Beating Activity Research Articles

Identification of Nicotinic Acetylcholine Receptor for N-Acetylcysteine to Rescue Nicotine-induced Injury Using BeatingCilia in Primary Tissue Derived Airway Organoids.

Smoking is one of the major contributors to airway injuries. N-acetylcysteine (NAC) has been proposed as a treatment or preventive measure for such injuries. However, the exact nature of the smoking-induced injury and the protective mechanism of NAC are not yet fully understood. Here, patient tissue-derived airway organoids for modeling smoking-induced injury, therapeutic investigation, and mechanism studies are developed. Airway organoids consist mainly of ciliated cells, together with basal cells, goblet cells, and myofibroblast-like cells. The organoids display apical-out and basal-in polarity and are enriched in beating cilia, which are sensitive to smoking challenge and NAC treatment. An algorithm is developed to measure ciliary beating activity by analyzing the altered beating pattern of cilia in response to nicotine challenge and NAC treatment. Nicotinic acetylcholine receptors (nAChRs) expressed by airway organoids are involved in the mechanisms of nicotine-induced injury through the nicotine-nAChR pathway. In contrast to the common understanding that NAC has an antioxidative effect that mitigates airway damage, it is elucidated that NAC binding to nicotine can abolish the binding capacity of nicotine to nAChRs and thus prevent nicotine-induced injury. This study focuses on the advances and potential of humanized organoids in understanding biological processes, mechanisms, and identifying therapeutic targets.

Improving the development of human engineered cardiac tissue by gold nanorods embedded extracellular matrix for long-term viability.

A myocardial infarction (MI), commonly called a heart attack, results in the death of cardiomyocytes (CMs) in the heart. Tissue engineering provides a promising strategy for the treatment of MI, but the maturation of human engineered cardiac tissue (hECT) still requires improvement. Conductive polymers and nanomaterials have been incorporated into the extracellular matrix to enhance the mechanical and electrical coupling between cardiac cells. Here we report a simple approach to incorporate gold nanorods (GNRs) into the fibrin hydrogel to form a GNR-fibrin matrix, which is used as the major component of the extracellular matrix for forming a 3D hECT construct suspended between two flexible posts. The hECTs made with GNR-fibrin hydrogel showed markers of maturation such as higher twitch force, synchronous beating activity, sarcomere maturation and alignment, t-tubule network development, and calcium handling improvement. Most importantly, the GNR-hECTs can survive over 9 months. We envision that the hECT with GNRs holds the potential to restore the functionality of the infarcted heart.

Unleashing Potential: First Vietnamese 3D in vitro Model using Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Advancements in studying cardiovascular diseases (CVDs) face challenges related to cell sources and utilizing animal models. To mitigate these limitations, three-dimensional (3D) in vitro modeling using human induced pluripotent stem cell-derived cardiomyocytes (iCMs) has emerged as a promising approach. In this study, human induced pluripotent stem cells (hiPSCs) were successfully differentiated into cardiomyocytes and subsequently used to generate spheroids via the hanging drop method. We comprehensively characterized the formation, fusion, and beating capability of the generated spheroids. The hiPSCs were efficiently differentiated into cardiomyocytes, exhibiting contractile activity and expressing cardiac-specific markers such as α-actinin and cardiac troponin. Subsequent results demonstrated the successful formation of iCM-derived cardiac spheroids within 48 hours, with beating activity observed after two days cultured in an ultra-low attachment 96-well plate. In addition, similar to the iCM monolayer, the established spheroids also positively expressed α-actinin. Notably, the spheroids exhibited rapid assembly and maintained survival and stable beating throughout the observation period. These findings highlight the potential of establishing a robust in vitro iCM-derived 3D spheroids model for drug testing and cardiovascular disease modeling, typically for Vietnamese people.

