scholarly journals Optical Investigation of Action Potential and Calcium Handling Maturation of hiPSC-Cardiomyocytes on Biomimetic Substrates

2019 ◽  
Vol 20 (15) ◽  
pp. 3799 ◽  
Author(s):  
Josè Manuel Pioner ◽  
Lorenzo Santini ◽  
Chiara Palandri ◽  
Daniele Martella ◽  
Flavia Lupi ◽  
...  
Keyword(s):  
Action Potential ◽  
Calcium Transient ◽  
Optical Measurements ◽  
Calcium Handling ◽  
Optical Investigation ◽  
Adrenergic Stimulation ◽  
Ventricular Tissue ◽  
Custom Made ◽  
Induced Pluripotent

Cardiomyocytes from human induced pluripotent stem cells (hiPSC-CMs) are the most promising human source with preserved genetic background of healthy individuals or patients. This study aimed to establish a systematic procedure for exploring development of hiPSC-CM functional output to predict genetic cardiomyopathy outcomes and identify molecular targets for therapy. Biomimetic substrates with microtopography and physiological stiffness can overcome the immaturity of hiPSC-CM function. We have developed a custom-made apparatus for simultaneous optical measurements of hiPSC-CM action potential and calcium transients to correlate these parameters at specific time points (day 60, 75 and 90 post differentiation) and under inotropic interventions. In later-stages, single hiPSC-CMs revealed prolonged action potential duration, increased calcium transient amplitude and shorter duration that closely resembled those of human adult cardiomyocytes from fresh ventricular tissue of patients. Thus, the major contribution of sarcoplasmic reticulum and positive inotropic response to β-adrenergic stimulation are time-dependent events underlying excitation contraction coupling (ECC) maturation of hiPSC-CM; biomimetic substrates can promote calcium-handling regulation towards adult-like kinetics. Simultaneous optical recordings of long-term cultured hiPSC-CMs on biomimetic substrates favor high-throughput electrophysiological analysis aimed at testing (mechanistic hypothesis on) disease progression and pharmacological interventions in patient-derived hiPSC-CMs.

Characterizing the contribution of voltage- and calcium-dependent coupling to action potential stability: implications for repolarization alternans

2007 ◽  
Vol 293 (4) ◽  
pp. H2109-H2118 ◽  
Author(s):  
Peter N. Jordan ◽  
David J. Christini
Keyword(s):  
Action Potential ◽  
Calcium Imaging ◽  
Calcium Transient ◽  
Calcium Handling ◽  
Calcium Dependent ◽  
Relative Contribution ◽  
Real Time Measurement ◽  
Transmembrane Voltage ◽  
Voltage Clamping ◽  
Potential Stability

Experiments have provided suggestive but inconclusive insights into the relative contributions of transmembrane voltage and intracellular calcium handling to the development of cardiac electrical instabilities such as repolarization alternans. In this study, we applied a novel combination of techniques (action potential voltage clamping, calcium-transient clamping, and stability analysis) to cardiac cell models to more clearly determine the roles that voltage- and calcium-dependent coupling play in regulating action potential stability and the development of alternans subsequent to the loss of stability. Using these techniques, we are able to demonstrate that voltage- and calcium-dependent coupling exhibit varying degrees of influence on action potential stability across models. Our results indicate that cellular dynamic instabilities such as alternans may be initiated by either voltage- or calcium-dependent mechanisms or by some combination of the two. Based on these modeling results, we propose novel single-cell experiments that incorporate action-potential voltage clamping, calcium imaging, and real-time measurement of action potential stability. These experiments will make it possible to experimentally determine the relative contribution of voltage coupling to the regulation of action potential stability in real cardiac myocytes, thereby providing further insights into the mechanism of alternans.

scholarly journals The fungicide Tebuconazole induces electromechanical cardiotoxicity in murine hearts

