Increase In Beat Frequency Research Articles

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Contraction pressure analysis using optical imaging in normal and MYBPC3-mutated hiPSC-derived cardiomyocytes grown on matrices with tunable stiffness

Current in vivo disease models and analysis methods for cardiac drug development have been insufficient in providing accurate and reliable predictions of drug efficacy and safety. Here, we propose a custom optical flow-based analysis method to quantitatively measure recordings of contracting cardiomyocytes on polydimethylsiloxane (PDMS), compatible with medium-throughput systems.Movement of the PDMS was examined by covalently bound fluorescent beads on the PDMS surface, differences caused by increased substrate stiffness were compared, and cells were stimulated with β-agonist. We further validated the system using cardiomyocytes treated with endothelin-1 and compared their contractions against control and cells incubated with receptor antagonist bosentan. After validation we examined two MYBPC3-mutant patient-derived cell lines.Recordings showed that higher substrate stiffness resulted in higher contractile pressure, while beating frequency remained similar to control. β-agonist stimulation resulted in both higher beating frequency as well as higher pressure values during contraction and relaxation. Cells treated with endothelin-1 showed an increased beating frequency, but a lower contraction pressure. Cells treated with both endothelin-1 and bosentan remained at control level of beating frequency and pressure. Lastly, both MYBPC3-mutant lines showed a higher beating frequency and lower contraction pressure.Our validated method is capable of automatically quantifying contraction of hiPSC-derived cardiomyocytes on a PDMS substrate of known shear modulus, returning an absolute value. Our method could have major benefits in a medium-throughput setting.

A 3-D human model of complex cardiac arrhythmias

Cardiac arrhythmias impact over 12 million people globally, with an increasing incidence of acquired arrhythmias. Although animal models have shed light onto fundamental arrhythmic mechanisms, species-specific differences and ethical concerns remain. Current human models using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) either lack the higher order tissue organization of the heart or implement unreliable arrhythmia induction techniques. Our goal was to develop a robust model of acquired arrhythmia by disrupting cardiomyocyte cell-cell signaling – one of the hallmarks of complex arrhythmias. Human 3D microtissues were generated by seeding hydrogel-embedded hiPSC-CMs and cardiac fibroblasts into an established microwell system designed to enable active and passive force assessment. Cell-cell signaling was disrupted using methyl-beta cyclodextrin (MBCD), previously shown to disassemble cardiac gap junctions. We demonstrate that arrhythmias were progressive and present in all microtissues within 5 days of treatment. Arrhythmic tissues exhibited reduced conduction velocity, an increased number of distinct action potentials, and reduced action potential cycle length. Arrhythmic tissues also showed significant reduction in contractile force generation, increased beating frequency, and increased passive tension and collagen deposition, in line with fibrosis. A subset of tissues with more complex arrhythmias exhibited 3D spatial differences in action potential propagation. Pharmacological and electrical defibrillation was successful. Transcriptomic data indicated an enrichment of genes consistent with cardiac arrhythmias. MBCD removal reversed the arrhythmic phenotype, resulting in synchronicity despite not resolving fibrosis. This innovative & reliable human-relevant 3D acquired arrhythmia model shows potential for improving our understanding of arrhythmic action potential conduction and furthering therapeutic development. Statement of significanceThis work describes a 3D human model of cardiac arrhythmia-on-a-chip with high reproducibility, fidelity, and extensive functional applicability. To mimic in vivo conditions, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts from healthy controls were combined in a biocompatible fibrin hydrogel and seeded between two deflectable polymeric rods. Using the innate functional properties of this 3D model as well as advanced optical imaging techniques we demonstrated dramatic changes in contraction rate, synchronicity, and electrophysiological conduction in arrhythmic tissues relative to controls. Taken together, these data demonstrate the distinctive potential of this new model for pathophysiological studies, and for arrhythmia drug testing applications.

