iPS Cardiomyocytes Research Articles

Abstract 18445: Rigorous Mechanics in Engineered Heart Tissues

Introduction: Heart failure can develop or be exacerbated by mechanical stressors, including increased diastolic strain (preload) and increased systolic impedance (afterload). Recent advances in human induced pluripotent stem cell (iPSC) technology and tissue engineering have enabled long term culture and maturation of engineered heart tissues (EHTs). Most current platforms do not allow for mechanical assessment under a range of loading conditions. Here, we develop a technique to engineer cardiac tissues that allow for non-destructive rigorous phenotyping of tissue mechanics. Methods: Custom tissue anchors were 3D printed using Boston Micro Fabrication HTL Resin and placed within polydimethylsiloxane tissue casting molds. iPS cardiomyocytes and ventricular fibroblasts were mixed 5:1 in 2 mg/mL collagen and cast in rectangular molds. Tissues were compacted around custom tissue anchors over 14 days. Tissues were dismounted from the molds and fixed for immunostaining or hooked onto a force transducer for length-tension, force-frequency, and work-loop analysis. Results: 3D printed tissue anchors accommodated EHT molding and compaction. EHTs spontaneously contracted after 3 days in culture, and compacted to 15.9±0.8% their initial tissue area by 14 days of culture. Engineered tissues were cell dense and mature, with bands of aligned sarcomeres. During isometric testing, tissues display a physiologic length-tension relationship, with linear increases in force with stretch between L 0 (slack length) and L max (length at maximum active force generation, 4.3±0.7 mN/mm 2 ; r 2 =0.96). These are coupled with increases in contraction and relaxation velocities. EHTs were capable of mimicking physiologic work loops. Conclusions: In summary, we have shown the ability to grow and mature three-dimensional human engineered heart tissues capable of rigorous mechanical assessment, enabling future studies on load-dependent cardiac remodeling.

Abstract P2187: SARS-CoV2 Nonstructural Protein, But Not The Omicron Ba.4/5 Mutant, Suppresses YAP Transcriptional Activity In The Heart

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV2, is a worldwide epidemic resulting in global healthcare crises and strained health resources. Currently, the omicron subvariants of SARS-CoV2, BA.4 and BA.5, have become the predominant variant in circulation around the world. Compared to previous variants, omicron BA.4 and BA.5 escape antibodies created by past COVID-19 vaccines and prior infections due to spike protein mutations. However, why omicron subvariants induce milder symptoms after infection are poorly understood. Here we screened SARS-CoV2 proteins and found that one nonstructural protein suppresses the transcriptional activity of YAP, the Hippo pathway signaling effector, independent of negative regulation by upstream Lats kinases in the heart. Our findings correlate with RNA sequencing data from COVID-19 patient lung samples and iPS cardiomyocytes showing decreased expression levels of YAP target genes, which suggested that SARS-CoV2 infection may affect lung epithelial cell and neonatal cardiomyocyte proliferation. Additionally, we found that the Omicron BA.4/5 variant of this viral protein suppresses YAP activity significantly less compared to the wildtype protein whose mechanism are under exploring now. These findings reveal the previously unknown function and mechanism of SARS-CoV2 nonstructural proteins in host cells. Considering the critical role of Hippo signaling in cell proliferation, apoptosis, and immune response, our data on SARS-CoV2 protein function may partially explain the lesser toxicity of Omicron BA.4/5 to the host cells.

The Junctophilin-2 Mutation p.(Thr161Lys) Is Associated with Hypertrophic Cardiomyopathy Using Patient-Specific iPS Cardiomyocytes and Demonstrates Prolonged Action Potential and Increased Arrhythmogenicity.

