Sox9 Inhibits Cochlear Hair Cell Fate by Upregulating Hey1 and HeyL Antagonists of Atoh1.

It is widely accepted that cell fate determination in the cochlea is tightly controlled by different transcription factors (TFs) that remain to be fully defined. Here, we show that Sox9, initially expressed in the entire sensory epithelium of the cochlea, progressively disappears from differentiating hair cells (HCs) and is finally restricted to supporting cells (SCs). By performing ex vivo electroporation of E13.5-E14.5 cochleae, we demonstrate that maintenance of Sox9 expression in the progenitors committed to HC fate blocks their differentiation, even if co-expressed with Atoh1, a transcription factor necessary and sufficient to form HC. Sox9 inhibits Atoh1 transcriptional activity by upregulating Hey1 and HeyL antagonists, and genetic ablation of these genes induces extra HCs along the cochlea. Although Sox9 suppression from sensory progenitors ex vivo leads to a modest increase in the number of HCs, it is not sufficient in vivo to induce supernumerary HC production in an inducible Sox9 knockout model. Taken together, these data show that Sox9 is downregulated from nascent HCs to allow the unfolding of their differentiation program. This may be critical for future strategies to promote fully mature HC formation in regeneration approaches.

Angiotensin II Type 1 Receptor Blocker Prevents Abdominal Aortic Aneurysm Progression in Osteoprotegerin-Deficient Mice via Upregulation of Angiotensin (1-7).

Background Angiotensin II type 1 receptor blockers (ARBs) have been shown to limit the growth of abdominal aortic aneurysm (AAA), but their efficacy is controversial. This study aimed to investigate the molecular mechanism underlying the protective effect of ARBs against AAA progression. Methods and Results Olmesartan, an ARB, was administered to wild-type and osteoprotegerin-knockout (Opg-KO) mice starting 2 weeks before direct application of CaCl2 to aortas to induce AAA. The protective effect of olmesartan against AAA in wild-type and Opg-KO mice was compared at 6 weeks after AAA induction. Olmesartan prevented AAA progression in Opg-KO mice, including excessive aortic dilatation and collapse of tunica media, but not in wild-type mice. Deficiency of the Opg gene is known to cause excessive activation of the tumor necrosis factor-related apoptosis-inducing ligand-induced c-Jun N-terminal kinase/matrix metalloproteinase 9 pathway, resulting in prolonged AAA progression. Olmesartan attenuated the upregulation of phosphorylated c-Jun N-terminal kinase and matrix metalloproteinase 9 expression in the aortic wall of Opg-KO mice. In cultured vascular smooth muscle cells, tumor necrosis factor-related apoptosis-inducing ligand-induced c-Jun N-terminal kinase phosphorylation and matrix metalloproteinase 9 expression were inhibited by angiotensin (1-7), the circulating levels of which are increased by ARBs. Furthermore, administering an angiotensin (1-7) antagonist to Opg-KO mice diminished the protective effect of olmesartan against AAA progression. Conclusions Olmesartan prevented AAA progression in Opg-KO mice by upregulating angiotensin (1-7), suggesting that angiotensin (1-7) may be a key factor that mediates the protective effect of ARBs.

Implication of satellite cell behaviors in capillary growth via VEGF expression-independent mechanism in response to mechanical loading in HeyL-null mice.