Abstract 16472: Deciphering a Mechanistic Link Between Enhanced Late Sodium Current and Triggered Atrial Fibrillation Using Patient-specific and Gene-corrected Induced Pluripotent Stem Cell-derived Atrial Cardiomyocytes

Introduction: Gain-of-function mutations in SCN5A, which encodes the cardiac sodium channel, have been linked with familial atrial fibrillation (AF). However, the mechanistic link between the late sodium current (I Na,L ) and triggered arrhythmia remains unclear. Hypothesis: To characterize the electrophysiological (EP) phenotype of gain-of-function AF-linked SCN5A mutations, elucidate the underlying cellular mechanisms using patient-specific and gene-corrected (GC) induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs). Methods: We generated iPSC-aCMs from two families carrying SCN5A mutations (E428K and N470K) and control subjects. Whole-cell patch clamp and multi-electrode arrays were recorded to assess the EP phenotypes of the atrial iPSC-CMs. We corrected the E428K iPSC-aCMs using CRISPR/Cas9 gene editing approach (isogenic control). Results: The SCN5A mutation lines displayed abnormal EP properties including increased beating frequency and irregularity with triggered beats characteristic of AF ( Fig. 1 ). E428K iPSC-aCMs displayed spontaneous arrhythmogenic activity with beat-to-beat irregularity ( Fig. 1 A-D ) with the prolonged APD ( Fig. 1 E-H ) associated with enhanced I Na,L ( Fig. 1 I-L ). In contrast, expression of SCN5A -E428K in heterologous expression system failed to show enhanced I Na,L . The gene-corrected E428K iPSC-aCMs normalized the aberrant EP phenotype. Gene expression profiling revealed differential expression of calcium and potassium channel homeostasis and nitric oxide mediated signal transduction which could result in EP remodeling of atrial CMs. Conclusions: Patient-specific and gene-corrected iPSC-aCMs exhibited striking ex-vivo EP phenotype of an AF-causing SCN5A gain-of-function mutation that produced minimal changes in-vitro . We established a mechanistic link between enhanced I Na,L , ion channel remodeling and nitric oxide signaling pathways, and triggered AF.

Experimental clearance rates of Aurelia coerulea ephyrae and medusae, and the predation impact on zooplankton in Jiaozhou Bay

The population explosion of jellyfish Aurelia coerulea occurred in Jiaozhou Bay, China in 2009. The potential predation impact of A. coerulea on zooplankton was investigated. Population clearance potential and residence time (t1/2) for copepods were calculated from laboratory clearance rates and measurements of jellyfish size and abundance from May to August 2009 in Jiaozhou Bay. Clearance rates varied widely with prey organisms, but they were not significantly different among various prey concentrations. Medusae captured rotifers, fish larvae and hydromedusae more efficiently than fish eggs, copepods and chaetognaths. Ephyrae captured rotifers and hydromedusae more efficiently than fish larvae and copepods. Clearance rate linearly increased with the cross sectional area of A. coerulea (size from 0.3 to 7.1 cm). Water temperature also had a marked effect on clearance rate and this was related to the increased beat frequency as water temperature increased. In early May 2009, A. coerulea potentially cleared the volume of water in the Bay less than 0.001 times a day, but this value was estimated to be more than 0.3 times a day in July. The t1/2 for copepods was less than 6 d in June and July. Abundances of copepods, hydromedusae and chaetognaths were extremely low in 2009 compared to 2008 and 2010 (jellyfish non-bloom years). Large predation pressure by the A. coerulea population occurred to control zooplankton communities in Jiaozhou Bay. A. coerulea, when present at a high population level, can be a keystone species in Jiaozhou Bay and control the trophic structure here.

Roles of Adenylate Cyclases in Ciliary Responses of Paramecium to Mechanical Stimulation.

Paramecium shows rapid forward swimming due to increased beat frequency of cilia in normal (forward swimming) direction in response to various kinds of stimuli applied to the cell surface that cause K+ -outflow accompanied by a membrane hyperpolarization. Some adenylate cyclases are known to be functional K+ channels in the membrane. Using gene-specific knockdown methods, we examined nine paralogues of adenylate cyclases in P.tetraurelia to ascertain whether and how they are involved in the mechanical stimulus-induced hyperpolarization-coupled acceleration of forward swimming. Results demonstrated that knockdown of the adenylate cyclase 1 (ac1)-gene and 2 (ac2)-gene inhibited the acceleration of forward swimming in response to mechanical stimulation of the cell, whereas that spared the acceleration response to external application of 8-Br-cAMP and dilution of extracellular [K+ ] induced hyperpolarization. Electrophysiological examination of the knockdown cells revealed that the hyperpolarization-activated inward K+ current is smaller than that of a normal cell. Our results suggest that AC1 and AC2 are involved in the mechanical stimulus-induced acceleration of ciliary beat in Paramecium.

