Kazuo Terai

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.

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.

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.

Effect of plaque debulking before stent implantation on in-stent neointimal proliferation: A serial 3-dimensional intravascular ultrasound study

BACKGROUND Recent intravascular ultrasound (IVUS) studies have suggested that plaque burden has a role in promoting intimal hyperplasia after stenting. We report on volumetric assessments of in-stent neointimal formation with 3-dimensional IVUS analysis, comparing directional coronary atherectomy (DCA) plus stenting (DCA/stenting) to stenting without DCA. METHODS Twenty-four patients (24 lesions) treated with DCA before stenting were matched to 24 patients (24 lesions) receiving stenting without DCA. All stents were a single Multilink stent. In both groups, serial IVUS was performed before and after intervention and during the 6-month follow-up period. The arterial segments that were analyzed with a computer-based contour detection program were the same as the stented segments analyzed on serial studies. These measurements were obtained: (1) lumen volume (LV), (2) stent volume (SV), (3) vessel volume (VV), (4) in-stent neointimal volume (ISV) calculated as SV-LV, and (5) percent in-stent neointimal volume (%ISV) calculated as ([SV-LV]/SV) x 100. RESULTS Baseline characteristics of the 2 groups were similar. After intervention, both groups achieved similar LV (140.0 mm(3) DCA/stenting vs 135.2 mm(3) stenting alone). However, the follow-up ISV and %ISV were significantly smaller in the DCA/stenting group (19.6 +/- 12.2 mm(3) DCA/stenting vs 44.6 +/- 29.5 mm(3) stenting alone; P =.00040; 15.3% +/- 10.6% DCA/stenting vs 31.5% +/- 17.7% stenting alone; P =.00040). Consequently, the DCA/stenting group showed a significantly greater follow-up LV (121.0 +/- 51.5 mm(3) DCA/stenting vs 91.5 +/- 26.7 mm(3) stenting alone; P =.016). CONCLUSIONS Plaque removal with DCA before stenting inhibits in-stent neointimal hyperplasia.

A Single-Center, Open-Label, Randomized, Parallel-Group Study Assessing the Differences Between an Angiotensin II Receptor Antagonist and an Angiotensin-Converting Enzyme Inhibitor in Hypertensive Patients with Congestive Heart Failure: The Research for Efficacy of Angiotensin II Receptor Antagonist in Hypertensive Patients with Congestive Heart Failure Study

BACKGROUND Angiotensin II receptor blockers (ARBs) and angiotensin-converting enzyme inhibitors (ACEIs) have been used to treat congestive heart failure (CHF). According to a MEDLINE search, however, few studies are available on the clinical differences between ARBs and ACEIs in CHF. OBJECTIVE To examine the clinical differences between an ARB (candesartan cilexetil) and an ACEI (lisinopril) in the treatment of CHF, we investigated exercise capacity, ventricular function, and neurohormonal levels in hypertensive patients with CHF before and after treatment with these agents. METHODS Patients with symptoms of CHF (New York Heart Association functional class II-III and left ventricular ejection fraction [LVEF] ≤45%) complicated by hypertension (systolic blood pressure [BP] ≥140 mm Hg or diastolic BP ≥90 mm Hg) were eligible for this single-center, open-label, randomized, parallel-group study. They were given either the ARB or the ACEI for 24 weeks. A cardiopulmonary exercise test and echocardiography were performed. Clinical findings and cardiac events in addition to the CHF symptoms were investigated. Neurohormonal levels were measured before and after 24 weeks of treatment with the study drug. The primary end point of this study was exercise capacity, which was measured using peak oxygen consumption (VO2). RESULTS Forty-two patients with CHF were enrolled and 38 (28 men, 10 women; mean [SD] age, 69.0 [8.2] years) completed the study. None of these patients had definite progression of the CHF symptoms. In the ARB-treated patients, mean (SD) peak VO2 (mL/min/kg) and LVEF (%) increased from 14.1 (2.9) to 15.3 (3.4) and from 34.4 (9.5) to 41.8 (9.5), respectively. In the ACEI group, the peak VO2 did not change, but the LVEF (%) increased from 34.2 (10.2) to 40.4 (13.0). However, the differences between ARB and ACEI were not clarified because of the possibility of a small sample size. CONCLUSIONS Although this study was not powered to show differences in efficacy between the ARB and ACEI in this study, our findings suggest that both ARB and ACEI had beneficial effects in hypertensive patients with CHF. Some unidentified differences in hemodynamic characteristics were found between the ARB and the ACEI groups.