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BACKGROUND: Device-related thrombus (DRT) following left atrial appendage occlusion (LAAO) can lead to adverse clinical outcomes. DRT rates and outcomes from randomized trials are limited.OBJECTIVES: This analysis investigated the incidence, predictors, and clinical outcomes of DRT following LAAO in the Amulet IDE (AMPLATZER Amulet LAA Occluder Trial) trial.METHODS: Successful implants occurred in 903 patients with an Amulet occluder (dual occlusive mechanism device) and 885 patients with a Watchman device (single occlusive mechanism device). These patients were then followed through 18 months and DRT was assessed by transesophageal echocardiography.RESULTS: The overall incidence of DRT was 3.9% (n = 70) with 3.4% (n = 30) in dual occlusive mechanism device patients and 4.8% (n = 40) in single occlusive mechanism device patients. Most DRTs (n = 19 of 31) were identified early (≤45 days) on the dual occlusive mechanism device, whereas most of the DRTs (n = 31 of 42) were identified late (>45 days) on the single occlusive mechanism device. Strong predictors of DRT included atrial fibrillation at time of procedure (HR: 2.44; 95% CI: 1.42-4.22; P < 0.01), female sex (HR: 1.65; 95% CI: 1.01-2.71; P = 0.04), and older age (HR: 1.04; 95% CI: 1.01-1.08; P = 0.02). There were no stroke events following DRT in the dual occlusive mechanism device group and 3 stroke events following DRT in the single occlusive mechanism device group. Patients with DRT were at a greater risk for cardiovascular mortality compared with non-DRT patients (8.7% vs 3.9%; HR: 2.33; 95% CI: 1.01-5.39; P = 0.04).CONCLUSIONS: Incidence of DRT following LAAO was low. Early DRTs are seen with the dual occlusive mechanism device and late DRTs are seen with the single occlusive mechanism device. Increased cardiovascular mortality risk in patients with DRT should be further investigated. (AMPLATZER Amulet LAA Occluder Trial; NCT02879448).

Background: Leadless pacemakers (LPs) can mitigate conventional pacemaker complications related to the transvenous leads and subcutaneous pocket surrounding the pulse generator. Although single-chamber leadless pacing has been established, multichamber pacing requires wireless bidirectional communication across multiple LPs to maintain synchrony. This preclinical study demonstrates the chronic performance of implant-to-implant (i2i) communication that achieves synchronous, dual-chamber pacing with 2 LPs. Methods: The i2i communication modality employs subthreshold electrical signals conducted between implanted LPs through the blood and myocardial tissue on a beat-by-beat basis. Right atrial and right ventricular LPs were implanted in 9 ovine subjects. The i2i transmission performance was evaluated 13 weeks after implant. Results: Right atrial and right ventricular LPs were implanted successfully and without complication in 9 ovine subjects. A total of 8715±457 right atrial-to-right ventricular and right ventricular-to-right atrial transmissions were sent per hour, with a success rate of 99.2±0.9%. Of periods with i2i communication failure when DDD pacing was not possible, 97.3±1.8% were resolved within 6 s. Conclusions: For the first time, synchronized, dual-chamber pacing has been demonstrated in a chronic preclinical feasibility study by 2 leadless pacemakers using beat-to-beat, wireless communication, achieving a success rate of 99.2%.

The future of the American Board of Internal Medicine Maintenance of Certification (MOC) program is at a crossroads. The current MOC program lacks a clear visible mission, adds to modern health care’s onerous bureaucracy, and thus pulls physicians from the most important humanistic aspects of their profession. The aim of the MOC program should be to promote the best patient care by ensuring certified physicians maintain core skills through continuous education and evaluation. The program should focus on education and be designed with the rigorous obligations of practicing physicians in mind. Moving forward, the American Board of Internal Medicine should cocreate MOC with the physician community and apply innovative adult education techniques. Over time, data-driven methods and member feedback should be used to provide continuous program improvement. This review describes the origins of the current state of MOC, explores its evidence base, provides examples of model programs for the maintenance of complex professional skills, and outlines guiding principles for the future of MOC.