Abstract 16686: A Novel Co-Culture 3D Cardiac Organoid Models Early Heart Development

Introduction: Embryoid body-derived cardiac organoids (CO), incorporating complex chamber-like structures, have been reported since 2021. By contrast, co-culture COs described so far, which have been assembled from primary or hESC derived-cardiomyocytes, endothelial and fibroblast cells, lack complex structures. Cells of the endocardial lineage have a critical role in heart septation and chamber formation in vivo , but their potential role in engineering complex COs is hitherto unreported. We present a co-culture CO method incorporating endocardial lineage cells, showing these cells' critical contribution in forming chambered and vascularised COs suitable to model heart development and congenital heart disease (CHD). Methods: hESC-cardiomyocytes (CM, Day12), hESC-endothelial cell (EC, Day16), hESC-cardiac fibroblasts (Fb, Day16), and hESC-endocardial like cells (ECC, Day12) are differentiated with 2D protocols. COs are formed by co-culturing cells (1-2x10 ^ 4 cells/organoid) in ultra low attachment plates. Mature COs contain CMs, ECs and Fbs (3:1:1). Developing COs contain CMs, ECs, and ECCs (2:1:2). After four weeks, COs are characterised by immunostaining and optical mapping. Results: Mature and Developing COs form in three days of co-culture, with beating activities restoring during 2-7 days. Fig1A shows that both mature COs and developing COs beat regularly. Mature COs show evenly distributed cell populations (Fig1B). Developing COs form chamber-like cavities with surrounding vascular lumens (Fig1C). The inside chamber is layered by endocardial cells (Fig1C); vascular lumens form networks (Fig1C) within the middle compartment of CMs (Fig1D). Fibroblasts mainly specify the outside layer of developing COs (Fig1D). Conclusions: Endocardial lineage is critical in forming chamber-like structures in COs. This lineage-controllable developing CO can apply to drug screening and model early heart development and CHDs.

Heart and kidney organoids maintain organ-specific function in a microfluidic system

Heart and kidney communicate with one another in an interdependent relationship and they influence each other's behavior reciprocally, as pathological changes in one organ can damage the other. Although independent human in vitro models for heart and kidney exist, they do not capture their dynamic crosstalk. We have developed a microfluidic system which can be used to study heart and kidney interaction in vitro. Cardiac microtissues (cMTs) and kidney organoids (kOs) derived from human induced pluripotent stem cells (hiPSCs) were generated and loaded into two separated communicating chambers of a perfusion chip. Static culture conditions were compared with dynamic culture under unidirectional flow. Tissue viability was maintained for minimally 72 h under both conditions, as indicated by the presence of sarcomeric structures coupled with beating activity in cMTs and the presence of nephron structures and albumin uptake in kOs. We concluded that this system enables the study of human cardiac and kidney organoid interaction in vitro while controlling parameters like fluidic flow speed and direction. Together, this “cardiorenal-unit” provides a new in vitro model to study the cardiorenal axis and it may be further developed to investigate diseases involving both two organs and their potential treatments.

Seasonal variation in activity and nearshore habitat use of Lake Trout in a subarctic lake

BackgroundIn lake ecosystems, predatory fish can move and forage across both nearshore and offshore habitats. This coupling of sub-habitats, which is important in stabilizing lake food webs, has largely been assessed from a dietary perspective and has not included movement data. As such, empirical estimates of the seasonal dynamics of these coupling movements by fish are rarely quantified, especially for northern lakes. Here we collect fine-scale fish movement data on Lake Trout (Salvelinus namaycush), a predatory cold-water fish known to link nearshore and offshore habitats, to test for seasonal drivers of activity, habitat use and diet in a subarctic lake.MethodsWe used an acoustic telemetry positioning array to track the depth and spatial movements of 43 Lake Trout in a subarctic lake over two years. From these data we estimated seasonal 50% home ranges, movements rates, tail beat activity, depth use, and nearshore habitat use. Additionally, we examined stomach contents to quantify seasonal diet. Data from water temperature and light loggers were used to monitor abiotic lake conditions and compare to telemetry data.ResultsLake Trout showed repeatable seasonal patterns of nearshore habitat use that peaked each spring and fall, were lower throughout the long winter, and least in summer when this habitat was above preferred temperatures. Stomach content data showed that Lake Trout acquired the most nearshore prey during the brief spring season, followed by fall, and winter, supporting telemetry results. Activity rates were highest in spring when feeding on invertebrates and least in summer when foraging offshore, presumably on large-bodied prey fish. High rates of nearshore activity in fall were associated with spawning. Nearshore habitat use was widespread and not localized to specific regions of the lake, although there was high overlap of winter nearshore core areas between years.ConclusionsWe provide empirical demonstrations of the seasonal extent to which a mobile top predator links nearshore and offshore habitats in a subarctic lake. Our findings suggest that the nearshore is an important foraging area for Lake Trout for much of the year, and the role of this zone for feeding should be considered in addition to its traditional importance as spawning habitat.

