Stephane Masse

Design and formulation of functional pluripotent stem cell-derived cardiac microtissues

Significance Robust and predictive in vitro models of human cardiac tissue function could have transformative impact on our ability to test new drugs and understand cardiac disease. Despite significant effort, the generation of high-fidelity adult-like human cardiac tissue analogs remains challenging. In this paper, we systematically explore the design criteria for pluripotent stem cell-derived engineered cardiac tissue. Parameters such as biomechanical stress during tissue remodeling, input-cell composition, electrical stimulation, and tissue geometry are evaluated. Our results suggest that a specified combination of a 3D matrix-based microenvironment, uniaxial mechanical stress, and mixtures of cardiomyocytes and fibroblasts improves the performance and maturation state of in vitro engineered cardiac tissue. Access to robust and information-rich human cardiac tissue models would accelerate drug-based strategies for treating heart disease. Despite significant effort, the generation of high-fidelity adult-like human cardiac tissue analogs remains challenging. We used computational modeling of tissue contraction and assembly mechanics in conjunction with microfabricated constraints to guide the design of aligned and functional 3D human pluripotent stem cell (hPSC)-derived cardiac microtissues that we term cardiac microwires (CMWs). Miniaturization of the platform circumvented the need for tissue vascularization and enabled higher-throughput image-based analysis of CMW drug responsiveness. CMW tissue properties could be tuned using electromechanical stimuli and cell composition. Specifically, controlling self-assembly of 3D tissues in aligned collagen, and pacing with point stimulation electrodes, were found to promote cardiac maturation-associated gene expression and in vivo-like electrical signal propagation. Furthermore, screening a range of hPSC-derived cardiac cell ratios identified that 75% NKX2 Homeobox 5 (NKX2-5)+ cardiomyocytes and 25% Cluster of Differentiation 90 OR (CD90)+ nonmyocytes optimized tissue remodeling dynamics and yielded enhanced structural and functional properties. Finally, we demonstrate the utility of the optimized platform in a tachycardic model of arrhythmogenesis, an aspect of cardiac electrophysiology not previously recapitulated in 3D in vitro hPSC-derived cardiac microtissue models. The design criteria identified with our CMW platform should accelerate the development of predictive in vitro assays of human heart tissue function.

Interrogating functional integration between injected pluripotent stem cell-derived cells and surrogate cardiac tissue

Myocardial infarction resulting in irreversible loss of cardiomyocytes (CMs) remains a leading cause of heart failure. Although cell transplantation has modestly improved cardiac function, major challenges including increasing cell survival, engraftment, and functional integration with host tissue, remain. Embryonic stem cells (ESCs), which can be differentiated into cardiac progenitors (CPs) and CMs, represent a candidate cell source for cardiac cell therapy. However, it is not known what specific cell type or condition is the most appropriate for transplantation. This problem is exasperated by the lack of efficient and predictive strategies to screen the large numbers of parameters that may impact cell transplantation. We used a cardiac tissue model, engineered heart tissue (EHT), and quantitative molecular and electrophysiological analyses, to test transplantation conditions and specific cell populations for their potential to functionally integrate with the host tissue. In this study, we validated our analytical platform using contractile mouse neonatal CMs (nCMs) and noncontractile cardiac fibroblasts (cFBs), and screened for the integration potential of ESC-derived CMs and CPs (ESC-CMs and -CPs). Consistent with previous in vivo studies, cFB injection interfered with electrical signal propagation, whereas injected nCMs improved tissue function. Purified bioreactor-generated ESC-CMs exhibited a diminished capacity for electrophysiological integration; a result correlated with lower (compared with nCMs) connexin 43 expression. ESC-CPs, however, appeared able to appropriately mature and integrate into EHT, enhancing the amplitude of tissue contraction. Our results support the use of EHT as a model system to accelerate development of cardiac cell therapy strategies.

