Beat Amplitude Research Articles

Modelling myocardial ischemia/reperfusion injury with inflammatory response in human ventricular cardiac organoids.

Current therapeutic drug exploring targeting at myocardial ischemia/reperfusion (I/R) injury is limited due to the lack of humanized cardiac models that resemble myocardial damage and inflammatory response. Herein, we develop ventricular cardiac organoids from human induced pluripotent stem cells (hiPSCs) and simulate I/R injury by hypoxia/reoxygenation (H/R), which results in increased cardiomyocytes apoptosis, elevated oxidative stress, disrupted morphological structure and decreased beat amplitude. RNA-seq reveals a potential role of type I interferon (IFN-I) in this I/R injury model. We then introduce THP-1 cells and reveal inflammatory responses between monocytes/macrophages and H/R-induced ventricular cardiac organoids. Furthermore, we demonstrate Anifrolumab, an FDA approved antagonist of IFN-I receptor, effectively decreases IFN-I secretion and related gene expression, attenuates H/R-induced inflammation and oxidative stress in the co-culture system. This study advances the modelling of myocardial I/R injury with inflammatory response in human cardiac organoids, which provides a reliable platform for preclinical study and drug screening.

Ciliary Motility Decreased by a CO2/HCO3--Free Solution in Ciliated Human Nasal Epithelial Cells Having a pH Elevated by Carbonic Anhydrase IV.

An application of CO2/HCO3--free solution (Zero-CO2) did not increase intracellular pH (pHi) in ciliated human nasal epithelial cells (c-hNECs), leading to no increase in frequency (CBF) or amplitude (CBA) of the ciliary beating. This study demonstrated that the pHi of c-hNECs expressing carbonic anhydrase IV (CAIV) is high (7.64), while the pHi of ciliated human bronchial epithelial cells (c-hBECs) expressing no CAIV is low (7.10). An extremely high pHi of c-hNECs caused pHi, CBF and CBA to decrease upon Zero-CO2 application, while a low pHi of c-hBECs caused them to increase. An extremely high pHi was generated by a high rate of HCO3- influx via interactions between CAIV and Na+/HCO3- cotransport (NBC) in c-hNECs. An NBC inhibitor (S0859) decreased pHi, CBF and CBA and increased CBF and CBA in c-hNECs upon Zero-CO2 application. In conclusion, the interactions of CAIV and NBC maximize HCO3- influx to increase pHi in c-hNECs. This novel mechanism causes pHi to decrease, leading to no increase in CBF and CBA in c-hNECs upon Zero-CO2 application, and appears to play a crucial role in maintaining pHi, CBF and CBA in c-hNECs periodically exposed to air (0.04% CO2) with respiration.

Abstract We023: P53 Activation Promotes Maturational Characteristics of Pluripotent Stem Cell-derived Cardiomyocytes in 3D Suspension Culture via FOXO-FOXM1 Regulation

Background: Current protocols can generate highly-pure populations of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro that recapitulate key characteristics of mature in vivo cardiomyocytes. Yet, there exists a risk of arrhythmias when hiPSC-CMs are injected into large animal models. This prompts further investigation into the mechanisms of hiPSC-CM maturation to facilitate clinical translation. Hypothesis: The forkhead box (FOX) family of transcription factors can regulate maturation in neonatal cardiomyocytes through a balance between FOXO and FOXM1. We also previously found that p53 activation could enhance hiPSC-CM maturation. Therefore, we hypothesized that p53 activation increases FOXO and decreases FOXM1 to promote hiPSC-CM maturation in three-dimensional (3D) suspension culture. Methodology: 3D cultures of hiPSC-CMs were treated with Nutlin-3a (p53 activator), LOM612 (FOXO activator), AS1842856 (FOXO inhibitor), or RCM-1 (FOXM1 inhibitor), starting 2 days after onset of beating. The hiPSC-CMs were assessed for maturation in metabolic, contractile, and electrophysiological properties, by Seahorse mito stress test, Multi electrode array, and VALA kinetic image cytometer respectively. Results: P53 activation promoted FOXO upregulation and FOXM1 downregulation in hiPSC-CMs, measured by RT-qPCR and immunostaining. Alongside this, p53 activation also promoted hiPSC-CM metabolic and contractile maturational characteristics, seen by increase in oxygen consumption and beat amplitude respectively. FOXO inhibition significantly decreased expression of cardiac-specific markers such as TNNT2 and eliminated spontaneous beating. In contrast, FOXO activation or FOXM1 inhibition promoted maturational characteristics of hiPSC-CMs such as increase in contractility, oxygen consumption, and voltage peak maximum upstroke velocity. Further, by single-cell RNAseq, FOXO activated groups showed increase in cardiac maturational pathways compared against DMSO control groups. Conclusions: These results show that p53 activation promotes FOXO and suppresses FOXM1, which modulate hiPSC-CM maturation in 3D suspension. Our study expands current understanding of hiPSC-CM maturational mechanisms in a clinically-relevant 3D culture system.

