Analysis of gene expression profiling of amyloidogenic immunoglobulin light-chains on cultured rat cardiomyocytes.

The present study aimed to investigate the toxic effects of different amyloidogenic light-chains (LCs) on cardiomyocytes, and demonstrate the differentially expressed genes (DEGs) and signaling pathways that participate in this process. Cultured cardiomyocytes were treated with recombinant κ LC peptide (AL-09) or with serum from a patient diagnosed with multiple myeloma (λ LC) with cardiac involvement. The 6xHis peptide or serum from healthy patients was used as peptide control or serum control, respectively. Cell viability was determined using CCK-8 assay and apoptosis was analyzed by flow cytometry. The DEGs were detected by RNA sequencing (RNA-Seq), followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Changes in gene expression levels were confirmed by reverse transcription-quantitative PCR. The cell viability in the AL-09 peptide-treated (0.2 mg/ml) and patient serum-treated (1:10 dilution) cardiomyocytes decreased to 42 and -72% of the corresponding control groups. The extent of cell apoptosis increased in AL-09-treated cardiomyocytes compared with the control group. RNA-Seq showed 256 DEGs co-existed in the two paired groups, including 127 upregulated and 88 downregulated genes. The KEGG pathways for upregulated expressed genes included the 'TGF-ß signaling pathway', the 'Hedgehog signaling pathway', the 'ErbB signaling pathway' and 'lysine degradation'. The higher mRNA expression of bone morphogenetic protein (Bmp) 4, Bmp6, prostaglandin G/H synthase (Ptgs)1, Ptgs2, epiregulin, Tgfa and procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 were confirmed. The KEGG pathways of downregulated expressed genes included genes involved with the 'p53 signaling pathway' and the 'cell cycle'. The mRNA expression levels of E3 ubiquitin-protein ligase CCNB1IP1 showed significant downregulation in the AL-09 peptide group compared with those in the 6xHis peptide group. In conclusion, cardiomyocytes treated with amyloidogenic λ and κ LCs presented with decreased cell viability compared with controls. Cell apoptosis increased in κ LC-treated cells compared with controls. The gene expression profiles associated with transforming growth factor-ß-bone morphogenetic protein, the receptor tyrosine-protein kinase erbB-2 signaling pathways, prostaglandins, collagen production, the p53 signaling pathway and the cell cycle were altered in light-chain-treated cardiomyocytes.

Cardiac Myosin-Binding Protein C Release Profile After Cardiac Surgery in Intensive Care Unit.

BACKGROUND: Cardiac surgical procedures produce iatrogenic myocardial cell injury with necrosis that result in an obligatory release of biomarkers. Cardiac myosin binding protein C (cMyBP-C) has recently emerged as a specific and sensitive biomarker in patients with acute myocardial injury. We therefore aimed to investigate the release profiles of cMyBP-C after cardiac surgical procedures. METHODS: Enzyme-linked immunosorbent assay to detect blood cMyBP-C was established by using two monoclonal antibodies against N-terminus of human cMyBP-C. Consecutive patients undergoing cardiac operations (N = 151) were recruited in this study. Blood cMyBP-C was assayed preoperatively, at intensive care unit arrival (0 hour after the operation), at 2 to 48 hours, and before discharge. The characteristics and detailed surgical procedure were recorded. RESULTS: The established immunoassay was capable of detecting human cMyBP-C (0 to 1000 ng/L). The released cMyBP-C peaked immediately after cardiac surgery (0 h), attaining 3.8-fold higher than before the operation, dropped abruptly within 24 hours, and stayed at a higher level until discharge. Postoperative cMyBP-C levels correlated positively with high-sensitivity cardiac troponin T (hs-cTnT), creatine kinase, myoglobin, and creatine kinase MB isoenzyme. Different cardiac surgical procedures were characterized by different levels of release of cardiac biomarkers. Isolated off-pump coronary artery bypass grafting was associated with the smaller amount of cMyBP-C release, whereas valve replacement/plasty surgery produced higher release, in particular the multiple-valve surgery. Both cMyBP-C and hs-cTnT correlated with surgical techniques, postoperative intensive care unit stay, and hospital stay. CONCLUSIONS: Circulating cMyBP-C is a promising novel biomarker for evaluating cardiac surgical trauma in patients undergoing a cardiac operation.

