scholarly journals Contribution of two-pore K+ channels to cardiac ventricular action potential revealed using human iPSC-derived cardiomyocytes

2017 ◽  
Vol 312 (6) ◽  
pp. H1144-H1153 ◽  
Author(s):  
Sam Chai ◽  
Xiaoping Wan ◽  
Drew M. Nassal ◽  
Haiyan Liu ◽  
Christine S. Moravec ◽  
...  
Keyword(s):  
Action Potential ◽  
Expression Profile ◽  
Cardiac Electrophysiology ◽  
Human Heart ◽  
Induced Pluripotent Stem Cell ◽  
Heart Tissue ◽  
Induced Pluripotent ◽  
Interfering Rna ◽  
Human Ipsc ◽  
Physiological Systems

Two-pore K+ (K2p) channels have been described in modulating background conductance as leak channels in different physiological systems. In the heart, the expression of K2p channels is heterogeneous with equivocation regarding their functional role. Our objective was to determine the K2p expression profile and their physiological and pathophysiological contribution to cardiac electrophysiology. Induced pluripotent stem cells (iPSCs) generated from humans were differentiated into cardiomyocytes (iPSC-CMs). mRNA was isolated from these cells, commercial iPSC-CM (iCells), control human heart ventricular tissue (cHVT), and ischemic (iHF) and nonischemic heart failure tissues (niHF). We detected 10 K2p channels in the heart. Comparing quantitative PCR expression of K2p channels between human heart tissue and iPSC-CMs revealed K2p1.1, K2p2.1, K2p5.1, and K2p17.1 to be higher expressed in cHVT, whereas K2p3.1 and K2p13.1 were higher in iPSC-CMs. Notably, K2p17.1 was significantly lower in niHF tissues compared with cHVT. Action potential recordings in iCells after K2p small interfering RNA knockdown revealed prolongations in action potential depolarization at 90% repolarization for K2p2.1, K2p3.1, K2p6.1, and K2p17.1. Here, we report the expression level of 10 human K2p channels in iPSC-CMs and how they compared with cHVT. Importantly, our functional electrophysiological data in human iPSC-CMs revealed a prominent role in cardiac ventricular repolarization for four of these channels. Finally, we also identified K2p17.1 as significantly reduced in niHF tissues and K2p4.1 as reduced in niHF compared with iHF. Thus, we advance the notion that K2p channels are emerging as novel players in cardiac ventricular electrophysiology that could also be remodeled in cardiac pathology and therefore contribute to arrhythmias. NEW & NOTEWORTHY Two-pore K+ (K2p) channels are traditionally regarded as merely background leak channels in myriad physiological systems. Here, we describe the expression profile of K2p channels in human-induced pluripotent stem cell-derived cardiomyocytes and outline a salient role in cardiac repolarization and pathology for multiple K2p channels.

scholarly journals An aged human heart tissue model showing age-related molecular and functional deterioration resembling the native heart

2018 ◽  
Author(s):  
Aylin Acun ◽  
Trung Dung Nguyen ◽  
Pinar Zorlutuna
Keyword(s):  
Human Heart ◽  
Induced Pluripotent Stem Cell ◽  
Heart Tissue ◽  
Tissue Model ◽  
Age Related ◽  
Ready Availability ◽  
Age Appropriate ◽  
Induced Pluripotent ◽  
And Function

AbstractDeaths attributed to ischemic heart disease increased by 41.7% from 1990 to 2013. This is primarily due to an increase in the aged population, however, research on cardiovascular disease (CVD) has been overlooking aging, a well-documented contributor to CVD. The field heavily depends on the use of young animals due to lower costs and ready availability, despite the prominent differences between young and aged heart structure and function. Here we present the first human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (iCM)-based, in vitro aged myocardial tissue model as an alternative research platform. Within 4 months, iCMs go through accelerated senescence and show cellular characteristics of aging. Furthermore, the model tissues fabricated using these aged iCMs, with stiffness resembling that of aged human heart, show functional and pharmacological deterioration specific to aged myocardium. Our novel tissue model with age-appropriate physiology and pathology presents a promising new platform for investigating CVD or other age-related diseases.

scholarly journals Syncytium cell growth increases IK1 contribution in human iPS-cardiomyocytes

2019 ◽  
Author(s):  
Weizhen Li ◽  
Emilia Entcheva
Keyword(s):  
Membrane Potential ◽  
Cardiac Electrophysiology ◽  
Induced Pluripotent Stem Cell ◽  
Heart Tissue ◽  
Protein Quantification ◽  
Resting Membrane Potential ◽  
Dependent Manner ◽  
All Optical ◽  
Spontaneous Frequency ◽  
Induced Pluripotent

