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(Circulation Research. 2005;96:1274.)
© 2005 American Heart Association, Inc.

Cellular Biology

Endothelin-1–Induced Arrhythmogenic Ca²⁺ Signaling Is Abolished in Atrial Myocytes of Inositol-1,4,5-Trisphosphate(IP₃)–Receptor Type 2–Deficient Mice

Xiaodong Li, Aleksey V. Zima, Farah Sheikh, Lothar A. Blatter, Ju Chen

From the Department of Medicine (H.L., F.S., J.C.), University of California San Diego, La Jolla, Calif; and the Department of Physiology (A.V.Z., L.A.B.), Loyola University of Chicago, Stritch School of Medicine, Maywood, Ill.

Correspondence to Ju Chen, Department of Medicine, University of California San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0641. E-mail juchen{at}ucsd.edu

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Recent studies have suggested that inositol-1,4,5-trisphosphate-receptor (IP₃R)–mediated Ca²⁺ release plays an important role in the modulation of excitation–contraction coupling (ECC) in atrial tissue and the generation of arrhythmias, specifically chronic atrial fibrillation (AF). IP₃R type-2 (IP₃R2) is the predominant IP₃R isoform expressed in atrial myocytes. To determine the role of IP₃R2 in atrial arrhythmogenesis and ECC, we generated IP₃R2-deficient mice. Our results revealed that endothelin-1 (ET-1) stimulation of wild-type (WT) atrial myocytes caused an increase in basal [Ca²⁺]_i, an enhancement of action potential (AP)-induced [Ca²⁺]_i transients, an improvement of the efficacy of ECC (increased fractional SR Ca²⁺ release), and the occurrence of spontaneous arrhythmogenic Ca²⁺ release events as the result of activation of IP₃R-dependent Ca²⁺ release. In contrast, ET-1 did not alter diastolic [Ca²⁺]_i or cause spontaneous Ca²⁺ release events in IP₃R2-deficient atrial myocytes. Under basal conditions the spatio-temporal properties (amplitude, rise-time, decay kinetics, and spatial spread) of [Ca²⁺]_i transients and fractional SR Ca²⁺ release were not different in WT and IP₃R2-deficient atrial myocytes. WT and IP₃R2-deficient atrial myocytes also showed a significant and very similar increase in the amplitude of AP-dependent [Ca²⁺]_i transients and Ca²⁺ spark frequency in response to isoproterenol stimulation, suggesting that both cell types maintained a strong inotropic reserve. No compensatory changes in Ca²⁺ regulatory protein expression (IP₃R1, IP₃R3, RyR2, NCX, SERCA2) or morphology of the atria could be detected between WT and IP₃R2-deficient mice. These results show that lack of IP₃R2 abolishes the positive inotropic effect of neurohumoral stimulation with ET-1 and protects from its arrhythmogenic effects.

Key Words: IP₃ receptor • intracellular calcium • atrial arrhythmias • excitation-contraction coupling • endothelin

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Chronic atrial fibrillation (AF) is the most common sustained form of cardiac arrhythmia. AF is characterized by an atrial activation rate of typically >400 beats per minute, and is associated with 2 major complications including cardiac dysfunction and thrombus formation, resulting in an increased risk of morbidity because of heart failure and stroke.^1,2 In recent years, considerable attention has focused on the cellular and molecular mechanisms involved in AF (for review see Nattel^3,4). Whereas a multitude of mechanisms, including ischemic, metabolic, inflammatory, and structural factors, contribute to the etiology of AF,^5,6 several studies have suggested that abnormal intracellular calcium homeostasis and perturbations in Ca²⁺ handling may be an important modulator of atrial arrhythmias,⁴ including an elevated sarcoplasmic reticulum (SR) Ca²⁺ load leading to arrhythmogenic intracellular Ca²⁺ release.^7,8

Two types of Ca²⁺ release channels serve to regulate intracellular Ca²⁺ concentration ([Ca²⁺]_i): the ryanodine receptor (RyR) and the IP₃R. The RyR2 is considered the major Ca²⁺ release channel required for excitation–contraction coupling (ECC) in the heart.^9–11 During ECC, action potential (AP)-induced membrane depolarization leads to the opening of voltage-gated Ca²⁺ channels and Ca²⁺ entry. Entering Ca²⁺ activates the RyR which leads to massive Ca²⁺ release from the SR by a mechanism known as Ca²⁺-induced Ca²⁺ release (CICR¹²), which is required for inducing contraction.