2021 ◽  
Author(s):  
Artur Santos-Miranda ◽  
Julliane V Joviano-Santos ◽  
Taynara Cruz-Nascimento ◽  
Diego Santos Souza ◽  
Leisiane Marques ◽  
...  
Keyword(s):  
Action Potential ◽  
Cardiac Tissue ◽  
Ex Vivo ◽  
Calcium Transient ◽  
Acute Exposure ◽  
Sodium Calcium Exchanger ◽  
Whole Cell Patch Clamp ◽  
Toxicological Studies ◽  
Qt Interval Duration

Tebuconazole (TEB) is an important fungicide that belongs to the triazole family. It is largely applied in agriculture and its use has increased in the last decade. Since TEB is stable in water and soil, long-term exposure of humans to this pesticide is a real threat. Acute toxicological studies to uncover the toxicity of TEB are limited, and there is evidence of an association between long-term exposure to TEB and damage of several biological systems, including hepatotoxicity and cardiotoxicity. In this paper, the effects of acute exposure of cardiomyocytes and murine hearts to TEB were addressed to elucidate its impact on electromechanical properties of the cardiac tissue. In whole-cell patch-clamp records, TEB inhibited both the total outward potassium current (IC50=5.7±1.5 μmol.l−1) and the L-type calcium current (IC50=33.2±7.4 μmol.l−1). Acute exposure to TEB at 30 μmol.l−1 prolonged the action potential duration as well as an induced out-of-pace action potential, and increased the sodium/calcium exchanger current in its forward and reverse modes. Moreover, sarcomere shortening and calcium transient in isolated cardiomyocytes was enhanced when cells were exposed to TEB at 30 μmol.l−1. In ex vivo experiments, TEB 30 μmol.l−1 caused significant electrocardiogram remodeling with prolonged PR, QRS, and QT interval duration. Accordingly, TEB exposure was prone to the appearance of arrhythmias. Combined, our results demonstrate that acute TEB exposure affects the cardiomyocyte's electro-contractile properties and triggers the appearance of ECG abnormalities, including conduction defects and arrhythmias.

scholarly journals Proarrhythmic Effects of Electrolyte Imbalance in Virtual Human Atrial and Ventricular Cardiomyocytes

2020 ◽  
Author(s):  
Sanjay R Kharche
Keyword(s):  
Action Potential ◽  
Action Potential Duration ◽  
Calcium Transient ◽  
Additional Treatment ◽  
Electrolyte Imbalance ◽  
Serum Electrolytes ◽  
Model Response ◽  
Arrhythmia Mechanisms ◽  
In Silico Modeling

Dialysis is prescribed to renal failure patients as a long-term chronic treatment. Whereas dialysis therapeutically normalizes serum electrolytes and removes small toxin molecules, it fails to alleviate fibroblast induced structural fibrosis, and unresponsive uremia. The simultaneous presence of altered electrolytes and fibrosis or uremia is thought to be pro-arrhythmogenic. This study explored potential arrhythmogenesis under pre-dialysis (high electrolyte levels) and post-dialysis (low physiological electrolyte levels) in the presence of fibrosis and uremia in human atrial and ventricular model cardiomyocytes.Two validated human cardiomyocyte models were used in this study that permitted simulation of cardiac atrial and ventricular detailed electrophysiology. Pathological conditions simulating active fibrosis and uremia were implemented in both models. Pre- and post-dialysis conditions were simulated using high and low electrolyte levels respectively. Arrythmogenesis was quantified by computing restitution curves that permitted identification of action potential duration and calcium transient alternans instabilities. In comparison to control conditions, fibrosis abbreviated action potential durations while uremia prolonged the same. Under pre-dialysis conditions, an elevation of serum electrolyte levels caused action potential durations to be abbreviated under both fibrosis and uremia. Alternans instability was observed in the ventricular cardiomyocyte model. Under post-dialysis conditions, lower levels of serum electrolytes promoted an abbreviated action potential duration under fibrosis but caused a large increase of the control and uremic action potential durations. Alternans instabilities were observed in the atrial cardiomyocyte model under post-dialysis conditions at physiological heart rates. The calcium transient restitution showed similar alternans instabilities. Co-existing conditions such as fibrosis and uremia in the presence of unphysiological electrolyte levels promote arrhythmogenesis and may require additional treatment to improve dialysis outcomes.Clinical Relevance. Knowledge of model response to clinically relevant conditions permits use of in silico modeling to better understand and dissect underlying arrhythmia mechanisms.

