The IP3 Receptor Regulates Cardiac Hypertrophy in Response to Select Stimuli

doi:10.1161/CIRCRESAHA.110.220038

Circ Res. Author manuscript; available in PMC 2011 September 3.

Published in final edited form as:

Circ Res. 2010 September 3; 107(5): 659–666.

Published online 2010 July 8. doi: 10.1161/CIRCRESAHA.110.220038

PMCID: PMC2933281

NIHMSID: NIHMS221767

The IP₃ Receptor Regulates Cardiac Hypertrophy in Response to Select Stimuli

Hiroyuki Nakayama, Ilona Bodi, Marjorie Maillet, Jaime DeSantiago, Timothy L. Domeier, Katsuhiko Mikoshiba, John N. Lorenz, Lothar A. Blatter, Donald M. Bers, and Jeffery D. Molkentin

Department of Pediatrics, Division of Molecular Cardiovascular Biology (H.N., I.B., M.M., J.D.M.), and the Howard Hughes Medical Institute (J.D.M), University of Cincinnati, Cincinnati Children’s Hospital Medical Center, 240 Albert Sabin Way, Cincinnati, Ohio 45229. Department of Pharmacology, University of California, Davis (J.D., D.M.B), 451 Health Sciences Drive, Davis, CA 95616 Department of Molecular Biophysics and Physiology, Rush University School of Medicine (L.A.B., T.L.D), 1750 W. Harrison Street, Chicago, IL 60612 RIKEN Brain Science Institute, 2-1 Hirosawa Wako-shi, Saitama, Japan (K.M.) Department of Molecular and Cellular Physiology, University of Cincinnati School of Medicine, 231 Albert Sabin Way, Cincinnati OH, 45267 (J.N.L)

Correspondence: Jeffery D. Molkentin Howard Hughes Medical Institute Cincinnati Children’s Hospital Medical Center 240 Albert Sabin Way Cincinnati, OH 45229-3039 ; Email: Jeff.Molkentin/at/cchmc.org

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Abstract

Rationale

Inositol 1,4,5-trisphosphate (IP₃) is a second messenger that regulates intracellular Ca²⁺ release through IP₃ receptors located in the sarco(endo)plasmic reticulum of cardiac myocytes. Many prohypertrophic G-protein-coupled receptor (GPCR) signaling events lead to IP₃ liberation, although its importance in transducing the hypertrophic response has not been established in vivo.

Objective

Here we generated conditional, heart-specific transgenic mice with both gain- and loss-of-function for IP₃ receptor signaling to examine its hypertrophic growth effects following pathologic and physiologic stimulation.

Methods and Results

Overexpression of the mouse type-2 IP₃ receptor (IP₃R2) in the heart generated mild baseline cardiac hypertrophy at 3 months of age. Isolated myocytes from overexpressing lines showed increased Ca²⁺ transients and arrhythmias in response to endothelin-1 stimulation. While low levels of IP₃R2 overexpression failed to augment/synergize cardiac hypertrophy following 2 weeks of pressure overload stimulation, it did enhance hypertrophy following 2 weeks of isoproterenol infusion, in response to Gαq overexpression, and/or in response to exercise stimulation. To inhibit IP₃ signaling in vivo we generated transgenic mice expressing an IP₃ chelating protein (IP₃-sponge). IP₃-sponge transgenic mice abrogated cardiac hypertrophy in response to isoproterenol and angiotensin II infusion, but not pressure overload stimulation. Mechanistically, IP₃R2-enhanced cardiac hypertrophy following isoproterenol infusion was significantly reduced in the calcineurin-Aβ null background.

Conclusion

These results indicate that IP₃-mediated Ca²⁺ release plays a central role in regulating cardiac hypertrophy downstream of GPCR signaling, in part, through a calcineurin-dependent mechanism.

