Ang II-induced Cardiac Hypertrophy Research Articles

Testosterone plays a permissive role in angiotensin II-induced hypertension and cardiac hypertrophy in male rats.

Sex hormones contribute to sex differences in blood pressure. Inappropriate activation of the renin-angiotensin system is involved in vascular dysfunction and hypertension. This study evaluated the role of androgens (testosterone) in angiotensin II (Ang II)-induced increase in blood pressure, vascular reactivity, and cardiac hypertrophy. Eight-week-old male Wistar rats underwent sham operation, castration, or castration with testosterone replacement. After 12 weeks of chronic changes in androgen status, Ang II (120 ng/kg per minute) or saline was infused for 28 days via subcutaneous miniosmotic pump, and changes in blood pressure was measured. Vascular reactivity and Ang II receptor levels were examined in mesenteric arteries. Heart weight, cardiac ANP mRNA levels, and fibrosis were also assessed. Ang II infusion increased arterial pressure in intact males. The Ang II-induced increase in hypertensive response was prevented in castrated males. Testosterone replacement in castrated males restored Ang II-induced hypertensive responses. Castration reduced vascular AT1R/AT2R ratio, an effect that was reversed by testosterone replacement. Ang II-induced hypertension was associated with increased contractile response of mesenteric arteries to Ang II and phenylephrine in intact and testosterone-replaced castrated males; these increases were prevented in castrated males. Ang II infusion induced increased left ventricle-to-body weight ratio and ANP mRNA expression, indicators of left ventricular hypertrophy, and fibrosis in intact and testosterone-replaced castrated males, and castration prevented the increase in these parameters caused by Ang II. This study demonstrates that testosterone plays a permissive role in development and maintenance of Ang II-induced vascular dysfunction, hypertension, and cardiac hypertrophy.

P2826Teneligliptin attenuated AngII-induced cardiac hypertrophy by inhibiting Nox4-HDAC4 axis

P2826Teneligliptin attenuated AngII-induced cardiac hypertrophy by inhibiting Nox4-HDAC4 axis

An essential role for Wnt/\u03b2-catenin signaling in mediating hypertensive heart disease

Activation of the renin-angiotensin system (RAS) is associated with hypertension and heart disease. However, how RAS activation causes cardiac lesions remains elusive. Here we report the involvement of Wnt/β-catenin signaling in this process. In rats with chronic infusion of angiotensin II (Ang II), eight Wnt ligands were induced and β-catenin activated in both cardiomyocytes and cardiac fibroblasts. Blockade of Wnt/β-catenin signaling by small molecule inhibitor ICG-001 restrained Ang II-induced cardiac hypertrophy by normalizing heart size and inhibiting hypertrophic marker genes. ICG-001 also attenuated myocardial fibrosis and inhibited α-smooth muscle actin, fibronectin and collagen I expression. These changes were accompanied by a reduced expression of atrial natriuretic peptide and B-type natriuretic peptide. Interestingly, ICG-001 also lowered blood pressure induced by Ang II. In vitro, Ang II induced multiple Wnt ligands and activated β-catenin in rat primary cardiomyocytes and fibroblasts. ICG-001 inhibited myocyte hypertrophy and Snail1, c-Myc and atrial natriuretic peptide expression, and abolished the fibrogenic effect of Ang II in cardiac fibroblasts. Finally, recombinant Wnt3a was sufficient to induce cardiomyocyte injury and fibroblast activation in vitro. Taken together, these results illustrate an essential role for Wnt/β-catenin in mediating hypertension, cardiac hypertrophy and myocardial fibrosis. Therefore, blockade of this pathway may be a novel strategy for ameliorating hypertensive heart disease.

C1QTNF1 attenuates angiotensin II-induced cardiac hypertrophy via activation of the AMPKa pathway.

Complement C1q tumor necrosis factor related proteins (C1QTNFs) have been reported to have diverse biological influence on the cardiovascular system. C1QTNF1 is a member of the CTRP superfamily. C1QTNF1 is expressed in the myocardium; however, its function in myocytes has not yet been investigated. To systematically investigate the roles of C1QTNF1 in angiotensin II (Ang II)-induced cardiac hypertrophy. C1QTNF1 knock-out mice were used with the aim of determining the role of C1QTNF1 in cardiac hypertrophy in the adult heart. Data from experiments showed that C1QTNF1 was up-regulated during cardiac hypertrophic processes, which were triggered by increased reactive oxygen species. C1QTNF1 deficiency accelerated cardiac hypertrophy, fibrosis, inflammation responses, and oxidative stress with deteriorating cardiac dysfunction in the Ang II-induced cardiac hypertrophy mouse model. We identified C1QTNF1 as a negative regulator of cardiomyocyte hypertrophy in Ang II-stimulated neonatal rat cardiomyocytes using the recombinant human globular domain of C1QTNF1 and C1QTNF1 siRNA. Injection of the recombinant human globular domain of C1QTNF1 also suppressed the Ang II-induced cardiac hypertrophic response in vivo. The anti-hypertrophic effects of C1QTNF1 rely on AMPKa activation, which inhibits mTOR P70S6K phosphorylation. An AMPKa inhibitor abrogated the anti-hypertrophic effects of the recombinant human globular domain of C1QTNF1 both in vivo and vitro. Moreover, C1QTNF1-mediated AMPKa activation was triggered by the inhibition of PDE1-4, which subsequently activated the cAMP/PKA/LKB1 pathway. Our results demonstrated that C1QTNF1 improves cardiac function and inhibits cardiac hypertrophy and fibrosis by increasing and activating AMPKa, suggesting that C1QTNF1 could be a therapeutic target for cardiac hypertrophy and heart failure.

