Absolute Refractory Period Research Articles

A Method for Electrical Stimulus Artifact Removal Exploiting Neural Refractoriness: Validation by Contrasting Cathodic and Anodic Stimulation.

To present a novel method for removing stimulus transient that exploits the absolute refractory period of electrically excitable neural tissues. Electrical stimulation often generates significant signal artifacts that can obscure important physiological signals. Removal of the artifact and understanding latent information from these signals could provide objective measures of circuit engagement, potentially driving advancements in neuromodulation research and therapies. We conducted intracranial physiology studies on five consecutive patients with Parkinson's disease who underwent deep brain stimulation (DBS) surgery as part of their routine care. Monopolar stimuli (either cathodic or anodic) were delivered in pairs through the DBS electrode across a range of inter-stimulus intervals. Recordings from adjacent unused electrode contacts used broadband sampling and precise synchronization to generate a robust template for the stimulus transient during the absolute refractory period. These templates of stimulus transient were then subtracted from recordings at different intervals to extract and analyze the residual neural potentials. After artifact removal, the residual signals exhibited absolute and relative refractory periods with timing indicative of neural activity. Cathodic and anodic DBS pulses generated distinct patterns of local tissue activation, showing phase independence from the prior stimulus. The earliest detectable neural responses occurred at short peak latencies (ranging from 0.19 to 0.38 ms post-stimulus) and were completely or partially obscured by the stimulus artifact prior to removal. Cathodic stimuli produced stronger local tissue responses than anodic stimuli, aligning with clinical observations of lower activation thresholds for cathodic stimulation. However, cathodic and anodic pulses induced artifact patterns that were equivalent but opposite. The proposed artifact removal technique enhances prior approaches by allowing direct measurement of local tissue responses without requirements for stimulus polarity reversal, template scaling, or specialized filters. This approach could be integrated into future neuromodulation systems to visualize stimulus-evoked neural potentials that would otherwise be obscured by stimulus artifacts.

The recovery cycle of excitability assessed by a conventional electrodiagnostic machine: A study in healthy volunteers and in Charcot-Marie-Tooth 1A patients

ObjectiveTo validate the ‘paired pulses’ technique with a conventional electrodiagnostic machine (CEM) for studying the axonal excitability recovery cycle (ERC). MethodsPaired pulses, with a variable inter-stimulus interval, were delivered at the wrist along the median nerve. The CEM repeatability was verified in a group of 15 healthy volunteers (test/retest analysis). ERC was then applied in 40 healthy volunteers and 10 patients with Charcot-Marie-Tooth type 1A (CMT1A), using both the threshold tracking (TT) reference method and CEM (basal condition, during and after ischemia). ResultsCEM parameters evaluating absolute refractory and supernormal periods were reproducible (interclass correlation coefficient > 0.75). CEM results were consistent with TT method and literature data. In CMT1A, refractory and superexcitable periods were significantly reduced. According to receiving operator characteristic analysis, the CEM supernormal period area was the most relevant parameter for discriminating CMT1A from healthy volunteers (area under the curve = 0.98). ConclusionsCEM was a valid procedure for studying ERC. CMT1A patients exhibited ERC alterations due to modifications in passive membrane properties and of nodal ion channel distribution resulting from demyelination. SignificanceStudying ERC with CEM could be performed in routine practice in patients with peripheral neuropathies to provide information on motor axonal excitability.

Cardiac contractility modulation in patients with heart failure - A review of the literature.

Experimental in vivo and in vitro studies showed that electric currents applied during the absolute refractory period can modulate cardiac contractility. In preclinical studies, cardiac contractility modulation (CCM) was found to improve calcium handling, reverse the foetal myocyte gene programming associated with heart failure (HF), and facilitate reverse remodeling. Randomized control trials and observational studies have provided evidence about the safety and efficacy of CCM in patients with HF. Clinically, CCM therapy is indicated to improve the 6-min hall walk, quality of life, and functional status of HF patients who remain symptomatic despite guideline-directed medical treatment without an indication for cardiac resynchronization therapy (CRT) and have a left ventricular ejection fraction (LVEF) ranging from 25 to 45%. Although there are promising results about the role of CCM in HF patients with preserved LVEF (HFpEF), further studies are needed to elucidate the role of CCM therapy in this population. Late gadolinium enhancement (LGE) assessment before CCM implantation has been proposed for guiding the lead placement. Furthermore, the optimal duration of CCM application needs further investigation. This review aims to present the existing evidence regarding the role of CCM therapy in HF patients and identify gaps and challenges that require further studies.