Keywords: Hypertrophy, Calcium, signaling, calcineurin

INTRODUCTION

Cardiac hypertrophy occurs as an adaptive response to various cardiovascular diseases such as hypertension, valvular insufficiency, ischemic heart disease, infectious agents or mutations in sarcomeric genes ¹. Ca²⁺ underlies excitation contraction-coupling (ECC) and it serves as a second messenger to induce cardiac hypertrophy, in part, by activating select Ca²⁺-dependent reactive signaling proteins such as calcineurin, calmodulin dependent kinase II (CaMKII), and protein kinase C ² ³. However, it remains unknown how Ca²⁺ activates these hypertrophic signaling effectors in the heart given ECC-mediated Ca²⁺ fluxing that bathes the entire cytoplasm of a cardiac myocyte ⁴. One possibility is that specialized pools of Ca²⁺ have evolved that are temporally and spatially distinct from the cytoplasmic Ca²⁺ transient in ECC. For example, CaMKII is activated in cardiomyocytes by a perinuclear Ca²⁺ pool associated with the inositol 1,4,5 trisphosphate (IP₃) receptor ⁵.

IP₃ is a second messenger generated by hydrolysis of membrane lipid phosphatidyl-inositol 4,5-bisphosphate by phospholipase C (PLC) in response to GPCR activation associated with growth factors and neuroendocrine agonists ⁶. Once generated, IP₃ causes Ca²⁺ release from intracellular stores by binding the IP₃ receptor (IP₃R), an intracellular Ca²⁺ release channel embedded in the sarcoplasmic reticulum (SR) and nuclear envelope. Cardiac hypertrophy has been associated with increased PLC activity and increased generation of IP₃⁷ ⁸. Moreover, expression of IP₃Rs is increased in both human and animal models of heart failure, suggesting that this form of Ca²⁺ release may be associated with pathology ⁹ ¹⁰.

The IP₃R family consists of three genes, IP₃R1, IP₃R2 and IP₃R3 ¹¹. IP₃R2 is thought to be the most prominent gene expressed in the heart ¹² ¹³, and its deletion in gene-targeted mice abolished positive inotropy and spontaneous Ca²⁺ release in atrial myocytes caused by endothelin-1 (ET-1) stimulation ¹³. Even though ventricular myocytes express much lower levels of IP₃Rs than atrial myocytes, these receptors, in some reports, can alter Ca²⁺ release and predispose to arrhythmia ¹⁴ ¹⁵ ¹⁶. While the IP₃Rs can affect Ca²⁺ release, it has not been possible to determine their necessity in regulating the cardiac hypertrophic response because all three receptor genes are expressed in the heart, complicating a gene-targeting approach, not withstanding lethality issues in IP₃R1 null mice. Here we generated transgenic mice with IP₃R2 overexpression and the inhibitory IP₃-sponge protein, demonstrating for the first time that the IP₃R functions as a hypertrophic effector in vivo.

MATERIALS AND METHODS

Generation of transgenic mice

cDNAs encoding mouse IP₃R2 and recombinant Flag-tagged IP₃-sponge protein ¹⁷ ¹⁸ were cloned into the murine inducible α-myosin heavy chain (α-MHC) promoter expression vector ¹⁹ (gift from Dr. Jeffrey Robbins, Children’s Hospital, Cincinnati, Ohio, USA). Gαq transgenic mice were kindly provided by Dr. Gerald Dorn II ²⁰. NFAT-luciferase reporter transgenic mice and calcineurin Aβ null mice (CnAβ^−/−) were previously reported ²¹ ²². To reduce strain effects the CnAβ null mice (C57BL/6) were backcrossed 6 generations into the IP₃R2 (FVB/N) background. Mice were given doxycycline (Sigma, Saint Louis, MO) at 1g/L in the drinking water for 3 weeks to shutdown protein expression from the inducible transgenes. All animal procedures were approved by the Institutional Animal Care and Use Committee of Cincinnati Children’s Hospital Medical Center.