Organ-specific model of simulated ischemia/reperfusion and hyperglycemia based on engineered heart tissue

Here we aimed to establish an in vitro engineered heart tissue (EHT) co-morbidity mimicking model of ischemia-reperfusion injury and diabetes. EHTs were generated from primary neonatal rat cardiomyocytes. Hyperglycemic conditions or hyperosmolar controls were applied for one day to model acute hyperglycemia and for seven days to model chronic hyperglycemia. 120 min' simulated ischemia (SI) was followed by 120 min' reperfusion (R) and 1-day follow-up reperfusion (FR). Normoxic controls (N) were not subjected to SI/R. Half of the EHTs was paced, the other half was left unpaced. To assess cell injury, lactate-dehydrogenase (LDH) concentration was measured. Beating force and activity (frequency) were monitored as cardiomyocyte functional parameters. LDH-release indicated relevant cell injury after SI/N in each experimental condition, with much higher effects in the chronically hyperglycemic/hyperosmolar groups. SI stopped beating of EHTs in each condition, which returned during reperfusion, with weaker recovery in chronic conditions than in acute conditions. Acutely treated EHTs showed small LDH-release and ∼80% recovery of force during reperfusion and follow-up, while chronically treated EHTs showed a marked LDH-release, only ∼30% recovery with reperfusion and complete loss of beating activity during 24 h follow-up reperfusion. We conclude that EHTs respond differently to SI/R injury in acute and chronic hyperglycemia/hyperosmolarity, and that our EHT model is a novel in vitro combination of diabetes and ischemia-reperfusion.

In vitro effect of hydroxychloroquine on pluripotent stem cells and their cardiomyocytes derivatives.

Introduction: Hydroxychloroquine (HDQ) is an antimalarial drug that has also shown its effectiveness in autoimmune diseases. Despite having side effects such as retinopathy, neuromyopathy and controversial cardiac toxicity, HDQ has been presented and now intensively studied for the treatment and prevention of coronavirus disease 2019 (COVID-19). Recent works revealed both beneficial and toxic effects during HDQ treatment. The cardiotoxic profile of HDQ remains unclear and identifying risk factors is challenging. Methods: Here, we used well-established cell-cultured to study the cytotoxic effect of HDQ, mouse induced pluripotent stem cells (miPSC) and their cardiomyocytes (CMs) derivatives were exposed to different concentrations of HDQ. Cell colony morphology was assessed by microscopy whereas cell viability was measured by flow cytometry and impedance-based methods. The effect of HDQ on beating activity of mouse and human induced pluripotent stem cell-derived CMs (miPSC-CMs and hiPSC-CMs, respectively) and mouse embryonic stem cell-derived CMs (mESC-CMs) were captured by the xCELLigence RTCA and microelectrode array (MEA) systems. Results and discussion: Our results revealed that 20µM of HDQ promotes proliferation of stem cells used suggesting that if appropriately monitored, HDQ may have a cardioprotective effect and may also represent a possible candidate for tissue repair. In addition, the field potential signals revealed that higher doses of this medication caused bradycardia that could be reversed with a higher concentration of ß-adrenergic agonist, Isoproterenol (Iso). On the contrary, HDQ caused an increase in the beating rate of hiPSC-CMs, which was further helped upon application of Isoproterenol (Iso) suggesting that HDQ and Iso may also work synergistically. These results indicate that HDQ is potentially toxic at high concentrations and can modulate the beating activity of cardiomyocytes. Moreover, HDQ could have a synergistic inotropic effect with isoproterenol on cardiac cells.

