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The vasoactive peptide angiotensin II (AngII) 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.
The in vivo role of the MEF2A target gene Xirp2 in AngII-mediated cardiac remodeling was investigated.
Here we demonstrate that the MEF2A target gene Xirp2 (also known as cardiomyopathy associated gene 3; CMYA3) is an important effector of the AngII 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 AngII. Initially, we characterized the Xirp2 promoter and demonstrate that AngII activates Xirp2 expression by stimulating MEF2A transcriptional activity. To further characterize the role of Xirp2 downstream of AngII 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 AngII, adult Xirp2 hypomorphic mice displayed cardiac hypertrophy and increased βMHC expression. Strikingly, Xirp2 hypomorphic mice chronically infused with AngII exhibited altered pathological cardiac remodeling including an attenuated hypertrophic response, as well as diminished fibrosis and apoptosis.
These findings reveal a novel MEF2A-Xirp2 pathway that functions downstream of AngII signaling to modulate its pathological effects in the heart.
Angiotensin II (AngII) is a potent hypertensive agonist that also promotes extensive myocardial damage even in the absence of hypertension (1). The repertoire of downstream effectors in AngII-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 up-regulated in hearts of mice treated with AngII but not in salt-induced hypertensive mice (4) suggesting that CMYA3 is directly regulated by AngII signaling. This gene, since named Xirp2 (also known as mXinβ and myomaxin), is a direct target of the MEF2A transcription factor and is markedly down-regulated 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 two-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 sub-cellular 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 AngII, 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 AngII infusion, hypomorphic mice displayed diminished cardiac hypertrophy, fibrosis, and apoptosis. Furthermore, we demonstrate that regulation of Xirp2 gene expression in response to AngII signaling is mediated by MEF2A. Our results suggest that MEF2A and Xirp2 are important downstream effectors in mediating pathological cardiac remodeling in response to AngII signaling.
Details of materials and experimental procedures can be found in the expanded Methods section in the Online Data Supplement.
Xirp2 loxP-neo targeted mice were generated by inGenious Targeting Laboratory Inc. (Stony Brook, NY).
Hearts were fixed in 4% paraformaldehyde, cryoprotected in sucrose, and placed in embedding compound (OCT). Whole-heart sections were stained with hematoxylin & eosin (H&E). Masson’s trichrome staining was performed to determine the extent of cardiac fibrosis. Apoptosis was assessed by terminal dUTP nick-end labeling (TUNEL) assay using the Promega DeadEnd™ Colorimetric TUNEL System kit. For immunofluorescence, heart cryosections were blocked in BSA prior to incubation with primary and secondary antibodies.
Angiotensin II (0.9µg/hr) was administered via subcutaneous osmotic mini-pumps (Alzet model 2004) for 14 days.
Transthoracic M-mode echocardiography was performed on mice at baseline (pre-treatment) and post-2week AngII infusion. Blood pressure analysis was performed using the non-invasive tail cuff method (Model BP 2000, Visitech Systems).
cDNA was prepared from total RNA isolated from either hindlimb or ventricular tissue using Trizol reagent (Invitrogen). Primers for qRT-PCR/RT-PCR can be found in the Online Data Supplement. For qRT-PCR, individual non-pooled 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 previously (15) and hybridized to the Mouse Gene 1.0 ST Array (Affymetrix) at the Boston University Microarray Facility.
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 PVDF membrane (Biorad) and immunoblotted using primary antibodies described in the Online Data Supplement.
COS1 cells were grown in Dulbecco’s modified Eagle medium with 10% Fetal Bovine Serum, 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’s t-test or Welch’s t-test was performed for each pair-wise comparison. A p-value of <0.05 was considered to be statistically significant.
To determine whether the AngII-mediated up-regulation of Xirp2 was a direct effect of the hormone on cardiomyocytes or due to secondary effects resulting from pressure overload, primary neonatal rat ventricular myocytes (NRVMs) were isolated and treated with AngII. As shown in Figure 1A, Xirp2 transcripts were induced in NRVMs upon AngII treatment indicating that Xirp2 is directly stimulated by the hormone.
The above results prompted us to map the AngII-responsive region in the proximal 1.5kb Xirp2 promoter (5). Due to the very high basal activity of this promoter and smaller deletion constructs in NRVMs, we were unable to detect enhanced activation by AngII in this system. Subsequently, we examined the responsiveness of various Xirp2 promoter constructs (Fig. 1B) to AngII in COS cells since these cells express the type I angiotensin receptor. In transiently transfected COS cells, the −1425 Xirp2 promoter was stimulated 2.5-fold by AngII (Fig. 1C). A truncated Xirp2 promoter (−285/+60) was similarly activated by AngII (Fig. 1C), indicating that the AngII-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 AngII in vascular smooth muscle (16,17) we reasoned that AngII-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. AngII activation of the −285 mutant promoter was significantly reduced, indicating that the MEF2 site functions as an AngII-responsive element in the Xirp2 promoter (Fig. 1C).