Abstract 407: Modeling Atrial Fibrillation in a Dish Using Atrial iPSC Derived Cardiomyocytes

Mutations in multiple genes have been linked with familial atrial fibrillation (AF) but the underlying pathophysiologic mechanisms and implications for therapy remain poorly understood. To characterize the electrophysiological phenotype of an AF-linked SCN5A mutation and assess novel mechanism-based therapies, we generated patient-specific atrial induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from a carrier with an SCN5A-E428K mutation and an unaffected family member using a differentiation protocol optimized for atrial differentiation generated atrial iPSC-CMs. The atrial iPSC-CMs carrying SCN5A-E428K displayed increased window and late Na+ current (INa,L), increased beating frequency and irregularity with triggered beats and prolongation of the action potential duration (APD) versus control atrial iPSC-CMs. The multi-electrode array (MEA) recordings of mutant atrial iPSC-CMs showed spontaneous arrhythmogenic activity with beat-to-beat irregularity. We further showed that targeted inhibition of the INa,L with ranolazine normalized the aberrant electrophysiologic phenotype in the SCN5A-E428K atrial iPSC-CMs and mexiletine reversed the sodium channel gating defects, and reduced beating frequency and irregularity. Our study illustrates the potential use of atrial iPSC-CMs for modeling AF in a dish, elucidating the underlying cellular mechanisms, and identifying novel mechanism-based therapies custom-tailored for individual patients.

The role of curvature feedback in the energetics and dynamics of lamprey swimming: A closed-loop model

Like other animals, lampreys have a central pattern generator (CPG) circuit that activates muscles for locomotion and also adjusts the activity to respond to sensory inputs from the environment. Such a feedback system is crucial for responding appropriately to unexpected perturbations, but it is also active during normal unperturbed steady swimming and influences the baseline swimming pattern. In this study, we investigate different functional forms of body curvature-based sensory feedback and evaluate their effects on steady swimming energetics and kinematics, since little is known experimentally about the functional form of curvature feedback. The distributed CPG is modeled as chains of coupled oscillators. Pairs of phase oscillators represent the left and right sides of segments along the lamprey body. These activate muscles that flex the body and move the lamprey through a fluid environment, which is simulated using a full Navier-Stokes model. The emergent curvature of the body then serves as an input to the CPG oscillators, closing the loop. We consider two forms of feedback, each consistent with experimental results on lamprey proprioceptive sensory receptors. The first, referred to as directional feedback, excites or inhibits the oscillators on the same side, depending on the sign of a chosen gain parameter, and has the opposite effect on oscillators on the opposite side. We find that directional feedback does not affect beat frequency, but does change the duration of muscle activity. The second feedback model, referred to as magnitude feedback, provides a symmetric excitatory or inhibitory effect to oscillators on both sides. This model tends to increase beat frequency and reduces the energetic cost to the lamprey when the gain is high and positive. With both types of feedback, the body curvature has a similar magnitude. Thus, these results indicate that the same magnitude of curvature-based feedback on the CPG with different functional forms can cause distinct differences in swimming performance.

Photoconductive antenna as local oscillator in terahertz frequency measurement: heterodyne efficiency and bias effect

In a photoconductive antenna (PCA), femtosecond-laser-excited carriers will form a broadband terahertz photon-carrier (PC) comb, and the terahertz PC comb can be used as a multi-frequency local oscillator to carry out heterodyne detection of continuous terahertz sources with high frequency accuracy. In this paper, the heterodyne efficiency and the bias effects of a PCA terahertz PC comb are investigated. The results show that the pair beat signals (with the beat frequencies lower than the repetition frequency of femtosecond laser) of a continuous terahertz source and the two adjacent comb teeth do not decrease with the increase of beat frequency. Applying a bias voltage to the PCA can effectively enhance the terahertz emission efficiency. However, such a bias voltage has no positive effects on the heterodyne detection responsivity because the heterodyne detection is intrinsically based on the terahertz rectification effect that is proportional to the photo-excited electrons. In addition, by using a reference terahertz source with high frequency stability, it is possible to measure the fluctuation and the drift of the repetition frequency of femtosecond lasers with higher accuracy. The results are helpful for improving the performance of terahertz frequency measurement system based on PCA PC combs.

