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(Circulation. 2005;111:1652-1659.)
© 2005 American Heart Association, Inc.

Molecular Cardiology

Serum and Glucocorticoid-Responsive Kinase-1 Regulates Cardiomyocyte Survival and Hypertrophic Response

Takuma Aoyama, MD, PhD; Takashi Matsui, MD, PhD; Mikhail Novikov, MD; Jongsun Park, PhD; Brian Hemmings, PhD; Anthony Rosenzweig, MD

From the Program in Cardiovascular Gene Therapy and Cardiology Division, MGH, Harvard Medical School, Boston, Mass (T.A., T.M., M.N., A.R.), and Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland (J.P., B.H.).

Correspondence to Anthony Rosenzweig, MD, MGH, 114 16th St, Room 2600, Charlestown, MA 02129. E-mail arosenzweig{at}partners.org

Received August 30, 2004; revision received November 15, 2004; accepted November 23, 2004.

	Abstract

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Background— Serum- and glucocorticoid-responsive kinase-1 (SGK1), a serine-threonine kinase that is highly expressed in the heart, has been previously reported to regulate sodium channels. Because SGK1 is a PI 3-kinase–dependent kinase with structural homology to Akt, we examined its regulation in the heart and its effects on cardiomyocyte (CM) apoptosis and hypertrophy in vitro.

Methods and Results— Rats were subjected to aortic banding, and expression of total and phosphorylated SGK1 was examined. Both phospho- and total SGK1 increased 2 to 7 days after banding. Phospho-SGK1 was also upregulated in CMs stimulated in vitro with IGF-I or phenylephrine. Infection of CMs with an adenoviral vector encoding constitutively active SGK1 (Ad.SGK1.CA) inhibited apoptosis after serum-deprivation or hypoxia (P<0.05), whereas expression of kinase-dead SGK1 (Ad.SGK1.KD) increased it and partially mitigated the protective effects of IGF-I (P<0.05). SGK1 activation was also sufficient to increase cell size, protein synthesis, sarcomere organization, and ANF expression both at baseline and in response to phenylephrine but was not necessary for the hypertrophic response to phenylephrine. Evaluation of potential downstream signaling pathways demonstrated that SGK1 induces phosphorylation of tuberin, p70s6kinase, and GSK3ß in CMs, which may contribute to its effects.

Conclusions— SGK1 is dynamically regulated during acute biomechanical stress in the heart and inhibits CM apoptosis while enhancing the hypertrophic response.

Key Words: apoptosis • hypertrophy • signal transduction

	Introduction

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Cardiomyocyte (CM) hypertrophy and apoptosis are seen in a wide variety of common cardiac pathologies, including remodeling after ischemic injury and heart failure. Thus, understanding the signaling mechanisms that control these processes in the heart may have significant clinical implications.

Previous work has identified phosphoinositide 3-kinase (P3K) and its downstream effector, Akt (or protein kinase B), as important determinants of CM survival both in vitro and in vivo.^1–4 In addition, PI3K and Akt appear to regulate cell and organ growth in many species and settings,^5–7 including the mammalian heart.^8–10 Although Akt itself has multiple downstream substrates relevant to these phenotypes, recent studies have implicated phosphorylation and inhibition of GSK3ß as an important mediator of effects on both hypertrophic growth^11–13 and CM survival.^14,15 Recent studies in our own laboratory have demonstrated the importance of PI3K-dependent but Akt-independent signaling pathways in cardioprotection,¹⁶ prompting us to search for such pathways in the heart.

SGK1 is a serine-threonine kinase initially identified as transcriptionally induced by glucocorticoids and serum¹⁷ that is highly expressed in the heart of developing embryos and adult animals.¹⁸ SGK1 is activated downstream of PI3K in response to growth factors or oxidative stress.^19,20 The catalytic domain of SGK1 is 54% identical to that of Akt,¹⁹ and SGK1 shares a variety of downstream substrates with Akt.^21,22 In other cell types, SGK1 has been reported to inhibit apoptosis.²³ At least 2 other isoforms of SGK—SGK2 and SGK3—have been identified after the initial characterization of SGK1.²⁴ Although both SGK1 and SGK3 are broadly expressed, SGK2 expression is more restricted and barely detectable in the heart.²⁴ Interestingly, SGK1 appears the most responsive to IGF-I stimulation in other systems²⁴ and has been previously characterized to share common downstream substrates with Akt.²¹ For these reasons, we focused our attention on the role of this isoform in CMs.