scholarly journals Arrhythmic Risk Assessment of Hypokalaemia Using Human Pluripotent Stem Cell-Derived Cardiac Anisotropic Sheets

2021 ◽  
Vol 9 ◽  
Author(s):  
Bimal Gurung ◽  
Gary Tse ◽  
Wendy Keung ◽  
Ronald A. Li ◽  
Wing Tak Wong
Keyword(s):  
Stem Cell ◽  
Action Potential ◽  
Pluripotent Stem Cells ◽  
Calcium Transient ◽  
Calcium Handling ◽  
Mapping Technique ◽  
Complex Structures ◽  
Human Pluripotent Stem Cell ◽  
Exact Test ◽  
Refractory Periods

Introduction: Hypokalaemia, defined as an extracellular concentration of K+ below 3.5 mM, can cause cardiac arrhythmias by triggered or re-entrant mechanisms. Whilst these effects have been reported in animal and human stem cell-based models, to date there has been no investigation in more complex structures such as the human ventricular cardiac anisotropic sheet (hvCAS). Here, we investigated arrhythmogenicity, electrophysiological, and calcium transient (CaT) changes induced by hypokalaemia using this bioengineered platform.Methods: An optical mapping technique was applied on hvCAS derived from human pluripotent stem cells to visualize electrophysiological and CaT changes under normokalaemic (5 mM KCl) and hypokalaemic (3 mM KCl) conditions.Results: Hypokalaemia significantly increased the proportion of preparations showing spontaneous arrhythmias from 0/14 to 7/14 (Fisher’s exact test, p = 0.003). Hypokalaemia reduced longitudinal conduction velocity (CV) from 7.81 to 7.18 cm⋅s−1 (n = 9, 7; p = 0.036), transverse CV from 5.72 to 4.69 cm⋅s−1 (n = 12, 11; p = 0.030), prolonged action potential at 90% repolarization (APD90) from 83.46 to 97.45 ms (n = 13, 15; p < 0.001), increased action potential amplitude from 0.888 to 1.195 ΔF (n = 12, 14; p < 0.001) and CaT amplitude from 0.76 to 1.37 ΔF (n = 12, 13; p < 0.001), and shortened effective refractory periods from 242 to 165 ms (n = 12, 13; p < 0.001).Conclusion: Hypokalaemia exerts pro-arrhythmic effects on hvCAS, which are associated with alterations in CV, repolarization, refractoriness, and calcium handling. These preparations provide a useful platform for investigating electrophysiological substrates and for conducting arrhythmia screening.

Characterization and modelling of the abnormal electrophysiological response under beta-adrenergic stimulation in hypertrophic cardiomyopathy

EP Europace ◽  
2021 ◽  
Vol 23 (Supplement_3) ◽  
Author(s):  
R Doste ◽  
R Coppini ◽  
A Bueno-Orovio
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Action Potential ◽  
Calcium Current ◽  
Potassium Currents ◽  
Calcium Transient ◽  
Measured Data ◽  
Inactivation Kinetics ◽  
Ion Currents ◽  
Adrenergic Stimulation ◽  
Abnormal Response

