Substrate Mapping Alters Ventricular Tachycardia Inducibility.

BACKGROUND: Initiation of ventricular tachycardia (VT) by programmed electrical stimulation (PES) has an important role to allow mapping and assess ablation end points. We hypothesized that substrate mapping may alter VT inducibility by mechanical bumping of critical sites. METHODS: Subjects with left ventricular scar-related VT that was inducible by PES who were undergoing ablation were included. PES was repeated after substrate mapping (Group I) or after time under sedation/anesthesia during which additional imaging and transeptal puncture were performed without substrate mapping (Group II). The response to the second PES was categorized as type I if the same VT was induced, type II if a different VT was induced, and type III if VT was not inducible. RESULTS: Twenty-eight patients (median age 66 years, 61% ischemic cardiomyopathy), 14 in Group I and 14 in Group II, were included. Age, time between initial and second PES, type of cardiomyopathy, ejection fraction, and anesthesia methods were not different between the 2 groups. Initial VT cycle length, however, was shorter in Group I (305 millisecond [range, 235-600] versus 350 millisecond [range, 235-600], P=0.016). Also, Group I required more extrastimuli to induce VT in PES 1 (2 [1-4] versus 2 [1-3], P=0.022). In Group I, following substrate mapping, the second PES induced the same VT in 3 patients (21%), a different VT in 9 (64%), and no VT in 2 (14%) patients. In contrast, in Group II the same VT was induced in 10 (71%) patients, a different VT in 3 (21%) and no VT in 1 (7%) patient (P=0.017). CONCLUSIONS: Mechanical effects of substrate mapping commonly alter inducibility of VT. This has important implications for catheter ablation procedure planning and acute assessment of outcome and can potentially account for some recurrent VTs that are not recognized at the time of the procedure.

A challenging VT ablation with a large cardiac tumor.

INTRODUCTION: Ventricular tachycardia (VT) normally occurs from an abnormal structural substrate. CASE: We report a case in which VT was caused by a large tumor in the interventricular septum. Surgical intervention was not an option due to the location of the tumor and its proximity to the coronary arteries. CONCLUSION: The patient underwent ablation and upgrade to CRT before ultimately receiving a heart transplant.

Risk Factors for Repeat Infection and Mortality After Extraction of Infected Cardiovascular Implantable Electronic Devices.

OBJECTIVES: This study sought to investigate the factors associated with repeat infection following lead extraction procedures. BACKGROUND: Although lead extraction is an essential therapy for patients with cardiovascular implantable electronic device (CIED) infection, repeat infection still occurs in some patients. METHODS: The authors reviewed data for consecutive patients who underwent extraction of infected CIEDs from August 2003 to May 2019. Repeat infection was defined as infective endocarditis, sepsis with no alternative focus, or pocket infection after extraction of infected CIEDs. RESULTS: Extraction of infected CIEDs was performed in 496 patients. The most commonly implicated pathogen was Staphylococcus aureus (n = 188). In 449 patients (90.5%), all leads were completely extracted using only transvenous techniques. Thirty-three patients (6.7%) underwent surgical lead extraction, and 14 (2.8%) had retained leads or lead components. After a median follow-up of 352 [40 to 1,255] days after CIED extraction, 144 patients (29.0%) died. Repeat infection occurred in 47 patients (9.5%) with the median time from the extraction to repeat infection of 103 [45 to 214] days. In multivariable analysis, presence of a left ventricular assist device, younger age at extraction, and S. aureus infection were independent predictors of repeat infection. Additionally, chronic kidney disease, congestive heart failure, presence of septic emboli, S. aureus infection, and occurrence of major complications were independent predictors of increased mortality. CONCLUSIONS: Patients with S. aureus infection have a high risk of repeat infection and poor prognosis after CIED extraction. Repeat infection is also predicted by younger age and the presence of a left ventricular assist device, whereas mortality was predicted by congestive heart failure, chronic kidney disease, and septic emboli.

Periaortic Ventricular Tachycardias in Nonischemic Cardiomyopathy: Substrate and Electrocardiographic Correlations.

