLC function that generates IP3 and diacylglycerol (DAG). IP3 stimulates IP3Rs and Ca²⁺ translocation from intracellular stores,^8,16,17 whereas DAG activates transient receptor potential channels (TRPCs),¹⁸ and protein kinase C (PKC) isoforms, phosphorylating a wide spectrum of ion channels and contractile proteins.^19,20,21 Additionally, GPCR ligands may initiate PLC-independent signaling mechanisms, modulating transmembrane ionic fluxes.^21,22 Thus, whether IP3Rs contribute to the functional consequences of GPCR stimulation in cardiomyocytes remains to be elucidated.

Based on this premise, we have investigated the role of IP3Rs in human LV myocytes obtained from normal donor hearts not used from transplantation. This analysis in human cells was complemented with a number of assays in mouse myocytes to define the role of IP3Rs in Ca²⁺ mobilization, electrical activity, arrhythmic events, and myocyte contractile behavior following GPCR stimulation.

Methods

An expanded description of the methods is available in the online-only Data Supplement.

Human Hearts and Myocyte Isolation

Donor hearts not used for transplantation and explanted failing hearts were utilized in this study (Table 1 in the online-only Data Supplement). The protocol was approved by the Institutional Review Board (#2010P002475). Donor and failing human hearts were shipped in cold cardioplegia solution, and were processed immediately upon receipt.

Myocardial samples, obtained at autopsy from patients who died from causes other than cardiovascular diseases, were fixed in formalin and used as controls for the quantitative evaluation of myocyte apoptosis by the TdT assay.¹² As previously performed in our laboratory,^12,13,23 large LV myocardial samples were used; a branch of the left main coronary artery was cannulated and the distal arteries were ligated. Initially, the tissue was perfused with a solution containing (mmol/L): NaCl 126, KCl 4.4, MgCl₂ 5, HEPES 5, glucose 22, taurine 20, creatine 5, Na pyruvate 5, NaH₂PO₄ 5, and 2,3-butanedione monoxime 10 (pH 7.4, adjusted with NaOH). A constant temperature of 37°C was maintained, and the buffer was gassed with 85% O₂ and 15% N₂. After ~10 minutes, 0.015 mmol/L CaCl₂, 274 units/ml collagenase (type 2, Worthington Biochemical Corp), and 0.57 units/ml protease (type XIV, Sigma) were added to the perfusate for enzymatic dissociation of the tissue. At completion of digestion, the myocardium was cut in small pieces, subjected to repeated pipetting to obtain a single cell preparation and re-suspended in Ca²⁺ 0.015 mmol/L solution. Aliquots of cell suspensions were centrifuged for 5 minutes at 20g, and myocytes were fixed in 4% paraformaldehyde, or frozen for immunocytochemistry and biochemical assays. For electrophysiological and mechanical studies only rod-shaped myocytes exhibiting cross striations, and showing no spontaneous contractions or contractures were selected; cells were used within 12 hours following organ acquisition.

Mouse Hearts and Myocyte Isolation

C57Bl/6 mice were maintained in accordance with the Guide for Care and Use of Laboratory Animals, and all animal experiments were approved by the local animal care committee (IACUC). For myocyte isolation, a protocol similar to that described above was employed.^24–26 Antibodies for FACS analysis, and primers for PCR studies are listed in Table 2 and Table 3 in the online-only Data Supplement.

Cell Shortening, Ca²⁺ Transients, and Patch-Clamp Studies

Isolated LV myocytes were placed in a bath located on the stage of an Axiovert (Zeiss), IX51 and IX71 (Olympus) inverted microscopes for measurements of contractility, Ca²⁺ transients and patch-clamp studies.^24–27 Experiments were performed at room temperature.

Data Analysis

The magnitude of sampling employed in each measurement is listed in Table 1 in the online-only Data Supplement. Data are presented as mean±SEM. Independent samples were compared using ANOVA analysis with Bonferroni correction or Fisher’s exact tests, as appropriate for continuous or categorical responses. The normality and homogeneity of variance of the data were checked first to meet the assumptions of ANOVA. Paired observations were evaluated with Wilcoxon signed-rank sum test or paired t-test as appropriate. For data with multiple measurements (myocytes) from the same donor (mouse/patient), linear mixed-effects models with patient/mouse as random effect, main effects for treatment group were applied to examine and compare response changes. The compound symmetry covariance matrix was specified to account for dependency among observations. Differences in mean changes between assessments were compared between groups and within each group by LSMEANS statement. Throughout, a P<0.05 was considered statistical significance and all tests were two sided with a type I error of 0.05. The statistical analyses were performed using SAS version 9.3 (SAS Institute).²⁸

