Di-5-ANEQ(F)PTEA Offers Better Performance than Di-4-ANEQ(F)PTEA for In-Situ Cardiac Optical Mapping.

Cardiac optical mapping has traditionally been performed in ex-vivo, motion-arrested hearts. Recently, in-situ cardiac optical mapping has been made possible by both motion correction techniques and long-wavelength voltage sensitive dyes (VSDs). However, VSDs have been observed to wash out quickly from blood-perfused in-situ hearts. In this study, we evaluate the performance of a newly developed VSD, di-5-ANEQ(F)PTEA, relative to an earlier VSD, di-4-ANEQ(F)PTEA. We find that di-5-ANEQ(F)PTEA persists over 3 times longer, produces improved signal-to-noise ratio, and does not prolong loading unacceptably.Clinical Relevance-Optical mapping has provided many insights into cardiac arrhythmias, but has traditionally been limited to ex-vivo preparations. The present findings extend the utility of optical mapping in the more realistic in-vivo setting and may eventually enable its use in patients.

Optical Mapping of Virtual Electrode Polarization Pattern and Its Relationship with Pacemaker Location during Gastric Pacing.

Gastric rhythmic contractions are regulated by bioelectrical events known as slow waves (SW). Abnormal SW activity is associated with gastric motility disorders. Gastric pacing is a potential treatment method to restore rhythmic SW activity. However, to date, the efficacy of gastric pacing is inconsistent and the underlying mechanisms of gastric pacing are poorly understood. Optical mapping is widely used in cardiac electrophysiology studies. Its immunity to pacing artifacts offers a distinct advantage over conventional electrical mapping for studying pacing. In the present study, we first found that optical mapping can image pacing-induced virtual electrode polarization patterns in the stomach (adjacent regions of depolarized and hyperpolarized tissue). Second, we found that elicited SWs usually (15 of 16) originated from the depolarized areas of the stimulated region (virtual cathodes). To our knowledge, this is the first direct observation of virtual electrode polarization patterns in the stomach. Conclusions: Optical mapping can image virtual electrode polarization patterns during gastric pacing with high spatial resolution.Clinical Relevance- Gastric pacing is a potential therapeutic method for gastric motility disorders. This study provides direct observation of virtual electrode polarization pattern during gastric pacing and improves our understanding of the mechanisms underlying gastric pacing..

An Inexpensive, Portable Shield to Improve Healthcare Worker Safety During Intubation Procedures.

Healthcare workers (HCW) are exposed to risk of infection during intubation procedures, in particular, in the prehospital setting. Here, we demonstrate a novel shield that can be used during intubation to block aerosols and droplets from reaching the HCW. The device is mounted on the patient's head and provides a barrier between patient and HCW. It incorporates a self-sealing port through which an endotracheal tube can be inserted. The port "floats" in the plane of the shield to facilitate maneuvering of the endotracheal tube. The shield is fabricated from transparent materials to enable the HCW to visualize the procedure. Using two complementary imaging methods, background oriented Schlieren imaging and laser sheet droplet imaging, we show that the device prevents detectable transmission of gas flow and droplets through the shield both before and after endotracheal tube insertion.Clinical Relevance- This device has the potential to protect HCWs from infections during intubation procedures, especially in the prehospital setting.

Optical mapping of cardiac electromechanics in beating in vivo hearts.