The hows and whys of amastigote flagellum motility in Trypanosoma cruzi.

The protist Trypanosoma cruzi exhibits several extracellular stages characterized by the presence of a long and motile flagellum and one intracellular life cycle stage termed amastigote, which possesses a tiny flagellum barely exiting the flagellar pocket. This stage was so far described as replicative but immotile cells. Unexpectedly, the recent work of M. M. Won, T. Krüger, M. Engstler, and B. A. Burleigh (mBio 14:e03556-22, 2023, https://doi.org/10.1128/mbio.03556-22) revealed that this short flagellum actually displays beating activity. This commentary explores how such a short flagellum could be constructed and why it could affect the parasite's survival inside the mammalian host.

PO-01-003 DIFFERENTIAL ENTRAINMENT PACING FOR A PRECISE DELINEATION OF THE CIRCUIT IN A COMPLEX MACROREENTRANT ATRIAL TACHYCARDIA WITH THE ATRIAL ACTIVATION DURATION EXCEEDING THE TACHYCARDIA CYCLE LENGTH

Precise delineation of macroreentrant AT circuits with an atrial activation duration (AAD) exceeding the TCL is challenging because huge bystander regions adjacent to reentrant circuits can exist that activate extremely late, almost simultaneously with early activation of the next beat within reentrant circuits. Thus, 3D-mapping systems cannot clearly annotate local electrograms outside the window of interest, and activation maps of these ATs often appear confusing with 2 sites of earliest activation, the true earliest site (TES) within the circuit and pseudo-earliest site (PES) in the bystander region. We hypothesized that differential entrainment pacing (DEP) from the TES and PES would be helpful to delineate the precise AT circuit differentiating the TES from the PES. DEP was applied to 9 macroreentrant ATs with TCLs of 258±48ms, in which the 3D-EAM exhibited the earliest activation at 2 remote sites. The AT circuit was determined as follows: mitral isthmus (MI) dependent (n=2), LA roof-dependent (n=1), right atrium scar-related reentry (n=1), PV-gap reentry (n=1), a bi-AT (n=1), and cavotricuspid isthmus dependent (n=3). Entrainment pacing from the TES exhibbited a PPI–TCL <30ms and orthodromic capture of the PES with a long stimulus-electrogram (S-EGM) interval >75% of the TCL in all cases. In contrast, entrainment pacing from the PES exhibited a PPI–TCL >30ms in 7 cases, AT terminated in 1, and failure to capture the TES due to unidirectional block of the PES in 1. A representative example is shown in the Figure. The activation map shows the 2 earliest sites, the LA anterior and posterior walls. An ablation catheter (pacing site 1) and Pentaray catheter (pacing site 2) were positioned at each earliest site, and DEP was performed from pacing sites 1 and then 2. The entrainment pacing from site 1 exhibited a PPI–TCL of 20ms and orthodromic capture of site 2 with a long S-EGM interval (262ms). In contrast, entrainment pacing from site 2 exhibited a PPI–TCL of 120ms and antidromic capture of site 1. Those results indicated that site 1 was the TES within the circuit and site 2 was the PES in a bystander area that activated extremely late. The diagnosis of MI dependent AT with a huge bystander region on the LA posterior wall was made. Correctly annotated, the AAD was 315ms, which was greater than the TCL. DEP from the TES and PES was helpful to delineate the precise circuit of an AT with an AAD exceeding the TCL.