A Three-Dimensional Display for Cardiac Activation Mapping

A three‐dimensional display is described that allows activation sequences from the epicardium and endocardium to be shown simultaneously on the same image. Three electrode arrays (epicardial sock, left ventricular balloon, right ventricular balloon) are represented in a three‐dimensional perspective by an array of dots that are intensified when activated. This arrangement requires fewer calculations and is easier to interpret than siiced‐isochronal maps but cannot represent a complete heart cycle in one image. The three‐dimensional display eliminates the distortion caused by two‐dimensional diagrams and facilitates activation correlation between electrode arrays. A standard, low cost microcomputer has been used to implement the activation display.

A comparison of unipolar and bipolar electrodes during cardiac mapping studies.

Controversy exists as to whether the unipolar or bipolar electrode configuration is superior in detecting local activations during cardiac mapping studies. However, the strengths and weaknesses of each mode suggest that they may provide complementary information. To examine therelative merits of unipolar and bipolar electrode configurations, recordings by each were simultaneously acquired during episodes of ventricular tachycardia in eight consecutive patients undergoing map guided arrhythmia surgery. Unipolar electrograms were classified as either unambiguous or ambiguous according to whether or not they were polyphasic in nature. The activation times from the unambiguous electrograms were compared with activation times from the corresponding bipolar signals where local activation was measured both at the signals peak amplitude (BI‐PK), and at the point at which the waveforms first major, rapid transient crossed baseline (BI‐TRN). Occurrences of discrete diastolic activations were also quantified from the unipolar and bipolar tracings. From a total of 415 unipolar electrograms, 301 unambiguous signals were identified as suitable for comparison with the bipolar signals. Both BI‐PK and BI‐TRN criteria for the determination of local activation were highly correlated with and not significantly different from the local activation from the unipolar electrogram. From 85 ambiguous unipolar electrograms, it was possible to determine local activation from the corresponding bipolar signal in 33% of the occurrences. From the eight patients, 64 diastolic potentials were recorded of which 42 were seen only in bipolar mode, 7 in only unipolar mode, and 15 were evident in both tracings. The prevalence of diastolic potentials was significantly greater in recordings made using bipolar mode. The results demonstrate that complementary information regarding local activations and diastolic potentials can be derived from unipolar and bipolar recordings and suggest that both electrode configurations should be used in multichannel cardiac mapping systems.

Dantrolene Improves Survival After Ventricular Fibrillation by Mitigating Impaired Calcium Handling in Animal Models

Background— Resistant ventricular fibrillation, refibrillation. and diminished myocardial contractility are important factors leading to poor survival after cardiac arrest. We hypothesized that dantrolene improves survival after ventricular fibrillation (VF) by rectifying the calcium dysregulation caused by VF. Methods and Results— VF was induced in 26 Yorkshire pigs for 4 minutes. Cardiopulmonary resuscitation was then commenced for 3 minutes, and dantrolene or isotonic saline was infused at the onset of cardiopulmonary resuscitation. Animals were defibrillated and observed for 30 minutes. To study the effect of VF on calcium handling and its modulation by dantrolene, hearts from 14 New Zealand rabbits were Langendorff-perfused. The inducibility of VF after dantrolene administration was documented. Optical mapping was performed to evaluate diastolic spontaneous calcium elevations as a measure of cytosolic calcium leak. The sustained return of spontaneous circulation (systolic blood pressure ≥60 mm Hg) was achieved in 85% of the dantrolene group in comparison with 39% of controls (P=0.02). return of spontaneous circulation was achieved earlier in dantrolene-treated pigs after successful defibrillation (21±6 s versus 181±57 s in controls, P=0.005). The median number of refibrillation episodes was lower in the dantrolene group (0 versus 1, P=0.04). In isolated rabbit hearts, the successful induction of VF was achieved in 83% of attempts in controls versus 41% in dantrolene-treated hearts (P=0.007). VF caused diastolic calcium leaks in the form of spontaneous calcium elevations. Administration of 20 &mgr;mol/L dantrolene significantly decreased spontaneous calcium elevation amplitude versus controls. (0.024±0.013 versus 0.12±0.02 arbitrary unit [200-ms cycle length], P=0.001). Conclusions— Dantrolene infusion during cardiopulmonary resuscitation facilitates successful defibrillation, improves hemodynamics postdefibrillation, decreases refibrillation, and thus improves survival after cardiac arrest. The effects are mediated through normalizing VF-induced dysfunctional calcium cycling.

