Increased InsP₃Rs in the junctional sarcoplasmic reticulum augment Ca²⁺ transients and arrhythmias associated with cardiac hypertrophy

Abstract

Cardiac hypertrophy is a growth response of the heart to increased hemodynamic demand or damage. Accompanying this heart enlargement is a remodeling of Ca²⁺ signaling. Due to its fundamental role in controlling cardiomyocyte contraction during every heartbeat, modifications in Ca²⁺ fluxes significantly impact on cardiac output and facilitate the development of arrhythmias. Using cardiomyocytes from spontaneously hypertensive rats (SHRs), we demonstrate that an increase in Ca²⁺ release through inositol 1,4,5-trisphosphate receptors (InsP₃Rs) contributes to the larger excitation contraction coupling (ECC)-mediated Ca²⁺ transients characteristic of hypertrophic myocytes and underlies the more potent enhancement of ECC-mediated Ca²⁺ transients and contraction elicited by InsP₃ or endothelin-1 (ET-1). Responsible for this is an increase in InsP₃R expression in the junctional sarcoplasmic reticulum. Due to their close proximity to ryanodine receptors (RyRs) in this region, enhanced Ca²⁺ release through InsP₃Rs served to sensitize RyRs, thereby increasing diastolic Ca²⁺ levels, the incidence of extra-systolic Ca²⁺ transients, and the induction of ECC-mediated Ca²⁺ elevations. Unlike the increase in InsP₃R expression and Ca²⁺ transient amplitude in the cytosol, InsP₃R expression and ECC-mediated Ca²⁺ transients in the nucleus were not altered during hypertrophy. Elevated InsP₃R2 expression was also detected in hearts from human patients with heart failure after ischemic dilated cardiomyopathy, as well as in aortic-banded hypertrophic mouse hearts. Our data establish that increased InsP₃R expression is a general mechanism that underlies remodeling of Ca²⁺ signaling during heart disease, and in particular, in triggering ventricular arrhythmia during hypertrophy.

In response to increased hemodynamic requirements or damage the heart undergoes a hypertrophic growth response. Hypertrophy is induced by physiological stimuli, such as exercise or pregnancy and by pathological conditions such as hypertension and ischemic heart disease. Although hypertrophy can initially be an adaptive compensatory response, chronically it may become decompensated. As a result, cardiac function is decreased and the heart exhibits an increased propensity for arrhythmias that together ultimately lead to heart failure and death (1).

Ca²⁺ is a fundamental regulator of cardiac function causing myocyte contraction via excitation-contraction coupling (ECC) (2), and stimulating the gene transcription that underlies hypertrophy (3). Accompanying cardiac hypertrophy and failure is a remodeling of Ca²⁺ signaling (4). Whilst enhanced Ca²⁺ transients facilitate greater myocyte contraction during adaptive hypertrophy, Ca²⁺ fluxes are diminished during heart failure and thereby contribute to decreased cardiac output (5). Remodeling of the Ca²⁺ signaling proteome also underlies the increased arrhythmias associated with hypertrophy and heart failure (6).

In addition to the RyRs that mediate ECC-dependent Ca²⁺ fluxes, cardiomyocytes also express InsP₃Rs, albeit outnumbered by RyRs at approximately 50:1 (7). Mammals have 3 InsP₃R isoforms (types 1–3) (8), with InsP₃R2 being the main isoform in cardiomyocytes (9, 10). Although Ca²⁺ flux via these InsP₃Rs is relatively small in comparison to the large Ca²⁺ transients occurring during every heartbeat, recent data suggests that InsP₃Rs have an important role in cardiac physiology. We, and others have shown, that Ca²⁺ release through InsP₃Rs contributes to the inotropic, arrhythmogenic, and hypertrophic effect of Gα_q-coupled agonists such as the vasoactive peptide ET-1 (11–16). Whether altered InsP₃R signaling also contributes to remodeling of Ca²⁺ homeostasis during cardiac hypertrophy is not yet determined. An increase in InsP₃R expression has however been reported during heart failure in humans (17). Moreover, InsP₃-induced Ca²⁺ release (IICR) is increased in SR microsomes prepared from hypertrophic myocytes (18).

