Association of epicardial and intramyocardial fat with ventricular arrhythmias.

BACKGROUND: Among patients with ischemic cardiomyopathy (ICM) and nonischemic cardiomyopathy (NICM), myocardial fibrosis is associated with an increased risk for ventricular arrhythmia (VA). Growing evidence suggests that myocardial fat contributes to ventricular arrhythmogenesis. However, little is known about the volume and distribution of epicardial adipose tissue and intramyocardial fat and their relationship with VAs. OBJECTIVE: The purpose of this study was to assess the association of contrast-enhanced computed tomography (CE-CT)-derived left ventricular (LV) tissue heterogeneity, epicardial adipose tissue volume, and intramyocardial fat volume with the risk of VA in ICM and NICM patients. METHODS: Patients enrolled in the PROSE-ICD registry who underwent CE-CT were included. Intramyocardial fat volume (voxels between -180 and -5 Hounsfield units [HU]), epicardial adipose tissue volume (between -200 and -50 HU), and LV tissue heterogeneity were calculated. The primary endpoint was appropriate ICD shocks or sudden arrhythmic death. RESULTS: Among 98 patients (47 ICM, 51 NICM), LV tissue heterogeneity was associated with VA (odds ratio [OR] 1.10; P = .01), particularly in the ICM cohort. In the NICM subgroup, epicardial adipose tissue and intramyocardial fat volume were associated with VA (OR 1.11, P = .01; and OR = 1.21, P = .01, respectively) but not in the ICM patients (OR 0.92, P =.22; and OR = 0.96, P =.19, respectively). CONCLUSION: In ICM patients, increased fat distribution heterogeneity is associated with VA. In NICM patients, an increased volume of intramyocardial fat and epicardial adipose tissue is associated with a higher risk for VA. Our findings suggest that fat's contribution to VAs depends on the underlying substrate.

Evaluation of a deep learning-enabled automated computational heart modelling workflow for personalized assessment of ventricular arrhythmias.

Personalized, image-based computational heart modelling is a powerful technology that can be used to improve patient-specific arrhythmia risk stratification and ventricular tachycardia (VT) ablation targeting. However, most state-of-the-art methods still require manual interactions by expert users. The goal of this study is to evaluate the feasibility of an automated, deep learning-based workflow for reconstructing personalized computational electrophysiological heart models to guide patient-specific treatment of VT. Contrast-enhanced computed tomography (CE-CT) images with expert ventricular myocardium segmentations were acquired from 111 patients across five cohorts from three different institutions. A deep convolutional neural network (CNN) for segmenting left ventricular myocardium from CE-CT was developed, trained and evaluated. From both CNN-based and expert segmentations in a subset of patients, personalized electrophysiological heart models were reconstructed and rapid pacing was used to induce VTs. CNN-based and expert segmentations were more concordant in the middle myocardium than in the heart's base or apex. Wavefront propagation during pacing was similar between CNN-based and original heart models. Between most sets of heart models, VT inducibility was the same, the number of induced VTs was strongly correlated, and VT circuits co-localized. Our results demonstrate that personalized computational heart models reconstructed from deep learning-based segmentations even with a small training set size can predict similar VT inducibility and circuit locations as those from expertly-derived heart models. Hence, a user-independent, automated framework for simulating arrhythmias in personalized heart models could feasibly be used in clinical settings to aid VT risk stratification and guide VT ablation therapy. KEY POINTS: Personalized electrophysiological heart modelling can aid in patient-specific ventricular tachycardia (VT) risk stratification and VT ablation targeting. Current state-of-the-art, image-based heart models for VT prediction require expert-dependent, manual interactions that may not be accessible across clinical settings. In this study, we develop an automated, deep learning-based workflow for reconstructing personalized heart models capable of simulating arrhythmias and compare its predictions with that of expert-generated heart models. The number and location of VTs was similar between heart models generated from the deep learning-based workflow and expert-generated heart models. These results demonstrate the feasibility of using an automated computational heart modelling workflow to aid in VT therapeutics and has implications for generalizing personalized computational heart technology to a broad range of clinical centres.

C-reactive Protein Elevation Is Associated With QTc Interval Prolongation in Patients Hospitalized With COVID-19.