Fluctuation-response relations for integrate-and-fire models with an absolute refractory period.

We study the problem of relating the spontaneous fluctuations of a stochastic integrate-and-fire (IF) model to the response of the instantaneous firing rate to time-dependent stimulation if the IF model is endowed with a non-vanishing refractory period and a finite (stereotypical) spike shape. This seemingly harmless addition to the model is shown to complicate the analysis put forward by Lindner Phys. Rev. Lett. (2022), i.e., the incorporation of the reset into the model equation, the Rice-like averaging of the stochastic differential equation, and the application of the Furutsu-Novikov theorem. We derive a still exact (although more complicated) fluctuation-response relation (FRR) for an IF model with refractory state and a white Gaussian background noise. We also briefly discuss an approximation for the case of a colored Gaussian noise and conclude with a summary and outlook on open problems.

Cost-utility of cardiac contractility modulation in patients with heart failure with reduced ejection fraction in Italy.

Cardiac contractility modulation (CCM) is a device therapy for heart failure, based on the delivery of high-voltage biphasic impulses to the right ventricular septum during the myocardial absolute refractory period. This study evaluated the cost-effectiveness of CCM therapy plus optimal medical therapy (OMT) vs. OMT alone in patients with heart failure with reduced ejection fraction. A Markov model with a lifespan time horizon was developed to assess the cost-utility using the FIX trials as main data sources. A deterministic sensitivity analysis and a probabilistic sensitivity analysis were run to analyse the decision uncertainty in the model through cost-effectiveness acceptability curve (CEAC) and cost-effectiveness acceptability frontier (CEAF). Value of information analysis was also conducted computing the expected value of perfect information (EVPI) and the expected value of partial perfect information. The base case results showed that the CCM plus OMT option was highly cost-effective compared with OMT alone with an incremental cost-utility ratio of €7034/quality-adjusted life year (QALY). The CEAC and CEAF illustrated that for all willingness to pay levels above €5600/QALY, tested up to €50000/QALY, CCM plus OMT alternative had the highest probability of being cost-effective. The EVPI per patient was estimated to be €124412 on a willingness to pay threshold of €30000/QALY. For patients with heart failure with reduced ejection fraction, CCM therapy could be cost-effective when taking a lifetime horizon. Further long-term, post-approval clinical studies are needed to verify these results in a real-world context, particularly concerning the effect of CCM therapy on mortality.

Inferring causal connectivity from pairwise recordings and optogenetics.

To understand the neural mechanisms underlying brain function, neuroscientists aim to quantify causal interactions between neurons, for instance by perturbing the activity of neuron A and measuring the effect on neuron B. Recently, manipulating neuron activity using light-sensitive opsins, optogenetics, has increased the specificity of neural perturbation. However, using widefield optogenetic interventions, multiple neurons are usually perturbed, producing a confound-any of the stimulated neurons can have affected the postsynaptic neuron making it challenging to discern which neurons produced the causal effect. Here, we show how such confounds produce large biases in interpretations. We explain how confounding can be reduced by combining instrumental variables (IV) and difference in differences (DiD) techniques from econometrics. Combined, these methods can estimate (causal) effective connectivity by exploiting the weak, approximately random signal resulting from the interaction between stimulation and the absolute refractory period of the neuron. In simulated neural networks, we find that estimates using ideas from IV and DiD outperform naïve techniques suggesting that methods from causal inference can be useful to disentangle neural interactions in the brain.

Neuronal plasticity features are independent of neuronal holding membrane potential

Dynamical reversible neuronal features in vitro are typically examined using a fixed holding membrane potential, imitating the physiological conditions of intact brains in an awake state. Here, a set of neuronal plasticity features in synaptic blocked cultures are found to be independent of the holding membrane potential in the range [−95, −50] mV. Specifically, dendritic maximal firing frequency and its absolute refractory period are independent of the holding membrane potential. In addition, the stimulation threshold is also independent of the holding membrane potential in neurons that do not show membrane depolarization in response to sub-threshold stimulations. These robust dendritic plasticity features are a prerequisite for neuronal modeling and for their utilization in interconnected neural networks to realize higher-order functionalities.