Here, we hypothesized that enhanced Ca²⁺ release via InsP₃Rs contributes to remodeling of ECC-mediated Ca²⁺ transients, and to the increased arrhythmogenic Ca²⁺ signals observed in ventricular cardiomyocytes during compensated hypertrophy. To test these hypotheses, in a model that reflects the slow development of hypertrophy in humans, Ca²⁺ fluxes and contractility were investigated in hypertrophic ventricular myocytes isolated form SHRs (19). We found that the increase in amplitude of ECC-mediated Ca²⁺ transients and propensity for extra-systolic spontaneous Ca²⁺ signals, characteristic of hypertrophic myocytes, was caused by augmented InsP₃R signaling. This profound effect of enhanced InsP₃R activity in hypertrophic myocytes was due to an increase in InsP₃R expression, specifically in the junctional SR membrane in close proximity to RyRs. At this location, Ca²⁺ release via InsP₃Rs acted to sensitize RyRs, thereby enhancing Ca²⁺ release during ECC and inducing spontaneous elementary Ca²⁺-release events and extra-systolic Ca²⁺ transients. InsP₃R2 expression was also increased in hypertrophic cardiomyocytes isolated from aortically banded mice and in human hearts displaying ischemic dilated cardiomyopathy. We propose that InsP₃Rs play a fundamental role in the physiology of hypertrophic hearts contributing to remodeled cardiac function and triggering ventricular arrhythmia.

Results

SHR Cardiomyocytes Develop Hypertrophy.

As previously described, at 6 months, cardiomyocytes from SHRs are hypertrophic (20). Cardiomyocyte width was increased in SHRs compared to WKY controls (Table S1) resulting in a decrease in the cellular length/width ratio (WKY: 3.41 ± 0.11 vs. SHR: 2.71 ± 0.12; P < 0.001). Messenger RNA levels of the hypertrophic marker atrial natriuretic factor (ANF) (21) were also greater in SHR cardiomyocytes at the age of 6 months than in WKY controls (12.68 ± 4.04 vs. 0.69 ± 0.32, P < 0.05). In 12-week-old animals, cell size and ANF mRNA levels were not different between the 2 strains (Table S1).

Ca²⁺ Release via InsP₃Rs Is Increased During Hypertrophy and Remodels ECC.

Consistent with previous observations (20), the amplitude of electrically evoked systolic Ca²⁺ transients was greater in cardiomyocytes from 6-month-old SHRs than in WKY controls (SHR: 0.70 ± 0.07 mM, WKY: 0.50 ± 0.06 mM; n = 48 and 42, respectively, P < 0.05). Fractional shortening of myocytes under basal conditions was however not significantly different between the 2 strains (WKY: 10.35 ± 1.50%, SHR: 7.59 ± 0.98%).

To reveal whether increased Ca²⁺ release via InsP₃Rs contributes to remodeling of Ca²⁺ signaling and myocyte function during hypertrophy, we measured global Ca²⁺ transients and cellular contraction under conditions where InsP₃Rs were either inhibited or activated. As Ca²⁺ transients are greater under basal conditions in hypertrophic SHR than in WKY myocytes, the systolic Ca²⁺ amplitude was normalized to that before treatment. Inhibition of InsP₃Rs with 2-APB (2 μM) (11) caused a greater reduction in ECC-mediated Ca²⁺ transient amplitude in SHR myocytes compared with WKY controls (Fig. 1A). Concurrently, 2-APB also decreased the magnitude of contraction in SHRs (Fig. 1Aii). These data suggested that Ca²⁺ release via InsP₃Rs contributes to the greater basal ECC-associated Ca²⁺ transients observed in SHR myocytes.

Fig. 1.

Systolic Ca²⁺ transient amplitude and cellular contraction recorded from indo-1 AM-loaded ventricular myocytes isolated from 6-month-old WKY rats and SHRs. Data presented were obtained 20 min after application of 2-APB or InsP₃ ester. Representative traces are shown in i; values normalized to pre-application are shown in ii. For better comparison, single traces have been time-shifted. (A) Effect of 2 μM 2-APB on Ca²⁺ transient amplitude and fractional shortening. (B) Effect of 10 μM InsP₃ ester ± 2 μM 2-APB on Ca²⁺ transient amplitude. (C) As in B on fractional shortening. N numbers are indicated. *, P < 0.05; Student's t test.

