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

Originally publishedhttps://doi.org/10.1161/CIRCULATIONAHA.113.002764Circulation. 2013;128:1286–1297

Abstract

Background—

Little is known about the function of inositol 1,4,5-trisphosphate 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 after Gαq-protein–coupled receptor 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 myocytes, and this analysis was integrated with assays in mouse myocytes to identify the mechanisms by which IP3Rs influence the electric and mechanical properties of the myocardium. We report that IP3Rs are expressed and operative in human left ventricular myocytes. After Gαq-protein–coupled receptor 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 afterdepolarizations. Ca2+ transient amplitude and cell shortening are enhanced, and extrasystolic 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 Gαq-protein–coupled receptor activation provides inotropic reserve, which is hampered by electric 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—

The Gαq-protein/coupled receptor/IP3R axis modulates the electromechanical properties of the human myocardium and its propensity to develop arrhythmias.

Introduction

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 After 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 reuptake in the SR by the sarcoendoplasmic Ca2+ pump 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,37 strengthening the notion that defects in Ca2+ homeostasis affect the electric properties of the heart.

Editorial see p 1273

Clinical Perspective on p 1297

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, inducing cell cycle reentry and modulating the fate of these cells, after 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 that 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 human cardiac stem cells 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–nuclear factor of activated T cell 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 extrasystolic 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 receptor (GPCR) agonists may involve IP3R-mediated Ca2+ mobilization, cytoplasmic Ca2+ overload, perturbations in transmembrane potential, or sensitization of RyRs, leading to elementary release of Ca2+ and electric instability.

Ca2+ mobilized by IP3Rs relies on intracellular levels of IP3, which is produced by phospholipase C (PLC). GPCR agonists promote PLC function, which generates IP3 and diacylglycerol. IP3 stimulates IP3Rs and Ca2+ translocation from intracellular stores,8,16,17 whereas diacylglycerol activates transient receptor potential channels18 and protein kinase C (PKC) isoforms, phosphorylating a wide spectrum of ion channels and contractile proteins.1921 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.

On the basis of 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, electric activity, arrhythmic events, and myocyte contractile behavior after 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 used in this study (Table I in the online-only Data Supplement). The protocol was approved by the Institutional Review Board (No. 2010P002475). Donor and failing human hearts were shipped in cold cardioplegia solution and were processed immediately on receipt.

Myocardial samples, obtained at autopsy from patients who died of causes other than cardiovascular diseases, were fixed in formalin and used as controls for the quantitative evaluation of myocyte apoptosis by the terminal deoxynucleotidyl transferase 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 U/mL collagenase (type 2; Worthington Biochemical Corp), and 0.57 U/mL protease (type XIV, Sigma) were added to the perfusate for enzymatic dissociation of the tissue. At completion of digestion, the myocardium was cut into small pieces, subjected to repeated pipetting to obtain a single-cell preparation, and resuspended 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 after organ acquisition.

Mouse Hearts and Myocyte Isolation

C57Bl/6 mice were maintained in accordance with the Guide for the Care and Use of Laboratory Animals, and all animal experiments were approved by the local animal care committee. For myocyte isolation, a protocol similar to that described above was used.2426 Antibodies for fluorescence-activated cell sorter analysis and primers for polymerase chain reaction studies are listed in Tables II and III 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 Axiovert (Zeiss), IX51, and IX71 (Olympus) inverted microscopes for measurements of contractility, Ca2+ transients, and patch-clamp studies.2427 Experiments were performed at room temperature.

Data Analysis

The magnitude of sampling used in each measurement is listed in Table I in the online-only Data Supplement. Data are presented as mean±SEM. Independent samples were compared by use of ANOVA analysis with Bonferroni correction or Fisher 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 the 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 least squares means (LSMEANS) statement. Throughout, a value of P<0.05 was considered statistically significant, and all tests were 2 sided with a type I error of 0.05. The statistical analyses were performed with SAS version 9.3 (SAS Institute).28

Results

Human Hearts

Human cardiomyocytes were obtained from the LV of 34 donor hearts from 14 men and 20 women with an average age of 44±4 years (Table I 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 3 randomly selected donor hearts and age-matched control hearts obtained at autopsy from patients who died of causes other than cardiovascular diseases (Figure I 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 nonischemic (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 II in the online-only Data Supplement). Transcripts for the 3 IP3R subtypes were identified by quantitative reverse transcription–polymerase chain reaction 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 III and IV in the online-only Data Supplement).

Figure 1.

