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Background: The accuracy of noninvasive arrhythmia source localization using a forward-solution computational mapping system has not yet been evaluated in blinded, multicenter analysis. This study tested the hypothesis that a computational mapping system incorporating a comprehensive arrhythmia simulation library would provide accurate localization of the site-of-origin for atrial and ventricular arrhythmias and pacing using 12-lead ECG data when compared with the gold standard of invasive electrophysiology study and ablation. Methods: The VMAP study (Vectorcardiographic Mapping of Arrhythmogenic Probability) was a blinded, multicenter evaluation with final data analysis performed by an independent core laboratory. Eligible episodes included atrial and ventricular: tachycardia, fibrillation, pacing, premature atrial and ventricular complexes, and orthodromic atrioventricular reentrant tachycardia. Mapping system results were compared with the gold standard site of successful ablation or pacing during electrophysiology study and ablation. Mapping time was assessed from time-stamped logs. Prespecified performance goals were used for statistical comparisons. Results: A total of 255 episodes from 225 patients were enrolled from 4 centers. Regional accuracy for ventricular tachycardia and premature ventricular complexes in patients without significant structural heart disease (n=75, primary end point) was 98.7% (95% CI, 96.0%–100%; P 0 P 0 P 0 Conclusions: Computational ECG mapping using a forward-solution approach exceeded prespecified accuracy goals for arrhythmia and pacing localization. Spatial accuracy analysis demonstrated clinically actionable results. This rapid, noninvasive mapping technology may facilitate catheter-based and noninvasive targeted arrhythmia therapies. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT04559061.

Background Relationships between right ventricular (RV) and left ventricular (LV) shape and function may be useful in determining optimal timing for pulmonary valve replacement in patients with repaired tetralogy of Fallot (rTOF). However, these are multivariate and difficult to quantify. We aimed to quantify variations in biventricular shape associated with pulmonary regurgitant volume (PRV) in rTOF using a biventricular atlas. Methods In this cross-sectional retrospective study, a biventricular shape model was customized to cardiovascular magnetic resonance (CMR) images from 88 rTOF patients (median age 16, inter-quartile range 11.8–24.3 years). Morphometric scores quantifying biventricular shape at end-diastole and end-systole were computed using principal component analysis. Multivariate linear regression was used to quantify biventricular shape associations with PRV, corrected for age, sex, height, and weight. Regional associations were confirmed by univariate correlations with distances and angles computed from the models, as well as global systolic strains computed from changes in arc length from end-diastole to end-systole. Results PRV was significantly associated with 5 biventricular morphometric scores, independent of covariates, and accounted for 12.3% of total shape variation (p Conclusion A biventricular atlas of rTOF patients quantified multivariate relationships between left–right ventricular morphometry and wall motion with pulmonary regurgitation. Regional RV dilation, LV reduction, LV septal-lateral flattening and increased RV strain were all associated with increased pulmonary regurgitant volume. Morphometric scores provide simple metrics linking mechanisms for structural and functional alteration with important clinical indices.

Modeling the hearts of patients with hypoplastic left heart syndrome has remained an ongoing challenge. Thus, in this work, we developed a HLHS-specific biventricular template to capture the single ventricle morphology in this cohort accurately and robustly. We have demonstrated that using the HLHS-specific template to reconstruct patient-specific models from cardiac magnetic resonance images result in significantly higher accuracy comparing to that of using the generic template. Additionally, element distortion in meshes created using the HLHS-specific template is compatible with meshes currently used for finite element analysis. Accurate 3D patient-specific models will serve as powerful tools for subsequent statistical shape and biomechanics analyses to provide insights into remodeling of ventricular shape and function and their potential association with clinical outcomes.

Electrical impulse generation and its conduction within cells or cellular networks are the cornerstone of electrophysiology. However, the advancement of the field is limited by sensing accuracy and the scalability of current recording technologies. Here we describe a scalable platform that enables accurate recording of transmembrane potentials in electrogenic cells. The platform employs a three-dimensional high-performance field-effect transistor array for minimally invasive cellular interfacing that produces faithful recordings, as validated by the gold standard patch clamp. Leveraging the high spatial and temporal resolutions of the field-effect transistors, we measured the intracellular signal conduction velocity of a cardiomyocyte to be 0.182 m s-1, which is about five times the intercellular velocity. We also demonstrate intracellular recordings in cardiac muscle tissue constructs and reveal the signal conduction paths. This platform could provide new capabilities in probing the electrical behaviours of single cells and cellular networks, which carries broad implications for understanding cellular physiology, pathology and cell-cell interactions.