BACKGROUND: Periaortic fibrotic ventricular tachycardia (VT) substrate is common in nonischemic cardiomyopathy (NICM), often intramural, and difficult to ablate. We sought to better characterize normal and abnormal periaortic voltage map parameters and NICM periaortic VTs. METHODS: In 15 patients without heart disease, the 5th percentile of endocardial voltage for increasing distance from the aortic valve ring was determined. In 53 consecutive patients with NICM (64±11 years; left ventricular ejection fraction 31±10%) undergoing ablation of recurrent VT, periaortic electrogram voltage and VT characteristics were analyzed. RESULTS: In healthy patients, the fifth percentile of the bipolar voltage increased proportional to the distance from the aortic valve ring, from 1.0 mV at 1 cm to 1.5 mV at 1.5 cm; the corresponding unipolar voltage cutoffs were 5.0 and 7.5 mV. A total of 160 VTs were induced in 53 patients with NICM, of which 28 VTs in 20 patients had periaortic origins. Periaortic VTs were associated with similar periaortic bipolar voltage, but lower UVs consistent with intramural fibrosis as an important substrate. Periaortic VTs could be divided into left and right bundle branch block forms with mapping showing right septal and lateral exits. Left bundle branch block VTs were more often acutely abolished with ablation (100% versus 69%; P=0.034), but with a 23% incidence of heart block. Greater extent of low voltage was associated with more induced VTs and worse acute outcome. CONCLUSIONS: Adjusting voltage parameters based on distance from the aortic valve may improve definition of left ventricular outflow tract arrhythmia substrate. Periaortic VTs are common in NICM, often associated with intramural substrate and can be divided into left bundle branch block and right bundle branch block types associated with different ablation outcomes and risks.

Novel Workflow for Conversion of Catheter-Based Electroanatomic Mapping to DICOM Imaging for Noninvasive Radioablation of Ventricular Tachycardia.

PURPOSE: A recent clinical trial has demonstrated that noninvasive radioablation (NIRA) has the potential to reduce recurrent ventricular tachycardia (VT) that is refractory to drugs and standard catheter ablation. Electroanatomic mapping (EAM) that would be useful for planning is obtained during catheter ablation, but incompatibility between EAM and DICOM formats required for radiation planning has impeded the use of existing catheter-based mapping to guide NIRA and is an important hurdle for its wider adoption. In this paper we define a process to facilitate the fusion of catheter-based EAM with DICOM imaging for radiation planning. METHOD AND MATERIALS: The raw data export of the CARTO3 EAM system (version 6.0.45.171, ".mesh" file) was processed with a MATLAB script to generate 3-dimensional (3D) visual took kit files containing X, Y, Z coordinates obtained during mapping and corresponding impedance, voltage, and other point-based information. The image could then be visualized with standard image processing software (3D Slicer) and the target outlined on the image surface. This structure was in turn converted to a DICOM image and fused with patient thoracic imaging using anatomic landmarks. Robustness of the workflow was assessed through implementation with a second magnetic resonance imaging based VT ablation planning system, ADAS-VT. RESULTS: This process facilitated the fusion of EAM and DICOM imaging to inform selection of NIRA targets. The workflow was found to be robust and compatible with a second VT ablation planning system. CONCLUSIONS: The conversion of catheter-based EAM to a DICOM compatible format permits the fusion of images for radiation planning and provides an avenue for the wider application of NIRA. Further improvements in the compatibility of these imaging formats would be expected to improve quality and reproducibility of image fusion.

Lead Integrity Alert Triggered by T-wave Oversensing.

T-wave oversensing (TWOS) is a relatively common occurrence in pacemakers and defibrillators that can lead to pauses and inappropriate implantable cardioverter-defibrillator shocks. We present a case of TWOS that triggered the Lead Integrity Alert (Medtronic, Minneapolis, MN, USA) without evidence of actual lead failure.

Genomic Instability Associated with p53 Knockdown in the Generation of Huntington's Disease Human Induced Pluripotent Stem Cells.