Angiogenesis and muscle satellite cell (SC)-mediated myonuclear accretion are considered essential for the robust response of contraction-induced muscle hypertrophy. Moreover, both myonucleus and SCs are physically adjacent to capillaries and are the major sites for the expression of proangiogenic factors, such as VEGF, in the skeletal muscle. Thus, events involving the addition of new myonuclei via activation of SCs may play an important role in angiogenesis during muscle hypertrophy. However, the relevance among myonuclei number, capillary supply, and angiogenesis factor is not demonstrated. The Notch effector HeyL is specifically expressed in SCs in the skeletal muscle and is crucial for SC proliferation by inhibiting MyoD in overload-induced muscle hypertrophy. Here, we tested whether the addition of new myonuclei by SC in overloaded muscle is associated with angiogenic adaptation by reanalyzing skeletal muscle from HeyL-knockout (KO) mice, which show blunted responses of SC proliferation, myonucleus addition, and overload-induced muscle hypertrophy. Reanalysis confirmed blunted SC proliferation and myonuclear accretion in the plantaris muscle of HeyL-KO mice 9 wk after synergist ablation. Interestingly, the increase in capillary-to-fiber ratio observed in wild-type (WT) mice was impaired in HeyL-KO mice. In both WT and HeyL-KO mice, the expression of VEGFA and VEGFB was similarly increased in response to overload. In addition, the expression pattern of TSP-1, a negative regulator of angiogenesis, was also not changed between WT and HeyL-KO mice. Collectively, these results suggest that SCs activation-myonuclear accretion plays a crucial role in angiogenesis during overload-induced muscle hypertrophy via independent of angiogenesis regulators.

S-1-Propenylcysteine promotes IL-10-induced M2c macrophage polarization through prolonged activation of IL-10R/STAT3 signaling.

Atherosclerosis is a chronic inflammatory disease that may lead to the development of serious cardiovascular diseases. Aged garlic extract (AGE) has been reported to ameliorate atherosclerosis, although its mode of action remains unclear. We found that AGE increased the mRNA or protein levels of arginase1 (Arg1), interleukin-10 (IL-10), CD206 and hypoxia-inducible factor 2α (HIF2α) and decreased that of CD68, HIF1α and inducible nitric oxide synthase in the aorta and spleen of apolipoprotein E knockout mice. We also found that S-1-propenylcysteine (S1PC), a characteristic sulfur compound in AGE, increased the level of IL-10-induced Arg1 mRNA and the extent of M2c-like macrophage polarization in vitro. In addition, S1PC increased the population of M2c-like macrophages, resulting in suppressed the population of M1-like macrophages and decreased lipopolysaccharide-induced production of pro-inflammatory cytokines. These effects were accompanied by prolonged phosphorylation of the IL-10 receptor α (IL-10Rα) and signal transducer and activator of transcription 3 (STAT3) that inhibited the interaction between IL-10Rα and Src homology-2-containing inositol 5'-phosphatase 1 (SHIP1). In addition, administration of S1PC elevated the M2c/M1 macrophage ratio in senescence-accelerated mice. These findings suggest that S1PC may help improve atherosclerosis due to its anti-inflammatory effect to promote IL-10-induced M2c macrophage polarization.

Smad4 regulates the nuclear translocation of Nkx2-5 in cardiac differentiation.

Bmp plays an important role in cardiomyocyte differentiation, but the function of Smad4 in Bmp signaling remains elusive. Here, we show that disruption of the Smad4 gene in cardiac progenitors expressing Sfrp5 led to embryonic lethality with hypoplastic heart formation. Although the expression of Nkx2-5 is regulated by Bmp signaling, expression of Nkx2-5 was weakly detected in the mutant heart. However, the nuclear translocation of Nkx2-5 was impaired. Expression of CK2 or PP1, which could alter the phosphorylation status of the NLS of Nkx2-5, was not affected, but Nkx2-5 was found to bind to Smad4 by co-immunoprecipitation experiments. Introduction of Smad4 into cells derived from Smad4 conditional knockout embryonic hearts restored the nuclear localization of Nkx2-5, and exogenous Nkx2-5 failed to translocate into the nucleus of Smad4-depleted fibroblasts. These results suggest that Smad4 plays an essential role in cardiomyocyte differentiation by controlling not only transcription but also the nuclear localization of Nkx2-5.

Disruption of Osteoprotegerin has complex effects on medial destruction and adventitial fibrosis during mouse abdominal aortic aneurysm formation.

