Hisao Hirota

AMP-activated protein kinase protects cardiomyocytes against hypoxic injury through attenuation of endoplasmic reticulum stress

ABSTRACT Oxygen deprivation leads to the accumulation of misfolded proteins in the endoplasmic reticulum (ER), causing ER stress. Under conditions of ER stress, inhibition of protein synthesis and up-regulation of ER chaperone expression reduce the misfolded proteins in the ER. AMP-activated protein kinase (AMPK) is a key regulatory enzyme involved in energy homeostasis during hypoxia. It has been shown that AMPK activation is associated with inhibition of protein synthesis via phosphorylation of elongation factor 2 (eEF2) in cardiomyocytes. We therefore examined whether AMPK attenuates hypoxia-induced ER stress in neonatal rat cardiomyocytes. We found that hypoxia induced ER stress, as assessed by the expression of CHOP and BiP and cleavage of caspase 12. Knockdown of CHOP or caspase 12 through small interfering RNA (siRNA) resulted in decreased expression of cleaved poly(ADP-ribose) polymerase following exposure to hypoxia. We also found that hypoxia-induced CHOP expression and cleavage of caspase 12 were significantly inhibited by pretreatment with 5-aminoimidazole-4-carboxyamide-1-β-d-ribofuranoside (AICAR), a pharmacological activator of AMPK. In parallel, adenovirus expressing dominant-negative AMPK significantly attenuated the cardioprotective effects of AICAR. Knockdown of eEF2 phosphorylation using eEF2 kinase siRNA abolished these cardioprotective effects of AICAR. Taken together, these findings demonstrate that activation of AMPK contributes to protection of the heart against hypoxic injury through attenuation of ER stress and that attenuation of protein synthesis via eEF2 inactivation may be the mechanism of cardioprotection by AMPK.

Signals through gp130 upregulate bcl-x gene expression via STAT1-binding cis-element in cardiac myocytes.

We described recently the activation of the Janus kinasesignal transducer and activator of transcription (JakSTAT) and mitogen-activated protein (MAP) kinase pathways by leukemia inhibitory factor (LIF) through gp130, a signal transducer of IL-6-related cytokines, that transduces hypertrophic signals in cardiac myocytes. In addition, stimulation of gp130 by IL-6-related cytokines is known to exert a cytoprotective effect. In the present study, we investigated the possibility that activation of gp130 initiates activation of the cytoprotective genes in cardiac myocytes. Incubation of cardiac myocytes with LIF induced the expression of bcl-x, and the isoform that was induced by LIF was identified as bcl-xL. Induction of bcl-xL protein was also identified by Western blotting. Antisense oligonucleotide against bcl-x mRNA inhibited protective effect of LIF accompanied with the reduction in bclxL protein. We constructed bcl-x promoter-luciferase reporter gene plasmids (-639/+10- or -161/+10-luciferase), and transfected them to cardiac myocytes. LIF stimulation increased the luciferase activity of -639/+10-luciferase plasmids. Although -161/+10-luciferase plasmids presented comparable responsiveness to LIF, the basal transcription level was impaired. The LIF-responsive cis-element was localized to a DNA fragment (positions -161 to +10) that contains an interferon-gamma activation site (GAS) motif (GGA) at position -41 of the bcl-x gene promoter. This motif bound to STAT1, not to STAT3, and site-directed mutagenesis revealed that this motif was essential for LIF-responsive promoter activity. These data suggest that LIF induces bcl-x mRNA via STAT1 binding cis-element in cardiac myocytes, presenting cytoprotective effect.

