Myofibrillogenesis regulator-1 attenuated hypoxia/reoxygenation-induced apoptosis by inhibiting the PERK/Nrf2 pathway in neonatal rat cardiomyocytes.

The purpose of this study was to investigate the role of myofibrillogenesis regulator-1 (MR-1) in cardiomyocyte apoptosis induced by hypoxia/reoxygenation (H/R), through protein kinase R-like ER kinase (PERK)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. To address this aim, an H/R model of neonatal rat cardiomyocytes was used. MR-1 was overexpressed using an adenoviral vector system and knocked down using MR-1 specific siRNA. Apoptosis was assessed by using Annexin V/PI double staining, terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling assay, and the Bcl-2/Bax ratio. Western blotting was used to detect the protein levels of MR-1, glucose-regulated protein 78 (GRP78), total and phosphorylated PERK, Nrf2, activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), Bcl-2 and Bax. Immunofluorescence staining was used to assess the subcellular location of Nrf2. We found that H/R induced significant apoptosis in neonatal rat cardiomyocytes. MR-1 overexpression attenuated H/R-induced apoptosis, decreased GRP78 (P < 0.01) and CHOP expression (P < 0.05), and increased the Bcl-2/Bax ratio (P < 0.01). MR-1 overexpression suppressed H/R-induced PERK phosphorylation, Nrf2 nuclear translocation, and ATF4 expression (P < 0.01). While MR-1 knockdown aggravated H/R-induced apoptosis, increased expression of GRP78 and CHOP (P < 0.05), and decreased the Bcl-2/Bax ratio (P < 0.01). MR-1 knockdown significantly increased H/R-induced PERK phosphorylation (P < 0.05), Nrf2 nuclear translocation, and ATF4 expression (P < 0.01). These findings suggest that MR-1 alleviates H/R-induced cardiomyocyte apoptosis through inhibition of the PERK/Nrf2 pathway.

Panax quinquefolium saponin attenuates cardiomyocyte apoptosis and opening of the mitochondrial permeability transition pore in a rat model of ischemia/reperfusion.

AIMS: Opening of the mitochondrial permeability transition pore (mPTP) is a critical event during ischemia/reperfusion (I/R) injury. Recently, we showed that Panax quinquefolium saponin (PQS) alleviates apoptosis of cardiomyocytes by suppressing excessive endoplasmic reticulum stress (ERS) during I/R injury. Here, we hypothesized that this anti-apoptotic effect might be mediated through inhibition of mPTP and the mitochondrial apoptotic pathway. METHODS: Ninety-six healthy male Sprague-Dawley rats were randomly divided into sham, I/R, I/R+PQS (200 mg/kg/d), Cyclosporine A (CsA, 10 mg/kg), I/R+CsA (10 mg/kg), and I/R+PQS+CsA. I/R was modeled in rats by ligating the left anterior descending artery (LAD) for 30 min followed by 120 min of reperfusion. To evaluate the cardioprotective function of PQS, we measured hemodynamics, serum content of creatine kinase-MB (CK-MB), myocardial infarct size, and myocardial apoptotic index (AI). We investigated the underlying mechanism by examining changes in the mitochondrial ultrastructure and membrane potential (ΔΨm), dynamics of mPTP opening, expression of cleaved caspase-3, cleaved caspase-9 in the myocardium, Bcl-2 and Bax in the mitochondria versus cytosol, and translocation of cytochrome c. RESULTS: Administration of PQS to I/R rats significantly reduced serum CK-MB level, infarct size and AI. In addition, PQS protected the mitochondrial structure, markedly inhibited mPTP opening and ΔΨm depolarization, led to upregulation of Bcl-2 and downregulation of Bax in the mitochondria compared to the cytosol, and suppressed the expression of cleaved caspase-9 and cleaved caspase-3, as well as I/R induced translocation of cytochrome c to the cytoplasm. CONCLUSION: Our results show that PQS can alleviate apoptosis of cardiomyocytes during I/R injury, possibly due to repressed mitochondrial apoptotic pathway associated with the opening of mPTP induced by myocardial I/R injury.

Cytosolic calreticulin inhibits microwave radiation-induced microvascular endothelial cell injury through the integrin-focal adhesion kinase pathway.

