Increase In Beating Rate Research Articles

Chamber-specific contractile responses of atrial and ventricular hiPSC-cardiomyocytes to GPCR and ion channel targeting compounds: A microphysiological system for cardiac drug development

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) have found utility for conducting in vitro drug screening and disease modelling to gain crucial insights into pharmacology or disease phenotype. However, diseases such as atrial fibrillation, affecting >33 M people worldwide, demonstrate the need for cardiac subtype-specific cells. Here, we sought to investigate the base characteristics and pharmacological differences between commercially available chamber-specific atrial or ventricular hiPSC-CMs seeded onto ultra-thin, flexible PDMS membranes to simultaneously measure contractility in a 96 multi-well format. We investigated the effects of GPCR agonists (acetylcholine and carbachol), a Ca2+ channel agonist (S-Bay K8644), an HCN channel antagonist (ivabradine) and K+ channel antagonists (4-AP and vernakalant). We observed differential effects between atrial and ventricular hiPSC-CMs on contractile properties including beat rate, beat duration, contractile force and evidence of arrhythmias at a range of concentrations.As an excerpt of the compound analysis, S-Bay K8644 treatment showed an induced concentration-dependent transient increase in beat duration of atrial hiPSC-CMs, whereas ventricular cells showed a physiological increase in beat rate over time. Carbachol treatment produced marked effects on atrial cells, such as increased beat duration alongside a decrease in beat rate over time, but only minimal effects on ventricular cardiomyocytes. In the context of this chamber-specific pharmacology, we not only add to contractile characterization of hiPSC-CMs but propose a multi-well platform for medium-throughput early compound screening.Overall, these insights illustrate the key pharmacological differences between chamber-specific cardiomyocytes and their application on a multi-well contractility platform to gain insights for in vitro cardiac liability studies and disease modelling.

Cardiac stereotactic body radiotherapy to treat malignant ventricular arrhythmias directly affects the cardiomyocyte electrophysiology

BackgroundPromising as a treatment option for life-threatening ventricular arrhythmias, cardiac stereotactic body radiotherapy (cSBRT) has demonstrated early antiarrhythmic effects within days of treatment. The mechanisms underlying the immediate and short-term antiarrhythmic effects are poorly understood. ObjectiveWe hypothesize that cSBRT has a direct antiarrhythmic effect on cellular electrophysiology through reprogramming of ion channel and gap junction protein expression. MethodsAfter exposure to 20 Gy of x-rays in a single fraction, neonatal rat ventricular cardiomyocytes were analyzed 24 and 96 hours postradiation to determine changes in conduction velocity, beating frequency, calcium transients, and action potential duration in both monolayers and single cells. In addition, the expression of gap junction proteins, ion channels, and calcium handling proteins was evaluated at protein and messenger RNA levels. ResultsAfter irradiation with 20 Gy, neonatal rat ventricular cardiomyocytes exhibited increased beat rate and conduction velocity 24 and 96 hours after treatment. Messenger RNA and protein levels of ion channels were altered, with the most significant changes observed at the 96-hour mark. Upregulation of Cacna1c (Cav1.2), Kcnd3 (Kv4.3), Kcnh2 (Kv11.1), Kcnq1 (Kv7.1), Kcnk2 (K2P2.1), Kcnj2 (Kir2.1), and Gja1 (Cx43) was noted, along with improved gap junctional coupling. Calcium handling was affected, with increased Ryr2 ryanodin-rezeptor 2 and Slc8a1 Na+/Ca2+ exchanger expression and altered properties 96 hours posttreatment. Fibroblast and myofibroblast levels remained unchanged. ConclusioncSBRT modulates the expression of various ion channels, calcium handling proteins, and gap junction proteins. The described alterations in cellular electrophysiology may be the underlying cause of the immediate antiarrhythmic effects observed after cSBRT.

Modulation of Cardiac Ryanodine Receptors by Chloride Intracellular Ion Channel 2

