Action potential variability in human pluripotent stem cell-derived cardiomyocytes obtained from healthy donors

Human pluripotent stem cells (PSC) have been used for disease modelling, after differentiation into the desired cell type. Electrophysiologic properties of cardiomyocytes derived from pluripotent stem cells are extensively used to model cardiac arrhythmias, in cardiomyopathies and channelopathies. This requires strict control of the multiple variables that can influence the electrical properties of these cells. In this article, we report the action potential variability of 780 cardiomyocytes derived from pluripotent stem cells obtained from six healthy donors. We analyze the overall distribution of action potential (AP) data, the distribution of action potential data per cell line, per differentiation protocol and batch. This analysis indicates that even using the same cell line and differentiation protocol, the differentiation batch still affects the results. This variability has important implications in modeling arrhythmias and imputing pathogenicity to variants encountered in patients with arrhythmic diseases. We conclude that even when using isogenic cell lines to ascertain pathogenicity to variants associated to arrythmias one should use cardiomyocytes derived from pluripotent stem cells using the same differentiation protocol and batch and pace the cells or use only cells that have very similar spontaneous beat rates. Otherwise, one may find phenotypic variability that is not attributable to pathogenic variants

R534C mutation in hERG causes a trafficking defect in iPSC-derived cardiomyocytes from patients with type 2 long QT syndrome

Patient-specific cardiomyocytes obtained from induced pluripotent stem cells (CM-iPSC) offer unprecedented mechanistic insights in the study of inherited cardiac diseases. The objective of this work was to study a type 2 long QT syndrome (LQTS2)-associated mutation (c.1600C > T in KCNH2, p.R534C in hERG) in CM-iPSC. Peripheral blood mononuclear cells were isolated from two patients with the R534C mutation and iPSCs were generated. In addition, the same mutation was inserted in a control iPSC line by genome editing using CRISPR/Cas9. Cells expressed pluripotency markers and showed spontaneous differentiation into the three embryonic germ layers. Electrophysiology demonstrated that action potential duration (APD) of LQTS2 CM-iPSC was significantly longer than that of the control line, as well as the triangulation of the action potentials (AP), implying a longer duration of phase 3. Treatment with the IKr inhibitor E4031 only caused APD prolongation in the control line. Patch clamp showed a reduction of IKr on LQTS2 CM-iPSC compared to control, but channel activation was not significantly affected. Immunofluorescence for hERG demonstrated perinuclear staining in LQTS2 CM-iPSC. In conclusion, CM-iPSC recapitulated the LQTS2 phenotype and our findings suggest that the R534C mutation in KCNH2 leads to a channel trafficking defect to the plasma membrane.Fil: Mesquita, Fernanda C. P.. Universidade Federal do Rio de Janeiro; BrasilFil: Arantes, Paulo C.. Universidade Federal do Rio de Janeiro; BrasilFil: Kasai Brunswick, Tais H.. Universidade Federal do Rio de Janeiro; BrasilFil: Araujo, Dayana S.. Universidade Federal do Rio de Janeiro; BrasilFil: Gubert, Fernanda. Universidade Federal do Rio de Janeiro; BrasilFil: Monnerat, Gustavo. Universidade Federal do Rio de Janeiro; BrasilFil: Silva dos Santos, Danúbia. Universidade Federal do Rio de Janeiro; BrasilFil: Neiman, Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Leitão, Isabela C.. Universidade Federal do Rio de Janeiro; BrasilFil: Barbosa, Raiana A. Q.. Universidade Federal do Rio de Janeiro; BrasilFil: Coutinho, Jorge L.. National Institute Of Cardiology; BrasilFil: Vaz, Isadora M.. Pontificia Universidad Catolica de Parana; BrasilFil: dos Santos, Marcus N.. Universidade Federal do Rio de Janeiro; BrasilFil: Borgonovo, Tamara. Pontificia Universidad Catolica de Parana; BrasilFil: Cruz, Fernando E. S.. National Institute of Cardiology; BrasilFil: Miriuka, Santiago Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Medei, Emiliano H.. Universidade Federal do Rio de Janeiro; BrasilFil: Campos de Carvalho, Antonio C.. Universidade Federal do Rio de Janeiro; Brasil. National Institute of Cardiology; Brasil. National Institute for Science and Technology in Regenerative Medicine; BrasilFil: Carvalho, Adriana B.. Universidade Federal do Rio de Janeiro; Brasil. National Institute for Science and Technology in Regenerative Medicine; Brasi