Activation of JAK-STAT and MAP Kinases by Leukemia Inhibitory Factor Through gp130 in Cardiac Myocytes

BACKGROUND Interleukin (IL)-6-related cytokines share gp130 as the signal-transducing protein. Downstream of gp130, two signal-transducing pathways have been recognized, the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway and the Ras-mitogen-activated protein kinase (MAPK) pathway. To determine whether these two signaling pathways through gp130 are present in cardiac myocytes, we examined their activation by using leukemia inhibitory factor (LIF), which is a member of the IL-6 cytokine family. METHODS AND RESULTS Lysates from neonatal rat cardiac myocytes were immunoprecipitated with anti-gp130, anti-JAK1, or anti-STAT3 antibody and blotted with anti-phosphotyrosine antibody. Tyrosine phosphorylation of gp130, JAK1, and STAT3 was observed after LIF stimulation in cardiac myocytes. MAPKs were maximally activated 5 minutes after LIF stimulation. Furthermore, anti-gp130 antibody significantly inhibited the LIF-induced activation of JAK1, STAT3, and MAPKs. To examine whether these signaling pathways were also activated in the adult heart in vivo, LIF was injected intravenously into a 6-week-old mouse, and the heart was examined subsequently. gp130, STAT3, and MAPKs were activated in the heart after LIF treatment. CONCLUSIONS These results demonstrate for the first time that a JAK-STAT pathway and a MAPK pathway are present down-stream of gp130 in cardiac myocytes and are rapidly activated by LIF both in vitro and in vivo. Activation of gp130 constitutes a novel signaling pathway in cardiac myocytes.

Activation of gp130 Transduces Hypertrophic Signal Through Interaction of Scaffolding/Docking Protein Gab1 With Tyrosine Phosphatase SHP2 in Cardiomyocytes

Abstract— Grb2-associated binder-1 (Gab1) is a scaffolding/docking protein and contains a Pleckstrin homology domain and potential binding sites for Src homology (SH) 2 and SH3 domains. Gab1 is tyrosine phosphorylated and associates with protein tyrosine phosphatase SHP2 and p85 phosphatidylinositol 3-kinase on stimulation with various cytokines and growth factors, including interleukin-6. We previously demonstrated that interleukin-6–related cytokine, leukemia inhibitory factor (LIF), induced cardiac hypertrophy through gp130. In this study, we report the role of Gab1 in gp130-mediated cardiac hypertrophy. Stimulation with LIF induced tyrosine phosphorylation of Gab1, and phosphorylated Gab1 interacted with SHP2 and p85 in cultured cardiomyocytes. We constructed three kinds of adenovirus vectors, those carrying wild-type Gab1 (AdGab1WT), mutated Gab1 lacking SHP2 binding site (AdGab1F627/659), and &bgr;-galactosidase (Ad&bgr;-gal). Compared with cardiomyocytes infected with Ad&bgr;-gal, longitudinal elongation of cardiomyocytes induced by LIF was enhanced in cardiomyocytes infected with AdGab1WT but inhibited in cardiomyocytes infected with AdGab1F627/659. Upregulation of BNP mRNA expression by LIF was evoked in cardiomyocytes infected with Ad&bgr;-gal and AdGab1WT but not in cardiomyocytes infected with AdGab1F627/659. In contrast, Gab1 repressed skeletal &agr;-actin mRNA expression through interaction with SHP2. Furthermore, activation of extracellular signal–regulated kinase 5 (ERK5) was enhanced in cardiomyocytes infected with AdGab1WT compared with cardiomyocytes infected with Ad&bgr;-gal but repressed in cardiomyocytes infected with AdGab1F627/659. Coinfection of AdGab1WT with adenovirus vector carrying dominant-negative ERK5 abrogated longitudinal elongation of cardiomyocytes induced by LIF. Taken together, these findings indicate that Gab1-SHP2 interaction plays a crucial role in gp130-dependent longitudinal elongation of cardiomyoctes through activation of ERK5.

Circulating interleukin-6 family cytokines and their receptors in patients with congestive heart failure.

Abstractgp130 is a common signal-transducing receptor subunit for the interleukin (IL)-6 cytokine family. Studies in genetically engineered animal models have demonstrated a critical role for the gp130-dependent cardiomyocyte survival pathway in the transition to heart failure. In the present study, we examined plasma levels of the IL-6 family of cytokines and the soluble form of their receptors in patients with congestive heart failure (CHF). Circulating levels of the IL-6 family of cytokines, soluble IL-6 receptor (sIL-6R), and soluble gp130 (sgp130) were examined in 48 patients with various degrees of CHF, including dilated cardiomyopathy (DCM), ischemic cardiomyopathy (ICM), and valvular cardiomyopathy (VCM). Circulating levels of IL-6, leukemia inhibitory factor (LIF), and sgp130 significantly increased in association with the severity of CHF. No significant difference was observed in the circulating levels of sIL-6R and IL-11 among these patients. Interestingly, DCM patients showed higher circulating sgp130 levels than patients with ICM or VCM. Our findings suggest that gp130 expression in the heart is likely to be dynamic, and that the IL-6 family of cytokines and their common receptor gp130 participates in the pathogenesis of CHF, especially in DCM.