OBJECTIVE: To determine the effects of cytosolic CRT on MR-induced MMEC injury, and the underlying mechanism. METHODS: MMECs were randomized into eight groups: control, AdCRT (infected with pAdCMV/V5-DEST-CRT adenovirus), stCRT (transfected with rCRT-siRNAs), Mock (transfected with scrambled siRNAs), MR (exposed to MR for six minutes), AdCRT + MR, stCRT + MR, and Mock + MR. The magnitude of cell injury were assessed by Annexin V-PI staining, LDH activity in culture medium, MMEC migration ability, ultrastructure and cytoskeletal stability. Subcellular colocalization of CRT and ConA or integrin were evaluated by immunocytochemistry. The mRNA and protein expression levels of target genes were examined by qRT-PCR and western blotting, respectively. RESULTS: MR-induced cytotoxicity was dose-dependent. Overexpression of cytosolic CRT suppressed MR injury, shown as decreased cell apoptosis, reduced LDH activity, enhanced cell migration capability, and maintenance of ultrastructure and cytoskeleton integrity. Conversely, CRT deficiency aggravated MR-induced injury. Exposure of AdCRT MMECs to MR promoted membrane translocation of CRT and the interaction of CRT-integrin-α. Correlation analysis revealed that integrin-α expression or FAK phosphorylation was positively associated with cytosolic CRT expression. CONCLUSIONS: Cytosolic CRT inhibits MR-induced MMEC injury through activation of the integrin-FAK pathway.

[Endoplasmic reticulum-mediated integrated stress response].

Integrated stress response (ISR) is a high conserved cell adaptive response, which is induced by oxidative stress, deprivation of acid aminos, and endoplasmic reticulum (ER) stress through eukaryotic translation initiator factor 2alpha (eIF2alpha) pathway. Recently, it is reported that protein kinase R-like ER kinase (PERK) , the upstream of eIF2alpha is the key molecule in ISR. PERK regulates protein synthesis, folding, autophagy and apoptosis through cross-talking with inositol-requiring enzyme-1 (IRE1) and activating transcription factor 6 (ATF6), another two signaling pathways in ER stress. We reviewed the factors induced ISR and its signaling pathways, summarized the physiological and pathophysiological role of endoplasmic reticulum-mediated integrated stress response.

Efficacy and Safety Evaluation of Taohong Siwu Decoction () for Patients with Angina Pectoris: A Meta-Analysis of Randomized Controlled Trials.

OBJECTIVE: To assess the efficacy and safety of Taohong Siwu Decoction (, TSD), a Chinese herbal compound prescription, in patients with angina pectoris (AP). METHODS: Randomized clinical trials (RCTs) comparing TSD plus conventional treatment (CT) with CT plus placebo or CT only in the patients with AP were searched in PubMed, Cochrane Library, Excerpta Medica Database, Chinese National Knowledge Infrastructure, Chinese Biomedical Literature Database, Wanfang Database, Chinese Scientific Journal Database, Chinese Clinical Trial Registry and International Clinical Trial Registry from their inception to March 2017. The primary outcomes include a composite event of death, acute myocardial infarction (AMI), and target vessel revascularization. The secondary outcomes include angina symptom, electrocardiogram (ECG) improvement and serum high-sensitivity C-reactive protein (hs-CRP), endothelin-1 (ET-1), triglycerides (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels. The methodological quality of included studies and extracted available data were assessed. RevMan 5.3 software was used to conduct statistical analysis. The relative risk (RR) and standardized mean difference (SMD) with 95% confidence interval (CI) was calculated. A funnel plot was used to evaluate the publication bias. RESULTS: Among 204 studies identified in the literature search, 12 trials including 959 patients with AP met the inclusion criteria. No studies reported the primary outcome including death, AMI and target vessel revascularization. TSD combined with CT showed significant improvement in relieving angina symptom [RR=3.70, 95% CI (2.42, 5.67)] and ECG [RR=3.20, 95% CI (2.20, 4.65)] compared with CT alone. TSD combined with CT reduced the serum hs-CRP, TG, TC and LDL-C levels compared with CT alone. No serious adverse events were reported in TSD combined with CT. CONCLUSIONS: TSD combined with CT has a potential benefit on relieving AP without significant adverse events. However, the efficacy on the cardiovascular events needs to be assessed by more rigorously-designed, largescale, and multi-center RCTs in future.