Direct activation of InsP₃Rs with InsP₃ ester (11) promoted a greater increase in Ca²⁺transient amplitude and inotropy in SHR compared to WKY myocytes (Fig. 1 B and C), which was abrogated in both strains by 2-APB (Fig. 1 Bii and Cii). No difference in ECC-mediated Ca²⁺ transient amplitude or cellular contraction was observed between myocytes isolated from 12-week-old WKY rats or SHRs (Fig. S1A).

InsP₃R2 Expression Is Increased in Hypertrophic Cardiomyocytes.

Next, we analyzed whether an increase in InsP₃R expression underlies altered InsP₃ signaling during hypertrophy. At 6 months, InsP₃R2 mRNA and protein levels were higher in SHR than in WKY myocytes, whereas at 12 weeks, InsP₃R2 mRNA and protein levels were lower in SHRs compared to WKY controls (Fig. 2 A and B). RyR2 protein levels were not different between the 2 strains at the age of 12 weeks or 6 months (Fig. 2B).

Fig. 2.

Expression of InsP₃R2 during hypertrophy. (A) Relative InsP₃R2 mRNA levels. Values for SHRs have been normalized to age-matched WKY rats. (Bi) Relative InsP₃R2 and RyR2 protein levels. SHR/WKY ratios have been determined for 12-week- and 6-month-old rats. Representative immunoblots for InsP₃R2 (ii) and RyR2 (iii) are shown. Seventy-five micrograms membrane proteins from ventricular cardiomyocytes were loaded per lane. (C) Immunofluorescent staining for InsP₃R2 and RyR2. DAPI was used to stain the nuclei. (Scale bar, 30 μm.) Pixels positive for InsP₃R2 and RyR2 are shown in white. (D) Ratio of cytosolic/perinuclear fluorescence intensity in a 3-μm ring in these 2 regions. (E) Pearson's coefficient for co-localization of RyR2 and InsP₃R2. (F) Profiles of RyR2 and InsP₃R2 fluorescence intensity sampled along the longitudinal axis of a WKY (i) or SHR (ii) myocyte, as depicted by the white lines on the overlay images in C. (G) Representative immunoblots detecting InsP₃R2 in hearts from mice that have undergone aortic banding (AOB) and control mice. (H) As in G in human disease hearts from patients showing ischemic cardiomyopathy (isch) as well as control hearts (ctrl). Forty micrograms membrane proteins from left ventricles were loaded per lane. Calnexin and calsequestrin were used as loading controls. N numbers are indicated. *, P < 0.05; Student's t test.

Immunofluorescent labeling revealed that in WKY myocytes, InsP₃R2 was predominantly expressed in the perinuclear regions with weaker staining along the SR membrane, where RyRs are localized (Fig. 2C). InsP₃R2 was also expressed in the perinuclear regions of SHR cardiomyocytes, but compared to WKY cells its expression was significantly greater along the RyR2-stained striations outside the nuclear region. Thus, the ratio of cytosolic/nuclear InsP₃R2 immunofluorescence was increased (Fig. 2D). No difference in RyR2 immunostaining between the 2 strains was observed (Fig. 2C). In both WKY and SHR myocytes, InsP₃R2 co-localized with RyR2s (intensity profile along indicated line, Fig. 2 C and F) and Pearson's coefficient (Fig. 2E), indicating that like RyR2s, InsP₃R2s are located at dyadic junctions alongside T-tubule membranes (Fig. 2C). The co-localization of these 2 channels was markedly increased in hypertrophic myocytes (Fig. 2 E and F). We concluded that, as a result of hypertrophic remodeling, the number of InsP₃R2s located in the junctional SR membrane is increased, thereby mediating their greater co-localization with RyR2s.

