scholarly journals The Use of iPSC-Derived Cardiomyocytes and Optical Mapping for Erythromycin Arrhythmogenicity Testing

2019 ◽  
Author(s):  
A.D. Podgurskaya ◽  
V.A. Tsvelaya ◽  
M.M. Slotvitsky ◽  
E.V. Dementyeva ◽  
K.R. Valetdinova ◽  
...  
Keyword(s):  
Conduction Velocity ◽  
Potassium Current ◽  
Optical Mapping ◽  
Capture Rate ◽  
Torsade De Pointes ◽  
Induced Pluripotent Stem Cell ◽  
Experimental Models ◽  
Mapping Method ◽  
Neonatal Rat ◽  
Induced Pluripotent

AbstractErythromycin is an antibiotic that prolongs the QT-interval and causes Torsade de Pointes (TdP) by blocking the rapid delayed rectifying potassium current (IKr) without affecting either the slow delayed rectifying potassium current (IKs) or inward rectifying potassium current (IK1). Erythromycin exerts this effect in the range of 1.5 μM–100 μM. However, the mechanism of action underlying its cardiotoxic effect and its role in the induction of arrhythmias, especially in multicellular cardiac experimental models, remain unclear. In this study the re-entry formation, conduction velocity, and maximum capture rate were investigated in a monolayer of human induced pluripotent stem cell (iPSC)-derived cardiomyocytes from a healthy donor and in a neonatal rat ventricular myocyte (NRVM) monolayer using the optical mapping method under erythromycin concentrations of 15, 30, and 45 μM. In the monolayer of human iPSC-derived cardiomyocytes, the conduction velocity (CV) varied up to 12±9% at concentrations of 15–45 μM as compared with that of the control, whereas the maximum capture rate (MCR) declined substantially up to 28±12% (p < 0.05). In contrast, the tests on the NRVM monolayer showed no significant effect on the MCR. The results of the arrhythmogenicity test provided evidence for a “window” of concentrations of the drug (15 to 30 μM) at which the probability of re-entry increased.

scholarly journals Doxorubicin induces caspase-mediated proteolysis of KV7.1

2018 ◽  
Author(s):  
Anne Strigli ◽  
Christian Raab ◽  
Sabine Hessler ◽  
Tobias Huth ◽  
Adam J. T. Schuldt ◽  
...  
Keyword(s):  
Posttranslational Modification ◽  
Potassium Current ◽  
Induced Pluripotent Stem Cell ◽  
Neonatal Rat ◽  
Loss Of Function ◽  
Long Qt ◽  
Human Cardiomyocytes ◽  
Induced Pluripotent ◽  
Lqt Syndrome ◽  
Cardiac Potassium Current

AbstractThe voltage-gated potassium channel Kv7.1 (KCNQ1) co-assembles with KCNE1 to generate the cardiac potassium current IKs. Gain- and loss-of-function mutations in KCNQ1 are associated with atrial fibrillation and long-QT (LQT) syndrome, respectively, highlighting the importance of modulating IKS activity for proper cardiac function. On a post-translational level, IKS can be regulated by phosphorylation, ubiquitination and sumoylation. Here, we report proteolysis of Kv7.1 as a novel, irreversible posttranslational modification. The identification of two C-terminal fragments (CTF1 and CTF2) of Kv7.1 led us to identify an aspartate critical for the generation of CTF2 and caspases as responsible for mediating Kv7.1 proteolysis. Activating caspases by apoptotic stimuli significantly reduced Kv7.1/KCNE1 currents, which was abrogated in cells expressing caspase-resistant Kv7.1 D459A/KCNE1 channels. An increase in cleavage of Kv7.1 could be detected in the case of LQT mutation G460S, which is located adjacent to the cleavage site. Application of apoptotic stimuli or doxorubicin-induced cardiotoxicity provoked caspase-mediated cleavage of endogenous Kv7.1 in human cardiomyocytes. In summary, our findings establish caspases as novel regulatory components for modulating Kv7.1 activity which may have important implications for the molecular mechanism of doxorubicin-induced cardiotoxicity.Non-standard Abbreviations and AcronymsCamcalmodulinEBCequilibrium buffer contentLQT syndromelong QT syndromeNRVMNeonatal rat ventricular cardiomyocyteshiPSC-CMshuman induced pluripotent stem cell-derived cardiomyocytes

scholarly journals Tissue engineering of functional cardiac muscle: molecular, structural, and electrophysiological studies