Abstract 287: Transgenic Overexpression of Dimethylarginine Dimethylaminohydrolase 1 Protects From Angiotensin II - Induced Cardiac Hypertrophy and Vascular Remodeling

Background: Dimethylarginine dimethylaminohydrolase 1 (DDAH1) hydrolyzes the endogenous inhibitor of nitric oxide synthases asymmetric dimethylarginine (ADMA). DDAH1 is also suggested to have ADMA-independent effects. DDAH1 overexpression lowers ADMA levels and protects from renal interstitial fibrosis and vascular oxidative stress in angiotensin-II-induced hypertension. The current study was designed to test the hypothesis that DDAH1 overexpression protects from angiotensin II-induced cardiac hypertrophy and vascular remodeling. Methods: Angiotensin II (AngII) was infused in the doses of 0.75 and 1.5 mg/kg/day, respectively, in DDAH1 transgenic mice (TG) and wild type (WT) littermates via osmotic minipumps. Echocardiography was performed in the first and fourth week after start of the infusion. Systolic blood pressure was measured by the tail-cuff method. Cardiac hypertrophy and vascular remodeling was assessed by histology after 4 weeks of AngII infusion. Results: TG mice had decreased plasma and tissue ADMA. Infusion of Ang II resulted in an increase in systolic blood pressure, which was similar between TG and WT mice at week 1, however, TG mice were protected from a further increase in blood pressure. After 4-weeks infusion of AngII TG mice had significantly higher left ventricular lumen to wall ratio, smaller size of cardiomyocytes and reduced myocardial collagen expression compared to WT littermates. TG mice had lower left ventricular posterior wall thickness in systole and diastole as compared to WT controls. The vasomotor function of aortic rings in response to acetylcholine was improved in the TG mice as compared to the WT mice. TG mice had less aortic hypertrophy and fibrosis and more elastin in aorta as compared to WT mice. Aortic infiltration of CD45 + , CD3 + , CD8 + and CD4 + T-cells was significantly lower in TG than in WT mice. Conclusion: This study shows that upregulation of DDAH1 protects from AngII-induced cardiac hypertrophy and vascular remodeling. Upregulation of DDAH1 might be a potential therapeutic approach for protection from AngII – induced end organ damage. We are currently investigating, whether protective effects of DDAH1 are ADMA-dependent or ADMA-independent.

FKBP12.6 protects heart from AngII-induced hypertrophy through inhibiting Ca2+ /calmodulin-mediated signalling pathways invivo and invitro.

We previously observed that disruption of FK506‐binding protein 12.6 (FKBP12.6) gene resulted in cardiac hypertrophy in male mice. Studies showed that overexpression of FKBP12.6 attenuated thoracic aortic constriction (TAC)‐induced cardiac hypertrophy in mice, whereas the adenovirus‐mediated overexpression of FKBP12.6 induced hypertrophy and apoptosis in cultured neonatal cardiomyocytes, indicating that the role of FKBP12.6 in cardiac hypertrophy is still controversial. In this study, we aimed to investigate the roles and mechanisms of FKBP12.6 in angiotensin II (AngII)‐induced cardiac hypertrophy using various transgenic mouse models in vivo and in vitro. FKBP12.6 knockout (FKBP12.6−/−) mice and cardiac‐specific FKBP12.6 overexpressing (FKBP12.6 TG) mice were infused with AngII (1500 ng/kg/min) for 14 days subcutaneously by implantation of an osmotic mini‐pump. The results showed that FKBP12.6 deficiency aggravated AngII‐induced cardiac hypertrophy, while cardiac‐specific overexpression of FKBP12.6 prevented hearts from the hypertrophic response to AngII stimulation in mice. Consistent with the results in vivo, overexpression of FKBP12.6 in H9c2 cells significantly repressed the AngII‐induced cardiomyocyte hypertrophy, seen as reductions in the cell sizes and the expressions of hypertrophic genes. Furthermore, we demonstrated that the protection of FKBP12.6 on AngII‐induced cardiac hypertrophy was involved in reducing the concentration of intracellular Ca2+ ([Ca2+]i), in which the protein significantly inhibited the key Ca2+/calmodulin‐dependent signalling pathways such as calcineurin/cardiac form of nuclear factor of activated T cells 4 (NFATc4), calmodulin kinaseII (CaMKII)/MEF‐2, AKT/Glycogen synthase kinase 3β (GSK3β)/NFATc4 and AKT/mTOR signalling pathways. Our study demonstrated that FKBP12.6 protects heart from AngII‐induced cardiac hypertrophy through inhibiting Ca2+/calmodulin‐mediated signalling pathways.