Results

Human Hearts

Human cardiomyocytes were obtained from the LV of 34 donor hearts; 14 males and 20 females with an average age of 44±4 years (Table 1 in the online-only Data Supplement). These hearts were declined for transplantation. In donor hearts, foci of replacement fibrosis and areas of diffuse interstitial fibrosis were not detected histologically.²³ Similarly, the coronary vessels had minimal atherosclerotic lesions with essentially no reduction in luminal diameter. Inflammatory infiltrates were not identified. Myocyte apoptosis was comparable in three randomly selected donor hearts and age-matched control hearts obtained at autopsy from patients who died from causes other than cardiovascular diseases (Figure 1 in the online-only Data Supplement). Thus, myocyte survival in donor hearts was not affected by the preservation protocol. End-stage failure in explanted hearts was of ischemic (n=6) and non-ischemic (n=9) origin.

IP3Rs, Ca²⁺ Transients, and Human Myocyte Contractility

Molecular assays were conducted in isolated myocyte preparations with minimal levels of contamination from fibroblasts, endothelial cells and smooth muscle cells (Figure 2 in the online-only Data Supplement). Transcripts for the three IP3R subtypes were identified by qRT-PCR in human LV myocytes. The expression of IP3R type-2 was also confirmed by Western blotting. Similarly, the presence of IP3Rs in mouse LV myocytes was documented by both methodologies (Figure 1 and Figures 3 and 4 in the online-only Data Supplement).

Figure 1.

IP3Rs in human and mouse cardiomyocytes. A, qRT-PCR show transcripts for the three IP3R subtypes in human and mouse LV myocytes. Representative curves and PCR products are shown together with the mRNA obtained from the human and mouse brain, that was used as positive control (red curves). The PCR products had the expected molecular weight. Human β2-microglobulin: housekeeping gene (see Figure 3 in the online-only Data Supplement). MW: molecular weight. For sequences see Figure 4 in the online-only Data Supplement. B, Expression of IP3R protein by Western blotting in human and mouse LV myocytes with IP3R-2 and pan-IP3R antibodies, respectively. GAPDH: loading condition.

The effect of IP3R activation on Ca²⁺ cycling and contractility of human LV myocytes was investigated by stimulating GPCRs with adenosine 5′-triphosphate (ATP) and endothelin-1 (ET-1), which both enhance the synthesis of IP3.^{8,10,11,14,17,29–31} ATP increased myocyte contractility, and this response was attenuated with the unspecific IP3R blocker 2-APB (Figure 2A). The enhanced cell performance mediated by GPCR activation was coupled with an increase in Ca²⁺ transient amplitude. Inhibition of IP3Rs with the selective blocker xestospongin-C (XeC) did not modify baseline myocyte mechanics, but abrogated the changes mediated by GPCR stimulation (Figure 2B and 2C). The increase in contractility with these agonists led to episodes of after-contractions during relaxation in 27% of human cardiomyocytes; however, spontaneous and sustained Ca²⁺ elevations were observed rarely (Figure 2D–2F). The effects of GPCR activation on cardiomyocytes were equally present in myocardial trabeculae dissected from the LV wall. ATP and ET-1 enhanced isometric developed tension, which was abolished by IP3R antagonists (Figure 2G and 2H). Thus, IP3R inhibition in human cardiomyocytes and myocardium attenuates the positive inotropic action of GPCR stimulation.

Figure 2.

IP3Rs, Ca²⁺ cycling, and contractility in human cardiomyocytes. A, Sarcomere shortening in a field stimulated human cardiomyocyte, exposed first to ATP (10 μmol/L) and then to the IP3R blocker 2-APB (10 μmol/L). Higher time resolution traces are shown in the inset. B, Ca²⁺ transients (red-traces) and sarcomere shortening (black-traces) in a field stimulated cardiomyocyte before (Tyrode) and after exposure to ET-1 (100 nmol/L). C, Quantitative data for Fluo-loaded cells are shown as mean±SEM. Tyr: Tyrode; XeC: xestospongin-C (2 μmol/L). *P<0.05 versus Tyr, †P<0.05 versus ET-1. D, After-contractions (arrowheads) in a LV myocyte exposed to ATP. E, Fraction of myocytes, 27%, with impaired relaxation (black portion of the bar). *P<0.05 versus Tyr. F, Ca²⁺ transients (red-traces) and sarcomere shortening (black-traces) in field stimulated cardiomyocyte exposed to ATP. An ectopic (arrows) and sustained Ca²⁺ elevation coupled with prolonged contraction is shown. G, Isometric tension in human LV trabeculae exposed to ATP (100 μmol/L) and then to the IP3R blocker 2-APB. H, Quantitative data are shown as mean±SEM. Base: baseline in the presence of Krebs-Henseleit buffer. *P<0.05 versus Base.