Aortic aneurysm refers to dilatation of the aorta due to loss of elasticity and degenerative weakening of its wall. A preventive role for osteoprotegerin (Opg) in the development of abdominal aortic aneurysm has been reported in the CaCl2-induced aneurysm model, whereas Opg was found to promote suprarenal aortic aneurysm in the AngII-induced ApoE knockout mouse aneurysm model. To determine whether there is a common underlying mechanism to explain the impact of Opg deficiency on the vascular structure of the two aneurysm models, we analyzed suprarenal aortic tissue of 6-month-old ApoE-/-Opg-/- mice after AngII infusion for 28 days. Less aortic dissection and aortic lumen dilatation, more adventitial thickening, and higher expression of collagen I and Trail were observed in ApoE-/-Opg-/- mice relative to ApoE-/-Opg+/+ mice. An accumulation of α-smooth muscle actin and vimentin double-positive myofibroblasts was noted in the thickened adventitia of ApoE-/-Opg-/- mice. Our results suggest that fibrotic remodeling of the aorta induced by myofibroblast accumulation might be an important pathological event which tends to limit AngII-induced aortic dilatation in ApoE -/-Opg-/- mice.

Sustained expression of HeyL is critical for the proliferation of muscle stem cells in overloaded muscle.

In overloaded and regenerating muscle, the generation of new myonuclei depends on muscle satellite cells (MuSCs). Because MuSC behaviors in these two environments have not been considered separately, MuSC behaviors in overloaded muscle remain unexamined. Here, we show that most MuSCs in overloaded muscle, unlike MuSCs in regenerating muscle, proliferate in the absence of MyoD expression. Mechanistically, MuSCs in overloaded muscle sustain the expression of Heyl, a Notch effector gene, to suppress MyoD expression, which allows effective MuSC proliferation on myofibers and beneath the basal lamina. Although Heyl-knockout mice show no impairment in an injury model, in a hypertrophy model, their muscles harbor fewer new MuSC-derived myonuclei due to increased MyoD expression and diminished proliferation, which ultimately causes blunted hypertrophy. Our results show that sustained HeyL expression is critical for MuSC proliferation specifically in overloaded muscle, and thus indicate that the MuSC-proliferation mechanism differs in overloaded and regenerating muscle.

Cell-autonomous and redundant roles of Hey1 and HeyL in muscle stem cells: HeyL requires Hes1 to bind diverse DNA sites.

The undifferentiated state of muscle stem (satellite) cells (MuSCs) is maintained by the canonical Notch pathway. Although three bHLH transcriptional factors, Hey1, HeyL and Hes1, are considered to be potential effectors of the Notch pathway exerting anti-myogenic effects, neither HeyL nor Hes1 inhibits myogenic differentiation of myogenic cell lines. Furthermore, whether these factors work redundantly or cooperatively is unknown. Here, we showed cell-autonomous functions of Hey1 and HeyL in MuSCs using conditional and genetic null mice. Analysis of cultured MuSCs revealed anti-myogenic activity of both HeyL and Hes1. We found that HeyL forms heterodimeric complexes with Hes1 in living cells. Moreover, our ChIP-seq experiments demonstrated that, compared with HeyL alone, the HeyL-Hes1 heterodimer binds with high affinity to specific sites in the chromatin, including the binding sites of Hey1. Finally, analyses of myogenin promoter activity showed that HeyL and Hes1 act synergistically to suppress myogenic differentiation. Collectively, these results suggest that HeyL and Hey1 function redundantly in MuSCs, and that HeyL requires Hes1 for effective DNA binding and biological activity.

The SGLT2 Inhibitor Luseogliflozin Rapidly Normalizes Aortic mRNA Levels of Inflammation-Related but Not Lipid-Metabolism-Related Genes and Suppresses Atherosclerosis in Diabetic ApoE KO Mice.

