Modulation of Angiotensin II–Mediated Cardiac Remodeling by the MEF2A Target Gene Xirp2
Rationale: The vasoactive peptide angiotensin II (Ang II) is a potent cardiotoxic hormone whose actions have been well studied, yet questions remain pertaining to the downstream factors that mediate its effects in cardiomyocytes.
Objective: The in vivo role of the myocyte enhancer factor (MEF)2A target gene Xirp2 in Ang II–mediated cardiac remodeling was investigated.
Methods and Results: Here we demonstrate that the MEF2A target gene Xirp2 (also known as cardiomyopathy associated gene 3 [CMYA3]) is an important effector of the Ang II signaling pathway in the heart. Xirp2 belongs to the evolutionarily conserved, muscle-specific, actin-binding Xin gene family and is significantly induced in the heart in response to systemic administration of Ang II. Initially, we characterized the Xirp2 promoter and demonstrate that Ang II activates Xirp2 expression by stimulating MEF2A transcriptional activity. To further characterize the role of Xirp2 downstream of Ang II signaling we generated mice harboring a hypomorphic allele of the Xirp2 gene that resulted in a marked reduction in its expression in the heart. In the absence of Ang II, adult Xirp2 hypomorphic mice displayed cardiac hypertrophy and increased β myosin heavy chain expression. Strikingly, Xirp2 hypomorphic mice chronically infused with Ang II exhibited altered pathological cardiac remodeling including an attenuated hypertrophic response, as well as diminished fibrosis and apoptosis.
Conclusions: These findings reveal a novel MEF2A-Xirp2 pathway that functions downstream of Ang II signaling to modulate its pathological effects in the heart.
Angiotensin II (Ang II) is a potent hypertensive agonist that also promotes extensive myocardial damage even in the absence of hypertension.1 The repertoire of downstream effectors in Ang II–mediated pathological cardiac remodeling, however, remains largely incomplete.2,3 A recent global gene expression study identified transcripts of a novel gene, named CMYA3 (cardiomyopathy associated gene 3), that were upregulated in hearts of mice treated with Ang II but not in salt-induced hypertensive mice,4 suggesting that CMYA3 is directly regulated by Ang II signaling. This gene, since named Xirp2 (also known as mXinβ and myomaxin), is a direct target of the MEF2A transcription factor and is markedly downregulated in hearts lacking MEF2A.5,6
Xirp2 belongs to the ancient, muscle-specific, actin-binding Xin gene family whose expression can be traced to ancestral vertebrates with a 2-chambered heart.7–9 Xirp2 is expressed in cardiac and skeletal muscle where it interacts with filamentous actin and α-actinin through the novel actin-binding motif, the Xin repeat.5,8 In striated muscle, Xirp2 localizes to the peripheral Z-disc region, or costamere,5 and the intercalated disk.10,11 The subcellular localization of Xirp2 is significant in that the costamere and intercalated disk harbor mechanical stress sensors that are critical for normal muscle function.12–14
Antisense knockdown of Xin in developing chick embryos, the sole Xin family member in this species, results in a severe disruption of cardiac looping morphogenesis.9 In mice, a loss-of-function mutation of mXinα, the mammalian ortholog of Xin, results in cardiomyopathy and conduction defects.11 In the present study we sought to determine the role of Xirp2 in cardiac development and/or function. Mice harboring a hypomorphic Xirp2 allele are viable but display cardiac hypertrophy. As Xirp2 is regulated by Ang II, we also examined cardiac pathology in hypomorphic mice with long-term administration of this hormone. In contrast to wild type mice exposed to a chronic Ang II infusion, hypomorphic mice displayed diminished cardiac hypertrophy, fibrosis, and apoptosis. Furthermore, we demonstrate that regulation of Xirp2 gene expression in response to Ang II signaling is mediated by MEF2A. Our results suggest that MEF2A and Xirp2 are important downstream effectors in mediating pathological cardiac remodeling in response to Ang II signaling.
Details of materials and experimental procedures can be found in the expanded Methods section in the Online Data Supplement at http://circres.ahajournals.org.