Western blot analysis

Western blot analysis for mouse ventricle homogenates were performed as previously reported ²³. Antibodies included IP₃R2, Gαq and α-tubulin (Santa Cruz), GAPDH (Research Diagnostics Inc., Flanders, NJ) and Flag M2 monoclonal (Sigma). Chemifluorescent detection was performed with the Vistra ECF reagent (Amersham Pharmacia Biotech, Piscataway, NJ) and scanned with a Storm 860 PhosphoImager (Molecular Dynamics, Piscataway, NJ).

Isolation of adult cardiomyocytes and Ca²⁺ measurements

Only Ca²⁺-tolerant cardiomyocytes were selected for Ca²⁺ measurements as previously reported ²⁴. Ca²⁺ transients were measured using Fluo-4 as previously described 25 or using Fura-2 fluorescence ratio at room temperature using a Delta Scan dual-bean spectrofluorophotometer (Photon Technology, Birmingham, NJ, USA), at an emission wavelength of 510 nM and excitation of 340 and 380 nM. The amplitude (Δ340/380 nM ratio) of the Ca²⁺ transients was measured before and after exposure to freshly prepared 100 nM endothelin-1 (ET-1, Calbiochem) or with 1 μM forskolin alone or in combination with 40 nM ET-1. Arrhythmias were classified by three or more extra-systolic Ca²⁺ release events over a 15 min recording period following previously reported criteria ¹⁶.

RT-PCR, luciferase assay, immunohistochemistry, and histological analysis

Reverse-transcriptase PCR was performed as previously described ²⁶. For histological analysis, hearts were collected at the indicated times, fixed in 10% formalin containing PBS, and embedded in paraffin. Myocyte cross-sectional areas were analyzed in slides stained with wheat-germ agglutinin-FITC ²⁴. Immunohistochemistry on adult myocytes was performed as described previously using an IP₃R2 antibody (Abcam) and α-actinin (Sigma) ²⁷.

Echocardiography, invasive hemodynamics, and surgical models

Mice were anesthetized with isoflurane, and echocardiography was performed using a Hewlett Packard 5500 instrument with a 15-MHz microprobe as previously described ²³. Invasive hemodynamics in the closed-chest mouse with a 1.4F Millar catheter was performed as described previously ²⁸. Transverse aortic constriction (TAC) to induce cardiac pressure overload hypertrophy was previously described ²³. Pressure gradients across the constriction were measured by Doppler echocardiography as previously described ²⁹. Alzet miniosmotic pumps (no. 2002; Alza Corp., Mountain View, California, USA) containing isoproterenol (Iso) (60 mg/kg/day), angiotensin II (2 mg/kg/day) or PBS were surgically inserted in 2 month-old mice as previously described ²⁶. Swimming for 20 days as a model of exercise-induced cardiac hypertrophy was described previously ²¹ ²⁴.

Statistical analysis

All results are presented as means plus or minus SEM. Statistical analysis was performed using SigmaPlot11.0 software for unpaired t-test (for two groups) and two-way ANOVA (for four-six groups).

RESULTS

Generation of IP₃ receptor transgenic mice

To determine the role of IP₃-mediated Ca²⁺ release in the heart we generated inducible, cardiac-specific transgenic mice expressing the mouse type-2 IP₃R ¹⁹. Responder transgenic lines were crossed with driver transgenic mice encoding the tetracycline transactivator (tTA) protein (double transgenic (DTG)) and a “high” (3.11) and “low” (4.9) line were selected based on western analysis (Figure 1A). TAC induced a 2.4-fold increase in IP₃R2 protein expression in the mouse heart, and relative to this, lines 3.11 and 4.9 showed 12- and 5-fold more protein expression, respectively. To examine the reliability of the inducible system, doxycycline (Dox) was administered to the low-expressing DTG mice, causing near complete extinguishment of IP₃R2 expression (Figure 1B). Importantly, ventricular myocytes isolated from adult IP₃R2 DTG mice showed a significant increase of amplitude in the Ca²⁺ transient and extrasystolic Ca²⁺ transients after ET-1 stimulation, while no effect was observed in tTA control ventricular myocytes (Figure 1C). Immunohistochemistry from DTG ventricular myocytes showed that the IP₃R2 was localized in a distinct sarcomeric pattern that is consistent with the SR (Figure 1D). Because of low endogenous expression, little IP₃R2 protein was detectable in control tTA ventricular myocytes (Figure 1D; the IP₃R2 antibody employed also reacted with a nucleoplasmic protein in all samples, masking the nuclear envelope localized IP₃R2).