Abstract Or111: Titin cleavage disrupts sarcomere-adhesion tensional homeostasis triggering cardiomyocyte arrhythmia in vitro

The cardiac tissue experiences several forms of mechanical stress, including contraction of cardiomyocytes and forces derived from the stiffness of extracellular matrix (ECM). Preserving homeostatic mechanical forces is crucial for a healthy heart, yet the mechanisms involved are not well-explored. Our study delves into cardiomyocyte responses following the sudden release of mechanical tension across titin, a giant sarcomere protein crucial for setting cardiomyocyte stiffness. Through the expression of virally transduced TEV protease (TEVp) in neonatal mouse cardiomyocytes (NMCMs), we induced the cleavage of mutant titin including the specific TEV protease recognition site (TEVs) within the mechanically active I-band region of the protein. Titin cleavage was validated by 1.8% polyacrylamide SDS-PAGE electrophoresis, revealing a primary band at ~2.2 MDa in TEV-positive NMCMs, corresponding to digested titin fragment A-M. This cleavage, confirmed at 2 days post-infection (dpi) with ~80% efficiency, reached 100% by 5 dpi. Consequently, the Young’s modulus of affected cardiomyocytes dropped by 50%, as determined by Atomic Force Spectroscopy. Sarcomere disarray increased over time, as evidenced by immunofluorescence, yet this effect did not induce cell death or proliferation. Cell viability was confirmed by the fact that affected cardiomyocytes are able to contract spontaneously, although titin cleavage resulted in reduced beating amplitude and induced arrhythmic behavior. Considering this arrhythmogenic situation, we tested cell-cell adhesion through dissociation assays, which revealed significantly reduced intercellular adhesion in affected cardiomyocytes. RNAseq confirmed involvement of pathways related to heart contraction, anchoring junctions, and cell junctions. Specifically, dysregulated genes associated with gap-junction and costamere complexes were observed at 5 dpi. Alterations of adhesion were further confirmed by reductions in connexin 43 and focal adhesion length in immunofluorescence experiments, which also showed mislocalization of important adhesive proteins. In conclusion, titin cleavage in neonatal cardiomyocytes leads to sarcomere disarray, perturbed cell-cell adhesion and arrhythmia. These findings highlight the importance of full mechanical integrity of the titin filament in living cardiomyocytes.

Boric acid toxic sugar bait suppresses male Aedes aegypti (Diptera: Culicidae): wing beat frequency and amplitude, flight activity, fecundity, insemination, and mate-finding Allee effect.

Controlling the spread of arboviral diseases remains a considerable challenge due to the rapid development of insecticide resistance in Aedes mosquitoes. This study evaluated the effects of boric acid-containing toxic sugar bait (TSB) on field populations of resistant Aedes aegypti mosquitoes. In addition, this study examined the flight activity and wing beat frequency and amplitude of males and the flight activity, fecundity, and insemination of females after pairing with males exposed to TSB. The population dynamics of Aedes mosquitoes under imbalanced sex ratios were examined to simulate realistic field conditions for male suppression under the effect of TSB. The mortality of male mosquitoes was consistently high within 24 h after exposure. By contrast, the mortality of female mosquitoes was inconsistent, with over 70% mortality observed at 168 h. The flight activity and wing beat amplitude of treated males were significantly lower than those of controls, but no significant difference in wing beat frequency was detected. The fecundity and insemination of treated female mosquitoes were lower than those of controls. A simulation study indicated that considerably low male population densities led to mating failures, triggering a mate-finding Allee effect and resulting in persistently low population levels. Boric acid-containing TSB could effectively complement current chemical intervention approaches to control resistant mosquito populations. TSB is effective in reducing field male populations and impairing male flight activity and female-seeking behavior, resulting in decreased fecundity and insemination. Male suppression due to TSB potentially results in a small mosquito population. © 2024 Society of Chemical Industry.