Role of PI3‐kinase in Angiotensin II‐induced Cardiac Hypertrophy: Shift from Class III to Class I

Cardiac hypertrophy is initially an adaptive response to increased hemodynamic workload or defective contractile machinery; however, in the long‐term, it contributes to heart failure and sudden cardiac death. The extracellular regulatory factors and intracellular signaling pathways involved are not fully clear. Here, we studied the chronic effect of Ang II infusion on cardiac hypertrophy and fibrosis, and evaluated the role of class I PI3‐kinase in these actions of Ang II using lentiviral vector‐mediated expression of a dominant negative form of Class I PI3‐kinase subunit p85α in the heart of rats. Subcutaneous 4‐week infusion of Ang II induced severe cardiac hypertrophy and perivascular fibrosis in the heart. Cardiac transduction of Lv‐DNp85 abolished Ang II‐induced phosphorylation of Akt in the heart, as well as Ang II‐induced cardiac hypertrophy and perivascular fibrosis. Next, we examined intracellular mechanisms underlying Ang II‐induced cardiac hypertrophy in cultured cardiomyocytes. It is well known that mitochondrial oxidative stress mediates Ang II‐induced cardiac hypertrophy and the major source of reactive oxygen species (ROS), damaged mitochondria, is cleared by autophagy (also called mitophagy) in cardiac myocytes. Treatment of cardiomyocytes with Ang II (10−9 to 10−5 M) significantly increased autophagy with two phases (increases at low doses, decreases at high doses, peaked at 10−7 M). Ang II (10−6 M)‐induced increases in autophagy were significantly potentiated by PI3‐kinase Class I inhibitor, LY294002 (1 μM) and dramatically attenuated by 3‐Methyladenine (3‐MA, 1 μM), a PI3‐kinase Class III inhibitor. Ang II‐induced mitochondrial ROS production, measured using MitoSOX, was attenuated by LY294002 and potentiated by 3‐MA significantly. PI3‐kinases activity were measured using ELISA in cardiomyocytes, demonstrating that Ang II (10−6 M, i.e. high dose) significantly increased the activity of both Class I and Class III PI3‐kinases; however, Ang II (10−9 M, i.e. low dose) selectively elevated PI3‐kinase Class III activity. Together these data suggest that Ang II could have dual effects in the heart: 1) a protective arm to prevent ROS over‐generation in physiological cardiac hypertrophy, which is mediated by a Class III PI3‐kinase/autophagy‐dependent pathway; and 2) a deleterious arm due to increased mitochondrial ROS production through inhibition of autophagy by a Class I PI3‐kinase pathway in cardiomyocytes.Support or Funding InformationSupported by NIH NS55008.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

H‐ ras deletion protects against angiotensin II–induced arterial hypertension and cardiac remodeling through protein kinase G‐Iβ pathway activation

Ras proteins regulate cell survival, growth, differentiation, blood pressure, and fibrosis in some organs. We have demonstrated that H- ras gene deletion produces mice hypotension via a soluble guanylate cyclase-protein kinase G (PKG)-dependent mechanism. In this study, we analyzed the consequences of H- ras deletion on cardiac remodeling induced by continuous angiotensin II (AngII) infusion and the molecular mechanisms implied. Left ventricular posterior wall thickness and mass and cardiomyocyte cross-sectional area were similar between AngII-treated H-Ras knockout (H -ras-/-) and control wild-type (H -ras+/+) mice, as were extracellular matrix protein expression. Increased cardiac PKG-Iβ protein expression in H -ras-/- mice suggests the involvement of this protein in heart protection. Ex vivo experiments on cardiac explants could support this mechanism, as PKG blockade blunted protection against AngII-induced cardiac hypertrophy and fibrosis markers in H -ras-/- mice. Genetic modulation studies in cardiomyocytes and cardiac and embryonic fibroblasts revealed that the lack of H-Ras down-regulates the B-RAF/MEK/ERK pathway, which induces the glycogen synthase kinase-3β-dependent activation of the transcription factor, cAMP response element-binding protein, which is responsible for PKG-Iβ overexpression in H -ras-/- mouse embryonic fibroblasts. This study demonstrates that H- ras deletion protects against AngII-induced cardiac remodeling, possibly via a mechanism in which PKG-Iβ overexpression could play a partial role, and points to H-Ras and/or downstream proteins as potential therapeutic targets in cardiovascular disease.-Martín-Sánchez, P., Luengo, A., Griera, M., Orea, M. J., López-Olañeta, M., Chiloeches, A., Lara-Pezzi, E., de Frutos, S., Rodríguez-Puyol, M., Calleros, L., Rodríguez-Puyol, D. H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Iβ pathway activation.