Pathogenic variants of the Chloride intracellular ion channel 2 (CLIC2) are implicated in hypertrophic cardiomyopathy (HCM), a diverse heart condition leading to arrhythmic sudden cardiac death and heart failure. CLIC2 is a unique class of metamorphic protein, related to the enzymatic glutathione s-transferase family and form functional ion channel. Artificial bilayer studies have shown that CLIC2 negatively modulates the activity of cardiac ryanodine receptor 2 (RyR2), implicating the sarcoplasmic/endoplasmic reticulum (SR/ER) Ca2+ release. However, the subsequent consequences of RyR2 and CLIC2 interaction in cardiac physiology remains unknown. Therefore, we ablated clic2 in human induced pluripotent stem cell (hiPSC) line using the CRISPR-Cas9 system and performed cardiomyocyte differentiation. clic2−/− hiPSC-derived cardiomyocytes (hiPSC-CMs) showed altered electrophysiological properties leading to impaired excitation-contraction coupling. clic2−/− hiPSC-CMs showed an increase in beat rate and field potential duration by 143.49±5.96% and 162.43±5.82%, respectively, whereas conduction velocity and spike amplitude decreased by 43.81±2.22% and 43.32±3.54%, respectively compared to WT hiPSC-CMs. Additionally, action potential duration measurements of clic2−/− hiPSC-CMs showed a decrease in APD30, APD50, and APD90 by 74.42%, 65.19%, and 66.46%, respectively compared to WT hiPSC-CMs. Moreover, clic2−/− hiPSC-CMs showed altered Ca2+ handling, with a decrease in peak amplitude (57.07±2.25%) and duration (38.29±1.30%) compared to the WT hiPSC-CMs. The caffeine induced Ca2+ release in clic2−/− hiPSC-CMs, attributed to the SR/ER Ca2+ overload which decreased by 72.19±3.51% compared to WT hiPSC-CMs. Transcriptomics analysis of clic2−/− hiPSC-CMs revealed 223 transcripts with significant changes in expression levels (89 upregulated and 134 downregulated), implicating CLIC2 in hypertrophy cardiomyopathy, dilated cardiomyopathy, and other heart diseases. Furthermore, the protein expression of CLIC2 increases in end-stage failing human heart samples compared to the non-failing heart. In conclusion, our study sheds light on the novel role of CLIC2 in the regulation of cardiac (patho)physiology. National Institutes of Health (NIH) (HL133050, and HL157453); American Heart Association-Transformational Project Award; National Center for advancing translational sciences (TR004178, understudied proteins and druggable targets) (HS). Presidential Predoctoral Fellowship; Margaret T. Nishikawara Merit Scholarship Endowment in Physiology; Graduate School’s Alumni Grants for Graduate Research and Scholarship Program, The Ohio State University (SS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract P1041: Multiplexed Cardiotoxicity And Efficacy Screening Using Kinetic Image Cytometry Of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes

Traditionally, cardiotoxicity testing used low-throughput animal models which may not fully recapitulate human physiology. Stem cell technology enables screening in a more physiologically-relevant model system in less time and with less cost, but most current high-throughput screening modalities rely on whole-well or whole-field averaged data, which obfuscate the responses of individual hiPSC-cardiomyocytes (CMs). By measuring individual hiPSC-CMs, heterogenous responses that would otherwise be “averaged out” can be quantified, thus improving sensitivity. We previously published that the single-cell Kinetic Image Cytometry (KIC) methods developed at Vala Sciences predicted arrhythmogenic liability with an accuracy of 90% using intracellular Ca 2+ kinetics as a readout. In this study, we expand upon this methodology by loading metabolically matured hiPSC-CMs with Cal-520 (a green Ca 2+ dye) and BeRST-1 (a far red membrane voltage dye), acquiring interleaved movies of Ca 2+ and membrane potential at 60 Hz (30 Hz per channel). We then created image analysis algorithms to measure Ca 2+ transient (CT) and action potential (AP) kinetics, and to derive contraction measurements from those movies. To validate our approach, we exposed cells to isoproterenol, nifedipine, dofetilide, and butadiene 2-monoxime (BDM) at various concentrations. Isoproterenol increased beat rate, decreased both AP and CT durations, increased CT magnitude, and elicited a lusitropic effect. Nifedipine increased beat rate, decreased AP and CT durations, and reduced CT magnitude and contraction distance. Dofetilide reduced beat rate, prolonged APs and CTs, and increased contraction distance. BDM had multiple inhibitor effects on APs and CTs, and strongly reduced contraction. Thus, this multiplexed assay can quantify multiple effects of drugs on APs, CTs, and contraction, providing an unprecedented suite of information relevant to cardiosafety and efficacy.

Radiation-induced electrophysiological remodelling in neonatal rat ventricular cardiomyocytes

Abstract Background Ventricular tachycardia (VT) carries a relatively high risk of recurrence and significantly impacts patients' quality of life, despite state of the art treatment including antiarrhythmic drug therapy and catheter ablation. Cardiac stereotactic body radiotherapy (SBRT) has emerged as a promising non-invasive treatment option for refractory VT. A marked reduction in VT episodes is observed within days of treating high-risk patients with a single fraction SBRT. The cellular mechanisms underlying the clinically demonstrated antiarrhythmic effect are still poorly understood. Radiation-induced fibrosis is observed several months after irradiation, suggesting other mechanisms that explain the immediate benefit of radiotherapy on the VT substrate. Purpose We hypothesized that early antiarrhythmic effects of single-fraction radiation are produced by direct molecular and functional electrophysiological changes, independent of fibrosis. Our aim was to investigate the effects of 20 Gy ionizing photon radiation on the electrophysiology of ventricular neonatal rat cardiomyocytes at time periods from 24 hours to 96 hours after irradiation. Methods Ventricular neonatal rat cardiomyocytes were isolated, cultivated for 24 to 96 hours and then exposed to photon beam with a single dose of 20 Gy. Expression changes of ion channels, calcium handling proteins and structural proteins affecting cellular electrophysiology were assessed via RT-qPCR and Western blotting. Beat frequency was evaluated by video analysis and action potentials (APD) were recorded using the whole-cell voltage clamp technique in current-clamp mode. Intracellular calcium transients in Fura-2 AM loaded cardiomyocytes were recorded with an IonOptix recording system. Results Irradiation lead to significant changes in the mRNA and protein level of ion channels, with most distinct changes being observed 96h after radiation. Increased mRNA and protein expression of (Cav1.2) and of the potassium channels Kcnd3 (Kv4.3), Kcnh2 (Kv11.1), Kcnq1 (Kv7.1), Kcnk2 (K2P2.1) and Kcnj2 (Kir2.1) was observed. Additionally, Connexin 43 (Cx43) was upregulated. Irradiated cardiomyocytes showed a significant increase in beat rate over time. APD was significantly shortened, with a significantly increased upstroke velocity. Regarding calcium handling, significant upregulations of Ryr2 (RYR2) and Slc8a1 (NCX) transcript and protein level was detected. Additionally, calcium handling properties were altered. Conclusion Our results suggest that acute irradiation effects are associated with electrophysiological remodelling in cardiomyocytes. Changes of cellular ion channel expression and calcium homeostasis lead to normalization and shortening of heart-failure associated APD prolongation. These results reveal new insights into acute antiarrhythmic effects on cardiomyocytes after SBRT-therapy. Funding Acknowledgement Type of funding sources: None.

