Difei Shen

Lysosomal cysteine peptidase cathepsin L protects against cardiac hypertrophy through blocking AKT/GSK3β signaling

The lysosomal cysteine peptidase cathepsin L (CTSL) is an important lysosomal proteinase involved in a variety of cellular functions including intracellular protein turnover, epidermal homeostasis, and hair development. Deficiency of CTSL in mice results in a progressive dilated cardiomyopathy. In the present study, we tested the hypothesis that cardiac overexpression of human CTSL in the murine heart would protect against cardiac hypertrophy in vivo. The effects of constitutive human CTSL expression on cardiac hypertrophy were investigated using in vitro and in vivo models. Cardiac hypertrophy was produced by aortic banding (AB) in CTSL transgenic mice and control animals. The extent of cardiac hypertrophy was quantitated by two-dimensional and M-mode echocardiography as well as by molecular and pathological analyses of heart samples. Constitutive overexpression of human CTSL in the murine heart attenuated the hypertrophic response, markedly reduced apoptosis, and fibrosis. Cardiac function was also preserved in hearts with increased CTSL levels in response to hypertrophic stimuli. These beneficial effects were associated with attenuation of the Akt/GSK3β signaling cascade. Our in vitro studies further confirmed that CTSL expression in cardiomyocytes blunts cardiac hypertrophy through blocking of Akt/GSK3β signaling. The study indicates that CTSL improves cardiac function and inhibits cardiac hypertrophy, inflammation, and fibrosis through blocking Akt/GSK3β signaling.

Activating transcription factor 3 deficiency promotes cardiac hypertrophy, dysfunction, and fibrosis induced by pressure overload.

Activating transcription factor 3 (ATF3), which is encoded by an adaptive-response gene induced by various stimuli, plays an important role in the cardiovascular system. However, the effect of ATF3 on cardiac hypertrophy induced by a pathological stimulus has not been determined. Here, we investigated the effects of ATF3 deficiency on cardiac hypertrophy using in vitro and in vivo models. Aortic banding (AB) was performed to induce cardiac hypertrophy in mice. Cardiac hypertrophy was estimated by echocardiographic and hemodynamic measurements and by pathological and molecular analysis. ATF3 deficiency promoted cardiac hypertrophy, dysfunction and fibrosis after 4 weeks of AB compared to the wild type (WT) mice. Furthermore, enhanced activation of the MEK-ERK1/2 and JNK pathways was found in ATF3-knockout (KO) mice compared to WT mice. In vitro studies performed in cultured neonatal mouse cardiomyocytes confirmed that ATF3 deficiency promotes cardiomyocyte hypertrophy induced by angiotensin II, which was associated with the amplification of MEK-ERK1/2 and JNK signaling. Our results suggested that ATF3 plays a crucial role in the development of cardiac hypertrophy via negative regulation of the MEK-ERK1/2 and JNK pathways.

Cellular repressor of E1A-stimulated genes attenuates cardiac hypertrophy and fibrosis.

Cellular repressor of E1A‐stimulated genes (CREG) is a secreted glycoprotein of 220 amino acids. It has been proposed that CREG acts as a ligand that enhances differentiation and/or reduces cell proliferation. CREG has been shown previously to attenuate cardiac hypertrophy in vitro. However, such a role has not been determined in vivo. In the present study, we tested the hypothesis that overexpression of CREG in the murine heart would protect against cardiac hypertrophy and fibrosis in vivo. The effects of constitutive human CREG expression on cardiac hypertrophy were investigated using both in vitro and in vivo models. Cardiac hypertrophy was produced by aortic banding and infusion of angiotensin II in CREG transgenic mice and control animals. The extent of cardiac hypertrophy was quantitated by two‐dimensional and M‐mode echocardiography as well as by molecular and pathological analyses of heart samples. Constitutive over‐expression of human CREG in the murine heart attenuated the hypertrophic response, markedly reduced inflammation. Cardiac function was also preserved in hearts with increased CREG levels in response to hypertrophic stimuli. These beneficial effects were associated with attenuation of the mitogen‐activated protein kinase (MAPK)‐extracellular signal‐regulated kinase 1 (MEK‐ERK1)/2‐dependent signalling cascade. In addition, CREG expression blocked fibrosis and collagen synthesis through blocking MEK‐ERK1/2‐dependent Smad 2/3 activation in vitro and in vivo. Therefore, the expression of CREG improves cardiac functions and inhibits cardiac hypertrophy, inflammation and fibrosis through blocking MEK‐ERK1/2‐dependent signalling.