Qi-Regulating and Blood Circulation-Promoting Therapy Improves Health Status of Stable Angina Pectoris Patients with Depressive Symptoms.

Depressive symptoms have been found to be highly prevalent among patients with coronary heart disease (CHD) and seriously affect the patients' quality of life. However, most psychotropic drugs have warnings about potential side effects. Accordingly, safer effective alternatives are urgently demanded. Angina pectoris of CHD is considered as "chest stuffiness and heartache syndrome" in traditional Chinese medicine, with the major syndrome type named Qi stagnation and blood stasis. Qi-regulating and blood circulation-promoting therapy has increasingly shown unique advantages in CHD patients. This study investigated the efficacy of Xuefu Zhuyu decoction, a representative prescription of Qi-regulating and blood circulation-promoting therapy, on angina pectoris patients with depressive symptoms. Depressive symptoms were stratified at baseline in 30 patients with stable angina pectoris who participated in both baseline and 12-week follow-up studies. After performing a stratified analysis, the angina pectoris-specific health status and traditional Chinese medicine "chest stuffiness and heartache syndrome" were evaluated by self-reports using the associated questionnaire scales, respectively. We measured serum concentrations of serotonin, brain-derived neurotrophic factor, and ATP, which are associated with the development of depression. We found that the Xuefu Zhuyu granule significantly improved the angina pectoris-specific health status in patients after 12 weeks of treatment; specifically, it had a better curative effect on patients with depressive symptoms. Xuefu Zhuyu granule also significantly improved the chest stuffiness and heartache syndrome in patients with depressive symptoms (efficacy index is 61.24%, P < 0.05 versus baseline). Interestingly, Xuefu Zhuyu granule has been found to be more susceptible to improving ATP levels in patients with depressive symptoms, indicating that the improvement in serum ATP levels might account for the better efficacy of Xuefu Zhuyu granule in patients with depressive symptoms. Our data provide prospective evidence that Xuefu Zhuyu granule improves angina pectoris-specific health status through regulating Qi and promoting blood circulation. This trial is registered with ChiCTR-IOR-15006989.

Postconditioning with Calreticulin Attenuates Myocardial Ischemia/Reperfusion Injury and Improves Autophagic Flux.

BACKGROUND: Impaired autophagic flux contributes to cardiomyocyte death in ischemia/reperfusion (I/R) injury. Restoring the impaired autophagic flux by using agents may be a promising strategy that alleviates myocardial I/R injury. The present study aimed to evaluate the effect of exogenous calreticulin (CRT) postconditioning on impaired autophagic flux induced by hypoxia/reoxygenation (H/R) injury in H9c2 cells. METHODS: Rat myocardial I/R injury model was prepared. CRT postconditionging was fulfilled by an intraperitoneal injection of CRT (0.5 mg/kg body weight) 5 min before reperfusion. Hemodynamics, serum lactate dehydrogenase (LDH) activity and Cardiac troponin T (TnT) content, and infarct size were measured. The H/R injury model of H9c2 cells was prepared. CRT postconditioning was performed by adding 25 pg/mL CRT to the medium at the onset of reoxygenation. Cell death rate, lactate dehydrogenase (LDH) leakage, intracellular reactive oxygen species (ROS), and malondialdehyde (MDA) were assessed. Autophagic flux was monitored by mRFP-GFP-LC3 adenovirus infection. The number of autophagosomes and autolysosomes in cells were determined by counting the fluorescence dots. Western blot assay was used to determine the expression of autophagy-related proteins. RESULTS: CRT postconditionging improved cardiac function, reduced serum LDH activity and TnT content, and limited myocardial infarct size after myocardial I/R injury in rat. H/R induced H9c2 cells injury and autophagosomes accumulation in cells. CRT postconditioning attenuated H/R-induced cell death, LDH leakage, and the increase of intracellular ROS and MDA. Meanwhile, CRT postconditioning suppressed H/R-induced excessive formation of autophagosomes, as shown by a decrease of autophagosomes and the ratio of LC3-II/LC3-I, LC3-II, and Beclin1. It also improved H/R-induced impaired autophagosomes clearance, as shown by an increase of autolysosomes and the level of LAMP-2, and a decrease of the level of p62. CONCLUSION: These findings suggested that CRT postconditioning reduced myocardial I/R injury. CRT postconditioning also inhibited the excessive formation of autophagosomes, promoted the clearance of autophagosomes, and resorted the autophagic flux, consequently reduced the H/R injury in H9c2 cells.