High-resolution microsphere sensor for monitoring the spatial mechanical beating of cardiomyocytes in high-noise multi-scene environments

Advanced measurement methods are critical for quantifying spatial systole and diastole of cardiomyocytes in heart disease research. However, restricted by traditional microscopic observation and measurement methods, current research on the kinetics of a single cardiomyocyte mainly focuses on the focal plane of the utilized microscope. This paper proposes a novel, non-invasive, multi-point, and high-resolution three dimensional (3D) measuring method based on microsphere tracking, which can be used to analyze the mechanical beating of cardiomyocytes at a single-cell level. An expansion rotation method was proposed to analyze off-focus imaging informatics for characterizing the contractile motion of a single cardiomyocyte, obviating the need for costly and complex equipment to monitor cardiomyocytes’ mechanical beating activity. Furthermore, to the best of our knowledge, a 3D beating curve of a single cardiomyocyte was drawn for the first time, and the actual spatial movement states of cardiomyocytes in different scenarios were recorded. The applicability of the motion characterization method was tested to determine the pharmacological effects of a typical cardiovascular drug, isoprenaline (1 μM and 5 μM). Within 18 days, the 3D beating curves indicated that the motion of cardiomyocytes tends to be smooth and regular during maturation. The findings offer insights into the contractile motion of single cardiomyocytes and a deep understanding of their kinetics at a single-cell level for multiple scenarios.

Effect of Ethanolic Extract of Vernonia amygdalina on the Proliferation, Viability and Function of Mouse Induced Pluripotent Stem Cells and Cardiomyocytes

Vernonia amygdalina (V. amygdalina) leaves are commonly used in traditional medicine around the world for the treatment of a plethora disorders, including heart disease. The aim of this study was to examine and evaluate the cardiac effect of V. amygdalina leaf extracts using mouse induced pluripotent stem cells (miPSCs) and their cardiomyocytes' (CMs) derivatives. We used a well-established stem cell culture to assess the effect of V. amygdalina extract on miPSC proliferation, EB formation and the beating activity of miPS cell-derived CMs. To study the cytotoxic effect of our extract, undifferentiating miPSCs were exposed to different concentrations of V. amygdalina. Cell colony formation and EB morphology were assessed using microscopy, whereas the cell viability was accessed with an impedance-based method and immunocytochemistry following treatment with different concentrations of V. amygdalina. Ethanolic extract of V. amygdalina induced toxicity in miPSCs, as revealed by a decrease in cell proliferation and colony formation, and an increase in cell death at a concentration of ≥20 mg/mL. At a concentration of 10 mg/mL, the rate of beating EBs was observed with no significant difference regarding the yield of cardiac cells. In addition, V. amygdalina did not affect the sarcomeric organization, but induced positive or negative effects on miPS cell-derived CMs' differentiation in a concentration-dependent manner. Taken together, our findings demonstrate that the ethanolic extract of V. amygdalina affected cell proliferation, colony forming and cardiac beating capacities in a concentration-dependent manner.

Abstract 14513: An Aberrant Friend - Using Bundle Branch Block for Septal Accessory Pathway Localization

Introduction: While changes in the ventriculoatrial (VA) time with RBBB or LBBB during ORT utilizing a free wall accessory pathway (AP) is well described, such a change in VA time can also be helpful in identifying the location of a septal AP. Objectives: To demonstrate the effect of RBBB and LBBB aberrancy on the VA time for ORT involving septal AP and thereby assist in identifying a posteroseptal AP. Results: A 51-year-old man with WPW syndrome was referred for EP study and ablation due to palpitations. With programmed atrial extrastimulation, an atypical echo beat was noted. While attempting to induce tachycardia, double atrial extrastimuli resulted in non-sustained ORT both with RBBB and LBBB aberrancies (Figure). Measurement of the VA time during non-sustained ORT demonstrated a VA time that was longer in the presence of LBBB aberrancy (110ms) than RBBB aberrancy (97ms) compared to narrow QRS (95ms). Electroanatomic mapping identified the earliest atrial activation of the echo beat on the right posteroseptum. Radiofrequency ablation at this site resulted in immediate loss of ventricular pre-excitation. Conclusions: Changes in the VA interval during AP-mediated SVT are especially important for pathway localization. As demonstrated in prior studies, for free wall APs, the difference between sinus and bundle branch block time (ΔVA) is usually &gt;35ms compared to &lt;35ms for septal APs. Furthermore, studies described that for septal APs, posteroseptal APs have a greater VA time with LBBB, anteroseptal APs with RBBB and no significant change in VA time with midseptal APs. By demonstrating both RBBB and LBBB aberrancy in this patient, this case uniquely validates the concept that the VA time with LBBB aberrancy is greater with posteroseptal APs.