Elevated thyroid-stimulating hormone (TSH) is associated with atrial fibrillation and directly modulates electrophysiological properties of atrial cardiomyocytes

Abstract Background Thyroid dysfunction has a strong association with cardiac arrhythmias. However, subclinical hypothyroidism (SH) is often neglected in studies. SH is characterized by increased values of TSH and normal values of T3/T4. Recent studies indicate that high TSH levels are related to an increased incidence of cardiac electrical abnormalities, especially atrial fibrillation (AF). Purpose The present work aims to investigate direct effects of TSH on ion channel remodelling in cardiomyocytes and a possible causative role in the increased occurrence of arrhythmia in SH. Methods A retrospective monocentric study analysed medical records, ECGs and echocardiographies of 3134 patients between 2007 to 2020. Inclusion criteria comprised patient age ≥18 years, normal fT4, TSH values 4–75 mU/l and available ECGs. The patient cohort was divided into two groups based on current treatment recommendations for SH (TSH level 4–9.9 mU/l; 10–75 mU/l). Direct effects on ion channel remodelling were analysed in HL-1 and neonatal cardiomyocytes with confirmed expression of TSH receptors. TSH concentrations of 15, 30, and 60 mU/l simulated different degrees of hypothyroidism. RT-qPCR analyses, western blots and immunofluorescence stainings were performed after incubation with respective TSH concentrations for 24h. In vitro video analyses of automatism in NRCM before and after TSH incubation were performed. Statistical comparisons were calculated using student's t-tests and the chi-square test. In addition, a multivariate analysis of factors affecting the occurrence of AF in patients with SH was carried out. Results Elevated TSH levels were associated with higher prevalence of AF. In the group of patients with a TSH value of 10–75 mU/l, AF was observed in 42.8% of cases compared to 31.7% in patients with lower TSH levels (p<0.0001). In vitro, TSH application to cardiomyocytes led to significant changes in mRNA and protein expression of key ion channels and regulatory proteins. Incubation of HL-1 cells with 15 mU/l TSH showed a decrease in Kcne1 protein, while 60 mU/l TSH caused an upregulation of K2P3.1 (TASK-1), Kv7.1, and KCa2.1 expression. To further elucidate the TSH signaling cascade in cardiomyocytes, we investigated TSH effects on PKAα, CaMKIIδ and CaMKIIγ. PKAα was upregulated after 24h incubation with 60 mU/l TSH and exhibited an increase of the phosphorylated state. Finally, neonatal cardiomyocytes showed an increased beating rate after incubation with TSH. Conclusion The prevalence of atrial fibrillation is significantly increased in patients with SH. Our findings further reveal a direct effect of TSH on ion channel expression in cardiomyocytes as potential proarrhythmic mechanism. TSH induced changes in the expression of ion channels that determine action potential duration. Subclinical hypothyroidism contributes to the development of atrial fibrillation. Underlying mechanisms and therapeutic implications require further research. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Heidelberg University Medical School - Olympia Morata

Active force generation contributes to the complexity of spontaneous activity and to the response to stretch of murine cardiomyocyte cultures.