Hypertrophic cardiomyopathy (HCM) is one of the most common genetic cardiac diseases; it is primarily caused by mutations in sarcomeric genes. However, HCM is also associated with mutations in non-sarcomeric proteins and a Finnish founder mutation for HCM in non-sarcomeric protein junctophilin-2 (JPH2) has been identified. This study aimed at assessing the issue of modelling the rare Finnish founder mutation in cardiomyocytes (CMs) differentiated from iPSCs; therefore, presenting the same cardiac abnormalities observed in the patients. To explore the abnormal functions in JPH2-HCM, skin fibroblasts from a Finnish patient with JPH2 p.(Thr161Lys) were reprogrammed into iPSCs and further differentiated into CMs. As a control line, an isogenic counterpart was generated using the CRISPR/Cas9 genome editing method. Finally, iPSC-CMs were evaluated for the morphological and functional characteristics associated with JPH2 mutation. JPH2-hiPSC-CMs displayed key HCM hallmarks (cellular hypertrophy, multi-nucleation, sarcomeric disarray). Moreover, JPH2-hiPSC-CMs exhibit a higher degree of arrhythmia and longer action potential duration associated with slower inactivation of calcium channels. Functional evaluation supported clinical observations, with differences in beating characteristics when compared with isogenic-hiPSC-CMs. Thus, the iPSC-derived, disease-specific cardiomyocytes could serve as a translationally relevant platform to study genetic cardiac diseases.

Development of theoretical model expressing self-beating deformation behaviour of human iPS cardiomyocytes tube

In recent years, a regenerative therapy method using human iPS cell-derived myocardial cells (hiPS-CM) has attracted attention. In the future, large-scale myocardial three-dimensional structures that apply tissue engineering technology are expected. In this study, we developed a three-dimensional structure with a pump function by the self-beating power of hiPS-CM. Then, the deformation behavior of the 3D hiPS-CM structure associated with self-pulsation was examined. The pulsatile behavior of hiPS-CM was quantitatively evaluated by using a high-speed optical measurement system with the digital image correlation method. Then, the experimental strain data was used to construct the stress-strain response with use of a theoretical active stress model originally developed by Guccione et al. A theoretical constitutive model was also developed by combining the Guccione's active stress model and the viscoelastic Maxwell model. As a result, this theoretical model stress-strain behavior showed a good agreement with the combination model of experimental strain and theoretical stress.

Circular RNA hsa_circ_105039 promotes cardiomyocyte differentiation by sponging miR‑17 to regulate cyclinD2 expression.

Previously it was found that hsa_circ_105039 was underexpressed in the heart tissue of patients with congenital heart disease (CHD). However, the function and mechanism of hsa_circ_105039 in CHD are unclear. In the present study, induced pluripotent stem (iPS) cells were differentiated into cardiomyocytes using 1% dimethyl sulfoxide (DMSO). Cell differentiation, viability, migration and apoptosis were measured before and following hsa_circ_105039 knockdown or overexpression. The results indicated that hsa_circ_105039 overexpression promoted cell differentiation, viability and migration; whereas apoptosis was simultaneously repressed. A luciferase reporter assay verified that hsa_circ_105039 acted as a sponge for microRNA (miR)-17 and that cyclinD2 was a direct target of miR-17. Furthermore, differentiation-related genes and proteins were analyzed by reverse transcription-quantitative PCR and western blotting, respectively. The results showed that hsa_circ_105039 could also upregulate the expression of differentiation-related genes and proteins, including natriuretic peptide A, cardiac troponin I, GATA-binding protein 4 and homobox transcription factor, in iPS cells. The results suggested that hsa_circ_105039 exerted a protective effect by promoting miR-17/cyclinD2 in DMSO-induced iPS cardiomyocytes, which indicated that hsa_circ_105039 is a potential key molecule for the diagnosis of CHD.

