FGF13 Is a Novel Regulator of NF-?B and Potentiates Pathological Cardiac Hypertrophy.
ABSTRACT: FGF13 is an intracellular FGF factor. Its role in cardiomyopathies has been rarely investigated. We revealed that endogenous FGF13 is up-regulated in cardiac hypertrophy accompanied by increased nuclear localization. The upregulation of FGF13 plays a deteriorating role both in hypertrophic cardiomyocytes and mouse hearts. Mechanistically, FGF13 directly interacts with p65 by its nuclear localization sequence and co-localizes with p65 in the nucleus in cardiac hypertrophy. FGF13 deficiency inhibits NF-?B activation in ISO-treated NRCMs and TAC-surgery mouse hearts, whereas FGF13 overexpression shows the opposite trend. Moreover, FGF13 overexpression alone is sufficient to activate NF-?B in cardiomyocytes. The interaction between FGF13 and p65 or the effects of FGF13 on NF-?B have nothing to do with I?B. Together, an I?B-independent mechanism for NF-?B regulation has been revealed in cardiomyocytes both under basal and stressful conditions, suggesting the promising application of FGF13 as a therapeutic target for pathological cardiac hypertrophy and heart failure.
Project description:The pathogenesis of cardiac hypertrophy is tightly associated with activation of intracellular hypertrophic signalling pathways, which leads to the synthesis of various proteins. Tripartite motif 10 (TRIM10) is an E3 ligase with important functions in protein quality control. However, its role in cardiac hypertrophy was unclear. In this study, neonatal rat cardiomyocytes (NRCMs) and TRIM10-knockout mice were subjected to phenylephrine (PE) stimulation or transverse aortic constriction (TAC) to induce cardiac hypertrophy in vitro and in vivo, respectively. Trim10 expression was significantly increased in hypertrophied murine hearts and PE-stimulated NRCMs. Knockdown of TRIM10 in NRCMs alleviated PE-induced changes in the size of cardiomyocytes and hypertrophy gene expression, whereas TRIM10 overexpression aggravated these changes. These results were further verified in TRIM10-knockout mice. Mechanistically, we found that TRIM10 knockout or knockdown decreased AKT phosphorylation. Furthermore, we found that TRIM10 knockout or knockdown increased ubiquitination of phosphatase and tensin homolog (PTEN), which negatively regulated AKT activation. The results of this study reveal the involvement of TRIM10 in pathological cardiac hypertrophy, which may occur by prompting of PTEN ubiquitination and subsequent activation of AKT signalling. Therefore, TRIM10 may be a promising target for treatment of cardiac hypertrophy.
Project description:Rapamycin, also known as sirolimus, is an immunosuppressant drug used to prevent rejection organ (especially kidney) transplantation. However, little is known about the role of Rapa in cardiac hypertrophy induced by isoproterenol and its underlying mechanism. In this study, Rapa was administrated intraperitoneally for one week after the rat model of cardiac hypertrophy induced by isoproterenol established. Rapa was demonstrated to attenuate isoproterenol-induced cardiac hypertrophy, maintain the structure integrity and functional performance of mitochondria, and upregulate genes related to fatty acid metabolism in hypertrophied hearts. To further study the implication of NF-?B in the protective role of Rapa, cardiomyocytes were pretreated with TNF-? or transfected with siRNA against NF-?B/p65 subunit. It was revealed that the upregulation of extracellular circulating proinflammatory cytokines induced by isoproterenol was able to be reversed by Rapa, which was dependent on NF-?B pathway. Furthermore, the regression of cardiac hypertrophy and maintaining energy homeostasis by Rapa in cardiomyocytes may be attributed to the inactivation of NF-?B. Our results shed new light on mechanisms underlying the protective role of Rapa against cardiac hypertrophy induced by isoproterenol, suggesting that blocking proinflammatory response by Rapa might contribute to the maintenance of energy homeostasis during the progression of cardiac hypertrophy.