Of note, there are also important distinctions between SGK1 and Akt. For example, SGK1 lacks the pleckstrin homology domain that is present in Akt and thought to account for its phosphoinositide-driven translocation to the cell membrane.¹⁷ Unlike Akt, SGK1 regulates sodium channels,²⁵ regulates renal salt handling,²⁶ and does not enhance sarcolemmal GLUT4 or glucose uptake.²² Even with some common substrates such as FOXO3a, SGK1 and Akt appear to preferentially phosphorylate different sites, raising the possibility of coordinate regulation by these 2 kinases.²¹

To better understand the potential role of SGK1, we investigated its regulation in the heart and its biological effects in CMs.

	Methods

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Aortic Banding
Biomechanical stress was achieved by banding the ascending aorta in the rat as described.²⁷

Cell Culture
Primary CM cultures were prepared as described.¹ For morphological studies, CMs were additionally purified on a Percoll gradient, fixed, and stained with Hoechest 33258 and anti-actinin antibody as previously described.^1,2 Apoptotic cells were scored by nuclear morphology from randomly selected fields. At least 100 cells per sample were examined in 3 independent experiments. Cell surface area was determined from enlarged digital micrographs with NIH image software. At least 50 cells from each sample in 3 independent experiments were used to determine mean cell surface area.

In Vitro Apoptosis Models
To induce apoptosis, CMs were placed in serum-free DMEM under either normoxic or hypoxic (95% N₂/5% CO₂) conditions as described.¹

Recombinant Adenoviruses
Adenoviral vectors (Ad) were generated and characterized as described.¹ Ad.EGFP contains cytomegalovirus-driven expression cassettes for ß-galactosidase and enhanced green fluorescent protein (EGFP).²⁸ Ad.SGK1.CA and Ad.SGK1.KD are structurally similar but encode HA-epitope–tagged, constitutively active (CA; S422D), and kinase-dead (KD; K127M) mutants, respectively,²⁰ instead of ß-galactosidase. CMs were infected with Ad at a multiplicity of infection of 20 three days after plating.

Immunoblotting
Protein extraction and immunoblotting were performed as described.¹ Antibodies for the following were used: phospho-(Thr256) and total SGK1 (Upstate Biotechnology); cleaved caspase-3, phospho-(Thr1462), and total tuberin; phospho-(Ser371) and total p70S6kinase; phospho-(Ser9) and total GSK3ß; phospho-(Thr202/Tyr204) and total ERK1/2 MAPK; phospho-(Thr183/Tyr185) and total SAPK/JNK; and phospho-(Thr180/Tyr182) and total p38 MAP kinase (all from Cell Signaling).

Northern Blotting
RNA was extracted from CMs with Trizol. Northern blotting was performed as previously described.²⁸

SGK1 Kinase Assay
Cells were lysed in an ice-cold buffer (20 mmol/L Tris [pH 7.5], 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L ß-glycerolphosphate, 1 mmol/L Na₃VO₄, 1 µg/mL leupeptin, 1 mmol/L PMSF). Precleared cell lysates were immunoprecipitated with anti-SGK1 antibody overnight at 4°C. Immune complexes were washed twice with lysis buffer and once with kinase buffer (25 mmol/L Tris [pH 7.5], 5 mmol/L ß-glycerolphosphate, 2 mmol/L DTT, 0.1 mmol/L Na₃VO₄, 10 mmol/L MgCl₂) and incubated in kinase buffer with 200 µmol/L ATP and 25 µg/mL GSK3 substrate peptide (Cell Signaling) for 30 minutes at 30°C. Reactions were terminated by the addition of SDS sample buffer, and supernatants were boiled for 5 minutes. Membranes were probed with phospho-GSK3ß(Ser 9) antibody overnight at 4°C.

Akt Kinase Assay
Akt kinase assay was performed with the Akt Kinase Kit (Cell Signaling) according to manufacturer’s instructions.