DKK3 overexpression attenuates cardiac hypertrophy and fibrosis in an angiotensin-perfused animal model by regulating the ADAM17/ACE2 and GSK-3β/β-catenin pathways

AimsCardiac pressure and humoral factors induce cardiac hypertrophy and fibrosis, which are characterized by increased stiffness, reduced contractility and altered perfusion. Angiotensin II (AngII) is well known to promote this pathology. Angiotensin-converting enzyme (ACE) 2, which cleaves AngII and forms Ang-(1–7), exerts protective anti-hypertrophy and anti-fibrosis effects. A disintegrin and metalloproteinase 17 (ADAM17), a membrane-bound enzyme reported to cleave ACE2, may participate in the pathological process of AngII perfusion-induced heart damage. However, researchers have not clearly determined whether dickkopf-3 (DKK3) regulates the ADAM17/ACE2 pathway and, if so, whether DKK3-mediated regulation is related to the glycogen synthase kinase-3β (GSK-3β)/β-catenin pathway. In this study, we explored whether DKK3 overexpression ameliorates the development of AngII-induced cardiac fibrosis and hypertrophy through the ADAM17/ACE2 and GSK-3β/β-catenin pathways. MethodsMice were injected with a DKK3-overexpressing adenovirus or vehicle and then infused with AngII or saline using subcutaneously implanted mini-pumps for four weeks. Hearts were stained with hematoxylin-eosin, Masson's trichrome and immunohistochemical markers for histology. Primary fibroblasts were treated with the adenovirus and AngII and then examined using western blotting, EdU (5-ethynyl-2′-deoxyuridine) assays and immunofluorescence. Additionally, siRNA silencing was performed to study the role of DKK3 and the involved pathways. ResultsAngII-induced cardiac hypertrophy and interstitial and perivascular fibrosis were less severe in DKK3-overexpressing mice than in control mice. Moreover, the expression levels of fibrotic genes, such as collagen I and III, and the hypertrophic genes atrial natriuretic peptide (ANP) and beta-myosin heavy chain (β-MHC) were decreased. DKK3 overexpression also exerted a protective effect by inhibiting ADAM17 phosphorylation, thus increasing ACE2 expression and subsequently promoting AngII degradation. Furthermore, this process was mediated by the inhibition of GSK-3β and β-catenin and decreased translocation of β-catenin to the nucleus. On the other hand, the DKK3 knockdown by siRNA achieved opposite results. ConclusionDKK3 overexpression substantially alleviated AngII infusion-induced cardiac hypertrophy and fibrosis by regulating ADAM17/ACE2 pathway activity and inhibiting the GSK-3β/β-catenin pathway.

Short-term hypoxia upregulated Mas receptor expression to repress the AT1 R signaling pathway and attenuate Ang II-induced cardiomyocyte apoptosis.

Hypertension-stimulated cardiac hypertrophy and apoptosis play critical roles in the progression of heart failure. Our previous study suggested that hypertensive angiotensin II (Ang II) enhanced insulin-like growth factor receptor II (IGF-IIR) expression and cardiomyocyte apoptosis, which are involved JNK activation, sirtuin1 (SIRT1) degradation, and heat-shock transcription factor 1 (HSF1) acetylation. Moreover, previous studies have implied that short-term hypoxia (STH) might exert cardioprotective effects. However, the effects of STH on Ang II-induced cardiomyocyte apoptosis remain unknown. In this study, we found that STH reduced myocardial apoptosis caused by Ang II via upregulation of the Mas receptor (MasR) to inhibit the AT1 R signaling pathway. STH activates MasR to counteract the Ang II pro-apoptotic signaling cascade by inhibiting IGF-IIR expression via downregulation of JNK activation and reduction of SIRT1 degradation. Hence, HSF could remain deacetylated, and repress IGF-IIR expression. These effects decrease the activation of downstream pro-apoptotic and hypertrophic cascades and protect cardiomyocytes from Ang II-induced injury. In addition, we also found that silencing MasR expression enhanced Ang II-induced cardiac hypertrophy and the apoptosis signaling pathway. These findings suggest a critical role for MasR in cardiomyocyte survival. Altogether, our findings indicate that STH protects cardiomyocytes from Ang II-stimulated apoptosis. The protective effects of STH are associated with the upregulation of MasR to inhibit AT1 R signaling. STH could be a potential therapeutic strategy for cardiac diseases in hypertensive patients.