Space microgravity improves proliferation of human iPSC-derived cardiomyocytes.

SummaryIn microgravity, cells undergo profound changes in their properties. However, how human cardiac progenitors respond to space microgravity is unknown. In this study, we evaluated the effect of space microgravity on differentiation of human induced pluripotent stem cell (hiPSC)-derived cardiac progenitors compared with 1G cultures on the International Space Station (ISS). Cryopreserved 3D cardiac progenitors were cultured for 3 weeks on the ISS. Compared with 1G cultures, the microgravity cultures had 3-fold larger sphere sizes, 20-fold higher counts of nuclei, and increased expression of proliferation markers. Highly enriched cardiomyocytes generated in space microgravity showed improved Ca2+ handling and increased expression of contraction-associated genes. Short-term exposure (3 days) of cardiac progenitors to space microgravity upregulated genes involved in cell proliferation, survival, cardiac differentiation, and contraction, consistent with improved microgravity cultures at the late stage. These results indicate that space microgravity increased proliferation of hiPSC-cardiomyocytes, which had appropriate structure and function.

A Recombinant Dimethylarginine Dimethylaminohydrolase-1–Based Biotherapeutics to Pharmacologically Lower Asymmetric Dimethyl Arginine, thus Improving Postischemic Cardiac Function and Cardiomyocyte Mitochondrial Activity

High serum levels of asymmetric dimethyl arginine (ADMA) are associated with cardiovascular disease and mortality. Pharmacological agents to specifically lower ADMA and their potential impact on cardiovascular complications are not known. In this study, we aimed to investigate the effect of specific lowering of ADMA on myocardial response to ischemia-reperfusion injury (I/R) and direct effects on cardiomyocyte function. Effects of recombinant dimethylarginine dimethylaminohydrolase (rDDAH)-1 on I/R injury were determined using isolated mouse heart preparation. Respiration capacity and mitochondrial reactive oxygen species (ROS) generation were determined on mouse cardiomyocytes. Our results show that lowering ADMA by rDDAH-1 treatment resulted in improved recovery of cardiac function and reduction in myocardial infarct size in mouse heart response to I/R injury (control 22.24 ±4.60% versus rDDAH-1 15.90 ±4.23%, P < 0.01). In mouse cardiomyocytes, rDDAH-1 treatment improved ADMA-induced dysregulation of respiration capacity and decreased mitochondrial ROS. Furthermore, in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes with impaired contractility under hypoxia and high ADMA, rDDAH-1 treatment improved recovery and beating frequency (P < 0.05). rDDAH-1 treatment selectively modified I/R-induced myocardial cytokine expression, resulting in reduction in proinflammatory cytokine IL-17A (P < 0.001) and increased expression of anti-inflammatory cytokines IL-10 and IL-13 (P < 0.01). Further in vitro studies showed that IL-17A was the predominant and common cytokine modulated by ADMA-DDAH pathway in heart, cardiomyocytes, and endothelial cells. These studies show that lowering ADMA by pharmacological treatment with rDDAH-1 reduced I/R injury, improved cardiac function, and ameliorated cardiomyocyte bioenergetics and beating activity. These effects may be attributable to ADMA lowering in cardiomyocytes and preservation of cardiomyocyte mitochondrial function. SIGNIFICANCE STATEMENT: The pathological role of asymmetric dimethyl arginine (ADMA) has been demonstrated by its association with cardiovascular disease and mortality. Currently, pharmacological drugs to specifically lower ADMA are not available. The present study provides the first evidence that lowering of ADMA by recombinant recombinant dimethylarginine dimethylaminohydrolase (rDDAH)-1 improved postischemic cardiac function and cardiomyocyte bioenergetics and beating activity. Our studies suggest that lowering of ADMA by pharmacologic treatment offers opportunity to develop new therapies for the treatment of cardiovascular and renal disease.

