Published online before print November 19, 2007, doi: 10.1161/CIRCULATIONAHA.107.711317
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(Circulation. 2007;116:2587-2596.)
© 2007 American Heart Association, Inc.
Molecular Cardiology |
Calsarcin-1 Protects Against Angiotensin-II–Induced Cardiac Hypertrophy
From the Department of Internal Medicine III (D.F., C.K., C.H., S.L., R.W., H.A.K., N.F.), University of Heidelberg, Germany; Medizinische Universitäts-Poliklinik (M.v.E.), Universitätsklinikum Bonn, Germany; and Sanofi-Aventis Pharma (D.G.), Frankfurt, Germany.
Correspondence to Dr Norbert Frey, Medizinische Universitätsklinik Heidelberg, Department of Internal Medicine III, Im Neuenheimer Feld 410, D-69120 Heidelberg, Germany. E-mail norbert.frey{at}med.uni-heidelberg.de
Received April 25, 2007; accepted September 21, 2007.
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Abstract |
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Background— We have previously shown that deficiency for the z-disc protein calsarcin-1 (CS1) sensitizes the heart to calcineurin signaling and to stimuli of pathological hypertrophy. In the present study we asked whether overexpression of CS1 might exhibit antihypertrophic effects, and therefore we tested this hypothesis both in vitro and in vivo.
Methods and Results— Adenoviral gene transfer of CS1 into neonatal cardiomyocytes inhibited hypertrophy as a result of Gq-agonist stimulation, including angiotensin-II (Ang-II), endothelin-1, and phenylephrine. Consistently, Adenoviral gene transfer of CS1 also led to the reduction of increased levels of atrial natriuretic factor (mRNA) and the calcineurin-sensitive gene MCIP1.4, suggesting that CS1 inhibits calcineurin-dependent signaling. Furthermore, we generated CS1-overexpressing transgenic mice (CS1Tg). Unchallenged CS1Tg mice did not exhibit a pathological phenotype as assessed by echocardiography and analysis of cardiac gene expression. Likewise, when subjected to long-term infusion of Ang-II, both CS1Tg and wild-type mice developed a similar degree of arterial hypertension. Yet, in contrast to wild-type mice, Ang-II–treated CS1Tg animals did not display cardiac hypertrophy. Despite the absence of hypertrophy, both fractional shortening and dP/dtmax were preserved in CS1Tg Ang-II–treated mice as assessed by echocardiography and cardiac catherization, respectively. Moreover, induction of the hypertrophic gene program (atrial natriuretic factor, brain natriuretic peptide) was markedly blunted, and expression of the calcineurin-dependent gene MCIP1.4 was significantly reduced in CS1Tg mice, again consistent with an inhibitory role of CS1 on calcineurin.
Conclusions— The sarcomeric protein CS1 prevents Ang-II–induced cardiomyocyte hypertrophy at least in part via inhibition of calcineurin signaling. Thus, overexpression of CS1 might represent a novel approach to attenuate pathological cardiac hypertrophy.