Cardiomyocyte cultures exhibit spontaneous electrical and contractile activity, as in a natural cardiac pacemaker. In such preparations, beat rate variability exhibits features similar to those of heart rate variability in vivo. Mechanical deformations and forces feed back on the electrical properties of cardiomyocytes, but it is not fully elucidated how this mechano‐electrical interplay affects beating variability in such preparations. Using stretchable microelectrode arrays, we assessed the effects of the myosin inhibitor blebbistatin and the non‐selective stretch‐activated channel blocker streptomycin on beating variability and on the response of neonatal or fetal murine ventricular cell cultures against deformation. Spontaneous electrical activity was recorded without stretch and upon predefined deformation protocols (5% uniaxial and 2% equibiaxial strain, applied repeatedly for 1 min every 3 min). Without stretch, spontaneous activity originated from the edge of the preparations, and its site of origin switched frequently in a complex manner across the cultures. Blebbistatin did not change mean beat rate, but it decreased the spatial complexity of spontaneous activity. In contrast, streptomycin did not exert any manifest effects. During the deformation protocols, beat rate increased transiently upon stretch but, paradoxically, also upon release. Blebbistatin attenuated the response to stretch, whereas this response was not affected by streptomycin. Therefore, our data support the notion that in a spontaneously firing network of cardiomyocytes, active force generation, rather than stretch‐activated channels, is involved mechanistically in the complexity of the spatiotemporal patterns of spontaneous activity and in the stretch‐induced acceleration of beating. Key points Monolayer cultures of cardiac cells exhibit spontaneous electrical and contractile activity, as in a natural cardiac pacemaker. Beating variability in these preparations recapitulates the power‐law behaviour of heart rate variability in vivo. However, the effects of mechano‐electrical feedback on beating variability are not yet fully understood.Using stretchable microelectrode arrays, we examined the effects of the contraction uncoupler blebbistatin and the non‐specific stretch‐activated channel blocker streptomycin on beating variability and on stretch‐induced changes of beat rate.Without stretch, blebbistatin decreased the spatial complexity of beating variability, whereas streptomycin had no effects.Both stretch and release increased beat rate transiently; blebbistatin attenuated the increase of beat rate upon stretch, whereas streptomycin had no effects.Active force generation contributes to the complexity of spatiotemporal patterns of beating variability and to the increase of beat rate upon mechanical deformation. Our study contributes to the understanding of how mechano‐electrical feedback influences heart rate variability.

Fibroblast Growth Factor 1 Reduces Pulmonary Vein and Atrium Arrhythmogenesis via Modification of Oxidative Stress and Sodium/Calcium Homeostasis.

RationaleAtrial fibrillation is a critical health burden. Targeting calcium (Ca2+) dysregulation and oxidative stress are potential upstream therapeutic strategies. Fibroblast growth factor (FGF) 1 can modulate Ca2+ homeostasis and has antioxidant activity. The aim of this study was to investigate whether FGF1 has anti-arrhythmic potential through modulating Ca2+ homeostasis and antioxidant activity of pulmonary vein (PV) and left atrium (LA) myocytes.MethodsPatch clamp, western blotting, confocal microscopy, cellular and mitochondrial oxidative stress studies were performed in isolated rabbit PV and LA myocytes treated with or without FGF1 (1 and 10 ng/mL). Conventional microelectrodes were used to record electrical activity in isolated rabbit PV and LA tissue preparations with and without FGF1 (3 μg/kg, i.v.).ResultsFGF1-treated rabbits had a slower heart rate than that observed in controls. PV and LA tissues in FGF1-treated rabbits had slower beating rates and longer action potential duration than those observed in controls. Isoproterenol (1 μM)-treated PV and LA tissues in the FGF1-treated rabbits showed less changes in the increased beating rate and a lower incidence of tachypacing (20 Hz)-induced burst firing than those observed in controls. FGF1 (10 ng/mL)-treated PV and LA myocytes had less oxidative stress and Ca2+ transient than those observed in controls. Compared to controls, FGF1 (10 ng/mL) decreased INa−L in PV myocytes and lowered Ito, IKr−tail in LA myocytes. Protein kinase C (PKC)ε inhibition abolished the effects of FGF1 on the ionic currents of LA and PV myocytes.ConclusionFGF1 changes PV and LA electrophysiological characteristics possibly via modulating oxidative stress, Na+/Ca2+ homeostasis, and the PKCε pathway.

Full-length dystrophin deficiency leads to contractile and calcium transient defects in human engineered heart tissues

Cardiomyopathy is currently the leading cause of death for patients with Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder affecting young boys. Animal models have provided insight into the mechanisms by which dystrophin protein deficiency causes cardiomyopathy, but there remains a need to develop human models of DMD to validate pathogenic mechanisms and identify therapeutic targets. Here, we have developed human engineered heart tissues (EHTs) from CRISPR-edited, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing a truncated dystrophin protein lacking part of the actin-binding domain. The 3D EHT platform enables direct measurement of contractile force, simultaneous monitoring of Ca2+ transients, and assessment of myofibril structure. Dystrophin-mutant EHTs produced less contractile force as well as delayed kinetics of force generation and relaxation, as compared to isogenic controls. Contractile dysfunction was accompanied by reduced sarcomere length, increased resting cytosolic Ca2+ levels, delayed Ca2+ release and reuptake, and increased beat rate irregularity. Transcriptomic analysis revealed clear differences between dystrophin-deficient and control EHTs, including downregulation of genes related to Ca2+ homeostasis and extracellular matrix organization, and upregulation of genes related to regulation of membrane potential, cardiac muscle development, and heart contraction. These findings indicate that the EHT platform provides the cues necessary to expose the clinically-relevant, functional phenotype of force production as well as mechanistic insights into the role of Ca2+ handling and transcriptomic dysregulation in dystrophic cardiac function, ultimately providing a powerful platform for further studies in disease modeling and drug discovery.