GPCRs and Electrical Instability

To establish whether the electrical properties of human cardiomyocytes were influenced by activation of GPCRs, membrane potential and [Ca²⁺]_i were measured simultaneously in current-clamped cells.²⁵ In human cardiomyocytes, ATP and ET-1 decreased resting membrane potential (RMP), prolonged the action potential (AP), and increased early after-depolarizations (EADs), together with an increase in Ca²⁺ transient amplitude and extra-systolic Ca²⁺ elevations. Comparable responses were observed with the IP3R agonist, thimerosal (Figure 3A–3D). Importantly, ATP and ET-1 had similar effects on the intact myocardium. Human samples were perfused in a Langendorff apparatus, and transmural pseudo-EKG and monophasic action potentials (MAPs) were recorded. GPCR agonists delayed the electrical recovery and prolonged MAPs (Figure 4A and 4B), mimicking the observations at the myocyte level. Moreover, tachycardia and premature ventricular contractions occurred (Figure 4C). To exclude the contribution of ischemia, potentially introduced during the preservation of the organ, measurements were obtained in the presence of K_ATP channel blockade, since these channels are activated by hypoxia. Inhibition of K_ATP channels had no consequences on MAPs (Figure 5 in the online-only Data Supplement), indicating that the electrical manifestations dictated by GPCR stimulation were independent from myocardial ischemia. Thus, GPCR stimulation prolongs the AP and promotes the electrical instability of cardiomyocytes and the myocardium.

Figure 3.

IP3Rs and electrical properties of human cardiomyocytes. A, APs (black-traces) and Ca²⁺ transients (red-traces) in a human myocyte exposed to ATP. In the lower panel, traces are shown at higher time resolution. B, AP (black-traces) and Ca²⁺ transients (red-traces) in a human myocyte before and after ET-1 exposure. C, EADs (black-traces) and Ca²⁺ transients (red-traces) of human myocytes with ATP, ET-1, and the IP3R-agonist, thimerosal (10 μmol/L). D, Quantitative data are shown as mean±SEM. *P<0.05 versus Tyr.

Figure 4.

IP3Rs and electrical properties of human myocardium. A, ET-1 prolongs the electrical activation (pseudo-EKG, red-traces) and the AP (black-traces) of paced human myocardium. B, Quantitative data are shown as mean±SEM. Base: Krebs-Henseleit buffer. *P<0.05 versus Base. C, Premature-ventricular-contractions (PVCs; red-arrowheads) and triggered-activity (red-dashed-lines) in human myocardium with ET-1 and ATP (20 μmol/L).

GPCR Simulation in the Failing Human Heart

Contractile reserve is severely reduced in heart failure (HF), and the GPCR/IP3R axis may represent an important signaling pathway modulating the inotropic state of the diseased myocardium. Thus, explanted failing hearts were studied (Table 1 in the online-only Data Supplement). By Western blotting, IP3R-2 expression was significantly increased in failing LV myocytes (Figure 5A). Importantly, stimulation of GPCRs with ATP in failing LV myocytes led to a reduction in RMP and prolongation of the AP (Figure 5B). Similarly, measurements of cell shortening in one failing LV cardiomyocyte revealed that this GPCR/IP3R activation increased contractility, elicited premature contraction and altered relaxation (Figure 5C).

Figure 5.

IP3Rs and GPCR activation in the failing human myocardium. A, Expression of IP3R-2 protein by Western blotting in normal and failing human LV myocytes. GAPDH: loading condition. Quantitative data are shown as mean±SEM. *P<0.05 versus Normal Hearts. B, APs of a failing human myocyte before (black-trace, Tyr) and after (blue trace) exposure to ATP. Quantitative data are shown as mean±SEM. Tyr: Tyrode; *P<0.05 versus Tyr, using paired t-test.. C, Cell shortening in a field stimulated human failing cardiomyocyte, before and following exposure to ATP. Arrow and arrowhead point to a premature contraction and altered relaxation, respectively. D and E, Quantitative data for isometrically twitching human LV trabeculae. Data are shown as mean±SEM. Iso: isoproterenol (100 nmol/L); Base: baseline in the presence of Krebs-Henseleit buffer. *P<0.05 versus Base, †P<0.05 versus Normal Heart in the presence of Iso.