Gab family proteins are essential for postnatal maintenance of cardiac function via neuregulin-1/ErbB signaling

Grb2-associated binder (Gab) family of scaffolding adaptor proteins coordinate signaling cascades downstream of growth factor and cytokine receptors. In the heart, among EGF family members, neuregulin-1beta (NRG-1beta, a paracrine factor produced from endothelium) induced remarkable tyrosine phosphorylation of Gab1 and Gab2 via erythroblastic leukemia viral oncogene (ErbB) receptors. We examined the role of Gab family proteins in NRG-1beta/ErbB-mediated signal in the heart by creating cardiomyocyte-specific Gab1/Gab2 double knockout mice (DKO mice). Although DKO mice were viable, they exhibited marked ventricular dilatation and reduced contractility with aging. DKO mice showed high mortality after birth because of heart failure. In addition, we noticed remarkable endocardial fibroelastosis and increase of abnormally dilated vessels in the ventricles of DKO mice. NRG-1beta induced activation of both ERK and AKT in the hearts of control mice but not in those of DKO mice. Using DNA microarray analysis, we found that stimulation with NRG-1beta upregulated expression of an endothelium-stabilizing factor, angiopoietin 1, in the hearts of control mice but not in those of DKO mice, which accounted for the pathological abnormalities in the DKO hearts. Taken together, our observations indicated that in the NRG-1beta/ErbB signaling, Gab1 and Gab2 of the myocardium are essential for both maintenance of myocardial function and stabilization of cardiac capillary and endocardial endothelium in the postnatal heart.

Smad1 Protects Cardiomyocytes From Ischemia-Reperfusion Injury

Background—We previously reported that bone morphogenetic protein 2 (BMP2) protected against apoptosis of serum-deprived cardiomyocytes via induction of Bcl-xL through the Smad1 pathway. To investigate whether Smad1 signaling promotes cell survival in the adult heart, we subjected transgenic mice with cardiac-specific overexpression of smad1 gene (Smad1TG) to ischemia-reperfusion (I/R) injury. Methods and Results—The effects of BMP2 or adenovirus-mediated transfection of smad1 on cardiomyocyte survival in hypoxia-reoxygenation were examined using rat neonatal cardiomyocytes. BMP2 and Smad1 each significantly promoted survival and diminished apoptotic death of cardiomyocytes during hypoxia-reoxygenation. Interestingly, Smad1 was found to be activated during I/R in normal mouse heart. To examine physiological and pathological roles of Smad1 in I/R, we generated Smad1TG using the α-myosin heavy chain gene promoter. Phosphorylation of Smad1 was found in all smad1 transgene–positive mouse hearts. To examine whether Smad1 prevents injury of cardiomyocytes in vivo, we subjected Smad1TG and age-matched wild-type mice (WT) to I/R injury induced by 1 hour of ligation of the left coronary artery and 1 hour of reperfusion. TUNEL and DNA ladder analyses showed that Smad1TG had significantly smaller myocardial infarctions and fewer apoptotic deaths of cardiomyocytes than did WT. Interestingly, increased expression of Bcl-xL and β-catenin was more remarkable whereas caspase3 was less activated in Smad1TG heart than in that of WT. Conclusions—These findings suggest that the Smad1 signaling pathway plays a role in cardioprotection against I/R injury.