Introduction: The ambulatory continuous ECG monitors can provide a comprehensive cardiac assessment in outpatient settings. Extended cardiac monitoring is often needed by physicians to assess the efficacy of treatment, evaluate symptoms, and to stratify patients at risk. In this study, we compare the impact of outpatient cardiac monitoring between 1-14 days and 15-30 days. Method: A retrospective analysis was done on ECG data collected from 2,679 patients using Zywie ECG monitors (Zywie, Inc., Georgia). These patients were monitored for 14-30 days. We evaluated three arrhythmias in this study: i) Atrial fibrillation (AF) of duration >15s ii) Ventricular tachycardia (VT) consisting of more than 3 heartbeats iii) Pause of duration >3s. We analyzed the first occurrence of arrhythmia episodes in every patient during their monitoring period. We identified at least one episode of AF in 486 patients, VT in 106 patients, and pause in 78 patients. Results: The incidence of the first episode of AF, VT, and pause was observed after 14 days of monitoring in 15.8%, 28.3%, and 23.1% patients, respectively (Figure 1). Furthermore, among the VT episodes, 85.3% episodes had a duration between 1-10s and 14.7% with a duration >10s. Similarly, 76% of identified pauses had a duration between 3-4s, 12% between 4-5s, and 12% with a duration >5s. Conclusion: The results demonstrate the incremental diagnostic yield of critical arrhythmic events when the monitoring is extended beyond 14 days. This study suggests that long-term outpatient monitoring can provide better diagnosis, especially for patients at a higher risk of developing critical arrhythmias.

Acute limb ischemia is defined as a sudden and rapidly progressing decrease in limb perfusion, usually producing new or worsening symptoms or signs, and often threatening limb viability. Acute limb ischemia is most commonly caused by an acute thrombotic occlusion of a preexisting stenotic arterial segment (60% of cases) or by embolus (30%). Prompt diagnosis and urgent medical and percutaneous/surgical interventions are warranted. If not identified and addressed immediately, decreased perfusion and thrombosis can result in a high degree of limb loss–related morbidity. This report focuses on a rare case of an acute lower extremity ischemia following a lower extremity femoral vein access site–related intervention.

Myocardial elastography (ME) is an ultrasound-based technique that can image 2-D myocardial strains. The objectives of this study were to show that 2-D myocardial strains can be imaged with diverging wave imaging and are different in average between normal and coronary artery disease (CAD) patients. In this study, 66 patients with symptoms of CAD were imaged with ME prior to a nuclear stress test or an invasive coronary angiography. Radial cumulative strains were estimated in all patients. The end-systolic radial strain in the total cross-section of the myocardium in normal subjects (17.9 ± 8.7%) was significantly higher than in patients with reversible perfusion defect (6.2 ± 9.3%, p

Introduction Wide Complex Tachyarrhythmias (WCT) are commonly observed arrhythmias in patients with heart failure. Automatic identification of WCT using mobile cardiac telemetry (MCT) monitors is critical for high-quality patient care due to their potentially lethal nature. There is very limited literature on validating an algorithm's ability in detecting WCTs in near real-time in the outpatient setting. This study demonstrates the efficacy of the ZywieAI® algorithm in detecting episodes of sustained and/or non-sustained WCTs like ventricular tachycardia, ventricular flutter, and ventricular fibrillation. Method We propose a three-pronged approach to identify WCT episodes in ECG recordings where the heart rate is ≥100 bpm for ≥3 consecutive beats. Firstly, the ECG frequency spectrum is analyzed to identify the time instants of QRS onset-offset and fiducial points in the signal. Secondly, four novel discrete features are extracted from the fiducial points and ectopic beats to get a beat-level assessment. Lastly, a rule-based decision classifier interprets input features to identify a true WCT episode. Results The ZywieAI® algorithm was evaluated by analyzing 299 WCT episodes in three public PhysioNet databases: MIT-BIH Arrhythmia (MITBA), MIT-BIH Malignant Ventricular Arrhythmia (MITMVA), and CU Ventricular Tachyarrhythmia (CUVT). These annotated databases contain ECG recordings with a wide variety of PQRST morphologies and arrhythmias contaminated with noise, which make the WCT detection a challenging problem. The proposed algorithm identified patients who experienced WCT with 100% accuracy in all three databases. At the individual episode-level, the algorithm had an average gross sensitivity (Se) and specificity (Sp) of 94.8% and 97.6% for MITBA and 94.1% and 89.1% for MITMVA databases, respectively. The episode-level Se for CUVT database was 100%. Furthermore, the Se of the algorithm in identifying any WCT episode with ≥ 10 beats was 100%, 99.8%, and 100% for the respective databases. Conclusion The results suggest that the ZywieAI® algorithm can robustly identify WCT episodes as short as 0.46 seconds. The proposed algorithm can be used to evaluate real-time ECG signals in remote cardiac monitoring studies, assisting clinicians in the timely diagnosis of patients with most lethal wide complex arrhythmias.