Wings of Change: aPKC/FoxP-dependent plasticity in steering motor neurons underlies operant self-learning in Drosophila

Background Motor learning is central to human existence, such as learning to speak or walk, sports moves, or rehabilitation after injury. Evidence suggests that all forms of motor learning share an evolutionarily conserved molecular plasticity pathway. Here, we present novel insights into the neural processes underlying operant self-learning, a form of motor learning in the fruit fly Drosophila. Methods We operantly trained wild type and transgenic Drosophila fruit flies, tethered at the torque meter, in a motor learning task that required them to initiate and maintain turning maneuvers around their vertical body axis (yaw torque). We combined this behavioral experiment with transgenic peptide expression, CRISPR/Cas9-mediated, spatio-temporally controlled gene knock-out and confocal microscopy. Results We find that expression of atypical protein kinase C (aPKC) in direct wing steering motoneurons co-expressing the transcription factor FoxP is necessary for this type of motor learning and that aPKC likely acts via non-canonical pathways. We also found that it takes more than a week for CRISPR/Cas9-mediated knockout of FoxP in adult animals to impair motor learning, suggesting that adult FoxP expression is required for operant self-learning. Conclusions Our experiments suggest that, for operant self-learning, a type of motor learning in Drosophila, co-expression of atypical protein kinase C (aPKC) and the transcription factor FoxP is necessary in direct wing steering motoneurons. Some of these neurons control the wing beat amplitude when generating optomotor responses, and we have discovered modulation of optomotor behavior after operant self-learning. We also discovered that aPKC likely acts via non-canonical pathways and that FoxP expression is also required in adult flies.

Wings of Change: aPKC/FoxP-dependent plasticity in steering motor neurons underlies operant self-learning in Drosophila.

Motor learning is central to human existence, such as learning to speak or walk, sports moves, or rehabilitation after injury. Evidence suggests that all forms of motor learning share an evolutionarily conserved molecular plasticity pathway. Here, we present novel insights into the neural processes underlying operant self-learning, a form of motor learning in the fruit fly Drosophila. We operantly trained wild type and transgenic Drosophila fruit flies, tethered at the torque meter, in a motor learning task that required them to initiate and maintain turning maneuvers around their vertical body axis (yaw torque). We combined this behavioral experiment with transgenic peptide expression, CRISPR/Cas9-mediated, spatio-temporally controlled gene knock-out and confocal microscopy. We find that expression of atypical protein kinase C (aPKC) in direct wing steering motoneurons co-expressing the transcription factor FoxP is necessary for this type of motor learning and that aPKC likely acts via non-canonical pathways. We also found that it takes more than a week for CRISPR/Cas9-mediated knockout of FoxP in adult animals to impair motor learning, suggesting that adult FoxP expression is required for operant self-learning. Our experiments suggest that, for operant self-learning, a type of motor learning in Drosophila, co-expression of atypical protein kinase C (aPKC) and the transcription factor FoxP is necessary in direct wing steering motoneurons. Some of these neurons control the wing beat amplitude when generating optomotor responses, and we have discovered modulation of optomotor behavior after operant self-learning. We also discovered that aPKC likely acts via non-canonical pathways and that FoxP expression is also required in adult flies.

Room-temperature quantum coherence of entangled multiexcitons in a metal-organic framework.

Singlet fission can generate an exchange-coupled quintet triplet pair state 5TT, which could lead to the realization of quantum computing and quantum sensing using entangled multiple qubits even at room temperature. However, the observation of the quantum coherence of 5TT has been limited to cryogenic temperatures, and the fundamental question is what kind of material design will enable its room-temperature quantum coherence. Here, we show that the quantum coherence of singlet fission-derived 5TT in a chromophore-integrated metal-organic framework can be over hundred nanoseconds at room temperature. The suppressed motion of the chromophores in ordered domains within the metal-organic framework leads to the enough fluctuation of the exchange interaction necessary for 5TT generation but, at the same time, does not cause severe 5TT decoherence. Furthermore, the phase and amplitude of quantum beating depend on the molecular motion, opening the way to room-temperature molecular quantum computing based on multiple quantum gate control.

MicroBundlePillarTrack: A Python package for automated segmentation, tracking, and analysis of pillar deflection in cardiac microbundles.