Key Words: angiotensin • heart failure • hypertension • hypertrophy • myocardium
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Introduction |
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Cardiac hypertrophy may develop in response to pressure and/or volume overload as well as neurohormonal activation. Although it is believed that cardiac hypertrophy initially represents an adaptive process, sustained hypertrophy can ultimately progress to heart failure and sudden death.1,2 In patients, cardiac hypertrophy is a powerful and independent predictor of subsequent morbidity and mortality.3,4
Clinical Perspective p 2596
Numerous molecular pathways have been shown to mediate hypertrophic signaling (reviewed by Frey et al2 and Molkentin et al5). During the last decade it has become evident that calcium/calmodulin-dependent signaling plays a pivotal role in the mediation of pathological cardiac hypertrophy.6,7 Calcineurin dephosphorylates transcription factors of the nuclear factor of activated T cells (NFAT) family, thereby facilitating their nuclear translocation and subsequent induction of the hypertrophic gene program. Constitutive activation of calcineurin in mouse hearts by transgenesis is sufficient to induce massive cardiac enlargement and heart failure.6 Conversely, genetic ablation of the most abundant calcineurin isoform in the heart (CnAβ) largely prevents pathological hypertrophy as a result of pressure overload, which implies that calcineurin is also necessary in this process.8 In several experimental studies, calcineurin inhibition resulted in a reduction of hypertrophy, yet no signs of cardiac compromise were observed.9–11 The present findings challenge the notion that hypertrophy is an essentially required adaptation to pressure overload. Interestingly, recent data suggest that calcineurin is preferentially activated in pathological hypertrophy (ie, caused by pressure overload), whereas its activity remains unchanged on stimuli of physiological hypertrophy, such as exercise.12
Because calcineurin is a ubiquitously expressed molecule, we have previously aimed to identify heart- and muscle-specific modulators of its activity. Using a yeast-2-hybrid approach, we have identified a novel family of calcineurin-interacting proteins termed calsarcins.13,14 Calsarcins localize to the z-disc of striated muscle cells, which only recently has been recognized as a nodal point in cardiac muscle signaling and disease.15,16 Moreover, several z-disc proteins, including muscle LIM protein17 and Melusin18 have been shown to be essential in the heart’s adaptation to pathological biomechanical stress.
Of the 3 calsarcins, only calsarcin-1 (CS1) is expressed in adult cardiac muscle, whereas calsarcin-2 (also known as myozenin19 or FATZ20) and calsarcin-3 are exclusively detected in skeletal muscle. To assess the function of calsarcin-1 in vivo, we recently generated mice with targeted ablation of the CS1 gene. Although these mice displayed no significant pathological cardiac phenotype under baseline conditions, pressure overload led to markedly exaggerated cardiac hypertrophy and superinduction of the fetal gene program.21 Likewise, when crossing CS1 nulls with mice overexpressing constitutively active calcineurin, these animals showed superhypertrophy associated with invariable premature death at 3 to 4 weeks of age.21 Molecular analyses revealed that markers of calcineurin activity, such as NFAT-DNA binding as well as mRNA expression of the calcineurin-dependent gene MCIP1 (modulatory calcineurin interacting protein) were significantly increased in knockout mice, which suggested that the absence of CS1 sensitizes the heart to calcineurin signaling. Although the present data imply that CS1 is required for proper adaptation to pathological hypertrophy, it still remains unclear whether overexpression of CS1 is also sufficient to inhibit calcineurin and, in turn, to attenuate cardiac hypertrophy. In the present report we show that overexpression of CS1 blunts cardiomyocyte hypertrophy in vitro induced by the Gq-coupled agonists angiotensin-II (Ang-II), phenylephrine (PE), as well as endothelin-1 (ET-1). Moreover, transgenic mice overexpressing calsarcin-1 were resistant to Ang-II–induced cardiac hypertrophy in vivo. Molecular analyses revealed that increased levels of CS1 inhibit calcineurin-dependent signaling, suggesting that calsarcin acts as a negative regulator of this pathway.
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Methods |
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Experimental procedures for preparation of neonatal cardiomyocytes, generation and treatment of transgenic mice, echocardiography and cardiac catherization, RNA and protein isolation, quantitative real-time PCR analyses, immunoblotting, as well as immunofluorescence experiments are provided in the online-only Methods supplement.
Statistical analyses of the data were carried out with 1- or 2-way ANOVA followed by Student-Newman-Keuls post hoc tests. If appropriate, Student t test was employed (2-sided, assuming similar variances). Probability values <0.05 were considered statistically significant.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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Calsarcin-1 Overexpression Inhibits Gq Agonist–Induced Cardiomyocyte Hypertrophy In Vitro
We have previously shown that deficiency of CS1 sensitizes mouse hearts for calcineurin signaling and thereby exacerbates pathological cardiac hypertrophy. To now investigate whether overexpression of CS1 is also sufficient to blunt hypertrophic growth, we performed in vitro experiments in neonatal cardiomyocytes. We first generated an adenovirus that encompassed a hemagglutinin-tagged CS1 cDNA (AdCS1). An enhanced green fluorescent protein–expressing adenovirus under control of the same (cytomegalovirus-) promoter served as control. Infection of neonatal cardiomyocytes with AdCS1 led to overexpression of CS1 as shown by Western blotting (Figure 1A). Moreover, overexpressed CS1 localized orthotopically to the z-disc (Figure 1B).