Generation of Xirp2 loxP-Targeted Mice
Xirp2 loxP-neo targeted mice were generated by inGenious Targeting Laboratory Inc (Stony Brook, NY).
Histology and Immunofluorescence
Hearts were fixed in 4% paraformaldehyde, cryoprotected in sucrose, and placed in embedding compound (OCT). Whole-heart sections were stained with hematoxylin/eosin. Masson’s trichrome staining was performed to determine the extent of cardiac fibrosis. Apoptosis was assessed by TUNEL assay using the Promega DeadEnd Colorimetric TUNEL System kit. For immunofluorescence, heart cryosections were blocked in BSA before incubation with primary and secondary antibodies.
Administration of Ang II
Ang II (0.9 μg/h) was administered via subcutaneous osmotic mini-pumps (Alzet model 2004) for 14 days.
Echocardiography and Blood Pressure Analysis
Transthoracic M-mode echocardiography was performed on mice at baseline (pretreatment) and post-2week Ang II infusion. Blood pressure analysis was performed using the noninvasive tail cuff method (Model BP 2000, Visitech Systems).
Microarray and Gene Expression Analysis
cDNA was prepared from total RNA isolated from either hindlimb or ventricular tissue using TRIzol reagent (Invitrogen). Primers for quantitative real-time PCR (qRT-PCR)/RT-PCR can be found in the Online Data Supplement. For qRT-PCR, individual nonpooled samples were run in triplicate wells. qRT-PCR was performed with SYBR Green master mix (Applied Biosystems) using the 7900 Sequence Detection System (Applied Biosystems). For microarray, samples were prepared as described previously15 and hybridized to the Mouse Gene 1.0 ST Array (Affymetrix) at the Boston University Microarray Facility.
Western Blot Analysis
To detect cardiac Xirp2 protein, ventricular muscle was snap-frozen in liquid nitrogen immediately following dissection, pulverized and resuspended in sample loading buffer. Protein concentrations were analyzed by Bradford assay. Samples were subjected to SDS-PAGE, transferred to poly(vinylidene difluoride) membrane (Bio-Rad) and immunoblotted using primary antibodies described in the Online Data Supplement.
Cell Culture, Luciferase Assays, and Plasmids
COS1 cells were grown in DMEM with 10% FBS, 1% penicillin/streptomycin, and 1% l-glutamine and transfected using Mirus TransIT-LT1 transfection reagent. Luciferase assays were performed using Luciferase Assay Reagent (Promega), and results were normalized by Bradford assay. For analysis of Xirp2 expression in primary neonatal rat ventricular myocytes (NRVMs), cells were isolated as described previously.5 All Xirp2 luciferase promoter constructs were cloned into the pGL3b-luciferase vector (Promega) except the −1425/−285 deletion mutant which was cloned into the pGL3p-luciferase construct (Promega).
Appropriate data sets were analyzed for significance using 2-way ANOVA. Variance of data sets was determined using the Bartlett’s test. Either a 2-tailed Student t test or Welch’s t test was performed for each pairwise comparison. A probability value of <0.05 was considered to be statistically significant.
Ang II Stimulates MEF2A Transcriptional Activity to Regulate Xirp2 Expression
To determine whether the Ang II–mediated upregulation of Xirp2 was a direct effect of the hormone on cardiomyocytes or was attributable to secondary effects resulting from pressure overload, primary neonatal rat ventricular myocytes (NRVMs) were isolated and treated with Ang II. As shown in Figure 1A, Xirp2 transcripts were induced in NRVMs upon Ang II treatment indicating that Xirp2 is directly stimulated by the hormone.