Eco-pharma research aimed at developing COVID-19 therapeutic agent

A new pandemic of pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged and its severe and prognostic symptoms have become a social problem. SARS-CoV-2 infection is mainly initiated by binding of its surface Spike protein to angiotensin converting enzyme 2 (ACE2) on the host cell membrane followed by internalization via endocytosis-mediated pathway. We eventually found that ACE2 expression levels were upregulated by the exposure to epidemiologically reported risk factors for COVID-19 severity; cigarette side-stream smoke, anti-cancer drug, diabetes and obesity, through formation of protein complex between transient receptor potential canonical (TRPC) 3 and NADPH oxidase (Nox) 2 on the rodent myocardial plasma membrane. We thus used 20 already approved drugs that has previously reported to have potency to attenuate the formation of TRPC3-Nox2 protein complex and evaluated the inhibitory effect on Spike protein-induced ACE2-GFP internalization in HEK293 cells. We found that several TRPC3-Nox2 complex inhibitors also have potency to inhibit the Spike protein-induced ACE2 internalization, and finally identified one drug that has the most potency to prevent SARS-CoV-2-induced infection and proliferation in TMPRRS2-expressing VeroE6 cells, and fluorescence-labeled S protein-induced pseudo infection in human iPS cardiomyocytes. These results provide a new concept that the inhibition of TRPC3-Nox2 complex formation will be a novel strategy to improve COVID-19 aggravation and sequelae by suppressing ACE2-mediated infection pathway.

Inhibition of RNA Helicase Activity Prevents Coxsackievirus B3-Induced Myocarditis in Human iPS Cardiomyocytes.

Aims: Coxsackievirus B3 (CVB3) is known to be an important cause of myocarditis and dilated cardiomyopathy. Enterovirus-2C (E2C) is a viral RNA helicase. It inhibits host protein synthesis. Based on these facts, we hypothesize that the inhibition of 2C may suppress virus replication and prevent enterovirus-mediated cardiomyopathy. Methods and Results: We generated a chemically modified enterovirus-2C inhibitor (E2CI). From the in vitro assay, E2CI was showed strong antiviral effects. For in vivo testing, mice were treated with E2CI intraperitoneally injected daily for three consecutive days at a dose of 8 mg/kg per day, after CVB3 post-infection (p.i) (CVB3 + E2CI, n = 33). For the infected controls (CVB3 only, n = 35), mice were injected with PBS (phosphate buffered saline) in a DBA/2 strain to establish chronic myocarditis. The four-week survival rate of E2CI-treated mice was significantly higher than that of controls (92% vs. 71%; p < 0.05). Virus titers and myocardial damage were significantly reduced in the E2CI treated group. In addition, echocardiography indicated that E2CI administration dramatically maintained mouse heart function compared to control at day 28 p.i chronic stage (LVIDD, 3.1 ± 0.08 vs. 3.9 ± 0.09, p < 0.01; LVDS, 2.0 ± 0.07 vs. 2.5 ± 0.07, p < 0.001; FS, 34.8 ± 1.6% vs. 28.5 ± 1.5%; EF, 67. 9 ± 2.9% vs. 54.7 ± 4.7%, p < 0.05; CVB3 + E2CI, n = 6 vs. CVB3, n = 4). Moreover, E2CI is effectively worked in human iPS (induced pluripotent stem cell) derived cardiomyocytes. Conclusion: Enterovirus-2C inhibitor (E2CI) was significantly reduced viral replication, chronic myocardium damage, and CVB3-induced mortality in DBA/2 mice. These results suggested that E2CI is a novel therapeutic agent for the treatment of enterovirus-mediated diseases.

Autotaxin‐LPA‐LPP3 axis regulates mitochondrial respiration prompting myocardial dysfunction