The Acute Chronotropic Effect of the Uremic Metabolite, Trimethylamine‐N‐Oxide (TMAO), on Mouse Cardiac Muscle

BackgroundWhile it is understood that patients with chronic kidney disease (CKD) have an increased propensity for developing cardiovascular disease, the exact mechanisms remain unclear. Growing evidence suggests that the gut microbiome and its byproducts, such as TMAO, may be important contributors to this pathologic process. This uremic metabolite is cleared through renal excretion, which is compromised in CKD patients. Therefore, CKD patients often possess elevated plasma levels of this substance. In a previous study, we found that TMAO at pathological concentrations directly increases cardiac contractility in isolated, paced hearts. In this study, we sought to determine if TMAO also has chronotropic effects on isolated, spontaneously beating mouse hearts.MethodsWhole mouse hearts were extracted from anesthetized CD1 adult mice and subsequently connected to a force transducer and bubbled with oxygen inside an organ bath. The atria and sinoatrial node were kept intact to allow for spontaneous beating without artificial pacing. Changes in heart rate (in beats per minute) were measured after treatment with TMAO (3,000 μM) or vehicle (Ringer's solution). Additionally, calcium imaging was performed on cultured spontaneously beating embryonic rat cardiomyocytes. Intracellular Ca2+ responses in rat primary cardiomyocytes were measured with the fluorescent Ca2+ indicator Fura‐2 AM in response to vehicle and TMAO (3,000 uM).ResultsAverage beating frequency (in beats per minutes) of isolated hearts increased by 27% following treatment with 3,000 μM TMAO (P < 0.05, n =3) while there was no change with vehicle. Regarding isolated myocytes, TMAO was found to have a direct chronotropic effect as it increased beating frequency (as measured by calcium waves) by 13% compared to vehicle treatment (P < 0.05, n =4–5).ConclusionsIn addition to our previously described inotropic effects in the mouse heart, pathologic concentrations of TMAO also have a direct and positive chronotropic effect. The direct effects we have observed on the heart may increase cardiac output in early stages of CKD. However, cardiac remodeling as a result of increased beating frequency and contractility may have pathologic consequences in those with chronic disease. Further research is needed in order to elucidate the effect that TMAO has on cardiovascular function and pathology during CKD.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Integrated transcriptomic and regulatory network analyses identify microRNA-200c as a novel repressor of human pluripotent stem cell-derived cardiomyocyte differentiation and maturation.

MicroRNAs (miRNAs) are crucial for the post-transcriptional control of protein-encoding genes and together with transcription factors (TFs) regulate gene expression; however, the regulatory activities of miRNAs during cardiac development are only partially understood. In this study, we tested the hypothesis that integrative computational approaches could identify miRNAs that experimentally could be shown to regulate cardiomyogenesis. We integrated expression profiles with bioinformatics analyses of miRNA and TF regulatory programs to identify candidate miRNAs involved with cardiac development. Expression profiling showed that miR-200c, which is not normally detected in adult heart, is progressively down-regulated both during cardiac development and in vitro differentiation of human embryonic stem cells (hESCs) to cardiomyocytes (CMs). We employed computational methodologies to predict target genes of both miR-200c and five key cardiac TFs to identify co-regulated gene networks. The inferred cardiac networks revealed that the cooperative action of miR-200c with these five key TFs, including three (GATA4, SRF and TBX5) targeted by miR-200c, should modulate key processes and pathways necessary for CM development and function. Experimentally, over-expression (OE) of miR-200c in hESC-CMs reduced the mRNA levels of GATA4, SRF and TBX5. Cardiac expression of Ca2+, K+ and Na+ ion channel genes (CACNA1C, KCNJ2 and SCN5A) were also significantly altered by knockdown or OE of miR-200c. Luciferase reporter assays validated miR-200c binding sites on the 3' untranslated region of CACNA1C. In hESC-CMs, elevated miR-200c increased beating frequency, and repressed both Ca2+ influx, mediated by the L-type Ca2+ channel and Ca2+ transients. Our analyses demonstrate that miR-200c represses hESC-CM differentiation and maturation. The integrative computation and experimental approaches described here, when applied more broadly, will enhance our understanding of the interplays between miRNAs and TFs in controlling cardiac development and disease processes.