The above results prompted us to map the Ang II–responsive region in the proximal 1.5kb Xirp2 promoter.5 Because of the very high basal activity of this promoter and smaller deletion constructs in NRVMs, we were unable to detect enhanced activation by Ang II in this system. Subsequently, we examined the responsiveness of various Xirp2 promoter constructs (Figure 1B) to Ang II in COS cells because these cells express the type I angiotensin receptor. In transiently transfected COS cells, the −1425 Xirp2 promoter was stimulated 2.5-fold by Ang II (Figure 1C). A truncated Xirp2 promoter (−285) was similarly activated by Ang II (Figure 1C), indicating that the Ang II–responsive region resides within the first 300 base pairs upstream of the transcription start site. This minimal region harbors an essential MEF2 site.5 Given that MEF2 activity is modulated by Ang II in vascular smooth muscle16,17 we reasoned that Ang II–induced Xirp2 expression is mediated by MEF2. To test this hypothesis, we transfected a mutant promoter construct that harbors a mutation in the −75 MEF2 site (−285mut) which disrupts MEF2 DNA binding. Ang II activation of the −285 mutant promoter was significantly reduced, indicating that the MEF2 site functions as an Ang II–responsive element in the Xirp2 promoter (Figure 1C).
To further investigate the role of MEF2 downstream of Ang II–mediated activation of the Xirp2 gene, the −1425 Xirp2 promoter was cotransfected in COS cells along with MEF2A in the presence or absence of Ang II. Ang II or MEF2A alone activated the −1425 promoter by 2.5-fold and 3.4-fold, respectively (Figure 1D). The combination of Ang II and MEF2A robustly activated the −1425 Xirp2 promoter 10.6-fold (Figure 1D). Similar results were observed with the −285 promoter construct (Figure 1D). This cooperative effect was severely attenuated in 3 different mutant Xirp2 promoters in which the −75 MEF2 site was either deleted (−1425/−285) or disrupted by point mutation (−1425mut and −285mut) (Figure 1D). However, the ability of Ang II to stimulate MEF2A was not mediated by enhanced binding to the MEF2 site (Online Figure I).
To reinforce the notion that MEF2A is an essential regulator of Xirp2 downstream of Ang II signaling in vivo, we examined the expression of Xirp2 in NRVMs in which MEF2A was knocked down by adenoviruses expressing MEF2A-specific short hairpin (sh)RNAs (Online Figure VIII and to be described in detail elsewhere). We failed to observe an induction of Xirp2 by Ang II in cells transduced with MEF2A shRNAs but not control lacZ shRNAs (Figure 1E, compare lanes 3 and 4 with lanes 5 and 6). These results demonstrate that Xirp2 is a novel, direct transcriptional target of Ang II whose induction is mediated by MEF2A.
Generation of Xirp2 Hypomorphic Mice
Having established that Xirp2 is directly regulated by Ang II, we wanted to determine the in vivo requirement of this gene in Ang II–induced cardiomyopathy. Therefore, we generated a conditional null allele of the Xirp2 gene which contained loxP sequences flanking exons 4 and 6, and a PGK-neomycin (PGK-neo) cassette in the intron between exons 6 and 7 (Figure 2A). To generate a complete loss-of-function allele, conditional Xirp2 mice were crossed to EIIa-Cre transgenic mice which removed exons 4 to 6 along with the loxP flanked PGK-neo cassette in the germline. These heterozygous Xirp2 loxP mice (+/loxP) were intercrossed resulting in homozygous Xirp2 loxP/loxP mice that were viable, fertile and genotyped at the expected Mendelian ratios. The excision of exons 4 to 6 was confirmed by RT-PCR analysis on cardiac muscle cDNA (Figure 2B). Sequencing of these truncated cDNAs revealed an in-frame splice between exons 3 and 7 (Figure 2B). This in-frame splice had no effect on Xirp2 expression in homozygous loxP/loxP mice (data not shown) and as a result, these mice have not been further characterized.
In parallel, we generated loxP-neo targeted Xirp2 homozygous mice (referred to as loxP-neo) that retained the PGK-neo cassette (Figure 2A). Homozygous loxP-neo mice were identified in the expected Mendelian ratios demonstrating that this allele, like the in-frame deletion, does not affect viability. As retention of PGK-neo can often interfere with expression of the targeted gene,18 we examined Xirp2 transcript levels in homozygous loxP-neo mice by quantitative real time RT-PCR (qRT-PCR). Using primers spanning exons 2 and 3, qRT-PCR analysis of cardiac and skeletal muscle cDNA revealed that these tissues express Xirp2 at only 15% to 20% of wild type levels (Figure 2C). RT-PCR analysis using multiple downstream primer sets demonstrated similar results suggesting that full length Xirp2 transcripts are being produced from the loxP-neo targeted allele (Online Figure II). In addition, Xirp2 protein is largely absent from both hindlimb and cardiac muscle extracts (Figure 2D). Unlike the upregulation of Xirp2 in Xin knockout hearts,11 there was no compensatory increase in Xin gene expression in Xirp2 hypomorphic hearts (Figure 2E). Given these exciting results, we focused on characterizing the cardiac phenotype of Xirp2 hypomorphic mice.