Background: The relationship between inflammation and corrected QT (QTc) interval prolongation is currently not well defined in patients with COVID-19. Objective: This study aimed to assess the effect of marked interval changes in the inflammatory marker C-reactive protein (CRP) on QTc interval in patients hospitalized with COVID-19. Methods: In this retrospective cohort study of hospitalized adult patients admitted with COVID-19 infection, we identified 85 patients who had markedly elevated CRP levels and serial measurements of an ECG and CRP during the same admission. We compared mean QTc interval duration, and other clinical and ECG characteristics between times when CRP values were high and low. We performed mixed-effects linear regression analysis to identify associations between CRP levels and QTc interval in univariable and adjusted models. Results: Mean age was 58 ± 16 years, of which 39% were women, 41% were Black, and 25% were White. On average, the QTc interval calculated via the Bazett formula was 15 ms higher when the CRP values were "high" vs. "low" [447 ms (IQR 427-472 ms) and 432 ms (IQR 412-452 ms), respectively]. A 100 mg/L increase in CRP was associated with a 1.5 ms increase in QTc interval [ß coefficient 0.15, 95% CI (0.06-0.24). In a fully adjusted model for sociodemographic, ECG, and clinical factors, the association remained significant (ß coefficient 0.14, 95% CI 0.05-0.23). Conclusion: An interval QTc interval prolongation is observed with a marked elevation in CRP levels in patients with COVID-19.

Association of left ventricular tissue heterogeneity and intramyocardial fat on computed tomography with ventricular arrhythmias in ischemic cardiomyopathy.

Background: Gray zone, a measure of tissue heterogeneity on late gadolinium enhanced-cardiac magnetic resonance (LGE-CMR) imaging, has been shown to predict ventricular arrhythmias (VAs) in ischemic cardiomyopathy (ICM) patients. However, no studies have described whether left ventricular (LV) tissue heterogeneity and intramyocardial fat mass on contrast-enhanced computed tomography (CE-CT), which provides greater spatial resolution, is useful for assessing the risk of VAs in ICM patients with LV systolic dysfunction and no previous VAs. Objective: The purpose of this proof-of-concept study was to determine the feasibility of measuring global LV tissue heterogeneity and intramyocardial fat mass by CE-CT for predicting the risk of VAs in ICM patients with LV systolic dysfunction and no previous history of VAs. Methods: Patients with left ventricular ejection fraction ≤35% and no previous VAs were enrolled in a prospective, observational registry and underwent LGE-CMR. From this cohort, patients with ICM who additionally received CE-CT were included in the present analysis. Gray zone on LGE-CMR was defined as myocardium with signal intensity (SI) > peak SI of healthy myocardium but <50% maximal SI. Tissue heterogeneity on CE-CT was defined as the standard deviation of the Hounsfield unit image gradients (HU/mm) within the myocardium. Intramyocardial fat on CE-CT was identified as regions of image pixels between -180 and -5 HU. The primary outcome was VAs, defined as appropriate implantable cardioverter-defibrillator shock or sudden arrhythmic death. Results: The study consisted of 47 ICM patients, 13 (27.7%) of whom experienced VA events during mean follow-up of 5.6 ± 3.4 years. Increasing tissue heterogeneity (per HU/mm) was significantly associated with VAs after multivariable adjustment, including for gray zone (odds ratio [OR] 1.22; P = .019). Consistently, patients with tissue heterogeneity values greater than or equal to the median (≥22.2 HU/mm) had >13-fold significantly increased risk of VA events, relative to patients with values lower than the median, after multivariable adjustment that included gray zone (OR 13.13; P = .028). The addition of tissue heterogeneity to gray zone improved prediction of VAs (area under receiver operating characteristic curve increased from 0.815 to 0.876). No association was found between intramyocardial fat mass on CE-CT and VAs (OR 1.00; P = .989). Conclusion: In ICM patients, CE-CT-derived LV tissue heterogeneity was independently associated with VAs and may represent a novel marker useful for risk stratification.

HDL: Fact, fiction, or function? HDL cholesterol and cardiovascular risk.

The measurement of high-density lipoprotein cholesterol is highly utilized by clinicians to help predict cardiovascular risk, but this measure is not causally associated with atherosclerotic cardiovascular disease events. The use of Mendelian randomization studies has led to a change in investigative attention from the high-density lipoprotein cholesterol concentration to its physiological functions. High-density lipoprotein plays key roles in important pathways related to the development of atherosclerotic disease including reverse cholesterol transport, oxidation and inflammation, and endothelial function as well as in other physiological systems including immune system modulation, cellular apoptosis, and endothelial progenitor cell homeostasis. The identification of dysfunctional high-density lipoprotein may better predict future cardiovascular events compared to numerical high-density lipoprotein cholesterol and aid in enhanced clinical risk stratification. The emergence of discrete physiological measurements of high-density lipoprotein, such as cholesterol efflux capacity and the high-density lipoprotein inflammatory index, may provide an opportunity for clinical application in the future. However, the validity of these measurements and their commercial availability remain barriers to a realistic transition to clinical medicine.