Project description:The intracellular fibroblast growth factors (iFGF/FHFs) bind directly to cardiac voltage gated Na+ channels, and modulate their function. Mutations that affect iFGF/FHF-Na+ channel interaction are associated with arrhythmia syndromes. Although suspected to modulate other ionic currents, such as Ca2+ channels based on acute knockdown experiments in isolated cardiomyocytes, the in vivo consequences of iFGF/FHF gene ablation on cardiac electrical activity are still unknown. We generated inducible, cardiomyocyte-restricted Fgf13 knockout mice to determine the resultant effects of Fgf13 gene ablation. Patch clamp recordings from ventricular myocytes isolated from Fgf13 knockout mice showed a ~25% reduction in peak Na+ channel current density and a hyperpolarizing shift in steady-state inactivation. Electrocardiograms on Fgf13 knockout mice showed a prolonged QRS duration. The Na+ channel blocker flecainide further prolonged QRS duration and triggered ventricular tachyarrhythmias only in Fgf13 knockout mice, suggesting that arrhythmia vulnerability resulted, at least in part, from a loss of functioning Na+ channels. Consistent with these effects on Na+ channels, action potentials in Fgf13 knockout mice, compared to Cre control mice, exhibited slower upstrokes and reduced amplitude, but unexpectedly had longer durations. We investigated candidate sources of the prolonged action potential durations in myocytes from Fgf13 knockout mice and found a reduction of the transient outward K+ current (Ito). Fgf13 knockout did not alter whole-cell protein levels of Kv4.2 and Kv4.3, the Ito pore-forming subunits, but did decrease Kv4.2 and Kv4.3 at the sarcolemma. No changes were seen in the sustained outward K+ current or voltage-gated Ca2+ current, other candidate contributors to the increased action potential duration. These results implicate that FGF13 is a critical cardiac Na+ channel modulator and Fgf13 knockout mice have increased arrhythmia susceptibility in the setting of Na+ channel blockade. The unanticipated effect on Ito revealed new FGF13 properties and the unexpected lack of an effect on voltage-gated Ca2+ channels highlight potential compensatory changes in vivo not readily revealed with acute Fgf13 knockdown in cultured cardiomyocytes.
Project description:Both nuclear factors of activated T cells (NFAT) and nuclear factor-?B (NF?B) are Rel homology domain (RHD)-containing transcription factors whose independent activities are critically involved in regulating cardiac hypertrophy and failure.To determine the potential functional interaction between NFAT and NF?B signaling pathways in cardiomyocytes and its role in cardiac hypertrophy and remodeling.We identified a novel transcriptional regulatory mechanism whereby NF?B and NFAT directly interact and synergistically promote transcriptional activation in cardiomyocytes. We show that the p65 subunit of NF?B coimmunoprecipitates with NFAT in cardiomyocytes, and this interaction maps to the RHD within p65. Overexpression of the p65-RHD disrupts the association between endogenous p65 and NFATc1, leading to reduced transcriptional activity. Overexpression of I?B kinase ? (IKK?) or p65-RHD causes nuclear translocation of NFATc1, and expression of a constitutively nuclear NFATc1-SA mutant similarly facilitated p65 nuclear translocation. Combined overexpression of p65 and NFATc1 promotes synergistic activation of NFAT transcriptional activity in cardiomyocytes, whereas inhibition of NF?B with I?B?M or dominant negative IKK? reduces NFAT activity. Importantly, agonist-induced NFAT activation is reduced in p65 null mouse embryonic fibroblasts (MEFs) compared with wild-type MEFs. In vivo, cardiac-specific deletion of p65 using a Cre-loxP system causes a ?50% reduction in NFAT activity in luciferase reporter mice. Moreover, ablation of p65 in the mouse heart decreases the hypertrophic response after pressure overload stimulation, reduces the degree of pathological remodeling, and preserves contractile function.Our results suggest a direct interaction between NFAT and NF?B that effectively integrates 2 disparate signaling pathways in promoting cardiac hypertrophy and ventricular remodeling.
Project description:Decreased autophagy has been reported to contribute to the progression of cardiac hypertrophy. Our previous research has demonstrated that endophilin A2 (EndoA2) attenuates H2O2-induced cardiomyocyte apoptosis by strengthening autophagy. However, the role of EndoA2 in the regulation of autophagy in cardiac hypertrophy is unknown. In this study, we tested the hypothesis that EndoA2 suppresses cardiac hypertrophy induced by isoproterenol (ISO) by activating autophagy. In vivo, we established a cardiac hypertrophy model by subcutaneous injection of ISO and used intramyocardial delivery of adenovirus vector harboring EndoA2 cDNA (Ad-EndoA2) to overexpress EndoA2. The cardiac hypertrophic response and autophagy level were measured. EndoA2 overexpression suppressed pathological cardiac hypertrophy and enhanced autophagy in rat hearts. In addition, the effects of EndoA2 on cardiac hypertrophy and autophagy were observed in cultured neonatal rat cardiomyocytes (NRCMs) with gain- and loss-of-function approaches to regulate EndoA2 expression. The results were consistent with those of the in vivo study. Furthermore, the involvement of EndoA2-mediated autophagy in the attenuation of ISO-induced cardiac hypertrophy was explored by pharmaceutical inhibition of autophagy. Pretreatment with 3-methyladenine (3-MA) clearly diminished the anti-hypertrophic effects of EndoA2 in ISO-treated NRCMs. The results presented here provide the first evidence that EndoA2 is involved in ISO-induced cardiac hypertrophy. The anti-hypertrophic effects of EndoA2 can be partially attributed to its regulation of autophagy.