Astragaloside IV Prevents Cardiac Remodeling in the Apolipoprotein E-Deficient Mice by Regulating Cardiac Homeostasis and Oxidative Stress.

BACKGROUND: Hypercholesterolemia is a risk factor for the development of cardiac hypertrophy. Astragaloside IV (AST-IV) possesses cardiovascular protective properties. We hypothesize that AST-IV prevents cardiac remodeling with hypercholesterolemia via modulating tissue homeostasis and alleviating oxidative stress. METHODS: The ApoE-/- mice were treated with AST-IV at 1 or 10 mg/kg for 8 weeks. The blood lipids tests, echocardiography, and TUNEL were performed. The mRNA expression profile was detected by real-time PCR. The myocytes size and number, and the expressions of proliferation (ki67), senescence (p16INK4a), oxidant (NADPH oxidase 4, NOX4) and antioxidant (superoxide dismutase, SOD) were observed by immunofluorescence staining. RESULTS: Neither 1 mg/kg nor 10 mg/kg AST-IV treatment could decrease blood lipids in ApoE-/- mice. However, the decreased left ventricular ejection fraction (LVEF) and fractional shortening (FS) in ApoE-/- mice were significantly improved after AST-IV treatment. The cardiac collagen volume fraction declined nearly in half after AST-IV treatment. The enlarged myocyte size was suppressed, and myocyte number was recovered, and the alterations of genes expressions linked to cell cycle, proliferation, senescence, p53-apoptosis pathway and oxidant-antioxidants in the hearts of ApoE-/- mice were reversed after AST-IV treatment. The decreased ki67 and increased p16INK4a in the hearts of ApoE-/- mice were recovered after AST-IV treatment. The percentages of apoptotic myocytes and NOX4-positive cells in AST-IV treated mice were decreased, which were consistent with the gene expressions. CONCLUSION: AST-IV treatment could prevent cardiac remodeling and recover the impaired ventricular function induced by hypercholesterolemia. The beneficial effect of AST-IV might partly be through regulating cardiac homeostasis and anti-oxidative stress.

Preparation of Monoclonal Antibodies and a Simple Myeloperoxidase-Immunosorbent Assay for Detecting Human Myeloperoxidase.

Myeloperoxidase (MPO), a leukocyte hemoprotein released from neutrophils, is thought to be a potential participant in plaque formation and plaque rupture. Therefore, MPO is regarded as an early marker predicting the risk for atherosclerosis, especially for coronary artery disease and acute coronary syndrome. We generated hybridoma clones 1E3 and 3E8 secreting monoclonal antibodies (mAbs) specific to human MPO. BALB/c mice were immunized with MPO protein purified from human neutrophils. Splenocytes from these mice were fused with the mouse myeloma cell line SP2/0. Based on isotyping of the mAbs, both clones 1E3 and 3E8 were referred to the IgG1 subclass. The specificities of 1E3 and 3E8 were assessed by enzyme-linked immunosorbent assay (ELISA), and only 3E8 was confirmed by western blot. We developed a simple MPO-immunosorbent assay (MPO-ISA) on microplate based on both the immune activity and peroxidase activity of MPO. The mAb secreted by clone 3E8 was chosen as coating antibody to capture the plasma MPO without interfering with the peroxidase activity of MPO. Then, tetramethylbenzidine substrate was added to the microwell directly, catalyzed by captured MPO, and a colored product was formed. The simple MPO-ISA test has a sensitivity of 3.68 ng/mL. The linear concentration of MPO-ISA for commercial MPO standard ranged to 250 ng/mL. The average recovery rate is 101.02%. The imprecision within-day was <10% at three different MPO levels. The imprecision between-day was <10% at low and middle MPO levels and varied to 14.61% at the high MPO level. We found that the established MPO-ISA can detect the plasma MPO from human and cavy, but not from mouse and rat. Compared with the commercial human MPO ELISA assay, the MPO-ISA can be used to detect the natural human MPO protein, but not recombinant MPO polypeptides. The generated mAbs and MPO-ISA test may be useful tools to assess risk for inflammation and cardiac events.

