Arrhythmias including atrial fibrillation and congenital heart disease in Kleefstra syndrome: a possible epigenetic link.

AIMS: Kleefstra syndrome (KS), often diagnosed in early childhood, is a rare genetic disorder due to haploinsufficiency of EHMT1 and is characterized by neuromuscular and intellectual developmental abnormalities. Although congenital heart disease (CHD) is common, the prevalence of arrhythmias and CHD subtypes in KS is unknown. METHODS AND RESULTS: Inspired by a novel case series of KS patients with atrial tachyarrhythmias in the USA, we evaluate the two largest known KS registries for arrhythmias and CHD: Radboudumc (50 patients) based on health record review at Radboud University Medical Center in the Netherlands and GenIDA (163 patients) based on worldwide surveys of patient families. Three KS patients (aged 17-25 years) presented with atrial tachyarrhythmias without manifest CHD. In the international KS registries, the median [interquartile range (IQR)] age was considerably younger: GenIDA/Radboudumc at 10/13.5 (12/13) years, respectively. Both registries had a 40% prevalence of cardiovascular abnormalities, the majority being CHD, including septal defects, vascular malformations, and valvular disease. Interestingly, 4 (8%) patients in the Radboudumc registry reported arrhythmias without CHD, including one atrial fibrillation (AF), two with supraventricular tachycardias, and one with non-sustained ventricular tachycardia. The GenIDA registry reported one patient with AF and another with chronic ectopic atrial tachycardia (AT). In total, atrial tachyarrhythmias were noted in six young KS patients (6/213 or 3%) with at least four (three AF and one AT) without structural heart disease. CONCLUSION: In addition to a high prevalence of CHD, evolving data reveal early-onset atrial tachyarrhythmias in young KS patients, including AF, even in the absence of structural heart disease.

Elucidating arrhythmogenic right ventricular cardiomyopathy with stem cells.

Human stems cells have sparked many novel strategies for treating heart disease and for elucidating their underlying mechanisms. For example, arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart muscle disorder that is associated with fatal arrhythmias often occurring in healthy young adults. Fibro-fatty infiltrate, a clinical hallmark, progresses with the disease and can develop across both ventricles. Pathogenic variants in genes have been identified, with most being responsible for encoding cardiac desmosome proteins that reside at myocyte boundaries that are critical for cell-to-cell coupling. Despite some understanding of the molecular signaling mechanisms associated with ARVC mutations, their relationship with arrhythmogenesis is complex and not well understood for a monogenetic disorder. This review article focuses on arrhythmia mechanisms in ARVC based on clinical and animal studies and their relationship with disease causing variants. We also discuss the ways in which stem cells can be leveraged to improve our understanding of the role cardiac myocytes, nonmyocytes, metabolic signals, and inflammatory mediators play in an early onset disease such as ARVC.

Adipogenic Signaling Promotes Arrhythmia Substrates before Structural Abnormalities in TMEM43 ARVC.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic disorder of desmosomal and structural proteins that is characterized by fibro-fatty infiltrate in the ventricles and fatal arrhythmia that can occur early before significant structural abnormalities. Most ARVC mutations interfere with ß-catenin-dependent transcription that enhances adipogenesis; however, the mechanistic pathway to arrhythmogenesis is not clear. We hypothesized that adipogenic conditions play an important role in the formation of arrhythmia substrates in ARVC. Cardiac myocyte monolayers co-cultured for 2-4 days with mesenchymal stem cells (MSC) were derived from human-induced pluripotent stem cells with the ARVC5 TMEM43 p.Ser358Leu mutation. The TMEM43 mutation in myocyte co-cultures alone had no significant effect on impulse conduction velocity (CV) or APD. In contrast, when co-cultures were exposed to pro-adipogenic factors for 2-4 days, CV and APD were significantly reduced compared to controls by 49% and 31%, respectively without evidence of adipogenesis. Additionally, these arrhythmia substrates coincided with a significant reduction in IGF-1 expression in MSCs and were mitigated by IGF-1 treatment. These findings suggest that the onset of enhanced adipogenic signaling may be a mechanism of early arrhythmogenesis, which could lead to personalized treatment for arrhythmias associated with TMEM43 and other ARVC mutations.

Postoperative Myocardial Injury in Patients Classified as Low Risk Preoperatively Is Associated With a Particularly Increased Risk of Long-Term Mortality After Noncardiac Surgery.