Figure 1

Generation of inducible transgenic mice with enhanced IP₃R2 expression. (A) Western blot analysis of IP₃R2 protein from hearts of two independent DTG lines (4.9 and 3.11) raised without Dox (induced state) at 3 months of age. α-tubulin is a loading (more ...)

High-expressing IP₃R2 transgenic mice display cardiac hypertrophy

Both high and low IP₃R2 DTG mice (no Dox) developed mild cardiac hypertrophy by three months of age (Figure 2A). Consistent with this phenotype, re-expression of the hypertrophy-related genes atrial natriuretic factor (ANF) and skeletal α-actin (αSkA) were observed in the hearts of high expressing DTG mice (Figure 2B). DTG mice demonstrated normal values of baseline ventricular performance by echocardiography (Figure 2C), baseline myocyte Ca²⁺ transient amplitudes, and SR Ca²⁺ content in isolated cells (data not shown). However, a more sensitive approach using a Millar catheter showed that high expressing IP₃R2 DTG mice had a reduced contractile response to dobutamine infusion, although no change in baseline function was observed (Figure 2D). Myocyte Ca²⁺ responses following Iso stimulation were also reduced, suggesting desensitization of β-adrenergic receptors (data not shown). Thus, outside a mild deficit in functional reserve, high expressing DTG mice showed no greater signs of heart disease past 10 months of age.

Figure 2

High-expressing IP₃R2 DTG mice show greater cardiac hypertrophy. (A) Heart weight (HW) normalized to body weight (BW) for control (tTA) and IP₃R2 DTG (both lines) mice at 3 months of age. *P<0.05 versus tTA controls. (B) RT-PCR for atrial natriuretic (more ...)

To further investigate the hypothesis that the IP₃R2 was involved in hypertrophic signaling, high-expressing DTG and littermate tTA mice were subjected to 2 weeks of TAC to induce hypertrophy. Indeed, high-expressing DTG mice at 9-10 weeks of age displayed enhanced cardiac hypertrophy after TAC with equivalent pressure gradients across the constrictions (Figure 2E, and data not shown). Assessment of myocyte cross sectional areas also showed significantly more hypertrophy in IP₃R2 DTG mice compared with tTA controls (Figure 2F).

As previously reported, β-adrenergic stimulation activates protein kinase A (PKA) leading to IP₃R2 phosphorylation and sensitized Ca²⁺ release to IP₃ ³⁰. Consistent with these observations, 2 weeks of Iso infusion with Alzet minipumps produced more cardiac hypertrophy in DTG mice compared with tTA controls (Figure 2G,H). To further examine the impact of increased IP₃R2 expression downstream of GPCR signaling, we crossed IP₃R2 DTG mice with transgenic mice overexpressing Gαq, a direct PLC effector and disease inducer ²⁰. As predicted, combined IP₃R2 and Gαq overexpression exacerbated the hypertrophic phenotype and pathology, such as greater increases in ventricle weight normalized to body weight and decreased fractional shortening assessed by echocardiography (Figure 2I,J). The inset in Figure 2I shows that Gαq protein was overexpressed by approximately 3-fold with the transgene when crossed into the IP₃R2 DTG background. Taken together these results suggest that IP₃R2 expression can enhance cardiac hypertrophy and disease downstream of multiple pathologic stimuli.