Duration of Discharge of Neuromuscular Incapacitating Device and Inappropriate Implantable Cardioverter-Defibrillator Detections

Neuromuscular-incapacitating devices (NID) are being used increasingly worldwide by law-enforcement authorities to restrain violent behavior.1 Electrical noise-related, inappropriate detection by implantable cardioverter-defibrillator (ICD) has been documented.2,3 Detection of NID discharge by ICD has been described,4,5 but the effects of duration of NID discharge and ICD detection and therapy have not been established. The authors hypothesized that a longer duration of NID energy might lead to shocks from the defibrillator.4 With the approval of the animal care committee of our institution, we tested this hypothesis in a pig implanted with Medtronic 7275 GEM III DR ICD. The method used for performing the experiment in a pig model has been described previously. …

Characteristics of Local Electrograms with Diastolic Potentials: Identification of Different Components of Return Pathways in Ventricular Tachycardia

Background:Diastolic potentials are often sought as a possible site for catheter ablation in post-infarct ventricular tachycardia. However, delivery of energy at such sites is often unsuccessful. The purpose of this study was to determine the characteristics of local electrograms with diastolic potentials and to identify activation pattern which might indicate the critical portion of the return path of the ventricular tachycardia reentry circuit.Methods: In 17 patients with post-myocardial infarction ventricular tachycardia, 30 ventricular tachycardias were mapped with an 112 bipolar endocardial balloon at the time of surgery. Diastolic mapping of the return tract in ventricular tachycardia was performed. Four activation patterns were observed (15 figure 8 patterns, 2 circular patterns, 2 biregional patterns and 11 monoregional patterns). Of 3,360 local electrograms, 207 (6.2%) demonstrated a diastolic potential in ventricular tachycardia. They were classified into following four categories, based on the appearance and timing of the systolic component. Type A-1 electrogram: systolic activation was of low amplitude (<2 mV) and was prolonged (≤100 msec), but preceded the onset of the surface QRS in ventricular tachycardia. Type A-2 electrogram: systolic activation was of low amplitude, was prolonged, but followed the onset of the surface QRS. Type B electrogram: systolic electrogram was fractionated, but relatively normal amplitude (2.0–3.6 mV). Type C electrogram: systolic electrogram was almost normal.Results: Of all electrograms with diastolic potentials, three type A-1 electrograms (1.4%) were located at the exit of the return pathway, 11 type A-1 electrograms (5.3%) were located at the pre-exit site. No type A-1 was found at an entrance/bystander area. 21 type A-2 electrograms (10.1%) were at the pre-exit and 83 type A-2 electrograms (40.2%) were located at the entrance/bystander area, but such electrograms were never found at the exit site. 71 type B electrograms (34.3%) and 18 type C electrograms (8.7%) were located at the entrance/bystander area. To distinguish the type A-2 electrograms at the pre-exit site from those at the entrance/bystander area, the diastolic potential to QRS interval was measured. This interval at the pre-exit was significantly shorter than that at the entrance/bystander area (−47.2 ± 10.7 vs −96.3 ± 31.3 msec, p = 0.0001).Conclusion: Type A-1 electrograms indicated the exit or pre-exit site of return pathway. Type A-2 electrograms with diastolic potential to QRS interval <−50 msec indicated the pre-exit site. However, the other types of local electrograms with diastolic potential did not indicate the critical portion of the ventricular tachycardia circuit. These observations may be helpful during catheter mapping and ablation of patients with post-infarct ventricular tachycardia.Condensed Abstract. Diastolic potentials are often sought to direct catheter ablation in post-infarct ventricular tachycardia. We investigated the characteristics of local electrograms showing diastolic activity in an attempt to determine whether critical portions of the ventricular tachycardia circuit could be identified by a typical “signature.” In 17 patients with a remote myocardial infarction, 30 ventricular tachycardias were mapped with 112 bipolar endocardial balloon at the time of surgery. Diastolic potentials in association with low amplitude (<2 mV) and prolonged (≤100 msec) systolic electrograms preceding the onset of QRS were found at the exit site and pre-exit site of return pathway. A similar systolic electrogram occurring after QRS onset with a diastolic potential to QRS interval of <−50 msec was found at the pre-exit site. However, other local electrograms with diastolic activity were at sites remote from the exit or pre-exit of the return pathway. These observations may be helpful during catheter mapping and ablation in patients with ventricular tachycardia.