The effects of GPCR stimulation on the myocardium were evaluated in an isometric system. In comparison with donor human hearts, the inotropic response of LV trabeculae to the β-adrenergic agonist isoproterenol was significantly attenuated in failing hearts (Figure 5D). In contrast, GPCR stimulation promoted an increase in developed tension in the diseased myocardium mimicking the findings obtained in healthy hearts (Figure 2H and Figure 5E). Thus, the inotropic reserve is decreased in the failing heart muscle, while the GPCR/IP3R axis remains operative.

IP3Rs and Mouse Cardiomyocytes

To define the role of IP3Rs in myocyte contractility following GPCR activation, a loss of function assay was introduced in LV mouse cardiomyocytes. Initial studies were performed to demonstrate that GPCRs agonists induce in mouse cardiomyocytes responses similar to those seen in human cells. ATP, ET-1, angiotensin II or phenylephrine stimulation led to an increase in Ca²⁺ transient amplitude and myocyte contractility. Diastolic Ca²⁺ was also elevated. Extra-systolic Ca²⁺ and sustained Ca²⁺ increases were apparent, resulting in after-contractions and prolonged contractures, respectively (Figure 6 in the online-only Data Supplement). Increased IP3R affinity by thimerosal led to comparable results. In contrast, IP3R blockade (XeC) or inhibition of IP3 synthesis (U-73122) abolished the effects of GPCR stimulation (Figure 6 in the online-only Data Supplement). By employing two-photon microscopy working in line-scan mode, it was possible to demonstrate that extra-systolic Ca²⁺ and sustained increases in peak Ca²⁺ were distributed synchronously in the myocyte cytoplasm; this pattern of Ca²⁺ changes excludes that local releases of Ca²⁺ from the SR, or Ca²⁺ waves were involved in the process (Figure 7 in the online-only Data Supplement).

ATP and ET-1 may stimulate other effector pathways involving phosholipase-C (PLC), which activates PKC isoforms, resulting in the phosphorylation of ion channels and contractile proteins.^19–21 To establish whether this mechanism was implicated in myocyte performance, studies in mouse LV myocytes were conducted in the presence of the PKC inhibitor chelerythrine.³² Chelerythrine failed to abrogate the inotropic response triggered by ATP and ET-1 (Figure 8 in the online-only Data Supplement), suggesting that PKC-independent pathways play a critical role in mediating the impact of GPCR stimulation in myocytes.

Based on this information, which strengthened the human results, the consequences of downregulation of IP3R type-2 on Ca²⁺ cycling in myocytes was tested using a small hairpin-RNA (sh-RNA) assay. An adeno-associated AAV9 vector carrying EGFP and sh-RNA targeting IP3R type-2 (shRNA-IP3R2-EGFP) was injected intravenously in mice (Figure 9 in the online-only Data Supplement). Three weeks later, EGFP-positive and EGFP-negative LV myocytes were isolated (Figure 6A) and loaded with the Ca²⁺ indicator Rhod-2. Ca²⁺ transient was measured in field stimulated cells, in the absence and presence of ATP or ET-1. GPCRs activation failed to increase Ca²⁺ transients in EGFP-positive mouse myocytes. Similarly, extra-systolic and sustained Ca²⁺ elevations were not detected. Conversely, these responses were preserved in EGFP-negative cells (Figure 6B and 6C, and Figure 10 in the online-only Data Supplement).

Figure 6.

Downregulation of IP3Rs, Ca²⁺ transients, and extra-systolic Ca²⁺ elevations. A, Left panel: EGFP-negative myocyte (arrowheads) and EGFP-positive myocyte (arrows); the EGFP-positive myocyte is shown again by native EGFP fluorescence in the right panel. These myocytes were collected from a mouse heart treated with AAV9 vector carrying EGFP and shRNA targeting IP3R-2. B, Ca²⁺ transients in EGFP-negative (black-traces) and EGFP-positive myocytes (green-traces) in the presence of ATP and ET-1. C, Fraction of myocytes with extra-systolic and sustained Ca²⁺ elevation (black portion of the bars). Quantitative data are shown as mean±SEM. *P<0.05 versus Tyr. †P<0.05 versus EGFP-negative myocytes. D, Electrical (black-traces) and Ca²⁺ transient (red-traces) properties of myocytes dialyzed with IP3 (50 μmol/L, phase-contrast micrograph). E, Quantitative data are shown as mean±SEM. *P<0.05 versus Base, using paired t-test.