PDE4B and PDE4D differentially regulate cardiac pacemaker activity

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agence Nationale de la Recherche (ANR) Background Heart rate (HR) is generated by spontaneous electrical activity in the sinoatrial node (SAN) and is modulated by the autonomic nervous system. During sympathetic stimulation, the activation of β-adrenergic receptors (βAR) increases cAMP levels, leading to positive chronotropic effect. Among the 6 cardiac cAMP-PDE families, PDE4 is critical for controlling excitation-contraction coupling (ECC) during β-stimulation in atrial and ventricular myocytes. PDE4 may also be important for automaticity. 3 genes encode for PDE4 in the heart (pde4a, 4b, 4d). We propose to investigate their respective contribution to the regulation of pacemaker activity. Methods Total PDE activity in mouse SAN was determined as the cAMP-hydrolytic activity measured in the absence of PDE inhibitor and the fraction corresponding to PDE4 activity was assessed by including the PDE4 inhibitor Ro-20-1724. The in vitro pacemaker activity was assessed by measuring spontaneous Ca2+ transients in Fluo4-loaded-SAN tissue from wild-type (WT) and PDE4KO mice. Images were obtained using confocal microscopy. Telemetry EKG was recorded in conscious mice in control (CTRL) conditions, after pharmacological denervation with atropine (1 mg/kg) and propranolol (2 mg/kg) and after β-stimulation with isoproterenol (ISO, 1.5 mg/kg). HR variability was evaluated by calculating the SDNN (standard deviation of RR intervals) parameter. Results Ro-20-1724 (10 µM) increased beating rate of intact SAN and increased PKA-phosphorylation of key ECC actors (ryanodine receptor, phospholamban and contractile proteins). PDE4 activity was found to account for 60% of total cAMP-PDE activity in SAN. PDE4A, 4B and 4D isoforms were found to be expressed in mouse SAN. In PDE4BKO SAN, the effect of ISO on SAN beating rate was higher than in WT. Ablation of PDE4D induced decreased beating rate in CTRL and ISO conditions and increased Ca2+ spark frequency compared to WT SAN. In vivo, PDE4BKO and PDE4DKO mice displayed increased resting HR during day and night. HR variability was decreased in PDE4BKO, but not in PDE4DKO mice during the day, and decreased in both genotypes at night compared to WT mice. After atropine + propranolol denervation, the rhythmic phenotype was only maintained in PDE4BKO but not in PDE4DKO mice. The response to β-AR stimulation with ISO was higher in PDE4BKO than in PDE4DKO. In addition, under ISO we observed an increased number of premature beats and atrioventricular blocks in PDE4DKO, but not in PDE4BKO mice. Conclusion PDE4B and PDE4D differentially regulate cardiac pacemaker activity. While PDE4B clearly controls intrinsic SAN automaticity, PDE4D might be important for ANS-mediated regulation of HR and conduction.

Type 4 Phosphodiesterase regulates cardiac pacemaker function

Numerous epidemiological and clinical studies have revealed a positive correlation between heart rate (HR) and cardiovascular morbimortality. The autonomic nervous system is the major extracardiac determinant of HR. During sympathetic stimulation, the activation of β-adrenergic receptors (βAR) induces an increase in cAMP levels, leading to positive chronotropic effect. Among the 5 cardiac cAMP-PDE families, PDE4 is critical for controlling excitation-contraction coupling (ECC) during β-stimulation in atrial and ventricular cells. PDE4 may also be important for automaticity. Three genes encode for PDE4: pde4a , 4b , 4d . Their respective contribution to the regulation of pacemaker activity remains unknown. Total PDE activity was determined in mouse sinoatrial node (SAN) tissue as the cAMP-hydrolytic activity measured in the absence of PDE inhibitor and the fraction corresponding to PDE4 activity was assessed by including the PDE4 inhibitor Ro-201724. The in vitro pacemaker activity was assessed by measuring spontaneous Ca 2+ transients in Fluo4-loaded-SAN tissue. Images were obtained using confocal microscopy. Ro-20-1724 increased beating rate of intact SAN and increased PKA-phosphorylation of key ECC actors (ryanodine receptor, phospholamban and contractile proteins). PDE4 activity was found to account for 60% of total cAMP-PDE activity in SAN ( n = 3). PDE4A, 4B and 4D isoforms were found to be expressed in mouse SAN ( n = 5 independent experiments). In 4D-, but not in 4BKO mice, Ca 2+ homeostasis was altered in control conditions (ctrl) and after β-stimulation with isoprenaline (iso). Indeed, ablation of PDE4D induced decreased beating rate (ctrl: 1.00 ± 0.08 s −1 vs. 1.57 ± 0.05 s −1 ; iso: 1.71 ± 0.17 s −1 vs. 2.39 ± 0.08 s −1 , P < 0.0001) and increased Ca 2+ spark frequency (ctrl: 15.9 ± 5.2 vs. 1.9 ± 0.4 sparks/s/100 μm; iso: 22.9 ± 7.1 vs. 0.6 ± 0.2 sparks/s/100 μm, P < 0.0001) ( Fig. 1 ). PDE4 controls pacemaker function and PDE4D ablation strongly perturbs normal SAN activity.

A Scalable Approach Reveals Functional Responses of iPSC Cardiomyocyte 3D Spheroids.

Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) provide an in vitro model of the human myocardium. Complex 3D scaffolded culture methods improve the phenotypical maturity of iPSC-CMs, although typically at the expense of throughput. We have developed a novel, scalable approach that enables the use of iPSC-CM 3D spheroid models in a label-free readout system in a standard 96-well plate-based format. Spheroids were accurately positioned onto recording electrodes using a magnetic gold-iron oxide nanoparticle approach. Remarkably, both contractility (impedance) and extracellular field potentials (EFPs) could be detected from the actively beating spheroids over long durations and after automated dosing with pharmacological agents. The effects on these parameters of factors, such as co-culture (including human primary cardiac fibroblasts), extracellular buffer composition, and electrical pacing, were investigated. Beat amplitudes were increased greater than 15-fold by co-culture with fibroblasts. Optimization of extracellular Ca2+ fluxes and electrical pacing promoted the proper physiological response to positive inotropic agonists of increased beat amplitude (force) rather than the increased beat rate often observed in iPSC-CM studies. Mechanistically divergent repolarizations in different spheroid models were indicated by their responses to BaCl2 compared with E-4031. These studies demonstrate a new method that enables the pharmacological responses of 3D iPSC-CM spheroids to be determined in a label-free, standardized, 96-well plate-based system. This approach could have discovery applications across cardiovascular efficacy and safety, where parameters typically sought as readouts of iPSC-CM maturity or physiological relevance have the potential to improve assay predictivity.

PDE4 regulates cardiac pacemaker function

Numerous epidemiological and clinical studies have revealed a positive correlation between heart rate (HR) and cardiovascular morbimortality. The autonomic nervous system is the major extracardiac determinant of HR. During sympathetic stimulation, the activation of β-adrenergic receptors (βAR) induces an increase in cAMP levels, leading to positive chronotropic effect. Among the 5 cardiac cAMP-PDE families, PDE4 is critical for controlling excitation-contraction coupling (ECC) during β-stimulation in atrial and ventricular cells. PDE4 may also be important for automaticity. 3 genes encode for PDE4: pde4a,4b,4d. Their respective contribution to the regulation of pacemaker activity remains unknown. Total PDE activity was determined in mouse sinoatrial node (SAN) tissue as the cAMP-hydrolytic activity measured in the absence of PDE inhibitor and the fraction corresponding to PDE4 activity was assessed by including the PDE4 inhibitor Ro-201724. The in vitro pacemaker activity was assessed by measuring spontaneous Ca2+ transients in Fluo4-loaded-SAN tissue. Images were obtained using confocal microscopy. Ro-20-1724 increased beating rate of intact SAN and increased PKA-phosphorylation of key ECC actors (ryanodine receptor, phospholamban and contractile proteins). PDE4 activity was found to account for 60% of total cAMP-PDE activity in SAN ( n = 3). PDE4A, 4B and 4D isoforms were found to be expressed in mouse SAN ( n = 5 independent experiments). In 4D-, but not in 4BKO mice, Ca2+ homeostasis was altered in control conditions (ctrl) and after β-stimulation with isoprenaline (iso). Indeed, ablation of PDE4D induced decreased beating rate (ctrl: 1.00 ± 0.08s-1 vs. 1.57 ± 0.05s-1; iso: 1.71 ± 0.17s-1 vs. 2.39 ± 0.08s-1, P < 0.0001) and increased Ca2+ spark frequency (ctrl: 15.9 ± 5.2 vs. 1.9 ± 0.4sparks/s/100 μm; iso: 22.9 ± 7.1 vs. 0.6 ± 0.2sparks/s/100 μm, P < 0.0001) ( Fig. 1 ). PDE4 controls pacemaker function in mice and PDE4D ablation strongly perturbs normal SAN activity.

P2558PDE4 regulates cardiac pacemaker function

Abstract Background Numerous epidemiological and clinical studies have revealed a positive correlation between heart rate (HR) and cardiovascular morbimortality. The autonomic nervous system is the major extracardiac determinant of HR. During sympathetic stimulation, the activation of β-adrenergic receptors (βAR) induces an increase in cAMP levels, leading to positive chronotropic effect. Among the 5 cardiac cAMP-PDE families, PDE4 is critical for controlling excitation-contraction coupling (ECC) during βAR stimulation in atrial and ventricular cells. PDE4 may also be important for automaticity. 3 genes encode for PDE4s: pde4a, pde4b, pde4d. Their respective contribution to the regulation of pacemaker activity remains ill-defined. Purpose Define the role of PDE4 isoforms in the regulation of cardiac pacemaker activity Methods Total PDE activity was determined in mouse sinoatrial node (SAN) tissue as the cAMP-hydrolytic activity measured in the absence of PDE inhibitor and the fraction corresponding to PDE4 activity was assessed by including the PDE4 inhibitor Ro-20-1724. The in vitro pacemaker activity was assessed by measuring spontaneous Ca2+ transients in Fluo4-loaded-SAN tissue. Images were obtained using confocal microscopy. Results Ro-20-1724 increased beating rate of intact SAN and increased PKA-phosphorylation of key ECC actors (ryanodine receptor, phospholamban and contractile proteins). PDE4 activity was found to account for 60% of the total cAMP-PDE activity in SAN (n=3 independent experiments). PDE4A, PDE4B and PDE4D isoforms were found to be expressed in mouse SAN (n=5 independent experiments). In PDE4D-, but not in PDE4B-deficient mice, Ca2+ homeostasis was altered in control conditions (ctrl) and after βAR stimulation with isoprenaline (iso). Indeed, ablation of PDE4D induced decreased beating rate (ctrl: 1.00±0.08 s–1 vs 1.57±0.05 s–1; iso: 1.71±0.17 s–1 vs 2.39±0.08 s–1, p&lt;0.0001) and increased Ca2+ spark frequency (ctrl: 15.9±5.2 sparks/s/100 μm vs 1.9±0.4 sparks/s/100 μm; iso: 22.9±7.1 sparks/s/100 μm vs 0.6±0.2 sparks/s/100 μm, p&lt;0.0001) (Figure). Calcium Homeostasis in SAN cells Conclusion PDE4 controls pacemaker function in mice and PDE4D ablation strongly perturbs normal SAN activity. Acknowledgement/Funding ANR, Fondation Lefoulon Delalande, CORDDIM