Much of our understanding of the regulatory mechanisms governing the cell cycle in mammals has relied heavily on methods that measure the aggregate state of a population of cells. While instrumental in shaping our current understanding of cell proliferation, these approaches mask the genetic signatures of rare subpopulations such as quiescent (G0) and very slowly dividing (SD) cells. Results described in this study and those of others using single-cell analysis reveal that even in clonally derived immortalized cancer cells, ∼1–5% of cells can exhibit G0 and SD phenotypes. Therefore to enable the study of these rare cell phenotypes we established an integrated molecular, computational, and imaging approach to track, isolate, and genetically perturb single cells as they proliferate. A genetically encoded cell-cycle reporter (K67p-FUCCI) was used to track single cells as they traversed the cell cycle. A set of R-scripts were written to quantify K67p-FUCCI over time. To enable the further study G0 and SD phenotypes, we retrofitted a live cell imaging system with a micromanipulator to enable single-cell targeting for functional validation studies. Single-cell analysis revealed HT1080 and MCF7 cells had a doubling time of ∼24 and ∼48 h, respectively, with high duration variability in G1 and G2 phases. Direct single-cell microinjection of mRNA encoding (GFP) achieves detectable GFP fluorescence within ∼5 h in both cell types. These findings coupled with the possibility of targeting several hundreds of single cells improves throughput and sensitivity over conventional methods to study rare cell subpopulations.

Background Adverse cardiac remodeling is an important precursor to anthracycline-related cardiac dysfunction, however conventional remodeling indices are limited. We sought to examine the utility of statistical atlas-derived measures of ventricular shape to improve the identification of adverse anthracycline-related remodeling in childhood cancer survivors. Methods We analyzed cardiac magnetic resonance imaging from a cross-sectional cohort of 20 childhood cancer survivors who were treated with low (2 [N = 10]) or high (≥250 mg/m2 [N = 10]) dose anthracyclines, matched 1:1 by sex and age between dose groups. We reconstructed 3D computational models of left ventricular end-diastolic shape for each subject and assessed the ability of conventional remodeling indices (volume, mass, and mass to volume ratio) vs. shape modes derived from a statistical shape atlas of an asymptomatic reference population to stratify anthracycline-related remodeling. We compared conventional parameters and five atlas-based shape modes: 1) between survivors and the reference population (N = 1991) using multivariable linear regression, and 2) within survivors by anthracycline dose (low versus high) using two-sided T-tests, multivariable logistic regression, and receiver operating characteristic curves. Results Compared with the reference population, survivors had differences in conventional measures (lower volume and mass) and shape modes (corresponding to lower overall size and lower sphericity; all p p = 0.039). Collectively, atlas-based shape modes in conjunction with conventional measures discriminated survivors who received low vs. high anthracycline dosage (area under the curve [AUC] 0.930, 95% confidence interval 0.816, 1.00) significantly better than conventional measures alone (AUC 0.710, 95% confidence interval 0.473, 0.947; AUC comparison p = 0.0498). Conclusions Compared with a reference population, heart size is smaller in anthracycline-exposed childhood cancer survivors. Atlas-based measures of left ventricular shape may improve the detection of anthracycline dose-related remodeling differences.