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): British Heart Foundation Introduction Hypertrophic Cardiomyopathy (HCM) is the most common inheritable heart pathology and the main cause of sudden cardiac death in young adults. HCM patients often present an enhanced arrhythmogenicity that can lead to lethal arrhythmias, especially during exercise. Recent studies have shown an abnormal response of HCM myocytes to β-adrenergic stimulation (β-ARS), with prolongation of their action potential duration (APD). The mechanisms underlying this aberrant response to sympathetic stimulation remain unknown. Purpose To investigate the key ionic mechanisms underlying the HCM abnormal response to β-ARS using human-based experimental and computational methodologies. Methods Experimental ionic currents, action potential and calcium transient were recorded in human adult cardiomyocytes from control and HCM patients. Isoproterenol (10-7 mol/L) was used to elicit β-ARS. Whole-cell ruptured patch voltage clamp experiments were conducted to characterise L-type calcium and potassium currents, with recordings performed before and after 3 min of drug exposure. The latest models of human ventricular electrophysiology and beta-adrenergic receptor signalling were integrated and calibrated using the human measured data. Simulations under isoproterenol were performed to quantify the effects of β-ARS on the action potential and calcium transient. The role of the main ion currents affected by β-ARS and by HCM remodelling was evaluated. Results In vitro, isoproterenol shortened APD (-16 ± 3%) in control, while prolonging APD in HCM myocytes (+23 ± 8%). Analysis of the measured data indicated two possible mechanisms contributing to APD prolongation in HCM myocytes. Firstly, a protracted L-type calcium current, presenting slower inactivation kinetics in HCM compared to control. The relative increase of potassium currents under β-ARS was also lower in HCM myocytes. The developed in silico models of β-ARS replicated the behaviour observed in the experimental data, based on slower L-type calcium current inactivation kinetics and a smaller increase of potassium currents in HCM. In absence of β-ARS, simulated HCM cardiomyocytes exhibited prolonged APD compared to control (525 ± 88 vs 281 ± 56 ms, p < 0.001). Under β-ARS, APD in control was reduced (-16.46%), whereas APD was prolonged in HCM (+11.63%). Further analysis showed that the reduction of the potassium currents increment under β-ARS was the main cause of the APD prolongation in HCM myocytes, with L-type calcium inactivation minimally contributing to APD prolongation. Conclusions In this study we assessed the effects of β-ARS on ion currents and APD in control and HCM myocytes. Our modelling results suggest that the increase of potassium repolarising currents under β-ARS is greatly reduced in HCM cardiomyocytes, being the main mechanism underlying their APD prolongation. This APD prolongation may have severe consequences in HCM patients, increasing the risk of exercise-induced arrhythmias. Abstract Figure.

scholarly journals Long-term effects of human induced pluripotent stem cell-derived retinal cell transplantation in Pde6b knockout rats

2021 ◽  
Author(s):  
Jee Myung Yang ◽  
Sunho Chung ◽  
KyungA Yun ◽  
Bora Kim ◽  
Seongjun So ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Transplantation ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Histological Evaluation ◽  
Long Term Effects ◽  
Retinal Cells ◽  
Rpe Cells ◽  
Induced Pluripotent

AbstractRetinal degenerative disorders, including age-related macular degeneration and retinitis pigmentosa (RP), are characterized by the irreversible loss of photoreceptor cells and retinal pigment epithelial (RPE) cells; however, the long-term effect of implanting both human induced pluripotent stem cell (hiPSC)-derived RPE and photoreceptor for retinal regeneration has not yet been investigated. In this study, we evaluated the long-term effects of hiPSC-derived RPE and photoreceptor cell transplantation in Pde6b knockout rats to study RP; cells were injected into the subretinal space of the right eyes of rats before the appearance of signs of retinal degeneration at 2–3 weeks of age. Ten months after transplantation, we evaluated the cells using fundus photography, optical coherence tomography, and histological evaluation, and no abnormal cell proliferation was observed. A relatively large number of transplanted cells persisted during the first 4 months; subsequently, the number of these cells decreased gradually. Notably, immunohistochemical analysis revealed that the hiPSC-derived retinal cells showed characteristics of both RPE cells and photoreceptors of human origin after transplantation. Functional analysis of vision by scotopic electroretinogram revealed significant preservation of vision after transplantation. Our study suggests that the transplantation of hiPSC-derived retinal cells, including RPE cells and photoreceptors, has a potential therapeutic effect against irreversible retinal degenerative diseases.

scholarly journals Recurrent contracted sockets treated with personalized, three-dimensionally printed conformers and buccal grafts

2021 ◽  
pp. 112067212110000
Author(s):  
Annabel LW Groot ◽  
Jelmer S Remmers ◽  
Roel JHM Kloos ◽  
Peerooz Saeed ◽  
Dyonne T Hartong
Keyword(s):  
Three Dimensional ◽  
Case Series ◽  
Secondary Outcome ◽  
Retrospective Case ◽  
Upper Eyelid ◽  
Ocular Prosthesis ◽  
Buccal Mucosal Graft ◽  
Custom Made ◽  
3D Printed