Cardiac Hypertrophy in Xirp2 Hypomorphic Mice
Because Xirp2 is enriched at the muscle costamere we reasoned that its reduction would adversely affect the normal growth and/or function of the heart. We measured heart weight : body weight (HW:BW) ratios in wild type and Xirp2 hypomorphic adult mice. Between 9 and 15 weeks postnatally Xirp2 hypomorphic mice displayed a modest increase in HW:BW ratio (19%) (Figure 3A). We performed morphometric analysis of ventricular myocytes in adult hypomorphic hearts and observed a significant increase in the cross-sectional area (CSA) (1.6-fold) (Figure 3B) but other hallmarks of cardiomyopathy such as fibrosis and apoptosis were not significantly altered (data not shown). Similarly, examination of cardiomyocytes by electron microscopy did not reveal any obvious perturbations in myofibrillar structure (data not shown). Echocardiographic assessment of cardiac function showed no significant differences in ejection fraction (%EF) or fractional shortening (%FS) in hypomorphic mice (Online Figure III). As Xirp2 is also expressed in skeletal muscle future studies will focus on the characterization of a possible phenotype in this tissue.
To further characterize the hypertrophic phenotype we examined the expression of hypertrophic marker genes by qRT-PCR. There was no significant change in expression of atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), or α myosin heavy chain (αMHC) genes (Figure 4A). However, Xirp2 hypomorphic hearts exhibited a significant activation (3.9-fold) of the β myosin heavy chain (βMHC) gene (Figure 4A).
Global Dysregulation of Cardiac Gene Expression in Xirp2 Hypomorphic Mice
To investigate the molecular mechanisms of the hypomorphic cardiac phenotype we performed microarray analysis of ventricular RNA from adult wild type and Xirp2 hypomorphic mice. We found a dysregulation of genes belonging to a broad spectrum of functional categories including metabolism (15%), muscle contraction (11%), calcium handling (9%), and cytoskeleton (5%) (Figure 4B). A subset of these genes was validated by qRT-PCR. MARCKS (myristoylated alanine-rich C kinase substrate), Pdlim3/ALP (α-actinin–interacting LIM protein), and lipocalin 2 genes were significantly upregulated, whereas RCAN1/MCIP1 (regulator of calcineurin) was significantly downregulated (Figure 4C). In addition, the downregulation of RCAN1/MCIP1 was confirmed by Western blot analysis (Online Figure IV). Interestingly, like Xirp2, both MARCKS and Pdlim3/ALP are involved in F-actin and α-actinin cross-linking dynamics, respectively.19,20 The lipocalin 2 gene encodes a glycoprotein involved in numerous cellular processes21 and its upregulation in hypomorphic hearts is consistent with the reported activation of this gene in human and rodent models of heart failure.22 One possible outcome of reduced RCAN1/MCIP1, a modulator of the prohypertrophic factor calcineurin,23 is an elevation in calcineurin activity, and consequently, increased cardiomyocyte size in hypomorphic hearts. Taken together, the above data are consistent with pathological cardiac hypertrophy in Xirp2 hypomorphic mice.
Diminished Cardiac Pathophysiology in Xirp2 Hypomorphic Mice Infused With Ang II
To determine whether Xirp2 is required for stress-induced cardiac remodeling in vivo we subjected hypomorphic mice to chronic Ang II infusion. The 2-week Ang II infusion in wild type mice resulted in a 20% increase in HW:BW (Figure 5A) which was confirmed by the 2.5-fold increase in ventricular myocyte CSA (Figure 5B). In contrast, Ang II treatment failed to induce a significant increase in HW:BW ratios in Ang II–infused hypomorphic mice (Figure 5A). This evidence of attenuated cardiac hypertrophy is supported by the less pronounced increase in hypomorphic cardiomyocyte CSA compared to that of wild type animals (1.7-fold compared to 2.7-fold respectively) (Figure 5B). These results indicate that the residual amount of Xirp2 in hypomorphic hearts is insufficient to fully induce the hypertrophic effects of Ang II.