Optical mapping has been widely used in the study of cardiac electrophysiology in motion-arrested, ex vivo heart preparations. Recent developments in motion artifact mitigation techniques have made it possible to optically map beating ex vivo hearts, enabling the study of cardiac electromechanics using optical mapping. However, the ex vivo setting imposes limitations on optical mapping such as altered metabolic states, oversimplified mechanical loads, and the absence of neurohormonal regulation. In this study, we demonstrate optical electromechanical mapping in an in vivo heart preparation. Swine hearts were exposed via median sternotomy. Voltage-sensitive dye, either di-4-ANEQ(F)PTEA or di-5-ANEQ(F)PTEA, was injected into the left anterior descending artery. Fluorescence was excited by alternating green and amber light for excitation ratiometry. Cardiac motion during sinus and paced rhythm was tracked using a marker-based method. Motion tracking and excitation ratiometry successfully corrected most motion artifact in the membrane potential signal. Marker-based motion tracking also allowed simultaneous measurement of epicardial deformation. Reconstructed membrane potential and mechanical deformation measurements were validated using monophasic action potentials and sonomicrometry, respectively. Di-5-ANEQ(F)PTEA produced longer working time and higher signal/noise ratio than di-4-ANEQ(F)PTEA. In addition, we demonstrate potential applications of the new optical mapping system including electromechanical mapping during vagal nerve stimulation, fibrillation/defibrillation. and acute regional ischemia. In conclusion, although some technical limitations remain, optical mapping experiments that simultaneously image electrical and mechanical function can be conducted in beating, in vivo hearts.

Ectopic foci do not co-locate with ventricular epicardial stretch during early acute regional ischemia in isolated pig hearts.

Ectopic activation during early acute regional ischemia may initiate fatal reentrant arrhythmias. However, the origin of this ectopy remains poorly understood. Studies suggest that systolic stretch arising from dyskinesia in ischemic tissue may cause ectopic depolarization due to cardiac mechanosensitivity. The aim of this study was to investigate the link between mechanical stretch and ectopic electrical activation during early acute regional ischemia. We used a recently developed optical mapping technique capable of simultaneous imaging of mechanical deformation and electrical activation in isolated hearts. Eight domestic swine hearts were prepared in left ventricular working mode (LVW), in which the left ventricle was loaded and contracting. In an additional eight non-working (NW) hearts, contraction was pharmacologically suppressed with blebbistatin and the left ventricle was not loaded. In both groups, the left anterior descending coronary artery was tied below the first diagonal branch. Positive mechanical stretch (bulging) during systole was observed in the ischemic zones of LVW, but not NW, hearts. During ischemia phase 1a (0-15 min post-occlusion), LVW hearts had more ectopic beats than NW hearts (median: 19, interquartile range: 10-28 vs. median: 2, interquartile range: 1-6; p = 0.02); but the difference during phase 1b (15-60 min post-occlusion) was not significant (median: 27, interquartile range: 22-42 vs. median: 16, interquartile range: 12-31; p = 0.37). Ectopic beats arose preferentially from the ischemic border zone in both groups (p < 0.01). In LVW hearts, local mechanical stretch was only occasionally co-located with ectopic foci (9 of 69 ectopic beats). Despite the higher rate of ectopy observed in LVW hearts during ischemia phase 1a, the ectopic beats generally did not arise by the hypothesized mechanism in which ectopic foci are generated by co-local epicardial mechanical stretch.

Gastric pacing response evaluated with simultaneous electrical and optical mapping.

Gastric pacing is an attractive therapeutic approach for correcting abnormal bioelectrical activity. While high-resolution (HR) electrical mapping techniques have largely contributed to the current understanding of the effect of pacing on the electrophysiological function, these mapping techniques are restricted to surface contact electrodes and the signal quality can be corrupted by pacing artifacts. Optical mapping of voltage sensitive dyes is an alternative approach used in cardiac research, and the signal quality is not affected by pacing artifacts. In this study, we simultaneously applied HR optical and electrical mapping techniques to evaluate the bioelectrical slow wave response to gastric pacing. The studies were conducted in vivo on porcine stomachs ( n=3) where the gastric electrical activity was entrained using high-energy pacing. The pacing response was optically tracked using voltage-sensitive fluorescent dyes and electrically tracked using surface contact electrodes positioned on adjacent regions. Slow waves were captured optically and electrically and were concordant in time and direction of propagation with comparable mean velocities ([Formula: see text]) and periods ([Formula: see text]). Importantly, the optical signals were free from pacing artifacts otherwise induced in electrical recordings highlighting an advantage of optical mapping. Clinical Relevance- Entrainment mapping of gastric pacing using optical techniques is a major advance for improving the preclinical understanding of the therapy. The findings can thereby inform the efficacy of gastric pacing in treating functional motility disorders.