Simultaneous Unipolar and Bipolar Recording of Cardiac Electrical Activity

An analog mapping system using a true bipolar left ventricular balloon electrode array is described, which enables simultaneous unipolar and bipolar recordings. It is an adaptation of a previous clinical analog mapping system used in the investigation of ventricular arrhythmias. The bipolar balloon array consists of 112 electrode pairs, each having a 2‐mm separation. The signals from the electrodes are sensed in parallel by separate unipolar and bipolar amplifier units, which then drive a common multiplexer bus. The bipolar recording unit consists of high quality instrumentation amplifiers with adjustable gain and exhibits a full bandwidth minimum common mode rejection of 78 dB. Using this combination, it is possible to record local cardiac micropotentials while still retaining the advantages of unipolar electrograms to track overall cardiac activation.

Optimizing cardiac resuscitation outcomes using wavelet analysis

Ventricular fibrillation (VF) is the most lethal of cardiac arrhythmias that leads to sudden cardiac death if untreated within minutes of its occurrence. Defibrillation using electric shock resets the heart to return to spontaneous circulation (ROSC) state, however the success of which depends on various factors such as the viability of myocardium and the time lag between the onset of VF to defibrillation. Recent studies have reported that performing cardio pulmonary resuscitation (CPR) procedure prior to applying shock increases the survival rate especially when VF is untreated for more than 5 minutes. Considering the limited time within which the VF has to be treated for better survival rates, the choice of the right therapy (shock parameters, shock first or CPR first, drug administration) is vital. In aiding this choice, it would be of immense help for emergency medical staff (EMS) if an objective feedback could be provided at near real-time rate on the VF characteristics and its relation to the shock outcomes. Existing works in the literature have demonstrated correlation between the characteristics of the VF waveform and the outcome (ROSC) of the defibrillation. The proposed work improves on this by attempting to arrive at a near real-time monitoring tool in aiding the EMS staff. Using data collected from 16 pigs during VF, the proposed wavelet methodology achieved an overall accuracy of 94% in successfully predicting the shock outcomes.

Wavelet-based markers of ventricular fibrillation in optimizing human cardiac resuscitation

During cardiac resuscitation from ventricular fibrillation (VF) it would be helpful if we could monitor and predict the optimal state of the heart to be shocked into a perfusing rhythm. Real-time feedback of this state to the emergency medical staff (EMS) could improve the survival rate after resuscitation. In this paper, using real world out-of-the-hospital human VF data obtained during resuscitation by EMS personnel, we present the results of applying wavelet markers in predicting the shock outcomes. We also performed comparative analysis of 5 existing techniques (spectral and correlation based approaches) against the proposed wavelet markers. A database of 29 human VF tracings was extracted from the defibrillator recordings collected by the EMS personnel and was used to validate the waveform markers. The results obtained by the comparison of the wavelet based features with other spectral, and correlation-based features indicates that the proposed wavelet features perform well with an overall accuracy of 79.3% in predicting the shock outcomes and hence demonstrate potential to provide near real-time feedback to EMS personnel in optimizing resuscitation outcomes.