Bcl-xl reduces doxorubicin-induced myocardial damage but fails to control cardiac gene downregulation

OBJECTIVE We recently reported that doxorubicin (Dox), an effective anti-cancer drug, induces apoptosis in cardiac myocytes in association with reduction of Bcl-xl expression. In the present study, we further examined whether overexpression of Bcl-xl ameliorates Dox-induced cardiac myocyte damage. METHODS AND RESULTS Overexpression of the Bcl-xl gene by adenovirus vector resulted in an 11-fold increase in Bcl-xl protein in neonatal rat cardiac myocytes (BCL) compared to that in cells with beta-galactosidase gene transfection (CTL). Although Dox treatment generated similar amounts of reactive oxygen species (ROS) in BCL and CTL, cell viability was maintained and the number of apoptotic cardiac myocytes was significantly decreased in BCL. Cytochrome c release and enhanced caspase-3 activity after Dox treatment were significantly suppressed and Bax expression level was decreased in BCL. Cardiac-specific gene expression is known to be inhibited by Dox. The expression of cardiac alpha-actin and sarcoplasmic reticulum Ca(2+)-ATPase 2a mRNA was equally inhibited in BCL and CTL after Dox treatment. CONCLUSIONS Overexpression of Bcl-xl in cardiac myocytes failed to regulate Dox-induced ROS generation and cardiac-specific gene downregulation but inhibited apoptosis accompanied by reduction of Bax protein.

Interaction of scaffolding adaptor protein Gab1 with tyrosine phosphatase SHP2 negatively regulates IGF-I-dependent myogenic differentiation via the ERK1/2 signaling pathway.

Grb2-associated binder 1 (Gab1) coordinates various receptor tyrosine kinase signaling pathways. Although skeletal muscle differentiation is regulated by some growth factors, it remains elusive whether Gab1 coordinates myogenic signals. Here, we examined the molecular mechanism of insulin-like growth factor-I (IGF-I)-mediated myogenic differentiation, focusing on Gab1 and its downstream signaling. Gab1 underwent tyrosine phosphorylation and subsequent complex formation with protein-tyrosine phosphatase SHP2 upon IGF-I stimulation in C2C12 myoblasts. On the other hand, Gab1 constitutively associated with phosphatidylinositol 3-kinase regulatory subunit p85. To delineate the role of Gab1 in IGF-I-dependent signaling, we examined the effect of adenovirus-mediated forced expression of wild-type Gab1 (Gab1WT), mutated Gab1 that is unable to bind SHP2 (Gab1ΔSHP2), or mutated Gab1 that is unable to bind p85 (Gab1Δp85), on the differentiation of C2C12 myoblasts. IGF-I-induced myogenic differentiation was enhanced in myoblasts overexpressing Gab1ΔSHP2, but inhibited in those overexpressing either Gab1WT or Gab1Δp85. Conversely, IGF-I-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation was significantly repressed in myoblasts overexpressing Gab1ΔSHP2 but enhanced in those overexpressing either Gab1WT or Gab1Δp85. Furthermore, small interference RNA-mediated Gab1 knockdown enhanced myogenic differentiation. Overexpression of catalytic-inactive SHP2 modulated IGF-I-induced myogenic differentiation and ERK1/2 activation similarly to that of Gab1ΔSHP2, suggesting that Gab1-SHP2 complex inhibits IGF-I-dependent myogenesis through ERK1/2. Consistently, the blockade of ERK1/2 pathway reversed the inhibitory effect of Gab1WT overexpression on myogenic differentiation, and constitutive activation of the ERK1/2 pathway suppressed the enhanced myogenic differentiation by overexpression of Gab1ΔSHP2. Collectively, these data suggest that the Gab1-SHP2-ERK1/2 signaling pathway comprises an inhibitory axis for IGF-I-dependent myogenic differentiation.

Aldosterone augments endothelin-1-induced cardiac myocyte hypertrophy with the reinforcement of the JNK pathway

Aldosterone is thought to regulate cardiac work independently of sodium retention, though the mechanisms remain to be known. In the present study, we have demonstrated that aldosterone reinforces endothelin‐mediated cardiac hypertrophy with the increase in cell surface area and upregulation of the transcripts characteristic of hypertrophy. We have also shown that aldosterone augments c‐Jun N‐terminal kinase activation induced by endothelin‐1. Taken together, it is suggested that aldosterone modulates cardiac hypertrophy, at least partially, synergistically with extracellular signals that have been shown to be involved in cardiac remodeling.