Recent clinical studies have revealed the treatment of diabetic patients with sodium glucose co-transporter2 (SGLT2) inhibitors to reduce the incidence of cardiovascular events. Using nicotinamide and streptozotocin (NA/STZ) -treated ApoE KO mice, we investigated the effects of short-term (seven days) treatment with the SGLT2 inhibitor luseogliflozin on mRNA levels related to atherosclerosis in the aorta, as well as examining the long-term (six months) effects on atherosclerosis development. Eight-week-old ApoE KO mice were treated with NA/STZ to induce diabetes mellitus, and then divided into two groups, either untreated, or treated with luseogliflozin. Seven days after the initiation of luseogliflozin administration, atherosclerosis-related mRNA levels in the aorta were compared among four groups; i.e., wild type C57/BL6J, native ApoE KO, and NA/STZ-treated ApoE KO mice, with or without luseogliflozin. Short-term luseogliflozin treatment normalized the expression of inflammation-related genes such as F4/80, TNFα, IL-1ß, IL-6, ICAM-1, PECAM-1, MMP2 and MMP9 in the NA/STZ-treated ApoE KO mice, which showed marked elevations as compared with untreated ApoE KO mice. In contrast, lipid metabolism-related genes were generally unaffected by luseogliflozin treatment. Furthermore, after six-month treatment with luseogliflozin, in contrast to the severe and widely distributed atherosclerotic changes in the aortas of NA/STZ-treated ApoE KO mice, luseogliflozin treatment markedly attenuated the progression of atherosclerosis, without affecting serum lipid parameters such as high density lipoprotein, low density lipoprotein and triglyceride levels. Given that luseogliflozin normalized the aortic mRNA levels of inflammation-related, but not lipid-related, genes soon after the initiation of treatment, it is not unreasonable to speculate that the anti-atherosclerotic effect of this SGLT2 inhibitor emerges rapidly, possibly via the prevention of inflammation rather than of hyperlipidemia.

Sfrp5 identifies murine cardiac progenitors for all myocardial structures except for the right ventricle.

Upon acquirement of pulmonary circulation, the ancestral heart may have been remodelled coincidently with, or accompanied by, the production and rearrangement of progenitor cells. However, the progenitor populations that give rise to the left ventricle (LV) and sinus venosus (SV) are still ambiguous. Here we show that the expression of Secreted frizzled-related protein Sfrp5 in the mouse identifies common progenitors for the outflow tract (OFT), LV, atrium and SV but not the right ventricle (RV). Sfrp5 expression begins at the lateral sides of the cardiac crescent, excluding early differentiating regions, and continues in the venous pole, which gives rise to the SV. Lineage-tracing analysis revealed that descendants of Sfrp5-expressing cells at E7.5 contribute not only to the SV but also to the LV, atria and OFT and are found also in the dorsal splanchnic mesoderm accompanied by the expression of the secondary heart field marker, Islet1. These findings provide insight into the arrangement of cardiac progenitors for systemic circulation.

EPA Prevents the Development of Abdominal Aortic Aneurysms through Gpr-120/Ffar-4.

Abdominal aortic aneurysms (AAAs), which commonly occur among elderly individuals, are accompanied by a risk of rupture with a high mortality rate. Although eicosapentaenoic acid (EPA) has been reported to prevent AAA formation, the mechanism by which EPA works on vascular smooth muscle cells is unknown. This study aimed to investigate the mechanism by which orally-administered EPA prevents the formation of severe AAAs that develop in Osteoprotegerin (Opg) knockout (KO) mice. In the CaCl2-induced AAA model, EPA attenuated the enhanced progression of AAAs in Opg-KO mice, including the increase in aortic diameter with destruction of elastic fibers in the media. Immunohistochemical analyses showed that EPA reduced the phosphorylation of transforming growth factor beta-activated kinase-1/Map3k7 (Tak-1) and c-Jun NH2-terminal kinase (JNK), as well as the expression of Matrix metalloproteinase-9 (Mmp-9) in the media of the aorta. In smooth muscle cell cultures, rh-TRAIL-induced activation of the Tak-1-JNK pathway and increase in Mmp-9 expression were inhibited by EPA. Moreover, GW9508, a specific ligand for G-protein coupled receptor (Gpr)-120/Free fatty acid receptor (Ffar)-4, mimicked the effects of EPA. The effects of EPA were abrogated by knockdown of the Gpr-120/Ffar-4 receptor gene. Our data demonstrate that the Trail-Tak-1-JNK-Mmp-9 pathway is responsible for the enhancement of AAAs in Opg-KO mice, and that EPA inhibits the Tak-1-JNK pathway by activating Gpr-120/Ffar-4, which results in the attenuation of AAA development.