Cardiac myocytes also contain IP₃R channels, however their functional importance in the heart has remained controversial. IP₃Rs release Ca²⁺ from intracellular Ca²⁺ stores when activated by IP₃, a product generated by phospholipase C (PLC) metabolism of phosphoinositol-4,5-bisphosphate (PIP₂) in response to G-protein–coupled receptor or receptor tyrosine kinase stimulation.¹³ In mammalian cells, there are 3 IP₃R subtypes referred to as type-1, -2, and -3, each transcribed by 3 distinct genes.¹⁴ Each IP₃R subtype has a different affinity for IP₃ and a differential tissue expression pattern.^15,16 IP₃R2 is the predominant IP₃R expressed in ventricular¹⁷ as well as in atrial myocytes,¹⁸ but its physiological role still remains unclear and controversial.^11,19,20

Recent reports have emphasized the importance of IP₃ signaling and IP₃-dependent Ca²⁺ release for ECC in the atria. Atrial myocytes express IP₃R2s at 6 to 10x higher levels than ventricular myocytes, and InsP₃Rs colocalize with RyRs in the subsarcolemmal space.^18,21 Furthermore, it was demonstrated that InsP₃-dependent Ca²⁺ release in atrial myocytes may serve to enhance Ca²⁺ spark frequency (Ca²⁺ sparks are referred to as the classical elementary Ca²⁺ release event from clusters of RyRs^22,23) and twitch [Ca²⁺]_i transient amplitude,^18,20,24 and thus may have a positive inotropic function. IP₃-dependent Ca²⁺ signaling has been implied in cardiac arrhythmias attributable to ischemia and reperfusion injury, inflammatory processes, and developing cardiac failure.^21,25 IP₃ receptors are upregulated in heart failure²⁶ and AF.²⁷ In atrial myocytes IP₃ caused spontaneous [Ca²⁺]_i transients, Ca²⁺ waves, and Ca²⁺ alternans,^20,21 and facilitated the generation of early (EADs) and delayed (DADs) after-depolarizations,²¹ all disturbances in Ca²⁺ signaling related to cardiac arrhythmias (see Kockscamper et al⁷). Notably these effects were absent in ventricular myocytes.²⁰

To study the role of IP₃-dependent Ca²⁺ release in heart at the cellular level has been difficult for several reasons, one of which is that the small IP₃-dependent Ca²⁺ signals are difficult to discern from the large [Ca²⁺]_i transients brought on by Ca²⁺ entry and Ca²⁺ release from RyRs during normal ECC.²⁰ Furthermore, many of the pharmacological activators and inhibitors of the IP₃R cannot be applied to intact cells or lack sufficient specificity to dissect and isolate contributions from IP₃Rs.²⁸ Therefore, we set out to generate IP₃R2-deficient mice to determine the role of IP₃R2 in the atrium and its potential role in AF.

Our results revealed that in response to endothelin-1 (ET-1), IP₃R2-deficient atrial myocytes failed to increase diastolic [Ca²⁺]_i and to have a positive inotropic response. ET-1 also failed to cause spontaneous Ca²⁺ release events in IP₃R2-deficient atrial myocytes. On the other hand, both wild-type (WT) and IP₃R2-deficient atrial myocytes showed a very similar strong positive inotropic response and enhanced Ca²⁺ spark activity after ß-adrenergic stimulation. No compensatory changes in the expression of Ca²⁺ regulatory proteins (IP₃R1, IP₃R3, RyR2, NCX, SERCA2) or morphology of the atria could be detected between WT and IP₃R2-deficient mice. These results show that IP₃R2 can be a major modulator of intracellular Ca²⁺ homeostasis in atrial myocytes and may play an important role for the initiation and perpetuation of AF.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Gene Targeting and Generation of IP₃R2-Deficient Mice
The IP₃R2 genomic DNA was isolated from a 129SVJ mouse genomic library (Stratagene) and used to construct the IP₃R2 targeting vector by standard techniques.²⁹ Briefly, 2 fragments of the IP₃R2 gene were cloned into a targeting vector that contained a neomycin selection cassette flanked by FRT sites. A 539-bp fragment containing exon 3 of IP₃R2 (116 bp) was inserted into 2 flanking LoxP sites (Figure 1A). The targeting vector was linearized with NotI and subsequently electroporated into R1 embryonic stem (ES) cells. G418-resistant ES clones (480 clones) were screened for homologous recombination by DNA blot analysis, as described below. Two independent homologous recombinant ES clones were microinjected into blastocysts from C57BL/6J mice to generate male chimeras. Male chimeras were bred with female Black Swiss mice to generate germline transmitted floxed heterozygous mice (IP₃R2^+/flox). IP₃R2^+/flox mice were subsequently intercrossed and crossed with Pro-Cre mice,³⁰ which restricted Cre expression to male germ cells undergoing spermatogenesis, to generate mice which were doubly heterozygous for IP₃R2 floxed allele and Pro-Cre allele (Pro-Cre, IP₃R2^+/flox). Pro-Cre, IP₃R2^+/flox males were crossed to female breeders to generate germline heterozygous null mutant offsprings (IP₃R2^+/–). Intercrosses of the IP₃R2^+/– were used to generate homozygous null mutant mice (IP₃R2^–/–).

