ABSTRACT
OBJECTIVE To study the role of phosphatidylinositol-3-kinase （PI3K） on sunitinib-induced myocardial systolic dysfunction. METHODS Using human-induced pluripotent stem cell-derived cardiomyocytes （hiPSC-CMS） as objects， the contractile force of cardiomyocytes was measured by CardioExcyte 96 system， and IC50 of sunitinib was calculated after hiPSC- CMS were treated with sunitinib at different concentrations [0 （control）， 0.5， 1， 3， 5， 10 μmol/L] for 24 hours. The effects of sunitinib （3.14 μmol/L） on the contractile frequency of cardiomyocytes， calcium transient amplitude and calcium transient recovery time course， mRNA expression of myocardial injury markers atrial natriuretic peptide （ANP）， brain natriuretic peptide （BNP） and β-myosin heavy chain （β-MHC） were detected. PI3K activator 3，4，5-triphosphate phos-phatidylinositol （PIP3， 1 μmol/L） and sunitinib were used to intervene in hiPSC-CMs jointly， so as to investigate the role of PI3K in the myocardial systolic dysfunction induced by sunitinib. RESULTS Sunitinib inhibited the contractile force of hiPSC-CMs in a concentration-dependent manner. IC50 of sunitinib was 3.14 μmol/L. After intervention with 3.14 μmol/L sunitinib， the contractile frequency of hiPSC-CMs and calcium transient amplitude were decreased significantly （P＜0.05 or P＜0.01）； the duration of calcium transient recovery was prolonged significantly （P＜0.05）， and mRNA expressions of ANP， BNP and β-MHC were significantly increased （P＜0.01）. After PI3K was activated with PIP3， the contractile force of hiPSC-CMs was increased significantly （P＜0.01）. CONCLUSIONS Activating PI3K activity is a potential molecular mechanism to improve myocardial toxicity induced by sunitinib.
ABSTRACT
【Objective】 To explore the effects of insulin on the QT interval and induced arrhythmias of cardiomyocytes derived from human induced pluripotent stem cells （hiPSC-CMs）. 【Methods】 Immunofluorescence staining and flow cytometry were used to analyze the purity of hiPSC-CMs. Microelectrode array （MEA） was utilized to detect the electrophysiological changes including heart rate （HR）， field potential duration （FPD， which is similar to QT interval in ECG）， FPDc （FPD corrected by HR）， conduction velocity （CV）， and spike amplitude before and after insulin treatment. The effects of E4031 on QT interval prolongation and induced arrhythmias of hiPSC-CMs were evaluated before and after treatment with insulin. 【Results】 hiPSC-CMs highly expressed myocardial specific marker cTnT. The purity of hiPSC-CMs was 97.1%. After 5-day insulin treatment of hiPSC-CMs， HR increased by （11.9±3.3）%， FPD shortened by （22.7±2.8）%， FPDc shortened by （15.6±1.6）%， and spike amplitude increased by （39.1±7.9）% when compared with untreated group， but CV remained unchanged. 10 nmol/L of E4031 could prolong the FPDc of hiPSC-CMs by （37.8±9.0）%， and 30 nmol/L of E4031 could induce arrhythmias. After insulin treatment， 10 nmol/L of E4031 prolonged the FPDc of hiPSC-CMs by （21.8±3.1）% （compared with the untreated group， insulin decreased FPDc prolongation by E4031， 37.8%±9.0% vs. 21.8%±3.1%， P<0.05）， while 30 nmol/L of E4031 did not induce arrhythmias. 【Conclusion】 Insulin can shorten the QT interval of hiPSC-CMs and significantly reduce the QT interval prolongation and the risk of arrhythmias induced by drugs.
ABSTRACT
Drug-induced arrhythmia is one of the main causes of failure in drug development, and it is also a major cause of drug withdrawal, therefore, accurate prediction of drug-induced arrhythmia in the non-clinical research stage is the best way to reduce cost. Literature was retrieved by formally searching PubMed, Metstr, CNKI and Baidu Scholar, 1 479 published articles were found through search method, 63 full-text articles were included. After reviewed the relevant literatures, the advantages and disadvantages of the different experimental cells and the related evaluation methods are assessed, in order to provide reference for toxicity evaluation.
