Thoracic Transverse Aortic Constriction Research Articles

The RING-domain E3 ubiquitin ligase RNF146 promotes cardiac hypertrophy by suppressing the LKB1/AMPK signaling pathway

The RING-domain E3 ubiquitin ligase RNF146 is an enzyme that plays an important role in ubiquitin-proteasomal protein degradation and participates in various pathophysiological processes. However, its role in cardiac hypertrophy is unclear. In the present work, thoracic transverse aortic constriction (TAC) was performed in transgenic mice with RNF146 knockout mice (KO) and wild-type mice, and neonatal rat cardiomyocytes (NRCMs) were subjected to angiotensin II (Ang II) stimulation to induce cardiac hypertrophy in vitro and in vivo. RNF146 expression was significantly increased in hypertrophied murine hearts and Ang II-stimulated NRCMs. RNF146-KO mice and knockdown of RNF146 NRCMs attenuated TAC- or Ang II-stimulated cardiac hypertrophy. Conversely, enforced expression of RNF146 aggravated these changes. Mechanistically, we found that RNF146 KO or knockdown increased the activation of the AMP-activated protein kinase (AMPK) pathway. Furthermore, we found that RNF146 KO or knockdown decreased ubiquitination of Liver kinase B1 (LKB1), which promoted the activation of the AMPK pathway in a dependent manner. In conclusion, RNF146 targets LKB1 protein for ubiquitin–proteasome degradation in cardiomyocytes and subsequently promotes cardiac hypertrophy by suppressing the activation of the AMPK signaling pathway.

HECT (Homologous to the E6-AP Carboxyl Terminus)-Type Ubiquitin E3 Ligase ITCH Attenuates Cardiac Hypertrophy by Suppressing the Wnt/β-Catenin Signaling Pathway.

The HECT (homologous to the E6-AP carboxyl terminus)-type ubiquitin E3 ligase ITCH is an enzyme that plays an important role in ubiquitin-proteasomal protein degradation. Disheveled proteins (Dvl1 [disheveled protein 1], Dvl2, and Dvl3) are the main components of the Wnt/β-catenin signaling pathway, which is involved in cardiac hypertrophy. The aim of this study was to examine the role of ITCH during development of cardiac hypertrophy. Thoracic transverse aortic constriction (TAC) was performed in transgenic mice with cardiac-specific overexpression of ITCH (ITCH-Tg) and wild-type mice. Cardiac hypertrophy after TAC was attenuated in ITCH-Tg mice, and the survival rate was higher for ITCH-Tg mice than for wild-type mice. Protein interaction between ITCH and Dvls was confirmed with immunoprecipitation in vivo and in vitro. Expression of key molecules of the Wnt/β-catenin signaling pathway (Dvl1, Dvl2, GSK3β [glycogen synthase kinase 3β], and β-catenin) was inhibited in ITCH-Tg mice compared with wild-type mice. Notably, the ubiquitination level of Dvl proteins increased in ITCH-Tg mice. Protein and mRNA expression levels of ITCH increased in response to Wnt3a stimulation in neonatal rat cardiomyocytes. Knockdown of ITCH using small-interfering RNA increased cardiomyocyte size and augmented protein expression levels of Dvl proteins, phospho-GSK3β, and β-catenin after Wnt3a stimulation in cardiomyocytes. Conversely, overexpression of ITCH attenuated cardiomyocyte hypertrophy and decreased protein expression levels of Dvl proteins, phospho-GSK3β and β-catenin. In conclusion, ITCH targets Dvl proteins for ubiquitin-proteasome degradation in cardiomyocytes and attenuates cardiac hypertrophy by suppressing the Wnt/β-catenin signaling pathway.

Beta 3 adrenoreceptors protect from hypertrophic remodelling through AMP-activated protein kinase and autophagy.