Movies of human induced pluripotent stem cell (hiPSC)-derived engineered cardiac tissue (microbundles) contain abundant information about structural and functional maturity. However, extracting these data in a reproducible and high-throughput manner remains a major challenge. Furthermore, it is not straightforward to make direct quantitative comparisons across the multiple in vitro experimental platforms employed to fabricate these tissues. Here, we present "MicroBundlePillarTrack," an open-source optical flow-based package developed in Python to track the deflection of pillars in cardiac microbundles grown on experimental platforms with two different pillar designs ("Type 1" and "Type 2" design). Our software is able to automatically segment the pillars, track their displacements, and output time-dependent metrics for contractility analysis, including beating amplitude and rate, contractile force, and tissue stress. Because this software is fully automated, it will allow for both faster and more reproducible analyses of larger datasets and it will enable more reliable cross-platform comparisons as compared to existing approaches that require manual steps and are tailored to a specific experimental platform. To complement this open-source software, we share a dataset of 1,540 brightfield example movies on which we have tested our software. Through sharing this data and software, our goal is to directly enable quantitative comparisons across labs, and facilitate future collective progress via the biomedical engineering open-source data and software ecosystem.

Numerical study on the influence of centroid‐to‐buoyancy center ratio on the motion stability of supercavitating vehicle

SummaryIn order to study and solve the influence of the change of the position of the centroid of the supercavitating vehicle on the motion stability, the influence law of the position of the centroid of the supercavitating vehicle on the motion stability is found, and the quantitative criterion that can guide the design of the supercavitating vehicle is summarized. On the basis of theoretical analysis, the dynamic mesh technology combined with the volume of fluid multiphase flow model and the rigid body six degrees of freedom motion calculation model is used to carry out the supercavitating vehicle motion and supercavitating flow field coupling numerical simulation research for supercavitating vehicles with different cavitator shapes. The results show that the position relationship between the centroid position and the buoyancy position has a great influence on the motion stability of the vehicle. When the center of buoyancy is before the center of mass, the vehicle is unstable; when the center of buoyancy is behind the center of mass, the vehicle sails stably, and when the position of buoyancy action coincides with the center of mass, the amplitude of the tail beat of the vehicle is the smallest, and the navigation is the most stable, which is the best position of the centroid. In this article, the concept of centroid‐to‐buoyancy center ratio is proposed. The so‐called centroid‐to‐buoyancy center ratio refers to the ratio of the length from the centroid to the top of the head of the supercavitating vehicle to the length from the center of buoyancy to the top of the head of the vehicle. When the centroid‐to‐buoyancy center ratio fluctuates in the range of [0.8–1], it is an ideal condition for the stable navigation of the vehicle. When the centroid‐to‐buoyancy center ratio is in the range of (0, 0.8), the tail beat frequency is high, which accelerates the kinetic energy consumption and the rapid instability of the vehicle. Therefore, under the same constraint conditions, it is not that the closer the center of mass is, the better the stability of the vehicle motion is. The optimal centroid position is not a certain “point,” but a “periodic fluctuation interval” that changes with the buoyancy center.

On the role of viscoelasticity in mucociliary clearance: a hydrodynamic continuum approach

We present numerical and analytical predictions of mucociliary clearance based on the continuum description of a viscoelastic mucus film, where momentum transfer from the beating cilia is represented via a Navier-slip boundary condition introduced by Bottier et al. (PLoS Comput. Biol., vol. 13, issue 7, 2017a, e1005552). Mucus viscoelasticity is represented via the Oldroyd-B model, where the relaxation time and the viscosity ratio have been fitted to experimental data for the storage and loss moduli of different types of real mucus, ranging from healthy to diseased conditions. We solve numerically the fully nonlinear governing equations for inertialess flow, and develop analytical solutions via asymptotic expansion in two limits: (i) weak viscoelasticity, i.e. low Deborah number; (ii) low cilia beat amplitude (CBA). All our approaches predict a drop in the mucus flow rate in relation to the Newtonian reference value, as the cilia beat frequency is increased. This relative drop increases with decreasing CBA and slip length. In diseased conditions, e.g. mucus properties characteristic of cystic fibrosis, the drop reaches 30 % in the physiological frequency range. In the case of healthy mucus, no significant drop is observed, even at very high frequency. This contrasts with the deterioration of microorganism propulsion predicted by the low-amplitude theory of Lauga (Phys. Fluids, vol. 19, issue 8, 2007, 083104), and is due to the larger beat amplitude and slip length associated with mucociliary clearance. In the physiological range of the cilia beat frequency, the low-amplitude prediction is accurate for both healthy and diseased conditions. Finally, we find that shear-thinning, modelled via a multi-mode Giesekus model, does not significantly alter our weakly viscoelastic and low-amplitude predictions based on the Oldroyd-B model.

Consonance Perception in Congenital Amusia: Behavioral and Brain Responses to Harmonicity and Beating Cues.