InsP₃R2 expression was also increased in hearts from mice after aortic banding (Fig. 2G) and in human patients with ischemic dilated cardiomyopathy (Fig. 2H). These data suggested that increased InsP₃R2 expression is a general hallmark of hypertrophy.

Increased InsP₃R Expression in the Junctional SR Causes a Spatially Restricted Remodeling of ECC-mediated Ca²⁺ Transients during Hypertrophy.

To establish how increased InsP₃R expression in the junctional SR membrane impacted on ECC-mediated Ca²⁺ signals, we performed confocal Ca²⁺ imaging experiments. In addition to directly stimulating InsP₃Rs with a membrane-permeant InsP₃ ester, we tested the effect of physiologically activating InsP₃Rs with InsP₃ generated following ET-1 stimulation (11, 22). In SHR myocytes, stimulation with ET-1 and InsP₃ ester increased the amplitude of nuclear and cytosolic Ca²⁺ transients during electrical pacing (Fig. 3 A–E). Contrastingly, in WKY myocytes, only nuclear systolic Ca²⁺ transients were augmented (Fig. 3 C and E). The enhancement of systolic Ca²⁺ transients by ET-1 and InsP₃ ester was sensitive to 2-APB, further indicating that this effect was mediated by InsP₃Rs (Fig. 3 Bii–Eii). The increase in nuclear Ca²⁺ transient amplitude in SHRs was comparable to that observed in WKY myocytes (Fig. 3 C and E). To accommodate for variation between cells in the absolute magnitude of Ca²⁺ changes, the ratio of nuclear to cytosolic Ca²⁺ transient amplitude was calculated. This ratio was increased in WKY myocytes following ET-1 or InsP₃ ester stimulation whereas no change was observed in SHR myocytes (Fig. S2A). The difference in ratio between the 2 strains is explained by restriction of the ET-1- and InsP₃ ester-stimulated increase in Ca²⁺ transient amplitude to the nuclear compartment in WKY myocytes, whereas, in SHR myocytes nuclear and cytosolic Ca²⁺ transient amplitude were both increased. These data indicate that in non-hypertrophied myocytes, Ca²⁺ release via InsP₃Rs impacts more profoundly on nuclear Ca²⁺ transients, whereas in hypertrophic myocytes, increased junctional InsP₃R expression specifically augments the cytosolic Ca²⁺ transients. There was no difference in the ratio of nuclear to cytoplasmic Ca²⁺ transients between 12-week-old WKY and SHRs under basal conditions, or during ET-1 or InsP₃ ester stimulation (Fig. S1B).

Fig. 3.

Confocal analysis of systolic nuclear and cytosolic Ca²⁺ transient amplitude in fluo-4 AM-loaded ventricular myocytes. Data presented were determined 15 min after application of ET-1; InsP₃ ester or 2-APB. Representative traces are shown in i; values normalized to pre application are shown in (ii). For better comparison, single traces have been slightly time-shifted. (A) Representative SHR myocyte displaying Ca²⁺ transients at a series of time points before and after application of 10 μM InsP₃ester. (Scale bar, 10 μm.) (B) Effect of 100 nM ET-1 ± 2 μM 2-APB on cytosolic peak amplitude. (C) As in B for nuclear peak amplitude. (D) Effect of 10 μM InsP₃ ester ± 2 μM 2-APB on cytosolic peak amplitude. (E) as in D for nuclear peak amplitude. N numbers are indicated. *, P < 0.05; Student's t test.

Maximal Ca²⁺ release from nuclear and cytosolic Ca²⁺ stores induced by 10 mM caffeine (RyR agonist) was not significantly different between WKY and SHR myocytes (Fig. S2B), indicating that differences in Ca²⁺ store content do not underlie the changes in ECC-associated Ca²⁺ transients during ET-1 and InsP₃ ester stimulation.

Extra-Systolic InsP₃-dependent Ca²⁺-release Events Are Increased in SHR Myocytes.