Project description:Postinfarct cardiac hypertrophy is an independent risk factor for heart failure and sudden death. Regression of cardiac hypertrophy has emerged as a promising strategy in the treatment of myocardial infarction (MI). Here we hypothesized that frizzled1 (FZD1), a receptor of the canonical Wnt signaling pathway, is a novel mediator of ischemia-associated cardiac hypertrophy. MI was induced in mice by left anterior descending (LAD) coronary occlusion. One week after MI, the expression of FZD1 was found to be notably increased in the left ventricles (LVs) of the MI-mice compared to shams. Mouse recombinant FZD1 protein (RFP) was subcutaneously injected in the mice to provoke autoimmunization response. Anti-FZD1 antibody titer was significantly increased in the plasma of RFP-treated mice. RFP significantly mitigated the MI-induced cardiac hypertrophy and improved cardiac function in the MI mouse hearts. Moreover, increased heart and LV weights, myocardial size and the expression of ?-myosin heavy chain in the MI-mice were also found to be attenuated by RFP. FZD1 was found to be significantly up-regulated in hypoxia-treated neonatal rat cardiomyocytes (NRCMs). Silencing FZD1 by siRNA transfection notably repressed the hypoxia-induced myocardial hypertrophy in NRCMs. Mechanistically, activation of canonical Wnt signaling induced by MI, e.g., ?-catenin and glycogen synthase kinase-3?, was restrained in the LVs of the MI-mice treated by RFP, these inhibition on canonical Wnt signaling was further confirmed in hypoxic NRCMs transfected with FZD1 siRNA. In conclusion, immunization of RFP attenuated cardiac hypertrophy and improved cardiac function in the MI mice via blocking the canonical Wnt signaling pathway.
Project description:BACKGROUND AND PURPOSE: Sirtuin 6 (SIRT6) is involved in regulation of glucose and fat metabolism. However, its possible contribution to cardiac dysfunction remains to be determined. In the present study, the effect of SIRT6 on cardiac hypertrophy induced by angiotensin II (AngII) and the underlying molecular mechanisms were investigated. EXPERIMENTAL APPROACH: The expression and deacetylase activity of SIRT6 were measured in hypertrophic cardiomyocytes induced by AngII. After SIRT6 overexpression by transfection, or depletion by RNA interference in neonatal rat cardiomyocytes, cellular hypertrophy was monitored by measuring cell surface area and the mRNA levels of hypertrophic biomarkers. Further, the interaction between SIRT6 and the transcription factor NF-?B was investigated by co-immunoprecipitation, confocal immunofluorescence microscopy and luciferase reporter gene assay. The expression and deacetylase activity of SIRT6 were measured in vivo, using the abdominal aortic constriction (AAC) model of cardiac hypertrophy in rats. KEY RESULTS: In AngII-induced hypertrophic cardiomyocytes and also in AAC-induced hypertrophic hearts, the expression of SIRT6 protein was upregulated, while its deacetylase activity was decreased. Overexpression of wild-type SIRT6 but not its catalytically inactive mutant, attenuated AngII-induced cardiomyocyte hypertrophy. We further demonstrated a physical interaction between SIRT6 and NF-?B catalytic subunit p65, whose transcriptional activity could be repressed by SIRT6 overexpression. CONCLUSIONS AND IMPLICATIONS: Our findings suggest that SIRT6 suppressed cardiomyocyte hypertrophy in vitro via inhibition of NF-?B-dependent transcriptional activity and that this effect was dependent on its deacetylase activity.