RETRACTED: LncRNA myocardial infarction-associated transcript (MIAT) contributed to cardiac hypertrophy by regulating TLR4 via miR-93

It has been reported that lncRNA myocardial infarction-associated transcript (MIAT) facilitated the pathological development in angiotensin II (AngII)-induced cardiac hypertrophy. Nevertheless, the underlying mechanism of MIAT involved in cardiac hypertrophy is largely unknown. In this study, AngII-treated cardiomyocytes were applied as a cardiac hypertrophy model in vitro. The expressions of MIAT and miR-93 were detected by qRT-PCR. The protein levels of toll-like receptor 4 (TLR4), atrial natriuretic factor (ANF), beta-myosin heavy chain (β-MHC), phosphoinositide-3 kinase (PI3K), protein kinase B (Akt), phosphorylated Akt (p-Akt), mammalian target of rapamycin (mTOR), and phosphorylated mTOR (p-mTOR) were determined by western blot. Luciferase reporter assay and RNA immunoprecipitation (RIP) were performed to explore the relationship between MIAT, TLR4 and miR-93. Hypertrophic response was assessed by measuring cell surface area and quantifying the expressions of ANF and β-MHC. The results demonstrated that MIAT was upregulated and miR-93 was downregulated in AngII-treated cardiomyocytes. MIAT functioned as a molecular sponge of miR-93 in cardiomyocytes. Additionally, TLR4 was identified as a target of miR-93 and MIAT promoted TLR4 expression by sponging miR-93. MIAT knockdown decreased cell surface area and the expression levels of ANF and β-MHC in AngII-treated cardiomyocytes by modulating miR-93. Moreover, enforced expression of TLR4 partially reversed the protective effect of miR-93 overexpression on AngII-induced cardiac hypertrophy. Furthermore, MIAT knockdown or miR-93 overexpression inactivated the PI3K/Akt/mTOR pathway via TLR4 in AngII-induced cardiac hypertrophy. Taken together, these data suggested that MIAT knockdown inhibited AngII-induced cardiac hypertrophy by regulating miR-93/TLR4 axis, highlighting a promising therapy target for cardiac hypertrophy.

SIRT2 Acts as a Cardioprotective Deacetylase in Pathological Cardiac Hypertrophy.

Pathological cardiac hypertrophy induced by stresses such as aging and neurohumoral activation is an independent risk factor for heart failure and is considered a target for the treatment of heart failure. However, the mechanisms underlying pathological cardiac hypertrophy remain largely unknown. We aimed to investigate the roles of SIRT2 in aging-related and angiotensin II (Ang II)-induced pathological cardiac hypertrophy. Male C57BL/6J wild-type and Sirt2 knockout mice were subjected to the investigation of aging-related cardiac hypertrophy. Cardiac hypertrophy was also induced by Ang II (1.3 mg/kg/d for 4 weeks) in male C57BL/6J Sirt2 knockout mice, cardiac-specific SIRT2 transgenic (SIRT2-Tg) mice, and their respective littermates (8 to ≈12 weeks old). Metformin (200 mg/kg/d) was used to treat wild-type and Sirt2 knockout mice infused with Ang II. Cardiac hypertrophy, fibrosis, and cardiac function were examined in these mice. SIRT2 protein expression levels were downregulated in hypertrophic hearts from mice. Sirt2 knockout markedly exaggerated cardiac hypertrophy and fibrosis and decreased cardiac ejection fraction and fractional shortening in aged (24-month-old) mice and Ang II-infused mice. Conversely, cardiac-specific SIRT2 overexpression protected the hearts against Ang II-induced cardiac hypertrophy and fibrosis and rescued cardiac function. Mechanistically, SIRT2 maintained the activity of AMP-activated protein kinase (AMPK) in aged and Ang II-induced hypertrophic hearts in vivo as well as in cardiomyocytes in vitro. We identified the liver kinase B1 (LKB1), the major upstream kinase of AMPK, as the direct target of SIRT2. SIRT2 bound to LKB1 and deacetylated it at lysine 48, which promoted the phosphorylation of LKB1 and the subsequent activation of LKB1-AMPK signaling. Remarkably, the loss of SIRT2 blunted the response of AMPK to metformin treatment in mice infused with Ang II and repressed the metformin-mediated reduction of cardiac hypertrophy and protection of cardiac function. SIRT2 promotes AMPK activation by deacetylating the kinase LKB1. Loss of SIRT2 reduces AMPK activation, promotes aging-related and Ang II-induced cardiac hypertrophy, and blunts metformin-mediated cardioprotective effects. These findings indicate that SIRT2 will be a potential target for therapeutic interventions in aging- and stress-induced cardiac hypertrophy.