Purpose: Recurrent contracted sockets are complex situations where previous surgeries have failed, disabling the wear of an ocular prosthesis. A combined method of surgery and long-term fixation using custom-made, three-dimensional (3D) printed conformers is evaluated. Methods: Retrospective case series of nine patients with recurrent excessive socket contraction and inability to wear a prosthesis, caused by chemical burns ( n = 3), fireworks ( n = 3), trauma ( n = 2) and enucleation and radiotherapy at childhood due to optic nerve glioma ( n = 1) with three average previous socket surgeries (range 2–6). Treatment consisted of a buccal mucosal graft and personalized 3D-printed conformer designed to be fixated to the periosteum and tarsal plates for minimal 2 months. Primary outcome was the retention of an ocular prosthesis. Secondary outcome was the need for additional surgeries. Results: Outcomes were measured at final follow-up between 7 and 36 months postoperatively (mean 20 months). Eight cases were able to wear an ocular prosthesis after 2 months. Three cases initially treated for only the upper or only the lower fornix needed subsequent surgery for the opposite fornix for functional reasons. Two cases had later surgery for cosmetic improvement of upper eyelid position. Despite pre-existing lid abnormalities (scar, entropion, lash deficiency), cosmetic outcome was judged highly acceptable in six cases because of symmetric contour and volume, and reasonably acceptable in the remaining two. Conclusions: Buccal mucosal transplant fixated with a personalized 3D-designed conformer enables retention of a well-fitted ocular prosthesis in previously failed socket surgeries. Initial treatment of both upper and lower fornices is recommended to avoid subsequent surgeries for functional reasons.

Cultured olfactory interneurons from Limax maximus: optical and electrophysiological studies of transmitter-evoked responses

1993 ◽  
Vol 69 (6) ◽  
pp. 1940-1947 ◽  
Author(s):  
L. D. Rhines ◽  
P. G. Sokolove ◽  
J. Flores ◽  
D. W. Tank ◽  
A. Gelperin
Keyword(s):  
Action Potential ◽  
Intracellular Calcium ◽  
Local Field ◽  
Local Field Potential ◽  
Field Potential ◽  
Optical Measurements ◽  
Optical Studies ◽  
Isolated Cells ◽  
Fura 2 ◽  
Limax Maximus

1. The olfactory processing network in the procerebral (PC) lobe of the terrestrial mollusk Limax maximus exhibits a coherent oscillation of local field potential that is modulated by odor input. To understand the cellular basis of this oscillation, we developed a cell culture preparation of isolated PC neurons and studied the responses of isolated cells to stimulation with neurotransmitters known to be present in the PC lobe. 2. The distribution of PC soma diameters suggests at least two different populations of neurons. Approximately 95% of isolated cells had soma diameters of 7-8 microns, with the remaining cells having larger diameters (10-15 microns). 3. Extracellular measurements of action potentials and optical measurements of intracellular calcium concentrations in fura-2-loaded cells were made. Serotonin and dopamine excited PC neurons and promoted transitions from steady to bursty activity. Both amines elicited increases in intracellular calcium, presumably concomitant with the increase in action-potential frequency. 4. Glutamate suppressed action-potential firing and reduced intracellular calcium. This effect was seen most clearly when glutamate was applied to cells excited by high potassium medium. Quisqualate is an effective glutamate agonist in this system, whereas kainate is not. 5. Combined with anatomic and biochemical data and with studies of the effects of these neurotransmitters on the oscillating local field potential of the intact PC network, the data from isolated PC neurons are consistent with the hypothesis that dopamine and serotonin modulate network dynamics, whereas glutamate is involved in generating the basic oscillation of local field potential in the PC. 6. The optical studies of fura-2-loaded cells showed that several treatments that increase the rate of action-potential production lead to elevations in intracellular calcium. Optical studies of intracellular calcium may be useful for multisite measurements of activity in the intact, oscillating PC lobe network.

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