Modeling IP[formula omitted]-induced Ca[formula omitted] signaling based on its interspike interval statistics

Inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ signaling is a second messenger system used by almost all eukaryotic cells. Recent research demonstrated randomness of Ca2+ signaling on all structural levels. We compile eight general properties of Ca2+ spiking common to all cell types investigated and suggest a theory of Ca2+ spiking starting from the random behavior of IP3 receptor channel clusters mediating the release of Ca2+ from the endoplasmic reticulum capturing all general properties and pathway-specific behavior. Spike generation begins after the absolute refractory period of the previous spike. According to its hierarchical spreading from initiating channel openings to cell level, we describe it as a first passage process from none to all clusters open while the cell recovers from the inhibition which terminated the previous spike. Our theory reproduces the exponential stimulation response relation of the average interspike interval Tav and its robustness properties, random spike timing with a linear moment relation between Tav and the interspike interval SD and its robustness properties, sensitive dependency of Tav on diffusion properties, and nonoscillatory local dynamics. We explain large cell variability of Tav observed in experiments by variability of channel cluster coupling by Ca2+-induced Ca2+ release, the number of clusters, and IP3 pathway component expression levels. We predict the relation between puff probability and agonist concentration and [IP3] and agonist concentration. Differences of spike behavior between cell types and stimulating agonists are explained by the different types of negative feedback terminating spikes. In summary, the hierarchical random character of spike generation explains all of the identified general properties.

P259 S–ICD OFFERS A POTENTIAL SYNERGISTIC THERAPY FOR HEART FAILURE PATIENTS WITH AN INDICATION FOR ICD IMPLANTATION AND CANDIDATES FOR CCM IMPLANTATION

Abstract CCM (cardiac contractility modulation) is a very promising therapy in heart failure patients as it allows, by increasing intracellular calcium handling, an improvement in cardiac contractility, ejection fraction, and patient quality of life. It delivers high–energy (7.5 V) and long–duration (about 20 milliseconds) pulses in absolute refractory period through the use of two leads on the right ventricular septum. It is indicated in patients with narrow QRS (< 130 msec) and ejection fraction between 25 and 45%. Therefore, a large proportion of patients suitable for CCM therapy also have an indication for ICD implantation. The coexistence of an ICD (single–chamber or dual–chamber) and CCM presupposes the presence of 3 leads in the right ventricle and possibly a catheter in the right atrium if a dual–chamber ICD is used. The presence of numerous leads increases the risk of infection, mechanical complications, and damage on the leads. In view of the above, the strategy we propose in patients with concomitant indication for ICD and CCM is the use of the subcutaneous defibrillator (S–ICD) in patients with potential indication for CCM, narrow QRS, no pacing indication, and ejection fraction <35%, thus limiting the number of leads and the risk of complications. In our center, we have started using this strategy with excellent clinical results and in the absence of complications. One of the cases treated with this strategy is a 60–year–old patient with nonischemic dilated myocardiopathy, ejection fraction 34%, QRS < 130 msec, NYHA class III, despite therapy with beta blockers, ARNI, SGLT2–i, MRA at the maximum dose. The patient, also considering the potential future need for CCM implantation, underwent S–ICD implantation. After 3 months, NYHA class III with reduced quality of life persisted (MLHFQ 82). Therefore, the patient successfully underwent CCM implantation, with marked improvement in symptoms (MLHFQ 71) and ejection fraction (43%) 3 months later. This strategy avoided the placement of additional leads in the right ventricle while ensuring protection from potentially fatal arrhythmias and improvement in contractile function and symptoms. We suggest the use of the subcutaneous defibrillator (S–ICD) in all patients in whom there might be an indication for the use of CCM and no need for pacing, ensuring maximum efficacy and reducing the risk of infection and leads related complications.