DNA Laddering
Genomic DNA (1.5 µg)was ³²P-labeled by Klenow polymerase and separated by gel electrophoresis as described.¹

MTT Assay
MTT assay was performed according to manufacturer’s protocol (R&D).

NFAT Activity
NFAT transcriptional activity was determined with an NFAT-dependent luciferase reporter encoded in a recombinant adenoviral vector²⁹ (kindly provided by Dr Jeffery Molkentin, Cincinnati Children’s Hospital). Luciferase activity was measured as previously described.²⁹

Leucine Incorporation
CMs were incubated with [³H]-leucine (1 µCi/mL) with or without phenylephrine (PE; 20 µmol/L) for 36 hours, and leucine incorporation was determined as described.³⁰

Statistical Analysis
All data are from 3 independent experiments and given as mean±SEM. ANOVA was used to determine statistical significance. The null hypothesis was rejected at P<0.05.

	Results

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Cardiac SGK1 Is Induced by Mechanical Stress In Vivo
SGK1 protein was detectable in hearts at baseline and increased modestly 6 hours after ascending aortic constriction (AAC). Two to 7 days after AAC, total SGK1 protein increased dramatically and was accompanied by a comparable increase in phosphorylated (activated) SGK1 (Figure 1A). Interestingly, although the initial increase in phospho-SGK1 occurred in association with increased Akt activation, SGK1 phosphorylation persisted longer and coincided with the observed sustained increase in GSK3ß phosphorylation.

View larger version (69K): [in this window] [in a new window]   Figure 1. SGK1 is dynamically regulated in heart in vivo and in CMs in vitro. A, Pressure-overload hypertrophy. At indicated time after AAC, hearts were harvested for immunoblotting of cardiac total cell lysates with indicated antibodies. B, C, Total and phospho-SGK1 levels in CMs. CMs were subjected to hypoxia, SD, H2O2 (20 µmol/L), IGF-I (100 nmol/L), or PE (20 µmol/L) for times indicated. Total cellular protein (50 µg/lane) was separated by SDS-PAGE and subjected to immunoblotting for phospho- and total SGK1 protein.

Phosphorylation of SGK1 Is Induced by IGF-I and PE
We examined the effects of a variety of pathophysiologically relevant stimuli on SGK1 in CMs. Both hypoxia and oxidative stress (H₂O₂) induced an increase in phospho- and total SGK1 protein in a time-dependent manner (Figure 1B). In contrast, serum deprivation (SD) modestly reduced levels of total and phospho-SGK1. Although IGF-I and PE did not increase the total levels of immunoreactive SGK1 protein, they both induced SGK1 phosphorylation, peaking 10 to 30 minutes after IGF-I (100 nmol/L) stimulation (Figure 1C, left) and 60 minutes after PE (20 µmol/L) stimulation (right).

Recombinant Adenoviral Vectors
To characterize the biological effects of SGK1 in CMs, we generated recombinant Ad carrying HA-tagged, CA (S422D) and KD (K127M) mutants of SGK1.²⁰ Infection of CMs with these viruses generated appropriately sized, immunoreactive proteins (data not shown); CMs infected with these were harvested 10 minutes after IGF-1 treatment (100 nmol/L) and subjected to kinase assay. In the absence of stimulation, Ad.SGK1.CA increased SGK1 activity 2.45±0.21-fold (P<0.05), whereas Ad.SGK1.KD reduced it (0.44±0.02-fold, P<0.05) compared with control-Ad–infected CMs (Figure 2). Stimulation of control virus–infected CMs with IGF-I induced a comparable, 2.3-fold increase in endogenous SGK1 activity. Treatment of Ad.SGK1.CA-infected CMs with IGF-I further increased SGK1 activity, whereas Ad. SGK1.KD inhibited it (2.26±0.07- and 0.40±0.02-fold, respectively; P<0.05 versus IGF-I–treated control cells). Thus, Ad.SGK1.CA infection is sufficient to increase SGK1 activity in unstimulated CMs but remains responsive to further stimulation, and Ad.SGK1.KD acts as a dominant negative to inhibit endogenous SGK1 activity.