AimsThe abundance of beta 3‐adrenergic receptors (β3‐ARs) is upregulated in diseased human myocardium. We previously showed that cardiac‐specific expression of β3‐AR inhibits the hypertrophic response to neurohormonal stimulation. Here, we further analysed signalling pathways involved in the anti‐hypertrophic effect of β3‐AR.Methods and results In vitro hypertrophic responses to phenylephrine (PE) were analysed in neonatal rat ventricular myocytes (NRVM) infected with a recombinant adenovirus expressing the human β3‐AR (AdVhβ3). We confirmed results in mice with cardiomyocyte‐specific moderate expression of human β3‐AR (β3‐TG) and wild‐type (WT) littermates submitted to thoracic transverse aortic constriction (TAC) for 9 weeks. We observed a colocalization of β3‐AR with the AMP‐activated protein kinase (AMPK) both in neonatal rat and in adult mouse cardiomyocytes. Treatment of NRVM with PE induced hypertrophy and a decrease in phosphorylation of Thr172‐AMPK (/2, P = 0.0487) and phosphorylation of Ser79‐acetyl‐CoA carboxylase (ACC) (/2.6, P = 0.0317), inducing an increase in phosphorylated Ser235/236 S6 protein (×2.5, P = 0.0367) known to be involved in protein synthesis. These effects were reproduced by TAC in WT mice but restored to basal levels in β3‐AR expressing cells/mice. siRNA targeting of AMPK partly abrogated the anti‐hypertrophic effect of β3‐AR in response to PE in NRVM. Concomitant with hypertrophy, autophagy was decreased by PE, as measured by microtubule‐associated protein 1 light chain 3 (LC3)‐II/LC3‐I ratio (/2.6, P = 0.0010) and p62 abundance (×3, P = 0.0016) in NRVM or by TAC in WT mice (LC3‐II/LC3‐I ratio: /5.4, P = 0.0159), but preserved in human β3‐AR expressing cells and mice, together with reduced hypertrophy.ConclusionsCardiac‐specific moderate expression of β3‐AR inhibits the hypertrophic response in part through AMPK activation followed by inhibition of protein synthesis and preservation of autophagy. Activation of the cardiac β3‐AR pathway may provide future therapeutic avenues for the modulation of hypertrophic remodelling.

P1615HECT-Type Ubiquitin E3 Ligase ITCH attenuates cardiac hypertrophy by suppressing Wnt signaling pathway

Abstract Background The homologous to the E6-AP carboxyl terminus (HECT)–type ubiquitin E3 ligase ITCH is an enzyme that plays an important role in ubiquitin proteasomal protein degradation. Dishevelled proteins (Dvl1, Dvl2 and Dvl3), which are involved in canonical Wnt/β catenin signaling pathway, play a role in cardiac hypertrophy. Purpose The aim of this study was to examine whether ITCH interacts with Dvls and prevents cardiac hypertrophy induced by pressure overload. Methods and results We confirmed the protein interaction between ITCH and Dvls in cardiomyocytes. Overexpression of ITCH decreased protein expression levels of Dvls, phospho-GSK3β and β-catenin. Conversely, knockdown of ITCH using small interfering RNA augmented canonical Wnt/β catenin signaling pathway. Thoracic transverse aortic constriction (TAC) was performed in transgenic mice with cardiac-specific overexpression of ITCH (ITCH-Tg) and wild-type (WT) mice. The canonical Wnt/β catenin signaling pathway was inhibited and cardiac hypertrophy was attenuated in ITCH-Tg mice compared with WT mice after TAC. Overexpression of ITCH in cardiomyocytes Conclusion We demonstrated that ITCH targets Dvls for ubiquitin-proteasome degradation in cardiomyocytes and ameliorates cardiac hypertrophy by suppressing canonical Wnt/β catenin signaling pathway.

Abstract 563: Cardiac TIGAR Reduces Myocardial Energetics and Cardiac Functionin the Pressure Overload Heart Failure Model

Background: Although metabolic alterations were observed in heart failure (HF), only recently have the mechanisms underlying these changes been identified. Tumor suppressor p53 responds to metabolic changes thorough several mechanisms. One of the p53 targets, TIGAR (TP53-induced glycolysis and apoptosis regulator) reduces glycolysis and suppresses autophagy, which augments ischemic damage, however its role on HF is unclear. Method and Results: In order to investigate TIGAR’s function in HF, we compared myocardial metabolic and functional outcomes between TIGAR deficient (TIGAR–/–) mice and wild-type (TIGAR+/+) mice subjected to chronic thoracic transverse aortic constriction (TAC), a pressure-overload HF model. In wild-type mice hearts, p53 and TIGAR increased markedly during HF development. Eight weeks after TAC surgery, the left ventricular (LV) dysfunction, fibrosis, oxidative damage, and myocyte apoptosis were significantly advanced in wild-type than in TIGAR–/– mouse heart. Further, myocardial high-energy phosphates in wild-type hearts were significantly decreased compared to those of TIGAR–/– mouse heart. Glucose oxidation and glycolysis rates were also reduced in isolated perfused wild-type hearts following TAC than those in TIGAR–/– hearts, which suggest that the upregulation of TIGAR in HF causes impaired myocardial energetics and function. The effects of TIGAR knockout on LV function were also replicated in tamoxifen (TAM)-inducible cardiac-specific TIGAR knockout mice (TIGARflox/flox/ Tg(Myh6-cre/Esr1) mice). Conclusion: The ablation of TIGAR during pressure-overload HF preserves myocardial function and energetics. Thus, cardiac TIGAR targeted therapy to increase glucose metabolism will be a novel strategy for HF.