Transcriptomic analyses of Hand2 transgenic embryos.

In this article, we further provide the data generated for the previously published research article "Specification of jaw identity by the Hand2 transcription factor." To better understand the downstream genes of the basic helix-loop-helix transcription factor Hand2, we generated double-transgenic mice (Hand2 (NC) ) by intercrossing CAG-floxed CAT-Hand2 mice with Wnt1-Cre mice for conditional activation of Hand2 expression in the neural crest. Altered expression of Hand2 induces transformation of the upper jaw to the lower jaw in Hand2 (NC) mutant mice. This data article provides Tables detailing the differentially expressed genes between wild-type and Hand2 (NC) mutant embryos. The raw array data of our transcriptomes as generated using Affymetrix microarrays are available on the NCBI Gene Expression Omnibus (GEO) browser (Reference number GSE75805).

Specification of jaw identity by the Hand2 transcription factor.

Acquisition of the lower jaw (mandible) was evolutionarily important for jawed vertebrates. In humans, syndromic craniofacial malformations often accompany jaw anomalies. The basic helix-loop-helix transcription factor Hand2, which is conserved among jawed vertebrates, is expressed in the neural crest in the mandibular process but not in the maxillary process of the first branchial arch. Here, we provide evidence that Hand2 is sufficient for upper jaw (maxilla)-to-mandible transformation by regulating the expression of homeobox transcription factors in mice. Altered Hand2 expression in the neural crest transformed the maxillae into mandibles with duplicated Meckel's cartilage, which resulted in an absence of the secondary palate. In Hand2-overexpressing mutants, non-Hox homeobox transcription factors were dysregulated. These results suggest that Hand2 regulates mandibular development through downstream genes of Hand2 and is therefore a major determinant of jaw identity. Hand2 may have influenced the evolutionary acquisition of the mandible and secondary palate.

Dazl is a target RNA suppressed by mammalian NANOS2 in sexually differentiating male germ cells.

Evolutionally conserved Nanos RNA-binding proteins play crucial roles in germ cell development. While a mammalian Nanos family protein, NANOS2, is required for sexual differentiation of male (XY) germ cells in mice, the underlying mechanisms and the identities of its target RNAs in vivo remain elusive. Using comprehensive microarray analysis and a bacterial artificial chromosome transgenic system, here we identify Dazl, a germ cell-specific gene encoding an RNA-binding protein implicated in translation, as a crucial target of NANOS2. Importantly, removal of the Dazl 3'-untranslated region in XY germ cells stabilizes the Dazl mRNA, resulting in elevated meiotic gene expression, abnormal resumption of the cell cycle and impaired processing-body formation, reminiscent of Nanos2-knockout phenotypes. Furthermore, our data suggest that NANOS2 acts as an antagonist of the DAZL protein. We propose a dual system of NANOS2-mediated suppression of Dazl expression as a pivotal molecular mechanism promoting sexual differentiation of XY germ cells.

The Transcription Factor Hand1 Is Involved In Runx2-Ihh-Regulated Endochondral Ossification.