Right ventricular insertion promotes reinitiation of ventricular fibrillation in defibrillation failure.

BACKGROUND: Shocks near defibrillation threshold (nDFT) strength commonly extinguish all ventricular fibrillation (VF) wavefronts, but a train of rapid, well-organized postshock activations (PAs) typically appears before sinus rhythm ensues. If one of the PA waves undergoes partial propagation block (wavebreak), reentry may be induced, causing VF to reinitiate and the shock to fail. OBJECTIVE: The purpose of this study was to determine whether wavebreak leading to VF reinititation following nDFT shocks occurs preferentially at the right ventricular insertion (RVI), which previous studies have identified as a key site for wavebreak. METHODS: We used panoramic optical mapping to image the ventricular epicardium of 6 isolated swine hearts during nDFT defibrillation episodes. After each experiment, the hearts were fixed and their geometry scanned with magnetic resonance imaging (MRI). The MRI and mapping datasets were spatially coregistered. For failed shocks, we identified the site of the first wavebreak of a PA wave during VF reinitiation. RESULTS: We recorded 59 nDFT failures. In 31 of these, the first wavebreak event occurred within 1 cm of the RVI centerline, most commonly on the anterior side of the right ventricular insertion (aRVI) (23/31). The aRVI region occupies 16.8% ± 2.5% of the epicardial surface and would be expected to account for only 10 wavebreaks if they were uniformly distributed. By χ2 analysis, aRVI wavebreaks were significantly overrepresented. CONCLUSION: The anterior RVI is a key site in promoting nDFT failure. Targeting this site to prevent wavebreak could convert defibrillation failure to success and improve defibrillation efficacy.

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

Cardiomyocytes from CCND2-overexpressing human induced-pluripotent stem cells repopulate the myocardial scar in mice: A 6-month study.

BACKGROUND: Cardiomyocytes that have been differentiated from CCND2-overexpressing human induced-pluripotent stem cells (hiPSC-CCND2OE CMs) can proliferate when transplanted into mouse hearts after myocardial infarction (MI). However, it is unknown whether remuscularization can replace the thin LV scar and if the large muscle graft can electrophysiologically synchronize to the recipient myocardium. Our objectives are to evaluate the structural and functional potential of hiPSC-CCND2OE CMs in replacing the LV thin scar. METHODS: NOD/SCID mice were treated with hiPSC-CCND2OE CMs (i.e., the CCND2OE group), hiPSC-CCND2WT CMs (the CCND2WT group), or an equal volume of PBS immediately after experimentally-induced myocardial infarction. The treatments were administered to one site in the infarcted zone (IZ), two sites in the border zone (BZ), and a fourth group of animals underwent Sham surgery. RESULTS: Six months later, engrafted cells occupied >50% of the scarred region in CCND2OE animals, and exceeded the number of engrafted cells in CCND2WT animals by ~8-fold. Engrafted cells were also more common in the IZ than in the BZ for both cell-treatment groups. Measurements of cardiac function, infarct size, wall thickness, and cardiomyocyte hypertrophy were significantly improved in CCND2OE animals compared to animals from the CCND2WT or PBS-treatment groups. Measurements in the CCND2WT and PBS groups were similar, and markers for cell cycle activation and proliferation were significantly higher in hiPSC-CCND2OE CMs than in hiPSC-CCND2WT CMs. Optical mapping of action potential propagation indicated that the engrafted hiPSC-CCND2OE CMs were electrically coupled to each other and to the cells of the native myocardium. No evidence of tumor formation was observed in any animals. CONCLUSIONS: Six months after the transplantation, CCND2-overexpressing hiPSC-CMs proliferated and replaced >50% of the myocardial scar tissue. The large graft hiPSC-CCND2OE CMs also electrically integrated with the host myocardium, which was accompanied by a significant improvement in LV function.

Convergences of Life Sciences and Engineering in Understanding and Treating Heart Failure.