Rationale: ZO-1 (Zonula occludens-1), a plasma membrane-associated scaffolding protein regulates signal transduction, transcription, and cellular communication. Global deletion of ZO-1 in the mouse is lethal by embryonic day 11.5. The function of ZO-1 in cardiac myocytes (CM) is largely unknown. Objective: To determine the function of CM ZO-1 in the intact heart, given its binding to other CM proteins that have been shown instrumental in normal cardiac conduction and function. Methods and Results: We generated ZO-1 CM-specific knockout (KO) mice using α-Myosin Heavy Chain-nuclear Cre (ZO-1cKO) and investigated physiological and electrophysiological function by echocardiography, surface ECG and conscious telemetry, intracardiac electrograms and pacing, and optical mapping studies. ZO-1cKO mice were viable, had normal Mendelian ratios, and had a normal lifespan. Ventricular morphometry and function were not significantly different between the ZO-1cKO versus control (CTL) mice, basally in young or aged mice, or even when hearts were subjected to hemodynamic loading. Atrial mass was increased in ZO-1cKO. Electrophysiological and optical mapping studies indicated high-grade atrioventricular (A-V) block in ZO-1cKO comparing to CTL hearts. While ZO-1-associated proteins such as vinculin, connexin 43, N-cadherin, and α-catenin showed no significant change with the loss of ZO-1, Connexin-45 and Coxsackie-adenovirus (CAR) proteins were reduced in atria of ZO-1cKO. Further, with loss of ZO-1, ZO-2 protein was increased significantly in ventricular CM in a presumed compensatory manner but was still not detected in the AV nodal myocytes. Importantly, the expression of the sodium channel protein NaV1.5 was altered in AV nodal cells of the ZO-1cKO versus CTL. Conclusions: ZO-1 protein has a unique physiological role in cardiac nodal tissue. This is in alignment with its known interaction with CAR and Cx45, and a new function in regulating the expression of NaV1.5 in AV node. Uniquely, ZO-1 is dispensable for function of the working myocardium.

Purpose Structure-guided total variation is a recently introduced prior that allows reconstruction of images using knowledge of the location and orientation of edges in a reference image. In this work, we demonstrate the advantages of a variant of structure-guided total variation known as directional total variation (DTV), over traditional total variation (TV), in the context of compressed-sensing reconstruction and super-resolution. Methods We compared TV and DTV in retrospectively undersampled ex vivo diffusion tensor imaging and diffusion spectrum imaging data from healthy, sham, and hypertrophic rat hearts. Results In compressed sensing at an undersampling factor of 8, the RMS error of mean diffusivity and fractional anisotropy relative to the fully sampled ground truth were 44% and 20% lower in DTV compared with TV. In super-resolution, these values were 29% and 14%, respectively. Similarly, we observed improvements in helix angle, transverse angle, sheetlet elevation, and sheetlet azimuth. The RMS error of the diffusion kurtosis in the undersampled data relative to the ground truth was uniformly lower (22% on average) with DTV compared to TV. Conclusion Acquiring one fully sampled non-diffusion-weighted image and 10 diffusion-weighted images at 8× undersampling would result in an 80% net reduction in data needed. We demonstrate efficacy of the DTV algorithm over TV in reducing data sampling requirements, which can be translated into higher apparent resolution and potentially shorter scan times. This method would be equally applicable in diffusion MRI applications outside the heart.

Heterogeneous mechanical dyskinesis has been implicated in many arrhythmogenic phenotypes. Strain-dependent perturbations to cardiomyocyte electrophysiology may contribute to this arrhythmogenesis through processes referred to as mechanoelectric feedback. Although the role of stretch-activated ion currents has been investigated using computational models, experimental studies suggest that mechanical strain may also promote arrhythmia by facilitating calcium wave propagation. To investigate whether strain-dependent changes in calcium affinity to the myofilament may promote arrhythmogenic intracellular calcium waves, we modified a mathematical model of rabbit excitation-contraction coupling coupled to a model of myofilament activation and force development. In a one-dimensional compartmental analysis, we bidirectionally coupled 50 sarcomere models in series to model calcium diffusion and stress transfer between adjacent sarcomeres. These considerations enabled the model to capture 1) the effects of mechanical feedback on calcium homeostasis at the sarcomeric level and 2) the combined effects of mechanical and calcium heterogeneities at the cellular level. The results suggest that in conditions of calcium overload, the vulnerable window of stretch-release to trigger suprathreshold delayed afterdepolarizations can be affected by heterogeneity in sarcomere length. Furthermore, stretch and sarcomere heterogeneity may modulate the susceptibility threshold for delayed afterdepolarizations and the aftercontraction wave propagation velocity.