SummaryHuman induced pluripotent stem-cell-derived cardiomyocytes (hiPS-CMs) enable cardiotoxicity testing and personalized medicine. However, their maturity is of concern, including relatively depolarized resting membrane potential and more spontaneous activity compared to adult cardiomyocytes, implicating low or lacking inward-rectifier potassium current (Ik1). Here, protein quantification confirms Ik1 expression in hiPS-CM syncytia, albeit several times lower than in adult heart tissue. We find that hiPS-CM cell culture density influences Ik1 expression and the associated electrophysiology phenotype. All-optical cardiac electrophysiology and pharmacological treatments reveal reduction of spontaneous and irregular activity in denser cultures. Blocking Ik1 with BaCl2 increased spontaneous frequency and blunted action potential upstrokes during pacing in a dose-dependent manner only in the highest-density cultures, in line with Ik1’s role in regulating the resting membrane potential. Our results emphasize the importance of syncytial growth of hiPS-CM for more physiologically-relevant phenotype and the power of all-optical electrophysiology to study cardiomyocytes in their multicellular setting.

scholarly journals Assessment of temporal functional changes and miRNA profiling of human iPSC-derived cardiomyocytes

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Naresh Kumar ◽  
Julie A. Dougherty ◽  
Heather R. Manring ◽  
Ibrahim Elmadbouh ◽  
Muhamad Mergaye ◽  
...  
Keyword(s):  
Cell Transplantation ◽  
Human Heart ◽  
Induced Pluripotent Stem Cell ◽  
Acid Oxidation ◽  
Mirna Profiling ◽  
Functional Changes ◽  
Ultrastructural Morphology ◽  
Induced Pluripotent ◽  
Human Ipsc

Abstract Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been developed for cardiac cell transplantation studies more than a decade ago. In order to establish the hiPSC-CM-based platform as an autologous source for cardiac repair and drug toxicity, it is vital to understand the functionality of cardiomyocytes. Therefore, the goal of this study was to assess functional physiology, ultrastructural morphology, gene expression, and microRNA (miRNA) profiling at Wk-1, Wk-2 & Wk-4 in hiPSC-CMs in vitro. Functional assessment of hiPSC-CMs was determined by multielectrode array (MEA), Ca2+ cycling and particle image velocimetry (PIV). Results demonstrated that Wk-4 cardiomyocytes showed enhanced synchronization and maturation as compared to Wk-1 & Wk-2. Furthermore, ultrastructural morphology of Wk-4 cardiomyocytes closely mimicked the non-failing (NF) adult human heart. Additionally, modulation of cardiac genes, cell cycle genes, and pluripotency markers were analyzed by real-time PCR and compared with NF human heart. Increasing expression of fatty acid oxidation enzymes at Wk-4 supported the switching to lipid metabolism. Differential regulation of 12 miRNAs was observed in Wk-1 vs Wk-4 cardiomyocytes. Overall, this study demonstrated that Wk-4 hiPSC-CMs showed improved functional, metabolic and ultrastructural maturation, which could play a crucial role in optimizing timing for cell transplantation studies and drug screening.

Abstract P309: Particulate Matter Increases Oxidative Stress And Shortens The Action Potential In IPS-derived Cardiomyocytes

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Mariana Argenziano ◽  
jiajia yang ◽  
Mariana Burgos Angulo ◽  
Thomas V McDonald
Keyword(s):  
Oxidative Stress ◽  
Particulate Matter ◽  
Action Potential ◽  
Cardiac Electrophysiology ◽  
Structural Changes ◽  
Hospital Admissions ◽  
Induced Pluripotent Stem Cell ◽  
Toxic Substances ◽  
Direct Role ◽  
Atp Production

Introduction: Air particulate matter (PM) represents one of the most critical environmental issues worldwide, causing more than 3 million deaths a year. In the US, hospital admissions due to heart failure (HF) increase by 0.8% for every 10 μg/m3 elevation in PM. However, the biological mechanisms behind the effects of PM on cardiovascular disease (CVD) remain poorly defined. Recent studies showed that PM 2.5 can translocate into the circulation, causing cumulative toxicity. With air pollution increasing due to human activity and the growing prevalence of HF, there is a critical need to understand PM's contributions to CVD to develop preventive treatments and novel therapeutic approaches. Hypothesis: We hypothesize that PM can exert its toxic effect by increasing oxidative stress and apoptosis and affecting cardiac electrophysiology. Methods: Three independent induced pluripotent stem cell lines (IPSC) were differentiated into cardiomyocytes (iCMs) and cultured for 30 days before treatment with 100 μg/ml of PM 2.5 for 48h. Experiments including immunostaining, qPCR, RNAseq and Multielectrode Array (MEA) were performed in control (CT) and PM-treated iCMs (PM). Results: Treatment with PM increased ROS and decreased ATP production (CT 9.9±1.2pmol vs PM 6.6±0.8pmol, p<0.01, n=20). Immunostaining showed mitochondrial fragmentation and increased expression of cleaved caspase3 without structural changes. Moreover, PM caused upregulation of the apoptotic markers P53 , PARP1 and CASP3, oxidative stress markers CYP1A1, CYP1B1 and MT2A, and cardiac markers CACNA1C together with downregulation of GJA1 . RNAseq analysis showed upregulation of Gene Ontology terms related to detoxification, response to toxic substances and oxidative stress. Upregulated KEGG pathways included oxidative phosphorylation, hypertrophic cardiomyopathy and dilated cardiomyopathy. MEA experiments revealed a decrease in the spike amplitude and conduction velocity, along with shortening of the action potential (APD90: CT 577±20ms vs. PM 489±16ms, p<0.05, n=20) and increased beat period irregularity (CT 3.2±0.7% vs. PM 13.1±1.6%, p<0.001, n=20). These electrophysiological changes were reversed by treatment with the antioxidant N-acetylcysteine. Conclusions: We conclude that PM plays a direct role in the development of CVD, causing an increase in oxidative stress and affecting the electrophysiology of the heart. Further functional studies in iCMs from HF patients will provide evidence of the effects of these changes on the phenotype of the disease.