2001 ◽  
Vol 280 (1) ◽  
pp. H168-H178 ◽  
Author(s):  
M. Papadaki ◽  
N. Bursac ◽  
R. Langer ◽  
J. Merok ◽  
G. Vunjak-Novakovic ◽  
...  
Keyword(s):  
Cardiac Muscle ◽  
Cardiac Myocytes ◽  
Conduction Velocity ◽  
Capture Rate ◽  
Signal Amplitude ◽  
Model System ◽  
Neonatal Rat ◽  
Excitation Threshold ◽  
Electrophysiological Properties

The primary aim of this study was to relate molecular and structural properties of in vitro reconstructed cardiac muscle with its electrophysiological function using an in vitro model system based on neonatal rat cardiac myocytes, three-dimensional polymeric scaffolds, and bioreactors. After 1 wk of cultivation, we found that engineered cardiac muscle contained a 120- to 160-μm-thick peripheral region with cardiac myocytes that were electrically connected through gap junctions and sustained macroscopically continuous impulse propagation over a distance of 5 mm. Molecular, structural, and electrophysiological properties were found to be interrelated and depended on specific model system parameters such as the tissue culture substrate, bioreactor, and culture medium. Native tissue and the best experimental group (engineered cardiac muscle cultivated using laminin-coated scaffolds, rotating bioreactors, and low-serum medium) were comparable with respect to the conduction velocity of propagated electrical impulses and spatial distribution of connexin43. Furthermore, the structural and electrophysiological properties of the engineered cardiac muscle, such as cellularity, conduction velocity, maximum signal amplitude, capture rate, and excitation threshold, were significantly improved compared with our previous studies.

scholarly journals Cardiomyocyte functional screening: interrogating comparative electrophysiology of high-throughput model cell systems

2019 ◽  
Vol 317 (6) ◽  
pp. C1256-C1267 ◽  
Author(s):  
Simon P. Wells ◽  
Helen M. Waddell ◽  
Choon Boon Sim ◽  
Shiang Y. Lim ◽  
Gabriel B. Bernasochi ◽  
...  
Keyword(s):  
Conduction Velocity ◽  
High Throughput ◽  
Microelectrode Array ◽  
Ventricular Myocytes ◽  
Field Potential ◽  
Induced Pluripotent Stem Cell ◽  
Neonatal Rat ◽  
Functional Screening ◽  
Electrophysiological Properties

Cardiac arrhythmias of both atrial and ventricular origin are an important feature of cardiovascular disease. Novel antiarrhythmic therapies are required to overcome current drug limitations related to effectiveness and pro-arrhythmia risk in some contexts. Cardiomyocyte culture models provide a high-throughput platform for screening antiarrhythmic compounds, but comparative information about electrophysiological properties of commonly used types of cardiomyocyte preparations is lacking. Standardization of cultured cardiomyocyte microelectrode array (MEA) experimentation is required for its application as a high-throughput platform for antiarrhythmic drug development. The aim of this study was to directly compare the electrophysiological properties and responses to isoproterenol of three commonly used cardiac cultures. Neonatal rat ventricular myocytes (NRVMs), immortalized atrial HL-1 cells, and custom-generated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were cultured on microelectrode arrays for 48–120 h. Extracellular field potentials were recorded, and conduction velocity was mapped in the presence/absence of the β-adrenoceptor agonist isoproterenol (1 µM). Field potential amplitude and conduction velocity were greatest in NRVMs and did not differ in cardiomyocytes isolated from male/female hearts. Both NRVMs and hiPSC-CMs exhibited longer field potential durations with rate dependence and were responsive to isoproterenol. In contrast, HL-1 cells exhibited slower conduction and shorter field potential durations and did not respond to 1 µM isoproterenol. This is the first study to compare the intrinsic electrophysiologic properties of cultured cardiomyocyte preparations commonly used for in vitro electrophysiology assessment. These findings offer important comparative data to inform methodological approaches in the use of MEA and other techniques relating to cardiomyocyte functional screening investigations of particular relevance to arrhythmogenesis.

scholarly journals An in vitro bioengineered model of the human arterial neurovascular unit to study neurodegenerative diseases

2020 ◽  
Author(s):  
Jerome Robert ◽  
Nicholas Weilinger ◽  
Li-Ping Zao ◽  
Stefano Cataldi ◽  
Emily Button ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Cortical Neurons ◽  
Neurovascular Unit ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Experimental Models ◽  
Flow Conditions ◽  
Anatomy And Physiology ◽  
Induced Pluripotent

