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-␤-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.

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 (AdGab1 WT ), mutated Gab1 lacking SHP2 binding site (AdGab1 F627/659 ), and ␤-galactosidase (Ad␤-gal). Compared with cardiomyocytes infected with Ad␤-gal, longitudinal elongation of cardiomyocytes induced by LIF was enhanced in cardiomyocytes infected with AdGab1WT but inhibited in cardiomyocytes infected with AdGab1 F627/659 . Upregulation of BNP mRNA expression by LIF was evoked in cardiomyocytes infected with Ad␤-gal and AdGab1WT but not in cardiomyocytes infected with AdGab1 F627/659 . In contrast, Gab1 repressed skeletal ␣-actin mRNA expression through interaction with SHP2. Furthermore, activation of extracellular signalregulated kinase 5 (ERK5) was enhanced in cardiomyocytes infected with AdGab1WT compared with cardiomyocytes infected with Ad␤-gal but repressed in cardiomyocytes infected with AdGab1 F627/659 . Coinfection of AdGab1 WT 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. Key Words: hypertrophy Ⅲ Gab1 Ⅲ SHP2 Ⅲ gp130 Ⅲ ERK5 G rb2-associated binder-1 (Gab1) is a member of the Gab family of scaffolding/docking proteins (Gab1, Gab2, and Gab3). 1,2 Gab1 contains a Pleckstrin homology (PH) domain in the amino-terminal region, as well as tyrosinebased motifs and proline-rich sequences, which are potential binding sites for various Src homology (SH) 2 domains and SH3 domains, respectively. 3 Gab1 is tyrosine phosphorylated on stimulation with various growth factors, cytokines, and G protein-coupled receptor (GPCR) agonists. [3][4][5][6][7] Gab1 interacts with multiple signaling molecules, such as protein tyrosine phosphatase SHP2, p85 phosphatidylinositol 3-kinase, phospholipase C-␥, and Grb2. 3-6 Among these, the major binding partner of Gab1 in cells stimulated with growth factors and cytokines is SHP2, a ubiquitously expressed protein tyrosine phosphatase with two SH2 domains. 8 Two tyrosine residues located in the most C-terminal ends of the Gab family proteins have been reported to fall within consensus binding motifs (YXXV/I/L)...

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 ␣-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 ␤-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.

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