Project description:Cardiac-specific metallothionein (MT) overexpression extends lifespan, but the mechanism underlying the effect of MT protection against age-associated cardiovascular diseases (CVD) remains elusive. To elucidate this, male wild-type and two lines of MT-transgenic (MT-TG) mice, MM and MT-1 (cardiac-specific overexpressing MT about 10- and 80-fold, respectively) at three representative ages (2-3, 9-10, and 18-20 months), were utilized. A stable human MT2A overexpressing cardiomyocytes (H9c2MT7) was also introduced.Histomorphology and echocardiographic analysis revealed that age-associated cardiac hypertrophy, remodeling, and dysfunction were ameliorated in MT-TG mice. Also, aging-accompanied NF-?B activation, characterized by increased nuclear p65 translocation, elevated DNA-binding activity, and upregulation of inflammatory cytokines, was largely attenuated by MT overexpression. Treatment of H9c2 cardiomyocytes with tumor necrosis factor-? (TNF-?), which mimicked an inflammatory environment, significantly increased NF-?B activity, and some age-related phenotypes appeared. The NF-?B activation was further proved to be pivotal for both age-associated and TNF-?-induced nitrative damage to cardiac 2-oxoglutarate dehydrogenase (2-OGD) by virtue of NF-?B p65 gene silencing. MT inhibited NF-?B activation and associated nitrative damage to cardiac 2-OGD in both old MT-TG hearts and TNF-?-treated H9c2MT7 cardiomyocytes; these protective effects were abolished in H9c2MT7 cardiomyocytes by MT-specific gene silencing. Innovation and Conclusion: Together, these findings indicate that the protective effects of MT against age-associated CVD can be attributed mainly to its role in NF-?B inhibition and resultant alleviation of nitrative damage to 2-OGD. Antioxid. Redox Signal. 25, 936-952.
Project description:BACKGROUND:The exact mechanism causing decreased expression of the dual specific phosphatase-1 ( DUSP-1) gene in diabetes-associated cardiac hypertrophy is not known. DNA promoter methylation is often associated with decreased gene expression in many diseases including cardiovascular diseases. So, we investigated whether epigenetic silencing via promoter methylation is involved in the decreased expression of DUSP-1 in diabetes-associated cardiac hypertrophy. METHODS:Real-time polymerase chain reaction (PCR) and Western blotting confirmed the down regulation of the DUSP-1 gene at transcriptional and translational levels. Bisulfite-converted DNA samples from myocardium of rat model of diabetic cardiomyopathy (DCM), high glucose (HG)-treated neonatal rat cardiomyocytes (NRCMs) and cardiac tissues from archived human myocardial DCM autopsies along with their respective controls were analyzed for methylation in the promoter region of the DUSP-1 gene. RESULTS:We observed no methylation in the promoter regions of the DUSP-1 gene in DCM rat hearts, in HG-treated NRCMs (between -355 bp and -174 bp) and in cardiac tissues from archived human myocardial DCM autopsies (between -274 bp and -73 bp). CONCLUSION:Methylation-mediated silencing of the DUSP-1 promoter does not appear to be associated with reduced expression, indicating the involvement of other factors in specific suppression of DUSP-1 in diabetes-associated cardiac hypertrophy.
Project description:Mitogen-activated protein kinase (MAPK) pathways provide a critical connection between extrinsic and intrinsic signals to cardiac hypertrophy. Extracellular signal-regulated protein kinase (ERK)5, an atypical MAPK is activated in the heart by pressure overload. However, the role of ERK5 plays in regulating hypertrophic growth and hypertrophy-induced apoptosis is not completely understood.Herein, we investigate the in vivo role and signaling mechanism whereby ERK5 regulates cardiac hypertrophy and hypertrophy-induced apoptosis.We generated and examined the phenotypes of mice with cardiomyocyte-specific deletion of the erk5 gene (ERK5(cko)). In response to hypertrophic stress, ERK5(cko) mice developed less hypertrophic growth and fibrosis than controls. However, increased apoptosis together with upregulated expression levels of p53 and Bad were observed in the mutant hearts. Consistently, we found that silencing ERK5 expression or specific inhibition of its kinase activity using BIX02189 in neonatal rat cardiomyocytes (NRCMs) reduced myocyte enhancer factor (MEF)2 transcriptional activity and blunted hypertrophic responses. Furthermore, the inhibition of MEF2 activity in NRCMs using a non-DNA binding mutant form of MEF2 was found to attenuate the ERK5-regulated hypertrophic response.These results reveal an important function of ERK5 in cardiac hypertrophic remodeling and cardiomyocyte survival. The role of ERK5 in hypertrophic remodeling is likely to be mediated via the regulation of MEF2 activity.