Atrial arrhythmias affect millions of people worldwide. Characterization and study of arrhythmias in the atria in the clinic is currently performed point by point using mapping catheters capable of generating maps of the electrical activation rate or cycle length. In this paper, we describe a new ultrasound-based mapping technique called electromechanical cycle length mapping (ECLM) capable of estimating the electromechanical activation rate, or cycle length, i.e., the rate of the mechanical activation of the myocardium which follows the electrical activation. ECLM relies on frequency analysis of the incremental strain within the atria and can be performed in a single acquisition. ECLM was validated in a canine model paced from the left atrial appendage, against pacing rates within the reported range of cycle lengths previously measured during atrial arrhythmias such as atrial fibrillation. Correlation between the global estimated electromechanical cycle lengths and pacing rates was shown to be excellent (slope = 0.983, intercept = 3.91, r(2) = 0.9999). The effect of the number of cardiac cycles on the performance of ECLM was also investigated and the reproducibility of ECLM was demonstrated (error between consecutive acquisitions for all pacing rates: 6.3 ± 4.3%). These findings indicate the potential of ECLM for noninvasively characterizing atrial arrhythmias and provide feedback on the treatment planning of catheter ablation procedures in the clinic.

Intracardiac echocardiography (ICE) is a useful imaging modality which is used during RF ablation procedures to identify anatomical structures. Utilizing ICE in conjunction with myocardial elastography (ME) can provide additional information on the mechanical properties of cardiac tissue and provide information on mechanical changes caused by ablation. The objective of this study was to demonstrate that ICE can be used at high frame rate using a diverging beam transmit sequence to image myocardial strain and differentiate myocardial tissue properties before, during, and after ablation for a clinical ablation procedure. In this feasibility study, three normal canines and eight patients with atrial fibrillation (AF) were studied in vivo. A 5.8-MHz ICE transducer was used to image the heart with a diverging beam transmit method achieving 1200 frames per second (fps). Cumulative axial displacement estimation was performed using 1-D cross-correlation with a window size of 2.7 mm and 95% overlap. Axial cumulative strains were estimated in the left atrium (LA) and right atrium (RA) using a least-squares estimator with a kernel of 2 mm on the axial displacements. In the canine case, radial thickening was detected in the lateral wall and in the interatrial septum during LA emptying. For AF patients, the mean absolute strain in the ablated region was lower (6.7 ± 3.1%) than before the ablation (17.4 ± 9.3%) in LA at the end of the LA emptying phase. In the cavotricuspid isthmus (CTI) region, mean absolute strain magnitude at the end of the RA emptying phase was found to be higher during ablation (43.0 ± 18.1%) compared with after ablation (33.7 ± 15.8%). Myocardial strains in the LA of an AF patient were approximately 2.6 times lower in the ablated region than before ablation. This initial feasibility indicates that ME can be used as a new imaging modality in conjunction with ICE in RF ablation guidance and lesion monitoring.

Case report A 56-year-old man was transferred to our institution with incessant ventricular tachycardia (VT). He had been diagnosed with nonischemic cardiomyopathy and New York Heart Association class I congestive heart failure 15 years previously, and he had a single-chamber implantable cardioverter-defibrillator (ICD) placed for primary prevention. He had been maintained on sotalol 80 mg twice a day for several episodes of VT in the previous 2 years. One month before admission, he suffered an ICD shock for polymorphic VT/ventricular fibrillation, and amiodarone was added. At that time, an echocardiogram showed a left ventricular ejection fraction of 0.20 with global hypokinesis and an end-diastolic diameter of 7.2 cm and an interventricular septum thickness of 1.0 cm. One week before the transfer, he presented to the hospital after suffering 3 ICD shocks for VT at 188–194 beats/min that was unresponsive to antitachycardia pacing, in the setting of an argument with his brother. Intravenous amiodarone and lidocaine were administered in addition to sotalol, but he continued to experience VT episodes, resulting in 7 more shocks for VT. He was transferred to our hospital for further management. On arrival to our intensive care unit, the patient’s rhythm was sinus with a left bundle branch block pattern, but multiple sustained episodes of monomorphic VT occurred with a rate of 140–150 beats/min. The VTwas not tolerated hemodynamically, frequently accelerated with antitachycardia pacing, and resulted in more ICD shocks. A representative 12-lead electrocardiogram