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P<0.001 for each comparison). HA indicates hemagglutinin.
Neonatal rat ventricular cardiomyocytes, either infected with AdCS1 or control virus (20 mol), were treated with the prohypertrophic agonists Ang-II (10 µmoi/L), PE (50 µmol/L), or ET-1 (1 µmol/L), respectively. After 24 hours, all substances induced significant cardiomyocyte hypertrophy (Figure 1C through 1E). Measurement of cell surface area (n
100 cells per group) revealed an increase of 98.1% in response to Ang-II (P<0.001) (Figure 1C and 1F), 50.6% in response to PE (P<0.001) (Figure 1D and 1G), and 56.0% after ET-1 treatment (P<0.001) (Figure 1E and 1H). Whereas infection with AdCS1 did not significantly alter the cell surface area of unstimulated neonatal rat ventricular cardiomyocytes, overexpression of CS1 significantly attenuated Ang-II–induced cardiomyocyte hypertrophy by 78.5% (P<0.001). Similarly, CS1 reduced PE-mediated hypertrophy by 78.1% (<0.001) and inhibited hypertrophy caused by ET-1 stimulation by 42.5% (P<0.001). Taken together, the present data show that calsarcin is able to suppress cardiomyocyte hypertrophy in response to several Gq-coupled agonists.
Adenoviral Overexpression of CS1 Blunts Calcineurin-Dependent Gene Expression in Response to Gq Agonist Stimulation
Next, we sought to determine whether the inhibition of cardiomyocyte hypertrophy was accompanied by attenuation of the hypertrophic (fetal) gene program. We thus analyzed the mRNA expression of atrial natriuretic factor (ANF) in unstimulated as well as Ang-II–treated neonatal rat ventricular cardiomyocytes. Ang-II led to a significant increase of ANF mRNA levels in cells infected with control virus (+53%, P<0.01) (Figure 2A). In contrast, in cardiomyocytes infected with AdCS1, ANF induction by Ang-II was completely abrogated.
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P<0.01, To assess whether calcineurin-dependent gene expression was altered by CS1, we determined the expression level of the 1.4 isoform of the MCIP gene (also known as RCAN1 and DSCR1). Because of an alternative promoter with a cluster of 15 NFAT binding sites,22 this isoform is exquisitely sensitive to increased calcineurin activity. Ang-II significantly induced MCIP1.4 in cardiomyocytes infected with control virus (+71.8%, P<0.01), which is consistent with previous observations that Ang-II activates calcineurin signaling.23 In contrast, overexpression of CS1 even led to a nonsignificant reduction of MCIP1.4 mRNA levels at baseline (–34.4%), which was unchanged under stimulation with Ang-II (–37.8%).
To further corroborate the present findings, we tested whether the results were also reproducible on the protein level. Therefore, we utilized a polyclonal antibody that simultaneously detects 2 MCIP1 isoforms, the calcineurin-sensitive 1.4 isoform as well as the calcineurin-insensitive 1.1 isoform.22,24,25 Consistent with the mRNA expression data, Ang-II was able to markedly upregulate the MCIP 1.4 isoform (+124.2%, P<0.001) (Figure 2C). Similarly, PE (+108.3%, P<0.001) and ET-1 (+39.9%, P<0.01) significantly induced expression of the MCIP1.4 (online-only Data Supplement Figure). In contrast, neither agonist was able to upregulate MCIP1.4 in neonatal cardiomyocytes that overexpress CS1 (Figure 2C; online-only Data Supplement Figure), again suggesting that CS1 inhibits calcineurin in cardiomyocytes in vitro.