Electrophysiological abnormalities in induced pluripotent stem cell-derived cardiomyocytes generated from Duchenne muscular dystrophy patients.

Duchenne muscular dystrophy (DMD) is an X‐linked progressive muscle degenerative disease, caused by mutations in the dystrophin gene and resulting in death because of respiratory or cardiac failure. To investigate the cardiac cellular manifestation of DMD, we generated induced pluripotent stem cells (iPSCs) and iPSC‐derived cardiomyocytes (iPSC‐CMs) from two DMD patients: a male and female manifesting heterozygous carrier. Dystrophin mRNA and protein expression were analysed by qRT‐PCR, RNAseq, Western blot and immunofluorescence staining. For comprehensive electrophysiological analysis, current and voltage clamp were used to record transmembrane action potentials and ion currents, respectively. Microelectrode array was used to record extracellular electrograms. X‐inactive specific transcript (XIST) and dystrophin expression analyses revealed that female iPSCs underwent X chromosome reactivation (XCR) or erosion of X chromosome inactivation, which was maintained in female iPSC‐CMs displaying mixed X chromosome expression of wild type (WT) and mutated alleles. Both DMD female and male iPSC‐CMs presented low spontaneous firing rate, arrhythmias and prolonged action potential duration. DMD female iPSC‐CMs displayed increased beat rate variability (BRV). DMD male iPSC‐CMs manifested decreased I f density, and DMD female and male iPSC‐CMs showed increased I Ca,L density. Our findings demonstrate cellular mechanisms underlying electrophysiological abnormalities and cardiac arrhythmias in DMD.

Fabrication of scaffold-free tubular cardiac constructs using a Bio-3D printer.

A major challenge in cardiac tissue engineering is the host’s immune response to artificial materials. To overcome this problem, we established a scaffold-free system for assembling cell constructs using an automated Bio-3D printer. This printer has previously been used to fabricate other three-dimensional (3D) constructs, including liver, blood vessels, and cartilage. In the present study, we tested the function in vivo of scaffold-free cardiac tubular construct fabricated using this system. Cardiomyocytes derived from induced pluripotent stem cells (iCells), endothelial cells, and fibroblasts were combined to make the spheroids. Subsequently, tubular cardiac constructs were fabricated by Bio-3D printer placing the spheroids on a needle array. Notably, the spheroid fusion and beat rate in the constructs were observed while still on the needle array. After removal from the needle array, electrical stimulation was used to test responsiveness of the constructs. An increased beat rate was observed during stimulation. Importantly, the constructs returned to their initial beat rate after stimulation was stopped. In addition, histological analysis shows cellular reorganization occurring in the cardiac constructs, which may mimic that observed during organ transplantation. Taken together, our results indicate that these engineered cardiac tubular constructs, which address both the limited supply of donor tissues as well as the immune-induced transplant rejection, has potential to be used for both clinical and drug testing applications. To our knowledge, this is the first time that cardiac tubular constructs have been produced using optimized Bio-3D printing technique and subsequently tested for their use as cardiac pumps.

Disruption of neonatal cardiomyocyte physiology following exposure to bisphenol-a

Bisphenol chemicals are commonly used in the manufacturing of polycarbonate plastics, polyvinyl chloride plastics, resins, and thermal printing applications. Humans are inadvertently exposed to bisphenols through contact with consumer products and/or medical devices. Recent reports have shown a link between bisphenol-a (BPA) exposure and adverse cardiovascular outcomes; although these studies have been limited to adult subjects and models. Since cardiac physiology differs significantly between the developing and adult heart, we aimed to assess the impact of BPA exposure on cardiac function, using a neonatal cardiomyocyte model. Neonatal rat ventricular myocytes were monitored to assess cell viability, spontaneous beating rate, beat rate variability, and calcium-handling parameters in the presence of control or bisphenol-supplemented media. A range of doses were tested to mimic environmental exposure (10−9–10−8M), maximum clinical exposure (10−5M), and supraphysiological exposure levels (10−4M). Acute BPA exposure altered cardiomyocyte functionality, resulting in a slowed spontaneous beating rate and increased beat rate variability. BPA exposure also impaired intracellular calcium handling, resulting in diminished calcium transient amplitudes, prolonged calcium transient upstroke and duration time. Alterations in calcium handling also increased the propensity for alternans and skipped beats. Notably, the effect of BPA-treatment on calcium handling was partially reversible. Our data suggest that acute BPA exposure could precipitate secondary adverse effects on contractile performance and/or electrical alternans, both of which are dependent on intracellular calcium homeostasis.