On March 1 and 2, 2018, the National Institutes of Health 2018 Progenitor Cell Translational Consortium, Cardiovascular Bioengineering Symposium, was held at the University of Alabama at Birmingham. Convergence of life sciences and engineering to advance the understanding and treatment of heart failure was the theme of the meeting. Over 150 attendees were present, and >40 scientists presented their latest work on engineering human functional myocardium for disease modeling, drug development, and heart failure research. The scientists, engineers, and physicians in the field of cardiovascular sciences met and discussed the most recent advances in their work and proposed future strategies for overcoming the major roadblocks of cardiovascular bioengineering and therapy. Particular emphasis was given for manipulation and using of stem/progenitor cells, biomaterials, and methods to provide molecular, chemical, and mechanical cues to cells to influence their identity and fate in vitro and in vivo. Collectively, these works are profoundly impacting and progressing toward deciphering the mechanisms and developing novel treatments for left ventricular dysfunction of failing hearts. Here, we present some important perspectives that emerged from this meeting.

High-resolution optical mapping of gastric slow wave propagation.

BACKGROUND: Improved understanding of the details of gastric slow wave propagation could potentially inform new diagnosis and treatment options for stomach motility disorders. Optical mapping has been used extensively in cardiac electrophysiology. Although optical mapping has a number of advantages relative to electrical mapping, optical signals are highly sensitive to motion artifact. We recently introduced a novel cardiac optical mapping method that corrects motion artifact and enables optical mapping to be performed in beating hearts. Here, we reengineer the method as an experimental tool to map gastric slow waves. METHODS: The method was developed and tested in 12 domestic farm pigs. Stomachs were exposed by laparotomy and stained with the voltage-sensitive fluorescence dye di-4-ANEPPS through a catheter placed in the gastroepiploic artery. Fiducial markers for motion tracking were attached to the serosa. The dye was excited by 450 or 505 nm light on alternate frames of an imaging camera running at 300 Hz. Emitted fluorescence was imaged between 607 and 695 nm. The optical slow wave signal was reconstructed using a combination of motion tracking and excitation ratiometry to suppress motion artifact. Optical slow wave signals were compared with simultaneously recorded bipolar electrograms and suction electrode signals, which approximate membrane potential. KEY RESULTS: The morphology of optical slow waves was consistent with previously published microelectrode recordings and simultaneously recorded suction electrode signals. The timing of the optical slow wave signals was consistent with the bipolar electrograms. CONCLUSIONS AND INFERENCES: Optical mapping of slow wave propagation in the stomach is feasible.

Effects of gadolinium on cardiac mechanosensitivity in whole isolated swine hearts.

Mechanical stimulation can elicit electrical activation of the heart. This mechanosensitivity can start life-threatening arrhythmias (commotio cordis) or terminate them (precordial thump). Mechanosensitivity may also be involved in arrhythmogenesis in other settings. Stretch-activated ion channels (SACs) are thought to be important in mechanosensitivity and a number of agents that block them have been identified. Such agents could potentially be used as tools in experimental investigation of mechanosensitivity. However, studies using them in intact-heart preparations have yielded inconsistent results. In the present study, we used isolated, perfused hearts from 25-35 kg pigs and a computer-controlled device that repeatably delivered focal mechanical stimuli. The concentration-dependent ability of the SAC blocker gadolinium to suppress mechanical activation was assessed by the success rate of mechanical stimulation and by the delay between successful mechanical stimulation and electrical activation. In six hearts, perfusate was recirculated. In an additional six hearts, perfusate was not recirculated to prevent gadolinium from forming complexes with metabolic waste and possibly precipitating. Gadolinium did not suppress mechanically-induced activation. Although gadolinium has been shown to be an effective SAC blocker in isolated cells, using it to probe the role of mechanical stimulation in whole heart preparations should be done with great caution.

Effects of combination of sotalol and verapamil on initiation, maintenance, and termination of ventricular fibrillation in swine hearts.