Ablation of cardiac TIGAR preserves myocardial energetics and cardiac function in the pressure overload heart failure model.

Despite the advances in medical therapy, the morbidity and mortality of heart failure (HF) remain unacceptably high. HF results from reduced metabolism-contraction coupling efficiency, so the modulation of cardiac metabolism may be an effective strategy for therapeutic interventions. Tumor suppressor p53 (TP53) and its downstream target TP53-induced glycolysis and apoptosis regulator (TIGAR) are known to modulate cardiac metabolism and cell fate. To investigate TIGAR's function in HF, we compared myocardial, metabolic, and functional outcomes between TIGAR knockout (TIGAR-/-) mice and wild-type (TIGAR+/+) mice subjected to chronic thoracic transverse aortic constriction (TAC), a pressure-overload HF model. In wild-type mice hearts, p53 and TIGAR increased markedly during HF development. Eight weeks after TAC surgery, the left ventricular (LV) dysfunction, fibrosis, oxidative damage, and myocyte apoptosis were significantly advanced in wild-type than in TIGAR-/- mouse heart. Further, myocardial high-energy phosphates in wild-type hearts were significantly decreased compared with those of TIGAR-/- mouse heart. Glucose oxidation and glycolysis rates were also reduced in isolated perfused wild-type hearts following TAC than those in TIGAR-/- hearts, which suggest that the upregulation of TIGAR in HF causes impaired myocardial energetics and function. The effects of TIGAR knockout on LV function were also replicated in tamoxifen (TAM)-inducible cardiac-specific TIGAR knockout mice (TIGARflox/flox/Tg(Myh6-cre/Esr1) mice). The ablation of TIGAR during pressure-overload HF preserves myocardial function and energetics. Thus, cardiac TIGAR-targeted therapy to increase glucose metabolism will be a novel strategy for HF. NEW & NOTEWORTHY The present study is the first to demonstrate that TP53-induced glycolysis and apoptosis regulator (TIGAR) is upregulated in the myocardium during experimental heart failure (HF) in mice and that TIGAR knockout can preserve the heart function and myocardial energetics during HF. Reducing TIGAR to enhance myocardial glycolytic energy production is a promising therapeutic strategy for HF.

Netrin-1 prevents the development of cardiac hypertrophy and heart failure.

The aim of the present study was to examine whether netrin-1 is involved in the development of cardiac hypertrophy, induced by pressure overload. For this investigation, thoracic transverse aortic constriction (TAC) was performed in mice. A total of 18 mice were divided into three groups (n=6 per group): Sham, TAC and TAC + recombinant netrin-1. Neonatal rat cardiomyocytes were stimulated with endothelin-1 (ET-1), and samples were collected to examine the expression levels of netrin‑1 by western blot analysis and the mRNA expression of A‑type natriuretic peptide by reverse transcription‑quantitative polymerase chain reaction. It was found that the expression of netrin‑1 was decreased in the TAC mice and in the neonatal rat cardiomyocytes in response to ET‑1 stimulation. Netrin‑1 eliminated ventricular remodeling, cardiac dysfunction and DNA damage during pressure overload. Furthermore, analysis of the signaling events indicated that netrin‑1‑mediated protection against cardiac hypertrophy was attributed to interruption of the activation of mitogen‑activated protein kinase kinase (MEK) kinase‑1 (K1)‑dependent MEK‑extracellular signal‑regulated protein kinase 1/2 (ERK1/2) and c‑Jun N‑terminal kinase 1/2 (JNK1/2). Therefore, netrin‑1 prevented cardiac hypertrophy and heart failure through the negative regulation of the MEKK1-dependent MEK‑ERK1/2 and JNK1/2 signaling pathways.