Abstract Introduction: The neurovascular unit (NVU) – the interaction between the neurons and the cerebrovasculature – is increasingly important to interrogate through human-based experimental models. Although advanced models of cerebral capillaries have been developed in the last decade, there is currently noin vitro3-dimensional (3D) perfusible model of the human cortical arterial NVU. Method: We used a tissue-engineering technique to develop a scaffold-directed, perfusible, 3D human NVU that is cultured in native-like flow conditions that mimics the anatomy and physiology of cortical penetrating arteries. Results: This system, composed of primary human vascular cells (endothelial cells, smooth muscle cells and astrocytes) and induced pluripotent stem cell (iPSC) derived neurons, demonstrates a physiological multilayer organization of the involved cell types. It also reproduces key characteristics of cortical neurons and astrocytes, as well as the formation of a selective and functional endothelial barrier. We further provide proof-of-principle that our in vitro human arterial NVU may be suitable to study neurodegenerative diseases such as Alzheimer’s disease (AD), as we report both phosphorylated tau and beta-amyloid accumulation in our model over time. Finally, we show that our arterial NVU model enables the study of neuronal and glial fluid biomarkers. Conclusion: This model is a suitable tool to investigate arterial NVU functions such as neuronal electrophysiology in health and disease. Further the design of platform allows culture under native-like flow conditions for extended periods of time and yields sufficient tissue and media for downstream immunohistochemistry and biochemistry analyses.

scholarly journals An in vitro bioengineered model of the human arterial neurovascular unit to study neurodegenerative diseases

2020 ◽  
Author(s):  
Jerome Robert ◽  
Nicholas Weilinger ◽  
Li-Ping Zao ◽  
Stefano Cataldi ◽  
Emily Button ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Cortical Neurons ◽  
Neurovascular Unit ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Experimental Models ◽  
Flow Conditions ◽  
Anatomy And Physiology ◽  
Induced Pluripotent

Abstract Introduction: The neurovascular unit (NVU) – the interaction between the neurons and the cerebrovasculature – is increasingly important to interrogate through human-based experimental models. Although advanced models of cerebral capillaries have been developed in the last decade, there is currently no in vitro 3-dimensional (3D) perfusible model of the human cortical arterial NVU.Method: We used a tissue-engineering technique to develop a scaffold-directed, perfusible, 3D human NVU that is cultured in native-like flow conditions that mimics the anatomy and physiology of cortical penetrating arteries.Results: This system, composed of primary human vascular cells (endothelial cells, smooth muscle cells and astrocytes) and induced pluripotent stem cell (iPSC) derived neurons, demonstrates a physiological multilayer organization of the involved cell types. It reproduces key characteristics of cortical neurons and astrocytes and enables formation of a selective and functional endothelial barrier. We provide proof-of-principle data showing that this in vitro human arterial NVU may be suitable to study neurovascular components of neurodegenerative diseases such as Alzheimer’s disease (AD), as endogenously produced phosphorylated tau and beta-amyloid accumulate in the model over time. Finally, neuronal and glial fluid biomarkers relevant to neurodegenerative diseases are measurable in our arterial NVU model.Conclusion: This model is a suitable research tool to investigate arterial NVU functions in healthy and disease states. Further, the design of the platform allows culture under native-like flow conditions for extended periods of time and yields sufficient tissue and media for downstream immunohistochemistry and biochemistry analyses.

Abstract 342: Serum Albumin Hydrogels Alter Excitation-Contraction Coupling in Neonatal Rat Myocytes and Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Eleanor J Humphrey ◽  
Manuel M Mazo ◽  
Nadav Amdursky ◽  
Nicholas S Peters ◽  
Molly M Stevens ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Serum Albumin ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Neonatal Rat ◽  
Calcium Handling ◽  
Time To Peak ◽  
Induced Pluripotent ◽  
Reduced Time