Generation of functional cardiomyocytes derived from human somatic cells and therapy for heart diseases

Background and HypothesisSuccessful generation of induced pluripotent stem cells (iPSCs) and their differentiation into cardiomyocytes (iCMCs) have created exciting possibilities, wherein iCMCs may offer a renewable cell source for therapy, disease modeling and drug discovery. The major problem remains to generate safe, homogeneous, highly efficient functional iCMCs from adult somatic cells for drug screening and cell therapy. Thus, we hypothesize that the iPSC‐derived functional iCMCs would be a good in vitro model for drug screening and a superior source of cells for therapy in a mouse heart failure model.MethodsRecently, we reported an approach of using highly efficient viral‐free combination of DNA and mRNA embryonic transcription factors to generate iPSCs and functional iCMCs from adult human cells. These differentiated iCMCs were characterized qualitatively and quantitatively by their genes and proteins expression. Furthermore, the contractility of chronotropic drugs treated and untreated iCMCs were measured and analyzed by our new particle image velocimetry (PIV) method followed by divergence and Fourier's spectrum analyses. We also compared our PIV technique with available standard confocal calcium transient imaging analysis. Finally, to see the therapeutic potential, we have transplanted these iCMCs intramyocardially in a mouse heart failure model.ResultsWe attained the following observations: We identified a new combination of mature CMC cell‐surface markers CD36 and γ‐sarcoglycan to isolate a homogenous population from the CMCs differentiated culture by FACS analysis. To follow the in vitro differentiation process of CMC cultures, we recorded and analyzed the same areas at various time points using high frame rate video microscopy. Our PIV image analysis technique provided beat patterns: time‐series data of tissue displacement, measured relative to a resting reference state. The beat patterns revealed that the contractility of early CMC nodes was asynchronous in space and irregular in time (days 8 and 13). During subsequent days as the CMCs matured, contractile centers became synchronous (day 15). Our optically recorded beat patterns were in good agreement with the calcium‐imaging signal. In our in vitro drug screening systems, isoproterenol resulted in a substantial and sustained increase in beat frequency without triggering arrhythmias, on an in vitro day 14 CMC culture. Our day 35 post‐echocardiography data showed a significant improvement in the cardiac functions in iCMCs treated mice over the untreated control mice. Moreover, the fluorescence imaging data showed that the injected cells were stained positive for human nuclear antigen, integrin‐β antigen and human cardiotroponin T (CTT), suggesting the presence of iCMCs at the 35‐day post‐transplanted mouse heart.ConclusionOverall, these in vitro data offer new opportunities to model cardiac diseases and allow efficient screening of drug effects at various stages of cardiac developments. Furthermore, our in vivo data showed that the human adult somatic cell‐derived iCMCs were engrafted well and also functional in the failing mouse heart, suggesting its therapeutic application.Support or Funding InformationAmerican Heart Association Grant‐in Aid

L-Type Calcium Channel Inhibition Contributes to the Proarrhythmic Effects of Aconitine in Human Cardiomyocytes.