Figure 1. Inositol 1,4,5-trisphosphate receptors (IP3Rs) in human and mouse cardiomyocytes. A, Quantitative reverse transcription–polymerase chain reaction shows transcripts for the 3 IP3R subtypes in human and mouse left ventricular (LV) myocytes. Representative curves and polymerase chain reaction products are shown, together with the mRNA obtained from the human and mouse brain that was used as positive control (red curves). The polymerase chain reaction products had the expected molecular weight (MW). Human β2-microglobulin is the housekeeping gene (see Figure III in the online-only Data Supplement). For sequences, see Figure IV 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 is the loading condition.

The effect of IP3R activation on Ca2+ cycling and contractility of human LV myocytes was investigated by stimulating GPCRs with ATP and endothelin-1 (ET-1), both of which enhance the synthesis of IP3.8,10,11,14,17,2931 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 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 aftercontractions 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.

Figure 2. Inositol 1,4,5-trisphosphate receptors (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 [Tyr]) and after exposure to endothelin-1 (ET-1; 100 nmol/L). C, Quantitative data for Fluo-loaded cells are shown as mean±SEM. XeC indicates xestospongin-C (2 µmol/L). *P<0.05 vs Tyrode, †P<0.05 vs ET-1. D, Aftercontractions (arrowheads) in a left ventricular (LV) myocyte exposed to ATP. E, Fraction of myocytes, 27%, with impaired relaxation (black portion of the bar). *P<0.05 vs Tyrode. F, Ca2+ transients (red traces) and sarcomere shortening (black traces) in a 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 indicates baseline in the presence of Krebs-Henseleit buffer. *P<0.05 vs baseline.

GPCRs and Electric Instability

To establish whether the electric properties of human cardiomyocytes were influenced by activation of GPCRs, membrane potential and [Ca]2+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 afterdepolarizations, together with an increase in Ca2+ transient amplitude and extrasystolic 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-ECG and monophasic APs were recorded. GPCR agonists delayed the electric recovery and prolonged monophasic APs (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 KATP channel blockade because these channels are activated by hypoxia. Inhibition of KATP channels had no consequences on monophasic APs (Figure V in the online-only Data Supplement), indicating that the electric manifestations dictated by GPCR stimulation were independent of myocardial ischemia. Thus, GPCR stimulation prolongs the AP and promotes the electric instability of cardiomyocytes and the myocardium.

Figure 3.

Figure 3. Inositol 1,4,5-trisphosphate receptors (IP3Rs) and electric properties of human cardiomyocytes. A, Action potentials (APs; black traces) and Ca2+ transients (red traces) in a human myocyte exposed to ATP. Bottom, Traces are shown at higher time resolution. B, AP (black traces) and Ca2+ transients (red traces) in a human myocyte before and after endothelin-1 (ET-1) exposure. C, Early afterdepolarizations (black traces) and Ca2+ transients (red traces) of human myocytes with ATP, ET-1, and the IP3R-agonist thimerosal (Thimer; 10 µmol/L). D, Quantitative data are shown as mean±SEM. *P<0.05 vs Tyrode (Tyr). RMP indicates resting membrane potential.

Figure 4.

Figure 4. Inositol 1,4,5-trisphosphate receptors (IP3Rs) and electric properties of human myocardium. A, Endothelin-1 (ET-1) prolongs the electric activation (pseudo-ECG, red traces) and the action potential (black traces) of paced human myocardium. B, Quantitative data are shown as mean±SEM. Base indicates Krebs-Henseleit buffer. *P<0.05 vs Base. C, Premature ventricular contractions (red arrowheads) and triggered activity (red dashed-lines) in human myocardium with ET-1 and ATP (20 µmol/L). MAP indicates monophasic action potential.

GPCR Simulation in the Failing Human Heart

Contractile reserve is severely reduced in heart failure, 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 I 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 1 failing LV cardiomyocyte revealed that this GPCR/IP3R activation increased contractility, elicited premature contraction, and altered relaxation (Figure 5C).

Figure 5.

Figure 5. Inositol 1,4,5-trisphosphate receptors (IP3Rs) and Gαq-protein–coupled receptor (GPCR) activation in the failing human myocardium. A, Expression of IP3R-2 protein by Western blotting in normal and failing human left ventricular (LV) myocytes. GAPDH is the loading condition. Quantitative data are shown as mean±SEM. *P<0.05 vs normal hearts. B, Action potentials (APs) of a failing human myocyte before (black trace; Tyrode [Tyr]) and after (blue trace) exposure to ATP. Quantitative data are shown as mean±SEM. *P<0.05 vs Tyrode, paired t test. C, Cell shortening in a field-stimulated human failing cardiomyocyte before and after 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. Base indicates baseline in the presence of Krebs-Henseleit buffer; Iso, isoproterenol (100 nmol/L); and RMP, resting membrane potential. *P<0.05 vs Base, †P<0.05 vs normal heart in the presence of Iso.