As in human cells, stimulation of GPCR function with ATP or ET-1, or enhanced IP3R affinity by thimerosal decreased RMP, prolonged the AP, and increased the frequency of EADs in mouse myocytes. Ca²⁺ transient amplitude increased, and extra-systolic Ca²⁺ elevations were apparent (Figure 11 in the online-only Data Supplement). Similarly, spontaneous sustained depolarization to the plateau and long-lasting [Ca²⁺]_i increases were detected (Figure 12 in the online-only Data Supplement). Importantly, direct activation of IP3Rs by dialysis with IP3 in the myocyte cytoplasm had effects comparable to those seen with GPCR agonists or thimerosal (Figure 6D and 6E).

IP3R blockade, in the absence of GPCR agonists, did not alter the AP, Ca²⁺ transients and myocyte contractility (Figure 13 in the online-only Data Supplement). Conversely, IP3R inhibition reversed the effects of ET-1 and ATP on cardiomyocytes (Figure 14 in the online-only Data Supplement). Electrical abnormalities similar to those observed in isolated myocytes were detected in the perfused mouse heart under conditions favoring IP3R function; the arrhythmic events triggered by regular pacing and programmed electrical stimulation¹³ were markedly enhanced (Figure 15 in the online-only Data Supplement). Thus, IP3Rs mediate partly the effects of GPCR stimulation on Ca²⁺ cycling and the electrical and mechanical properties of adult human and mouse cardiomyocytes.

GPCR stimulation and Ca²⁺ Release from RyRs

The increase in Ca²⁺ transient amplitude and extra-systolic Ca²⁺ elevations induced by GPCR agonists in myocytes may result from the prolongation of the AP and altered repolarization phase. Alternatively, Ca²⁺ mobilized by IP3Rs may sensitize RyRs, enhancing excitation contraction-coupling gain and promoting spontaneous Ca²⁺ releases, which, in turn, may affect myocyte electrical properties.¹¹

To establish the role of the AP profile on the properties of Ca²⁺ transients following GPCR stimulation, AP-clamp studies were performed.²⁵ AP waveforms in Tyrode solution (Ctrl-APs) or with GPCR agonists (GPCR-APs) were employed as voltage-clamp commands, while monitoring intracellular Ca²⁺. Despite GPCR stimulation, Ctrl-APs were characterized by physiological Ca²⁺ transients with no extra-systolic Ca²⁺ elevations (n=23/23) (Figure 7A and 7B, and Figure 16 in the online-only Data Supplement). Conversely, GPCR-APs showed increased Ca²⁺ transients in the presence (n=27/27) or absence (n=8/8) of GPCR agonists (Figure 7B and Figure 16 and 17 in the online-only Data Supplement). Thus, the alterations in Ca²⁺ transients following GPCR stimulation are secondary to changes in AP profile.

Figure 7.

The profile of the AP conditions the effects of GPCR activation on Ca²⁺ transients, and IP3R activation does not alter ryanodine receptor function. A and B, APs (V_membrane: membrane potential, black-traces) and Ca²⁺ transients (Fluo-3, red-traces) in a mouse cardiomyocyte exposed to ET-1. APs with variable profiles were recorded in current-clamp mode (A) before (Ctrl-AP) and after activation of GPCRs (GPCR-AP) and used as voltage-clamp commands (V_command, blue-traces) (B); Ca²⁺ transients (red-traces) and membrane currents (black-traces) are shown. C, I_CaL (black-traces) and Ca²⁺ transients (red-traces) elicited by depolarizing steps (blue-traces) by patch-clamp in the presence of ET-1 and ATP. D, Voltage relations for I_CaL, Ca²⁺ transient amplitude and ECC gain in Tyr and after IP3R stimulation. E, Transmebrane potential (black traces) and cytosolic [Ca²⁺]_i (red traces) in myocytes exposed to ryanodine before and after ATP stimulation. F, Effects of IP3R activation on the AP following blockade of RyRs (ryanodine, 10 μmol/L). Quantitative data are shown as mean±SEM. *P<0.05 versus ryanodine alone.

To test whether RyRs were sensitized by GPCR activation, Fluo-3 loaded myocytes were exposed to a family of depolarizing steps in a voltage-clamp mode²⁵ to progressively activate L-type Ca²⁺ current (I_CaL) and release of Ca²⁺ from RyRs. I_CaL, Ca²⁺ transient amplitude, and the ability of Ca²⁺ influx to induce RyR-Ca²⁺ release (excitation contraction-coupling gain) did not change with activation of GPCRs (Figure 7C and 7D). These results tend to exclude that the release of Ca²⁺ following activation of IP3Rs enhances the sensitivity of RyRs.