View larger version (19K): [in this window] [in a new window]   Figure 1. Targeted generation of IP3R2-deficient mice. A, Targeting strategy, restriction map of the relevant genomic gene of IP3R2 is shown in the top panel, the targeting construct is shown in the center, and the mutated locus after recombination is shown at the bottom. The targeting construct was generated by flanking exon 3 of IP3R2 with 2 loxP sites and flanking the neo-cassette by frt sites. B indicates BamHI; K, KpnI; N, NotI, Neo represents neomycin resistance gene; arrowheads represent LoxP sites; and the long boxes represent frt sites. B, Detection of WT and targeted alleles by Southern blot analysis, DNAs isolated from 3 neo positive electroporated ES cells (lane 1 to lane 3) were digested with Acc65I and analyzed by DNA blot analysis with the probe as shown in A. The 14- and 7-kb bands represent WT and mutant allele, respectively. C, Protein analysis of IP3R2 in WT and IP3R2-deficient mice. Protein extracts from atria, ventricle, and lungs from mice were subjected to protein analysis, using IP3R2 specific antibodies. Antibodies specific for sarcomeric -actinin were used to normalize for protein loading.

DNA Analyses
Genomic DNA was extracted from G418-resistant ES cell clones and mouse tails, as previously described.³¹ ES cell DNA was digested using Acc65I, electrophoresed on a 1% (wt/vol) agarose gel, and subsequently blotted onto a nitrocellulose membrane. A 400-bp fragment was generated by polymerase chain reaction (PCR) using mouse genomic DNA and specific IP₃R2 primers (forward, TGGAAAGGTCTGGGTGAGTC; reverse, GGCTGCTGAGATGGACAAG). The PCR product was subsequently radiolabeled using -[³²P]dATP by random priming (Invitrogen). DNA blots were hybridized with the radiolabeled probe and visualized by autoradiography.

Offspring from intercrosses were genotyped by PCR analysis using mouse tail DNA and WT (forward, ACCCTGATGAGGGAAGGTCT; reverse, ATCGATTCATAGGGCACACC) and mutant allele specific primers (neo-specific primer: forward, AATGGGCTGACCGCTTCCTCGT; reverse, TCTGAGAGTGCCTGGCTTTT). PCR products were visualized by ethidium bromide staining.

Protein Blot Analysis
Total protein extracts were prepared from cardiac atria, ventricle, and lung from WT as well as IP₃R2-deficient (4-week-old) mice and protein analysis was performed, as previously described.²⁹ Immunodetection of IP₃R2 (270 kDa) was performed in cardiac atria, ventricular, and lung samples by using a rabbit polyclonal antibody to IP₃R2 (1:2000), as previously described.³² Immunodetection of -actinin (100 kDa) was performed on samples by using a mouse monoclonal antibody to sarcomeric -actinin (1:2000; Sigma). Immunodetection of IP₃R1 (274 kDa), IP₃R3 (267 kDa), ryanodine receptor type 2 (RyR2; 565 kDa), sodium-calcium exchanger (NCX;160 and 120 kDa), and sarcoplasmic reticulum Ca²⁺ ATPase (SERCA2a; 110 kDa) was performed on atrial samples by using rabbit polyclonal antibodies to IP₃R1 (1:2000), IP₃R3 (1:2000), and SERCA2a (1:10 000) or mouse monoclonal antibodies to RyR2 (1:3000; Affinity BioReagents) and NCX (1:500; Swant), as previously described.³³ Membranes were subsequently incubated with horseradish peroxidase–conjugated anti-rabbit Ig (1:3000; Sigma) or anti-mouse Ig (1:3000; Zymed). All results were visualized by using enhanced chemiluminescence (Amersham).