Early Effects of Extracellular Vesicles Secreted by Adipose Tissue Mesenchymal Cells in Renal Ischemia Followed by Reperfusion: Mechanisms Rely on a Decrease in Mitochondrial Anion Superoxide Production

Acute kidney injury (AKI) caused by ischemia followed by reperfusion (I/R) is characterized by intense anion superoxide (O2•−) production and oxidative damage. We investigated whether extracellular vesicles secreted by adipose tissue mesenchymal cells (EVs) administered during reperfusion can suppress the exacerbated mitochondrial O2•− formation after I/R. We used Wistar rats subjected to bilateral renal arterial clamping (30 min) followed by 24 h of reperfusion. The animals received EVs (I/R + EVs group) or saline (I/R group) in the kidney subcapsular space. The third group consisted of false-operated rats (SHAM). Mitochondria were isolated from proximal tubule cells and used immediately. Amplex Red™ was used to measure mitochondrial O2•− formation and MitoTracker™ Orange to evaluate inner mitochondrial membrane potential (Δψ). In vitro studies were carried out on human renal proximal tubular cells (HK-2) co-cultured or not with EVs under hypoxic conditions. Administration of EVs restored O2•− formation to SHAM levels in all mitochondrial functional conditions. The gene expression of catalase and superoxide dismutase-1 remained unmodified; transcription of heme oxygenase-1 (HO-1) was upregulated. The co-cultures of HK-2 cells with EVs revealed an intense decrease in apoptosis. We conclude that the mechanisms by which EVs favor long-term recovery of renal structures and functions after I/R rely on a decrease of mitochondrial O2•− formation with the aid of the upregulated antioxidant HO-1/Nuclear factor erythroid 2-related factor 2 system, thus opening new vistas for the treatment of AKI

Adipose-Derived Stromal Cell Therapy Improves Cardiac Function After Coronary Occlusion in Rats

Recent studies have identified adipose tissue as a new source of mesenchymal stem cells for therapy. The purpose of this study was to investigate the therapy with adipose-derived stromal cells (ASCs) in a rat model of healed myocardial infarction (MI). ASCs from inguinal subcutaneous adipose tissue of male Wistar rats were isolated by enzymatic digestion and filtration. Cells were then cultured until passage 3. Four weeks after ligation of the left coronary artery of female rats, a suspension of either 100 mu l with phosphate-buffered saline (PBS)+Matrigel+2x10(6) ASCs labeled with Hoechst (n=11) or 100 mu l of PBS+Matrigel (n=10) was injected along the borders of the ventricular wall scar tissue. A sham-operated group (n=5) was submitted to the same surgical procedure except permanent ligation of left coronary artery. Cardiac performance was assessed by electro- and echocardiogram. Echo was performed prior to injections (baseline. BL) and 6 weeks after injections (follow-up, FU), and values after treatment were normalized by values obtained before treatment. Hemodynamic measurements were performed 6 weeks after injections. All infarcted animals exhibited cardiac function impairment. Ejection fraction (EF), shortening fractional area (SFA), and left ventricular akinesia (LVA) were similar between infarcted groups before treatment. Six weeks after therapy. ASC group showed significant improvement in all three ECHO indices in comparison to vehicle group. In anesthetized animals dp/dt(+) was also significantly higher in ASCs when compared to vehicle. In agreement with functional improvement, scar area was diminished in the ASC group. We conclude that ASCs improve cardiac function in infarcted rats when administered directly to the myocardium

Granulocyte-colony Stimulating Factor Treatment of Chronic Myocardial Infarction

The aim of this study was to investigate the impact of granulocyte-colony stimulating factor (G-CSF) administration on cardiac function of rats with chronic myocardial infarction through two different protocols: high dose short term and low dose long term protocols. Wistar rats were submitted to MI surgery and after 4 weeks they received recombinant human G-CSF (Filgrastim) or vehicle subcutaneously. We tested the classical protocol (50 A mu g/kg/day during 7 days) and the long term low dose treatment (four cycles of 5 days of 10 A mu g/kg/day). Cardiac performance was evaluated before, 4 and 6 weeks after G-CSF injections by electro- and echocardiography, hemodynamic and treadmill exercise test. All infarcted groups exhibited impaired function compared to sham operated animals. Moreover, all cardiac functional parameter were not different between G-CSF and Vehicle group at resting conditions as well as after treadmill exercise stress test, despite intense white blood cell mobilization in both protocols at all time points. Hypertrophy was not different and infarct size was similar in histological analysis These data clearly show that G-CSF treatment was unable to restore cardiac function impaired by myocardial infarction either with classical approach or long term low dose administration