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Objective: To investigate the role of estrogen-related receptor alpha (ERRα) in regulating the adenosine triphosphate (ATP) synthesis in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Methods: Undifferentiated human induced pluripotent stem cells (hiPSCs) were induced to differentiate into cardiomyocytes by sequential transient activation/inhibition of Wnt signaling pathway. Immunofluorescence was used to detect the expression of pluripotency markers sex determining region Y-box 2 (SOX2), stage specific embryonic antigen 4 (SSEA4) and tumor resistance antigen 1-60 (TRA-1-60) in hiPSCs and the expression of cardiac specific markers cardiac troponin T (cTnT) and connexin 43 (Cx43) in hiPSCCMs, respectively; RT-qPCR was used to detect the mRNA expression levels of cardiac troponin T2 (TNNT2) and myosin heavy chain 6 (MYH6) in hiPSCs and hiPSCs-CMs; Western blotting was performed to detect the protein expression levels of ERRα, cytochrome C (CytC) and mitochondrial pyruvate carrier 1 (MPC1) in hiPSCs-CMs. Additionally, the ERRα-specific inhibitor XCT790 was used to treat the hiPSC-CMs, and then the protein expressions of ERRα, CytC and MPC1 were detected by Western blotting, and the changes of cell viability, intracellular ATP content and mitochondrial membrane potential were measured by assay kits. Results: Immunofluorescence results showed that hiPSCs expressed SOX2, SSEA4 and TRA-1-60, while hiPSC-CMs expressed cTnT and Cx43; compared with hiPSCs, the mRNA levels of TNNT2 and MYH6 in hiPSC-CMs increased significantly (82.820 ± 2.005 vs. 1.001 ± 0.029, 90982.000 ± 1968.000 vs. 1.003 ± 0.053, respectively, P<0.05), and intracellular ATP content and protein expression levels of ERRα, CytC and MPC1 also increased significantly [(9.905 ± 1.286) nmol/mg protein vs. (4.582 ± 0.141) nmol/mg protein, 5.392 ± 0.313 vs. 1.050 ± 0.076, 8.954 ± 0.293 vs. 1.071 ± 0.067, 2.605 ± 0.088 vs. 1.031 ± 0.091, respectively] with significant differences (P<0.05). Furthermore, compared with the control group, 10 μmol/L XCT790 could effectively inhibit the protein activity of ERRα in hiPSC-CMs without cytotoxicity, and reduced intracellular ATP content and mitochondrial membrane potential [(4.903 ± 1.158) nmol/mg protein vs. (9.310 ± 0.980) nmol/mg protein, 1.407 ± 0.022 vs. 1.977 ± 0.093, respectively], meanwhile down-regulated the protein expression levels of MPC1 and CytC in hiPSC-CMs (0.705 ± 0.019 vs. 0.897 ± 0.011, 0.594 ± 0.021 vs. 0.797 ± 0.025, respectively, P<0.05). Conclusions: The increase of ATP content after differentiation of hiPSCs into cardiomyocytes is related to the increase of ERRα expression. In hiPSC-CMs, ERRα may regulate the ATP synthesis though regulating the mitochondrial membrane potential and the protein expression of CytC and MPC1.
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Objective To comparatively study the characteristics of 3 kinds of culture substrates of human odontogenic induced pluripotent stem cells(iPSCs).Methods The human odontogenic iPSCs were cultured by 3 kinds of substrates:mouse embryonic fibroblasts(MEF),matrigel and recombinant human vitronectin(VTN-N).The iPSCs growth situation was compared among three groups.Results The preparation time of these 3 kinds of substrates was 14,3,1 hlespectively,and,the difference was statistically significant (P＜0.05).The iPSCs reprogramming time was (30± 1.6),(26 ± 2.1),(27 ± 1.4) d,lespectively,wht that in the MEF group significantly higer than in other two groups (P＜0.05).The reprogramming efficiencies were 0.3 ％ ± 0.03 ％,0.56 ％ ± 0.08 ％,0.7 ％ ± 0.02 ％ respectively (P＜ 0.05).Three kinds of substrate could better support iPSCs growth and make them to maintain un-differentiation status.Conclusion with no heterologous animal components,and the adrantaga of simple pleparation,oonfrollable standard and shorter gramming time is easy to prepare,the standard is controllable and the reprogramming time is shorter,which is an ideal substrate for supporting iPSCs growth.