In atrial cardiomyocytes, which express approximately 6 fold more InsP₃Rs than ventricular myocytes, Ca²⁺ release via InsP₃Rs underlies the induction of extra-systolic Ca²⁺ transients (11, 12). We therefore investigated whether the increased InsP₃R expression and activity observed in SHR ventricular myocytes caused them to exhibit more extra-systolic Ca²⁺-release events. Extra-systolic events were determined as rises in Ca²⁺ concentration that were temporally distinguished from signals induced by field stimulation and that also impacted on contraction (see arrows Fig. 4A). Stimulation with ET-1 or InsP₃ ester caused a 2-APB-sensitive increase in the number of extra-systolic Ca²⁺ transients in both SHR and WKY myocytes (Fig. 4B and Table S2). However, the number of cells that exhibited extra-systolic Ca²⁺ transients and the frequency of events per cell were greater in SHRs than WKYs (ET-1: WKY: 26% vs. SHR: 50%; InsP₃ ester: WKY: 29% vs. SHR: 57%, Table S2 and Fig. 4B). In both strains, extra-systolic Ca²⁺-release events began to occur within a few minutes of InsP₃ ester or ET-1 stimulation and increased throughout the time-course of the experiment (Fig. 4B). The rate at which the frequency of the extra-systolic Ca²⁺ transients increased following InsP₃ ester or ET-1 stimulation was greater for SHR myocytes than WKY myocytes. After 1,000 s, the incidence of extra-systolic Ca²⁺ transients was significantly higher in SHRs than WKY cells (Fig. S3). No difference in the frequency of extra-systolic Ca²⁺ transients was observed between the 2 strains at 12 weeks (Fig. S1 C and D). These data indicate that activation of InsP₃Rs was responsible for the initiation of the extra-systolic Ca²⁺ transients and provides an explanation for the increased frequency of extra-systolic Ca²⁺ transients during hypertrophy.

Fig. 4.

Analysis of extra-systolic Ca²⁺ release events in indo-1 AM-loaded ventricular myocytes during hypertrophy. (A) Representative traces for global Ca²⁺ transients and cellular contraction recorded from SHR myocytes before and after stimulation with 100 nM ET-1 or 10 μM IP₃ ester. Arrows indicate extra-systolic events. (B) Extra-systolic Ca²⁺ release events per cell during stimulation with 100 nM ET-1 or 10 μM InsP₃ ester.

Enhanced Ca²⁺ Release via InsP₃Rs Increases the Rate of Rise of Systolic Ca²⁺ Transients and Elevates Diastolic [Ca²⁺] in SHRs.

A hypertrophy-associated increase in InsP₃R-mediated Ca²⁺ flux via junctional InsP₃Rs acting to induce Ca²⁺ release via neighboring RyRs could provide a mechanism to accentuate Ca²⁺ signaling during ECC. To test this hypothesis, the effect of ET-1 and InsP₃ ester on the rate of rise of pacing-evoked systolic Ca²⁺ transients was measured. During both ET-1 and InsP₃ ester stimulation, the rate of rise of the Ca²⁺ transient was faster in hypertrophic SHR than in WKY cells (Fig. 5A). The effects of ET-1 and InsP₃ ester were abrogated by adenoviral-mediated expression of a cherry fluorescent protein-tagged InsP₃ 5′-phosphatase, which disrupts InsP₃ signaling (5′P; Fig. 5B) (16). There was no difference in the rate of rise of the systolic Ca²⁺ transient in myocytes from 12-week-old WKY and SHRs (Fig. S1E).

Fig. 5.

Analysis of global Ca²⁺ transient kinetics, diastolic [Ca²⁺], and elementary Ca²⁺ release during hypertrophy. (A) Rate of rise of Ca²⁺ transient (peak amplitude/time to peak) after 20 min stimulation with 100 nM ET-1 or 10 μM InsP₃ ester (InsP₃). Representative traces are shown in i; values normalized to pre application are shown in ii. Cells have been loaded with indo-1 a.m. and electrically paced at 0.3 Hz. (B) As in Aii for ventricular myocytes infected with control cherry virus or InsP₃ 5′-phosphatase (5′P) virus. (C) Changes in diastolic [Ca²⁺] during application of 100 nM ET-1 ± 2 μM 2-APB or 10 μM InsP₃ ester ± 2 μM 2-APB, normalized to before application. (D) As in C for ventricular myocytes that have been infected with control cherry virus or 5′P virus. (E) Surface plot of representative elementary Ca²⁺ release events during normal pacing (control) and during pacing in the presence of 1 mM tetracaine + 10 μM InsP₃ ester. F/F₀ traces are shown below. (F) Schematic indicating how InsP₃R-mediated Ca²⁺ release augments systolic Ca²⁺ transients during hypertrophy. (I) InsP₃R2; R, RyR2. N numbers are indicated: *, P < 0.05; Student's t test.