Low-expressing IP₃R2 transgenic mice show increased cardiac hypertrophy to Iso and exercise

We also repeated our entire analysis with low expressing IP₃R2 DTG mice. Remarkably, 2 weeks of TAC stimulation in low-expressing IP₃R2 DTG mice failed to show augmented cardiac hypertrophy compared to tTA control mice (Figure 3A,B). Pressure gradients across the aortic constriction were not different between the 2 groups of TAC mice (data not shown). However, low-expressing IP₃R2 DTG mice did show significantly greater cardiac hypertrophy following 2 weeks of Iso infusion, similar to the enhancement observed in high expressing DTG mice (Figure 3C,D). In addition, the enhanced hypertrophic response in low–expressing DTG mice caused by β-adrenergic stimulation was reversed when the transgene was shutdown by administration of Dox (Figure 3E,F). These results in lower expressing DTG mice suggest that IP₃R-augmented Ca²⁺ release activated by β-adrenergic receptor stimulation is likely a physiologically important aspect of signaling in cardiac myocytes.

Figure 3

Low–expressing IP₃R2 DTG mice demonstrate enhanced cardiac hypertrophy only to Iso infusion and swimming. (A,B) HW/BW and myocyte cross-sectional areas from control (tTA) and IP₃R2 DTG (line 4.9) mice 2 weeks after sham or TAC without Dox (induced). (more ...)

We also employed a model of physiologic cardiac hypertrophy due to forced swimming exercise over 20 days in IP₃R2 DTG and tTA control mice. Surprisingly, both low and high expressing IP₃R2 DTG mice demonstrated significantly greater cardiac hypertrophy after 20 days of swimming compared with tTA controls (Figure 3G,H). Forced swimming likely induces a concomitant catecholamine response in the mice, which would be consistent with the enhanced hypertrophy profile associated with Iso infusion, collectively suggesting that IP₃-mediated Ca²⁺ release is of physiologic relevance in regulating cardiac hypertrophy to select stimuli.

The results presented above suggested that Gαq and cAMP pathways might converge on the IP₃R to regulate hypertrophy. To further address this assertion, we measured Ca²⁺ transients and arrhythmia in adult myocytes from IP₃R2 DTG mice using forskolin in combination with low dose ET-1 (40 nM). We were not able to use Iso given the β-receptor desensitization discussed above, but forskolin elevated cAMP and increased the Ca²⁺ transient and induced mild arrhythmia (Online Figure I). While low dosage ET-1 was without effect, when used in combination with forskolin, even greater Ca²⁺ release and arrhythmias were observed in adult myocytes from IP₃R2 DTG mice (Online Figure I).

Inhibition of IP₃ signaling reduces cardiac hypertrophy to Iso and angiotensin II infusion

Here we generated inducible IP₃-sponge transgenic mice to reduce endogenous IP₃ signaling in the adult mouse to examine its necessity in programming hypertrophy. The IP₃-sponge is a truncated and soluble IP₃ receptor that binds free IP₃ with exceedingly high affinity ¹⁸. Two responder transgenic lines were obtained, although just the higher expressing line (line 22.3) was analyzed (Figure 4A). To determine the effectiveness of IP₃-chelating activity in vivo we crossed IP₃-sponge mice with high expressing IP₃R2 DTG mice, after which adult myocytes were isolated and stimulated with ET-1. Importantly, triple transgenic mice (tTA driver, IP₃-sponge and IP₃R2 responder transgenes) showed no loss in protein expression for either the IP₃-sponge or IP₃R2 (Figure 2B). Myocytes from IP₃R2 high overexpressing mice showed a robust increase in Ca²⁺ transients and arrhythmic events, which was completely blocked by the IP₃-sponge (Figure 4C,D). Thus, targeted expression of the IP₃-sponge in the mouse heart eliminates functional IP₃-dependent Ca²⁺ release in isolated ventricular myocytes.

Figure 4

Generation and analysis of IP₃-inhibited transgenic mice. (A) Western blot analysis for Flag-tagged IP₃-sponge protein from the hearts of tTA controls and IP₃-sponge DTG mice. (B) Western blot analysis for IP₃R2 and IP₃-sponge (IP₃-SP) protein levels (more ...)