Generation of Mice With Cardiac-Specific Overexpression of CS1
In order to validate the antihypertrophic properties of CS1 in vivo, we generated transgenic mice with cardiac-specific overexpression of CS1 with the use of an N-terminally hemagglutinin-tagged murine CS1 cDNA linked to the 
-MHC-promoter26 (Figure 3A). Transgenesis resulted in 5 transgenic lines with stable overexpression of CS1 on the protein level. Quantitative analysis revealed the highest expression levels of transgenic CS1 in line 5 (140% overexpression compared with endogenous protein). Of note, levels of endogenous CS1 remained unchanged, suggesting a lack of compensatory downregulation (Figure 3C). To verify that the transgenic protein colocalizes with endogenous CS1, we carried out immunofluorescence experiments. Costaining of cardiac cryosections with antibodies against CS1 as well as the hemagglutinin-tag of the transgenic protein revealed that transgenic CS1 localizes orthotopically to the sarcomeric z-disc (Figure 3D).
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Absence of a Pathological Baseline Phenotype in CS1-Transgenic Mice
Morphometric analysis of transgenic mice revealed no obvious pathological phenotype. As shown in Figure 4A, the gross morphology of transgenic hearts did not differ from wild-type (WT) mice. Likewise, heart weight to body weight ratios in 2 independent transgenic lines (Figure 4B) did not differ significantly from WT mice (mean wt, 5.7 mg/g; CS1tg#3, 5.3 mg/g; CS1tg#5, 5.4 mg/g). Accordingly, absolute heart weights (wt, 145 mg; CS1tg#3, 149 mg; CS1tg#5, 148 mg) and body weights (wt, 25.7 g; CS1tg#3, 28.1 g; CS1tg#5, 27.7 g; n=6 to 26 per group) were not different (data not shown). We also analyzed older animals (17 to 19 weeks of age) and again observed no difference compared with WT littermates (data not shown).
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The transgenic line with the highest expression of the transgene (line 5) was chosen for further analyses. To assess cardiac function and dimension in vivo, we performed echocardiography on transgenic mice and WT controls (Figure 4C). Neither fractional shortening (Figure 4D) nor left ventricular end-diastolic diameters differed significantly between groups (n=14 to 17 mice per genotype) (Figure 4E). Finally, mRNA expression levels of members of the hypertrophic gene program were determined. Again, no significant difference was found for ANF, β-MHC (β-myosin heavy chain, not shown) as well as for the calcineurin-responsive transcript MCIP1.4 (Figure 4F and 4G). In summary, CS1-transgenic mice did not display a detectable baseline phenotype, despite significant overexpression of transgenic protein.
Calsarcin-1 Inhibits Ang-II–Induced Cardiac Hypertrophy In Vivo
Next, we aimed to assess whether CS1 is also able to suppress cardiac hypertrophy in vivo. Therefore, 8-week-old CS1-transgenic mice were subjected to long-term stimulation with Ang-II (500 ng/kg per min) using subcutaneous minipumps. The administration of Ang-II led to a comparable increase in systolic blood pressure in transgenic mice and WT controls. As displayed in Figure 5A, WT animals showed an increase of 26.1% (from 115.0 to 145.0 mm Hg, P<0.05) and transgenic mice had a rise in blood pressure by 24.1% (from 118.9 to 148.9 mm Hg, P<0.01). Despite similar effects on blood pressure, only WT mice developed significant cardiac hypertrophy (Figure 5B and 5C). In WT animals, the left ventricle weight to tibia length ratio increased by 40.2% (from 5.8 to 8.1 mg/mm, P<0.01), whereas CS1-transgenic mice merely revealed an increase of 9.5% (from 6.3 to 6.9 mg/mm, P=NS), resulting in a significant difference between Ang-II–stimulated WT compared with CS1-transgenic mice (P<0.05; n=4 to 10 mice per group).