Functional and structural phenotyping of cardiomyocytes in the 3D organization of embryoid bodies exposed to arsenic trioxide

Chronic exposure to environmental pollutants threatens human health. Arsenic, a world-wide diffused toxicant, is associated to cardiac pathology in the adult and to congenital heart defects in the foetus. Poorly known are its effects on perinatal cardiomyocytes. Here, bioinformatic image-analysis tools were coupled with cellular and molecular analyses to obtain functional and structural quantitative metrics of the impairment induced by 0.1, 0.5 or 1.0 µM arsenic trioxide exposure on the perinatal-like cardiomyocyte component of mouse embryoid bodies, within their 3D complex cell organization. With this approach, we quantified alterations to the (a) beating activity; (b) sarcomere organization (texture, edge, repetitiveness, height and width of the Z bands); (c) cardiomyocyte size and shape; (d) volume occupied by cardiomyocytes within the EBs. Sarcomere organization and cell morphology impairment are paralleled by differential expression of sarcomeric α-actin and Tropomyosin proteins and of acta2, myh6 and myh7 genes. Also, significant increase of Cx40, Cx43 and Cx45 connexin genes and of Cx43 protein expression profiles is paralleled by large Cx43 immunofluorescence signals. These results provide new insights into the role of arsenic in impairing cytoskeletal components of perinatal-like cardiomyocytes which, in turn, affect cell size, shape and beating capacity.

Assessment of the In Vitro Cytotoxicity Effects of the Leaf Methanol Extract of Crinum zeylanicum on Mouse Induced Pluripotent Stem Cells and Their Cardiomyocytes Derivatives.

Crinum zeylanicum (C. zeylanicum) is commonly used in African folk medicine to treat cardiovascular ailments. In the present study, we investigated the cytotoxic effect of the leaf methanol extract of C. zeylanicum (CZE) using mouse pluripotent stem cells (mPSCs). mPSCs and their cardiomyocytes (CMs) derivatives were exposed to CZE at different concentrations. Cell proliferation, differentiation capacity, and beating activity were assessed using xCELLigence system and microscopy for embryoid body (EB) morphology. Expression of markers associated with major cardiac cell types was examined by immunofluorescence and quantitative RT-PCR. Intracellular reactive oxygen species (ROS) levels were assessed by dichlorodihydrofluorescein diacetate staining. The results showed that the plant extract significantly reduced cell proliferation and viability in a concentration- and time-dependent manner. This was accompanied by a decrease in EB size and an increase in intracellular ROS. High concentrations of CZE decreased the expression of some important cardiac biomarkers. In addition, CZE treatment was associated with poor sarcomere structural organization of CMs and significantly decreased the amplitude and beating rate of CMs, without affecting CMs viability. These results indicate that CZE might be toxic at high concentrations in the embryonic stages of stem cells and could modulate the contracting activity of CMs.

Steady state-evoked potentials of subjective beat perception in musical rhythms.