To establish whether RyRs contribute to the changes in the electrical properties of myocytes after GPCR stimulation, these Ca²⁺ channels were blocked by ryanodine and the effects of ATP were studied. Under this condition, Ca²⁺ transients and myocyte shortening were inhibited (Figure 18 in the online-only Data Supplement); however, RMP decreased, the AP was prolonged, and arrhythmic events occurred (Figure 7E and 7F and Figure 19 in the online-only Data Supplement). These findings suggest that RyRs are not implicated in the electrical instability of cardiomyocytes mediate by GCPR activation.

IP3Rs and Electrical Properties Following GPCR Stimulation

Although GPCR stimulation prolongs the AP favoring an increase in Ca²⁺ transients, the mechanism(s) involved in the changes of the electrical activity of cardiomyocytes have not been defined as yet. Based on the fact that (a) IP3 dialysis or (b) enhanced IP3R affinity to IP3 recapitulate the effects of GPCR stimulation, and that (c) IP3R downregulation or (d) IP3R inhibition attenuate the effects of ATP and ET-1, the possibility was raised that mobilization of Ca²⁺ from the SR by IP3Rs contributes to the altered electrical properties of myocytes in the presence of GPCR agonists. Ca²⁺ translocation from the SR may enhance cytosolic Ca²⁺ load, which may promote Ca²⁺ extrusion via forward mode NCX; this process may contribute to the depolarization of the RMP, and the delayed repolarization phase of the AP.^33,34

To test this hypothesis, voltage-clamp studies were performed in myocytes loaded with Fluo-3. IP3R function led to elevations in diastolic Ca²⁺ that were followed by sustained and transient inward currents. Importantly, the increase in diastolic Ca²⁺ developed slowly over a period of several seconds, potentially reflecting IP3R-mediated Ca²⁺ release (Figure 8A). The changes in resting Ca²⁺ and inward currents were preserved, despite inhibition of RyRs (Figure 8B). Conversely, blockade of the NCX prevented inward currents in the presence of increasedcytosolic Ca²⁺ (Figure 8C).

Figure 8.

IP3Rs activate the NCX through changes in [Ca²⁺]_i. A, Transient and sustained inward currents (black-traces) and slowly developing diastolic [Ca²⁺]_i elevations (red-traces) in mouse myocytes exposed to thimerosal. Membrane potential was held at −70 mV; short (5 ms) depolarizing steps to 0 mV were used to trigger Ca²⁺ transients. The Ca²⁺ fluorescent signal is reported at higher magnification in the lower traces. B, Transient and sustained inward currents (black-traces) and slowly developing [Ca²⁺]_i elevations (red-traces) in mouse myocytes held at −70 mV following exposure to ATP and ryanodine. C, Slowly developing diastolic [Ca²⁺]_i elevations (red-traces) in a mouse myocytes exposed to ATP and ryanodine in the presence of nickel (2.5 mmol/L), fail to induce transient and sustained inward currents (black-traces). Membrane potential was held at −70 mV; short (5 ms) depolarizing steps to 0 mV were employed. D, Current voltage relations for the nickel (Ni)-sensitive current measured in voltage-clamp in the absence of IP3R stimulation and in the presence of ATP, ET-1, or IP3 dialysis (left). On the right, superimposed curves indicate that the Ni-sensitive current is enhanced following IP3R stimulation. To prevent RyR-Ca²⁺ release, ryanodine was employed. E, In the presence of buffered [Ca²⁺]_i, ATP and ET-1 fail to alter the myocyte AP profile. Quantitative data are shown as mean±SEM.

To define the role of NCX following activation of IP3Rs, a nickel sensitive current, indicative of NCX activity, was measured.³⁵ ATP, ET-1, and dialysis with IP3 induced large inward currents, consistent with enhanced Ca²⁺ extrusion via forward mode NCX (Figure 8D). Additionally, to strengthen the possibility that Ca²⁺ extrusion was enhanced following Ca²⁺ mobilization by IP3Rs, myocytes were voltage-clamped at −70 mV; Ca²⁺ transient was triggered by short depolarizing steps and caffeine puffs to monitor intracellular SR Ca²⁺ stores. In the presence of ATP or ET-1, this protocol resulted in a gradual decrease in Ca²⁺ transients (Figure 20 in the online-only Data Supplement), supporting the notion that Ca²⁺ leak occurs under conditions favoring IP3R function.