To further investigate the role of MEF2 downstream of AngII-mediated activation of the Xirp2 gene, the −1425 Xirp2 promoter was co-transfected in COS cells along with MEF2A in the presence or absence of AngII. AngII or MEF2A alone activated the −1425 promoter by 2.5-fold and 3.4-fold, respectively (Fig. 1D). The combination of AngII and MEF2A robustly activated the −1425 Xirp2 promoter 10.6-fold (Fig. 1D). Similar results were observed with the −285 promoter construct (Fig. 1D). This cooperative effect was severely attenuated in three different mutant Xirp2 promoters in which the −75 MEF2 site was either deleted (−1425/−285) or disrupted by point mutation (−1425mut and −285mut) (Fig. 1D). However, the ability of AngII to stimulate MEF2A was not mediated by enhanced binding to the MEF2 site (Online Fig. I).
To reinforce the notion that MEF2A is an essential regulator of Xirp2 downstream of AngII signaling in vivo, we examined the expression of Xirp2 in NRVMs in which MEF2A was knocked down by adenoviruses expressing MEF2A-specific short hairpin RNAs (shRNAs) (Online Fig. IX and to be described in detail elsewhere). We failed to observe an induction of Xirp2 by AngII in cells transduced with MEF2A shRNAs but not control lacZ shRNAs (Fig. 1E, compare lanes 3 and 4 to lanes 5 and 6). These results demonstrate that Xirp2 is a novel, direct transcriptional target of AngII whose induction is mediated by MEF2A.
Having established that Xirp2 is directly regulated by AngII, we wanted to determine the in vivo requirement of this gene in AngII-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 (Fig. 2A). To generate a complete loss-of-function allele, conditional Xirp2 mice were crossed to EIIa-Cre transgenic mice which removed exons 4–6 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 4–6 was confirmed by RT-PCR analysis on cardiac muscle cDNA (Fig. 2B). Sequencing of these truncated cDNAs revealed an in-frame splice between exons 3 and 7 (Fig. 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 (Fig. 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 15–20% of wild type levels (Fig. 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 Fig. II). In addition, Xirp2 protein is largely absent from both hindlimb and cardiac muscle extracts (Fig. 2D). Unlike the up-regulation of Xirp2 in Xin knockout hearts (11), there was no compensatory increase in Xin gene expression in Xirp2 hypomorphic hearts (Fig. 2E). Given these exciting results we focused on characterizing the cardiac phenotype of 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 post-natally Xirp2 hypomorphic mice displayed a modest increase in HW:BW ratio (19%) (Fig. 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) (Fig. 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 Fig. 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 alpha myosin heavy chain (αMHC) genes (Fig. 4A). However, Xirp2 hypomorphic hearts exhibited a significant activation (3.9-fold) of the beta myosin heavy chain (βMHC) gene (Fig. 4A).
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%) (Fig. 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 up-regulated, whereas RCAN1/MCIP1 (regulator of calcineurin) was significantly down-regulated (Fig. 4C). In addition, the down-regulation of RCAN1/MCIP1 was confirmed by Western blot analysis (Online Fig. 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 processes (21) and its up-regulation 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 pro-hypertrophic 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 pathologic cardiac hypertrophy in Xirp2 hypomorphic mice.
To determine whether Xirp2 is required for stress-induced cardiac remodeling in vivo we subjected hypomorphic mice to chronic AngII infusion. The 2-week AngII infusion in wild type mice resulted in a 20% increase in HW:BW (Fig. 5A) which was confirmed by the 2.5-fold increase in ventricular myocyte CSA (Fig. 5B). In contrast, AngII treatment failed to induce a significant increase in HW:BW ratios in AngII-infused hypomorphic mice (Fig. 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) (Fig. 5B). These results indicate that the residual amount of Xirp2 in hypomorphic hearts is insufficient to fully induce the hypertrophic effects of AngII.
Since chronic AngII administration induces cardiac interstitial fibrosis, we subjected hearts from wild type and hypomorphic mice to Masson’s trichrome staining. Upon treatment with AngII, wild type mice exhibited a 2.8-fold increase in fibrosis relative to sham-operated animals (Fig. 5C). In striking contrast, chronic AngII infusion was unable to stimulate an increase in fibrosis in Xirp2 hypomorphic mice (Fig. 5C). We subsequently performed TUNEL assay to assess the extent of apoptosis in AngII-infused hearts. AngII-infused wild type mice showed a 3.3-fold increase in the amount of TUNEL-positive cells in the heart (Fig. 5D). However, AngII-infused Xirp2 hypomorphic mice showed no significant increase in myocardial apoptosis (Fig. 5D). Finally, the AngII dose used in this study induced hypertension in wild type and hypomorphic mice without any significant difference between the two groups (Online Fig. V).