Tissue engineering provides a promising method of introducing functional cardiomyocytes (CMs) to damaged myocardium after myocardial infarction; however, finding a biocompatible construct with the chemical and mechanical properties capable of supporting CM function is challenging. Serum albumin hydrogels are novel autogenic scaffolds with elastic properties that can be tailored to mimic the stiffness of native adult myocardium. We assessed the hypothesis that culturing immature CMs on these serum albumin hydrogels would affect CM gene expression and calcium handling. Neonatal cardiomyocyte (NRVM) viability was maintained for at least 14 days on the hydrogels, with clear sarcomeric striations. Cardiac gene expression was quantified using RT-qPCR and demonstrated an up regulation in many genes of cells cultured on hydrogels compared to glass (e.g. relative expression (log 2-ΔΔCt) of ryanodine receptor 2: glass= -2.3±0.5, hydrogel= -0.3±0.1,p<0.01; connexin 43:glass= -1.7±0.5, hydrogel= 0.3±0.1,p<0.01,n=4-6). Compared to glass, NRVMs on hydrogels have an increased time to peak of the calcium transients measured using Fluo-4AM and field stimulated at 1 Hz (tp glass=38±3 ms, tp hydrogel= 54±2 ms, p<0.01,n=4-6). Compared to glass the hydrogels also have a reduced time 50% decay (t50 glass=108±13 ms, t50 hydrogel=78±6 ms, p<0.05,n=4-6) and 80% decay (t80 glass=217±19 ms, t80 hydrogel= 152±10 ms,p<0.05,n=4-6). Human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) were cultured on the hydrogels for up to 28 days. Calcium handling was faster in the iPSC-CMs cultured on the hydrogels in comparison to glass with a reduced time to peak (tp glass=281±43 ms, tp hydrogel= 186±8 ms, p<0.05, n=4) and time to 50% decay (t50 glass=269±15 ms, t50 hydrogel=204±10 ms,p<0.01,n=4) and 90% decay (t90 glass=535±33 ms, t90 hydrogel=397±19 ms, p<0.01,n=4). The serum albumin hydrogels are compatible with NRVM and iPSC-CM culture for at least 28 days. We demonstrate that the serum albumin hydrogels have significant effects on CM calcium cycling and have the potential for use in myocardial repair. Further study is required to determine the mechanisms involved in calcium handling alterations and then assess this engineered patch in vivo for cardiac repair.

Abstract 477: Modeling the Effect of TBX5 Downregulation on Human Atrial Conduction Using Induced Pluripotent Stem Cell-derived Cardiomyocytes

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Sherri M Biendarra-tiegs ◽  
Sergey Yechikov ◽  
Laura Houshmand ◽  
R. E Gonzalez ◽  
Zhi Hong Lu ◽  
...  
Keyword(s):  
Stem Cell ◽  
Conduction Velocity ◽  
Heart Development ◽  
Pluripotent Stem Cell ◽  
Microelectrode Array ◽  
Association Studies ◽  
Induced Pluripotent Stem Cell ◽  
Immunofluorescent Staining ◽  
Genome Wide Association Studies ◽  
Induced Pluripotent

Atrial fibrillation (AF) poses a notable healthcare burden due to a high incidence in the increasing population over age 65 and limitations of current treatment approaches. One challenge to effectively treat AF is patient-to-patient heterogeneity in the underlying mechanisms of disease. Therefore, a better understanding of AF pathogenesis and more personalized approaches to therapy could reduce risk of side effects and improve therapeutic efficacy. Genome wide association studies (GWAS) have revealed several candidate genes for AF including TBX5 , which encodes for a transcription factor involved in heart development. While work in animal models suggests that loss of TBX5 promotes atrial arrythmias, experimental evidence in human cells is lacking. We created an in vitro model of human atrial conduction using day 60+ induced pluripotent stem cell-derived atrial-like cardiomyocytes (iPSC-aCMs) differentiated from three established healthy iPSC lines. Over 90% atrial-like purity (out of 350+ alpha-actinin positive cardiomyocytes) could be achieved based on MLC2v-/MLC2a+ immunofluorescent staining. TBX5 knockdown via esiRNA resulted in downregulation of genes related to conduction velocity ( GJA5 and SCN5A ), consistent with an enhanced risk of AF. Single cell optical electrophysiology demonstrated slightly reduced action potential amplitude and upstroke velocity for TBX5 knockdown cells versus GFP esiRNA controls, suggesting a functional effect of SCN5A downregulation. Additionally, microelectrode array studies have revealed a trend towards slowed conduction velocity with TBX5 knockdown compared to GFP esiRNA controls (13.1±3.0 cm/s vs 17.0±3.8 cm/s respectively). By further investigating the functional effects of modulating transcription factors such as TBX5 in iPSC-aCMs, our results provide enhanced insight into the regulation of atrial conduction and identify potential AF-related pathways for therapeutic targeting.

scholarly journals Human Erbb2-induced Erk Activity Robustly Stimulates Cycling and Functional Remodeling of Rat and Human Cardiomyocytes

2020 ◽  
Author(s):  
Nicholas Strash ◽  
Sophia DeLuca ◽  
Geovanni Janer Carattini ◽  
Soon Chul Heo ◽  
Ryne Gorsuch ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Induced Pluripotent Stem Cell ◽  
Neonatal Rat ◽  
Mutant Form ◽  
Cardiac Tissues ◽  
Human Cardiomyocytes ◽  
Heart Repair ◽  
Induced Pluripotent ◽  
Species Specific