P246 HOW TO OPTIMIZE THE EFFICACY OF CCM THERAPY: MULTIPARAMETRIC EVALUATION AND CASE REPORT

Abstract We implanted a Cardiac Contractility Modulation (CCM) device in a 66–year–old man with congestive acute heart failure (AHF) refractory to optimized medical therapy and non–responder to CRT–D since 2015. The patient was evaluated multiple times and declared ineligible for a new heart by several heart transplant centers. Effective periodic Levosimendan treatments were performed before the implantation.. However hospitalizations still occurred monthly. The Echo exam, on the day of the implant, showed a left ventricular ejection (LVEF) fraction of 25%, PAPS of 30 mmHg, and E/E’ of 26. Interrogation of the CRT–D revealed an AF burden of 35% and several episodes of tachyarrhythmia at high frequency (130 bpm). The benefits of CCM therapy were almost immediate with the disappearance of breathlessness and the absence of hospitalizations for seven months. Only in June 2022, the patient was admitted to the ER for HF symptoms. At the admission, the heart rate was 125 bpm and, by performing the follow–up of the CCM device, the percentage of delivered therapy was low (<20%) due to the high heart rate. The interrogation of the ICD showed a reduced AF Burden (19 %). However, in the two weeks before the admission, several episodes of organized supraventricular tachycardia (SVT) conducted 1:1, at a rate of around 140 bpm, were recorded and stopped the CCM the device from delivering any impulse, in the absolute refractory period. In the HF Diagnostics implemented in the ICD, we also noticed an indication of fluid overload consistent with a steep reduction of intrathoracic impedance in the exact same time frame. Consequently, we increased the dosage of BB to reduce the heart rate and allow CCM delivery to reach a percentage higher than 80% within the 24 hours timespan. The clinical status of the patient improved immediately in the next following days. The HF diagnostics of ICD, after three weeks from discharge, revealed an increase in intrathoracic impedance and a decrease in hypervolemia which were consistent with the improved symptoms reported by the patient. Finally, the echocardiographic exam at 6 months from the implant showed an EF of 32, PAPS of 30 mmHg, and E/E’ of 12 which justified a decrease in the score of the Minnesota Living with Heart Failure Questionnaire (MLWHF) from 80 (before CCM) to 36 after the implantation. A multiparametric evaluation of CCM delivery must be performed during the patient‘s follow–up to maximize the efficacy of the therapy itself.

Cardiac contractility modulation: an effective treatment strategy for heart failure beyond reduced left ventricular ejection fraction?

Heart failure (HF) with preserved ejection fraction (HFpEF) causes a progressive limitation of functional capacity, poor quality of life (QoL) and increased mortality, yet unlike HF with reduced ejection fraction (HFrEF) there are no effective device-based therapies. Both HFrEF and HFpEF are associated with dysregulations in myocardial cellular calcium homeostasis and modifications in calcium-handling proteins, leading to abnormal myocardial contractility and pathological remodelling. Cardiac contractility modulation (CCM) therapy, based on a pacemaker-like implanted device, applies extracellular electrical stimulation to myocytes during the absolute refractory period of the action potential, which leads to an increase in cytosolic peak calcium concentrations and thereby the force of isometric contraction promoting positive inotropism. Subgroup analysis of CCM trials in HFrEF has demonstrated particular benefits in patients with LVEF between 35% and 45%, suggesting its potential effectiveness also in patients with higher LVEF values. Available evidence on CCM in HFpEF is still preliminary, but improvements in terms of symptoms and QoL have been observed. Future large, dedicated, prospective studies are needed to evaluate the safety and efficacy of this therapy in patients with HFpEF.