Aconitine (ACO) is well-known for causing lethal ventricular tachyarrhythmias. While cardiac Na+ channel opening during repolarization has long been documented in animal cardiac myocytes, the cellular effects and mechanism of ACO in human remain unexplored. This study aimed to assess the proarrhythmic effects of ACO in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). ACO concentration-dependently (0.3 ~ 3.0 μM) shortened the action potentials (AP) durations (APD) in ventricular-like hiPSC-CMs by > 40% and induced delayed after-depolarization. Laser-scanning confocal calcium imaging analysis showed that ACO decreased the duration and amplitude of [Ca2+]i transients and increased in the beating frequencies by over 60%. Moreover, ACO was found to markedly reduce the L-type calcium channel (LTCC) currents (ICa,L) in hiPSC-CMs associated with a positive-shift of activation and a negative shift of inactivation. ACO failed to alter the peak and late Na+ currents (INa) in hiPSC-CMs while it drastically increased the late INa in Guinea-pig ventricular myocytes associated with enhanced activation/delayed inactivation of INa at -55 mV~ -85 mV. Further, the effects of ACO on ICa,L, INa and the rapid delayed rectifier potassium current (Ikr) were validated in heterologous expression systems by automated voltage-clamping assays and a moderate suppression of Ikr was observed in addition to concentration-dependent ICa,L inhibition. Lastly, increased beating frequency, decreased Ca2+ wave and shortened field potential duration were recorded from hiPSC-CMs by microelectrode arrays assay. In summary, our data demonstrated that LTCC inhibition could play a main role in the proarrhythmic action of ACO in human cardiomyocytes.

Sheared-beam imaging of object with depth information

Sheared-beam imaging technique is a non-conventional imaging method which can be used to image remote objects through atmospheric turbulence without needing any adaptive optics. In this imaging technique, the target is coherently illuminated by three laser beams which are laterally sheared at the transmitter plane and arranged into an L shape. In addition, each beam is modulated by a slight frequency shift. The speckle intensity signals scattered from the target are received by a detector array, and then the image of target can be reconstructed by computer algorithm. By far, most of studies in this field have focused on two-dimensional imaging. In real conditions, however, the surface of targets we are concerned about reveals that different depths introduce various phase delays in the scattering signal from target. This delay causes the phase-shift errors to appear between the ideal target Fourier spectrum and the Fourier spectrum received by detector array. Finally, this would result in poor image quality and low resolution. In this study, a three-dimensional target imaging model is established based on the two-dimensional target imaging model. The influence of modulated beat frequency between sheared beam and reference beam is studied on the objects with depth information, and the result shows that large beat frequency may have an adverse effect on reconstructed images. The simulation we have developed for this three-dimensional imaging model uses three targets with different shapes. Each target is divided into several sub-blocks, and we set different depth values (within 10 m) for these blocks. Then beat frequencies are increased from 5 Hz to about 1 MHz, respectively. At each pair of frequencies, the reconstructed image is recorded. Srehl ratio is used as the measure of the imaging quality. Computer simulation results show that the Srehl ratio of reconstructed images descends with the increase of beat frequency, which is fully consistent with the theory of three-dimensional target imaging proposed before. Meanwhile, we find that the depth distribution of target also has an effect on imaging quality. As for actual space targets, the maximum depth is usually not more than 10 m. Compared with the influence caused by beat frequencies, the effect produced by depth distribution is negligible. Therefore when a space target is imaged, beat frequencies play the major role in reconstructing high-quality image. The results presented in this paper indicate that in order to achieve better imaging quality in the practical application, it is necessary to select the smallest beat frequency according to the detector performance and keep the candidate frequencies away from the low-frequency noise of the detector.

3D cardiac μtissues within a microfluidic device with real-time contractile stress readout.

We present the development of three-dimensional (3D) cardiac microtissues within a microfluidic device with the ability to quantify real-time contractile stress measurements in situ. Using a 3D patterning technology that allows for the precise spatial distribution of cells within the device, we created an array of 3D cardiac microtissues from neonatal mouse cardiomyocytes. We integrated the 3D micropatterning technology with microfluidics to achieve perfused cell-laden structures. The cells were encapsulated within a degradable gelatin methacrylate hydrogel, which was sandwiched between two polyacrylamide hydrogels. The polyacrylamide hydrogels were used as "stress sensors" to acquire the contractile stresses generated by the beating cardiac cells. The cardiac-specific response of the engineered 3D system was examined by exposing it to epinephrine, an adrenergic neurotransmitter known to increase the magnitude and frequency of cardiac contractions. In response to exogenous epinephrine the engineered cardiac tissues exhibited an increased beating frequency and stress magnitude. Such cost-effective and easy-to-adapt 3D cardiac systems with real-time functional readout could be an attractive technological platform for drug discovery and development.

Ultrasound-induced modulation of cardiac rhythm in neonatal rat ventricular cardiomyocytes.