Multiple mitogenic pathways capable of promoting mammalian cardiomyocyte (CM) proliferation have been identified as potential candidates for functional heart repair following myocardial infarction. However, it is unclear whether the effects of these mitogens are species-specific and how they directly compare in the same cardiac setting. Here, we examined how CM-specific lentiviral expression of various candidate mitogens affects human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and neonatal rat ventricular myocytes (NRVMs) in vitro. In 2D-cultured CMs from both species, and in highly mature 3D-engineered cardiac tissues generated from NRVMs, a constitutively-active mutant form of the human gene Erbb2 (cahErbb2) was the most potent tested mitogen. Persistent expression of cahErbb2 induced CM proliferation, sarcomere loss, and remodeling of tissue structure and function, which were attenuated by small molecule inhibitors of Erk signaling. These results suggest transient activation of Erbb2/Erk axis in cardiomyocytes as a potential strategy for regenerative heart repair.

scholarly journals An in vitro bioengineered model of the human arterial neurovascular unit to study neurodegenerative diseases

2020 ◽  
Vol 15 (1) ◽  
Author(s):  
Jerome Robert ◽  
Nicholas L. Weilinger ◽  
Li-Ping Cao ◽  
Stefano Cataldi ◽  
Emily B. Button ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Cortical Neurons ◽  
Neurovascular Unit ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Experimental Models ◽  
Flow Conditions ◽  
Anatomy And Physiology ◽  
Induced Pluripotent

Abstract Introduction The neurovascular unit (NVU) – the interaction between the neurons and the cerebrovasculature – is increasingly important to interrogate through human-based experimental models. Although advanced models of cerebral capillaries have been developed in the last decade, there is currently no in vitro 3-dimensional (3D) perfusible model of the human cortical arterial NVU. Method We used a tissue-engineering technique to develop a scaffold-directed, perfusible, 3D human NVU that is cultured in native-like flow conditions that mimics the anatomy and physiology of cortical penetrating arteries. Results This system, composed of primary human vascular cells (endothelial cells, smooth muscle cells and astrocytes) and induced pluripotent stem cell (iPSC) derived neurons, demonstrates a physiological multilayer organization of the involved cell types. It reproduces key characteristics of cortical neurons and astrocytes and enables formation of a selective and functional endothelial barrier. We provide proof-of-principle data showing that this in vitro human arterial NVU may be suitable to study neurovascular components of neurodegenerative diseases such as Alzheimer’s disease (AD), as endogenously produced phosphorylated tau and beta-amyloid accumulate in the model over time. Finally, neuronal and glial fluid biomarkers relevant to neurodegenerative diseases are measurable in our arterial NVU model. Conclusion This model is a suitable research tool to investigate arterial NVU functions in healthy and disease states. Further, the design of the platform allows culture under native-like flow conditions for extended periods of time and yields sufficient tissue and media for downstream immunohistochemistry and biochemistry analyses.

scholarly journals Human Erbb2-induced Erk activity robustly stimulates cycling and functional remodeling of rat and human cardiomyocytes

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Nicholas Strash ◽  
Sophia DeLuca ◽  
Geovanni L Janer Carattini ◽  
Soon Chul Heo ◽  
Ryne Gorsuch ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Induced Pluripotent Stem Cell ◽  
Neonatal Rat ◽  
Mutant Form ◽  
Cardiac Tissues ◽  
Human Cardiomyocytes ◽  
Heart Repair ◽  
Induced Pluripotent ◽  
Species Specific

Multiple mitogenic pathways capable of promoting mammalian cardiomyocyte (CM) proliferation have been identified as potential candidates for functional heart repair following myocardial infarction. However, it is unclear whether the effects of these mitogens are species-specific and how they directly compare in the same cardiac setting. Here, we examined how CM-specific lentiviral expression of various candidate mitogens affects human induced pluripotent stem cell-derived CMs (hiPSC-CMs) and neonatal rat ventricular myocytes (NRVMs) in vitro. In 2D-cultured CMs from both species, and in highly mature 3D-engineered cardiac tissues generated from NRVMs, a constitutively-active mutant form of the human gene Erbb2 (cahErbb2) was the most potent tested mitogen. Persistent expression of cahErbb2 induced CM proliferation, sarcomere loss, and remodeling of tissue structure and function, which were attenuated by small molecule inhibitors of Erk signaling. These results suggest transient activation of Erbb2/Erk axis in cardiomyocytes as a potential strategy for regenerative heart repair.

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