INTRODUCTION: Sotalol and verapamil alone reduce reentry incidence during ventricular fibrillation (VF). We tested whether the combination of these two drugs had a synergistic effect on initiation, maintenance, and termination of VF. METHODS: Six open-chest pigs received intravenous sotalol (1.5 mg/kg) followed by verapamil (0.136 mg/kg). VF threshold (VFT) was determined by a burst pacing protocol. Two 20 seconds episodes of VF were recorded from a 21 × 24 unipolar electrode plaque on the lateral posterior left ventricular epicardium before and after each drug. VF activation patterns were quantified. The duration of long duration VF (LDVF) maintenance was compared to our previously published data. RESULTS: Sotalol alone and combined with verapamil significantly increased the VFT from 12.3 ± 4.1 to 20.3 ± 7.1 and 26.7 ± 8.6 mA compared with baseline (P < .05). Sotalol decreased the number of wavefronts by 20%, VF activation rate by 17% and conduction velocity 11%, while the addition of verapamil neutralized these effects. Addition of verapamil to sotalol further decreased the fractionation incidence from 14% to 29% and multiplicity from 24% to 31% compared with baseline. The combination of the two drugs increased the VF cycle length, decreased synchronicity, increased regularity index and shortened the duration of LDVF maintenance compared with our previous data of verapamil alone or no drug. Synchronicity index was lower and regularity index was higher in animals in which VF spontaneously terminated earlier than 10 minutes than in animals in which VF terminated longer than 10 minutes. CONCLUSION: The combination of sotalol and verapamil increased VFT but accelerated LDVF termination.

Optical Mapping of Membrane Potential and Epicardial Deformation in Beating Hearts.

Cardiac optical mapping uses potentiometric fluorescent dyes to image membrane potential (Vm). An important limitation of conventional optical mapping is that contraction is usually arrested pharmacologically to prevent motion artifacts from obscuring Vm signals. However, these agents may alter electrophysiology, and by abolishing contraction, also prevent optical mapping from being used to study coupling between electrical and mechanical function. Here, we present a method to simultaneously map Vm and epicardial contraction in the beating heart. Isolated perfused swine hearts were stained with di-4-ANEPPS and fiducial markers were glued to the epicardium for motion tracking. The heart was imaged at 750 Hz with a video camera. Fluorescence was excited with cyan or blue LEDs on alternating camera frames, thus providing a 375-Hz effective sampling rate. Marker tracking enabled the pixel(s) imaging any epicardial site within the marked region to be identified in each camera frame. Cyan- and blue-elicited fluorescence have different sensitivities to Vm, but other signal features, primarily motion artifacts, are common. Thus, taking the ratio of fluorescence emitted by a motion-tracked epicardial site in adjacent frames removes artifacts, leaving Vm (excitation ratiometry). Reconstructed Vm signals were validated by comparison to monophasic action potentials and to conventional optical mapping signals. Binocular imaging with additional video cameras enabled marker motion to be tracked in three dimensions. From these data, epicardial deformation during the cardiac cycle was quantified by computing finite strain fields. We show that the method can simultaneously map Vm and strain in a left-sided working heart preparation and can image changes in both electrical and mechanical function 5 min after the induction of regional ischemia. By allowing high-resolution optical mapping in the absence of electromechanical uncoupling agents, the method relieves a long-standing limitation of optical mapping and has potential to enhance new studies in coupled cardiac electromechanics.

Verapamil reduces incidence of reentry during ventricular fibrillation in pigs.