View larger version (42K): [in this window] [in a new window]   Figure 2. SGK1 activity. SGK1 activity was measured in CMs infected with Ad.EGFP, Ad.SGK1.CA, or Ad.SGK1.KD in absence or presence of IGF-I (10 minutes, 100 nmol/L). Representative assay and densitometric quantification from 3 independent experiments are shown. P<0.05 vs Ad.EGFP-infected CMs under same conditions.

SGK1 Inhibits CM Apoptosis
We next examined the effects of SGK1 on CM apoptosis after SD and hypoxia. Neither Ad.SGK1.CA nor Ad.SGK1.KD had significant effects on CM apoptosis as determined by nuclear morphology in unstimulated CMs (Figure 3A); however, Ad.SGK1.CA substantially protected CMs against both SD- and hypoxia-induced apoptosis (Figure 3A). In contrast, inhibition of SGK1 with Ad.SGK1.KD significantly increased the number of apoptotic nuclei compared with control virus–infected CMs in both models (Figure 3A). These changes in nuclear morphology correlated well with DNA laddering, the biochemical hallmark of apoptosis, and cleavage (activation) of caspase-3 (Figure 3B and 3C). Moreover, there were parallel effects on overall cell viability, as indicated by the MTT assay (Figure 3D). To determine whether SGK1 is necessary for IGF-I–mediated cardioprotection, CMs were subjected to hypoxia/SD and in the presence or absence of IGF-I (100 nmol/L) (Figure 3E). IGF-I substantially reduced CM apoptosis, and this effect was significantly but incompletely reversed by expression of SGK1.KD (Figure 3E). Together, these data suggest that SGK1 activation is both necessary and sufficient to protect CMs from apoptosis resulting from SD or hypoxia and that SGK1 contributes to but does not fully explain the cardioprotective effects of IGF-I.

View larger version (47K): [in this window] [in a new window]   Figure 3. SGK1 modulates CM apoptosis. A, Morphological analysis of apoptosis. Inset, Morphological features quantified in bar graph. CMs infected with control virus under normoxic (a) or hypoxic (b) conditions were stained with Hoechst 33258 (white) for nuclear morphology and anti–-actinin (red) to identify CMs. Bar graphs show quantification of apoptotic CMs (arrows) under indicated conditions. P<0.05 vs Ad.EGFP-infected CMs under SD () or hypoxic () conditions. B, DNA fragmentation. DNA from CMs exposed to SD (48 hours) or hypoxia (24 hours) was extracted, radiolabeled, and electrophoresed. Representative DNA ladder and quantification of 3 independent experiments are shown. P<0.05 vs Ad.EGFP-infected CMs under SD () or hypoxic () conditions. C, Caspase-3. CMs infected with indicated Ad constructs were subjected to SD or hypoxia for 6 hours, followed by assessment of procaspase-3 processing. Representative assay and densitometric quantification from 3 independent experiments are shown. P<0.05 vs Ad.EGFP-infected CMs under SD () or hypoxic () conditions. D, MTT assay. CMs were exposed to SD or hypoxia for 24 hours, and overall viability was assessed by MTT. P<0.01 vs Ad.EGFP-infected CMs under SD; P<0.01, P<0.05 under hypoxic conditions. Results are representative of 8 independent experiments. E, SGK1.KD suppresses cardioprotective effects of IGF-I. CMs infected with or without SGK1.KD were subjected to hypoxia for 24 hours in presence or absence of IGF-I (100 nmol/L) as indicated. CMs with morphological change of apoptosis were quantified as above. Results are representative of 3 independent experiments. P<0.05 vs uninfected hypoxic CMs without IGF-I treatment;.P<0.05 vs IGF-I–stimulated, hypoxic CM without SGK1.KD expression.

SGK1 Modulates the Hypertrophic Response of CMs
To investigate the role of SGK1 in the hypertrophic response, we first examined the effects of SGK1 on protein synthesis in CMs that were either untreated or stimulated with PE for 36 hours. Ad.SGK1.CA infection increased [³H]-leucine incorporation in unstimulated CMs (42.1±9.6% versus Ad.EGFP-infected CM, P<0.05; Figure 4A) to a level comparable to that seen in control CMs stimulated with PE for 36 hours. Stimulation of Ad.SGK1.CA-infected CMs with PE further increased protein synthesis. In contrast to our results with apoptosis, Ad.SGK1.KD did not inhibit protein synthesis either at baseline or in response to PE. Similarly, SGK1 activation increased cell size and sarcomere organization in CMs (Figure 4B), as well as mRNA levels of ANF, a marker of the hypertrophic response (Figure 4C), but inhibition of SGK1 did not block these responses. Thus, although SGK1 activation is sufficient to enhance CM hypertrophy both at baseline and after PE stimulation, it is not necessary for the hypertrophic response to PE.