Cell Isolation
The procedure for cell isolation was in full compliance with the guidelines approved by the Institutional Animal Care and Use Committee of Loyola University Chicago, Stritch School of Medicine. Mice were anesthetized in a gas chamber with 3% to 5% isoflurane in a 100% O₂ atmosphere. Hearts were excised from adult IP₃R2-deficient and age- and strain-matched WT mice. Hearts were mounted on a Langendorff-perfusion apparatus and perfused for 6 minutes at 37°C with nominally Ca²⁺-free Dulbecco’s modified Eagle’s medium (DMEM; GIBCO type 22300) gassed with a 95% O₂/5% CO₂ mixture. Perfusion was then switched to the same solution containing 0.5 to 0.8 mg/mL collagenase (type B, Boehringer Mannheim) and 20 µmol/L Ca²⁺ with perfusion continuing until the heart became flaccid (7 to 12 minutes). Atria were removed, enzyme activity was stopped by switching the solution to DMEM containing 0.5% to 1% BSA, and the atria were cut into smaller pieces and gently triturated with a glass pipette to separate individual cells. Isolated myocytes were plated on glass cover slips which formed the bottom of the experimental superfusion chamber, mounted on an inverted microscope for [Ca²⁺]_i measurements. All experiments were performed at room temperature (22 to 24°C).

[Ca²⁺]_i Measurements
[Ca²⁺]_i was measured with fluorescence laser scanning confocal microscopy. Intact atrial myocytes were loaded with the Ca²⁺ indicator fluo-4x20 minutes incubation in Tyrode solution containing 20 µmol/L fluo-4 acetoxymethyl ester (fluo-4/AM; Molecular Probes) at room temperature. Cells were superfused continuously with normal Tyrode solution (in mM: NaCl 140; KCl 4; CaCl₂ 1; MgCl₂ 1; glucose 10; HEPES 10; pH 7.4). Fifteen to 20 minutes was allowed for deesterification of the dye. The laser scanning confocal microscope was a Radiance 2000 MP (BioRad) attached to a Nikon TE300 inverted microscope and equipped with a 40x oil-immersion objective lens (Nikon CFI Plan Fluor; N.A.=1.3). Fluo-4 was excited with the 488 nm line of an argon ion laser and fluorescence was measured at wavelengths >515 nm. Images were acquired in the transverse linescan mode (3 or 6 ms/scan; pixel size 0.1 µm). [Ca²⁺]_i transients are presented as background-subtracted normalized fluorescence (F/F₀) where F is the fluorescence intensity and F₀ is resting fluorescence recorded under steady-state conditions at the beginning of an experiment. [Ca²⁺]_i transients were evoked by electrical field stimulation (0.5 Hz). Ca²⁺ spark frequencies are expressed as number of observed sparks per second and per 100 µm of scanned distance in the confocal linescan mode (sparks* s^–1*(100 µm)^–1)

Drugs
Endothelin-1, 2-aminoethoxydiphenyl borate (2-APB) and isoproterenol were obtained from Sigma.

Statistics
Data are presented as means±SEM obtained from n different cells. Statistical differences between data sets were evaluated by Student t test.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Targeted Disruption of the Mouse IP₃R2 Gene
The IP₃R2 gene replacement targeting construct is depicted in Figure 1A (middle). The targeting construct was designed to flank exon 3 of the IP₃R2 gene by loxP sites (triangles) as well as to contain the neomycin (Neo) selection cassette flanked by FRT sites (black rectangles; Figure 1A, middle). Targeted ES cells were identified by Southern blot hybridization analysis, giving the expected 7-kb mutant fragment compared with the WT 14-kb fragment (Figure 1B). Two of 480 neo resistant ES cells were identified as targeted ES cells. These 2 ES cell clones were independently injected into blastocysts which gave rise to chimeric mice that were then used to breed to generate germline-transmitting heterozygous floxed mice (IP₃R2^+/flox). The IP₃R2^+/flox mice were crossed with protamine-cre (Pro-Cre) mice,³⁰ generating mice which were doubly heterozygous (Pro-Cre, IP₃R2^+/flox). Cre expression in Pro-Cre mice is restricted to male germ cells undergoing spermatogenesis. Therefore, Pro-Cre, IP₃R2^+/flox males were crossed with female breeders to generate germline heterozygous null mutant offsprings (IP₃R2^+/–). Intercrosses of the IP₃R2^+/– mice were used to generate homozygous null mutant mice (IP₃R2^–/–).

To ensure that the mutant allele is a true null allele, protein blot analysis was performed using protein samples from cardiac atria, ventricle, and lung extracts from WT and IP₃R2-deficient hearts, using specific IP₃R2 antibodies. As shown in Figure 1C, no IP₃R2 protein could be detected in the IP₃R2-deficient tissues. Furthermore, the data revealed that IP₃R2s appear to be more abundant in atrial tissue when compared with ventricle.