As RyR opening is controlled by [Ca²⁺]_i, we next tested whether Ca²⁺ release via InsP₃Rs modulated the efficiency of Ca²⁺-induced Ca²⁺ release (CICR) by changing diastolic [Ca²⁺] levels. Under basal conditions, diastolic Ca²⁺ levels were not different between strains (WKY: 103.39 ± 8.86 nM vs. SHR: 95.74 ± 14.21 nM). Following stimulation with ET-1 or InsP₃ ester, diastolic [Ca²⁺] was increased in SHR myocytes (ET-1: 96.1 ± 5.6 nM to 132.6 ± 15.2 nM, InsP₃ ester: 69.5 ± 19.9 nM to 184.5 ± 36.7 nM, Fig. 5C), whereas no change was seen in WKY cells (Fig. 5C). 2-APB or 5′P expression abrogated the increase in diastolic [Ca²⁺] caused by InsP₃ ester or ET-1 in SHRs (Fig. 5 C and D) without effecting diastolic [Ca²⁺] under basal conditions. At 12 weeks of age, there was no difference in diastolic [Ca²⁺] during stimulation of SHR myocytes with ET-1 or InsP₃ ester (Fig. S1F).

Frequency of Elementary InsP₃-dependent Ca²⁺-release Events Is Increased during Hypertrophy.

To further resolve the consequences of increased InsP₃R expression for Ca²⁺ signaling, elementary Ca²⁺-release events were analyzed. Under normal paced conditions, Ca²⁺ events during the diastolic period were of greater amplitude in the hypertrophic SHR myocytes than in WKY cells (WKY: ΔF/F₀ = 0.26 ± 0.01 vs. SHR: 0.34 ± 0.04, Table S3 and Fig. 5E). Under conditions where RyRs were blocked with 1 mM tetracaine, InsP₃ ester application stimulated elementary Ca²⁺-release events (Fig. 5E) that occurred at a greater frequency in hypertrophic myocytes (WKY: 2.12 ± 0.46 vs. SHR: 6.84 ± 0.65, Table S3). These data suggest that InsP₃R-mediated Ca²⁺ signals contribute to the greater amplitude of diastolic Ca²⁺ events observed in SHR myocytes and may underlie the elevated diastolic [Ca²⁺] observed in SHR myocytes stimulated with InsP₃ or ET-1.

Our data suggest a model to explain the enhanced ECC-mediated Ca²⁺ signals and increased extra-systolic Ca²⁺-release events observed during hypertrophy (Fig. 5F). Key to this model is a hypertrophy-associated increase in InsP₃R expression in the dyadic region. Thus, more InsP₃Rs are in close proximity to RyRs in the SR membrane (Fig. 2 E and F). Ca²⁺ released via these InsP₃Rs sensitizes their adjacent RyRs, bringing them closer to threshold for activation. Under conditions of increased [InsP₃], elementary InsP₃-dependent Ca²⁺-release events are increased in frequency and diastolic [Ca²⁺] is elevated. Consequently, RyRs are triggered to generate extra-systolic Ca²⁺ signals and to accelerate the rate of rise of pacing-evoked Ca²⁺ transients (Fig. 5F).

Discussion

Here we demonstrate that enhanced Ca²⁺ signaling via InsP₃Rs located in the dyadic cleft remodels Ca²⁺ signaling during hypertrophy.

In agreement with previous data, we found that the amplitude of ECC-mediated Ca²⁺ transients under basal conditions was significantly greater as a result of hypertrophy in SHR myocytes (in the absence of any other stimulation) (20). Significantly, we determined that this increased amplitude of basal ECC-mediated Ca²⁺ transients was due to augmented Ca²⁺ release via InsP₃Rs. These data demonstrated that InsP₃Rs could contribute to ECC-mediated Ca²⁺ fluxes without additional neurohormonal input, thereby modifying myocyte Ca²⁺ signaling.