Immunotherapy for the prevention of atherosclerotic cardiovascular disease: Promise and possibilities.

Cardiovascular disease remains the leading cause of death worldwide with coronary atherosclerotic heart disease being the largest contributor. The mechanisms behind the presence and progression of atherosclerosis remain an area of intense scientific focus. Immune dysregulation and inflammation are key contributors to the development of an atherosclerotic plaque and its progression to acute coronary syndromes. Increased circulating levels of biomarkers of systemic inflammation including hsCRP are correlated with a higher cardiovascular risk. Targeting specific inflammatory pathways implicated in atherosclerotic plaque formation is an exciting area of ongoing research. Target specific therapies directed at pro-inflammatory cytokines such as IL-1ß, IL-6, TNFα, and CCL2 have demonstrated slowing in the progression of atherosclerosis in animal models and improved cardiovascular outcomes in human subjects. Most notably, treatment with the monoclonal antibody canakinumab, which directly targets and neutralizes IL-1ß, was recently shown to be associated with reduced risk of adverse cardiovascular events compared to placebo in a randomized, placebo-controlled trial. Several other therapies including colchicine, methotrexate and leukotriene inhibitors demonstrate the potential for lowering cardiovascular risk through immunomodulation, though further studies are needed. Understanding the role of inflammation in atherosclerosis and the development of targeted immunotherapies continues to be an evolving area of research that is rapidly becoming clinically relevant for the 21st century cardiac patient.

Generation and primary characterization of iAM-1, a versatile new line of conditionally immortalized atrial myocytes with preserved cardiomyogenic differentiation capacity.

Aims: The generation of homogeneous cardiomyocyte populations from fresh tissue or stem cells is laborious and costly. A potential solution to this problem would be to establish lines of immortalized cardiomyocytes. However, as proliferation and (terminal) differentiation of cardiomyocytes are mutually exclusive processes, their permanent immortalization causes loss of electrical and mechanical functions. We therefore aimed at developing conditionally immortalized atrial myocyte (iAM) lines allowing toggling between proliferative and contractile phenotypes by a single-component change in culture medium composition. Methods and results: Freshly isolated neonatal rat atrial cardiomyocytes (AMs) were transduced with a lentiviral vector conferring doxycycline (dox)-controlled expression of simian virus 40 large T antigen. Under proliferative conditions (i.e. in the presence of dox), the resulting cells lost most cardiomyocyte traits and doubled every 38 h. Under differentiation conditions (i.e. in the absence of dox), the cells stopped dividing and spontaneously reacquired a phenotype very similar to that of primary AMs (pAMs) in gene expression profile, sarcomeric organization, contractile behaviour, electrical properties, and response to ion channel-modulating compounds (as assessed by patch-clamp and optical voltage mapping). Moreover, differentiated iAMs had much narrower action potentials and propagated them at >10-fold higher speeds than the widely used murine atrial HL-1 cells. High-frequency electrical stimulation of confluent monolayers of differentiated iAMs resulted in re-entrant conduction resembling atrial fibrillation, which could be prevented by tertiapin treatment, just like in monolayers of pAMs. Conclusion: Through controlled expansion and differentiation of AMs, large numbers of functional cardiomyocytes were generated with properties superior to the differentiated progeny of existing cardiomyocyte lines. iAMs provide an attractive new model system for studying cardiomyocyte proliferation, differentiation, metabolism, and (electro)physiology as well as to investigate cardiac diseases and drug responses, without using animals.

Islands of spatially discordant APD alternans underlie arrhythmogenesis by promoting electrotonic dyssynchrony in models of fibrotic rat ventricular myocardium.

Fibrosis and altered gap junctional coupling are key features of ventricular remodelling and are associated with abnormal electrical impulse generation and propagation. Such abnormalities predispose to reentrant electrical activity in the heart. In the absence of tissue heterogeneity, high-frequency impulse generation can also induce dynamic electrical instabilities leading to reentrant arrhythmias. However, because of the complexity and stochastic nature of such arrhythmias, the combined effects of tissue heterogeneity and dynamical instabilities in these arrhythmias have not been explored in detail. Here, arrhythmogenesis was studied using in vitro and in silico monolayer models of neonatal rat ventricular tissue with 30% randomly distributed cardiac myofibroblasts and systematically lowered intercellular coupling achieved in vitro through graded knockdown of connexin43 expression. Arrhythmia incidence and complexity increased with decreasing intercellular coupling efficiency. This coincided with the onset of a specialized type of spatially discordant action potential duration alternans characterized by island-like areas of opposite alternans phase, which positively correlated with the degree of connexinx43 knockdown and arrhythmia complexity. At higher myofibroblast densities, more of these islands were formed and reentrant arrhythmias were more easily induced. This is the first study exploring the combinatorial effects of myocardial fibrosis and dynamic electrical instabilities on reentrant arrhythmia initiation and complexity.