PO-02-206 A MECHANISM FOR PACING-INDUCED REGIONAL CONTRACTILE DYSFUNCTION MEDIATED BY RELEASED CATECHOLAMINES

We have previously shown that non-excitatory epicardial stimulation (NES) increases measures of cardiac function mediated by local release of norepinephrine. In this further investigation we focused on the effects of NES on the local myocardial contractile pattern. Farm pigs were anesthetized and instrumented with a LV high-fidelity pressure/volume conductance transducer, thoracic aortic pressure transducer, left atrial (LA)/ LV epicardial electrodes, and sonomicrometer segment length (SL) gauges placed proximal (lateral base) and distal (posterior mid LV) to the LV stimulation site. During constant LA pacing, LV pulses (0.8ms, 2-3x threshold volts) were applied during the absolute refractory period before and after β-receptor blockade (BB, metoprolol). Regional contractile function was analyzed by constructing LVP vs. SL loops in a beat-by-beat fashion thus allowing characterization of local cardiac function in relation to LV pressure over the entire cardiac cycle. NES resulted in a local contractile dysfunction characterized by an early shortening, subsequent lengthening, and re-shortening pattern during the cardiac cycle in the SL gauge proximal (middle tracing), but not remote to the stimulation site (bottom tracing). The onset of early lengthening in the proximal SL coincided with peak pressure generation, followed by re-shortening as LV pressure fell. The LVP vs. SL loop was markedly altered. This contractile effect rapidly waned within 10-15 seconds after cessation of electrical stimulation and was abolished by BB. The NES induced dysfunction developed within an average of 5-10 seconds of stimulation onset and was accompanied by increases in several global indices of cardiac function (dP/dt, peak LVP, early aortic valve opening) which were also abrogated by B-blockade. These findings demonstrate that pacing elicits local catecholamine release that may cause dyssynergy and mimic the regional pacing-induced contractile dysfunction seen at pacing sites in man.

PO-04-018 RECOGNITION AND INTERPRETATION OF IMPLANTABLE CARDIOVERTER-DEFIBRILLATOR INTERVAL CHANGES ON REMOTE MONITORING FOLLOWING A CARDIAC CONTRACTILITY MODULATION IMPLANT.

Cardiac contractility modulation (CCM) devices aim to improve the quality of life in chronic heart failure patients by delivering an electrical stimulus during the absolute myocardial refractory period in hopes of improving contractility without additional myocardial oxygen consumption. With 7,000 patients receiving the benefits of CCM by May 2022, it becomes increasingly important to remain educated regarding purpose, functionality, and potential for implantable device interactions. Investigate abnormal implantable cardioverter-defibrillator (ICD) remote monitoring (RM) transmissions to eliminate device interactions. Patient (73 yr) received an ICD in 2017 for non-ischemic cardiomyopathy and ventricular tachycardia and is compliant with RM and in-person follow-up. A CCM device was implanted December 2021 with no ICD re-programming post-iterative testing. Multiple RM transmissions reviewed January – May 2022 noted changes (Table 1), including “noise” visualized on both atrial and ventricular channels; however, only atrial over-sensing occurred (Figure 1). The RM team inquired about possible external stimuli in May 2022’s transmission and from clinic correspondence, learned of the patient’s CCM implant. Patient was seen in-clinic with appropriate company representatives present. ICD re-programming completed: Post Ventricular Atrial Blanking shortened to 110 ms; PVAB Method to Absolute; V Blanking post VS increased to 140 ms, and VF detection decreased to 214 bpm. CCM changes made: 8 hours to 9 hours and pulse delivery from 40 ms to 35 ms. ICD changes observed via RM over 5-month period post-CCM implant include false-positive (FP) AT/AF detections, short V-V intervals, and PVC burden (unclear if accurate or FP due to RV over-sensing). The increased sensed R-waves initially assumed to be an inappropriate measurement of CCM output were 15mV via in-office automatic testing. Changes recognized via RM allowed the clinic to non-urgently schedule simultaneous assessment and adjustment of both implanted devices with the hope of preventing over-sensing, specifically RV over-sensing resulting in inappropriate ICD therapies. While an in-office ICD evaluation did occur immediately post CCM implant, it is imperative to continue RM to assess for concomitant device interactions.Tabled 1Table 1DateRV SensingShort interval counter (SIC)PVC Runs/Singles CountEpisodes11/26/21 2:28 am RM3.6mV0Runs 7.3Singles 126.6 since 11/07/21No episodes01/18/22 in-clinic4.1mVnot available (n/a)n/aNo episodes01/18/22 8:09 pm RM4.1mV127 since 12/16/21Runs 5.6Singles 108.7 since 12/18/21No episodes01/18/22 8:12 pm RM4.1mV127 since 12/16/21Runs 5.6Singles 108.7 since 12/18/21No episodes01/20/22 6:11 pm RM3.5mV10 since 01/18/22Runs 2.2Singles 59.5 since 01/18/22No episodes02/25/22 1:23 am RM3.9mV158 since 01/18/22Runs 7.1Singles 110.8 since 01/20/221 AT/AF (FP)05/26/22 3:34 am RM15.6mV1060 since 01/18/22Runs 18.4Singles 161.3 since 02/25/221 high rate NST (true positive)4 AT/AF (FP)05/31/22 in clinic16.5mVn/aRuns 15.9Singles 150.7 since 01/18/22n/a Open table in a new tab

