Chunlei Liu

Salubrinal protects against tunicamycin and hypoxia induced cardiomyocyte apoptosis via the PERK-eIF2α signaling pathway.

Objectives This study examined the protective effect of salubrinal and the mechanism underlying this protection against tunicamycin (TM)- and hypoxia-induced apoptosis in rat cardiomyocytes. Methods Neonatal rat cardiomyocytes were cultured from the ventricles of 1-day-old Wistar rats. Cells were exposed to different concentrations of salubrinal (10, 20, and 40 µmol/L) for 30 min followed by TM treatment or hypoxia for 36 h. Apoptosis was measured by a multiparameter HCS (high content screening) apoptosis assay, TUNEL assay and flow cytometry. The phosphorylation of eukaryotic translation initiation factor 2 subunit alpha (eIF2α) and the expression of cleaved caspase-12 were determined by Western blotting. C/EBP homologous protein (CHOP) was detected by immunocytochemistry. Results HCS, TUNEL assays and flow cytometry showed that salubrinal protected cardiomyocytes against apoptosis induced by TM or hypoxia. Western blotting showed that salubrinal protected cardiomyocytes against apoptosis by inducing eIF2α phosphorylation and down-regulating the expression of the endoplasmic reticulum stress-mediated apoptotic proteins, CHOP and cleaved caspase-12. Conclusions Our study suggests that salubrinal protects rat cardiomyocytes against TM- or hypoxia-associated apoptosis via a mechanism involving the inhibition of ER stress-mediated apoptosis.

Salubrinal protects cardiomyocytes against apoptosis in a rat myocardial infarction model via suppressing the dephosphorylation of eukaryotic translation initiation factor 2α

The aim of the present study was to examine the role of eIF2α in cardiomyocyte apoptosis and evaluate the cardioprotective role of salubrinal in a rat myocardial infarction (MI) model. Rat left anterior descending coronary arteries were ligated and the classical proteins involved in the endoplasmic reticulum stress (ERS)-induced apoptotic pathway were analyzed using quantitative polymerase chain reaction and western blot analysis. Salubrinal was administered to the rats and cardiomyocyte apoptosis and infarct size were evaluated by a specific staining method. Compared with the sham surgery group, the rate of cardiomyocyte apoptosis in the MI group was increased with the development of the disease. It was also demonstrated that the mRNA and protein levels of GRP78, caspase-12, CHOP and the protein expression of p-eIF2α were increased in the MI group. Furthermore, the results showed that treatment with salubrinal can decrease cardiomyocyte apoptosis and infarct size by increasing eIF2α phosphorylation and decreasing the expression of caspase-12 and CHOP. The present study suggests that salubrinal protects against ER stress-induced rat cadiomyocyte apoptosis via suppressing the dephosphorylation of eIF2α in the ERS-associated pathway.

Inhibition of serine/threonine protein phosphatase PP1 protects cardiomyocytes from tunicamycin-induced apoptosis and I/R through the upregulation of p-eIF2α.

The serine/threonine protein phosphatase PP1 mediates the dephosphorylation of phosphorylated eukaryotic translation initiation factor 2 subunit α (p-eIF2α), which is a central regulator of protein synthesis. In the present study, we examined the protective effects of PP1–12 (an inhibitor of the serine/threonine protein phosphatase PP1) against tunicamycin (TM)-induced apoptosis in cultured cardiomyocytes in vitro, as well as in an in vivo model of ischemia/reperfusion (I/R) injury in rat hearts. Neonatal cardiomyocytes cultured from the ventricles of the hearts of 1-day-old Wistar rats were exposed to various concentrations of PP1–12 (0.3, 1 and 3 μmol/l) for 30 min, followed by treatment with TM for 36 h. Cell viability was assessed by adenosine triphosphate (ATP) bioluminescence, and the results revealed that pre-treatment with PP1–12 protected cell viability. Western blot analysis revealed that PP1–12 induced eIF2α phosphorylation and immuncytochemistry indicated that PP1–12 downregulated the expression of C/EBP homologous protein (CHOP), which is related to apoptosis. PP1–12 suppressed cell apoptosis, with maximum protective effects displayed at the concentration of 3 μmol/l. For the in vivo experiments, male Sprague-Dawley rats were randomly divided into 5 groups: i) sham-operated; ii) vehicle (I/R + DMSO); iii) I/R + 1 mg/kg/day PP1–12; iv) I/R + 3 mg/kg/day PP1–12; and v) I/R + 10 mg/kg/day PP1–12. PP1–12 reduced the expression of cleaved caspase-12 and increased the phosphorylation of eIF2α, as revealed by western blot analysis. By calculating the apoptotic index (AI), we found that 10 mg/kg/day PP1–12 exerted the most pronounced anti-apoptotic effect. The infarction area was significantly decreased following treatment with this concentration of PP1–12, as revealed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Taken together, these data suggest that PP1–12 protects cardiomyocytes from TM- and I/R-induced apoptosis, and this effect is achieved at least in part through the inhibition of cell apoptosis and the induction of eIF2α phosphorylation.