LIM and Cysteine-Rich Domains 1 Regulates Cardiac Hypertrophy by Targeting Calcineurin/Nuclear Factor of Activated T Cells Signaling

LIM domain proteins are important regulators in cell growth, cell fate determination, cell differentiation, and remodeling of the cell cytoskeleton. LIM and cysteine-rich domains 1 (Lmcd1) is a novel protein that contain 2 LIM domains with regular spacing in the carboxy-terminal region. However, its roles in cardiac growth remain unknown. Here, we investigated whether Lmcd1 regulates cardiac hypertrophy in vitro and in vivo and elucidated the underlying molecular mechanisms. We used primary cultured cardiac myocytes and cardiac-specific Lmcd1 transgenic mice. In wild-type mice subjected to the aortic banding, cardiac hypertrophy was evident at 8 weeks. In transgenic mice, however, cardiac hypertrophy was significantly greater than that in wild-type mice, as estimated by heart weight:body weight ratio, cardiomyocyte area, and echocardiographic measurements, as well as cardiac atrial natriuretic peptide and B-type natriuretic peptide mRNA and protein levels. Our results further showed that cardiac fibrosis observed in wild-type aortic banding mice was augmented in transgenic aortic banding mice. Importantly, calcineurin activity and nuclear factor of activated T cells activation level were increased more in transgenic mice than those in wild-type mice after 8-week aortic banding. In vitro experiments in cardiac myocytes further revealed that angiotensin II–induced calcineurin activity and nuclear factor of activated T cells activation were enhanced by overexpression but blunted by downregulation of Lmcd1. In conclusion, our results suggest that Lmcd1 plays a critical role in the development of cardiac hypertrophy via activation of calcineurin/nuclear factor of activated T cells signaling pathway.

Tetrandrine blocks cardiac hypertrophy by disrupting reactive oxygen species-dependent ERK1/2 signalling.

Background and purpose:  Tetrandrine, a well‐known naturally occurring calcium antagonist with anti‐inflammatory, antioxidant and anti‐fibrogenetic activities, has long been used clinically for treatment of cardiovascular diseases such as hypertension and arrhythmia. However, little is known about the effect of tetrandrine on cardiac hypertrophy. The aims of the present study were to determine whether tetrandrine could attenuate cardiac hypertrophy and to clarify the underlying molecular mechanisms.

Loss of Regulator of G Protein Signaling 5 Exacerbates Obesity, Hepatic Steatosis, Inflammation and Insulin Resistance

Background The effect of regulator of G protein signaling 5 (RGS5) on cardiac hypertrophy, atherosclerosis and angiogenesis has been well demonstrated, but the role in the development of obesity and insulin resistance remains completely unknown. We determined the effect of RGS5 deficiency on obesity, hepatic steatosis, inflammation and insulin resistance in mice fed either a normal-chow diet (NC) or a high-fat diet (HF). Methodology/Principal Findings Male, 8-week-old RGS5 knockout (KO) and littermate control mice were fed an NC or an HF for 24 weeks and were phenotyped accordingly. RGS5 KO mice exhibited increased obesity, fat mass and ectopic lipid deposition in the liver compared with littermate control mice, regardless of diet. When fed an HF, RGS5 KO mice had a markedly exacerbated metabolic dysfunction and inflammatory state in the blood serum. Meanwhile, macrophage recruitment and inflammation were increased and these increases were associated with the significant activation of JNK, IκBα and NF-κBp65 in the adipose tissue, liver and skeletal muscle of RGS5 KO mice fed an HF relative to control mice. These exacerbated metabolic dysfunction and inflammation are accompanied with decreased systemic insulin sensitivity in the adipose tissue, liver and skeletal muscle of RGS5 KO mice, reflected by weakened Akt/GSK3β phosphorylation. Conclusions/Significance Our data suggest that loss of RGS5 exacerbates HF-induced obesity, hepatic steatosis, inflammation and insulin resistance.

Stem Cell Antigen 1 Protects Against Cardiac Hypertrophy and Fibrosis After Pressure Overload

Stem cell antigen (Sca) 1, a glycosyl phosphatidylinositol-anchored protein localized to lipid rafts, is upregulated in the heart during myocardial infarction and renovascular hypertension-induced cardiac hypertrophy. It has been suggested that Sca-1 plays an important role in myocardial infarction. To investigate the role of Sca-1 in cardiac hypertrophy, we performed aortic banding in Sca-1 cardiac-specific transgenic mice, Sca-1 knockout mice, and their wild-type littermates. Cardiac hypertrophy was evaluated by echocardiographic, hemodynamic, pathological, and molecular analyses. Sca-1 expression was upregulated and detected in cardiomyocytes after aortic banding surgery in wild-type mice. Sca-1 transgenic mice exhibited significantly attenuated cardiac hypertrophy and fibrosis and preserved cardiac function compared with wild-type mice after 4 weeks of aortic banding. Conversely, Sca-1 knockout dramatically worsened cardiac hypertrophy, fibrosis, and dysfunction after pressure overload. Furthermore, aortic banding–induced activation of Src, mitogen-activated protein kinases, and Akt was blunted by Sca-1 overexpression and enhanced by Sca-1 deficiency. Our results suggest that Sca-1 protects against cardiac hypertrophy and fibrosis via regulation of multiple pathways in cardiomyocytes.