Congenital amusia is a neurodevelopmental disorder characterized by difficulties in the perception and production of music, including the perception of consonance and dissonance, or the judgment of certain combinations of pitches as more pleasant than others. Two perceptual cues for dissonance are inharmonicity (the lack of a common fundamental frequency between components) and beating (amplitude fluctuations produced by close, interacting frequency components). Amusic individuals have previously been reported to be insensitive to inharmonicity, but to exhibit normal sensitivity to beats. In the present study, we measured adaptive discrimination thresholds in amusic participants and found elevated thresholds for both cues. We recorded EEG and measured the MMN in evoked potentials to consonance and dissonance deviants in an oddball paradigm. The amplitude of the MMN response was similar overall for amusic and control participants; however, in controls, there was a tendency toward larger MMNs for inharmonicity than for beating cues, whereas the opposite tendency was observed for the amusic participants. These findings suggest that initial encoding of consonance cues may be intact in amusia despite impaired behavioral performance, but that the relative weight of nonspectral (beating) cues may be increased for amusic individuals.

Quartz Crystal Microbalance Technology Coupled with Impedance for the Dynamic Monitoring of the Cardiomyocyte Beating Function and Drug Screening

The main sensing techniques used to study myocardial pulsation are electrical impedance sensing (EIS) and by quartz crystal microbalance (QCM). While electrical impedance technology is the gold standard for the study of myocardial pulsation, the clinical application of drugs is being followed up in real time additionally, thus, QCM technology needs to be further developed as a very important class of quality sensor technology. Moreover, the application of EIS, in combination with the QCM, for monitoring myocardial pulsation, has been rarely reported. In this paper, a series of cell growth and adhesion conditions were optimized using rat primary cardiomyocytes, and QCM was used in combination with EIS to monitor the adhesion and the myocardial pulsation ability of the cells in real time. Furthermore, cardiomyocytes that adhered to the QCM and EIS were treated with isoprenaline (ISO), a positive inotropic drug, and verapamil (VRP), a negative inotropic drug. Next, the cell index (CI)-time (T) plots, beating amplitude (BA) and beating rate (BR) of the cardiomyocytes were calculated and changes in these parameters, before and after, dosing were evaluated. The results showed that the QCM technique results were not only consistent with the results obtained with EIS, but also that the QCM technique had a certain degree of sensitivity for the calculation of cardiomyocyte beating. Thus, our findings validate the reliability and validity of the QCM technique for measuring cardiomyocyte beating and drug testing. We hope that further studies would evaluate the application of the QCM technology for clinical use.

Potential for cardiac toxicity with methylimidazolium ionic liquids

Methylimidazolium ionic liquids (MILs) are solvent chemicals used in industry. Recent work suggests that MILs are beginning to contaminate the environment and lead to exposure in the general population. In this study, the potential for MILs to cause cardiac toxicity has been examined. The effects of 5 chloride MIL salts possessing increasing alkyl chain lengths (2 C, EMI; 4 C, BMI; 6 C; HMI, 8 C, M8OI; 10 C, DMI) on rat neonatal cardiomyocyte beat rate, beat amplitude and cell survival were initially examined. Increasing alkyl chain length resulted in increasing adverse effects, with effects seen at 10−5 M at all endpoints with M8OI and DMI, the lowest concentration tested. A limited sub-acute toxicity study in rats identified potential cardiotoxic effects with longer chain MILs (HMI, M8OI and DMI) based on clinical chemistry. A 5 month oral/drinking water study with these MILs confirmed cardiotoxicity based on histopathology and clinical chemistry endpoints. Since previous studies in mice did not identify the heart as a target organ, the likely cause of the species difference was investigated. qRT-PCR and Western blotting identified a marked higher expression of p-glycoprotein-3 (also known as ABCB4 or MDR2) and the breast cancer related protein transporter BCRP (also known as ABCG2) in mouse, compared to rat heart. Addition of the BCRP inhibitor Ko143 – but not the p-glycoproteins inhibitor cyclosporin A - increased mouse cardiomyocyte HL-1 cell sensitivity to longer chain MILs to a limited extent. MILs therefore have a potential for cardiotoxicity in rats. Mice may be less sensitive to cardiotoxicity from MILs due in part, to increased excretion via higher levels of cardiac BCRP expression and/or function. MILs alone, therefore may represent a hazard in man in the future, particularly if use levels increase. The impact that MILs exposure has on sensitivity to cardiotoxic drugs, heart disease and other chronic diseases is unknown.

Differential requirements of IQUB for the assembly of radial spoke 1 and the motility of mouse cilia and flagella.