To test whether the mobilization of Ca²⁺ from the SR via IP3Rs was implicated in the prolongation of the AP, decreased RMP, and increased EADs, Ca²⁺ from the SR was depleted by inhibition of SERCA and exposure to caffeine.^36,37 By this approach, ATP and ET-1 showed a markedly reduced effect on RMP, AP duration, and arrhythmia (Figure 21 in the online-only Data Supplement). Additionally, to establish the impact of increased Ca²⁺ load on the electrical instability of cardiomyocytes, these cells were studied under [Ca²⁺]_i buffered conditions, to prevent changes in cytosolic Ca²⁺.³⁸ GPCR agonists had no effects on RMP and AP (Figure 8E). Collectively, these observations indicate that Ca²⁺ mobilization from the SR via IP3Rs contributes to the alterations in the electrical properties of cardiomyocytes mediated by GPCR stimulation.

Discussion

The results of the current study provide a characterization of the electrical and mechanical properties of human LV myocytes and emphasize the critical role that the GPCR/IP3R axis has in regulating Ca²⁺ homeostasis, contractile performance and the electrical stability of the human heart. IP3Rs are expressed and functional in human LV myocytes, and the Ca²⁺ mobilized from the SR by IP3Rs contributes to the decrease in RMP, prolongation of the AP, and to the occurrence of EADs and sustained depolarizations following GPCR stimulation. Ca²⁺ transient amplitude and cell shortening are enhanced, and extra-systolic and dysregulated Ca²⁺ elevations and contractions become apparent. These alterations in the electromechanical behavior of human cardiomyocytes are coupled with increased isometric twitch of the myocardium and arrhythmic events, indicating that the GPCR/IP3R effector pathway offers inotropic reserve, which is hampered by electrical instability and contractile abnormalities.

IP3Rs are present in hCSCs⁸ and are preserved in the formed myocyte progeny. However, the growth promoting effects of IP3Rs in hCSCs are lost in post-mitotic human cardiomyocytes.³⁹ In both cases, IP3Rs modulate the release of Ca²⁺ from the SR, but its distal effect varies dramatically; the increase in cytosolic Ca²⁺ favors cell cycle reentry and asymmetric division of hCSCs,⁸ while, as shown here, the release of Ca²⁺ in adult human cardiomyocytes enhances cell contractile performance. Whether a link between these two distinct mechanisms actually exists is currently unknown, although IP3Rs have a critical independent biological function in the mother stem cell and the derived differentiated cardiomyocytes.

The contribution of IP3Rs to the electromechanical behavior of ventricular myocytes in small and large mammals is controversial.^10,39 Our findings support the notion that in the human heart IP3Rs represent an important substrate able to increase myocyte contractility and the propensity for electrical instability coupled with GPCR agonists. Increases in the circulating levels of neurohumoral transmitters and cytokines physiologically and pathologically^40,41 may involve IP3R activation predisposing the heart to arrhythmic events. The increased incidence of arrhythmias and sudden death in patients with chronic heart failure^42,43 may be related partly to upregulation of IP3Rs in the hypertrophied, dysfunctional cardiomyocytes.^44,45

The GPCR agonists ATP, ET-1, and angiotensin II stimulate IP3Rs in human cardiomyocytes, but other signaling pathways may be upregulated as well. These GPCR agonists activate PLC that generates not only IP3 but also DAG, which activates TRPCs and PKC isoforms.^18,21 PKC phosphorylates ion channels and myofilament proteins, affecting the electrical and mechanical properties of cardiomyocytes.^19–21 Additionally, ATP and ET-1 may enhance PLC-independent molecular events,^21,22 which may trigger transmembrane ionic fluxes. Importantly, the effects of ATP and ET-1 on myocyte contractility were maintained in the presence of a PKC inhibitor. Similarly, the alterations in the electrical properties of cardiomyocytes were recapitulated by direct stimulation with IP3 or by enhancing IP3R affinity. Conversely, the effects of GPCR agonists were abrogated by: (a) downregulation of IP3Rs; (b) inhibition of IP3R function or reduced IP3 production; (c) depletion of Ca²⁺ from the SR; and (d) buffered intracellular Ca²⁺. Collectively, our data support the notion that the Ca²⁺ mobilized by IP3Rs is a critical determinant of the electrical abnormalities dictated by stimulation of GPCRs. However, we cannot exclude that DAG-sensitive and PLC-independent signaling pathways may participate in the electromechanical changes initiated by IP3R function. GPCR agonists modulate I_CaL and the delayed rectifier K⁺ currents, which can alter the AP profile.^21,22 Moreover, changes in membrane potential may result from TRPCs and store-operated Ca²⁺ channels activated, respectively, by DAG and SR Ca²⁺ levels.^18,46