Alterations in DNA damage response and repair have been observed in Huntington's disease (HD). We generated induced pluripotent stem cells (iPSC) from primary dermal fibroblasts of 5 patients with HD and 5 control subjects. A significant fraction of the HD iPSC lines had genomic abnormalities as assessed by karyotype analysis, while none of our control lines had detectable genomic abnormalities. We demonstrate a statistically significant increase in genomic instability in HD cells during reprogramming. We also report a significant association with repeat length and severity of this instability. Our karyotypically normal HD iPSCs also have elevated ATM-p53 signaling as shown by elevated levels of phosphorylated p53 and H2AX, indicating either elevated DNA damage or hypersensitive DNA damage signaling in HD iPSCs. Thus, increased DNA damage responses in the HD genotype is coincidental with the observed chromosomal aberrations. We conclude that the disease causing mutation in HD increases the propensity of chromosomal instability relative to control fibroblasts specifically during reprogramming to a pluripotent state by a commonly used episomal-based method that includes p53 knockdown.

PARK2 patient neuroprogenitors show increased mitochondrial sensitivity to copper.

Poorly-defined interactions between environmental and genetic risk factors underlie Parkinson's disease (PD) etiology. Here we tested the hypothesis that human stem cell derived forebrain neuroprogenitors from patients with known familial risk for early onset PD will exhibit enhanced sensitivity to PD environmental risk factors compared to healthy control subjects without a family history of PD. Two male siblings (SM and PM) with biallelic loss-of-function mutations in PARK2 were identified. Human induced pluripotent stem cells (hiPSCs) from SM, PM, and four control subjects with no known family histories of PD or related neurodegenerative diseases were utilized. We tested the hypothesis that hiPSC-derived neuroprogenitors from patients with PARK2 mutations would show heightened cell death, mitochondrial dysfunction, and reactive oxygen species generation compared to control cells as a result of exposure to heavy metals (PD environmental risk factors). We report that PARK2 mutant neuroprogenitors showed increased cytotoxicity with copper (Cu) and cadmium (Cd) exposure but not manganese (Mn) or methyl mercury (MeHg) relative to control neuroprogenitors. PARK2 mutant neuroprogenitors also showed a substantial increase in mitochondrial fragmentation, initial ROS generation, and loss of mitochondrial membrane potential following Cu exposure. Our data substantiate Cu exposure as an environmental risk factor for PD. Furthermore, we report a shift in the lowest observable effect level (LOEL) for greater sensitivity to Cu-dependent mitochondrial dysfunction in patients SM and PM relative to controls, correlating with their increased genetic risk for PD.

A novel manganese-dependent ATM-p53 signaling pathway is selectively impaired in patient-based neuroprogenitor and murine striatal models of Huntington's disease.

The essential micronutrient manganese is enriched in brain, especially in the basal ganglia. We sought to identify neuronal signaling pathways responsive to neurologically relevant manganese levels, as previous data suggested that alterations in striatal manganese handling occur in Huntington's disease (HD) models. We found that p53 phosphorylation at serine 15 is the most responsive cell signaling event to manganese exposure (of 18 tested) in human neuroprogenitors and a mouse striatal cell line. Manganese-dependent activation of p53 was severely diminished in HD cells. Inhibitors of ataxia telangiectasia mutated (ATM) kinase decreased manganese-dependent phosphorylation of p53. Likewise, analysis of ATM autophosphorylation and additional ATM kinase targets, H2AX and CHK2, support a role for ATM in the activation of p53 by manganese and that a defect in this process occurs in HD. Furthermore, the deficit in Mn-dependent activation of ATM kinase in HD neuroprogenitors was highly selective, as DNA damage and oxidative injury, canonical activators of ATM, did not show similar deficits. We assessed cellular manganese handling to test for correlations with the ATM-p53 pathway, and we observed reduced Mn accumulation in HD human neuroprogenitors and HD mouse striatal cells at manganese exposures associated with altered p53 activation. To determine if this phenotype contributes to the deficit in manganese-dependent ATM activation, we used pharmacological manipulation to equalize manganese levels between HD and control mouse striatal cells and rescued the ATM-p53 signaling deficit. Collectively, our data demonstrate selective alterations in manganese biology in cellular models of HD manifest in ATM-p53 signaling.