Forced fusion of human ventricular scar cells with cardiomyocytes suppresses arrhythmogenicity in a co-culture model.

AIMS: Fibrosis increases arrhythmogenicity in myocardial tissue by causing structural and functional disruptions in the cardiac syncytium. Forced fusion of fibroblastic cells with adjacent cardiomyocytes may theoretically resolve these disruptions. Therefore, the electrophysiological effects of such electrical and structural integration of fibroblastic cells into a cardiac syncytium were studied. METHODS AND RESULTS: Human ventricular scar cells (hVSCs) were transduced with lentiviral vectors encoding enhanced green fluorescent protein alone (eGFP↑-hVSCs) or together with the fusogenic vesicular stomatitis virus G protein (VSV-G/eGFP↑-hVSCs) and subsequently co-cultured (1:4 ratio) with neonatal rat ventricular cardiomyocytes (NRVMs) in confluent monolayers yielding eGFP↑- and VSV-G/eGFP↑-co-cultures, respectively. Cellular fusion was induced by brief exposure to pH = 6.0 medium. Optical mapping experiments showed eGFP↑-co-cultures to be highly arrhythmogenic [43.3% early afterdepolarization (EAD) incidence vs. 7.7% in control NRVM cultures, P < 0.0001], with heterogeneous prolongation of action potential (AP) duration (APD). Fused VSV-G/eGFP↑-co-cultures displayed markedly lower EAD incidence (4.6%, P < 0.001) than unfused co-cultures, associated with decreases in APD, APD dispersion, and decay time of cytosolic Ca(2+) waves. Heterokaryons strongly expressed connexin43 (Cx43). Also, maximum diastolic potential in co-cultures was more negative after fusion, while heterokaryons exhibited diverse mixed NRVM/hVSC whole-cell current profiles, but consistently showed increased outward Kv currents compared with NRVMs or hVSCs. Inhibition of Kv channels by tetraethylammonium chloride abrogated the anti-arrhythmic effects of fusion in VSV-G/eGFP↑-co-cultures raising EAD incidence from 7.9 to 34.2% (P < 0.001). CONCLUSION: Forced fusion of cultured hVSCs with NRVMs yields electrically functional heterokaryons and reduces arrhythmogenicity by preventing EADs, which is, at least partly, attributable to increased repolarization force.

Fetal Mammalian Heart Generates a Robust Compensatory Response to Cell Loss.

BACKGROUND: Heart development is tightly regulated by signaling events acting on a defined number of progenitor and differentiated cardiac cells. Although loss of function of these signaling pathways leads to congenital malformation, the consequences of cardiac progenitor cell or embryonic cardiomyocyte loss are less clear. In this study, we tested the hypothesis that embryonic mouse hearts exhibit a robust mechanism for regeneration after extensive cell loss. METHODS AND RESULTS: By combining a conditional cell ablation approach with a novel blastocyst complementation strategy, we generated murine embryos that exhibit a full spectrum of cardiac progenitor cell or cardiomyocyte ablation. Remarkably, ablation of up to 60% of cardiac progenitor cells at embryonic day 7.5 was well tolerated and permitted embryo survival. Ablation of embryonic cardiomyocytes to a similar degree (50% to 60%) at embryonic day 9.0 could be fully rescued by residual myocytes with no obvious adult cardiac functional deficit. In both ablation models, an increase in cardiomyocyte proliferation rate was detected and accounted for at least some of the rapid recovery of myocardial cellularity and heart size. CONCLUSION: Our study defines the threshold for cell loss in the embryonic mammalian heart and reveals a robust cardiomyocyte compensatory response that sustains normal fetal development.

Light-induced termination of spiral wave arrhythmias by optogenetic engineering of atrial cardiomyocytes.