Environmental chemicals can disrupt neonatal cardiomyocyte physiology

BackgroundThe ubiquitous nature of plastics has raised concerns pertaining to continuous exposure to plastic polymers and human health risks. Of particular concern is the use of endocrine‐disrupting chemicals in plastic production, including bisphenol A (BPA). Widespread and continuous exposure to BPA can occur through dietary intake, inhalation, dermal and intravenous exposure via consumer products and medical devices. Recent studies have shown a link between BPA exposure and adverse cardiovascular outcomes. This is particularly concerning for vulnerable populations who are at risk of high BPA exposure due to medical treatment, including pediatric patients. Despite significant differences in the cardiac physiology of the developing and adult heart, our current understanding of BPA‐induced cardiac dysfunction has been limited to adult models.ObjectiveCardiac physiology differs significantly between the pediatric and adult heart. We aimed to determine the extent to which BPA exposure contributes to cardiovascular dysfunction, using a neonatal cardiomyocyte model.MethodsNeonatal rat ventricular myocytes (NRVM) were monitored to assess cell viability, spontaneous beating rate, beat rate variability, and calcium‐handling in the presence of control or bisphenol‐supplemented media. A range of environmental and clinically‐relevant exposure levels were tested [10−9 – 10−5M].ResultsAcute BPA exposure (15 – 20 min) altered cardiomyocyte functionality, resulting in a slowed spontaneous beating rate and increased beat rate variability beginning at low concentrations (10−8M BPA). BPA exposure also impaired intracellular calcium handling, resulting in diminished calcium transient amplitudes and prolonged calcium transient duration time, which replicated our previously published studies in isolated‐whole hearts. BPA‐induced changes in calcium handling increased the propensity for calcium transient alternans, beginning at low BPA concentrations (10−9M). The observed alterations in calcium kinetics following BPA exposure were not the result of cytotoxicity or altered cell viability.ConclusionOur studies show that acute BPA chemical exposure can alter neonatal cardiomyocyte physiology. Additional studies are necessary to determine the effects of bisphenol exposure on pediatric heart electrophysiology and mechanical function, as well as the impact of long‐term treatment that more closely mimics clinical exposure.Support or Funding InformationThis publication was supported by the National Institutes of Health (R00ES023477, UL1TR000075), Children's Research Institute and Children's National Heart Institute.

Performance and in-house validation of a bioassay for the determination of beta1-autoantibodies found in patients with cardiomyopathy.

BackgroundAutoantibodies specific for the adrenergic beta1-receptor were identified to be an essential factor for the pathogenesis of dilated cardiomyopathy. For the detection of these autoantibodies, a bioassay was developed and has been used, measuring the positive chronotropic effect on spontaneously beating neonatal rat cardiomyocytes. In order to use this bioassay as an analytical tool to monitor the effectiveness of autoantibody neutralizing therapy in a regulated field, there is a need to assess its analytical performance and validate it according to current guidelines.MethodsUsing standard autoantibody samples, the increased beat rate compared to the basal rate [delta beats/min] was recorded when investigating guideline required assay performance parameters.ResultsThe analytical specificity and sensitivity of the bioassay was demonstrated. The limit of detection and positivity cut-off level were determined to be 3.56 and 7.97 delta beats/min, respectively. The coefficient of variation (CV) of all tested single values (four technical replicates each) was ≤15.2%. The CV of precision within each measuring series did not exceed 20%. Furthermore, the sample stability under a variety of different storage conditions was assessed, as well as the robustness of the cardiomyocyte preparations, which were both given.ConclusionThis bioassay fulfilled guideline determined quality requirements and proved to be appropriate for its application in clinical trials.

Cardiac drug-drug interaction between HCV-NS5B pronucleotide inhibitors and amiodarone is determined by their specific diastereochemistry

Severe bradycardia/bradyarrhythmia following coadministration of the HCV-NS5B prodrug sofosbuvir with amiodarone was recently reported. Our previous preclinical in vivo experiments demonstrated that only certain HCV-NS5B prodrugs elicit bradycardia when combined with amiodarone. In this study, we evaluate the impact of HCV-NS5B prodrug phosphoramidate diastereochemistry (D-/L-alanine, R-/S-phosphoryl) in vitro and in vivo. Co-applied with amiodarone, L-ala,SP prodrugs increased beating rate and decreased beat amplitude in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), but D-ala,RP produgs, including MK-3682, did not. Stereochemical selectivity on emerging bradycardia was confirmed in vivo. Diastereomer pairs entered cells equally well, and there was no difference in intracellular accumulation of L-ala,SP metabolites ± amiodarone, but no D-ala,RP metabolites were detected. Cathepsin A (CatA) inhibitors attenuated L-ala,SP prodrug metabolite formation, yet exacerbated L-ala,SP + amiodarone effects, implicating the prodrugs in these effects. Experiments indicate that pharmacological effects and metabolic conversion to UTP analog are L-ala,SP prodrug-dependent in cardiomyocytes.