Cellular manganese content is developmentally regulated in human dopaminergic neurons.

Manganese (Mn) is both an essential biological cofactor and neurotoxicant. Disruption of Mn biology in the basal ganglia has been implicated in the pathogenesis of neurodegenerative disorders, such as parkinsonism and Huntington's disease. Handling of other essential metals (e.g. iron and zinc) occurs via complex intracellular signaling networks that link metal detection and transport systems. However, beyond several non-selective transporters, little is known about the intracellular processes regulating neuronal Mn homeostasis. We hypothesized that small molecules that modulate intracellular Mn could provide insight into cell-level Mn regulatory mechanisms. We performed a high throughput screen of 40,167 small molecules for modifiers of cellular Mn content in a mouse striatal neuron cell line. Following stringent validation assays and chemical informatics, we obtained a chemical 'toolbox' of 41 small molecules with diverse structure-activity relationships that can alter intracellular Mn levels under biologically relevant Mn exposures. We utilized this toolbox to test for differential regulation of Mn handling in human floor-plate lineage dopaminergic neurons, a lineage especially vulnerable to environmental Mn exposure. We report differential Mn accumulation between developmental stages and stage-specific differences in the Mn-altering activity of individual small molecules. This work demonstrates cell-level regulation of Mn content across neuronal differentiation.

Optimization of fluorescence assay of cellular manganese status for high throughput screening.

The advent of high throughput screening (HTS) technology permits identification of compounds that influence various cellular phenotypes. However, screening for small molecule chemical modifiers of neurotoxicants has been limited by the scalability of existing phenotyping assays. Furthermore, the adaptation of existing cellular assays to HTS format requires substantial modification of experimental parameters and analysis methodology to meet the necessary statistical requirements. Here we describe the successful optimization of the Cellular Fura-2 Manganese Extraction Assay (CFMEA) for HTS. By optimizing cellular density, manganese (Mn) exposure conditions, and extraction parameters, the sensitivity and dynamic range of the fura-2 Mn response was enhanced to permit detection of positive and negative modulators of cellular manganese status. Finally, we quantify and report strategies to control sources of intra- and interplate variability by batch level and plate-geometric level analysis. Our goal is to enable HTS with the CFMEA to identify novel modulators of Mn transport.

Gammaretroviral vector encoding a fluorescent marker to facilitate detection of reprogrammed human fibroblasts during iPSC generation.

Induced pluripotent stem cells (iPSCs) are becoming mainstream tools to study mechanisms of development and disease. They have a broad range of applications in understanding disease processes, in vitro testing of novel therapies, and potential utility in regenerative medicine. Although the techniques for generating iPSCs are becoming more straightforward, scientists can expend considerable resources and time to establish this technology. A major hurdle is the accurate determination of valid iPSC-like colonies that can be selected for further cloning and characterization. In this study, we describe the use of a gammaretroviral vector encoding a fluorescent marker, mRFP1, to not only monitor the efficiency of initial transduction but also to identify putative iPSC colonies through silencing of mRFP1 gene as a consequence of successful reprogramming.

Genetic risk for Parkinson's disease correlates with alterations in neuronal manganese sensitivity between two human subjects.

Manganese (Mn) is an environmental risk factor for Parkinson's disease (PD). Recessive inheritance of PARK2 mutations is strongly associated with early onset PD (EOPD). It is widely assumed that the influence of PD environmental risk factors may be enhanced by the presence of PD genetic risk factors in the genetic background of individuals. However, such interactions may be difficult to predict owing to the complexities of genetic and environmental interactions. Here we examine the potential of human induced pluripotent stem (iPS) cell-derived early neural progenitor cells (NPCs) to model differences in Mn neurotoxicity between a control subject (CA) with no known PD genetic risk factors and a subject (SM) with biallelic loss-of-function mutations in PARK2 and family history of PD but no evidence of PD by neurological exam. Human iPS cells were generated from primary dermal fibroblasts of both subjects. We assessed several outcome measures associated with Mn toxicity and PD. No difference in sensitivity to Mn cytotoxicity or mitochondrial fragmentation was observed between SM and CA NPCs. However, we found that Mn exposure was associated with significantly higher reactive oxygen species (ROS) generation in SM compared to CA NPCs despite significantly less intracellular Mn accumulation. Thus, this report offers the first example of human subject-specific differences in PD-relevant environmental health related phenotypes that are consistent with pathogenic interactions between known genetic and environmental risk factors for PD.