Role of high mobility group protein B-1 in the protective effect of fenofibrate against cardiac hypertrophy

Objective To investigate whether the regulation of high mobility group protein B-1 (HMGB1) by fenofibrate could affect the development of cardiac hypertrophy,and provide a preliminary study mechanism.Methods Primary cultured neonatal rat cardiomyocytes were used as the experimental subjects.The impacts of fenofibrarte on the level of HMGB1 expression and translocation in cardiomyocytes were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.Cardiac hypertrophy was induced in mice by thoracic transverse aortic constriction.The mice were divided into two groups:experimental group,fenofibrate administration (n =5),and control group,normal saline given (n =5).HMGB1 expression and translocation,as well as the progress of cardiac hypertrophy in the two groups were compared.Results Fenofibrate could down-regulate basal and LPS-stimulated HMGB1 expression (RNA level:P ＜0.05; Protein level:P ＜0.05),as well as the secretion of HMGB1 in cardiomyocytes (P ＜ 0.05).In addition,administration of fenofibrate to mice prevented the development of cardiac hypertrophy induced by thoracic transverse aortic constriction while increased levels of nuclear HMGB1.Conclusion Fenofibrate may inhibit the development of cardiac hypertrophy by regulating HMGB1 expression,which provides a new potential strategy to treat cardiac hypertrophy. Key words: Peroxisome proliferator activated receptor α; Fenofibrate; High mobility group protein B-1 ; Cardiomyocytes; Cardiac hypertrophy

HMGB1 Is Involved in the Protective Effect of the PPARαAgonist Fenofibrate against Cardiac Hypertrophy

High mobility group box 1 (HMGB1) is a ubiquitous nuclear DNA-binding protein whose function is dependent on its cellular location. Extracellular HMGB1 is regarded as a delayed mediator of proinflammatory cytokines for initiating and amplifying inflammatory responses, whereas nuclear HMGB1 has been found to prevent cardiac hypertrophy and heart failure. Because fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, has shown both protective effects against cardiac hypertrophy and inhibitory effects against inflammation, the potential modulation of HMGB1 expression and secretion by fenofibrate is of great interest. We herein provide evidence that fenofibrate modulates basal and LPS-stimulated HMGB1 expression and localization in addition to secretion of HMGB1 in cardiomyocytes. In addition, administration of fenofibrate to mice prevented the development of cardiac hypertrophy induced by thoracic transverse aortic constriction (TAC) while increasing levels of nuclear HMGB1. Altogether, these data suggest that fenofibrate may inhibit the development of cardiac hypertrophy by regulating HMGB1 expression, which provides a new potential strategy to treat cardiac hypertrophy.

Atrial endothelial impairment through Toll-like receptor 4 signaling causes atrial thrombogenesis

Low-grade inflammation associated with heart failure (HF) is known to deteriorate cardioembolic stroke in patients with atrial fibrillation (AF). Little is known about the relationship between atrial endothelial impairment induced by innate immunity and thrombus formation. We examined whether atrial endothelial impairment through Toll-like receptor (TLR) 4 signaling causes atrial thrombogenesis. TLR4, heat shock protein 60, and vascular cell adhesion molecule (VCAM)-1 expression were higher in the atrium of AF patients who underwent valve replacement surgery with HF compared with those without it (p < 0.05). We created thoracic transverse aortic constriction (TAC) in TLR4 knock-out (KO) and wild-type (WT) mice. Atrial thrombosis was observed less frequently in TLR4 KO mice (4/15) than in WT mice (16/20) 4 weeks after TAC despite similar severity of heart failure. The decrease in endothelial nitric oxide synthase (eNOS) phosphorylation and increase in VCAM-1 and plasminogen activator inhibitor (PAI)-1 expression, observed in the atrium of WT mice following TAC, were significantly attenuated in TLR4 KO mice (p < 0.05). Nuclear factor-κB (NF-κB) activation after TAC was attenuated in TLR4 KO mice compared with WT mice. Activation of mitogen-activated protein kinase p38 (p38) after TAC was also attenuated in TLR4 KO mice (p < 0.05). Thus, increased VCAM-1 and PAI-1, and decreased eNOS phosphorylation through the TLR4/NFκB/p38 pathway, may be associated with atrial thrombogenesis in the heart failure mice model. Atrial endothelial impairment through the TLR4 signaling may play a role in atrial thrombogenesis in AF patients with HF.