Background Prior studies have shown an association between myocardial injury after noncardiac surgery (MINS) and all-cause mortality in patients following noncardiac surgery. However, the association between preoperative risk assessments, Revised Cardiac Risk Index and American College of Surgeons National Surgical Quality Improvement Program, and postoperative troponin elevations and long-term mortality is unknown. Methods and Results A retrospective chart review identified 548 patients who had a troponin I level drawn within 14 days of noncardiac surgery that required an overnight hospital stay. Patients aged 40 to 80 years with at least 2 cardiovascular risk factors were included, while those with trauma, pulmonary embolism, and neurosurgery were excluded. Kaplan-Meier survival and odds ratio (OR) with sensitivity/specificity analysis were performed to assess the association between preoperative risk and postoperative troponin elevation and all-cause mortality at 1 year. Overall, 69%/31% were classified as low-risk/high-risk per the Revised Cardiac Risk Index and 66%/34% per American College of Surgeons National Surgical Quality Improvement Program. Comparing the low-risk versus high-risk groups, preoperative risk assessment was not associated with either postoperative troponin elevation or 1-year mortality. MINS portended a 1-year mortality of OR, 3.9 (95% CI, 2.44-6.33) in the total population. Patients classified as low risk preoperatively with MINS had the highest risk of 1-year mortality (OR, 9.6; 95% CI, 4.27-24.38), with a low prevalence of statin use. Conclusions Current preoperative risk stratification tools do not prognosticate the risk of postoperative troponin elevation and all-cause mortality at 1 year. Interestingly, patients classified as low risk preoperatively with MINS had a markedly higher 1-year mortality risk compared with the general population, and most of them are not taking a statin. Our results suggest that evaluating preoperatively low-risk patients for MINS presents an opportunity for prognostication, risk reclassification, and initiating therapies such as statins to mitigate long-term risk.

Validation of independent component analysis for rapid spike sorting of optical recording data.

Independent component analysis (ICA) is a technique that can be used to extract the source signals from sets of signal mixtures where the sources themselves are unknown. The analysis of optical recordings of invertebrate neuronal networks with fast voltage-sensitive dyes could benefit greatly from ICA. These experiments can generate hundreds of voltage traces containing both redundant and mixed recordings of action potentials originating from unknown numbers of neurons. ICA can be used as a method for converting such complex data sets into single-neuron traces, but its accuracy for doing so has never been empirically evaluated. Here, we tested the accuracy of ICA for such blind source separation by simultaneously performing sharp electrode intracellular recording and fast voltage-sensitive dye imaging of neurons located in the central ganglia of Tritonia diomedea and Aplysia californica, using a 464-element photodiode array. After running ICA on the optical data sets, we found that in 34 of 34 cases the intracellularly recorded action potentials corresponded 100% to the spiking activity of one of the independent components returned by ICA. We also show that ICA can accurately sort action potentials into single neuron traces from a series of optical data files obtained at different times from the same preparation, allowing one to monitor the network participation of large numbers of individually identifiable neurons over several recording episodes. Our validation of the accuracy of ICA for extracting the neural activity of many individual neurons from noisy, mixed, and redundant optical recording data sets should enable the use of this powerful large-scale imaging approach for studies of invertebrate and suitable vertebrate neuronal networks.

Human Cardiac Mesenchymal Stem Cells Remodel in Disease and Can Regulate Arrhythmia Substrates.