A significant issue concerned whether, following GPCR stimulation, Ca²⁺ mobilization from the SR via IP3Rs alters the electrical properties of myocytes directly, or indirectly by Ca²⁺ release from RyR channels.^10,11 The contribution of RyRs to the process was analyzed by combining Ca²⁺ imaging with electrophysiological measurements. Our findings indicate that IP3Rs contribute to the alteration of the AP, independently from an increase in sensitivity of the RyRs. Excitation contraction-coupling gain was preserved in the presence of GPCR agonists, documenting that RyR function was not enhanced by the changes in Ca²⁺.

Despite inhibition of RyRs, cytosolic Ca²⁺, NCX forward mode, and electrical abnormalities increased in LV myocytes following stimulation of the GPCR/IP3R axis. Collectively, the cascade of events initiated by GPCR ligands involves promotion of PLC enzymatic activity, IP3 production, opening of IP3R channels, and Ca²⁺ translocation from the SR to the cytoplasm. The latter favors the electrogenic extrusion of this cation via NCX, resulting in depolarization of the RMP and prolonged repolarization of the AP (Figure 22 in the online-only Data Supplement). These electrical changes positively impact on the process of Ca²⁺-induced Ca²⁺ release since the delayed repolarization of the AP sustains Ca²⁺ influx, amplifying Ca²⁺ transients and cell shortening.²⁵ By AP-clamp, remodeled APs, in the absence of GPCR ligands, elicit Ca²⁺ transients comparable to those associated with GPCR agonists. These findings indicate that the altered electrical activity has a critical role in the process, rather than suggesting a direct interaction between IP3R and RyR function. Moreover, the enhanced Ca²⁺ entry by the protracted AP may counteract the excess of Ca²⁺ extruded during diastole via NCX, restoring intracellular SR Ca²⁺ stores. This may account for the prolonged effects of IP3R in the presence of GPCR agonists. Conversely, with stable (time constrained) depolarizations in voltage-clamp, GPCR agonists are coupled with a progressive decrease in Ca²⁺ transient amplitude and SR Ca²⁺ load, emphasizing the importance that the changes in the AP profile have in mediating a sustained positive inotropic effect.

The extra-systolic elevations in Ca²⁺ and after-contractions detected in cardiomyocytes with GPCR agonists appear to be due to EADs, which may be the product of the alteration in the AP.⁴³ In fact, changes in AP profile alone were sufficient to trigger alone extra-systolic Ca²⁺ elevations, pointing to the alterations in the electrical properties of cardiomyocytes as the primary mediator of Ca²⁺ cycling abnormalities. However, the ionic basis for the sustained membrane depolarization, protracted Ca²⁺ increases and myocyte contractures remains to be completely defined. IP3-independent effector pathways may participate in the process, although the cellular alterations were abrogated by attenuating IP3R function.

The spatiotemporal dynamics of Ca²⁺ mobilized by IP3Rs is dictated by the hierarchical recruitment of elementary Ca²⁺ release when [IP3]_i is increased; this condition originates Ca²⁺ blips, puffs, and global regenerative waves.¹⁶ In the absence of GPCR agonists, blockade of IP3R channels has little or no impact on the mechanical and electrical behavior of cardiomyocytes, indicating that extracellular activators of PLC are needed to increase [IP3]_i and trigger an IP3R response. As in other cell types,^8,16,17 IP3R activation results in a time-dependent increase incytosolic Ca²⁺ of cardiomyocytes that develops slowly over a number of seconds. With GPCR agonists, the temporal dynamic of IP3R-mediated Ca²⁺ release provides the basis for the sustained changes in the AP, cellular contractility, and incidence of electrical disorders. A similar sequence of events appears to be operative in failing human cardiomyocytes, in which IP3R transcripts⁴⁷ and proteins are up-regulated, providing a potential etiology for arrhythmias and sudden death in this patient population.

Ca²⁺ release channels are differentially expressed during cardiac development and postnatally;^39,48,49 in cardiomyocytes, the contribution of the ubiquitous IP3R to intracellular Ca²⁺ mobilization is progressively attenuated from the fetal to the neonatal and adult life, in favor of the more specialized function of RyRs.^39,49 Thus, the up-regulation of IP3Rs may be viewed as a component of a larger fetal reprogramming in the failing myocardium, together with the multiple pathological changes that characterize the progression of the disease state.