Cardiac nuclear high mobility group box 1 prevents the development of cardiac hypertrophy and heart failure

AimsHigh mobility group box 1 (HMGB1) is an abundant and ubiquitous nuclear DNA-binding protein that has multiple functions dependent on its cellular location. HMGB1 binds to DNA, facilitating numerous nuclear functions including maintenance of genome stability, transcription, and repair. However, little is known about the effects of nuclear HMGB1 on cardiac hypertrophy and heart failure. The aim of this study was to examine whether nuclear HMGB1 plays a role in the development of cardiac hypertrophy induced by pressure overload.Methods and resultsAnalysis of human biopsy samples by immunohistochemistry showed decreased nuclear HMGB1 expression in failing hearts compared with normal hearts. Nuclear HMGB1 decreased in response to both endothelin-1 (ET-1) and angiotensin II (Ang II) stimulation in neonatal rat cardiomyocytes, where nuclear HMGB1 was acetylated and translocated to the cytoplasm. Overexpression of nuclear HMGB1 attenuated ET-1 induced cardiomyocyte hypertrophy. Thoracic transverse aortic constriction (TAC) was performed in transgenic mice with cardiac-specific overexpression of HMGB1 (HMGB1-Tg) and wild-type (WT) mice. Cardiac hypertrophy after TAC was attenuated in HMGB1-Tg mice and the survival rate after TAC was higher in HMGB1-Tg mice than in WT mice. Induction of foetal cardiac genes was decreased in HMGB1-Tg mice compared with WT mice. Nuclear HMGB1 expression was preserved in HMGB1-Tg mice compared with WT mice and significantly attenuated DNA damage after TAC was attenuated in HMGB1-TG mice.ConclusionThese results suggest that the maintenance of stable nuclear HMGB1 levels prevents hypertrophy and heart failure by inhibiting DNA damage.

Abstract 5170: Atrial Endothelial Impairment Induced by Toll-Like Receptor-4 Signaling May Cause Atrial Thrombogenesis

Background Recently atrial endothelial dysfunction has gained more attention as an important cause for atrial thrombogenesis in patients with atrial fibrillation (AF). It was reported that AF patients with left ventricular hypertrophy or heart failure (HF) have higher thromboembolic risks than those without. Several reports showed that angiotensin II receptor blockers prevented occurrences of AF and thrombogenesis by inhibiting inflammation. However, little is known how AF causes atrial inflammation and thrombogenesis. We examined whether atrial inflammation through toll-like receptor (TLR)-4 signaling causes atrial thrombogenesis. Methods and result First, we examined serum and atrial tissue heat shock protein 60 (HSP60) levels, which have been known as a TLR-4 ligand, in patients with and without AF. Atrial HSP60 increased in HF patients with AF compared to those without AF whereas serum HSP60 level was lower in HF patients with AF compared to those without AF (NYHA class I and II). Then, we created thoracic transverse aortic constriction (TAC) model in TLR-4 knockout (KO) mice (n=15) and wild-type (WT) mice (n=20). We performed echocardiography at 4 weeks after TAC surgery. Then, the atrium was excised, and vascular cell adhesion molecule (VCAM)-1, endothelial nitric oxide synthase (eNOS), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and mitogen activated protein kinase p38 (p38) were analyzed by Western blotting. We observed less frequent occurrence of atrial thrombosis in TLR-4 KO-TAC mice (4/15) than WT-TAC mice (16/20). Decrease in eNOS phosphorylation and increase in VCAM-1 expression in WT-TAC mice were significantly attenuated in TLR-4 KO-TAC mice (P&lt;0.05). Nuclear factor-kappa B (NFkB) was attenuated in TLR-4 KO-TAC mice compared to WT-TAC mice by electrophoretic mobility shift assay analysis. Phosphorylation of p38, but ERK and JNK was also attenuated in TLR-4 KO-TAC mice (P&lt;0.05). These results suggested that activation of VCAM-1 and decreased eNOS through TLR-4/p38 and NFkB signaling may be associated with atrial thrombogenesis in AF. Conclusion Atrial inflammation caused by TLR-4 signaling may play a role in atrial thrombogenesis in heart failure mice model.