An improved vocoder algorithm based on music harmonics and time sampling

Music plays an essential role in the healthy life of humans. However, few sound processor algorithms effectively encode the fine structure cues of music, resulting in inferior music perception for cochlear implant (CI) users, especially in pitch and melody. In this study, an improved music vocoder algorithm is proposed based on the conjunction of harmonic and time sampling (HTS). The algorithm includes two branches: the first, the pitch (i.e., fundamental frequency, F0) and important harmonics of music signal are extracted. Second, on the existing CI channels, the music signal is split into multiple sub-bands and the relevant envelopes are respectively matched to the F0 and important lower harmonics, then modulated and aligned with appropriate intervals which are not less than the auditory nerve response absolute refractory period (ANR-ARP). The violin sounds were synthesized and experimented with the CI tone vocoder, and the HTS algorithm was compared and evaluated with the classical continuous interleaved sampling (CIS) algorithm. Twenty normal hearing (NH) subjects were recruited for audiometry experiments. The results showed that the pitch ranking scores of the HTS were obviously better than that of the CIS, and in quiet and noisy conditions the melody recognition rates of the HTS were 46.4% and 49% higher than that of the CIS, respectively. And further results showed that the HTS algorithm also increased the timbre perception of CI vocoder music. It is suggested that the HTS algorithm has the potential to enhance the music perception of CI users.

376 CARDIAC CONTRACTILITY MODULATION: A NEW THERAPY FOR HEART FAILURE

Abstract Introduction Cardiac Contractility Modulation is a therapy for chronic Heart Failure patients with EF 25-45% in sinus rhythm with QRS < 130 ms clinically symptomatic (NYHA Class > II) despite optimised medical therapy. It is made possible by the implantation of the OPTIMIZER SMART® device. The CCM works by delivering a non-excitatory high-energy bipolar signal, synchronised with local electrical activity (or cooperating with another pre-existing CIED), in the absolute ventricular refractory period, by means of two actively fixating leads, positioned in the interventricular septum and spaced at least 1-2 cm apart. Purpose Assessment and feedback of the CCM therapy process in patients with dilated cardiomyopathy. Educational update for health professionals. Patient education. Method A Pubmed search correlated with clinical data and field experience was performed. Results Patients with severe Dilated Cardiomyopathy already on optimised medical therapy. The mechanism of action demonstrated a remodelling of the left ventricle with a so-called 'metabolic' therapy. In CCM therapy, electrical stimulation is delivered to the heart muscle during the absolute refractory period. In this phase, electrical signals activate the mobilisation of calcium ions in cardiomyocytes. CCM signals applied during the absolute refractory period cause an increase in cytosolic calcium during systole, resulting in improved cardiac contraction without an increase in oxygen consumption. Conclusions CCM therapy has shown to improve contractility and cardiac performance improving the quality of life of patients undergoing this treatment by reducing symptoms of fatigue and dyspnoea; it has reduced the number of hospitalisations for moderate to severe heart failure. Healthcare professionals recognise The typical ECG of a patient implanted with a CCM device shows a 'spike' in the absolute refractory period of the cardiac cycle: the 'R wave' of the QRS complex without being alarmed. The patient has been trained to be autonomous in charging. OPTIMIZER SMART® is recharged externally with a weekly rechargeable battery through a mini induction charger, rechargeable by itself.

Acute effects of cardiac contractility modulation stimulation in conventional 2D and 3D human induced pluripotent stem cell-derived cardiomyocyte models.