Usefulness of the Neutrophil-to-Lymphocyte Ratio in Predicting Adverse Events in Elderly Patients With Chronic Heart Failure

The neutrophil-to-lymphocyte (N/L) ratio has been associated with poor outcomes in patients with cardiovascular diseases, but it has not been studied in elderly patients with chronic heart failure (CHF).In this study, we analyzed 1355 elderly patients admitted with CHF. A multivariate logistic regression model was used to assess the independent association of the N/L ratio with chronic kidney disease (CKD). The patients were then divided into tertiles according to the N/L ratios. We used Cox regression analysis to assess the association between the N/L ratio and subsequent major cardiovascular events (MCE), including cardiac death and rehospitalization for heart failure.In the multiple logistic regression analysis, the N/L ratio was identified as a risk factor for CKD in elderly patients with CHF (odds ratio [OR] = 1.170, 95% confidence interval [CI] 1.054 to 1.298, P = 0.003). The median follow-up period was 18 months. In a multivariate analysis with the lowest tertile as the reference, the highest tertile of the N/L ratio remained significantly associated with MCE (hazard ratio [HR] = 1.425, 95% CI 1.109 to 1.832, P = 0.006), cardiac death (HR = 1.747, 95% CI 1.032 to 2.958, P = 0.038), and rehospitalization for heart failure (HR = 1.461, 95% CI 1.108 to 1.927, P = 0.007).In elderly patients with CHF, the N/L ratio is one of the important risk factors for CKD and the highest tertile of the N/L ratio is associated with an increased risk for MCE.

Usefulness of S100A12 as a prognostic biomarker for adverse events in patients with heart failure.

OBJECTIVES S100A12 has been proposed as a novel pivotal factor in inflammation produced by granulocytes. The purpose of this study was to investigate the relationship between S100A12 and chronic heart failure (CHF). DESIGN AND METHODS One hundred and seventy-seven patients with CHF and 66 subjects without CHF were included in this study. Plasma levels of S100A12 and high-sensitivity C-reactive protein (hs-CRP) were measured in all participants. After a follow-up period of 18months for CHF patients, major cardiovascular events (MCE), including cardiac death and rehospitalization for heart failure, were recorded. RESULTS Plasma levels of S100A12 were significantly higher in CHF patients than in control subjects (P<0.001) and positively correlated with hs-CRP (r=0.316, P<0.001). S100A12 levels were also higher in MCE patients than in MCE-free patients. The occurrence of MCE increased with advancing plasma S100A12 levels by stratification according to quartiles (Q4 vs Q1, P=0.015). Cox proportional hazards regression analysis revealed that S100A12 was an independent risk factor for MCE in CHF patients (P=0.009). CONCLUSIONS S100A12 is a potential biomarker of CHF that may provide important information regarding the prediction of MCE in patients with CHF.

Effects of PP1-12, a novel protein phosphatase-1 inhibitor, on ventricular function and remodeling after myocardial infarction in rats.

Abstract: PP1-12, a new protein phosphatase-1 inhibitor, is designed and synthesized to modulate the endoplasmic reticulum (ER) stress apoptotic pathway, which is involved in various cardiovascular diseases. In this study, we examined the effect of PP1-12 on ventricular remodeling and heart function after myocardial infarction. Rats that survived within 24 hours after coronary ligation were randomly divided into 6 groups and treated with normal saline, vehicle, PP1-12 at 1, 3, and 10 mg·kg−1·d−1 and perindopril at 2 mg·kg−1·d−1 for 4 weeks, respectively. At the end of the follow-up point, we evaluated echocardiographic and hemodynamic parameters, myocardial pathomorphology, apoptosis, and interstitial fibrosis, as well as the expression levels of important proteins involved in ER stress and apoptosis. Left ventricular geometry and function were ameliorated by PP1-12. PP1-12 inhibited interstitial fibrosis and reduced apoptosis of cardiomyocytes in a dose-dependent manner. PP1-12 decreased GRP78 and caspase-12 expression and increased p-eIF2&agr; and Bcl-2/Bax expression. These results suggest that PP1-12 efficiently inhibits left ventricular remodeling and improves heart function. The mechanism involved may be associated with the ability of PP1-12 to depress myocardial apoptosis induced by ER stress.