Isolated neonatal rat ventricular cardiomyocytes were used to study the influence of ultrasound on the chronotropic response in a tissue culture model. The beat frequency of the cells, varying from 40 to 90 beats/min, was measured based upon the translocation of the nuclear membrane captured by a high-speed camera. Ultrasound pulses (frequency = 2.5 MHz) were delivered at 300-ms intervals [3.33 Hz pulse repetition frequency (PRF)], in turn corresponding to 200 pulses/min. The intensity of acoustic energy and pulse duration were made variable, 0.02-0.87 W/cm(2) and 1-5 ms, respectively. In 57 of 99 trials, there was a noted average increase in beat frequency of 25% with 8-s exposures to ultrasonic pulses. Applied ultrasound energy with a spatial peak time average acoustic intensity (Ispta) of 0.02 W/cm(2) and pulse duration of 1 ms effectively increased the contraction rate of cardiomyocytes (P < 0.05). Of the acoustic power tested, the lowest level of acoustic intensity and shortest pulse duration proved most effective at increasing the electrophysiological responsiveness and beat frequency of cardiomyocytes. Determining the optimal conditions for delivery of ultrasound will be essential to developing new models for understanding mechanoelectrical coupling (MEC) and understanding novel nonelectrical pacing modalities for clinical applications.

Phosphoregulation of Cardiac Inotropy via Myosin Binding Protein-C During Increased Pacing Frequency or β1-Adrenergic Stimulation.

Mammalian hearts exhibit positive inotropic responses to β-adrenergic stimulation as a consequence of protein kinase A-mediated phosphorylation or as a result of increased beat frequency (the Bowditch effect). Several membrane and myofibrillar proteins are phosphorylated under these conditions, but the relative contributions of these to increased contractility are not known. Phosphorylation of cardiac myosin-binding protein-C (cMyBP-C) by protein kinase A accelerates the kinetics of force development in permeabilized heart muscle, but its role in vivo is unknown. Such understanding is important because adrenergic responsiveness of the heart and the Bowditch effect are both depressed in heart failure. The roles of cMyBP-C phosphorylation were studied using mice in which either WT or nonphosphorylatable forms of cMyBP-C [ser273ala, ser282ala, ser302ala: cMyBP-C(t3SA)] were expressed at similar levels on a cMyBP-C null background. Force and [Ca(2+)]in measurements in isolated papillary muscles showed that the increased force and twitch kinetics because increased pacing or β1-adrenergic stimulation were nearly absent in cMyBP-C(t3SA) myocardium, even though [Ca(2+)]in transients under each condition were similar to WT. Biochemical measurements confirmed that protein kinase A phosphorylated ser273, ser282, and ser302 in WT cMyBP-C. In contrast, CaMKIIδ, which is activated by increased pacing, phosphorylated ser302 principally, ser282 to a lesser degree, and ser273 not at all. Phosphorylation of cMyBP-C increases the force and kinetics of twitches in living cardiac muscle. Further, cMyBP-C is a principal mediator of increased contractility observed with β-adrenergic stimulation or increased pacing because of protein kinase A and CaMKIIδ phosphorylations of cMyB-C.

FRET Measurements of cAMP Dynamics in HL1 Cells Support the Key Role of Constitutive AC Activity in Cardiac Pacemaking

Spontaneously beating HL1 cardiac cells have evolved as a very useful tool to study regulation of pacemaking cardiac activity because of the ability to knock down specific molecules without generating KO mice in vivo. HL1 cells, however, have not been fully characterized and their pacemaker mechanisms may differ from those in cardiac pacemaking cells derived from adult heart. Basal constitutive adenylate cyclase activity is an obligate factor for normal spontaneous firing in adult sinoatrial nodal pacemaker cells.We expressed FRET sensors in spontaneously beating HL1 cardiac cells to measure cAMP concentration. The PDE inhibitor IBMX (100 μM) increased the basal concentration of cAMP more than twofold and increased beating frequency by 70%. AC1 knockdown with siRNA completely inhibited the IBMX-induced increase in cAMP production in cytoplasm and plasma membrane, and decreased the beating frequency of HL1 cells. Pharmacological inhibition of both plasma membrane AC (2′-5’ dideoxyadenosine) and cytoplasmic AC (KH7) selective blockers completely abolished the IBMX induced increase of cAMP concentration and resulted in complete cessation of spontaneous beating in HL1cardiac cells. Thus, as in adult sinoatrial nodal pacemaker cells, basal AC activity is required for the spontaneous beating of HL1 cardiac cells.