Synchronization of movement to music is a seemingly universal human capacity that depends on sustained beat perception. Previous research has suggested that listener's conscious perception of the musical structure (e.g., beat and meter) might be reflected in neural responses that follow the frequency of the beat. However, the extent to which these neural responses directly reflect concurrent, listener-reported perception of musical beat versus stimulus-driven activity is understudied. We investigated whether steady state-evoked potentials (SSEPs), measured using electroencephalography (EEG), reflect conscious perception of beat by holding the stimulus constant while contextually manipulating listeners' perception and measuring perceptual responses on every trial. Listeners with minimal music training heard a musical excerpt that strongly supported one of two beat patterns (context phase), followed by a rhythm consistent with either beat pattern (ambiguous phase). During the final phase, listeners indicated whether or not a superimposed drum matched the perceived beat (probe phase). Participants were more likely to indicate that the probe matched the music when that probe matched the original context, suggesting an ability to maintain the beat percept through the ambiguous phase. Likewise, we observed that the spectral amplitude during the ambiguous phase was higher at frequencies that matched the beat of the preceding context. Exploratory analyses investigated whether EEG amplitude at the beat-related SSEPs (steady state-evoked potentials) predicted performance on the beat induction task on a single-trial basis, but were inconclusive. Our findings substantiate the claim that auditory SSEPs reflect conscious perception of musical beat and not just stimulus features.

Versatile Cell and Animal Models for Advanced Investigation of Lead Poisoning.

The heavy metal, lead (Pb) can irreversibly damage the human nervous system. To help understand Pb-induced damage, we applied a genetically encoded Förster resonance energy transfer (FRET)-based Pb biosensor Met-lead 1.44 M1 to two living systems to monitor the concentration of Pb: induced pluripotent stem cell (iPSC)-derived cardiomyocytes as a semi-tissue platform and Drosophila melanogaster fruit flies as an in vivo animal model. Different FRET imaging modalities were used to obtain FRET signals, which represented the presence of Pb in the tested samples in different spatial dimensions. Using iPSC-derived cardiomyocytes, the relationship between beating activity (20–24 beats per minute, bpm) determined from the fluctuation of fluorescent signals and the concentrations of Pb represented by the FRET emission ratio values of Met-lead 1.44 M1 was revealed from simultaneous measurements. Pb (50 μM) affected the beating activity of cardiomyocytes, whereas two drugs that stop the entry of Pb differentially affected this beating activity: verapamil (2 μM) did not reverse the cessation of beating, whereas 2-APB (50 μM) partially restored this activity (16 bpm). The results clearly demonstrate the potential of this biosensor system as an anti-Pb drug screening application. In the Drosophila model, Pb was detected within the adult brain or larval central nervous system (Cha-gal4 > UAS-Met-lead 1.44 M1) using fast epifluorescence and high-resolution two-photon 3D FRET ratio image systems. The tissue-specific expression of Pb biosensors provides an excellent opportunity to explore the possible Pb-specific populations within living organisms. We believe that this integrated Pb biosensor system can be applied to the prevention of Pb poisoning and advanced research on Pb neurotoxicology.

IIIG9 inhibition in adult ependymal cells changes adherens junctions structure and induces cellular detachment

Ependymal cells have multiple apical cilia that line the ventricular surfaces and the central canal of spinal cord. In cancer, the loss of ependymal cell polarity promotes the formation of different types of tumors, such as supratentorial anaplastic ependymomas, which are highly aggressive in children. IIIG9 (PPP1R32) is a protein restricted to adult ependymal cells located in cilia and in the apical cytoplasm and has unknown function. In this work, we studied the expression and localization of IIIG9 in the adherens junctions (cadherin/β-catenin-positive junctions) of adult brain ependymal cells using confocal and transmission electron microscopy. Through in vivo loss-of-function studies, ependymal denudation (single-dose injection experiments of inhibitory adenovirus) was observed, inducing the formation of ependymal cells with a “balloon-like” morphology. These cells had reduced cadherin expression (and/or delocalization) and cleavage of the cell death marker caspase-3, with “cilia rigidity” morphology (probably vibrational beating activity) and ventriculomegaly occurring prior to these events. Finally, after performing continuous infusions of adenovirus for 14 days, we observed total cell denudation and reactive parenchymal astrogliosis. Our data confirmed that IIIG9 is essential for the maintenance of adherens junctions of polarized ependymal cells. Eventually, altered levels of this protein in ependymal cell differentiation may increase ventricular pathologies, such as hydrocephalus or neoplastic transformation.