Downregulation of ferritin heavy chain increases labile iron pool, oxidative stress and cell death in cardiomyocytes

Ferritin heavy chain (FHC) protein was significantly reduced in murine failing hearts following left coronary ligation or thoracic transverse aortic constriction. The mRNA expression of FHC was not significantly altered in failing hearts, compared to that in control sham-operated hearts. Prussian blue staining revealed spotty iron depositions in myocardial infarct failing hearts. Oxidative stress was enhanced in the myocardial infarct failing hearts, as evidenced by increases in 4-hydroxy-2-nonenal and 8-hydroxy-2′-deoxyguanosine immunoreactivity. To clarify the functional significance of FHC downregulation in hearts, we infected rat neonatal cardiomyocytes with adenoviral vector expressing short hairpin RNA targeted to FHC (Ad-FHC-RNAi). The downregulation of FHC induced a reduction in the viability of cardiomyocytes. The relative number of iron deposition-, 4-hydroxy-2-nonenal- or 8-hydroxy-2′-deoxyguanosine-positive cardiomyocytes was significantly higher in Ad-FHC-RNAi-infected cardiomyocytes than in control vector-infected cardiomyocytes. Treatment of Ad-FHC-RNAi-infected cardiomyocytes with desferrioxamine, an iron chelator, significantly reduced the number of iron, 4-hydroxy-2-nonenal or 8-hydroxy-2′-deoxyguanosine-positive cells, and increased viability. In addition, treatment with N-acetyl cysteine, an antioxidant, significantly reduced the number of 4-hydroxy-2-nonenal- or 8-hydroxy-2′-deoxyguanosine-positive cells. Reduced viability in Ad-FHC-RNAi-infected cardiomyocytes was significantly improved with N-acetyl cysteine treatment. These findings indicate that excessive free iron and the resultant enhanced oxidative stress caused by downregulation of FHC lead to cardiomyocyte death. The decrease in FHC expression in failing hearts may play an important role in the pathogenesis of heart failure.

Diacylglycerol kinase-ε restores cardiac dysfunction under chronic pressure overload: a new specific regulator of Gαqsignaling cascade

Galpha(q) protein-coupled receptor (GPCR) signaling pathway, which includes diacylglycerol (DAG) and protein kinase C (PKC), plays a critical role in cardiac hypertrophy. DAG kinase (DGK) catalyzes DAG phosphorylation and controls cellular DAG levels, thus acting as a regulator of GPCR signaling. It has been reported that DGKepsilon acts specifically on DAG produced by inositol cycling. In this study, we examined whether DGKepsilon prevents cardiac hypertrophy and progression to heart failure under chronic pressure overload. We generated transgenic mice with cardiac-specific overexpression of DGKepsilon (DGKepsilon-TG) using an alpha-myosin heavy chain promoter. There were no differences in cardiac morphology and function between wild-type (WT) and DGKepsilon-TG mice at the basal condition. Either continuous phenylephrine infusion or thoracic transverse aortic constriction (TAC) was performed in WT and DGKepsilon-TG mice. Increases in heart weight after phenylephrine infusion and TAC were abolished in DGKepsilon-TG mice compared with WT mice. Cardiac dysfunction after TAC was prevented in DGKepsilon-TG mice, and the survival rate after TAC was higher in DGKepsilon-TG mice than in WT mice. Phenylephrine- and TAC-induced DAG accumulation, the translocation of PKC isoforms, and the induction of fetal genes were blocked in DGKepsilon-TG mouse hearts. The upregulation of transient receptor potential channel (TRPC)-6 expression after TAC was attenuated in DGKepsilon-TG mice. In conclusion, these results demonstrate the first evidence that DGKepsilon restores cardiac dysfunction and improves survival under chronic pressure overload by controlling cellular DAG levels and TRPC-6 expression. DGKepsilon may be a novel therapeutic target to prevent cardiac hypertrophy and progression to heart failure.

Diacylglycerol Kinase .XI. Attenuates Pressure Overload-Induced Cardiac Hypertrophy