To assess the impact of IP₃ signaling inhibition on pressure overload hypertrophy, IP₃-sponge DTG and tTA mice were subject to TAC stimulation. Consistent with the results observed in low expressing IP₃R2 DTG mice, IP₃₊-sponge overexpression showed no ability to reduce the cardiac hypertrophic response after 2 weeks of TAC stimulation compared with tTA controls (Figure 4E,F). However, IP₃-sponge DTG mice showed significantly less hypertrophy following 2 weeks of Iso infusion (Figure 4G,H). No reduction in fractional shortening was noted over 2 weeks of Iso infusion, and levels of induced fibrosis were similar between tTA and IP₃-sponge DTG hearts (Online Figure II). We also observed that the hypertrophic response caused by angiotensin II infusion was significantly reduced in IP₃-sponge DTG mice (Figure 4I). Taken together, these results indicate that IP₃ signaling plays a necessary role in select forms of hypertrophic stimulation in adult mouse heart (see discussion).

Calcineurin signaling underlies IP₃-regulated hypertrophy

Calcineurin is Ca²⁺/calmodulin-dependent protein phosphatase that functions as a central regulator of cardiac hypertrophy, in part, by activating a transcription factor family known as nuclear factor of activated T-cells (NFAT) ³¹. Here we hypothesized that the calcineurin-NFAT pathway might respond to IP₃R signaling in mediating the cardiac hypertrophic response. To address this issue high and low IP₃R2 DTG mice were crossed with NFAT-luciferase reporter transgenic mice, showing a 3.5- and 2.4-fold increase in NFAT activity, respectively, compared with tTA mice at 8 weeks of age (Figure 5A). Expression of the IP₃-sponge did not affect NFAT-luciferase activity at baseline in the heart (Figure 5A). To extend these results and determine if calcineurin was necessary for IP₃R2 enhanced hypertrophy we crossed the IP₃R2 responder and tTA driver transgenes into the CnAβ null background. CnAβ^+/+ (wildtype) controls were also generated from the same backcross. Importantly, cardiac expression level of IP₃R2 protein directed by the transgene was similar between the CnAβ^−/− and CnAβ^+/+ backgrounds (Figure 5B). Remarkably, the augmented hypertrophy profile observed in IP₃R2 DTG mice following 2 weeks of Iso stimulation was blocked in the CnAβ^−/− background, but once again, was significantly augmented in the wildtype background (Figure 5C,D). Collectively, these results indicate that calcineurin-NFAT serve as a downstream effector of IP₃R-mediated Ca²⁺ signaling in the heart.

Figure 5

Calcineurin-NFAT signaling mediate IP₃R-dependent cardiac hypertrophy. (A) NFAT transcriptional activity in hearts of NFAT-luciferase reporter transgenic mice crossed with the tTA, IP₃R2 high (3.11), IP₃R low (4.9) and IP₃-sponge transgenes.* P<0.05 (more ...)

DISCUSSION

IP₃Rs are intracellular ligand-gated Ca²⁺ release channels that are activated by IP₃ binding where they function downstream of growth factors and GPCR signaling events ⁶. The physiological role of Ca²⁺ release from the IP₃R in ventricular myocytes has been a controversial issue, with some reports suggesting no effect on ECC, while others have observed a small but significant effect on spontaneous Ca²⁺ release in the form of sparks and enhanced Ca²⁺ transients ¹⁵^,³²^-³⁴. In atrial myocytes, IP₃-mediated Ca²⁺ release appears to be a more prominent event that modulates ECC and SR Ca²⁺ release ³⁵ ³⁶, likely because of higher endogenous IP₃R expression levels ³⁴ ¹⁵. Consistent with these reports, transgene-directed overexpression of IP₃R2 in ventricular myocytes had a prominent effect on ECC and even arrhythmia upon ET-1 stimulation, further suggesting that IP₃Rs are positioned within the junctional SR where they can affect ryanodine receptor (RyR) activity.