Introduction: Current indications for pulmonary valve replacement (PVR) in repaired tetralogy of Fallot (rTOF) rely on global ventricular measurements but are inconsistently applied, lead to mixed outcomes, and are widely debated. Hypothesis: We aimed to test the hypothesis that specific regional markers of biventricular shape can discriminate differences in ventricular remodeling between rTOF patients that were and were not designated for follow-up PVR better than standard clinical indices. Methods: In this cross-sectional retrospective study, biventricular shape models were fit to CMR images from 84 rTOF patients. A statistical atlas of end-diastolic shape was constructed using principal component analysis. Multivariate regression was used to quantify shape marker and clinical index associations with subsequent intervention status (PVR, n=48 vs. No-PVR, n=36) while accounting for confounders. Clustering analysis was then used to test the ability of statistically significant shape markers and clinical indices to discriminate PVR status as evaluated by Matthews correlation coefficient (MCC). Geometric strain analysis was also conducted to assess shape marker associations with systolic function. Results: PVR status correlated with shape markers associated with RV apical dilation and LV dilation (p Conclusions: Biventricular shape markers discriminated PVR status better than standard and current use clinical indices for PVR. These specific, regional features of cardiac morphology can provide mechanistic insight into adaptive vs. maladaptive types of ventricular remodeling that provide the foundation for clinical decision-making but may be overlooked when relying solely on global ventricular measurements.

Patients with repaired tetralogy of Fallot (rTOF) are at risk of long-term left ventricular (LV) dysfunction associated with poor outcomes. In this study, we examined the association of LV end-diastolic (ED) shape with components of systolic wall motion (SWM) that contribute to global systolic dysfunction in an rTOF patient cohort. Features of LV shape associated with conicity and septal wall curvature correlated with components of SWM. The effect of ED shape perturbations on SWM were examined in a finite element analysis of systolic ventricular mechanics. Variations in the combination of ED shape and myocardial contractility were able to match predicted measures of SWM. From these results, we hypothesize that greater LV conicity and a flatter septal wall are markers of reduced global myocardial contractility and should be examined further for clinical prognostic utility to improve patient outcomes.

Increased availability and access to medical image data has enabled more quantitative approaches to clinical diagnosis, prognosis, and treatment planning for congenital heart disease. Here we present an overview of long-term clinical management of tetralogy of Fallot (TOF) and its intersection with novel computational and data science approaches to discovering biomarkers of functional and prognostic importance. Efforts in translational medicine that seek to address the clinical challenges associated with cardiovascular diseases using personalized and precision-based approaches are then discussed. The considerations and challenges of translational cardiovascular medicine are reviewed, and examples of digital platforms with collaborative, cloud-based, and scalable design are provided.

Background Stereotactic ablative radiotherapy (SAbR) is an emerging therapy for refractory ventricular tachycardia (VT). However, the current workflow is complicated, and the precision and safety in patients with significant cardiorespiratory motion and VT targets near the stomach may be suboptimal. Objective We hypothesized that automated 12-lead electrocardiogram (ECG) mapping and respiratory-gated therapy may improve the ease and precision of SAbR planning and facilitate safe radiation delivery in patients with refractory VT. Methods Consecutive patients with refractory VT were studied at 2 hospitals. VT exit sites were localized using a 3-D computational ECG algorithm noninvasively and compared to available prior invasive mapping. Radiotherapy (25 Gy) was delivered at end-expiration when cardiac respiratory motion was ≥0.6 cm or targets were ≤2 cm from the stomach. Results In 6 patients (ejection fraction 29% ± 13%), 4.2 ± 2.3 VT morphologies per patient were mapped. Overall, 7 out of 7 computational ECG mappings (100%) colocalized to the identical cardiac segment when prior invasive electrophysiology study was available. Respiratory gating was associated with smaller planning target volumes compared to nongated volumes (71 ± 7 vs 153 ± 35 cc, P < .01). In 2 patients with inferior wall VT targets close to the stomach (6 mm proximity) or significant respiratory motion (22 mm excursion), no GI complications were observed at 9- and 12-month follow-up. Implantable cardioverter-defibrillator shocks decreased from 23 ± 12 shocks/patient to 0.67 ± 1.0 (P < .001) post-SAbR at 6.0 ± 4.9 months follow-up. Conclusions A workflow including computational ECG mapping and protocol-guided respiratory gating is feasible, is safe, and may improve the ease of SAbR planning. Studies to validate this workflow in larger populations are required., Graphical abstract