The effects of different initiation time of exercise training on left ventricular remodeling and cardiopulmonary rehabilitation in patients with left ventricular dysfunction after myocardial infarction.

PURPOSE: The purpose of this study was to determine whether different initiation of exercise training (ET) produces different effect sizes for left ventricular (LV) remodeling and cardiopulmonary rehabilitation in patients with LV dysfunction after myocardial infarction (MI). METHOD: Trials evaluating ET outcomes identified by searches in OVID MEDLINE, EMBASE, PubMed and WEB OF SCIENCE were used. Meta-analysis was conducted with the use of the software STATA 11.0. The results were expressed as the standardized mean difference (SMD), with corresponding 95% CI and p value. RESULTS: The largest changes in LV remodeling and cardiopulmonary capacity rehabilitation were obtained when programs began the acute phase after MI. With the healing of MI, the beneficial effects of ET on LV ejection fraction (LVEF), LV end-systolic diameter (LVDs) and peak VO2 were gradually weakened even worse. The incidence of major adverse cardiac events was not significantly increased in acute phase post-MI. Sensitivity analyses show that ET still had significant effect in reducing LVDs and increasing peak VO2, while ET no longer had statistical effect in increasing LVEF but showed favorable trends when the same research institution's works were excluded. CONCLUSIONS: ET has favorable effects on LV remodeling and cardiopulmonary rehabilitation in LV dysfunction post-MI patients. The greatest benefits are obtained when ET starts at the acute phase following MI. IMPLICATIONS FOR REHABILITATION: Early exercise training is safe and feasible in acute and healing phase after myocardial infarction. Early exercise training could attenuate LV remodeling and improve cardiopulmonary capacity in patients with myocardial infarction after hospital discharge (around one week post-MI). Exercise training has favorable effects on LV remodeling and cardiopulmonary capacity rehabilitation. Exercise training should be treated to have the same roles with drugs in secondary prevention of myocardial infarction.

Cardiac troponin I is abnormally expressed in non-small cell lung cancer tissues and human cancer cells.

Cardiac troponin I (cTnI) is the only sarcomeric protein identified to date that is expressed exclusively in cardiac muscle. Its expression in cancer tissues has not been reported. Herein, we examined cTnI expression in non-small cell lung cancer (NSCLC) tissues, human adenocarcinoma cells SPCA-1 (lung) and BGC 823 (gastric) by immunohistochemistry, western blot analysis and real-time PCR. Immunopositivity for cTnI was demonstrated in 69.4% (34/49) NSCLC tissues evaluated, and was strong intensity in 35.3% (6/17) lung squamous cell carcinoma cases. The non-cancer-bearing lung tissues except tuberculosis (9/9, 100%) showed negative staining for cTnI. Seven monoclonal antibodies (mAbs) against human cTnI were applied in immunofluorescence. The result showed that the staining pattern within SPCA-1 and BGC 823 was dependent on the epitope of the cTnI mAbs. The membrane and nucleus of cancer cells were stained by mAbs against N-terminal peptides of cTnI, and cytoplasm was stained by mAbs against the middle and C-terminal peptides of cTnI. A ~25 kD band was identified by anti-cTnI mAb in SPCA-1 and BGC 823 extracts by western blot, as well as in cardiomyocyte extracts. The cTnI mRNA expressions in SPCA-1 and BGC 823 cells were about ten thousand times less than that in cardiomyocytes. Our study shows for the first time that cTnI protein and mRNA were abnormally expressed in NSCLC tissues, SPCA-1 and BGC 823 cells. These findings challenge the conventional view of cTnI as a cardiac-specific protein, enabling the potential use of cTnI as a diagnostic marker or targeted therapy for cancer.

Targeted efficacy of dihydroartemisinin for translationally controlled protein expression in a lung cancer model.