The characteristics of reentrant circuits during short duration ventricular fibrillation (SDVF; 20 s in duration) and the role of Ca(++) and rapid-activating delayed rectifier potassium currents during long duration ventricular fibrillation (LDVF; up to 10 min in duration) were investigated using verapamil and sotalol. Activation mapping of the LV epicardium with a 21 × 24 electrode plaque was performed in 12 open-chest pigs. Pigs were given either verapamil (0.136 mg/kg) or sotalol (1.5 mg/kg) and verapamil. Reentry patterns were quantified for SDVF, and, for LDVF, activation patterns were compared with our previously reported control LDVF data. Verapamil significantly increased conduction velocity around the reentrant core by 10% and reduced the reentrant cycle length by 15%, with a net reduction in reentry incidence of 70%. Sotolol had an opposite effect of decreasing the conduction velocity around the core by 6% but increasing the reentrant cycle length by 13%, with a net reduction of reentry incidence of 50%. After 200 s of VF, verapamil significantly slowed wavefront conduction velocity and activation rate compared with control data. Verapamil decreased the incidence of reentry in SDVF by accelerating conduction velocity to increase the likelihood of conduction block, possibly through increased sympathetic tone. The drug slowed activation rate and conduction velocity after 200 s of VF, suggesting that L-type Ca(++) channels remain active and may be important in the maintenance of LDVF. Sotalol in addition to verapamil caused no additional antiarrhythmic effect.

The importance of Purkinje activation in long duration ventricular fibrillation.

BACKGROUND: The mechanisms that maintain long duration ventricular fibrillation (LDVF) are unclear. The difference in distribution of the Purkinje system in dogs and pigs was explored to determine if Purkinje activation propagates to stimulate working myocardium (WM) during LDVF and WM pacing. METHODS AND RESULTS: In-vivo extracellular recordings were made from 1044 intramural plunge and epicardial plaque electrodes in 6 pig and 6 dog hearts. Sinus activation propagated sequentially from the endocardium to the epicardium in dogs but not pigs. During epicardial pacing, activation propagated along the endocardium and traversed the LV wall almost parallel to the epicardium in dogs, but in pigs propagated away from the pacing site approximately perpendicular to the epicardium. After 1 minute of VF, activation rate near the endocardium was significantly faster than near the epicardium in dogs (P<0.01) but not pigs (P>0.05). From 2 to 10 minutes of LDVF, recordings exhibiting Purkinje activations were near the endocardium in dogs (P<0.01) but were scattered transmurally in pigs, and the WM activation rate in recordings in which Purkinje activations were present was significantly faster than the WM activation rate in recordings in which Purkinje activations were absent (P<0.01). In 10 isolated perfused dog hearts, the LV endocardium was exposed and 2 microelectrodes were inserted into Purkinje and adjacent myocardial cells. After 5 minutes of LDVF, mean Purkinje activation rate was significantly faster than mean WM activation rate (P<0.01). CONCLUSION: These extracellular and intracellular findings about activation support the hypothesis that Purkinje activation propagates to stimulate WM during sinus rhythm, pacing, and LDVF.

Panoramic optical mapping shows wavebreak at a consistent anatomical site at the onset of ventricular fibrillation.

AIMS: The first seconds of ventricular fibrillation (VF) are well organized and can consist of just one to two rotating waves (rotors). New rotors are spawned when local propagation block causes wave fragmentation. We hypothesized that this process, which leads to fully developed VF, begins at a consistent anatomic site. METHODS AND RESULTS: We initiated VF with a stimulus timed to the local T-wave in 10 isolated pig hearts. Hearts were stained with a voltage-sensitive dye and four video cameras recorded electrical propagation panoramically across the epicardium. In each VF episode, we identified the position of the first wavebreak event that produced new rotor(s) that persisted for at least one cycle. The first such wavebreak occurred along the anterior right ventricular insertion (ARVI) in 26 of 32 VF episodes. In these episodes, wavebreak sites were 6 ± 4 mm from the midline of the ARVI. In the remaining 6 episodes, wavebreak sites were 24 ± 5 mm from the midline on either the LV or RV. During rapid pacing, conduction speed was locally depressed at the ARVI when waves crossed parallel to the midline. Action potential duration (APD) was slightly longer (2.2 ± 2.1 ms) at the ARVI compared with other sites (P< 0.01). Temporal APD alternans were small and not unique to the break site, suggesting that dynamic APD properties were not the cause of wavebreak. CONCLUSION: The ARVI is the dominant site for wavebreak at the onset of VF in normal myocardium. This may be due to the anatomic complexity of the region.