Disruption of Tumor Necrosis Factor Receptor Associated Factor 5 Exacerbates Pressure Overload Cardiac Hypertrophy and Fibrosis

The cytoplasmic signaling protein tumor necrosis factor (TNF) receptor‐associated factor 5 (TRAF5), which was identified as a signal transducer for members of the TNF receptor super‐family, has been implicated in several biological functions in T/B lymphocytes and the innate immune response against viral infection. However, the role of TRAF5 in cardiac hypertrophy has not been reported. In the present study, we investigated the effect of TRAF5 on the development of pathological cardiac hypertrophy induced by transthoracic aorta constriction (TAC) and further explored the underlying molecular mechanisms. Cardiac hypertrophy and function were evaluated with echocardiography, hemodynamic measurements, pathological and molecular analyses. For the first time, we found that TRAF5 deficiency substantially aggravated cardiac hypertrophy, cardiac dysfunction and fibrosis in response to pressure overload after 4 weeks of TAC compared to wild‐type (WT) mice. Moreover, the mitogen‐activated protein/extracellular signal‐regulated kinase kinase (MEK)‐extracellular signal‐regulated kinases 1/2 (ERK1/2) signaling pathway was more activated in TRAF5‐deficient mice than WT mice. In conclusion, our results suggest that as an intrinsic cardioprotective factor, TRAF5 plays a crucial role in the development of cardiac hypertrophy through the negative regulation of the MEK‐ERK1/2 pathway. J. Cell. Biochem. 115: 349–358, 2014.

IKKi Deficiency Promotes Pressure Overload-Induced Cardiac Hypertrophy and Fibrosis

The inducible IκB kinase (IKKi/IKKε) is a recently described serine-threonine IKK-related kinase. Previous studies have reported the role of IKKi in infectious diseases and cancer. However, its role in the cardiac response to pressure overload remains elusive. In this study, we investigated the effects of IKKi deficiency on the development of pathological cardiac hypertrophy using in vitro and in vivo models. First, we developed mouse models of pressure overload cardiac hypertrophy induced by pressure overload using aortic banding (AB). Four weeks after AB, cardiac function was then assessed through echocardiographic and hemodynamic measurements. Western blotting, real-time PCR and histological analyses were used to assess the pathological and molecular mechanisms. We observed that IKKi-deficient mice showed significantly enhanced cardiac hypertrophy, cardiac dysfunction, apoptosis and fibrosis compared with WT mice. Furthermore, we recently revealed that the IKKi-deficient mice spontaneously develop cardiac hypertrophy. Moreover, in vivo experiments showed that IKKi deficiency-induced cardiac hypertrophy was associated with the activation of the AKT and NF-κB signaling pathway in response to AB. In cultured cells, IKKi overexpression suppressed the activation of this pathway. In conclusion, we demonstrate that IKKi deficiency exacerbates cardiac hypertrophy by regulating the AKT and NF-κB signaling pathway.

Mnk1 (Mitogen-Activated Protein Kinase–Interacting Kinase 1) Deficiency Aggravates Cardiac Remodeling in Mice

Identifying the key factor involved in cardiac remodeling is critically important for developing novel strategies to protect against heart failure. Here, the role of Mnk1 (mitogen-activated protein kinase–interacting kinase 1) in cardiac remodeling was clarified. Cardiac remodeling was induced by transverse aortic constriction in Mnk1-knockout mice and their wild-type control mice. After 4 weeks of transverse aortic constriction, Mnk1-knockout mice developed exaggerated cardiac hypertrophy, fibrosis, dysfunction, and cardiomyocyte apoptosis and showed increased ERK1/2 (extracellular signal–regulated kinase 1/2) activation along with reduced sprouty2 expression. In line with the in vivo studies, Mnk1 knockdown by Mnk1 siRNA transfection induced exaggerated angiotensin II–induced cardiomyocyte hypertrophy in neonatal rat ventricular myocytes (NRVMs). Moreover, adenovirus-mediated overexpression of Mnk1 in NRVMs protected cardiomyocytes from angiotensin II–induced hypertrophy. In addition, overexpression of sprouty2 rescued NRVMs with Mnk1 knockdown from angiotensin II–induced hypertrophy. In accordance with the in vivo studies, as compared with the control group, Mnk1 knockdown led to hyperphosphorylation of ERK1/2 and suppression of the sprouty2 expression in angiotensin II–treated NRVMs; furthermore, Mnk1 overexpression led to hypophosphorylation of ERK1/2 in angiotensin II–treated NRVMs. In addition, sprouty2 overexpression suppressed the activation of ERK1/2 in angiotensin II–treated NRVMs with Mnk1 knockdown. Impressively, MnK1-knockout mice with overexpression of sprouty2 exhibited signs of a blunted cardiac hypertrophic response. Mnk1 likely carries out a suppressive function in cardiac hypertrophy via regulating the sprouty2/ERK1/2 pathway. It implicates Mnk1 in the development of cardiac remodeling.