Electromechanical wave imaging (EWI) has been show capable of directly and entirely non-invasively mapping the trans mural electromechanical activation in all four cardiac chambers in vivo. In this study, we assessed EWI repeatability and reproducibility, as well as its capability of localizing electronic and, for the first time, biological pacing locations in closed-chest, conscious canines. Electromechanical activation was obtained in six conscious animals during normal sinus rhythm (NSR) and idioventricular rhythms occurring in dogs with complete heart block instrumented with electronic and biologic pacemakers (EPM and BPM respectively). After atrioventricular node ablation, dogs were implanted with an EPM in the right ventricular (RV) endocardial apex (n = 4) and two additionally received a BPM at the left ventricular (LV) epicardial base (n = 2). EWI was performed trans thoracically during NSR, BPM and EPM pacing, in conscious dogs, using an unfocused transmit sequence at 2000 frames/s. During NSR, the EW originated at the right atrium (RA), propagated to the left atrium (LA) and emerged from multiple sources in both ventricles. During EPM, the EW originated at the RV apex and propagated throughout both ventricles. During BPM, the EW originated from the LV basal lateral wall and subsequently propagated throughout the ventricles. EWI differentiated BPM from EPM and NSR and identified the distinct pacing origins. Isochrone comparison indicated that EWI was repeatable and reliable. These findings thus indicate the potential for EWI to serve as a simple, non-invasive and direct imaging technology for mapping and characterizing arrhythmias as well as the treatments thereof.

Background When atrial fibrillation (AF) is incessant, imaging during a prolonged ventricular RR interval may improve image quality. It was hypothesized that long RR intervals could be predicted from preceding RR values.

Background: Although local electrograms during atrial fibrillation (AF) are often spectrally analyzed over 8-second (8s) intervals, changes may be common over intervals as short as 2s. We sought to determine whether averaged 2s measurements of electrogram spectral parameters were similar to 8s measurements, and whether the 2s intervals could provide an estimate of the temporal stability of the signal frequency content in paroxysmal versus persistent AF. Methods: Complex fractionated atrial electrograms (CFAEs) were acquired outside the pulmonary vein ostia and from free wall sites in nine paroxysmal and 10 longstanding persistent AF patients. Using a 2s sliding calculation window, a frequency spectrum was computed every 100 ms over an interval of 8.4 seconds (82 spectra in total). The dominant frequency (DF), the dominant amplitude (DA), and the mean spectral profile (MP) were measured. The 2s measurements were compared to single 8.4-second interval measurements. Coefficients of variation (COV) were computed from the 82 spectra for each CFAE recording to determine temporal variability of parameters. Results: Over the sliding 2s computation intervals, as for fixed 8.4-second computation intervals, mean DA and DF were significantly higher in longstanding persistent AF while MP was significantly higher in paroxysmal AF (P ≤ 0.001). The COV was significantly higher for the DF parameter in paroxysmal AF (P < 0.001) and significantly higher for the MP parameter in persistent AF (P < 0.02). Conclusions: For both paroxysmal and persistent AF data, the 2s sliding window averages provide similarresultstosingle8.4-secondintervals,andinformationregardingtemporalstabilitywasadditionally obtained in the process. (PACE 2014; 37:79‐89)

Background Cardiac resynchronization therapy (CRT) and atrial ablation procedures currently lack a noninvasive imaging modality for reliable treatment planning and monitoring. Electromechanical wave imaging (EWI) is an ultrasound-based method that has previously been shown to be capable of noninvasively and transmurally mapping the activation sequence of the heart in animal studies by estimating and imaging the electromechanical wave, that is, the transient strains occurring in response to the electrical activation, at both high temporal and spatial resolutions. Objective To demonstrate the feasibility of transthoracic EWI for mapping the activation sequence during different cardiac rhythms in humans. Methods EWI was perfor`med in patients undergoing CRT and a left bundle branch block (LBBB) during sinus rhythm, left ventricular pacing, and right ventricular pacing, as well as in patients with atrial flutter (AFL) before intervention, EWI findings from patients with AFL were subsequently correlated with results from invasive intracardiac electrical mapping studies during intervention. In addition, the feasibility of single-heartbeat EWI at 2000 frames/s is demonstrated in humans for the first time in a patient with both AFL and right bundle branch block (RBBB). Results The electromechanical activation maps demonstrated the capability of EWI to localize the pacing sites and characterize the bundle branch block activation sequence transmurally in patients with CRT. In patients with AFL, the EWI propagation patterns obtained with EWI were in excellent agreement with those obtained from invasive intracardiac mapping studies. Conclusions Our findings demonstrate the potential capability of EWI to aid in the assessment and follow-up of patients undergoing CRT pacing therapy and atrial ablation, with preliminary validation in vivo.