The effects of GPCR stimulation on the myocardium were evaluated in an isometric system. Compared 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, whereas the GPCR/IP3R axis remains operative.

IP3Rs and Mouse Cardiomyocytes

To define the role of IP3Rs in myocyte contractility after GPCR activation, a loss-of-function assay was introduced in LV mouse cardiomyocytes. Initial studies were performed to demonstrate that GPCR 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. Extrasystolic Ca2+ and sustained Ca2+ increases were apparent, resulting in aftercontractions and prolonged contractures, respectively (Figure VI in the online-only Data Supplement). Increased IP3R affinity by thimerosal led to comparable results. In contrast, IP3R blockade (xestospongin-C) or inhibition of IP3 synthesis (U-73122) abolished the effects of GPCR stimulation (Figure VI in the online-only Data Supplement). By using 2-photon microscopy working in line-scan mode, we were able to demonstrate that extrasystolic 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 VII in the online-only Data Supplement).

ATP and ET-1 may stimulate other effector pathways involving PLC, which activates PKC isoforms, resulting in the phosphorylation of ion channels and contractile proteins.1921 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 VIII in the online-only Data Supplement), suggesting that PKC-independent pathways play a critical role in mediating the impact of GPCR stimulation in myocytes.

On the basis of this information, which strengthened the human results, the consequences of downregulation of IP3R-2 on Ca2+ cycling in myocytes were tested with a small hairpin-RNA (shRNA) assay. An adeno-associated AAV9 vector carrying enhanced green fluorescent protein (EGFP) and shRNA targeting IP3R type-2 (shRNA-IP3R2-EGFP) was injected intravenously in mice (Figure IX 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. GPCR activation failed to increase Ca2+ transients in EGFP-positive mouse myocytes. Similarly, extrasystolic and sustained Ca2+ elevations were not detected. Conversely, these responses were preserved in EGFP-negative cells (Figure 6B and 6C and Figure X in the online-only Data Supplement).

Figure 6.

Figure 6. Downregulation of inositol 1,4,5-trisphosphate receptors (IP3Rs), Ca2+ transients, and extrasystolic Ca2+ elevations. A, Left, enhanced green fluorescent protein (EGFP)–negative myocyte (arrowheads) and EGFP-positive myocyte (arrows). The EGFP-positive myocyte is shown again by native EGFP fluorescence on the right. 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 extrasystolic and sustained Ca2+ elevation (black portion of the bars). Quantitative data are shown as mean±SEM. *P<0.05 vs Tyrode (Tyr), †P<0.05 vs EGFP-negative myocytes. D, Electric (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 vs Base, paired t test. Ctrl indicates control; ET-1, endothelin-1.

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 early afterdepolarizations in mouse myocytes. Ca2+ transient amplitude increased, and extrasystolic Ca2+ elevations were apparent (Figure XI in the online-only Data Supplement). Similarly, spontaneous sustained depolarization to the plateau and longlasting [Ca]2+i increases were detected (Figure XII 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 XIII in the online-only Data Supplement). Conversely, IP3R inhibition reversed the effects of ET-1 and ATP on cardiomyocytes (Figure XIV in the online-only Data Supplement). Electric 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 electric stimulation13 were markedly enhanced (Figure XV in the online-only Data Supplement). Thus, IP3Rs partly mediate the effects of GPCR stimulation on Ca2+ cycling and the electric and mechanical properties of adult human and mouse cardiomyocytes.

GPCR Stimulation and Ca2+ Release From RyRs

The increase in Ca2+ transient amplitude and extrasystolic 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 electric properties.11

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

Figure 7.