Familial cardiomyopathy is a precursor of heart failure and sudden cardiac death. Over the past several decades, researchers have discovered numerous gene mutations primarily in sarcomeric and cytoskeletal proteins causing two different disease phenotypes: hypertrophic (HCM) and dilated (DCM) cardiomyopathies. However, molecular mechanisms linking genotype to phenotype remain unclear. Here, we employ a systems approach by integrating experimental findings from preclinical studies (e.g., murine data) into a cohesive signaling network to scrutinize genotype to phenotype mechanisms. We developed an HCM/DCM signaling network model utilizing a logic-based differential equations approach and evaluated model performance in predicting experimental data from four contexts (HCM, DCM, pressure overload, and volume overload). The model has an overall prediction accuracy of 83.8%, with higher accuracy in the HCM context (90%) than DCM (75%). Global sensitivity analysis identifies key signaling reactions, with calcium-mediated myofilament force development and calcium-calmodulin kinase signaling ranking the highest. A structural revision analysis indicates potential missing interactions that primarily control calcium regulatory proteins, increasing model prediction accuracy. Combination pharmacotherapy analysis suggests that downregulation of signaling components such as calcium, titin and its associated proteins, growth factor receptors, ERK1/2, and PI3K-AKT could inhibit myocyte growth in HCM. In experiments with patient-specific iPSC-derived cardiomyocytes (MLP-W4R;MYH7-R723C iPSC-CMs), combined inhibition of ERK1/2 and PI3K-AKT rescued the HCM phenotype, as predicted by the model. In DCM, PI3K-AKT-NFAT downregulation combined with upregulation of Ras/ERK1/2 or titin or Gq protein could ameliorate cardiomyocyte morphology. The model results suggest that HCM mutations that increase active force through elevated calcium sensitivity could increase ERK activity and decrease eccentricity through parallel growth factors, Gq-mediated, and titin pathways. Moreover, the model simulated the influence of existing medications on cardiac growth in HCM and DCM contexts. This HCM/DCM signaling model demonstrates utility in investigating genotype to phenotype mechanisms in familial cardiomyopathy.

PURPOSE: Patient-specific models of the heart can be used to improve the diagnosis of cardiac diseases, but practical application of these models can be impeded by the computational costs and numerical uncertainties of fitting mechanistic models to clinical measurements from individual patients. Reliable and efficient tuning of these models within clinically appropriate error bounds is a requirement for practical deployment in the time-constrained environment of the clinic. METHODS: We developed an optimization framework to tune parameters of patient-specific mechanistic models using routinely-acquired non-invasive patient data more efficiently than manual methods. We employ a hybrid particle swarm and pattern search optimization algorithm, but the framework can be readily adapted to use other optimization algorithms. RESULTS: We apply the proposed framework to tune full-cycle lumped parameter circulatory models using clinical data. We show that our framework can be easily adapted to optimize cross-species models by tuning the parameters of the same circulation model to four canine subjects. CONCLUSIONS: This work will facilitate the use of biomechanics and circulatory cardiac models in both clinical and research environments by ameliorating the tedious process of manually fitting the parameters.

Myocardial hypertrophy is the result of sustained perturbations to the mechanical and/or neurohormonal homeostasis of cardiac cells and is driven by integrated, multiscale biophysical and biochemical processes that are currently not well defined. In this brief review, we highlight recent computational and experimental models of cardiac hypertrophy that span mechanisms from the molecular level to the tissue level. Specifically, we focus on: (i) molecular-level models of the structural dynamics of sarcomere proteins in hypertrophic hearts, (ii) cellular-level models of excitation-contraction coupling and mechanosensitive signaling in disease-state myocytes, and (iii) organ-level models of myocardial growth kinematics and predictors thereof. Finally, we discuss how spanning these scales and combining multiple experimental/computational models will provide new information about the processes governing hypertrophy and potential methods to prevent or reverse them.