O45-6 - Teneligliptin Attenuated AngII-induced Cardiac Hypertrophy by Inhibiting Nox4-HDAC4 Axis

Background: NADPH oxidase 4 (Nox4) mediates nuclear export of histone deacetylase 4 (HDAC4), thereby inducing cardiac hypertrophy in response to hypertrophic stimuli including phenylephrine and pressure overload. Recent studies suggested that teneligliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, inhibited Nox4 expression in aorta and adipocytes. The purpose of this study was to determine whether teneligliptin ameliorates angiotensin II (AngII)-induced cardiac hypertrophy by suppression of Nox4-HDAC4 axis. Methods and Results: AngII (1.44 mg/kg/day) or saline was continuously infused into C57BL/6J mice with or without teneligliptin (30 mg/kg/day) by oral gavage for 1 week. AngII significantly increased blood pressure compared with control mice (125 ± 4 vs. 102 ± 2 mmHg; P < .01). AngII increased heart weight/body weight (LV/BW) and left ventricular wall thickness (LVWT), which were significantly attenuated by teneligliptin (LV/BW 3.9 ± 0.1 vs. 3.6 ± 0.1 mg/g; P < .05, LVWT 0.93 ± 0.04 vs. 0.78 ± 0.05 mm; P < .01) with no significant changes in aortic blood pressure and blood glucose. Histological analysis demonstrated that teneligliptin suppressed AngII-induced increase in cardiomyocyte cross-sectional-area. It also inhibited AngII-induced increases in expression levels of Nox4 mRNA by 22% (P < .05) and phosphorylated HDAC4, a cytosolic localized form of HDAC4, by 40% (P < .05). In cultured neonatal rat ventricular myocytes, teneligliptin or GKT137831, a specific inhibitor of Nox4, directly inhibited AngII-induced phosphorylation of HDAC4 by 39% and 60% (P < .01), respectively. Conclusions: Teneligliptin attenuated AngII-induced cardiac hypertrophy, at least in part, by inhibiting Nox4-HDAC4 axis.

Abstract 030: Angiotensin II-induced Hypertension and Cardiac Hypertrophy are Mediated Differentially by TLR3- and TLR4-dependent Pathways

The innate immune system plays a key role in onset and maintenance of hypertension. We showed that the toll-like receptor (TLR) adaptor protein TRIF (toll interleukin receptor domain-containing adaptor-inducing interferon-b), but not MyD88 (myeloid differentiation 88), is required for angiotensin II (Ang II) induced hypertension in mice. TRIF transduces signals from TLR3 and TLR4, to induce inflammatory gene expression. In this study, we sought to determine which TLR is responsible for TRIF-mediated hypertensive effects of Ang II. We used a TLR4 deficient mice (TLR4-del) that have TLR4 gene deleted and the wild type (WT) control strain (C57BL/10). We also used a TLR3-knockout mice (TLR3ko) that have the exon 1 of the Tlr3 gene deleted. Saline or Ang II (1000 ng/kg/min) was infused subcutaneously for 3 weeks using mini-osmotic pumps and systolic blood pressure (SBP) was measured by tail cuff. Ang II increased peak SBP in WT (from 108 ± 1.3 mmHg to 136.0 ± 12.7 mmHg, n= 3) and TLR4-del (from 115.6 ± 2.7 mmHg to 154.4 ± 3.3 mmHg, n=3) mice. In contrast, peak SBP was not increased significantly with Ang II infusion in TLR3ko mice (from 111.3 ± 4.5 to 122.2 ± 18.6 mmHg, n=6) nor was it increased in WT mice after selective inhibition of TLR3 signaling with CU CPT 4a. Thus, the TLR3/TRIF, but not TLR4, pathway is essential for Ang II hypertension. In parallel with the pressure responses to Ang II, renal expression of Nox4 was significantly decreased in TLR3ko mice but was unchanged in TLR4-del and WT mice. In contrast to Ang II-induced hypertension, AngII-induced cardiac hypertrophy, measured as heart weight to body weight ratio, after 3 weeks of Ang II infusion was reduced in both TLR4-del (5.38 ± 0.19 mg/g) and TLR3ko (4.99 ± 0.15 mg/g) compared to WT (7.14 ± 0.49 mg/g). Thus, cardiac hypertrophy is abrogated in TLR4-del despite a robust pressor response to Ang II. The cardiac pro-inflammatory gene expression of tumor necrosis factor alpha ( Tnfa ), NADPH oxidase 4 ( Nox4 ), and matrix metalloproteinase 9 ( Mmp9 ) was increased in WT heart but attenuated in both TLR4-del and TLR3ko hearts. The results indicate a selective dependence of Ang II hypertension on TLR3/TRIF pathway whereas Ang II-induced cardiac hypertrophy depends on both TLR3 and TLR4, likely through the common TRIF adaptor protein.

Inhibition of Mid-chain HETEs Protects Against Angiotensin II-induced Cardiac Hypertrophy.