Homozygous IP₃R2-deficient mice were viable and fertile. Assessment of Mendelian ratios from IP₃R2 intercrosses revealed that of the 327 offspring analyzed, 82 (25.1%) were homozygous for the disrupted allele and 91 (27.8%) were WT, indicating that IP₃R2 is not essential for normal embryonic development and survival. This is in contrast to IP₃R1-deficient mice, as the majority of IP₃R1 homozygous mutants are embryonic lethal and those surviving have severe ataxia and tonic/tonic-clonic seizures and die by the weaning period.³⁴ These data suggest that IP₃R subtypes have distinct functional roles already during development.

Action Potential Induced [Ca²⁺]_i Transients in Intact WT and IP₃R2-Deficient Atrial Myocytes
To study the functional consequences of IP₃R2 deficiency in cardiac ECC and Ca²⁺ signaling, we focused on atrial myocytes, because previous studies have demonstrated IP₃R-dependent Ca²⁺ release in this tissue,^20,21,24 but not in ventricular cells.²⁰ Single atrial myocytes were isolated from IP₃R2-deficient (IP₃R2-KO) and WT littermate mice and loaded with the Ca²⁺-sensitive indicator fluo-4/AM to record changes in [Ca²⁺]_i by confocal microscopy. AP-dependent [Ca²⁺]_i transient were evoked by field stimulation (0.5 Hz; Figure 2A and 2C, left). As summarized in supplemental Table I (available online at http://circres.ahajournals.org) the spatiotemporal properties of [Ca²⁺]_i transients (amplitude, rise time, decay kinetics, and spatial spread from the periphery to the center of the cells) did not differ significantly between WT and IP₃R2-KO atrial myocytes. The data suggest that under basal conditions the absence of the IP₃R2 had little or no effect on ECC.

View larger version (40K): [in this window] [in a new window]   Figure 2. Effects of ET-1 on Ca2+ signaling in intact WT and IP3R2-deficient atrial myocytes. Confocal linescan (raw fluo-4 fluorescence) images and spatially averaged [Ca2+]i (F/F0) transients recorded from field-stimulated (0.5 Hz) WT (A and B) and IP3R2-deficient (KO) (C) atrial myocytes. The traces were spatially averaged over the region marked by the red boxes to the left of the images. A, [Ca2+]i transients under control conditions (left) and during exposure to 100 nM ET-1 (right) in WT cells. Arrowheads and asterisks indicate spontaneous (nontriggered) Ca2+ release attributable to after-depolarizations and Ca2+ sparks, respectively. B, [Ca2+]i transients under control conditions (left) and during exposure to 100 nM ET-1 (right) in WT cells in the presence of the IP3R blocker 2-APB (5 µmol/L). C, [Ca2+]i transients under control conditions (left) and during exposure to 100 nM ET-1 in IP3R2-KO atrial myocytes. Average changes in diastolic [Ca2+]i (D) and electrically evoked [Ca2+]i transient amplitude (E) in the presence of ET-1, normalized to control conditions (100%, dashed line). Statistical significance levels: *P<0.05; +P<0.01; #P<0.001.

Endothelin-1 Effects on Ca²⁺ Signaling in Intact IP₃R2-KO and WT Atrial Myocytes
ET-1 has been shown to increase endogenous [IP₃] in atrial cells³⁵ and to cause IP₃-dependent Ca²⁺ release.^20,21 Consequently, we used ET-1 to assess the effects of IP₃R2 deficiency on Ca²⁺ signaling in atrial myocytes. In electrically stimulated WT cells (Figure 2A), exposure to ET-1 (100 nM) resulted in a significant increase in diastolic [Ca²⁺]_i (146±5% compared with control; n=17 cells; P<0.001; Figure 2D) and AP-dependent [Ca²⁺]_i transient amplitude (135±6% compared with control; n=17; P<0.001; Figure 2E). Furthermore, signs of spontaneous Ca²⁺ release activity were observed in WT atrial myocytes in the presence of ET-1. Specifically, WT atrial myocytes revealed spontaneous Ca²⁺ sparks (asterisks in Figure 2A, right; see also Figures 3 and 4) and Ca²⁺ release events triggered by spontaneous APs or delayed after depolarizations (DAD) (Figure 2A; arrowheads). The effects of ET-1 on diastolic [Ca²⁺]_i and [Ca²⁺]_i transient amplitude could be abolished by preincubation with the membrane permeant IP₃R antagonist 2-APB (Figure 2B, 2D, and 2E) that has been used successfully to inhibit IP₃R-dependent Ca²⁺ release in cardiac cells.^20,21,36