Human induced pluripotent stem cell - derived cardiomyocytes (iPSC-CMs) are regarded as a promising cell source for establishing in-vitro personalized cardiac tissue models and developing therapeutics. However, analyzing cardiac force and drug response using mature human iPSC-CMs in a high-throughput format still remains a great challenge. Here we describe a rapid light-based 3D printing system for fabricating micro-scale force gauge arrays suitable for 24-well and 96-well plates that enable scalable tissue formation and measurement of cardiac force generation in human iPSC-CMs. We demonstrate consistent tissue band formation around the force gauge pillars with aligned sarcomeres. Among the different maturation treatment protocols we explored, 3D aligned cultures on force gauge arrays with in-culture pacing produced the highest expression of mature cardiac marker genes. We further demonstrated the utility of these micro-tissues to develop significantly increased contractile forces in response to treatment with isoproterenol, levosimendan, and omecamtiv mecarbil. Overall, this new 3D printing system allows for high flexibility in force gauge design and can be optimized to achieve miniaturization and promote cardiac tissue maturation with great potential for high-throughput in-vitro drug screening applications. STATEMENT OF SIGNIFICANCE: The application of iPSC-derived cardiac tissues in translatable drug screening is currently limited by the challenges in forming mature cardiac tissue and analyzing cardiac forces in a high-throughput format. We demonstrate the use of a rapid light-based 3D printing system to build a micro-scale force gauge array that enables scalable cardiac tissue formation from iPSC-CMs and measurement of contractile force development. With the capability to provide great flexibility over force gauge design as well as optimization to achieve miniaturization, our 3D printing system serves as a promising tool to build cardiac tissues for high-throughput in-vitro drug screening applications.

The intact heart undergoes complex and multiscale remodelling processes in response to altered mechanical cues. Remodelling of the myocardium is regulated by a combination of myocyte and non-myocyte responses to mechanosensitive pathways, which can alter gene expression and therefore function in these cells. Cellular mechanotransduction and its downstream effects on gene expression are initially compensatory mechanisms during adaptations to the altered mechanical environment, but under prolonged and abnormal loading conditions, they can become maladaptive, leading to impaired function and cardiac pathologies. In this Review, we summarize mechanoregulated pathways in cardiac myocytes and fibroblasts that lead to altered gene expression and cell remodelling under physiological and pathophysiological conditions. Developments in systems modelling of the networks that regulate gene expression in response to mechanical stimuli should improve integrative understanding of their roles in vivo and help to discover new combinations of drugs and device therapies targeting mechanosignalling in heart disease. In this Review, the authors summarize mechanoregulated pathways in cardiac myocytes and fibroblasts that lead to altered gene expression and cell remodelling under physiological and pathophysiological conditions. They also discuss the use of systems modelling to discover new therapies to target mechanosignalling in heart disease.

Key points Mutations in the caveolae scaffolding protein, caveolin-3 (Cav3), have been linked to the long QT type 9 inherited arrhythmia syndrome (LQT9) and the cause of underlying action potential duration prolongation is incompletely understood. In the present study, we show that LQT9 Cav3 mutations, F97C and S141R, cause mutation-specific gain of function effects on Cav 1.2-encoded L-type Ca2+ channels responsible for ICa,L and also cause loss of function effects on heterologously expressed Kv 4.2 and Kv 4.3 channels responsible for Ito . A computational model of the human ventricular myocyte action potential suggests that the major ionic current change causing action potential duration prolongation in the presence of Cav3-F97C is the slowly inactivating ICa,L but, for Cav3-S141R, both increased ICa,L and increased late Na+ current contribute equally to action potential duration prolongation. Overall, the LQT9 Cav3-F97C and Cav3-S141R mutations differentially impact multiple ionic currents, highlighting the complexity of Cav3 regulation of cardiac excitability and suggesting mutation-specific therapeutic approaches. Abstract Mutations in the CAV3 gene encoding caveolin-3 (Cav3), a scaffolding protein integral to caveolae in cardiomyocytes, have been associated with the congenital long-QT syndrome (LQT9). Initial studies demonstrated that LQT9-associated Cav3 mutations, F97C and S141R, increase late sodium current as a potential mechanism to prolong action potential duration (APD) and cause LQT9. Whether these Cav3 LQT9 mutations impact other caveolae related ion channels remains unknown. We used the whole-cell, patch clamp technique to characterize the effect of Cav3-F97C and Cav3-S141R mutations on heterologously expressed Cav 1.2+Cav β2cN4 channels, as well as Kv 4.2 and Kv 4.3 channels, in HEK 293 cells. Expression of Cav3-S141R increased ICa,L density without changes in gating properties, whereas expression of Cav3-F97C reduced Ca2+ -dependent inactivation of ICa,L without changing current density. The Cav3-F97C mutation reduced current density and altered the kinetics of IKv4.2 and IKv4.3 and also slowed recovery from inactivation. Cav3-S141R decreased current density and also slowed activation kinetics and recovery from inactivation of IKv4.2 but had no effect on IKv4.3 . Using the O'Hara-Rudy computational model of the human ventricular myocyte action potential, the Cav3 mutation-induced changes in Ito are predicted to have negligible effect on APD, whereas blunted Ca2+ -dependent inactivation of ICa,L by Cav3-F97C is predicted to be primarily responsible for APD prolongation, although increased ICa,L and late INa by Cav3-S141R contribute equally to APD prolongation. Thus, LQT9 Cav3-associated mutations, F97C and S141R, produce mutation-specific changes in multiple ionic currents leading to different primary causes of APD prolongation, which suggests the use of mutation-specific therapeutic approaches in the future.