The T-shaped radial spoke (RS) is a protein complex attached to the A microtubule of the outer doublet microtubules, and it extends toward the central pair (CP). It modulates the beat frequency, amplitude, and waveform of the flagella and cilia by serving as a mechanochemical signal transducer between the CP and dyneins. In humans, RS defects cause primary ciliary dyskinesia, but the structural components of triple RSs (RS1, RS2, and RS3) in mammals remain to be elucidated in detail. Here, we introduce a mouse model that lacked the entire RS1 in sperm flagella, due to the deletion of Iqub, while the tracheal cilia possessed intact triple RSs. Furthermore, the absence of IQUB only resulted in male infertility, owing to the sperm motility defect. Based on the mouse model, the RS1 compositions are identified in sperm flagella. In summary, this study elucidates the RS1 components and function in mammalian flagella.

Simultaneous distance and vibration mapping of FMCW-LiDAR with akinetic external cavity diode laser

In this study, the simultaneous distance and vibration mapping of frequency-modulated continuous-wave light detection and ranging (FMCW-LiDAR) is performed using an electro-optical external cavity diode laser (ECDL). Owing to the akinetic operation regime of the ECDL, a uniform (R2 = 1.0000) interference signal can be generated over a bandwidth of 1 GHz at an optical frequency sweep rate of 10 kHz without any postprocessing or system for linearization. Furthermore, the proposed ECDL exhibits a high optical frequency sweep rate of 200 kHz with a considerable linearity of R2 = 0.99975. Based on a point sensing experiment using the FMCW-LiDAR system, three measurands, namely, distance, vibration frequency, and vibration velocity change, are demonstrated with a highly linear relationship between the beat frequency and amplitude of each individual parameter. Furthermore, temporal variations in multiple targets with rapidly frequency-varying vibrations, such as those occurring in music or voice sounds, can be successfully acquired by tracking the temporal change in the beat frequency of each target. Moreover, three-dimensional (3D) mapping of distance, vibration frequency, and vibration velocity change is simultaneously implemented in the same 3D space. The results show that the proposed FMCW-LiDAR system can be used for the 3D visualized display of various measurands within the same space frame.

Abstract P2031: Evaluation Of Adenosine Monophosphate-activated Protein Kinase Activator MK-8722 In Human Induced Pluripotent Stem Cell Derived Cardiomyocyte Monolayer And Microtissue (Biowire II) Models

MK-8722, a systemic pan-AMPK activator improves glucose homeostasis in animal models of diabetes, but causes cardiac hypertrophy # . Two- and 3-dimensional tissue (2D,3D) constructs of human induced pluripotent stem cell derived cardiomyocytes (CM) (iCells, Fujifilm/CDI) were used to probe the translatability of cardiac hypertrophy and the role of AMPK in cardiomyocyte function. Chronic 10-day exposure (2 μM and 10 μM MK-8722) of iCell cardiomyocyte 2D monolayers, probed via monitoring of cellular impedance (CardioECR, Agilent), revealed a decrease in beat amplitude (-13%,2μM;-49%,10μM) as well as an increase in excitability, based on the ability to follow higher pacing frequencies (+81%,2uM;+34%,10uM). Direct force measurements in 3D iCell CM tissues (Biowire II, TARA Biosystems) reported loss of contractility when stimulated at 1Hz (-74%,2μM;-90%,10 μM), as well as changes in passive resting tension (+18%,2μM;-75%,10 μM) over chronic six-week exposure. MK-8722 caused a marked increase in Biowire excitability as revealed by a large drop in the voltage threshold of entrainment of paced tissues (-36%,2μM;-43%,10 μM). The increased excitability of both 2D and 3D CM models suggests a cellular correlate of the increased electrocardiographic QRS amplitude observed in rhesus monkeys upon chronic dosing with MK-8722 # . Hypertrophy of the Biowires could be clearly observed at the end of the 6-week incubation and presented as increased microtissue widths (+5%,2μM; +10%,10μM) and thickened regions of attachment to the chamber force sensors at each end of the Biowire. Volume estimation revealed increases (+22.3%, 2μM; +31.4%, 10 μM) in Biowire tissue volumes that were comparable to heart hypertrophic changes in preclinical test species # (mouse, rat, and rhesus monkey). The effect of 10μM MK-8722 was more pronounced when treatment began before tissues had completed a multi-week electrical stimulation maturation protocol, rather than after. The functional effects in both 2D and 3D CM models, and the contractile and structural readouts of the 3D Biowire model were highly informative in the interpretation of the clinical applicability of the pleiotropic cardiac effects of MK-8722 observed in preclinical testing. # Myers, et al. Science 357, p 507-511 (2017)

Aquatic Walking and Swimming Kinematics of Neonate and Juvenile Epaulette Sharks.