View larger version (43K): [in this window] [in a new window]   Figure 4. SGK1 modulates the hypertrophic response of CMs. A, Protein synthesis. CMs infected with indicated viruses were treated with PE (20 µmol/L) or vehicle for 36 hours. Leucine incorporation was determined as described in Methods. P<0.05 vs Ad.EGFP-infected CMs under same conditions. B, Cell size and sarcomere organization. Inset, Representative CMs infected with Ad.EGFP, Ad.SGK1.CA, or Ad.SGK1.KD as indicated and treated with vehicle alone (top) or PE (20 µmol/L) for 48 hours. CMs were stained for -actinin, and nuclei were stained with Hoechest 33258. PE and activation of SGK1 led to increased cell size and more prominent sarcomere organization. Bar graph shows quantification cell surface area. P<0.05 vs Ad.EGFP-infected CMs under same conditions. Results are representative of 3 independent experiments. C, ANF expression. CMs were infected with indicated virus and harvested 48 hours after stimulation. ANF mRNA expression is shown in top panel and ethidium bromide staining for ribosomal RNA is shown in the bottom panel as loading control. PE treatment and SGK1 activation significantly increased ANF mRNA levels in CMs.

We examined the effects of SGK1 activation on a variety of signaling pathways relevant to cardiac hypertrophy.^31,32 We found no substantial effect of SGK1 on MAPK signaling, including ERK1/2, SAPK/JNK, and p38, in either the presence or absence of PE (Figure 5A). Similarly, the transcriptional activity of NFAT was not altered by SGK1 activation or inhibition (Figure 5B). In contrast, SGK1 enhanced phosphorylation (activation) of tuberin, p70S6K, and GSK3ß both at baseline and after PE stimulation (Figure 5C).

View larger version (64K): [in this window] [in a new window]   Figure 5. Signaling pathways. A, MAP kinase profile. CMs were infected with indicated virus and stimulated with PE (20 µmol/L) for 60 minutes as marked. Total cellular protein (50 µg/lane) was separated by SDS-PAGE and subjected to immunoblotting for phospho- and total MAPK proteins. B, NFAT transcription activity. CMs were infected with Ad.NFAT-luc combined with Ad.EGFP, Ad.SGK1.CA, or Ad.SGK1.KD as indicated, treated with vehicle or PE as above, and assayed for luciferase activity (n=6). Results were expressed as fold increase vs unstimulated, control virus–infected CMs. SGK1 did not affect NFAT reporter activity. C, Downstream signaling pathways. CMs were infected with indicated virus and treated with vehicle or PE for 60 minutes as indicated. Protein samples were subjected to immunoblotting for phospho- or total tuberin, p70S6kinase, and GSK3b.

	Discussion

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PI3K plays an important role in regulating growth and survival in many settings,³³ and in the heart, these effects have been linked to activation of the serine-threonine kinase Akt^1–3,9,10; however, Akt is a member of a family of closely related kinases activated downstream of PI3K, including SGK, and it may be difficult to distinguish their roles with traditional pharmacological approaches. Moreover, recent studies in our laboratory suggest that PI3K-dependent but Akt-independent signaling pathways play an important role in cardioprotection.¹⁶ For these reasons, we examined the regulation and role of SGK1 in CMs.

We found that both expression and activation of SGK1 increased in hearts subjected to pressure overload through AAC. Interestingly, the increase in phosphorylation of SGK1 more closely paralleled the increase in GSK3ß phosphorylation than did Akt activation, raising the possibility that SGK1 could contribute to sustained GSK3ß phosphorylation, particularly at times when Akt activation has begun to recede. This hypothesis is consistent with the observation that SGK1 activation induced GSK3ß phosphorylation in CMs in vitro. SGK1 was also activated in CMs by the prosurvival and hypertrophic agonists IGF-I and PE in vitro. Together, these data demonstrate that SGK1 is dynamically regulated in CMs and in the heart in vivo.