Background and ObjectiveAcute myocardial infarction (AMI) and ischemic heart disease are the two most prevalent causes of morbidity and mortality in the western world. Lysophosphatidic acid (LPA) a pro‐inflammatory, pro‐thrombotic bioactive glycerophospholipid plays a well‐known role in atherosclerotic disease; its role in myocardial dysfunction remains virtually unexplored. Following AMI, serum LPA concentration increases six‐fold over control subjects. Since LPA has a rapid turnover in the circulation, LPA production involves hydrolysis of lysophosphatidylcholine by the secreted enzyme autotaxin.In contrast, LPP3 catalyzes LPA dephosphorylation to generate lipid products that are not receptor active. The steady state of local and circulating LPA is balanced between these opposing pathways of LPA synthesis and degradation. The goal of this study was to determine the role of the autotaxin‐LPA‐LPP3 axis in myocardial dysfunction along with cardiomyocyte responses.MethodsTo study the role of the autotaxin‐LPA‐LPP3 axis in the myocardium, we used human induced pluripotent stem cell (IPS) cardiomyocytes and generated a cardiac‐specific Plpp3 deficient mouse strain. Mitochondrial bioenergetics was measured using the Seahorse XF24 analyzer.ResultsOur results show that exposure to LPA significantly decreased oxygen consumption rate (OCR; P&lt;0.001) and increased extracellular acidification rate (ECAR; P&lt;0.001) in IPS cardiomyocytes. Similar results were seen after subjecting the IPS cardiomyocytes to hypoxia where in the addition of autotaxin inhibitors had the reverse effect on OCR (P&lt;0.001) and ECAR (P&lt;0.001). LPA receptors blockade with pan‐Brp‐LPA restored the mitochondrial OCR (P&lt;0.001) and decreased the ECAR (P&lt;0.001). Generation of Plpp3 knockout mice (Plpp3Δ) showed increased mortality ~8 months. Plpp3Δ myocardium showed a distorted intercalated disc with damaged mitochondria with disorganized cristae and amorphous matrix densities as compared to lpp3fl in transmission electron microscopy. Lack of LPP3 resulted in deteriorating cardiac function. Plpp3Δ cardiomyocyte exposure to LPA significantly decreased oxygen consumption rate (OCR; P&lt;0.001) and increased extracellular acidification rate (ECAR; P&lt;0.001). Together these findings show that the absence of LPP3 increases LPA signaling and results in impaired mitochondrial function.Discussion and ConclusionsThe results presented in this study indicate that the autotaxin‐LPA‐LPP3 axis plays a critical role in the mitochondrial respiration and loss of LPP3 in cardiomyocytes from Plpp3Δ mice further confirms the impairment of myocardial function. Importantly, we demonstrate that autotaxin‐LPA‐LPP3 axis regulates mitochondrial respiration to limit myocardial function.Support or Funding InformationNational Institutes of Health Grants 1P20GM121307‐01A1 (Dr. Kevil) Sub‐Project ID: 8571 to Dr. Panchatcharam.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of electrical field stimulation on intracellular sodium concentration in human iPS cardiomyocytes

Effect of electrical field stimulation on intracellular sodium concentration in human iPS cardiomyocytes

Cytotoxicity monitoring for safety assessments: iPS cardiomyocytes and cancer cells

Cytotoxicity monitoring for safety assessments: iPS cardiomyocytes and cancer cells

Human iPS Cardiomyocytes with Genetically-encoded, Fluorescent Ca2 +, Voltage and cAMP Sensors Reveal Novel Kinetics Around cAMP Signaling: Implications for Drug Safety Screening

Human iPS Cardiomyocytes with Genetically-encoded, Fluorescent Ca2 +, Voltage and cAMP Sensors Reveal Novel Kinetics Around cAMP Signaling: Implications for Drug Safety Screening

In Silico Modeling of Human iPS Cardiomyocytes: Effect of Drugs and Comparison to Human Adult Cardiomyocytes

In Silico Modeling of Human iPS Cardiomyocytes: Effect of Drugs and Comparison to Human Adult Cardiomyocytes

Optical Stimulation of iPS Cardiomyocytes allows Brand New Insights into Contractility and Electropyhsiology Conjunctions

Optical Stimulation of iPS Cardiomyocytes allows Brand New Insights into Contractility and Electropyhsiology Conjunctions

Impedance based Assay on iPS cardiomyocytes: An efficient way of discriminating between cardioactive and non cardioactive drugs

Impedance based Assay on iPS cardiomyocytes: An efficient way of discriminating between cardioactive and non cardioactive drugs

Impedance and Combined Extracellular Field Potential Recordings of CiPA Reference Compounds on IPS Cardiomyocytes