Because chronic Ang II administration induces cardiac interstitial fibrosis, we subjected hearts from wild type and hypomorphic mice to Masson’s trichrome staining. At treatment with Ang II, wild type mice exhibited a 2.8-fold increase in fibrosis relative to sham-operated animals (Figure 5C). In striking contrast, chronic Ang II infusion was unable to stimulate an increase in fibrosis in Xirp2 hypomorphic mice (Figure 5C). We subsequently performed TUNEL assay to assess the extent of apoptosis in Ang II–infused hearts. Ang II–infused wild type mice showed a 3.3-fold increase in the amount of TUNEL-positive cells in the heart (Figure 5D). However, Ang II–infused Xirp2 hypomorphic mice showed no significant increase in myocardial apoptosis (Figure 5D). Finally, the Ang II dose used in this study induced hypertension in wild type and hypomorphic mice without any significant difference between the 2 groups (Online Figure V).
Analysis of Hypertrophic Markers in Xirp2 Hypomorphic Mice Upon Ang II Infusion
Given the dampened cardiomyopathy in Ang II treated hypomorphic mice we examined the expression of hypertrophic markers in hearts with long term administration of Ang II. Wild type animals displayed an increase in βMHC (5.7-fold) and ANF (5.1-fold) expression (Figure 6A). In contrast, βMHC expression was not significantly increased in Xirp2 hypomorphic hearts upon Ang II infusion, whereas ANF displayed responsiveness to Ang II. Expression of αMHC and BNP was not significantly dysregulated in either wild type or hypomorphic hearts upon Ang II infusion (Figure 6A). These results show a differential response of βMHC to Ang II signaling in stressed hypomorphic hearts which correlates with attenuated cardiac hypertrophy in these animals.
To further understand the mechanisms behind the attenuated cardiomyopathy in stressed hypomorphic mice we examined phosphorylation levels of intracellular signaling molecules known to function downstream of Ang II. By Western blot analysis, we found no significant difference in the phosphorylation of the MAPK components, Erk1/2, p38, and JNK, or protein kinase D1 (PKD1)24 in Ang II–infused hypomorphic hearts (data not shown). Also, we found no difference in the transcript or protein levels of the type I angiotensin receptor (AT1R) (Online Figure VI). In contrast, glycogen synthase kinase (GSK)-3β serine-9 phosphorylation was significantly reduced in Ang II–infused Xirp2 hypomorphic hearts (Figure 6B). This effect does not appear to be mediated by Akt, an upstream kinase of GSK-3β, because Western blot analysis did not detect differences in its activity in hypomorphic hearts (Online Figure VII). Inhibition of GSK-3β kinase activity, a well established hypertrophic antagonist, through increased phosphorylation on serine-9, is associated with enhanced hypertrophy.25 A major target of active GSK-3β is β-catenin, which is phosphorylated by GSK-3β and is subsequently targeted for ubiquitination and degradation.26 Western blot analysis revealed that β-catenin levels are significantly diminished in Ang II–treated hypomorphic mice (Figure 6C). Thus, the reduction in GSK-3β serine-9 phosphorylation in Ang II–treated hypomorphic mice is consistent with diminished cardiac hypertrophy.
In the present study we report for the first time that the novel MEF2A target gene, Xirp2, is an essential mediator of Ang II–induced pathological cardiac remodeling in vivo. We generated a Xirp2 hypomorphic allele which resulted in a marked reduction in its expression in skeletal and cardiac muscle in mice. Although these mice are viable, unstressed Xirp2 hypomorphic mice display cardiac hypertrophy. Paradoxically, hearts from hypomorphic mice infused with Ang II displayed attenuated cardiac hypertrophy, interstitial fibrosis and cardiomyocyte apoptosis.