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To test whether this differential hypertrophic response was also present on the cellular level, we measured cardiomyocyte cross-sectional areas in transverse sections of the papillary muscles (Figure 5D). Cardiomyocyte size from unchallenged CS1-transgenic mice did not differ from WT controls (Figure 5E). However, on Ang-II stimulation only WT hearts displayed significant hypertrophy (+18.1%; n=131 to 159 from 3 animals each, P<0.001), whereas no significant difference was observed in CS1-transgenic animals (+2.5% versus sham, P>0.05). Interestingly, both WT and transgenic mice revealed a virtually identical increase in ventricular performance as assessed by cardiac catheterization (Figure 5F). Ang-II–stimulated WT animals reached a +dP/dtmax of 10 936 mm Hg/sec compared with 10 296 mm Hg/sec in CS1-transgenic mice (P=n.s).
To further support the present findings, we also analyzed an independent cohort of animals at older age (24 to 26 weeks old, n=4 to 7 per group), which were subjected to the same Ang-II treatment protocol. Consistent with the data in younger animals, WT and CS1-overexpressing mice showed an increase in blood pressure, which was not significantly different between genotypes. Again, only WT hearts displayed significant hypertrophy in response to Ang-II (+31.4%, P<0.01), whereas no significant difference was observed in CS1-transgenic animals (+11.8% versus WT sham, P=NS). In addition, assessment of fractional shortening by echocardiography revealed that infusion of Ang-II induced a comparable increase in the ventricular performance in both groups (WT: 60.5% versus 54.3%, P<0.05; CS1-transgenic: 63.2% versus 50.5%, P<0.001) (Figure 5G). Of note, neither untreated nor Ang-II–infused animals displayed a significant difference between genotypes. Likewise, left ventricular diameters remained unchanged (Figure 5H). All details of morphometry and echocardiographical measurements are provided in supplementary Tables IV and V. Thus, overexpression of CS1 results in suppression of cardiac hypertrophy in response to Gq agonist stimulation without impairment of contractile function.
Blunted Hypertrophic Response of Calsarcin-1–Transgenic Mice Is Accompanied by Suppression of the Hypertrophic Gene Program and Calcineurin Signaling
To test whether attenuation of the hypertrophic response to Ang-II was also accompanied by inhibition of hypertrophic gene expression, mRNA levels of ANF and brain natriuretic peptide were analyzed by quantitative real-time PCR (Figure 6A). Consistent with previous findings,27 in WT mice Ang-II stimulation induced ANF 3.7-fold (P<0.01). In contrast, CS1-transgenic mice failed to induce ANF (0.8 fold, P=NS). Similarly, brain natriuretic peptide was significantly induced in WT mice on Ang-II stimulation (2.6x, P<0.05), whereas CS1-transgenic mice revealed a significantly blunted upregulation (1.6x, P<0.05 versus WT Ang-II, Figure 5B).
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Again, the abundance of the MCIP1.4 transcript was determined to examine whether calsarcin inhibits calcineurin-dependent signaling in vivo. Consistent with ANF and brain natriuretic peptide expression, MCIP1.4 mRNA was markedly induced in Ang-II–treated WT mice (5.2x, P<0.01), whereas transgenic overexpression of CS1 reduced MCIP1.4 mRNA levels by 47.8% (P<0.05, Figure 5C).
To further corroborate the present findings, we also tested whether MCIP1.4 was upregulated on the protein level. As shown in a representative Western blot (Figure 6D), the MCIP1.4 isoform was significantly upregulated in WT hearts, whereas CS1-transgenic mice displayed a considerably blunted induction. Densitometry (Figure 6E) revealed an upregulation of MCIP1.4 of 150.2% in Ang-II–treated WT mice (P<0.001). In accordance with the mRNA data, overexpressed CS1 significantly attenuated MCIP1.4 induction by 36.3% (P<0.05).
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Suppression of Gq-Coupled Receptor Agonists Induced Hypertrophy by Calsarcin-1
Signaling of the phosphatase calcineurin via its downstream target transcription factors of the NFAT family is both sufficient6 and necessary28 for the induction of cardiac hypertrophy. Because calcineurin is ubiquitously expressed, we have previously hypothesized that tissue-specific modulators of its activity might exist. In addition to the MCIPs, which are also expressed at significant levels in the brain,29 we have recently identified a novel, highly striated, muscle-specific protein family, termed calsarcins, that directly interact with calcineurin.13 Mice lacking the calsarcin-1 gene are sensitized to calcineurin and display exaggerated cardiac hypertrophy in response to calcineurin activation,21 suggesting that calsarcin might be an endogenous inhibitor of calcineurin signaling. Thus, we now aimed to test whether overexpression of CS1 is sufficient to attenuate calcineurin-mediated cardiomyocyte hypertrophy both in vitro and in vivo.