The developing long bone is a model of endochondral ossification that displays the morphological layers of chondrocytes toward the ossification center of the diaphysis. Indian hedgehog (Ihh), a member of the hedgehog family of secreted molecules, regulates chondrocyte proliferation and differentiation, as well as osteoblast differentiation, through the process of endochondral ossification. Here, we report that the basic helix-loop-helix transcription factor Hand1, which is expressed in the cartilage primordia, is involved in proper osteogenesis of the bone collar via its control of Ihh production. Genetic overexpression of Hand1 in the osteochondral progenitors resulted in prenatal hypoplastic or aplastic ossification in the diaphyses, mimicking an Ihh loss-of-function phenotype. Ihh expression was downregulated in femur epiphyses of Hand1-overexpressing mice. We also confirmed that Hand1 downregulated Ihh gene expression in vitro by inhibiting Runx2 transactivation of the Ihh proximal promoter. These results demonstrate that Hand1 in chondrocytes regulates endochondral ossification, at least in part through the Runx2-Ihh axis.

Osteoprotegerin Prevents Development of Abdominal Aortic Aneurysms.

Abdominal aortic aneurysms (AAAs), which commonly occur among elderly individuals, are accompanied by a risk of rupture and subsequent high mortality. Establishment of medical therapies for the prevention of AAAs requires further understanding of the molecular pathogenesis of this condition. This report details the possible involvement of Osteoprotegerin (OPG) in the prevention of AAAs through inhibition of Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In CaCl2-induced AAA models, both internal and external diameters were significantly increased with destruction of elastic fibers in the media in Opg knockout (KO) mice, as compared to wild-type mice. Moreover, up-regulation of TRAIL expression was observed in the media by immunohistochemical analyses. Using a culture system, both the TRAIL-induced expression of matrix metalloproteinase-9 in smooth muscle cells (SMCs) and the chemoattractive effect of TRAIL on SMCs were inhibited by OPG. These data suggest that Opg may play a preventive role in the development of AAA through its antagonistic effect on Trail.

Evidence of Notch-Hesr-Nrf2 Axis in Muscle Stem Cells, but Absence of Nrf2 Has No Effect on Their Quiescent and Undifferentiated State.

Nrf2 is a master regulator of oxidative stresses through the induction of anti-oxidative genes. Nrf2 plays roles in maintaining murine hematopoietic stem cells and fly intestinal stem cells. The canonical Notch signaling pathway is also crucial for maintaining several types of adult stem cells including muscle stem cells (satellite cells). Here, we show that Dll1 induced Nrf2 expression in myogenic cells. In addition, primary targets of Notch signaling, Hesr1 and Hesr3, were involved in the up-regulation of Nrf2 mRNA and expression of its target genes. In vitro, Nrf2 had anti-myogenic and anti-proliferative effects on primary myoblasts. In vivo, although Nrf2-knockout mice showed decreased expression of its target genes in muscle stem cells, adult muscle stem cells of Nrf2-knockout mice did not exhibit the phenotype. Taken together, in muscle stem cells, the Notch-Hesr-Nrf2 axis is a pathway potentially inducing anti-oxidative genes, but muscle stem cells either do not require Nrf2-mediated anti-oxidative gene expression or they have a complementary system compensating for the loss of Nrf2.

Enhanced prepulse inhibition and low sensitivity to a dopamine agonist in HESR1 knockout mice.