The establishment of rat model in myocardial ischemia with psychological stress.

BACKGROUND: Psychological stress can provoke and aggravate myocardial ischemia, and this stress can even trigger acute coronary syndromes or sudden cardiac death. Therefore, for the first time, this study aimed to investigate the method for establishing a rat model of myocardial ischemia with psychological stress and its evaluation. METHODS: Forty male Wistar rats were randomly divided into the sham (S, n=10), myocardial infarct (MI, n=10), psychological stress (MODEL, n=10), and myocardial infarct with psychological stress (MI + MODEL, n=10) groups. The rat model of psychological stress was established by measuring the data from activity restriction for 6 hours and followed by tail clamp stimulation for 5 minutes every day for 14 days. The rat model of the myocardial infarct with psychological stress was established by occluding the left coronary anterior descending artery in the MODEL rats. The body weight of rats was measured daily, the behavior parameters were evaluated via open-field test and elevated plus-maze, tongue color and sublingual vein were observed, rats' acral blood flow perfusion was detected by PIM II (Perfusion Imager II), mesenteric microcirculation was measured by capillaroscopy, and hemodynamics was measured by a polygraph system. An automatic biochemical analyzer determined the content of serum cTnT (cardiac troponin T), Hcy (homocysteine), and activity of LDH (lactate dehydrogenase). Myocardial infarct size was measured with TTC (triphenyhetrazolium chloride) staining. RESULTS: We found that rats in the psychological stress (MODEL) group were characterized by coarse hair, dark mucosa of the lips and claw, low spirit, decreased body weight, and increased anxiety. Compared with rats in the sham group, rats in the MODEL + MI group showed decreased mesenteric blood flow, narrowed arteriole and venule diameter, reduced acral blood flow perfusion, and LV ±dp/dtmax (the maximal rate of the increased and decrease of left ventricular pressure), as well as increased serum content of cTnT, Hcy, and LDH activity. Compared with the MI group, rats in the MODEL + MI group showed deteriorated microcirculation dysfunction manifested as a dark tongue color of deep purple, prominently extended and varicose sublingual vein, and aggravated myocardial damage in the form of increased infarct size and LDH leakage. CONCLUSIONS: In conclusion, the rat model of myocardial ischemia with psychological stress was successfully established, and manifested as aggravating behavioral disorder, mesenteric microcirculation and left ventricular dysfunction, and myocardial damage.

Calreticulin Ameliorates Hypoxia/Reoxygenation-Induced Human Microvascular Endothelial Cell Injury By Inhibiting Autophagy.

BACKGROUND: Autophagy has been found to be involved in myocardial ischemia/reperfusion injury. However, the underlying mechanism and significance of autophagy in reperfusion injury remain unclear. Herein, we evaluated the effects of exogenous calreticulin (CRT) on autophagy in hypoxia/reoxygenation (H/R)-treated human microvascular endothelial cells (MECs). METHODS: Human MECs were pretreated with CRT (25 pg/mL) for 30 min, followed by exposure in an incubator filled with a gas mixture of 90% N2, 5% O2, and 5% CO2 for 8-h hypoxia. The cells were then placed back in the normoxic CO2 incubator for 16-h reoxygenation. Cell injury was assessed by the cell counting kit-8 assay. Autophagosomes were detected by transmission electron microscopy and immunofluorescence staining. Western blot analysis was performed to detect phosphorylated mammalian target of rapamycin (p-mTOR), Beclin 1, and microtubule-associated protein 1 light chain 3 (LC3). RESULTS: H/R induced marked autophagy through the mTOR pathway. CRT suppressed rapamycin- and H/R-induced autophagosome formation, the LC3-II/LC3-I ratio, and Beclin 1 expression in human MECs by upregulating mTOR phosphorylation, consequently attenuating H/R-induced human MEC injury. CONCLUSIONS: Exogenous CRT attenuated H/R-induced human MEC injury by inhibiting autophagy.