Cardiac contractility modulation (CCM) is a medical device therapy whereby non-excitatory electrical stimulations are delivered to the myocardium during the absolute refractory period to enhance cardiac function. We previously evaluated the effects of the standard CCM pulse parameters in isolated rabbit ventricular cardiomyocytes and 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, on flexible substrate. In the present study, we sought to extend these results to human 3D microphysiological systems to develop a robust model to evaluate various clinical CCM pulse parameters in vitro. HiPSC-CMs were studied in conventional 2D monolayer format, on stiff substrate (i.e., glass), and as 3D human engineered cardiac tissues (ECTs). Cardiac contractile properties were evaluated by video (i.e., pixel) and force-based analysis. CCM pulses were assessed at varying electrical 'doses' using a commercial pulse generator. A robust CCM contractile response was observed for 3D ECTs. Under comparable conditions, conventional 2D monolayer hiPSC-CMs, on stiff substrate, displayed no contractile response. 3D ECTs displayed enhanced contractile properties including increased contraction amplitude (i.e., force), and accelerated contraction and relaxation slopes under standard acute CCM stimulation. Moreover, 3D ECTs displayed enhanced contractility in a CCM pulse parameter-dependent manner by adjustment of CCM pulse delay, duration, amplitude, and number relative to baseline. The observed acute effects subsided when the CCM stimulation was stopped and gradually returned to baseline. These data represent the first study of CCM in 3D hiPSC-CM models and provide a nonclinical tool to assess various CCM device signals in 3D human cardiac tissues prior to in vivo animal studies. Moreover, this work provides a foundation to evaluate the effects of additional cardiac medical devices in 3D ECTs.

Abstract 11951: Acute Cardiac Contractility Modulation Assessment in Conventional 2D Monolayers and 3D Engineered Cardiac Tissues Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Introduction: Cardiac contractility modulation (CCM) is a medical device therapy whereby non-excitatory electrical simulations are delivered to the myocardium during the absolute refractory period. We previously evaluated the effects of the standard CCM pulse parameters in isolated rabbit ventricular cardiomyocytes and 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, on flexible substrate. Hypothesis: In the present study, we sought to extend these results to 3D microphysiological systems to develop a human-based model to evaluate various clinical CCM pulse parameters in vitro. Methods: HiPSC-CMs were studied in conventional 2D monolayer format, on stiff substrate (i.e., glass), and as 3D human engineered cardiac tissues (ECTs). Cardiac contractile properties were evaluated by video-based analysis and custom force analysis. CCM pulses were assessed at varying clinical ‘doses’ using a commercial pulse generator. Robust response was observed at physiological Ca concentrations [1.8 mM] for 3D ECTs. Results: Under standard acute CCM stimulation 3D ECTs displayed enhanced contractile properties including increased peak contraction amplitude (i.e., force), and faster contraction and relaxation kinetics. Moreover, 3D ECTs displayed enhanced contractility in a CCM pulse parameter dependent manner. The observed effects subsided when the acute CCM stimulation was stopped and gradually returned to baseline. Under comparable conditions, conventional 2D monolayer hiPSC-CMs, on stiff substrate, displayed neutral response. Conclusions: These data represent the first study of acute CCM stimulation in a 3D hiPSC-CM model and provides a preclinical model to assess various CCM signals in human cardiac tissues prior to in vivo animal studies.

Abstract 12821: Progressive Improvement in Left Ventricular Performance Associated With Progressive Regional Dyssynchrony

Background : We have shown that non-excitatory epicardial stimulation (NES) increases measures of cardiac function mediated by local release of norepinephrine. In this further investigation we determined whether NES is accompanied by improved indices of left ventricular (LV) performance, reflected in PV loops and timing of aortic valve opening and closing. Methods : Anesthetized pigs (n=6) were instrumented with a LV high-fidelity pressure/volume conductance transducer, thoracic aortic pressure transducer, left atrial (LA)/ LV epicardial electrodes, and sonomicrometer segment length (SL) gauges placed proximal (lateral base) and distal (posterior mid LV) to the LV stimulation site. During constant LA pacing, LV pulses (0.8ms, 2-3x threshold volts) were applied during the absolute refractory period for 3 minutes (3x), before and after β-receptor blockade (BB, metoprolol). Data were analyzed at baseline, and at an estimated 30%, 60% and 100% of developed ventricular dyssynchrony (DVD) contraction pattern. Results: Consistent with earlier findings, NES resulted in an early shortening and relaxation pattern of the proximal SL manifest as an 80% decrease in the LVP vs. SL loop area in the absence of change in the remote segment. This was accompanied by an increase in peak systolic pressure and LV dP/dt max. Although NES did not change the area of the LVP vs. LV volume loop (stroke work) or alter the interval from LV electrical activation (R wave) to the onset of LV pressure increase, aortic valve opening occurred up to 14.1 ± 2.8 ms earlier (p <0.01) compared to baseline. The duration of an open aortic valve slightly increased (6.0 ± 3.9 ms) whereas ejection duration from valve opening to peak Pao decreased 18.2 ± 3.0 ms (p < 0.01). Conclusion : These findings demonstrate that NES elicits a local contractile dysynchrony resulting in an enhanced cardiac inotropic state characterized by more rapid global LV contraction with earlier aortic valve opening with no change in stroke work.