View larger version (40K): [in this window] [in a new window]   Figure 3. SR functon in WT and IP3R2-deficient atrial myocytes. A, Fluo-4 linescan images and F/F0 traces of electrically evoked [Ca2+]i transients, followed by caffeine (10 mmol/L) application to estimate SR Ca2+ load. B, Average fractional SR Ca2+ release in WT and IP3R2-KO atrial myocytes. [ET-1]=100 nM, [2-APB]=5 µmol/L. C, Fluo-4 linescan images and F/F0 traces of electrically evoked [Ca2+]i transients recorded under control conditions and in the presence of isoproterenol (ISO; 1 µmol/L) in WT (top) and IP3R2-deficient (KO; bottom) atrial myocytes. D, Average normalized increase in [Ca2+]i transient amplitude by ISO in WT and IP3R2-deficient (KO) atrial myocytes (100%=control). Whole-cell [Ca2+]i transients were obtained by averaging the entire cellular fluorescence signal from the line scanned.

View larger version (98K): [in this window] [in a new window]   Figure 4. Ca2+ sparks in WT (A and B) and IP3R2-deficient (C through E) atrial myocytes. Fluo-4 linescan images and selected plots of subcellular [Ca2+]i transients recorded from the areas marked by the red boxes. The images shown represent periods between 2 subsequent evoked [Ca2+]i transient. A, Basal spark activity under control conditions and in the presence of ET-1 (100 nM) in WT atrial myoctes. B, Effect of ET-1 in the presence of the IP3R blocker 2-APB (5 µmol/L) in WT cells. Ca2+ sparks in IP3R2-KO atrial myocyes in the presence of (C) ET-1, (D) isoproterenol (ISO, 1 µmol/L), and (E) after a period of high frequency (2 Hz) stimulation.

ET-1 exposure failed to significantly increase diastolic [Ca²⁺]_i in atrial myocytes from IP₃R2-deficient mice (Figure 2C and 2D), and no signs of spontaneous Ca²⁺ release were observed. In fact, ET-1 stimulation caused a decrease in [Ca²⁺]_i transient amplitude in IP₃R2-deficient atrial myocytes (Figure 2E). The [Ca²⁺]_i transient amplitude recovered after removal of ET-1 (data not shown). The same decrease in [Ca²⁺]_i transient amplitude was observed in WT myocytes pretreated with 2-APB. The latter suggests that basal IP₃ levels may have a small positive inotropic effect under control conditions. This is consistent with the slightly smaller [Ca²⁺]_i transient amplitude of IP₃R2-KO myocytes (supplemental Table I) even though this difference was not statistically significant.

SR Function in WT and IP₃R2-KO Atrial Myocytes
To assess whether the failure of IP₃R2-deficient atrial myocytes to respond to ET-1 was caused by differences in the efficacy of ECC, we measured SR Ca²⁺ load and fractional SR Ca²⁺ release. For this purpose SR Ca²⁺ load was measured by exposure of WT and IP₃R-KO cells to 10 mmol/L caffeine (Figure 3A). The amplitude of the caffeine-induced [Ca²⁺]_i transient was not significantly different between WT and IP₃R-KO mice (supplemental Table I). Fractional release, defined as the fraction (%) of Ca²⁺ release during an AP-induced twitch compared with total releasable Ca²⁺, is approximated by comparing the amplitudes of AP-induced [Ca²⁺]_i transients with caffeine-induced [Ca²⁺]_i transients ([Ca²⁺]_i,twitch/[Ca²⁺]_i,caffeine). The average fractional release under control conditions was 55±4% in WT (n=6) and 56±4% in IP₃R2-KO (n=9) myocytes, respectively (Figure 3B). Whereas there was no significant difference in fractional SR Ca²⁺ release under control conditions, the fractional release increased significantly (68±3%; n=6; P<0.01) in WT cells in the presence of ET-1 (Figure 3B). In contrast, ET-1 had no effect of fractional release in IP₃R2-KO myocytes (58±4%; n=5). Furthermore, 2-APB prevented the effect of ET-1 on fractional release in WT cells (59±7%; n=3; Figure 3B). The data revealed that in WT atrial cells, ET-1 exerts its positive inotropic effect through improvement of the efficacy of ECC. Because this effect was absent in IP₃R2-KO cells and was abolished by 2-APB in WT cells, we concluded that IP₃-dependent Ca²⁺ signaling plays a crucial role in mediating the ET-1 effect.