Recent data demonstrated the role of CYP1B1 in cardiovascular disease. It was, therefore, necessary to examine whether the inhibition of CYP1B1 and hence inhibiting the formation of its metabolites, using 2,4,3',5'-tetramethoxystilbene (TMS), would have a cardioprotective effect against angiotensin II (Ang II)-induced cardiac hypertrophy. For this purpose, male Sprague Dawley rats were treated with Ang II with or without TMS (300 μg/kg every third day i.p.). Thereafter, cardiac hypertrophy and the formation of mid-chain HETEs and arachidonic acid were assessed. In vitro, RL-14 cells were treated with Ang II (10 μM) in the presence and absence of TMS (0.5 μM). Then, reactive oxygen species, mitogen-activated protein kinase phosphorylation levels, and nuclear factor-kappa B-binding activity were determined. Our results demonstrated that TMS protects against Ang II-induced cardiac hypertrophy as indicated by the improvement in cardiac functions shown by the echocardiography as well as by reversing the increase in heart weight to tibial length ratio caused by Ang II. In addition, the cardioprotective effect of TMS was associated with a significant decrease in cardiac mid-chain HETEs levels. Mechanistically, TMS inhibited reactive oxygen species formation, the phosphorylation of ERK1/2, p38 mitogen-activated protein kinase, and the binding of p65 NF-κB.

HSF1 phosphorylation by ERK/GSK3 suppresses RNF126 to sustain IGF-IIR expression for hypertension-induced cardiomyocyte hypertrophy.

Hypertension-induced cardiac hypertrophy and apoptosis are major characteristics of early-stage heart failure (HF). Inhibition of extracellular signal-regulated kinases (ERK) efficaciously suppressed angiotensin II (ANG II)-induced cardiomyocyte hypertrophy and apoptosis by blocking insulin-like growth factor II receptor (IGF-IIR) signaling. However, the detailed mechanism by which ANG II induces ERK-mediated IGF-IIR signaling remains elusive. Here, we found that ANG II activated ERK to upregulate IGF-IIR expression via the angiotensin II type I receptor (AT1 R). ERK activation subsequently phosphorylates HSF1 at serine 307, leading to a secondary phosphorylation by glycogen synthase kinase III (GSK3) at serine 303. Moreover, we found that ANG II mediated ERK/GSK3-induced IGF-IIR protein stability by downregulating the E3 ubiquitin ligase of IGF-IIR RING finger protein CXXVI (RNF126). The expression of RNF126 decreased following ANG II-induced HSF1S303 phosphorylation, resulting in IGF-IIR protein stability and increased cardiomyocyte injury. Inhibition of GSK3 significantly alleviated ANG II-induced cardiac hypertrophy in vivo and in vitro. Taken together, these results suggest that HSF1 phosphorylation stabilizes IGF-IIR protein stability by downregulating RNF126 during cardiac hypertrophy. ANG II activates ERK/GSK3 to phosphorylate HSF1, resulting in RNF126 degradation, which stabilizes IGF-IIR protein expression and eventually results in cardiac hypertrophy. HSF1 could be a valuable therapeutic target for cardiac diseases among hypertensive patients.

218 Knockout p47phox reduces angiotensin ii-induced cardiac oxidative stress and hypertrophy

Oxidative stress due to the activation of a Nox2-containing NADPH oxidase is involved in Angiotensin II (AngII)-induced cardiovascular dysfunction. p47phox is a key regulatory subunit of Nox2. However, the role...

Exogenous cathepsin V protein protects human cardiomyocytes HCM from angiotensin Ⅱ-Induced hypertrophy

Angiotensin (Ang) Ⅱ-induced cardiac hypertrophy can deteriorate to heart failure, a leading cause of mortality. Endogenous Cathepsin V (CTSV) has been reported to be cardioprotective against hypertrophy. However, little is known about the effect of exogenous CTSV on cardiac hypertrophy. We used the human cardiomyocytes HCM as a cell model to investigate the effects of exogenous CTSV on Ang Ⅱ-induced cardiac cell hypertrophy. Cell surface area and expression of classical markers of hypertrophy were analyzed. We further explored the mechanism of CTSV cardioprotective by assessing the levels and activities of PI3K/Akt/mTOR and MAPK signaling pathway proteins. We found that pre-treating cardiomyocytes with CTSV could significantly inhibit Ang Ⅱ-induced hypertrophy. The mRNA expression of hypertrophy markers ANP, BNP and β-MHC was obviously elevated in Ang Ⅱ-treated cardiac cells. Whereas, exogenous CTSV effectively halted this elevation. Further study revealed that the protective effects of exogenous CTSV might be mediated by repressing the phosphorylation of proteins in the PI3K/Akt/mTOR and MAPK pathways. Based on our results, we concluded that exogenous CTSV inhibited Ang Ⅱ-induced hypertrophy in HCM cells by inhibiting PI3K/Akt/mTOR. This study provides experimental evidence for the application of CTSV protein for the treatment of cardiac hypertrophy.