Given the dampened cardiomyopathy in AngII treated hypomorphic mice we examined the expression of hypertrophic markers in hearts with long term administration of AngII. Wild type animals displayed an increase in βMHC (5.7-fold) and ANF (5.1-fold) expression (Fig. 6A). In contrast, βMHC expression was not significantly increased in Xirp2 hypomorphic hearts upon AngII-infusion, whereas ANF displayed responsiveness to AngII. Expression of αMHC and BNP was not significantly dysregulated in either wild type or hypomorphic hearts upon AngII-infusion (Fig. 6A). These results show a differential response of βMHC to AngII 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 AngII. 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 AngII-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 Fig. VI). In contrast, GSK-3β serine-9 phosphorylation was significantly reduced in AngII-infused Xirp2 hypomorphic hearts (Fig. 6B). This effect does not appear to be mediated by Akt, an upstream kinase of GSK-3β, since Western blot analysis did not detect differences in its activity in hypomorphic hearts (Online Fig. 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 AngII-treated hypomorphic mice (Fig. 6C). Thus, the reduction in GSK-3β serine-9 phosphorylation in AngII-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 AngII-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 AngII displayed attenuated cardiac hypertrophy, interstitial fibrosis and cardiomyocyte apoptosis.
It is well documented that AngII 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 AngII signaling in cardiac muscle. Further, the activation of the Xirp2 gene by AngII is controlled, in part, by the MEF2A transcription factor. The related Xin gene is also a MEF2 target (9) yet expression of this gene was not significantly induced in the heart by AngII. These observations suggest a tightly controlled regulation of the Xin gene family involving the AngII 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, since a reduction in its expression resulted in enlarged cardiomyocyte size. Cardiac hypertrophy in hypomorphic mice was accompanied by an up-regulation of the hypertrophic marker gene, βMHC, and a down-regulation of the calcineurin modulatory gene, RCAN1/MCIP1. The down-regulation 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 up-regulation of the hypertrophic marker, βMHC, but not other fetal cardiac genes in unstressed Xirp2 hypomorphic mice suggests an unconventional, but not unprecedented, mechanism of pathologic cardiac remodeling. Transgenic mice overexpressing either the beta2 adrenergic receptor (β2AR) or an inhibitor of beta adrenergic receptor kinase 1 (βARK1ct) in the heart displayed elevated levels of the βMHC but not the ANF or skeletal α-actin genes (28). While the significance of this specific pattern of hypertrophic gene dysregulation is not entirely clear these observations reveal that the coordinate up-regulation 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 AngII-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-3 and thus reduced β-catenin levels. Expression of the βMHC gene is sensitive to cardiac stress (29), and the failure to further up-regulate βMHC expression in AngII treated hypomorphic mice is likely a direct indication of the diminished hypertrophy. It is known that active GSK-3 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 un-phosphorylatable form of GSK-3β (GSK-3βS9A) in cardiomyocytes suppresses hypertrophy (30,31). Thus, reduced GSK-3βS9 phosphorylation in AngII treated hypomorphic mice may provide a mechanism for the dampened cardiac hypertrophy. Further, the concomitant reduction in β-catenin levels in AngII-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 AngII 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 pathologic cardiac remodeling in response to stress.
Surprisingly, the pre-existing cardiac hypertrophy in unstressed hypomorphic mice was not exacerbated by long-term administration of AngII. The attenuated cardiac remodeling in AngII treated hypomorphic mice may point to a unique, additional role for Xirp2 in the modulation of AngII 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 on AngII treated hypomorphic mice (Online Fig. VIII) revealed that the global profile of dysregulated genes in unstressed hypomorphic mice was largely distinct from the dysregulated gene program in AngII treated hypomorphic mice (hypo vs. AngII-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 two 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 AngII-induced cardiomyopathy.
What is known?
What new information does this article contribute?
In this manuscript we report that in the hear the evolutionarily conserved,actin-binding protein, Xirp2, functions downstream of angiotensin II (AngII) signaling
Prior to 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 pathologic cardiac hypertrophy in adult, unstressed mice. Interestingly, these mice display a blunted response to AngII-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 AngII-induced pathological cardiac remodeling. Furthermore, we demonstrate that the MEF2A transcription factor acts directly downstream of the AngII 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.
We are grateful to Jun Sadoshima (UMDNJ, Newark, NJ) for the total and phospho-S9-GSK-3β antibodies, Timothy McKinsey (Gilead, Westminster, CO) for the PKD1 antibodies, Jeffery Molkentin (HHMI, Cincinnati, OH) 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 of Ubixum Inc. (Palo Alto, CA) for assistance with Matlab image processing and Susan Kandarian (Boston University) for use of Metamorph software.
Sources of Funding
This work was supported by a grant from the NIH/NHLBI and Muscular Dystrophy Association (F.J.N.), a Clare Boothe Luce Fellowship (S.A.M)., a post-doctoral NIH Cardiovascular Training Grant (S.A.), and 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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