Our results show that overexpressed CS1 is indeed able to significantly blunt hypertrophy induced by the Gq/G11-coupled receptor agonists Ang-II, PE, as well as ET-1. Moreover, the present findings could be confirmed in vivo because transgenic overexpression of CS1 was sufficient to markedly reduce the hypertrophic response to long-term Ang-II infusion.
Overexpression of Calsarcin-1 Inhibits Calcineurin Signaling In Vitro and In Vivo
It is well established that the Gq/G11-agonists Ang-II, ET-1, and PE mediate prohypertrophic signals through several molecular pathways,30 in particular through calcineurin/NFAT.23,31 Conversely, combined ablation of the downstream G proteins Gq and G11 in genetically engineered mice results in complete resistance against cardiac hypertrophy.32 In cardiomyocytes infected with a CS1 encoding virus, the hypertrophic response to the Ang-II, PE, and ET-1 was significantly attenuated if not completely abolished. Moreover, a comparable reduction in Ang-II–induced cardiac hypertrophy was observed in CS1-overexpressing mice. In both CS1-treated cardiomyocytes as well as in homogenates from CS1-transgenic myocardium, we detected a marked reduction in the levels of MCIP1.4 mRNA and protein. The MCIP1.4 isoform has been shown to be tightly controlled by an alternative promoter containing 15 NFAT binding sites in the intron located upstream of MCIP1 exon 4.22 Thus, MCIP1.4 expression levels can serve as a highly sensitive readout for calcineurin activity in vivo.21,25,33 CS1 was sufficient to reverse MCIP induction on agonist stimulation, which suggests that inhibition of calcineurin is the underlying mechanism for attenuation of cardiomyocyte hypertrophy. Consistent with this notion, we also observed downregulation of hypertrophic genes such as ANF both in calsarcin-adenovirus treated cardiomyocytes as well as CS1-transgenic animals subjected to hypertrophic stimulation. Of note, ANF expression has been shown to be calcineurin-dependent,23,28 again implying that calsarcin inhibits hypertrophy and hypertrophic gene expression via inhibition of the calcineurin-/NFAT-pathway.
The z-Disc as a Nodal Point in Hypertrophic Signaling: Role of Calsarcin-1
The z-disc has traditionally been considered to serve a purely mechanical role in maintaining sarcomere and (via the costamere) membrane integrity. More recently however, the z-disc has also emerged as a nodal point in cardiomyocyte signaling.16 For example, loss of the z-disc muscle LIM protein in genetically engineered mice results in severe cardiomyopathy.34 Moreover, muscle LIM protein–deficient cardiomyocytes are unable to upregulate the hypertrophic gene program (ie, ANF, brain natriuretic peptide) in response to biomechanical stress, which supports the notion that the z-disc is a critical integrator of hypertrophic signals.17 Similar findings have been reported for the z-disc protein FHL-2, which negatively regulates cardiomyocyte hypertrophy via inhibition of ERK2 nuclear translocation.35 Likewise, MuRF-1 (muscle ring finger protein), another z-disc associated protein, blunts hypertrophy via inhibition of PKC-
and ERK1/2.36 In regard to calcineurin signaling, we and others have shown that calcineurin can be detected at the sarcomeric z-disc.10,13,37,38 Moreover, the calcineurin-dependent transcription factor NFAT has been detected at the z-disc in unstimulated striated muscle cells.39 Thus, calsarcin resides in close proximity to several molecules implicated in hypertrophic signaling, including calcineurin, and it is conceivable that calsarcins inhibit calcineurin activation at the level of the z-disc. Of note, calcineurin has also been detected in the nucleus of neonatal rat cardiomyocytes.13,40 Furthermore, Hallhuber and colleagues demonstrated that nuclear translocation of calcineurin is necessary for its prohypertrophic effects,40 whereas inhibition of nuclear import appeared to inhibit cardiomyocyte hypertrophy. Taking the present findings into account, we tested whether CS1 is also subject to nuclear shuttling after stimulation with Ang-II, PE, or ET-1. However, neither endogenous nor overexpressed CS1 showed a nuclear localization, regardless of the hypertrophic stimulus or the presence of the nuclear export inhibitor leptomycin B (data not shown). A possible explanation to reconcile the present findings is that different intracellular pools of calcineurin exist, eg, one at the z-disc, one in the nucleus. In this view, CS1 would specifically inhibit activation of sarcomeric calcineurin. Alternatively, overexpression of calsarcin might prevent nuclear translocation of calcineurin and thereby attenuate cardiomyocyte hypertrophy in response to upstream activators.