The epaulette shark, Hemiscyllium ocellatum, is a small, reef-dwelling, benthic shark that-using its paired fins-can walk, both in and out of water. Within the reef flats, this species experiences short periods of elevated CO2 and hypoxia as well as fluctuating temperatures as reef flats become isolated with the outgoing tide. Past studies have shown that this species is robust (i.e., respiratory and metabolic performance, behavior) to climate change-relevant elevated CO2 levels as well as hypoxia and anoxia tolerant. However, epaulette shark embryos reared under ocean warming conditions hatch earlier and smaller, with altered patterns and coloration, and with higher metabolic costs than their current-day counterparts. Findings to date suggest that this species has adaptations to tolerate some, but perhaps not all, of the challenging conditions predicted for the 21st century. As such, the epaulette shark is emerging as a model system to understand vertebrate physiology in changing oceans. Yet, few studies have investigated the kinematics of walking and swimming, which may be vital to their biological fitness, considering their habitat and propensity for challenging environmental conditions. Given that neonates retain embryonic nutrition via an internalized yolk sac, resulting in a bulbous abdomen, while juveniles actively forage for worms, crustaceans, and small fishes, we hypothesized that difference in body shape over early ontogeny would affect locomotor performance. To test this, we examined neonate and juvenile locomotor kinematics during the three aquatic gaits they utilize-slow-to-medium walking, fast walking, and swimming-using 13 anatomical landmarks along the fins, girdles, and body midline. We found that differences in body shape did not alter kinematics between neonates and juveniles. Overall velocity, fin rotation, axial bending, and tail beat frequency and amplitude were consistent between early life stages. Data suggest that the locomotor kinematics are maintained between neonate and juvenile epaulette sharks, even as their feeding strategy changes. Studying epaulette shark locomotion allows us to understand this-and perhaps related-species' ability to move within and away from challenging conditions in their habitats. Such locomotor traits may not only be key to survival, in general, as a small, benthic mesopredator (i.e., movements required to maneuver into small reef crevices to avoid aerial and aquatic predators), but also be related to their sustained physiological performance under challenging environmental conditions, including those associated with climate change-a topic worthy of future investigation.

Membrane muscles tension bat wings for faster flight

Watching a bat flit through the night sky, you might think that the delicate membrane stretched between the animal's elbow, long splayed digit bones and down its side to the hind limbs is no more than an inert flexible surface that catches air passively like fabric covering the struts of a kite. Yet, the elegant wing membrane is spanned by thin thread-like muscles. Could the muscles, known as plagiopatagiales proprii, tense and relax the membrane to fine-tune how the structure bows and catches the air as the animals swoop and flutter? Jorn Cheney, Jeremy Rehm, Sharon Swartz and Kenny Breuer from Brown University, USA, decided to find out.But first, the team need to know how Jamaican fruit bats (Artibeus jamaicensis) fly when they had full use of the wing membrane muscles. Cheney and Rehm tempted the animals with guava juice to fly through still air in a wind tunnel and into a 5 m s−1 head wind, filming the animals with six high-speed cameras to reconstruct the wing shape during their distinctive wingbeats to find out how much the membrane bowed as the animals propelled themselves forward. Then the pair painstakingly paralysed all 25 of the fine muscles in each wing by individually injecting botulism toxin into each, inactivating them temporarily. ‘We use tiny, microliter amounts of dilute Botox, much less than would be used in a cosmetic procedure to minimize a single forehead wrinkle’, says Swartz. Once the animals had recovered, Cheney and Rehm then encouraged the bats to fly again in the still air and headwind, to find out how loss of muscle control affected the animals. The animals with the paralysed wing membrane muscles struggled to remain aloft in the motionless air. Even though they could still fly at 6.2 m s−1 into the head wind – which was 1 m s−1 slower than they had managed with full use of the wing membrane muscles – the animals struggled when there was no headwind, descending rapidly to the ground as they tried to remain aloft. And, when the team compared the bats’ wing membranes as they flew into the head wind, it was clear that the membranes of the paralysed bats were slacker, bowing 12% more than the wing membranes of the bats that still had the use of their plagiopatagiales proprii, also increasing the amount of air drag on the wing to slow the animals by 16%. However, the bats with the immobilised wing membrane muscles managed to compensate for the increased drag by increasing their wing beat amplitude from ∼60 to ∼75 deg and tipping the wing more to sweep it through a less vertical angle.‘Reduced forward speed, reduced ability for low-speed flight and changes in kinematics [when the plagiopatagiales were inactive] are consistent with the plagiopatagiales playing a crucial role in reducing drag through active control over membrane tension indirectly controlling camber’, says Cheney, explaining that using the muscles to tension the wing membrane helps bats to fly at higher speeds. However, the team points out that the bats with the paralysed muscles could have taken advantage of their slacker wing membranes to select slower preferred speeds; so why didn't they? They suspect that the animals may not be able to provide the additional flight power required to remain aloft at the lower speeds caused by bowed wing membranes. The researchers conclude, ‘Jamaican fruit bats likely always employ their wing membrane muscles during sustained flight to control camber [bowing] and to enhance flight efficiency’. And, happily, the bats soon recovered full use of their plagiopatagiales muscles after their wing slackening experience to regain full bat mobility.