Drug induced arrhythmia is one of the most common causes of drug development failure. Human induced pluripotent stem cell-derived cardiomyocytes (iPSCMs) show great promise for cardiovascular research and predictive in-vitro cardiac safety screening.We present data recorded on the CardioExcyte 96, a hybrid screening instrument that combines impedance (cell contractility) with MEA-like extracellular field potential (EFP) recordings. A case study showing impaired excitation-contraction coupling will be presented. This indicates the importance of testing compounds in assays complementary to patch clamp electrophysiology to provide a more complete safety profile. Furthermore, in accordance with the Comprehensive In Vitro Proarrhythmia Assay (CiPA) guidelines, standard reference compounds were tested on iPS-derived cardiomyocytes. Changes in the impedance signal indicate effects on cell contractility and shape whereas the field potential parameters provide information about the electrophysiological activity of the beating network of cells. Pharmacological effects of a number of reference compounds including those designated high risk (e.g. Dofetilide), intermediate risk (e.g. Cisapride) or low risk (e.g. Verapamil) were evaluated and summarized in this study.The CardioExcyte 96 system provides a non-invasive, label-free, high temporal resolution approach for safety screening. It can be either used in an incubator or, by utilizing the dedicated incubation system, directly on a lab bench. Furthermore, the system is capable of electrically pacing the cells, which allows for screening of cells which beat with individual frequencies and, in addition, investigations of frequency dependent compound inhibition.

Combined impedance and extracellular field potential recordings on iPS cardiomyocytes

Combined impedance and extracellular field potential recordings on iPS cardiomyocytes

Transmembrane potential measurements of 3D cardiac microtissues derived from iPS cardiomyocytes

Transmembrane potential measurements of 3D cardiac microtissues derived from iPS cardiomyocytes

Functional and genetic characterization of human iPS cardiomyocytes for cardiac safety screening

Functional and genetic characterization of human iPS cardiomyocytes for cardiac safety screening

Efficient and cost-effective batch analysis of multi-electrode array (MEA) data from human iPS cardiomyocytes using Intelligent Waveform Service (IWS)

• Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) with multi-electrode array (MEA) technology could be used to develop assays for predictive electrophysiological safety screening. • Large volumes of MEA data are generated and analysis using existing tools is time consuming. There is a need to improve timeliness and cost-effectiveness of data reporting. • Neural ID’s Intelligent Waveform Service (IWS) is propriety enterprise software that uses pattern-based learning of waveforms. • IWS was used to analyze MEA data generated from hiPSC-CMs treated with Terfenadine and results were compared to existing analysis tools. • We show that analysis and reporting times using IWS can be reduced by up to five times and employing pattern recognition technology will improve accuracy and facilitate greater use of MEAs in compound screening. Methods Results • The non-sedating anti-histamine Terfenadine was chosen to validate the IWS software. • Data was recorded from spontaneously beating hiPSCCMs (Cellular Dynamics International) using the MEA1060 system (Multi Channel Systems). Data was filtered (1Hz high pass, 3kHz low pass) and sampled at 10kHz. • After baseline recordings, Terfenadine was cumulatively added to the bath. The last minute of recording at baseline and each Terfenadine concentration was used for analysis. • IWS was used to analyze the following parameters: Field potential duration (FPD), total spike amplitude, beat rate and conduction time. • Data analysis is conducted on the server using templates which are batch processed for efficient resource utilization. • Gradient markers are defined to determine spike interval and context of targeted patterns. • Based on the marker locations, patterns are defined graphically (Fig. 2) to detect repolarization wave location and measure its peak value. • IWS creates an empirical data model and does not rely on a predetermined library of patterns, leading to higher recognition rates than industry standards and the ability to analyze a broader range of data. • By simply clicking and dragging, additional pattern examples can be added to the knowledge database (Fig. 3). • The same process is used to detect other types of patterns, such as spikes, with amplitude (maximum/minimum) measurements applied (Fig. 4). Intelligent Waveform Service (IWS) IWS is proprietary enterprise software (Fig. 1) that: • Leverages machine learning to provide waveform analytics of biosignals. • Automates and accelerates discovery & preclinical research, providing more complete results at a fraction of the time and cost. • Learns example patterns, then runs batch jobs against multiple file sets and data formats, making analytics shareable among researchers and groups. Figure 1 – IWS Architecture Figure 4 – Detecting Spike Patterns and Measuring Amplitude Figure 3 – Adding Pattern Examples to the Template Figure 2 – Pattern Creation and Metrology Set-Up