AIMS: Atrial fibrillation (AF) is the most common cardiac arrhythmia and often involves reentrant electrical activation (e.g. spiral waves). Drug therapy for AF can have serious side effects including proarrhythmia, while electrical shock therapy is associated with discomfort and tissue damage. Hypothetically, forced expression and subsequent activation of light-gated cation channels in cardiomyocytes might deliver a depolarizing force sufficient for defibrillation, thereby circumventing the aforementioned drawbacks. We therefore investigated the feasibility of light-induced spiral wave termination through cardiac optogenetics. METHODS AND RESULTS: Neonatal rat atrial cardiomyocyte monolayers were transduced with lentiviral vectors encoding light-activated Ca(2+)-translocating channelrhodopsin (CatCh; LV.CatCh∼eYFP↑) or eYFP (LV.eYFP↑) as control, and burst-paced to induce spiral waves rotating around functional cores. Effects of CatCh activation on reentry were investigated by optical and multi-electrode array (MEA) mapping. Western blot analyses and immunocytology confirmed transgene expression. Brief blue light pulses (10 ms/470 nm) triggered action potentials only in LV.CatCh∼eYFP↑-transduced cultures, confirming functional CatCh-mediated current. Prolonged light pulses (500 ms) resulted in reentry termination in 100% of LV.CatCh∼eYFP↑-transduced cultures (n = 31) vs. 0% of LV.eYFP↑-transduced cultures (n = 11). Here, CatCh activation caused uniform depolarization, thereby decreasing overall excitability (MEA peak-to-peak amplitude decreased 251.3 ± 217.1 vs. 9.2 ± 9.5 µV in controls). Consequently, functional coresize increased and phase singularities (PSs) drifted, leading to reentry termination by PS-PS or PS-boundary collisions. CONCLUSION: This study shows that spiral waves in atrial cardiomyocyte monolayers can be terminated effectively by a light-induced depolarizing current, produced by the arrhythmogenic substrate itself, upon optogenetic engineering. These results provide proof-of-concept for shockless defibrillation.

Insulin-like growth factor promotes cardiac lineage induction in vitro by selective expansion of early mesoderm.

A thorough understanding of the developmental signals that direct pluripotent stem cells (PSCs) toward a cardiac fate is essential for translational applications in disease modeling and therapy. We screened a panel of 44 cytokines/signaling molecules for their ability to enhance Nkx2.5(+) cardiac progenitor cell (CPC) formation during in vitro embryonic stem cell (ESC) differentiation. Treatment of murine ESCs with insulin or insulin-like growth factors (IGF1/2) during early differentiation increased mesodermal cell proliferation and, consequently, CPC formation. Furthermore, we show that downstream mediators of IGF signaling (e.g., phospho-Akt and mTOR) are required for this effect. These data support a novel role for IGF family ligands to expand the developing mesoderm and promote cardiac differentiation. Insulin or IGF treatment could provide an effective strategy to increase the PSC-based generation of CPCs and cardiomyocytes for applications in regenerative medicine.

Inefficient reprogramming of fibroblasts into cardiomyocytes using Gata4, Mef2c, and Tbx5.

RATIONALE: Direct reprogramming of fibroblasts into cardiomyocytes is a novel strategy for cardiac regeneration. However, the key determinants involved in this process are unknown. OBJECTIVE: To assess the efficiency of direct fibroblast reprogramming via viral overexpression of GATA4, Mef2c, and Tbx5 (GMT). METHODS AND RESULTS: We induced GMT overexpression in murine tail tip fibroblasts (TTFs) and cardiac fibroblasts (CFs) from multiple lines of transgenic mice carrying different cardiomyocyte lineage reporters. We found that the induction of GMT overexpression in TTFs and CFs is inefficient at inducing molecular and electrophysiological phenotypes of mature cardiomyocytes. In addition, transplantation of GMT infected CFs into injured mouse hearts resulted in decreased cell survival with minimal induction of cardiomyocyte genes. CONCLUSIONS: Significant challenges remain in our ability to convert fibroblasts into cardiomyocyte-like cells and a greater understanding of cardiovascular epigenetics is needed to increase the translational potential of this strategy.

Reprogramming of mouse, rat, pig, and human fibroblasts into iPS cells.

The induction of pluripotency in somatic cells by transcription-factor overexpression has been widely regarded as one of the major breakthroughs in stem cell biology within this decade. The generation of these induced pluripotent stem cells (iPSCs) has enabled investigators to develop in vitro disease models for biological discovery and drug screening, and in the future, patient-specific therapy for tissue or organ regeneration. While new technologies for reprogramming are continually being discovered, the availability of iPSCs from different species is also increasing rapidly. Comparison of iPSCs across species may provide new insights into key aspects of pluripotency and early embryonic development. iPSCs from large animals may enable the generation of genetically modified large animal models or potentially transplantable donor tissues or organs. This unit describes the procedure for the generation of iPSCs from mouse, rat, pig and human fibroblasts.