To affect ECC, the IP₃Rs need to be positioned within the proper functional domains of the SR. Previous analysis of this issue demonstrated that IP₃R2 is enriched at the nuclear envelop, which is contiguous with the SR/ER network in adult cardiac myocytes ¹² ³⁷ ³⁸. However, localization to the nuclear envelope should not affect ECC, but instead appears to control nuclear Ca²⁺ signaling ³⁹ and a local pool of CaMKII ⁵. In addition to the nuclear envelope, the IP₃Rs are prominently localized to the SR, in similar regions as the RyR ¹⁶. Indeed, immunohistochemistry of adult myocytes from IP₃R2 DTG overexpressing mice versus tTA controls unequivocally showed IP₃R2 localization within the entire expanse of the SR and around the nucleus.

Another aspect of the controversy surrounding a functional role for IP₃Rs in cardiac myocytes is that generation of IP₃ appears to be relatively weak compared with other celltypes ⁴⁰ ⁴¹. For example, cardiomyocytes from mouse or human display only a 1.5- to 2-fold activation of PLC in response to α1-adrenergic receptor stimulation, achieving a level of approximately 30 nM ⁴² ⁴³ ⁴⁴ ⁴⁵. However, phosphorylation of IP₃R2 by PKA sensitizes the channel and enhances IP₃-mediated Ca²⁺ release at lower IP₃ concentrations ³⁰. In addition to this mechanism, Iso stimulation in cultured cardiomyocytes enhances IP₃ generation through an ET-1 paracrine/autocrine signaling circuit ³⁸. β-adrenergic stimulation also enhances SR Ca²⁺ levels and sensitizes the RyR, together leading to augmented Ca²⁺ release. In support of this contention, low levels of ET-1, which did not change Ca²⁺ release or induce arrhythmia, did synergistically increase arrhythmia when forskolin was also used to elevate cAMP. We also believe that physiologic exercise-induced cardiac hypertrophy, which appears to elicit a strong fear response in mice, augmented IP₃R Ca²⁺ release through an associated β-adrenergic co-stimulation effect. These concepts are also consistent with the vast array of neurohumoral mediators that underlie pathologic cardiac hypertrophy and failure, where multiple Gαq-coupled receptor agonists likely synergize with cAMP elevation afforded by β-receptor signaling to induce pathology.

Interestingly, only the high IP₃R2 DTG line showed augmented cardiac hypertrophy with pressure overload stimulation. In contrast, the low overexpressors and the IP₃-sponge mice each failed to show an effect with pressure overload stimulation. The simplest interpretation of these results is that a requirement for endogenous IP₃Rs is easily bypassed with extreme hypertrophic stimulation, such as afforded by pressure overload. Indeed, TRPC channels are also activated in pressure-overloaded hearts where they could easily compensate and provide local Ca²⁺ entry in the absence of IP₃R signaling to maintain calcineurin activation ⁴⁶. In contrast, Iso infusion engages a more restricted set of signaling pathways, such as PKA activation through elevated cAMP. As discussed earlier, β-adrenergic/PKA stimulation uniquely primes IP₃R2 activation and Ca²⁺ release, possibly explaining why Iso infusion in low expressing DTG mice and in IP₃-sponge mice demonstrated a positive effect versus tTA control mice.

The increase in Ca²⁺ release mediated by IP₃Rs at the level of the SR could induce the hypertrophic response through a number of different signaling mechanisms. Our working hypothesis is that IP₃R Ca²⁺ release generates a local Ca²⁺ signaling effect at the level of the T-tubular-SR junctional complex. Indeed, calcineurin is anchored at the Z-lines to calsarcin and α-actinin, in immediate proximity to the SR junctions ⁴⁷. Calcineurin was also shown to respond to IP₃-mediated Ca²⁺ release from a perinuclear/nuclear location in neonatal cardiomyocytes to induce hypertrophy ³⁸. Consistent with these results, we observed prominent NFAT activation in IP₃R2 DTG hearts and that deletion of CnAβ^−/− blocked the ability of Iso to enhance hypertrophy through the IP₃R2 transgene. This IP₃R-dependent release of Ca²⁺ at the T-tubular-SR junctions could also stimulate RyR Ca²⁺ leak, providing additional regional Ca²⁺ elevations to affect calcineurin. Thus, antagonism of the IP₃R may be a clinically relevant target, as it might reduce both arrhythmia and hypertrophic growth propensity.