Transcription factor Hesr family genes are important in neuronal development. We demonstrated previously that HESR1 and HESR2 modified expression of the dopamine transporter (DAT) reporter gene. HESR-family genes have been investigated in development, but their functions, especially in relation to behaviors regulated by dopamine, in adult animals remain unclear. In the present study, we investigated the effects of Hesr1 and Hesr2 on behavior. A behavioral test battery to examine spontaneous activity, anxiety-like behavior, aggressive behavior, pain sensitivity, and sensorimotor gating was conducted in Hesr1 and Hesr2 knockout (KO) mice. Enhanced prepulse inhibition (PPI), which is a form of sensorimotor gating, was observed in only Hesr1 KO mice; other behavioral traits were mostly comparable to wild-type animals in both the Hesr1 and the Hesr2 KO lines. Next, we used a dopamine agonist, apomorphine, to confirm the involvement of the dopaminergic system. Injection of apomorphine reduced the enhanced PPI in Hesr1 KO mice. Additionally, dose-dependent sensitivity to the agonist was lower in the Hesr1 KO mice than in wild-type mice, suggesting that the enhanced PPI resulted from this alteration in dopamine sensitivity. Furthermore, DAT mRNA was downregulated in Hesr1 KO mice, whereas the dopamine D1 and D2 receptors were comparable. These findings suggest Hesr1 to be a novel factor that affects dopamine sensitivity and the sensorimotor gating system.

Induction of Timp1 in smooth muscle cells during development of abdominal aortic aneurysms.

Abdominal aortic aneurysm (AAA) is known to develop mainly by the increased diameter of aorta through metalloproteinases (MMPs). Although activities of MMPs are tightly regulated by the presence of tissue inhibitor of MMPs (TIMPs) and imbalances between MMPs and TIMPs may serve to fragility of arterial wall, little is known about TIMPs behavior in aneurysmal formation. Here, we utilized a murine experimental AAA model, and found that by immunohistochemical analysis, Timp1 as and Timp1 mRNA levels was also revealed in aortic tissue in AAA by RT-PCR. In cultured vascular smooth muscle cells (SMCs), Tumor Necrosis Factor (TNF)-alpha significantly activated both Mmp9 and Timp1 expression, and they were blocked by Jun kinase inhibitor (SP600125) in a dose-dependent manner. Interestingly, a proteasome inhibitor (MG132), which is known as an agent for inhibition of the nuclear factor-kappa B (NF-kappaB), significantly inhibited the TNF-alpha-induced expression of Timp1, whereas MG132, which also works as an activator of c-Jun/AP-1 pathway, strongly increased Mmp9. Taken together, inflammatory cytokines, including TNF-alpha, may simultaneously induce MMPs and TIMPs for the remodeling of the medial layer, leading to the increased diameter of the aorta, the aneurysm.

Hesr2 knockout mice develop aortic valve disease with advancing age.

OBJECTIVE: Acquired heart diseases, such as valve disease, are major causes of human morbidity and mortality. However, the pathological mechanisms underlying these diseases are largely unknown. Our aim is to identify the role of the hairy and enhancer of split-related (Hesr)-2 gene in the adult heart. METHODS AND RESULTS: Echocardiography detected heart dysfunctions indicative of aortic valve anomalies, stenosis, and regurgitation, in ≈59% of >12-month-old Hesr2 knockout survivor mice. Morphological and histological analyses revealed thickened semilunar valves with increased fibrotic areas, indicating that sclerotic degeneration of valves is the main cause of aortic valve disease. The expression of osteogenic genes, such as osteopontin and sclerostin, were upregulated in the mutants, and the overexpression of sclerostin in endothelial cells resulted in thickened semilunar valves with increased fibrotic areas, similar to that seen in the Hesr2 knockout mice, suggesting that Hesr2 can regulate osteogenic gene expression in valves. Reduced left ventricular function, which may be caused by increased ventricular interstitial fibrosis, and enlarged myocardial cell size without ventricular wall thickening were found in both aortic valve stenosis/regurgitation-positive (33%) and aortic valve stenosis/regurgitation-negative (38%) subpopulations in 12-month-old survivor mice. Dilated left ventricular internal dimensions were specifically detected in the aortic valve stenosis/regurgitation-positive subpopulation, thus suggesting that the degeneration of cardiomyocytes is influenced by irregular hemodynamics. CONCLUSIONS: These data revealed that survivor mice lacking the Hesr2 gene exhibit fibrosis in the aortic valve and ventricle in adulthood, thus suggesting that Hesr2 plays an important role in maintaining the homeostasis of the aortic valve and ventricle.