A human heart cell type–specific cis-regulatory element atlas reveals gene regulatory mechanisms of cardiovascular diseases., Misregulated gene expression in human hearts can result in cardiovascular diseases that are leading causes of mortality worldwide. However, the limited information on the genomic location of candidate cis-regulatory elements (cCREs) such as enhancers and promoters in distinct cardiac cell types has restricted the understanding of these diseases. Here, we defined >287,000 cCREs in the four chambers of the human heart at single-cell resolution, which revealed cCREs and candidate transcription factors associated with cardiac cell types in a region-dependent manner and during heart failure. We further found cardiovascular disease–associated genetic variants enriched within these cCREs including 38 candidate causal atrial fibrillation variants localized to cardiomyocyte cCREs. Additional functional studies revealed that two of these variants affect a cCRE controlling KCNH2/HERG expression and action potential repolarization. Overall, this atlas of human cardiac cCREs provides the foundation for illuminating cell type–specific gene regulation in human hearts during health and disease.

How ultrastructural alterations in cardiomyocytes lead to heart disease remains unclear. Therefore, there is interest in simulate cardiac myocytes with realistic 3-D models of myocyte shape and structure. Using deep learning and image processing, we developed an efficient method to generate 3-D models of cardiac myocytes without the need for manual segmentation. Image processing was used to segment borders between mitochondria in two series of serial-block-face scanning electron microscopy (SBF-SEM) images of healthy mouse ventricular cardiomyocytes. Computed mitochondrial size and morphologies were in good agreement with published measurements. These new automated methods will facilitate more realistic simulation studies of cardiac myocytes and enable new investigations of the links between altered mitochondrial morphology and heart diseases.

Mitochondrial morphological defects are a common feature of diseased cardiac myocytes. However, quantitative assessment of mitochondrial morphology is limited by the time-consuming manual segmentation of electron micrograph (EM) images. To advance understanding of the relation between morphological defects and dysfunction, an efficient morphological reconstruction method is desired to enable isolation and reconstruction of mitochondria from EM images. We propose a new method for isolating and reconstructing single mitochondria from serial block-face scanning EM (SBEM) images. CDeep3M, a cloud-based deep learning network for EM images, was used to segment mitochondrial interior volumes and boundaries. Post-processing was performed using both the predicted interior volume and exterior boundary to isolate and reconstruct individual mitochondria. Series of SBEM images from two separate cardiac myocytes were processed. The highest F1-score was 95% using 50 training datasets, greater than that for previously reported automated methods and comparable to manual segmentations. Accuracy of separation of individual mitochondria was 80% on a pixel basis. A total of 2315 mitochondria in the two series of SBEM images were evaluated with a mean volume of 0.78 µm3. The volume distribution was very broad and skewed; the most frequent mitochondria were 0.04–0.06 µm3, but mitochondria larger than 2.0 µm3 accounted for more than 10% of the total number. The average short-axis length was 0.47 µm. Primarily longitudinal mitochondria (0–30 degrees) were dominant (54%). This new automated segmentation and separation method can help quantitate mitochondrial morphology and improve understanding of myocyte structure–function relationships.