DMH1, a highly selective small molecule BMP inhibitor promotes neurogenesis of hiPSCs: comparison of PAX6 and SOX1 expression during neural induction.

Recent successes in deriving human-induced pluripotent stem cells (hiPSCs) allow for the possibility of studying human neurons derived from patients with neurological diseases. Concomitant inhibition of the BMP and TGF-ß1 branches of the TGF-ß signaling pathways by the endogenous antagonist, Noggin, and the small molecule SB431542, respectively, induces efficient neuralization of hiPSCs, a method known as dual-SMAD inhibition. The use of small molecule inhibitors instead of their endogenous counterparts has several advantages including lower cost, consistent activity, and the maintenance of xeno-free culture conditions. We tested the efficacy of DMH1, a highly selective small molecule BMP-inhibitor for its potential to replace Noggin in the neuralization of hiPSCs. We compare Noggin and DMH1-induced neuralization of hiPSCs by measuring protein and mRNA levels of pluripotency and neural precursor markers over a period of seven days. The regulation of five of the six markers assessed was indistinguishable in the presence of concentrations of Noggin or DMH1 that have been shown to effectively inhibit BMP signaling in other systems. We observed that by varying the DMH1 or Noggin concentration, we could selectively modulate the number of SOX1 expressing cells, whereas PAX6, another neural precursor marker, remained the same. The level and timing of SOX1 expression have been shown to affect neural induction as well as neural lineage. Our observations, therefore, suggest that BMP-inhibitor concentrations need to be carefully monitored to ensure appropriate expression levels of all transcription factors necessary for the induction of a particular neuronal lineage. We further demonstrate that DMH1-induced neural progenitors can be differentiated into ß3-tubulin expressing neurons, a subset of which also express tyrosine hydroxylase. Thus, the combined use of DMH1, a highly specific BMP-pathway inhibitor, and SB431542, a TGF-ß1-pathway specific inhibitor, provides us with the tools to independently regulate these two pathways through the exclusive use of small molecule inhibitors.

The potential of induced pluripotent stem cells as a translational model for neurotoxicological risk.

An important goal of neurotoxicological research is to provide relevant and accurate risk assessment of environmental and pharmacological agents for populations and individuals. Owing to the challenges of human subject research and the real possibility of species specific toxicological responses, neuronal lineages derived from human embryonic stem cells (hESCs) and human neuronal precursors have been offered as a potential solution for validation of neurotoxicological data from model organism systems in humans. More recently, with the advent of induced pluripotent stem cell (iPSC) technology, there is now the possibility of personalized toxicological risk assessment, the ability to predict individual susceptibility to specific environmental agents, by this approach. This critical advance is widely expected to facilitate analysis of cellular physiological pathways in the context of human neurons and the underlying genetic factors that lead to disease. Thus this technology opens the opportunity, for the first time, to characterize the physiological, toxicological, pharmacological and molecular properties of living human neurons with identical genetic determinants as human patients. Furthermore, armed with a complete clinical history of the patients, human iPSC (hiPSC) studies can theoretically compare patients and at risk groups with distinct sensitivities to particular environmental agents, divergent clinical outcomes, differing co-morbidities, and so forth. Thus iPSCs and neuronal lineages derived from them may reflect the unique genetic blueprint of the individuals from which they are generated. Indeed, iPSC technology has the potential to revolutionize scientific approaches to human health. However, before this overarching goal can be reached a number of technical and theoretical challenges must be overcome. This review seeks to provide a realistic assessment of hiPSC technology and its application to risk assessment and mechanistic studies in the area of neurotoxicology. We seek to identify, prioritize, and detail the primary hurdles that need to be overcome if personalized toxicological risk assessment using patient-derived iPSCs is to succeed.