A greater role for InsP₃Rs in regulating ECC-mediated Ca²⁺ transients during hypertrophy was revealed following their direct activation with cell-permeant InsP₃ ester. These data showed that increased activation of InsP₃Rs could augment the amplitude of ECC-mediated Ca²⁺ transients mediated via RyRs even further. Consistent with previous reports (20, 23, 24), no increase in SR releasable Ca²⁺ was observed in hypertrophic SHR myocytes, thereby indicating that the enhancement of ECC-associated Ca²⁺ flux by InsP₃Rs was not due to an increase in store loading. InsP₃R2 expression was elevated as a result of hypertrophy thereby providing a mechanism for increased Ca²⁺ release via InsP₃Rs. InsP₃R expression in the heart has previously been reported to be modified following disease. In particular, InsP₃R expression is increased in atrial myocytes of humans and dogs during atrial fibrillation (AF) (25, 26). Furthermore, elevated InsP₃R levels and increased InsP₃ binding was reported in the left ventricle during human heart failure (17). Consistent with these reports and our observations in rats, we found that InsP₃R2 expression was significantly elevated in cardiac tissue from aortically-banded hypertrophic mice and from human hearts showing ischemic dilated cardiomyopathy. Due to its very low expression and insensitivity to hypertrophy in rat cardiac fibroblasts (Fig. S4), we considered that the changes in InsP₃R2 expression detected in human and mouse cardiac tissue was due solely to InsP₃R2 in cardiac myocytes. Our findings in rats, mice, and humans therefore suggested that increased InsP₃R expression is a general feature of cardiac disease, raising the possibility that increased Ca²⁺ release via InsP₃Rs contributes to pathological changes in Ca²⁺ signaling.

The enhanced InsP₃R2 expression had a striking spatial aspect in that InsP₃R expression was specifically increased in the junctional SR. Detailed analysis showed that these junctional InsP₃Rs co-localized with RyRs, which reside primarily in the dyadic cleft. This profound remodeling in InsP₃Rs expression and distribution had significant functional consequences. In particular, the increased number of dyadic InsP₃Rs augmented the amplitude of the cytosolic ECC-mediated Ca²⁺ transients and enhanced the positive inotropic effect of InsP₃ ester. Similarly, cytosolic ECC-mediated Ca²⁺ transient amplitude and contraction were enhanced when InsP₃Rs were engaged by InsP₃ generated following application of ET-1. Given that ET-1 is a potent pro-hypertrophic agonist, and its levels are elevated during heart failure, these findings have significant implications for cardiac function during hypertrophy (16, 22, 27). The activation of InsP₃Rs in SHR myocytes by ET-1 is in agreement with data from our laboratory and elsewhere showing that stimulation of the InsP₃ signaling cascade in cardiomyocytes with ET-1 modifies Ca²⁺ fluxes and contractility (11, 13, 28). The increase in nuclear Ca²⁺ transient amplitude during ECC by ET-1 and InsP₃ ester was not altered during hypertrophy reflecting the lack of a change in InsP₃R expression in this region. Together, these data suggested that Ca²⁺ release via InsP₃Rs in the dyadic region primed ECC-mediated Ca²⁺-induced Ca²⁺ release via RyRs (see Fig. 5F). Specifically, Ca²⁺ release via InsP₃Rs could elevate diastolic [Ca²⁺] closer to the threshold for activation of RyRs. Thus, we established that increased Ca²⁺ release via InsP₃Rs in hypertrophic myocytes can significantly contribute to remodel ECC-mediated Ca²⁺ signals.

