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
, 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 (β2
AR) 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
). 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
). It follows that expression of an un-phosphorylatable form of GSK-3β (GSK-3βS9A) in cardiomyocytes suppresses hypertrophy (30
). 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
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.
Novelty and Significance
What is known?
- The hormone angiotensin 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 angiotensin 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 angiotensin II.
- Angiotensin II regulates Xirp2 through the MEF2A transcription factor.
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.