Salubrinal attenuates right ventricular hypertrophy and dysfunction in hypoxic pulmonary hypertension of rats.

The phosphorylation of eukaryotic translation initiation factor 2 alpha (p-eIF2α) is essential for cell survival during hypoxia. The aim of this study was to investigate whether salubrinal, an inhibitor of p-eIF2α dephosphorylation could attenuate pulmonary arterial hypertension (PAH) and right ventricular (RV) hypertrophy in rats exposed to hypobaric hypoxia. PAH of rats was induced by hypobaric hypoxia. Salubrinal supplemented was randomized in either a prevention or a reversal protocol. At the end of the follow-up point, we measured echocardiography, hemodynamics, hematoxylin-eosin and Massons trichrome stainings. RNA-seq analysis is explored to identify changes in gene expression associated with hypobaric hypoxia with or without salubrinal. Compared with vehicle-treatment rats exposed to hypobaric hypoxia, salubrinal prevented and partly reversed the increase of the mean pulmonary artery pressure and RV hypertrophy. Whats more, salubrinal reduced the percentage wall thickness (WT%) of pulmonary artery and RV collagen volume fraction (CVF) in both prevention and reversal protocols. We also found that salubrinal was capable of reducing endoplasmic reticulum stress and oxidative stress. The result of RNA-seq analysis revealed that chronic hypoxia stimulated the differential expression of a series of genes involved in cell cycle regulation and ventricular hypertrophy and so on. Some of these genes could be ameliorated by salubrinal. These results indicate that salubrinal could prevent and reverse well-established RV remodeling, and restore the genes and pathways altered in the right ventricles of rats exposed to hypobaric hypoxia.

Sirt3 attenuates post-infarction cardiac injury via inhibiting mitochondrial fission and normalization of AMPK-Drp1 pathways

Mitochondrial damage is involved in the pathogenesis of post-infarction cardiac injury. However, the upstream regulators of mitochondrial damage have not yet been identified. The aim of our study is to explore the role of Sirt3 in post-infarction cardiac injury with a particular focus on mitochondrial fission and AMPK-Drp1 pathways. Our results indicated that Sirt3 was downregulated in the progression of post-infarction cardiac injury. Overexpression of Sirt3 attenuated cardiac fibrosis, sustained myocardial function, inhibited the inflammatory response, and reduced cardiomyocyte death. Functional studies illustrated that chronic post-infarction cardiac injury was characterized by increased mitochondrial fission, which triggered mitochondrial oxidative stress, metabolic disorders, mitochondrial potential reduction and caspase-9 apoptosis in cardiomyocytes. However, Sirt3 overexpression attenuated mitochondrial fission and thus preserved mitochondrial homeostasis and cardiomyocyte viability. Furthermore, our results confirmed that Sirt3 repressed mitochondrial fission via normalizing AMPK-Drp1 pathways. Inhibition of AMPK activity re-activated Drp1 and thus abrogated the inhibitory effect of Sirt3 on mitochondrial fission. Altogether, our results indicate that Sirt3 enhancement could be an effective approach to retard the development of post-infarction cardiac injury via disrupting mitochondrial fission and normalizing the AMPK-Drp1 axis.

High-content screening identifies inhibitors of the nuclear translocation of ATF6.

Activating transcription factor 6 (ATF6) is a transmembrane protein that consists of a cytoplasmic domain and an endoplasmic reticulum (ER) luminal domain. As unfolded protein levels arise in the ER, the ER cytoplasmic domain of ATF6 moves to the nucleus, where it activates the transcription of a range of genes, including those involved in apoptosis. As ATF6 only becomes functional once it has moved to the nucleus, compounds that inhibit its re-localization are of therapeutic interest. The aim of the present study was to rapidly and accurately identify such compounds using a novel image‑based, high‑content screening (HCS) technique. The results from the HCS analysis were then confirmed by luciferase reporter assays, western blot analysis and the measurement of cell viability. We found that HCS identified compounds which inhibited ATF6 nuclear translocation with high specificity, as confirmed by the luciferase reporter assay and western blot analysis. Moreover, we demonstrated that 3 of the 80 identified compounds impaired ATF6-mediated induced cell death. The data from this study support the theory that HCS is a novel, high throughput method which can be used for accurate and rapid compound screening.