Figure 7. The profile of the action potential (AP) conditions the effects of Gαq-protein–coupled receptor (GPCR) activation on Ca2+ transients, and inositol 1,4,5-trisphosphate receptor (IP3R) activation does not alter ryanodine receptor (RyR) function. A and B, APs (Vmembrane indicates membrane potential; black traces) and Ca2+ transients (Fluo-3; red traces) in a mouse cardiomyocyte exposed to endothelin-1 (ET-1). APs with variable profiles were recorded in current-clamp mode (A) before (Ctrl-AP) and after (GPCR-AP) activation of GPCRs and used as voltage-clamp commands (Vcommand; blue traces; B); Ca2+ transients (red traces) and membrane currents (black traces) are shown. C,ICaL (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 ICaL, Ca2+ transient amplitude, and excitation-contraction coupling gain in Tyrode [Tyr] and after IP3R stimulation. E, Transmembrane potential (black traces) and cytosolic [Ca]2+i (red traces) in myocytes exposed to ryanodine before and after ATP stimulation. F, Effects of IP3R activation on the AP after blockade of RyRs (ryanodine, 10 µmol/L). Quantitative data are shown as mean±SEM. *P<0.05 vs 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 (ICaL) and release of Ca2+ from RyRs. ICaL, 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+ after activation of IP3Rs enhances the sensitivity of RyRs.

To establish whether RyRs contribute to the changes in the electric 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 XVIII in the online-only Data Supplement); however, RMP decreased, the AP was prolonged, and arrhythmic events occurred (Figure 7E and 7F and Figure IX in the online-only Data Supplement). These findings suggest that RyRs are not implicated in the electric instability of cardiomyocytes mediated by GCPR activation.

IP3Rs and Electric Properties After GPCR Stimulation

Although GPCR stimulation prolongs the AP favoring an increase in Ca2+ transients, the mechanisms involved in the changes in the electric activity of cardiomyocytes have not been defined as yet. On the basis of the fact that IP3 dialysis or enhanced IP3R affinity to IP3 recapitulates the effects of GPCR stimulation and that IP3R downregulation or IP3R inhibition attenuates the effects of ATP and ET-1, the possibility was raised that mobilization of Ca2+ from the SR by IP3Rs contributes to the altered electric 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 increased cytosolic Ca2+ (Figure 8C).

Figure 8.

Figure 8. Inositol 1,4,5-trisphosphate receptors (IP3Rs) activate the Na+/Ca2+ exchanger (NCX) through changes in [Ca]2+i. A, Transient and sustained inward currents (black traces) and slowly developing diastolic [Ca]2+i elevations (red traces) in mouse myocytes exposed to thimerosal. Membrane potential was held at −70 mV; short (5-millisecond) depolarizing steps to 0 mV were used to trigger Ca2+ transients. The Ca2+ fluorescent signal is reported at higher magnification in the bottom traces. B, Transient and sustained inward currents (black traces) and slowly developing [Ca]2+i elevations (red traces) in mouse myocytes held at −70 mV after exposure to ATP and ryanodine. C, Slowly developing diastolic [Ca]2+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-millisecond) depolarizing steps to 0 mV were used. D, Current-voltage relations for the nickel-sensitive current measured in voltage clamp in the absence of IP3R stimulation and in the presence of ATP, enthelin-1 (ET-1), or IP3 dialysis (left). Right, Superimposed curves indicate that the nickel-sensitive current is enhanced after IP3R stimulation. To prevent ryanodine receptor–Ca2+ release, ryanodine was used. E, In the presence of buffered [Ca]2+i, ATP and ET-1 fail to alter the myocyte action potential (AP) profile. Quantitative data are shown as mean±SEM. RMP indicates resting membrane potential.

To define the role of NCX after 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 after 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 XX 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 early afterdepolarizations, Ca2+ from the SR was depleted by inhibition of sarcoendoplasmic Ca2+ pump 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 XXI in the online-only Data Supplement). Additionally, to establish the impact of increased Ca2+ load on the electric instability of cardiomyocytes, these cells were studied under [Ca]2+i-buffered conditions to prevent changes in cytosolic Ca.2+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 electric properties of cardiomyocytes mediated by GPCR stimulation.

Discussion

The results of the present study provide a characterization of the electric 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 electric 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 occurrence of early afterdepolarizations and sustained depolarizations after GPCR stimulation. Ca2+ transient amplitude and cell shortening are enhanced, and extrasystolic 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 electric instability and contractile abnormalities.