Background The Gaq protein-coupled receptor (GPCR) signaling pathway, which includes diacylglycerol (DAG) and protein kinase C (PKC), plays a critical role in the development of cardiac hypertrophy and heart failure. DAG kinase (DGK) phosphorylates DAG and controls cellular DAG levels, thus acting as a regulator of GPCR signaling. It has been previously reported that DGK inhibited GPCR agonist-induced activation of the DAG-PKC signaling and subsequent cardiomyocyte hypertrophy, so the purpose of this study was to examine whether DGK modifies the development of cardiac hypertrophy induced by pressure overload. Methods and Results Thoracic transverse aortic constriction (TAC) was created in transgenic mice with cardiac-specific overexpression of DGKξ (DGKξ-TG) and wild-type (WT) mice. Increases in heart weight at 4 weeks after TAC were attenuated in DGKξ-TG mice compared with WT mice. Increases in interventricular septal thickness, dilatation of the left ventricular cavity, and decreases in left ventricular systolic function in WT mice were observed with echocardiography at 4 weeks after TAC surgery. However, these structural and functional changes after TAC were attenuated in DGKξ-TG mice. In WT mice, cardiac fibrosis and up-regulation of profibrotic genes, such as transforming growth factor-β1, collagen type I, and collagen type III, were observed at 4 weeks after TAC. However, cardiac fibrosis and gene induction of type I and type III collagens, but not transforming growth factor-β1, were blocked in DGKξ-TG mice. Conclusion These results are the first in vivo evidence that DGKξ suppresses cardiac hypertrophy and fibrosis and prevents impaired left ventricular systolic function caused by pressure overload. (Circ J 2007; 71: 276 - 282)

Pressure Overload Induces Cardiac Dysfunction and Dilation in Signal Transducer and Activator of Transcription 6–Deficient Mice

Signal transducer and activator of transcription (STAT) proteins constitute a family of transcription factors that mediate many cytokine-induced responses. STAT6 is activated by angiotensin II and in rat hypertrophied hearts and in human hearts with dilated cardiomyopathy. This suggests that STAT6 may be involved in the pathogenesis of cardiac hypertrophy and heart failure. For this study we used STAT6-deficient (STAT6-/-) mice to examine the in vivo role of STAT6. STAT6-/- hearts showed no morphological, histological, or functional defects. We examined left ventricular structural and functional remodeling 1 week after thoracic transverse aortic constriction (TAC). Western blot and immunohistochemical analyses showed increased STAT6 activity after TAC in the heart of wild-type mice. STAT6-/- mice showed a significant increase in end-diastolic left ventricular internal dimension accompanied by impaired contractility compared with wild-type mice but no differences in hypertrophic parameters. The number of terminal deoxynucleotidyl transferase-mediated biotin dUTP nick-end labeling-positive myocytes after TAC had increased in STAT6-/- compared with wild-type mice. Prolonged induction of tumor necrosis factor-alpha (TNF-alpha) mRNA was observed in STAT6-/- hearts, whereas TNF-alpha mRNA was only transiently induced in wild-type mice. Tristetraprolin was induced after TAC in wild-type mice but not in STAT6-/- mice. Tristetraprolin reporter assay with the use of isolated neonatal cardiomyocyte indicated that the promoter was significantly activated by endothelin-1 in wild-type but not in STAT6-/- cardiomyocytes. The lack of promoter activation by endothelin-1 in STAT6-/- cardiomyocytes was rescued by forced expression of STAT6. STAT6 plays a protective role against hemodynamic stress in hearts.

Targeted deletion of apoptosis signal-regulating kinase 1 attenuates left ventricular remodeling.

Left ventricular remodeling that occurs after myocardial infarction (MI) and pressure overload is generally accepted as a determinant of the clinical course of heart failure. The molecular mechanism of this process, however, remains to be elucidated. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that plays an important role in stress-induced apoptosis. We used ASK1 knockout mice (ASK-/-) to test the hypothesis that ASK1 is involved in development of left ventricular remodeling. ASK-/- hearts showed no morphological or histological defects. Echocardiography and cardiac catheterization revealed normal global structure and function. Left ventricular structural and functional remodeling were determined 4 weeks after coronary artery ligation or thoracic transverse aortic constriction (TAC). ASK-/- had significantly smaller increases in left ventricular end-diastolic and end-systolic ventricular dimensions and smaller decreases in fractional shortening in both experimental models compared with WT mice. The number of terminal deoxynucleotidyl transferase biotin-dUDP nick end-labeling-positive myocytes after MI or TAC was decreased in ASK-/- compared with that in WT mice. Overexpression of a constitutively active mutant of ASK1 induced apoptosis in isolated rat neonatal cardiomyocytes, whereas neonatal ASK-/- cardiomyocytes were resistant to H2O2-induced apoptosis. An in vitro kinase assay showed increased ASK1 activity in heart after MI or TAC in WT mice. Thus, ASK1 plays an important role in regulating left ventricular remodeling by promoting apoptosis.