Inhibition of Cardiac Hypertrophy as a Therapeutic Goal: Potential Role of Calsarcin-1
Cardiac hypertrophy is considered to be an adaptive response to adjust the heart to increased biomechanical stress caused by arterial hypertension or valvular heart disease. At the same time, a wealth of data shows that cardiac hypertrophy is a strong and independent predictor of an adverse prognosis.3,4,41 Interestingly, several experiments in genetically modified animals revealed that inhibition of the hypertrophic response is not necessarily associated with a decrease in cardiac function,30 which suggests that inhibition of hypertrophy might be beneficial. Finally, new concepts are emerging that, at the molecular level, physiological hypertrophy (ie, hypertrophy caused by exercise) fundamentally differs from pathological hypertrophy. In this context it is worth noting that calcineurin/NFAT-signaling has predominantly been implicated in pathological hypertrophy. This has convincingly been shown by Wilkins and co-workers who used a transgenic reporter mouse that expressed luciferase under control of an NFAT-dependent promoter.12 Whereas experimental myocardial infarctions or pressure overload resulted in robust activation of the reporter in these mice, long-term exercise rather decreased NFAT activity, implying that calcineurin/NFAT selectively mediates pathological growth signals. Consistent with this notion, we have previously shown that the lack of CS1 aggravates pathological hypertrophy, whereas calsarcin-deficient mice subjected to exercise exhibited no differential hypertrophic growth.21
Thus, calcineurin/NFAT signaling may represent an attractive therapeutic target for the inhibition of hypertrophy and subsequent heart failure. Yet, because of the ubiquitous expression pattern of calcineurin, pharmacological strategies to inhibit its activity in the heart have been hampered by severe systemic side effects.42 It remains to be seen if it will be feasible to express CS1 at sufficient levels (ie, using a viral approach) in vivo to inhibit cardiac calcineurin/NFAT in the heart. Nevertheless, it is reassuring that CS1-transgenic mice abrogated the hypertrophic response to agonist stimulation whereas contractile function remained unaltered.
In summary, we provide evidence that overexpression of the novel sarcomeric protein CS1 is sufficient to inhibit Gq-agonist induced cardiac hypertrophy as well as hypertrophic gene expression both in vitro and in vivo. Mechanistically, CS1 attenuates calcineurin signaling in the heart (Figure 7), thus preventing pathological hypertrophy. Future studies will have to show whether this effect can be exploited therapeutically.
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Acknowledgments |
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We are grateful to Ulrike Oehl, Jutta Krebs (Heidelberg), and Daniel Hein (Frankfurt) for excellent technical assistance.
Sources of Funding
Dr Frank was supported by the Young Investigator Program of the University of Heidelberg. Dr Frey was supported by the Bundesministerium für Bildung und Forschung, Germany (NGFN2-Nationales Genomforschungsnetz), as well as by the German Research Foundation (DFG).
Disclosures
None.
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Footnotes |
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*Dr Frank and Dr Kuhn contributed equally to this article.
The online-only Data Supplement, consisting of tables, figures, and expanded Methods, is available online with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.107.711317/DC1.
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