P-094 Fundamental understanding of sperm motion in viscoelastic media

Abstract Study question How does viscosity influence the flagellar beating behaviour of free-swimming bull, mouse, and human sperm? Summary answer Sperm flagellar beating behaviour exhibits a transition from an irregular three-dimensional (3D) beating at 5 mPa·s to an organized two-dimensional (2D) waveform at 20 mPa·s. What is known already Sperm migrate in a complex viscoelastic environment through the female reproductive tract. The viscoelastic properties of the oviductal fluid significantly influence the progressive motility of sperm, acting as one of the key guidance mechanisms in vivo. However, the biomechanics of sperm flagellar activity in response to varying viscosity of the oviductal fluid is poorly understood. Understanding sperm flagellar behaviour in physiologically relevant environments is crucial to understanding reproduction and may help to describe unknown causes of infertility. Lack of high-speed high-resolution imaging techniques and automated image-analysis capabilities have been the main barriers to fully describe the flagellar beating behaviour. Study design, size, duration We used a custom-built high-speed high-resolution dark-field microscopy platform to resolve the flagellar dynamics of human, bull, and mouse sperm near surfaces in viscoelastic media ranging in viscosity from 1 to 250 mPa·s. The imaging system includes an automated image analysis algorithm to quantify sperm flagellar waveform and motility characteristics by extracting the flagellar centreline, reconstructing the waveform and calculating tangent-angle profiles. 20 sperm from 3 different bull, mice and humans were analyzed. Participants/materials, setting, methods Bull, mouse, and human sperm were used in this study. In each experiment, a diluted sperm sample in a buffer supplemented with methylcellulose was used and free-swimming sperm were imaged using dark-field microscopy at 200 frames per second. An automated image analysis algorithm was used to extract sperm flagellar centreline and Proper Orthogonal Decomposition (POD) was then used to study sperm flagellar waveform. Statistical analysis was performed using one-way ANOVA. Main results and the role of chance The reconstructed flagellar beating pattern was different for sperm swimming in low and high-viscosity media. Bull sperm exhibited a lower flagellar beating amplitude along the end piece when swimming in a high-viscosity media, a potential energy-efficient strategy to navigate a high viscosity fluid. The first two dominant POD modes (shape modes) describe more than 90% of the beating pattern for all species. Bull sperm exhibited a transition mode with irregular loops in 5 mPa·s buffer, but the flagellar shape cycle created an organised repetitive circular cycle in 1 mPa.s (3D beating) buffer and at viscosities higher than 5 mPa·s (2D beating). Human sperm also indicated a similar behaviour but with the transition happening at higher viscosities. Mouse sperm in high-viscosity media had a lower flagellar beating amplitude across the principal piece and higher beating amplitude across the end piece. The flagellar shape cycle in mouse sperm showed a periodic flagellar beating behaviour at high-viscosities (>20 mPa·s), but a shape cycle with distorted loops at lower viscosities. Our results showed in quantitative detail that increasing viscosity alters sperm flagellar beating pattern, and how sperm migration behaviour in low viscosity media can be distinct from their swimming behaviour in vivo. Limitations, reasons for caution A more comprehensive study of sperm motility parameters such as curvilinear velocity, average path velocity and straight line velocity with a larger sample size is required to fully characterise sperm swimming behaviour as a function of viscosity. Wider implications of the findings The increasing viscosity of the oviductal fluid regulates the sperm flagellar beating behaviour to switch from a 3D swimming behaviour with irregular shape cycles at lower viscosities to a 2D slithering mode with repetitive circular shape cycles at higher viscosities to achieve a more energy-efficient beating pattern for navigation. Trial registration number Not applicable