Spectral Profiles of CFAE.Spectral analysis of complex fractionated atrial electrograms (CFAE) may be useful for gaining insight into mechanisms underlying paroxysmal and longstanding atrial fibrillation (AF). The commonly used dominant frequency (DF) measurement has limitations.CFAE recordings were acquired from outside the 4 pulmonary vein ostia and at 2 left atrial free wall sites in 10 paroxysmal and 10 persistent AF patients. Two consecutive 8s-series were analyzed from recordings16s in duration. Power spectra were computed for each 8s-series in the range 3-12 Hz and normalized. The mean and standard deviation of normalized power spectra (MPS and SPS, respectively) were compared for paroxysmal versus persistent CFAE. Also, the DF and its peak amplitude (ADF) were compared for pulmonary vein sites only. Power spectra were computed using ensemble average and Fourier methods.No significant changes occurred in any parameter from the first to second recording sequence. For both sequences, MPS and SPS were significantly greater, and DF and ADF were significantly less, in paroxysmals versus persistents. The MPS and ADF measurements from ensemble spectra produced the most significant differences in paroxysmals versus persistents (P0.0001). DF differences were less significant, which can be attributed to the relatively high variability of DF in paroxysmals. The MPS was correlated to the duration of uninterrupted persistent AF prior to electrophysiologic study (P = 0.01), and to left atrial volume for all AF (P0.05).The MPS and ADF measurements introduced in this study are probably superior to DF for discerning power spectral differences in paroxysmal versus longstanding CFAE. (J Cardiovasc Electrophysiol, Vol. 23, pp. 971-979, September 2012).

Electromechanical wave imaging (EWI) is a novel ultrasound-based imaging modality for mapping of the electromechanical wave (EW), i.e. the transient deformations occurring in immediate response to the electrical activation. The correlation between the EW and the electrical activation has been established in prior studies. However, the methods used previously to map the EW required the reconstruction of images over multiple cardiac cycles, precluding the application of EWI for non-periodic arrhythmias such as fibrillation. In this study, new imaging sequences are developed and applied based on flash- and wide-beam emissions to image the entire heart at very high frame rates (2000 fps) during free breathing in a single heartbeat. The methods are first validated by imaging the heart of an open-chest canine while simultaneously mapping the electrical activation using a 64-electrode basket catheter. Feasibility is then assessed by imaging the atria and ventricles of closed-chest, conscious canines during sinus rhythm and during right-ventricular pacing following atrio-ventricular dissociation, i.e., during a non-periodic rhythm. The EW was validated against electrode measurements in the open-chest case, and followed the expected electrical propagation pattern in the closed-chest setting. These results indicate that EWI can be used for the characterization of non-periodic arrhythmias in conditions similar to the clinical setting, in a single heartbeat, and during free breathing.

Background— Complex fractionated atrial electrograms (CFAE) are morphologically more uniform in persistent longstanding as compared with paroxysmal atrial fibrillation (AF). It was hypothesized that this may result from a greater degree of repetitiveness in CFAE patterns at disparate left atrial (LA) sites in longstanding AF. Methods and Results— CFAEs were obtained from recording sites outside the 4 pulmonary vein (PV) ostia and at a posterior and an anterior LA site during paroxysmal and longstanding persistent AF (10 patients each, 120 sequences total). To quantify repetitiveness in CFAE, the dominant frequency was measured from ensemble spectra using 8.4-second sequences, and repetitiveness was calculated by 2 novel techniques: linear prediction and Fourier reconstruction methods. Lower prediction and reconstruction errors were considered indicative of increasing repetitiveness and decreasing randomness. In patients with paroxysmal AF, CFAE pattern repetitiveness was significantly lower (randomness higher) at antral sites outside PV ostia as compared with LA free wall sites ( P P Conclusions— In paroxysmal AF, CFAE repetitiveness is low and randomness high outside the PVs, particularly the left superior PV. As evolution to persistent longstanding AF occurs, CFAE repetitiveness becomes more uniformly distributed at disparate sites, possibly signifying an increasing number of drivers from remote PVs.