We also tested the availability of releasable Ca²⁺ from the SR through ß-adrenergic stimulation (isoproterenol, 1 µmol/L). Isoproterenol causes a positive inotropic effect by affecting [Ca²⁺]_i regulation and ECC through various mechanisms.¹⁹ In electrically stimulated WT and IP₃R2-deficient atrial myocytes, isoproterenol increased the amplitude of the [Ca²⁺]_i transient to 300% to 350% of control (Figure 3C and 3D). The relative increase in transient amplitude was not significantly different between WT and IP₃R2-deficient atrial myocytes, indicating that both cell types have the same significant inotropic reserve.

Ca²⁺ Sparks in IP₃R2-Deficient and WT Atrial Myoctes
Ca²⁺ sparks are considered the elementary Ca²⁺ release events during ECC and represent a localized nonpropagating elevation of [Ca²⁺]_i that results from the concerted opening of a group of clustered RyRs in the SR membrane.^22,23 In WT atrial myocytes (Figure 4A), ET-1 increased the Ca²⁺ spark frequency 4-fold (from 0.49±0.20 in control [n=12] to 2.14±0.64 sparks*s^–1*[100 µm]^–1 in the presence of ET-1 [n=10]; P<0.01; Figure 5). This effect was abolished in the presence of the IP₃R blocker 2-APB (n=5; Figures 4B and 5). 2-APB alone had very little effect on Ca²⁺ sparks (n=5). In IP₃R2-deficient atrial myocytes, the overall Ca²⁺ spark frequency was >5-fold lower (0.09±0.06 sparks*s^–1*[100 µm]^–1; n=5) compared with WT cells, and ET-1 had no significant effect on Ca²⁺ spark frequency. However, different interventions had a profound effect on Ca²⁺ spark frequency also in IP₃R2-KO myocytes. ß-adrenergic stimulation with isoproterenol ([ISO]=1 µmol/L) significantly increased the Ca²⁺ spark frequency to 2.45±0.46 sparks*s^–1*(100 µm)^–1. In WT myocytes, ISO stimulation caused a virtually identical increase in spark frequency (2.48±1.00 sparks*s^–1*[100 µm]^–1; Figure 5). Electrical stimulation at higher frequency (2 Hz) was followed by a period of intense spark activity (HF in Figures 4E and 5). The data suggest that in conditions that lead to increased SR Ca²⁺ load (ß-adrenergic and HF stimulation) IP₃R2-KO atrial myocytes are capable of higher Ca²⁺ spark activity, despite the lack of IP₃R2. The results further confirm that the effect of ET-1 on Ca²⁺ sparks in WT cells critically depend on a functional IP₃R2-dependent Ca²⁺ release. In WT atrial myocytes, IP₃-dependent Ca²⁺ release has been shown to facilitate Ca²⁺ sparks from RyRs through a mechanism by which Ca²⁺ release from IP₃Rs increases [Ca²⁺]_i in the vicinity of RyRs and thereby increases the probability of CICR.^18,20 Our observation that IP₃R2-deficient atrial myocytes revealed an overall lower spark activity may suggest that in WT cells, basal levels of IP₃ cause a small background release of Ca²⁺ from IP₃Rs which enhances the propensity of spontaneous Ca²⁺ sparks.

View larger version (14K): [in this window] [in a new window]   Figure 5. Ca2+ spark frequencies. Ca2+ spark frequencies measured in WT (black bars) and IP3R2-KO (gray bars) atrial myocytes. Spark frequency is expressed as the number of sparks per second and per 100 µm of scanned distance (sparks*s–1*[100 µm]–1). Experimental conditions: ET-1, 100 nM; ISO, 1 µmol/L; 2-ABP, 5 µmol/L; HF, high frequency stimulation at 2 Hz. Statistical significance levels: *P<0.05; +P<0.01; #P<0.001.

Expression of Ca²⁺ Regulatory Proteins in IP₃R2-Deficient and WT Atria
To assess whether the failure of IP₃R2-deficient atrial myocytes to respond to ET-1 could be attributable to perturbations in the baseline expression of other IP₃R subtypes or well-established Ca²⁺ regulatory proteins in WT and IP₃R2-deficient atria, we performed protein blot analysis on cardiac atria extracts to assess for IP₃R1, IP₃R3, RyR2, NCX, and SERCA2 expression, using specific antibodies. As shown in Figure 6, no compensatory changes in IP₃R1 and IP₃R3 protein levels could be detected in the IP₃R2-deficient atria. In addition, no significant changes in RyR2, NCX, and SERCA2 protein could be detected between WT and IP₃R2-deficient atria (Figure 6), suggesting that IP₃R2 deficiency does not affect the expression of other Ca²⁺ regulatory proteins relevant for ECC. Furthermore, no significant changes in atria weight/body weight (BW) ratios were observed between WT (left atria/BW: 0.013±0.001; right atria/BW: 0.011±0.0006; n=5), and IP₃R2-deficient (left atria/BW: 0.012±0.0008; right atria/BW: 0.012±0.0009; n=7) atria, suggesting that IP₃R2-deficient atria were not grossly morphologically different from WT atria. As a result, the failure of IP₃R2-deficient atrial myocytes to respond to ET-1 was not attributable to changes in atrial morphology or expression profile of Ca²⁺ regulatory proteins in IP₃R2-deficient atria.