To explore the functional role of SGK1 in CMs, we used recombinant adenoviral vectors carrying constitutively active or dominant negative mutants of SGK1. Activation of SGK1 was both necessary and sufficient to protect CMs from apoptosis after either SD or hypoxia in vitro. The specificity of studies of nuclear morphology was confirmed by analysis of DNA laddering and caspase-3 activation. These effects paralleled changes in overall cell viability as indicated by a reduction in the tetrazolium compound MTT. These studies are consistent with prior observations that SGK1 has cytoprotective effects in other systems.²³ Of note, SGK1.KD significantly but incompletely attenuated the protective effects of IGF-I. This finding is consistent with the well-documented cardioprotective effects of other PI3K effectors such as Akt¹ and suggests a model in which multiple PI3K-dependent pathways may each confer some survival advantage and be necessary for full cardioprotection (Figure 6).

View larger version (11K): [in this window] [in a new window]   Figure 6. Schematic of signaling model. We propose model in which multiple PI3K-dependent pathways modulate cell growth and survival. These pathways include Akt and SGK1, which appear to mediate convergent signaling on some targets (including GSK3ß, among others) but probably also signal through distinct downstream effectors.

Although a cytoprotective role for SGK1 has previously been documented in other settings,²³ SGK1 has not previously been implicated in control of hypertrophy or cell growth. In CMs, SGK1 activation enhanced hypertrophy both at baseline and after PE stimulation. Thus, the SGK1 activation observed after AAC or in response to hypertrophic agonists may well contribute to the hypertrophic phenotype, although the present study did not directly examine its role in vivo. In contrast to the apoptosis data discussed earlier, expression of SGK1.KD did not inhibit CM hypertrophy in response to PE. Thus, SGK1 does not appear necessary for CM hypertrophy, likely reflecting persistent signaling through parallel pathways involved in this response.^11,12

Multiple signaling pathways relevant to the hypertrophic response, including phosphorylation of ERK, SAPK/JNK, and p38, and NFAT-dependent transcription were not significantly altered by SGK1; however, we did identify SGK1-induced phosphorylation of several downstream effectors thought to play important roles in the cellular growth response (although previously linked to Akt), including tuberin, p70S6K, and GSK3ß.^11,12,34 All 3 of these substrates are potentially relevant to survival and growth signaling. Of note, we are not aware of any prior demonstration that SGK1 modulates activation of tuberin or p70S6K. The observation that GSK3ß phosphorylation is increased is particularly intriguing because GSK3ß has been closely connected to regulation of both CM hypertrophy and survival^11–15 and could function as an integrator of input from Akt and SGK1. This functional overlap could plausibly account for our observation that SGK1 was not necessary for the hypertrophic response (because Akt-mediated phosphorylation of GSK3ß would still occur). By the same logic, the observation that SGK1.KD attenuates the protective effect of IGF-I raises the possibility that SGK1 also has cardioprotective targets distinct from the downstream signaling if Akt (Figure 6).

In summary, the present study demonstrates that SGK1 is dynamically regulated in the heart and CMs and acts to modulate CM survival and the hypertrophic response. Of note, the functional consequences of SGK1 were examined only in vitro in the present study, and it seems likely that a complete understanding of the role of SGK1 in the heart and its relationship to Akt signaling will require analyses of in vivo genetic models. Nevertheless, we believe an appreciation of these parallel signaling pathways provides a necessary foundation for a more thorough understanding of the mechanisms of cardioprotection and growth in the heart.

	Acknowledgments

 
This work has been supported in part by grants from the NIH to Dr Rosenzweig (HL-59521, HL-61557, HL-073363) and to Dr Matsui (HL-04250) and by a Banyu Fellowship and Northeast Affiliate AHA Fellowship to Dr Aoyama. Friedrich Miescher Institute is funded by the Novartis Research Foundation. We thank Dr Jeffery Molkentin for ever-helpful scientific discussions and use of the NFAT reporter virus; we also thank Dr Tomohisa Nagoshi for his scientific input and help with the schematic illustration.

	References

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