Human in vitro neurocardiac coculture (ivNCC) assay development for evaluating cardiac contractility modulation.

Two of the most prominent organ systems, the nervous and the cardiovascular systems, are intricately connected to maintain homeostasis in mammals. Recent years have shown tremendous efforts toward therapeutic modulation of cardiac contractility and electrophysiology by electrical stimulation. Neuronal innervation and cardiac ganglia regulation are often overlooked when developing in vitro models for cardiac devices, but it is likely that peripheral nervous system plays a role in the clinical effects. We developed an in vitro neurocardiac coculture (ivNCC) model system to study cardiac and neuronal interplay using human induced pluripotent stem cell (hiPSC) technology. We demonstrated significant expression and colocalization of cardiac markers including troponin, α-actinin, and neuronal marker peripherin in neurocardiac coculture. To assess functional coupling between the cardiomyocytes and neurons, we evaluated nicotine-induced β-adrenergic norepinephrine effect and found beat rate was significantly increased in ivNCC as compared to monoculture alone. The developed platform was used as a nonclinical model for the assessment of cardiac medical devices that deliver nonexcitatory electrical pulses to the heart during the absolute refractory period of the cardiac cycle, that is, cardiac contractility modulation (CCM) therapy. Robust coculture response was observed at 14 V/cm (5V, 64 mA), monophasic, 2ms pulse duration for pacing and 20 V/cm (7V, 90 mA) phase amplitude, biphasic, 5.14 ms pulse duration for CCM. We observed that the CCM effect and kinetics were more pronounced in coculture as compared to cardiac monoculture, supporting a hypothesis that some part of CCM mechanism of action can be attributed to peripheral nervous system stimulation. This study provides novel characterization of CCM effects on hiPSC-derived neurocardiac cocultures. This innervated human heart model can be further extended to investigate arrhythmic mechanisms, neurocardiac safety, and toxicity post-chronic exposure to materials, drugs, and medical devices. We present data on acute CCM electrical stimulation effects on a functional and optimized coculture using commercially available hiPSC-derived cardiomyocytes and neurons. Moreover, this study provides an in vitro human heart model to evaluate neuronal innervation and cardiac ganglia regulation of contractility by applying CCM pulse parameters that closely resemble clinical setting. This ivNCC platform provides a potential tool for investigating aspects of cardiac and neurological device safety and performance.

The Effect of Doxapram on Proprioceptive Neurons: Invertebrate Model.

The resting membrane potential enables neurons to rapidly initiate and conduct electrical signals. K2p channels are key in maintaining this membrane potential and electrical excitability. They direct the resting membrane potential toward the K+ equilibrium potential. Doxapram is a known blocker for a subset of K2p channels that are pH sensitive. We assessed the effects of 0.1 and 5 mM doxapram on the neural activity within the propodite-dactylopodite (PD) proprioceptive sensory organ in the walking legs of blue crabs (Callinectes sapidus). Results indicate that 0.1 mM doxapram enhances excitation, while the higher concentration 5 mM may over-excite the neurons and promote a sustained absolute refractory period until the compound is removed. The effect of 5 mM doxapram mimics the effect of 40 mM K+ exposure. Verapamil, another known K2p channel blocker as well as an L-type Ca2+ channel blocker, reduces neural activity at both 0.1 and 5 mM. Verapamil may block stretch activated channels in sensory endings, in addition to reducing the amplitude of the compound action potential with whole nerve preparations. These findings are notable as they demonstrate that doxapram has acute effects on neurons of crustaceans, suggesting a targeted K2p channel. The actions of verapamil are complex due to the potential of affecting multiple ion channels in this preparation. Crustacean neurons can aid in understanding the mechanisms of action of various pharmacological agents as more information is gained.