View larger version (71K): [in this window] [in a new window]   Figure 6. Protein analysis of IP3R1, IP3R3, RyR2, NCX, and SERCA2 in WT and IP3R2-deficient mice. Atrial extracts from WT and IP3R2-deficient mice were subjected to protein analysis, using IP3R1, IP3R2, IP3R3, RyR2, NCX, and SERCA2 specific antibodies. Antibodies specific for sarcomeric -actinin were used to normalize for protein loading (data not shown).

Summary and Conclusions
Neurohormonal agents (ET-1, angiotensin II, catecholamines), which activate the G protein–PLC signaling pathway and IP₃ production, have been implicated in the generation of atrial arrhythmias and fibrillation.^25,37 Recent studies have suggested that IP₃R expression and IP₃R-dependent Ca²⁺ release play an important role in the generation of arrhythmias and atrial fibrillation (see introduction), however many aspects of the underlying mechanisms linking IP₃R/Ca²⁺ signaling and AF have remained elusive. Nonetheless, in cardiac hypertrophy and heart failure, not only IP₃Rs are upregulated, but also levels of neurohumoral factors, including ET-1 levels, are elevated and ET-receptor expression is altered.³⁸ IP₃R-dependent Ca²⁺ release has been shown to enhance ECC and to have positive inotropic effects. This positive inotropic effect occurs through IP₃R-dependent Ca²⁺ release in the vicinity of clusters of RyRs, which in turn facilitates CICR from RyRs and consequently enhances contraction. Whereas this, in principal, represents an additional mechanism of inotropic reserve and therefore can be considered beneficial, it also can become detrimental to normal Ca²⁺ release and ECC: enhanced IP₃-signaling and IP₃-dependent Ca²⁺ release also increases the likelihood of spontaneous Ca²⁺ release (Ca²⁺ sparks and waves), leads to Ca²⁺ alternans (a proarrhythmic condition;⁷), and to spontaneous action potentials and spontaneous [Ca²⁺]_i transients. Thus, through such a mechanism IP₃-dependent Ca²⁺ release can enhance the propensity of atrial arrhythmias^18,20,21 which are common in cardiac hypertrophy and heart failure.

Whereas previous studies on the role of IP₃R-dependent Ca²⁺ signaling for atrial arrhythmias relied heavily (and almost exclusively) on pharmacological tools to perturb the IP₃ signaling cascade, we present in this study a new animal model exquisitely suitable to study IP₃R2-dependent Ca²⁺ signaling for cardiac function. The IP₃R2-deficient mice, which we generated, are viable and develop phenotypically normal. Under basal conditions the Ca²⁺ signals during ECC in IP₃R2-KO atrial cells appear to be indistinguishable from WT cells, and both cell types reveal a similar responsiveness ([Ca²⁺]_i transients and Ca²⁺ sparks) to ß-adrenergic stimulation. Furthermore, no compensatory changes in Ca²⁺ regulatory protein expression (IP₃R1, IP₃R3, RyR2, NCX, SERCA2) or morphology of the atria could be detected in IP₃R2-deficient mice. However, the responsiveness to the neurohumoral agonist ET-1 is altered, and atrial cells from IP₃R2-deficient mice are significantly less prone to develop proarrhythmic disturbances in Ca²⁺ signaling. IP₃R2-deficient mice represent an important model for future studies of IP₃R2 signaling in cardiac ECC and arrhythmias.

	Acknowledgments

 
This work was supported by the National Institutes of Health (R01HL66100 to J.C. and R01HL62231 to L.A.B.) and the Potts Estate, Loyola University Chicago (RFC 107457 to A.V.Z.). We thank Dr Gregory A. Mignery for providing us the IP₃R1, IP₃R2, and IP₃R3 antibodies and Dr Wolfgang H. Dillmann (University of California, San Diego) for providing us with the SERCA2 antibody. We thank Brian French for excellent assistance with the myocyte isolation, and Drs Donald M. Bers and Gregory A. Mignery (Loyola University of Chicago) for valuable comments on the manuscript.

	Footnotes

 
Original received March 8, 2005; resubmission received May 11, 2005; accepted May 24, 2005.

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