IP3Rs are present in human cardiac stem cells8 and are preserved in the formed myocyte progeny. However, the growth-promoting effects of IP3Rs in human cardiac stem cells are lost in postmitotic 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 asymmetrical division of human cardiac stem cells,8 whereas, as shown here, the release of Ca2+ in adult human cardiomyocytes enhances cell contractile performance. Whether a link between these 2 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 electric instability coupled with GPCR agonists. Increases in the circulating levels of neurohumoral transmitters and cytokines physiologically and pathologically40,41 may involve IP3R activation predisposing the heart to arrhythmic events. The increased incidence of arrhythmias and sudden death in patients with chronic heart failure42,43 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 also be upregulated. These GPCR agonists activate PLC, which generates not only IP3 but also diacylglycerol, which activates transient receptor potential channels and PKC isoforms.18,21 PKC phosphorylates ion channels and myofilament proteins, affecting the electric and mechanical properties of cardiomyocytes.1921 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 electric properties of cardiomyocytes were recapitulated by direct stimulation with IP3 or by enhancing IP3R affinity. Conversely, the effects of GPCR agonists were abrogated by downregulation of IP3Rs, inhibition of IP3R function or reduced IP3 production, depletion of Ca2+ from the SR, and buffered intracellular Ca.2+ Collectively, our data support the notion that the Ca2+ mobilized by IP3Rs is a critical determinant of the electric abnormalities dictated by the stimulation of GPCRs. However, we cannot exclude that diacylglycerol-sensitive and PLC-independent signaling pathways may participate in the electromechanical changes initiated by IP3R function. GPCR agonists modulate ICaL and the delayed rectifier K+ currents, which can alter the AP profile.21,22 Moreover, changes in membrane potential may result from transient receptor potential channels and store-operated Ca2+ channels activated by diacylglycerol and SR Ca2+ levels, respectively.18,46

A significant issue concerned whether, after GPCR stimulation, Ca2+ mobilization from the SR via IP3Rs alters the electric 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 of 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 Ca.2+

Despite inhibition of RyRs, cytosolic Ca,2+ NCX forward mode, and electric abnormalities increased in LV myocytes after 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 last event favors the electrogenic extrusion of this cation via NCX, resulting in depolarization of the RMP and prolonged repolarization of the AP (Figure XXII in the online-only Data Supplement). These electric changes positively affect the process of Ca2+-induced Ca2+ release because 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 electric 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 extrasystolic elevations in Ca2+ and aftercontractions detected in cardiomyocytes with GPCR agonists appear to be attributable to early afterdepolarizations, which may be the product of the alteration in the AP.43 In fact, changes in AP profile alone were sufficient to trigger extrasystolic Ca2+ elevations, pointing to the alterations in the electric 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 electric behavior of cardiomyocytes, indicating that extracellular activators of PLC are needed to increase [IP3]i and to trigger an IP3R response. As in other cell types,8,16,17 IP3R activation results in a time-dependent increase in cytosolic Ca2+ of cardiomyocytes that develops slowly over a number of seconds. With GPCR agonists, the temporal dynamics of IP3R-mediated Ca2+ release provides the basis for the sustained changes in the AP, cellular contractility, and incidence of electric disorders. A similar sequence of events appears to be operative in failing human cardiomyocytes, in which IP3R transcripts47 and proteins are upregulated, providing a potential origin for arrhythmias and sudden death in this patient population.

Ca2+ release channels are differentially expressed during cardiac development and postnatally39,48,49; 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 upregulation 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.

Footnotes

S. Signore, A. Sorrentino, and Dr Ferreira-Martins contributed equally.

Guest Editor for this article was Barry London, MD, PhD.

The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.113.002764/-/DC1.

Correspondence to Marcello Rota, PhD, Department of Anesthesia and Medicine and Division of Cardiovascular Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115. E-mail

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Clinical Perspective

The function of inositol 1,4,5-trisphosphate receptors (IP3Rs) in the human heart is largely unknown and may have important implications in the modulation of myocardial performance and rhythm disturbances. IP3Rs may partly regulate the electric and mechanical properties of cardiomyocytes, contributing to the control of the electric stability and inotropic state of the myocardium. In this study, we have identified that IP3Rs are expressed and operative in nonfailing human left ventricular myocytes and are upregulated in failing human left ventricular myocytes. After Gαq-protein–coupled receptor activation, the Ca2+ mobilized from the sarcoplasmic reticulum by IP3Rs contributes to the decrease in resting membrane potential, prolongation of the action potential, and incidence of early afterdepolarizations. Ca2+ transient amplitude and myocyte shortening are enhanced, and extrasystolic and dysregulated Ca2+ elevations and contractions are apparent. These defects in the electric and mechanical behavior of human cardiomyocytes result in increased developed tension and electric disorders of the myocardium. Thus, the Gαq-protein–coupled receptor/IP3R effector pathway offers inotropic reserve, which is hindered by electric instability and contractile abnormalities.