BACKGROUND: The mesenchymal stem cell (MSC), known to remodel in disease and have an extensive secretome, has recently been isolated from the human heart. However, the effects of normal and diseased cardiac MSCs on myocyte electrophysiology remain unclear. We hypothesize that in disease the inflammatory secretome of cardiac human MSCs (hMSCs) remodels and can regulate arrhythmia substrates. METHODS: hMSCs were isolated from patients with or without heart failure from tissue attached to extracted device leads and from samples taken from explanted/donor hearts. Failing hMSCs or nonfailing hMSCs were cocultured with normal human cardiac myocytes derived from induced pluripotent stem cells. Using fluorescent indicators, action potential duration, Ca2+ alternans, and spontaneous calcium release (SCR) incidence were determined. RESULTS: Failing and nonfailing hMSCs from both sources exhibited similar trilineage differentiation potential and cell surface marker expression as bone marrow hMSCs. Compared with nonfailing hMSCs, failing hMSCs prolonged action potential duration by 24% (P<0.001, n=15), increased Ca2+ alternans by 300% (P<0.001, n=18), and promoted spontaneous calcium release activity (n=14, P<0.013) in human cardiac myocytes derived from induced pluripotent stem cells. Failing hMSCs exhibited increased secretion of inflammatory cytokines IL (interleukin)-1ß (98%, P<0.0001) and IL-6 (460%, P<0.02) compared with nonfailing hMSCs. IL-1ß or IL-6 in the absence of hMSCs prolonged action potential duration but only IL-6 increased Ca2+ alternans and promoted spontaneous calcium release activity in human cardiac myocytes derived from induced pluripotent stem cells, replicating the effects of failing hMSCs. In contrast, nonfailing hMSCs prevented Ca2+ alternans in human cardiac myocytes derived from induced pluripotent stem cells during oxidative stress. Finally, nonfailing hMSCs exhibited >25× higher secretion of IGF (insulin-like growth factor)-1 compared with failing hMSCs. Importantly, IGF-1 supplementation or anti-IL-6 treatment rescued the arrhythmia substrates induced by failing hMSCs. CONCLUSIONS: We identified device leads as a novel source of cardiac hMSCs. Our findings show that cardiac hMSCs can regulate arrhythmia substrates by remodeling their secretome in disease. Importantly, therapy inhibiting (anti-IL-6) or mimicking (IGF-1) the cardiac hMSC secretome can rescue arrhythmia substrates.

Postmortem MRI of human brain hemispheres: T2 relaxation times during formaldehyde fixation.

Unlike in vivo imaging, postmortem MRI allows for invasive examination of the tissue specimen immediately after the MR scan. However, natural tissue decomposition and chemical fixation cause the postmortem tissue's MRI properties to be different from those found in vivo. Moreover, these properties change as postmortem fixation time elapses. The goal of this study was to characterize the T(2) relaxation changes that occur over time in cadaveric human brain hemispheres during fixation. Five hemispheres immersed in formaldehyde solution were scanned on a weekly basis for 3 months postmortem, and once again at 6 months postmortem. The T(2) relaxation times were measured throughout the hemispheres. Over time, T(2) values near the edges of the hemispheres decreased rapidly after death, while T(2) values of deep tissue decreased more slowly. This difference is likely due to the relatively large distance from the hemisphere surface, and other barriers limiting diffusion of formaldehyde molecules to deep tissues. In addition, T(2) values in deep tissue did not continuously decay to a plateau, but instead reached a minimum and then increased to a plateau. This final increase may be due to the effects of prolonged tissue decomposition, a hypothesis that is supported by numerical simulations of the fixation process.

Quantification of image texture in X-ray phase-contrast-enhanced projection images of in vivo mouse lungs observed at varied inflation pressures.

To date, there are very limited noninvasive, regional assays of in vivo lung microstructure near the alveolar level. It has been suggested that x-ray phase-contrast enhanced imaging reveals information about the air volume of the lung; however, the image texture information in these images remains underutilized. Projection images of in vivo mouse lungs were acquired via a tabletop, propagation-based, X-ray phase-contrast imaging system. Anesthetized mice were mechanically ventilated in an upright position. Consistent with previously published studies, a distinct image texture was observed uniquely within lung regions. Lung regions were automatically identified using supervised machine learning applied to summary measures of the image texture data. It was found that an unsupervised clustering within predefined lung regions colocates with expected differences in anatomy along the cranial-caudal axis in upright mice. It was also found that specifically selected inflation pressures-here, a purposeful surrogate of distinct states of mechanical expansion-can be predicted from the lung image texture alone, that the prediction model itself varies from apex to base and that prediction is accurate regardless of overlap with nonpulmonary structures such as the ribs, mediastinum, and heart. Cross-validation analysis indicated low inter-animal variation in the image texture classifications. Together, these results suggest that the image texture observed in a single X-ray phase-contrast-enhanced projection image could be used across a range of pressure states to study regional variations in regional lung function.

Watching a memory form-VSD imaging reveals a novel memory mechanism.