OBJECTIVE: Lung cancer is one of the malignant tumors with greatest morbidity and mortality around the world. The keys to targeted therapy are discovery of lung cancer biomarkers to facilitate improvement of survival and quality of life for the patients with lung cancer. Translationally controlled tumor protein (TCTP) is one of the most overexpressed proteins in human lung cancer cells by comparison to the normal cells, suggesting that it might be a good biomarker for lung cancer. MATERIALS AND METHODS: In the present study, the targeted efficacy of dihydroartemisinin (DHA) on TCTP expression in the A549 lung cancer cell model was explored. RESULTS AND CONCLUSIONS: DHA could inhibit A549 lung cancer cell proliferation, and simultaneously up-regulate the expression of TCTP mRNA, but down-regulate its protein expression in A549 cells. In addition, it promoted TCTP protein secretion. Therefore, TCTP might be used as a potential biomarker and therapeutic target for non-small cell lung cancers.

Astragaloside IV alleviates hypoxia/reoxygenation-induced neonatal rat cardiomyocyte injury via the protein kinase a pathway.

BACKGROUND: Astragaloside IV (As-IV) exerts beneficial effects on hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury, possibly through normalization of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) function. The exact mechanisms remain unknown. This study was designed to investigate the role of protein kinase A (PKA) in the protective effect of As-IV on SERCA2a function. METHODS: Cultured cardiomyocytes from neonatal rats were exposed to 6 h of hypoxia followed by 3 h of reoxygenation (H/R) with or without As-IV treatment. Myocyte injury was determined by the creatine kinase (CK)-MB fraction in supernatant. Myocardial SERCA2a activity and PKA kinase activity were assessed. PKA subunit mRNA expression and Ser(16) phosphorylated phospholamban (Ser(16)-PLN) protein expression were detected by real-time PCR and Western blot, respectively. RESULTS: The administration of As-IV significantly decreased CK-MB release and restored SERCA2a activity in H/R cardiomyocytes. The mRNAs of PKA subunits, PKA-RIα, PKA-RIIα, PKA-RIIß, PKA-Cα and PKA-Cß, were downregulated in H/R cardiomyocytes. However, PKA-Cα mRNA expression was significantly increased after As-IV treatment. Meanwhile, there was a tendency to recovery of the H/R-induced PKA kinase activity decrease after As-IV treatment. The expression of Ser(16)-PLN protein, which is specifically phosphorylated by PKA, was upregulated in As-IV-treated H/R cardiomyocytes. CONCLUSIONS: These results suggest that the cardioprotection of As-IV may be through the upregulation of PKA and Ser(16)-PLN, thereby restoring SERCA2a function in H/R injury.

[Reduced Ca2+ current in rat cardiomyocytes transfected with troponin I R145W mutation gene].

OBJECTIVE: To investigate the effects of cardiac troponin I R145W mutation, detected in Chinese patients with hypertrophic cardiomyopathy, on Ca(2+) current modulation. METHODS: R146W mutation (resemble R145W in human) was introduced into rat cardiac troponin I cDNA by site-directed mutagenesis. With EGFP as a reporter gene, replication-defective adenovirus containing the wild or mutant cTnI gene was constructed. Adult rat cardiomyocytes, were isolated by Langendorff perfusion and cultured with serum-free medium and transduced with the recombinant adenoviruses. Western blot was used to determine the recombinant proteins. Whole cell patch clamp was employed to record L-type Ca(2+) currents on cultured myocytes. Intracellular free Ca(2+) and caffeine-induced sarcoplasmic reticulum (SR) Ca(2+) release were determined after the cells incubated with Fura-2/AM. RESULTS: DNA sequencing confirmed that R146W mutation was generated in rat cTnI cDNA. Bright green fluorescence was observed in the cultured cardiomyocytes at 48 h after transduction. The recombinant proteins could be identified with cTnI or GFP monoclonal antibody. The peak current of L-type Ca(2+) channel in cells transduced with cTnI R146W was significantly decreased compared to control cells and cells transfected with wild cTnI. Intracellular free Ca(2+) concentrations and caffeine-induced SR Ca(2+) release determined by Fura-2/AM were similar among various cells. CONCLUSION: Reduced peak current of L-type Ca(2+) channel in cells transduced with cTnI R146W might contribute to the disease-causing mechanism of this mutation in patients with hypertrophic cardiomyopathy.