Novelty and Significance

What Is Known

IP₃R signaling has been implicated in regulating cardiac hypertrophy.
IP₃R mediated calcium release has been implicated in regulating excitation contraction-coupling in the heart.
IP₃R signaling has been implicated in regulating calcineurin-NFAT signaling.

What New Information Does this Article Contribute

The first description of IP₃R2 overexpression in the heart of transgenic mice, which definitively shows that it can regulate cardiac hypertrophy.
The first description of IP₃ inhibition in the mouse heart and the observation that this mediator is required for isoproterenol induced hypertrophy.
The first proof that calcineurin/NFAT signaling mediate IP₃R2 signaling in vivo to control cardiac hypertrophy.

This study was designed to evaluate the importance of IP₃R2 calcium release and signaling in mediating the cardiac hypertrophic response in vivo. We examined both pressure overload induced hypertrophy and neurohormonal-regulated hypertrophy in IP₃R2 TG mice and mice expressing the IP₃-sponge to block this calcium release pathway. Our results definitely show that IP₃R2 mediated calcium release in response to neurohormonal stimulation can underlie the cardiac hypertrophic response, in part, by activating calcineurin/NFAT signaling.

Supplementary Material

Online Figure I. Assessment of elevated cAMP on Ca²⁺ handling in IP₃R2 DTG hearts with or without a small ET-1 stimulus at 40 nM. (A,B) Percent increase in the amplitude of the Ca²⁺ transient after forskolin (A) or ET-1 co-stimulation (B) in isolated adult cardiomyocytes from control tTA or IP₃R2 DTG hearts. Forskolin equally increased the Ca²⁺ transient in both genotypes, although co-stimulation with ET-1 gave a larger effect in the DTG myocytes. (C) Arrhythmic Ca²⁺ transients (%) in adult myocytes from DTG hearts with forskolin or forskolin with ET-1. ET-1 was used at 40 nM which is too low to induce arrhythmia in wildtype or IP₃R2 myocytes, but it can with forskolin in IP₃R2 DTG myocytes. (D,E) Representative Fura-2 ratio fluorescence tracings from IP₃R2 DTG (line 3.11) adult cardiomyocytes with forskolin (D) or forskolin with low ET-1 (E). Arrow shows compound addition.

Online Figure II. IP₃-sponge expression and suppression of IP₃R signaling does not promote pathologic hypertrophy with isoproterenol infusion. (A) Echocardiography to assess ventricular fractional shortening (FS) in tTA controls and IP₃-sponge DTG mice infused with vehicle or Iso for 2 weeks. No reductions in FS were observed in any group. Number of mice analyzed is shown in the bars. (B) Metamorph quantitation of ventricular fibrosis from Masson’s trichrome-stained histological sections in the mice treated as described in A. *P<0.05 versus tTA Veh. The IP₃-sponge did not worsen fibrosis with ISO infusion.

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Acknowledgments

Sources of funding This work was supported by grants from the National Institutes of Health (J.D.M., L.A.B., D.M.B), the Fondation Leducq (J.D.M), and the Howard Hughes Medical Institute (J.D.M.).

Non-standard abbreviations


αMHC	α-myosin heavy chain

CaMKII	calmodulin-dependent kinase II

CnA	calcineurin A subunit

DOX	doxycycline

DTG	double transgenic

ET-1	endothelin-1

GPCR	G-protein-coupled receptor

IP₃	inositol 1,4,5-trisphosphate

IP₃R	IP₃ receptor

Iso	isoproterenol

NFAT	nuclear factor of activated T-cells

PKA	protein kinase A

PLC	phospholipase C

RyR	ryanodine receptor

TAC	transverse aortic constriction

SR	sarcoplasmic reticulum

WT	wildtype

Footnotes

Disclosures: None

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