Studies of the mechanisms underlying memory formation have largely focused on the synapse. However, recent evidence suggests that additional, non-synaptic, mechanisms also play important roles in this process. We recently described a novel memory mechanism whereby a particular class of neurons was recruited into the Tritonia escape swim network with sensitization, a non-associative form of learning. Neurons that in the naïve state were loosely-affiliated with the network were rapidly recruited in, transitioning from variably bursting (VB) to reliably bursting (RB). Even after the memory had faded some new neurons remained, and some original members had left, leaving the network in an altered state. Further, we identified a candidate cellular mechanism underlying these network changes. Our study supports the view that brain networks may have surprisingly fluid functional structures and adds to the growing body of evidence that non-synaptic mechanisms often operate synergistically with changes at the synapse to mediate memory formation.

Methodology Considerations in Phase Mapping of Human Cardiac Arrhythmias.

BACKGROUND: Phase analysis of cardiac arrhythmias, particularly atrial fibrillation, has gained interest because of the ability to detect organized stable drivers (rotors) and target them for therapy. However, the lack of methodology details in publications on the topic has resulted in ongoing debate over the phase mapping technique. By comparing phase maps and activation maps, we examined advantages and limitations of phase mapping. METHODS AND RESULTS: Seven subjects were enrolled. We generated phase maps and activation maps from electrocardiographic imaging-reconstructed epicardial unipolar electrograms. For ventricular signals, phase was computed with (1) pseudoempirical mode decomposition detrending and (2) a novel Moving Average (MVG) detrending approach. For atrial fibrillation signals, MVG was modified to incorporate dynamic cycle length (DCL) changes (MVG-DCL). Phase maps were visually analyzed to study phase singularity points and rotors. Results show that phase is sensitive to cycle length choice, a limitation that was addressed by the MVG-DCL algorithm. MVG-DCL was optimal for atrial fibrillation analysis. Phase maps helped to highlight high-curvature wavefronts and rotors. However, for some activation patterns, phase generated nonrotational singularity points and false rotors. CONCLUSIONS: Phase mapping computes singularity points and visually highlights rotors. As such, it can help to provide a clearer picture of the spatiotemporal activation characteristics during atrial fibrillation. However, it is advisable to incorporate electrogram characteristics and the time-domain activation sequence in the analysis, to prevent misinterpretation and false rotor detection. Therefore, for mapping complex arrhythmias, a combined time-domain activation and phase mapping with variable cycle length seems to be the most reliable method.

Memory Formation in Tritonia via Recruitment of Variably Committed Neurons.

Prior studies have found that functional networks can rapidly add neurons as they build short-term memories, yet little is known about the principles underlying this process. Using voltage-sensitive dye imaging, we found that short-term sensitization of Tritonia's swim motor program involves rapid expansion of the number of participating neurons. Tracking neurons across trials revealed that this involves the conversion of recently discovered variably participating neurons to reliable status. Further, we identify a candidate serotonergic cellular mechanism mediating this process. Our findings reveal a new mechanism for memory formation, involving recruitment of pre-positioned, variably committed neurons into memory networks. This represents a shift from the field's long-term focus on synaptic plasticity, toward a view that certain neurons have characteristics that predispose them to join networks with learning.

Variable neuronal participation in stereotypic motor programs.

To what extent are motor networks underlying rhythmic behaviors rigidly hard-wired versus fluid and dynamic entities? Do the members of motor networks change from moment-to-moment or from motor program episode-to-episode? These are questions that can only be addressed in systems where it is possible to monitor the spiking activity of networks of neurons during the production of motor programs. We used large-scale voltage-sensitive dye (VSD) imaging followed by Independent Component Analysis spike-sorting to examine the extent to which the neuronal network underlying the escape swim behavior of Tritonia diomedea is hard-wired versus fluid from a moment-to-moment perspective. We found that while most neurons were dedicated to the swim network, a small but significant proportion of neurons participated in a surprisingly variable manner. These neurons joined the swim motor program late, left early, burst only on some cycles or skipped cycles of the motor program. We confirmed that this variable neuronal participation was not due to effects of the VSD by finding such neurons with intracellular recording in dye-free saline. Further, these neurons markedly varied their level of participation in the network from swim episode-to-episode. The generality of such unreliably bursting neurons was confirmed by their presence in the rhythmic escape networks of two other molluscan species, Tritonia festiva and Aplysia californica. Our observations support a view that neuronal networks, even those underlying rhythmic and stereotyped motor programs, may be more variable in structure than widely appreciated.