It is well documented that Ang II promotes myocardial damage, thus the identification of novel mediators of this signaling pathway in the heart is an important goal. We now provide evidence that Xirp2 is a direct transcriptional target of Ang II signaling in cardiac muscle. Further, the activation of the Xirp2 gene by Ang II is controlled, in part, by the MEF2A transcription factor. The related Xin gene is also a MEF2 target9 yet expression of this gene was not significantly induced in the heart by Ang II. These observations suggest a tightly controlled regulation of the Xin gene family involving the Ang II signaling pathway and MEF2.
By generating mice with a hypomorphic Xirp2 allele we were able to establish that Xirp2 is required for the proper physiological growth of the heart, because a reduction in its expression resulted in enlarged cardiomyocyte size. Cardiac hypertrophy in hypomorphic mice was accompanied by an upregulation of the hypertrophic marker gene, βMHC, and a downregulation of the calcineurin modulatory gene, RCAN1/MCIP1. The downregulation of a calcineurin modulator provides a plausible mechanism by which unstressed hypomorphic mice develop myocyte hypertrophy through increased calcineurin activity.27 Furthermore, the upregulation of Pdlim3/ALP and MARCKS, which encode cytoarchitectural proteins involved in actin dynamics localized to costameres and focal adhesions, respectively, may indicate a compensatory response to the reduction of Xirp2 at these structures. The cardiac phenotype displayed by unstressed Xirp2 hypomorphic mice is reminiscent of Xin knockout mice which also develop adult onset hypertrophy.11 These findings suggest that Xirp2 and Xin have partially overlapping functions in unstressed cardiomyocytes. In the future it will be of interest to determine the consequences in cardiac development and/or function in mice lacking both Xin family members.
The upregulation of the hypertrophic marker, βMHC, but not other fetal cardiac genes in unstressed Xirp2 hypomorphic mice suggests an unconventional, but not unprecedented, mechanism of pathological cardiac remodeling. Transgenic mice overexpressing either the β2 adrenergic receptor or an inhibitor of β adrenergic receptor kinase 1 (βARK1ct) in the heart displayed elevated levels of the βMHC but not the ANF or skeletal α-actin genes.28 Although the significance of this specific pattern of hypertrophic gene dysregulation is not entirely clear, these observations reveal that the coordinate upregulation of fetal cardiac genes is not a universal pathway and does not apply to all models of cardiomyopathy.
Our data also reveal an attenuation of Ang II–induced pathological cardiac remodeling in Xirp2 hypomorphic mice. The attenuated hypertrophy, fibrosis, and apoptosis were accompanied by compromised activation of βMHC expression and reduced phosphorylation of GSK-3β, and thus reduced β-catenin levels. Expression of the βMHC gene is sensitive to cardiac stress,29 and the failure to further upregulate βMHC expression in Ang II treated hypomorphic mice is likely a direct indication of the diminished hypertrophy. It is known that active GSK-3β functions as a hypertrophic antagonist and that phosphorylation of the kinase at serine-9 is an inactivating modification.25,26 It follows that expression of an unphosphorylatable form of GSK-3β (GSK-3βS9A) in cardiomyocytes suppresses hypertrophy.30,31 Thus, reduced GSK-3βS9 phosphorylation in Ang II treated hypomorphic mice may provide a mechanism for the dampened cardiac hypertrophy. Further, the concomitant reduction in β-catenin levels in Ang II–treated hypomorphic hearts is consistent with reports that depletion or reduction of β-catenin in the heart results in blunted pathological cardiac remodeling in response to stress.32,33
The reduced fibrosis and apoptosis in Ang II treated hypomorphic mice demonstrates that Xirp2 is required to promote these hallmarks of pathological remodeling in the heart downstream of this hormone. These results provide the first evidence that Xirp2 may be involved in cell survival pathways in cardiac stress signaling. As myocyte cell death and interstitial fibrosis are major contributors to end stage heart failure, minimizing the extent of these abnormalities in the diseased heart would be expected to significantly improve cardiac function. It is tempting to speculate that modulating Xirp2 expression through pharmacological strategies could identify an optimal level of Xirp2 activity that does not induce hypertrophy under normal physiological conditions but blunts pathological cardiac remodeling in response to stress.