Optogenetic GS Activation in Cardiomyocytes Enhances Pacemaker Activity

Stimulation of GS-protein coupled receptors leads to an increase of the second messenger cyclic adenosine monophosphate (cAMP). In cardiomyocytes the GS-signaling cascade is involved in positive regulation of chronotropy and contractility but chronic GS stimulation can also induce cardiac hypertrophy or arrhythmia.Experimentally, the GS-signaling cascade can be activated by s-receptor agonists but diffusion of drugs does not allow the precise control of location and timing. To overcome these limitations we used the optogenetic protein JellyOp, a directly GS-coupled, light-sensitive receptor (Bailes et al. PloS One, 2012) to activate GS-signaling by light.Illumination of JellyOp expressing HEK 293 cells resulted in elevation of cAMP levels without detectable dark activity. Cardiomyocytes were differentiated within embryoid bodies (EBs) from transgenic mouse embryonic stem cells that express JellyOp under control of the ubiquitous chicken s-actin promoter. Spontaneously beating EBs were analyzed at day 13 of differentiation by infrared video microscopy. Brief illumination (20 sec, 470 nm, 166.7 nW/mm2) increased beating frequency to 1239±349% of baseline (n=3) which returned to baseline after termination of illumination. The lowest effective light intensity was of 9.1 nW/mm2 resulting in frequency acceleration to 428±131% of baseline and the shortest effective illumination was 1 sec. Similar to dose-response-curves of receptor agonists, light-induced frequency acceleration showed a sigmoid dependence on light-intensity with a half maximal light intensity of 33.5 nW/mm2. Direct comparison showed that the rate of frequency increment was much faster using illumination (13.9±3.1%/s) than using perfusion with the s-receptor agonist isoprenaline (2.7±1.5%/s) but both stimulations led to a similar response in frequency elevation.In summary optogenetic JellyOp activation in cardiomyocytes enables the stimulation of the GS-signaling pathway with high temporal precision and will be useful to investigate temporal and site-specific effects of physiological and pathophysiological GS-activation.

Neural correlates of settlement in veliger larvae of the gastropod, Crepidula fornicata

AbstractSettlement behavior of molluscan veliger larvae prior to metamorphosis requires cessation of swimming, accomplished by arrest of prototrochal cilia on the margin of the velum (the larval swimming organ). Ciliary arrest in larvae of gastropods is mediated by an action potential that occurs synchronously across the velum as a consequence of electrical coupling between the prototrochal ciliated cells. We developed a preparation for extracellular recording of such ciliary arrest spikes from intact swimming and crawling veliger larvae of the caenogastropod Crepidula fornicata, using a fine wire electrode. Ciliary arrest spike rates during bouts of substrate crawling were significantly higher than those recorded during preceding swimming periods in larvae that were competent for metamorphosis, but not in precompetent larvae. Spike rates were similar on clean polystyrene substrates, and on substrates that had been coated with a natural cue for metamorphosis (mucus from conspecific adults). We used immunohistochemical methods to localize neuromodulators that might regulate the function of velar cilia. Labeled terminals for serotonin, FMRFamide, and tyrosine hydroxylase (an enzyme for catecholamine synthesis) were located in positions consistent with modulatory effects on the prototrochal ciliated cells. Prototrochal ciliary arrest spike rates and beat frequencies were measured in isolated velar lobes from competent larvae, which were exposed to serotonin, FMRFamide, and dopamine (10−5 mol L−1). Serotonin abolished arrest spiking and increased beat frequency; dopamine also increased beat frequency, and FMRFamide depressed it. Competent larvae tested in a small static water column swam to the top of the column when exposed to serotonin, but occupied lower positions than controls when in the presence of dopamine and FMRFamide. The larval nervous system appears to regulate velar functions that are critical for settlement behavior, and is likely to do so by integrating different sensory modalities in an age‐dependent manner.