scholarly journals Robust Expression of Functional NMDA Receptors in Human Induced Pluripotent Stem Cell-Derived Neuronal Cultures Using an Accelerated Protocol

2021 ◽  
Vol 14 ◽  
Author(s):  
Jacob B. Ruden ◽  
Mrinalini Dixit ◽  
José C. Zepeda ◽  
Brad A. Grueter ◽  
Laura L. Dugan
Keyword(s):  
Nmda Receptors ◽  
Neural Progenitor Cells ◽  
Induced Pluripotent Stem Cell ◽  
Calcium Flux ◽  
Neuronal Cultures ◽  
Mature Neurons ◽  
Health And Disease ◽  
Nervous System Function ◽  
Induced Pluripotent ◽  
Human Ipsc

N-methyl-D-aspartate (NMDA) receptors are critical for higher-order nervous system function, but in previously published protocols to convert human induced pluripotent stem cells (iPSCs) to mature neurons, functional NMDA receptors (NMDARs) are often either not reported or take an extended time to develop. Here, we describe a protocol to convert human iPSC-derived neural progenitor cells (NPCs) to mature neurons in only 37 days. We demonstrate that the mature neurons express functional NMDARs exhibiting ligand-activated calcium flux, and we document the presence of NMDAR-mediated electrically evoked postsynaptic current. In addition to being more rapid than previous procedures, our protocol is straightforward, does not produce organoids which are difficult to image, and does not involve co-culture with rodent astrocytes. This could enhance our ability to study primate/human-specific aspects of NMDAR function and signaling in health and disease.

scholarly journals Potential Consequences of the Red Blood Cell Storage Lesion on Cardiac Electrophysiology

2020 ◽  
Vol 9 (21) ◽  
Author(s):  
Marissa Reilly ◽  
Chantal D. Bruno ◽  
Tomas M. Prudencio ◽  
Nina Ciccarelli ◽  
Devon Guerrelli ◽  
...  
Keyword(s):  
Stem Cell ◽  
Blood Cell ◽  
Red Blood Cell ◽  
Cardiac Electrophysiology ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Cardiac Complications ◽  
Blood Products ◽  
Storage Lesion ◽  
Induced Pluripotent

Background The red blood cell (RBC) storage lesion is a series of morphological, functional, and metabolic changes that RBCs undergo following collection, processing, and refrigerated storage for clinical use. Since the biochemical attributes of the RBC unit shifts with time, transfusion of older blood products may contribute to cardiac complications, including hyperkalemia and cardiac arrest. We measured the direct effect of storage age on cardiac electrophysiology and compared it with hyperkalemia, a prominent biomarker of storage lesion severity. Methods and Results Donor RBCs were processed using standard blood‐banking techniques. The supernatant was collected from RBC units, 7 to 50 days after donor collection, for evaluation using Langendorff‐heart preparations (rat) or human induced pluripotent stem cell–derived cardiomyocytes. Cardiac parameters remained stable following exposure to “fresh” supernatant from red blood cell units (day 7: 5.8±0.2 mM K + ), but older blood products (day 40: 9.3±0.3 mM K + ) caused bradycardia (baseline: 279±5 versus day 40: 216±18 beats per minute), delayed sinus node recovery (baseline: 243±8 versus day 40: 354±23 ms), and increased the effective refractory period of the atrioventricular node (baseline: 77±2 versus day 40: 93±7 ms) and ventricle (baseline: 50±3 versus day 40: 98±10 ms) in perfused hearts. Beating rate was also slowed in human induced pluripotent stem cell–derived cardiomyocytes after exposure to older supernatant from red blood cell units (−75±9%, day 40 versus control). Similar effects on automaticity and electrical conduction were observed with hyperkalemia (10–12 mM K + ). Conclusions This is the first study to demonstrate that “older” blood products directly impact cardiac electrophysiology, using experimental models. These effects are likely caused by biochemical alterations in the supernatant from red blood cell units that occur over time, including, but not limited to hyperkalemia. Patients receiving large volume and/or rapid transfusions may be sensitive to these effects.

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