[Association between cardiac troponin I phosphorylation/degradation and cardiomyopathies].

OBJECTIVE: To investigate the association between cTnI phosphorylation/degradation and cardiomyopathies in extransplanted myocardium. METHODS: cTnI phosphorylation and degradation as well as PKC (beta1, beta2) expressions were determined in extransplanted hearts from patients with cardiomyopathies (n = 8) and from traffic accidents (n = 6) by Western blot. RESULTS: The cTnI bands were observed in LV myocardium of cardiomyopathy patients and normal myocardium while and cTnI degradation bands were only detected in LV myocardium from patients with cardiomyopathies. The phosphorylated cTnI bands were significantly upregulated in LV myocardium of cardiomyopathy patients compared to normal myocardium (P < 0.05). There was no myocardial PKCbeta1, PKCbeta2 expression in all examined hearts. CONCLUSION: The cTnI degradation products and increased phosphorylated cTnI expression are likely involved in the pathogenesis and development of cardiomyopathy.

[Assessment of cardiac structure and function by echocardiographic values for male Balb/c mice].

AIM: To assess the parameters of cardiac structure and function of male Balb/c mice by the echocardiography. METHODS: A total of 27 male Balb/c mice (from five to seven week old) were examined with a 13-MHz transthoracic linear-array transducer, hearts were removed from mice anesthetized with Nembutal, and the left ventricular (LV) mass were weighed. RESULTS: Complete 2-dimensional echocardiography for cardiac structure and function were obtained. Hemodynamic parameters were recorded. A correlation existed between LV weight (x) and echocardiographic LV mass (y) with the 2D) guided M-mode method: y = 1.15x + 3.26, (r = 0.80). CONCLUSION: Echocardiography appears to be a promising approach for noninvasively assessing LV mass and function in mice.

Oxygen-derived free radicals mediate endothelium-dependent contractions to acetylcholine in aortas from spontaneously hypertensive rats.

Experiments were designed to investigate whether or not oxygen-derived free radicals mediate endothelium-dependent contractions to acetylcholine in the aorta of spontaneously hypertensive rat (SHR). Isometric tension was measured in aortic rings taken from adult male SHR and Wistar-Kyoto rat (WKY) in the presence of NG-nitro-L-arginine. Endothelium-dependent contractions to acetylcholine were significantly greater in rings from SHR compared to WKY. Oxygen-derived free radicals, generated from xanthine plus xanthine oxidase, induced contractions that were larger in aortas from SHR than from WKY. Contractions to acetylcholine and free radicals were abolished by a selective TP-receptor antagonist, S 18886, and a preferential inhibitor of cyclo-oxygenase-1, valeryl salicylate, but not by a preferential inhibitor of cyclo-oxygenase-2, NS-398. Allopurinol, deferoxamine and the combination of superoxide dismutase plus catalase inhibited the contractions to oxygen-derived free radicals but did not significantly affect those to acetylcholine. In contrast, diethyldithiocarbamic acid, an inhibitor of superoxide dismutase, or Tiron, a scavenger of superoxide anion, reduced endothelium-dependent contractions to acetylcholine in aortas from SHR. The effect of these two drugs was additive. In SHR chronically treated with dimethylthiourea endothelium-dependent contractions to acetylcholine were decreased, and reduced further by acute in vitro exposure to deferoxamine or the combination of superoxide dismutase plus catalase. These results suggest that in the SHR aorta acetylcholine-induced endothelium-dependent contractions involve endothelial superoxide anion production and the subsequent dismutation into hydroxyl radicals and/or hydrogen peroxide. The free radicals activate cyclo-oxygenase-1, most likely to produce endoperoxides. Activation of TP-receptors is required to observe endothelium-dependent contractions to acetylcholine or endothelium-independent contractions in response to free radical generation.