Complex fractionated atrial electrograms (CFAEs) may represent a phenomenon associated with sources of atrial fibrillation (AF) and are being used increasingly as targets of catheter ablation. However, current methods have limited efficacy for characterizing CFAEs important to substrate arrhythmogenicity and do not measure electrogram morphology.The purpose of this study was to develop a methodology for quantifying the degree of morphologic heterogeneity in CFAE deflections, and to determine whether there are differences in this measurement between paroxysmal and persistent AF patients.Two successive bipolar CFAEs of length 8.4 seconds each were acquired during AF from two sites each at the ostia of the four pulmonary veins (PVs) and from the anterior and posterior left atrial free wall in patients with paroxysmal AF (N = 10) and long-standing persistent AF (N = 10). Extrinsic and intrinsic features of electrogram shape were used to characterize fractionation in CFAE sequences. The extrinsic parameters were the amplitude, upslope, downslope, and width of each deflection. The intrinsic parameter was the voltage profile as characterized by the sum of absolute values. These measurements were compared to the mean interval between CFAE deflections, a standard fractionation indicator.The variability of intrinsic/extrinsic morphologic parameters was higher in paroxysmal than persistent AF at the left superior PV (Por =.003), the posterior left atrial free wall, anterior left atrial free wall, left inferior PV, and right superior PV (P.05 for most parameters), and the right inferior PV (not significant). Mean CFAE deflection intervals were longer at all locations in paroxysmal AF but were significant only at the left superior PV and posterior left atrial free wall (P.05). Quantitative morphologic parameters were not well correlated with dominant frequency (r(2)0.32); thus, our new measures are robust to changes in activation rate.A novel method for quantifying CFAEs, independent of activation rate, has been developed. The method demonstrates greater significance in the difference between CFAE morphology in paroxysmal and long-standing AF compared with mean interval between CFAE deflections. The differences identified suggest that CFAE morphology may evolve as AF persists.

Background Accurate determination of regional areas of arrhythmic triggers is of key interest to diagnose arrhythmias and optimize their treatment. Electromechanical wave imaging (EWI) is an ultrasound technique that can image the transient deformation in the myocardium after electrical activation and therefore has the potential to detect and characterize location of triggers of arrhythmias. Objectives The objectives of this study were to investigate the relationship between the electromechanical and the electrical activation of the left ventricular (LV) endocardial surface during epicardial and endocardial pacing and during sinus rhythm as well as to map the distribution of electromechanical delays. Methods In this study, 6 canines were investigated. Two external electrodes were sutured onto the epicardial surface of the LV. A 64-electrode basket catheter was inserted through the apex of the LV. Ultrasound channel data were acquired at 2000 frames/s during epicardial and endocardial pacing and during sinus rhythm. Electromechanical and electrical activation maps were synchronously obtained from the ultrasound data and the basket catheter, respectively. Results The mean correlation coefficient between electromechanical and electrical activation was 0.81 for epicardial anterior pacing, 0.79 for epicardial lateral pacing, 0.69 for endocardial pacing, and 0.56 for sinus rhythm. Conclusion The electromechanical activation sequence determined by EWI follows the electrical activation sequence and more specifically in the case of pacing. This finding is of key interest in the role that EWI can play in the detection of the anatomical source of arrhythmias and the planning of pacing therapies such as cardiovascular resynchronization therapy.

The basic effector domain of myristoylated alanine-rich C kinase substrate (MARCKS), a major protein kinase C substrate, binds electrostatically to acidic lipids on the inner leaflet of the plasma membrane; interaction with Ca2+/calmodulin or protein kinase C phosphorylation reverses this binding. Our working hypothesis is that the effector domain of MARCKS reversibly sequesters a significant fraction of the l-α-phosphatidyl-d-myo-inositol 4,5-bisphosphate (PIP2) on the plasma membrane. To test this, we utilize three techniques that measure the ability of a peptide corresponding to its effector domain, MARCKS(151–175), to sequester PIP2 in model membranes containing physiologically relevant fractions (15–30%) of the monovalent acidic lipid phosphatidylserine. First, we measure fluorescence resonance energy transfer from Bodipy-TMR-PIP2 to Texas Red MARCKS(151–175) adsorbed to large unilamellar vesicles. Second, we detect quenching of Bodipy-TMR-PIP2 in large unilamellar vesicles when unlabeled MARCKS(151–175) binds to vesicles. Third, we identify line broadening in the electron paramagnetic resonance spectra of spin-labeled PIP2 as unlabeled MARCKS(151–175) adsorbs to vesicles. Theoretical calculations (applying the Poisson-Boltzmann relation to atomic models of the peptide and bilayer) and experimental results (fluorescence resonance energy transfer and quenching at different salt concentrations) suggest that nonspecific electrostatic interactions produce this sequestration. Finally, we show that the PLC-δ1-catalyzed hydrolysis of PIP2, but not binding of its PH domain to PIP2, decreases markedly as MARCKS(151–175) sequesters most of the PIP2.