At the most fundamental level, in the absence of RyR activity, SHR myocytes exhibited an increased frequency of elementary InsP₃-dependent Ca²⁺-release events. Interestingly, the amplitudes of those events were no different between WKY and SHR myocytes. This is not surprising given that Ca²⁺ puffs are fundamental Ca²⁺ signals that are conserved between cells as diverse as Xenopus oocytes and HeLa human epithelial cells (29). At the molecular level, Ca²⁺ puffs arise via the stochastic recruitment of neighboring InsP₃Rs (a cluster) until a threshold number required for puff generation is reached (30). Thus, it is plausible that greater InsP₃R expression in SHR myocytes simply increases the probability of recruiting this puff-generating threshold number of receptors without altering the properties of puffs. As a result, only the frequency of elementary events is increased in SHRs. The greater abundance of these elementary events may explain the elevated diastolic [Ca²⁺] observed in SHR myocytes stimulated with InsP₃ ester and ET-1. These data are consistent with the requirement for InsP₃Rs for the ET-1-stimulated increase in diastolic [Ca²⁺] observed in atrial myocytes (which have ≈6-fold greater InsP₃R expression than ventricular myocytes) (10, 12). As elementary Ca²⁺-release events (Ca²⁺ sparks and puffs) are the building blocks of higher order Ca²⁺ transients, it was not surprising that SHR myocytes also exhibited an increased frequency of extra-systolic Ca²⁺ transients. Similarly, stimulation of atrial myocytes with InsP₃ or InsP₃-generating agonists such as ET-1, potently induced arrhythmogenic Ca²⁺-release events that were dependent on InsP₃R2 expression (11–13, 28).

By bringing Ca²⁺ levels closer to the threshold for activation of RyRs, InsP₃-mediated sensitization of RyRs also served to increase the rate of rise of ECC-mediated Ca²⁺ transients. This may remediate the deterioration in Ca²⁺ signaling that occurs as hypertrophy progresses to failure. In particular, extra Ca²⁺ release via dyadic InsP₃Rs may compensate for the decreased coupling efficiency between L-type Ca²⁺ channels and RyRs due to a deterioration in the T-tubular network and increased width of the dyadic cleft that occurs during disease (31).

The arrhythmogenic effect of InsP₃R activity in the ventricles may have profound consequences. Coupled with increased systemic levels of InsP₃-generating agonists, such as ET-1 during hypertension and heart failure, it provides a possible mechanistic explanation why hypertrophic hearts are more likely to develop potentially lethal ventricular arrhythmias (32).

As InsP₃R2 is increased during cardiac hypertrophy, yet is dispensable for the normal physiological function of the healthy heart (12), it may represent an ideal target to which pharmacological modulators could be developed to intervene in both the induction of the hypertrophic gene program and the generation of arrhythmias.

Materials and Methods

Detailed methods for myocyte isolation, adenoviral infection, photometric, and confocal measurements of [Ca²⁺]_i, immunoblotting, immunofluorescence, quantitative RT-PCR, and cell length measurements are provided elsewhere (14, 33) and in SI Methods and Fig. S5.

Animal Models.

Male SHRs and normotensive Wistar-Kyoto (WKY) rats were obtained from Harlan and were housed under control conditions with ad libitum food and water. All experiments were performed in accordance with the guidelines from the code of practice for humane killing under Schedule 1 of the Animals (Scientific Procedures) Act 1986. Constriction of the transverse thoracic aorta was performed on 3-month-old male mice as described in SI Methods. The sham procedure was identical but without aortic ligation.

Patients.

Left ventricular tissue samples of human failing hearts were from individuals undergoing heart transplantation due to end-stage heart failure. All samples were obtained from male caucasians, aged 41–62. Samples from non-failing donor hearts were provided by the U.K. Human Tissue Bank. After cardiectomy, left ventricular samples were frozen in liquid nitrogen and stored at −80 °C. Detailed information about the patients can be found in SI Methods. All experiments involving human tissue samples have been approved by the Cambridgeshire Research Ethics Committee.

Recordings of Myocyte Contraction and [Ca²⁺]_i.

All experiments, unless otherwise stated, were performed at 22 °C on myocytes electrically paced with field electrodes at 0.33 Hz. This condition is referred to as the basal condition. Detailed procedures can be found in SI Methods.

Statistics.

Data are expressed as mean ± SEM. Statistical comparisons were carried out with Student's t test or 2-way ANOVA. Statistically significance was accepted at P < 0.05.