Angiotensin-(1-7) attenuates angiotensin II-induced cardiac hypertrophy via a Sirt3-dependent mechanism.

The objectives of the present study were to investigate the effect of ANG-(1-7) on the development of cardiac hypertrophy and to identify the intracellular mechanism underlying this action of ANG-(1-7). Blood pressure and heart rate were recorded using radiotelemetry before and after chronic subcutaneous infusion of control (PBS), ANG II, ANG-(1-7), or ANG II + ANG-(1-7) for 4 wk in normotensive rats. Chronic administration of ANG-(1-7) did not affect either basal blood pressure or the ANG II-induced elevation in blood pressure. However, ANG-(1-7) significantly attenuated ANG II-induced cardiac hypertrophy and perivascular fibrosis in these rats. These effects of ANG-(1-7) were confirmed in cultured cardiomyocytes, in which ANG-(1-7) significantly attenuated ANG II-induced increases in cell size. This protective effect of ANG-(1-7) was significantly attenuated by pretreatment with A779 (a Mas receptor antagonist) or Mito-TEMPO (a mitochondria-targeting superoxide scavenger) as well as blockade of Sirt3 (a deacetylation-acting protein) by viral vector-mediated overexpression of sirtuin (Sirt)3 short hairpin (sh)RNA. Western blot analysis demonstrated that treatment with ANG-(1-7) dramatically increased Sirt3 expression. In addition, ANG-(1-7) attenuated the ANG II-induced increase in mitochondrial ROS generation, an effect that was abolished by A779 or Sirt3 shRNA. Moreover, ANG-(1-7) increased FoxO3a deacetylation and SOD2 expression, and these effects were blocked by Sirt3 shRNA. In summary, the protective effects of ANG-(1-7) on ANG II-induced cardiac hypertrophy and increased mitochondrial ROS production are mediated by elevated SOD2 expression via stimulation of Sirt3-dependent deacetylation of FoxO3a in cardiomyocytes. Thus, activation of the ANG-(1-7)/Sirt3 signaling pathway could be a novel therapeutic strategy in the management of cardiac hypertrophy and associated complications.NEW & NOTEWORTHY Chronic subcutaneous ANG-(1-7) has no effect on ANG II-induced elevations in blood pressure but significantly attenuates ANG II-induced cardiac hypertrophy and fibrosis by a mitochondrial ROS-dependent mechanism. This protective effect of ANG-(1-7) against the action of ANG II action is mediated by stimulation of sirtuin-3-mediated deacetylation of FoxO3a, which triggers SOD2 expression.

Synthesis of the novel PARP-1 inhibitor AG-690/11026014 and its protective effects on angiotensin II-induced mouse cardiac remodeling.

We previously identified AG-690/11026014 (6014) as a novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor that effectively prevented angiotensin II (Ang II)-induced cardiomyocyte hypertrophy. In the present study, we reported a new synthesis route for 6014, and investigated its protective effects on Ang II-induced cardiac remodeling and cardiac dysfunction and the underlying mechanisms in mice. We designed a new synthesis route to obtain a sufficient quantity of 6014 for this in vivo study. C57BL/6J mice were infused with Ang II and treated with 6014 (10, 30, 90 mg·kg-1·d-1, ig) for 4 weeks. Then two-dimensional echocardiography was performed to assess the cardiac function and structure. Histological changes of the hearts were examined with HE staining and Masson's trichrome staining. The protein expression was evaluated by Western blot, immunohistochemistry and immunofluorescence assays. The activities of sirtuin-1 (SIRT-1) and the content of NAD+ were detected with the corresponding test kits. Treatment with 6014 dose-dependently improved cardiac function, including LVEF, CO and SV and reversed the changes of cardiac structure in Ang II-infused mice: it significantly ameliorated Ang II-induced cardiac hypertrophy evidenced by attenuating the enlargement of cardiomyocytes, decreased HW/BW and LVW/BW, and decreased expression of hypertrophic markers ANF, BNP and β-MHC; it also prevented Ang II-induced cardiac fibrosis, as implied by the decrease in excess accumulation of extracellular matrix (ECM) components collagen I, collagen III and FN. Further studies revealed that treatment with 6014 did not affect the expression levels of PARP-1, but dose-dependently inhibited the activity of PARP-1 and subsequently restored the activity of SIRT-1 in heart tissues due to the decreased consumption of NAD+ and attenuated Poly-ADP-ribosylation (PARylation) of SIRT-1. In conclusion, the novel PARP-1 inhibitor 6014 effectively protects mice against AngII-induced cardiac remodeling and improves cardiac function. Thus, 6014 might be a potential therapeutic agent for heart diseases..