A peptide corresponding to the basic (+13), unstructured effector domain of myristoylated alanine-rich C kinase substrate (MARCKS) binds strongly to membranes containing phosphatidylinositol 4,5-bisphosphate (PIP(2)). Although aromatic residues contribute to the binding, three experiments suggest the binding is driven mainly by nonspecific local electrostatic interactions. First, peptides with 13 basic residues, Lys-13 and Arg-13, bind to PIP(2)-containing vesicles with the same high affinity as the effector domain peptide. Second, removing basic residues from the effector domain peptide reduces the binding energy by an amount that correlates with the number of charges removed. Third, peptides corresponding to a basic region in GAP43 and MARCKS effector domain-like regions in other proteins (e.g. MacMARCKS, adducin, Drosophila A kinase anchor protein 200, and N-methyl-d-aspartate receptor) also bind with an energy that correlates with the number of basic residues. Kinetic measurements suggest the effector domain binds to several PIP(2). Theoretical calculations show the effector domain produces a local positive potential, even when bound to a bilayer with 33% monovalent acidic lipids, and should thus sequester PIP(2) laterally. This electrostatic sequestration was observed experimentally using a phospholipase C assay. Our results are consistent with the hypothesis that MARCKS could reversibly sequester much of the PIP(2) in the plasma membrane.

▪ Abstract We review the physical properties of phosphatidylinositol 4,5-bisphosphate (PIP2) that determine both its specific interactions with protein domains of known structure and its nonspecific electrostatic sequestration by unstructured domains. Several investigators have postulated the existence of distinct pools of PIP2 within the cell to account for the myriad functions of this lipid. Recent experimental work indicates certain regions of the plasma membrane—membrane ruffles and nascent phagosomes—do indeed concentrate PIP2. We consider two mechanisms that could account for this phenomenon: local synthesis and electrostatic sequestration. We conclude by considering the hypothesis that proteins such as MARCKS bind a significant fraction of the PIP2 in a cell, helping to sequester it in lateral membrane domains, then release this lipid in response to local signals such as an increased concentration of Ca++/calmodulin or activation of protein kinase C.

A 70-year-old man with nonischemic dilated cardiomyopathy initially diagnosed in 2001 and history of cardiac resynchronization therapy-defibrillator implant was admitted to our institution for advanced heart failure therapies. He required hospitalization in December 2013 for marked volume overload, at which time cardiac imaging showed his left ventricular (LV) ejection fraction to be 10% to 15% and his LV end-diastolic dimension 7.9 cm. The patient underwent implant of a Heartmate II LV assist device (LVAD; Thoratec, Pleasanton, CA) with concurrent mitral valve ring repair, tricuspid valve ring repair, and aortic valve repair. However, on postoperative day 9, he developed repetitive monomorphic ventricular tachycardia (VT) with rate 141 bpm (Figure 1). During VT, a decrease in pulsatility index was noted on LVAD interrogation. A transesophageal echocardiogram demonstrated excellent cannula position, and no evidence of LVAD-cannula–induced suction events was observed. The VT was unresponsive to antitachycardia pacing, and despite treatment with amiodarone, lidocaine, and esmolol infusions, VT remained incessant. Figure 1. ECG of ventricular tachycardia, consistent with apical site of origin. An initial catheter ablation was performed using an endocardial approach. A 3-dimensional electroanatomic map (CARTO; Biosense Webster, Diamond Bar, CA) of the left and right ventricles with a 3.5-mm open-irrigated catheter (Thermocool SF; Biosense Webster) demonstrated a small area of …

The interaction of heptalysine with vesicles formed from mixtures of the acidic lipid phosphatidylserine (PS) and the zwitterionic lipid phosphatidylcholine (PC) was examined experimentally and theoretically. Three types of experiments showed that smeared charge theories (e.g., Gouy-Chapman-Stern) underestimate the membrane association when the peptide concentration is high. First, the zeta potential of PC/PS vesicles in 100mM KCl solution increased more rapidly with heptalysine concentration (14.5mV per decade) than predicted by a smeared charge theory (6.0mV per decade). Second, changing the net surface charge density of vesicles by the same amount in two distinct ways produced dramatically different effects: the molar partition coefficient decreased 1000-fold when the mole percentage of PS was decreased from 17% to 4%, but decreased only 10-fold when the peptide concentration was increased to 1μM. Third, high concentrations of basic peptides reversed the charge on PS and PC/PS vesicles. Calculations based on finite difference solutions to the Poisson-Boltzmann equation applied to atomic models of heptalysine and PC/PS membranes provide a molecular explanation for the observations: a peptide adsorbing to the membrane in the presence of other surface-adsorbed peptides senses a local potential more negative than the average potential. The biological implications of these “discreteness-of-charge” effects are discussed.