Surprisingly, the preexisting cardiac hypertrophy in unstressed hypomorphic mice was not exacerbated by long-term administration of Ang II. The attenuated cardiac remodeling in Ang II treated hypomorphic mice may point to a unique, additional role for Xirp2 in the modulation of Ang II signals that is not dependent on, and largely separable from, its basal function in cardiac development and homeostasis. In support of this hypothesis, microarray analysis upon Ang II treated hypomorphic mice (Online Figure VIII) revealed that the global profile of dysregulated genes in unstressed hypomorphic mice was largely distinct from the dysregulated gene program in Ang II treated hypomorphic mice (hypo versus Ang II-hypo). These data argue against a common gene program triggered by the reduction of Xirp2 in the absence and presence of cardiac stress.
Collectively, our data support the notion that Xirp2 possesses 2 distinct functions in cardiomyocytes, such that its reduced levels in unstressed conditions is deleterious to the heart, but in the presence of stress, limiting amounts of Xirp2 appear to be beneficial. We previously reported that Xirp2 expression in NRVMs is induced by additional hypertrophic stimuli such as phenylephrine and serum.5 Therefore, it will be important to investigate whether a reduction in Xirp2 can also influence cardiac remodeling in response to additional neurohormonal insults and biomechanical stressors, or whether Xirp2 functions specifically as a mediator of Ang II–induced cardiomyopathy.
We are grateful to Junichi Sadoshima (University of Medicine and Dentistry of New Jersey, Newark) for the total and phospho-S9-GSK-3β antibodies, Timothy McKinsey (Gilead, Westminster, Colo) for the PKD1 antibodies, Jeffery Molkentin (Howard Hughes Medical Institute, Cincinnati, Ohio) for MCIP1 antibodies, Isabel Dominguez (Boston University) for β-catenin antibodies, and Geoffrey Copper (Boston University) for total and phospho-Akt antibodies. We also thank Andrew Betts (Ubixum Inc, Palo Alto, Calif) for assistance with Matlab image processing and Susan Kandarian (Boston University) for use of Metamorph software.
Sources of Funding
This work was supported by the NIH/National Heart, Blood, and Lung Institute and the Muscular Dystrophy Association (to F.J.N.), a Clare Boothe Luce Fellowship (to S.A.M.), a postdoctoral NIH Cardiovascular Training grant (to S.A.), undergraduate research fellowships from the American Heart Association (to D.D. and M.M.), and a Boston University Undergraduate Research Opportunities (UROP) fellowship (to K.D.).
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Novelty and Significance
What Is Known?
The hormone Ang II has widespread damaging effects on the heart but only a few downstream genes are known to mediate its effects.
The muscle-specific, actin-binding Xirp2 gene is regulated by Ang II.
The Xirp2 gene is regulated by the MEF2A transcription factor.
What New Information Does This Article Contribute?
A novel mouse model with reduced expression of Xirp2 in the heart results in cardiac hypertrophy.
Hearts with reduced Xirp2 expression display less myocardial damage when exposed to Ang II.
Ang II regulates Xirp2 through the MEF2A transcription factor.
Before this report, no information existed pertaining to the in vivo function of Xirp2 in the heart. To our knowledge this study is the first to describe the cardiac phenotype of a mouse knockdown model of Xirp2. We show that a reduction in Xirp2 expression in the heart results in pathological cardiac hypertrophy in adult, unstressed mice. Interestingly, these mice display a blunted response to angiotensin II (Ang II)–induced myocardial damage. This study demonstrates for the first time that the MEF2A target gene, Xirp2, plays an essential role in cardiomyocytes in vivo by mediating Ang II–induced pathological cardiac remodeling. Furthermore, we demonstrate that the MEF2A transcription factor acts directly downstream of the Ang II signaling pathway to regulate Xirp2 gene expression. Our findings have broad implications regarding muscle-specific, actin-binding genes that modulate cardiac muscle function in health and disease. In this article, we report that in the heart the evolutionarily conserved, actin-binding protein, Xirp2, functions downstream of Ang II signaling.
Original received September 11, 2009; revision received December 31, 2009; accepted January 7, 2010.