|Home | About | Journals | Submit | Contact Us | Français|
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.
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.
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.
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.
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.
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 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).
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.
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.
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.
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).
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).
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.
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.
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.
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).
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.
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 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.
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.
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.
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.
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.).
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.