Several biologically important peripheral (e.g., myristoylated alanine-rich C kinase substrate) and integral (e.g., the epidermal growth factor receptor) membrane proteins contain clusters of basic residues that interact with acidic lipids in the plasma membrane. Previous measurements demonstrate that the polyvalent acidic lipid phosphatidylinositol 4,5-bisphosphate is bound electrostatically (i.e., sequestered) by membrane-adsorbed basic peptides corresponding to these clusters. We report here three experimental observations that suggest monovalent acidic lipids are not sequestered by membrane-bound basic peptides. 1), Binding of basic peptides to vesicles does not decrease when the temperature is lowered below the fluid-to-gel phase transition. 2), The binding energy of Lys-13 to lipid vesicles increases linearly with the fraction of monovalent acidic lipids. 3), Binding of basic peptides to vesicles produces no self-quenching of fluorescent monovalent acidic lipids. One potential explanation for these results is that membrane-bound basic peptides diffuse too rapidly for the monovalent lipids to be sequestered. Indeed, our fluorescence correlation spectroscopy measurements show basic peptides bound to phosphatidylcholine/phosphatidylserine membranes have a diffusion coefficient approximately twofold higher than that of lipids, and those bound to phosphatidylcholine/phosphatidylinositol 4,5-bisphosphate membranes have a diffusion coefficient comparable to that of lipids.

The multivalent acidic phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) plays a key role in many biological processes. Recent studies show that unstructured clusters of basic residues from a number of peripheral proteins can laterally sequester PI(4,5)P2 in membranes. Specifically, experiments suggest that the basic effector domain of the myristoylated alanine-rich C kinase substrate (MARCKS), or a peptide corresponding to this domain, MARCKS(151–175), sequesters several PI(4,5)P2 and that this sequestration is due to nonspecific electrostatic interactions. Here, we use the finite difference Poisson-Boltzmann method to test this hypothesis by calculating the electrostatic free energy of lateral sequestration of PI(4,5)P2 by membrane-adsorbed basic peptides: Lys-7, Lys-13, and FA-MARCKS(151–175), a peptide based on MARCKS(151–175). In agreement with experiments, we find that the electrostatic free energy becomes more favorable when: 1), Lys-13 and FA-MARCKS(151–175) sequester several PI(4,5)P2; 2), the linear charge density of the basic peptide increases; 3), the mol percent monovalent acidic lipid in the membrane decreases; and 4), the ionic strength of the solution decreases. In addition, the electrostatic sequestration free energy is in excess of the entropic penalty associated with localizing PI(4,5)P2. Our calculations, thus, provide a structural and quantitative description of the observed interaction of PI(4,5)P2 with membrane-adsorbed basic sequences.

A wide variety of topics in nuclear physics can be taught in the undergraduate laboratory using just a few metallic samples for activation in a neutron howitzer. Rather than scorned for its low neutron flux, the neutron howitzer can be appreciated for its safety in the physics teaching laboratory and its usefulness in conjunction with high‐resolution gamma spectroscopy.

Intracardiac echocardiography (ICE) is commonly used during radio-frequency (RF) ablation procedures for procedural guidance. Besides its imaging function, ICE could be used to assess mechanical properties of the myocardium to improve the ablation outcome. The objective of this study was to demonstrate the feasibility of imaging myocardial strains in vivo within the same imaging plane as ICE at high temporal resolution. A 5.8-MHz center frequency ICE probe was used to image the heart of two humans with atrial arrhythmias in vivo before and after RF ablation at high frame rates (1200 Hz), and the channel data were acquired on a clinical ultrasound system. The RF signals were reconstructed on a 9cm depth and 90° field of view region and axial cumulative displacement estimation was performed using 1-D cross-correlation using a window size of 2.6 mm and 95% overlap. Cumulative axial strains were obtained from the displacements using a least-squares estimator with a kernel of 5.1 mm. Cumulative axial strains i...

Noise driven transients in single mode semiconductor, solid state, and molecular gas lasers after they are ``switched on'' have strong (nearly linear) correlations between the time at which the laser intensity becomes significantly different from zero and the peak intensity of the first large pulse emitted by laser. We present evidence of a clear difference in the signs of the slopes of the linear correlations for these lasers for transients resulting from gain switching and loss switching and demonstrate the physical origin for this difference.