Myofibrillogenesis Regulator 1 Rescues Renal Ischemia/Reperfusion Injury by Recruitment of PI3K-Dependent P-AKT to Mitochondria.

To investigate whether myofibrillogenesis regulator 1 (MR-1) attenuates renal ischemia/reperfusion (I/R) injury via inhibiting phosphorylated Akt (p-Akt) mitochondrial translocation-mediated opening of the mitochondrial permeability transition pore (mPTP), we injected adenovirus containing MR-1 gene or its siRNAs to the left kidney subcapsular areas of Sprague-Dawley rats, which subsequently underwent experimental renal I/R injury. Renal functions and the severity of the tubular injury were evaluated by the serum creatinine and blood urea nitrogen levels and the pathological scores. We also examined the mitochondrial morphology and functions. Total/p-Akt were assessed by western blot using the mitochondrial and the cytosolic fractions of cortex of renal tissue, respectively. We found that mitochondrial and cytosolic MR-1 levels and mitochondrial p-Akt decreased, and cytosolic p-Akt increased after reperfusion. Subcapsular injection of adenovirus led to higher MR-1 expression in the mitochondria/cytosol, inhibited mPTP opening, and alleviated renal I/R injury; adenovirus injection also upregulated mitochondrial total and p-Akt levels more prominently compared with the normal saline (NS) group. Subcapsular injection of MR-1 siRNAs significantly lowered MR-1 expression and induced renal injury, with increased mPTP opening and mitochondrial damage, similar to I/R injury. MR-1 interacted with Akt in renal cortex homogenate. Wortmannin, a phosphatidylinositol 3 kinase (PI3K) inhibitor, abolished both mitochondrial p-Akt recruitment and the protective effect of MR-1 overexpression on I/R injury. To conclude, MR-1 protects kidney against I/R injury through inhibiting mPTP opening and maintaining mitochondrial integrity, through the recruitment of PI3K-dependent p-Akt to the mitochondria. MR-1 could be a new therapeutic strategy for renal I/R injury.

Panax quinquefolium saponin attenuates cardiomyocyte apoptosis induced by thapsigargin through inhibition of endoplasmic reticulum stress.

BACKGROUND: Endoplasmic reticulum (ER) stress-related apoptosis is involved in the pathophysiology of many cardiovascular diseases, and Panax quinquefolium saponin (PQS) is able to inhibit excessive ER stress-related apoptosis of cardiomyocytes following hypoxia/reoxygenation and myocardial infarction. However, the pathway by which PQS inhibits the ER stress-related apoptosis is not well understood. To further investigate the protective effect of PQS against ER stress-related apoptosis, primary cultured cardiomyocytes were stimulated with thapsigargin (TG), which is widely used to model cellular ER stress, and it could induce apoptotic cell death in sufficient concentration. METHODS: Primary cultured cardiomyocytes from neonatal rats were exposed to TG (1 µmol/L) treatment for 24 h, following PQS pre-treatment (160 µg/mL) for 24 h or pre-treatment with small interfering RNA directed against protein kinase-like endoplasmic reticulum kinase (Si-PERK) for 6 h. The viability and apoptosis rate of cardiomyocytes were detected by cell counting kit-8 and flow cytometry respectively. ER stress-related protein expression, such as glucose-regulated protein 78 (GRP78), calreticulin, PERK, eukaryotic translation initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) were assayed by western blotting. RESULTS: Both PQS pre-treatment and PERK knockdown remarkably inhibited the cardiomyocyte apoptosis induced by TG, increased cell viability, decreased phosphorylation of both PERK and eIF2α, and decreased protein levels of both ATF4 and CHOP. There was no statistically significant difference between PQS pre-treatment and PERK knockdown in the cardioprotective effect. CONCLUSIONS: Our data indicate that the PERK-eIF2α-ATF4-CHOP pathway of ER stress is involved in the apoptosis induced by TG, and PQS might prevent TG-induced cardiomyocyte apoptosis through a mechanism involving the suppression of this pathway. These findings provide novel data regarding the molecular mechanisms by which PQS inhibits cardiomyocyte apoptosis.