Cardiotoxic Drugs Research Articles

Cancer survivors and cardiovascular diseases: from preventive strategies to treatment.

During the last decades, progress in the treatment of oncological diseases has led to an increase in the survival of cancer patients: cancer survivors (CS). Thus, the incidence of CS has increased enormously, in both adult CS and childhood and adolescent CS. Unfortunately, CS treated with anthracyclines, chest radiotherapy (RT) and other potentially cardiotoxic drugs have a higher risk of cardiovascular (CV) toxicity: heart failure with reduced ejection fraction (HFrEF), valve diseases, coronary artery diseases, vascular diseases and pericardial diseases. In fact, chest irradiation can cause coronary artery diseases that can be latent until at least 10 years after exposure; also, valvular heart diseases can appear after >20 years following irradiation; heart failure may appear later, several years after anticancer drugs or RT. Therefore, it is very important to stratify the CV risk of cancer patients at the end of cardiotoxic drugs, to plan the most appropriate long-term surveillance program, in accordance with 2022 ESC Guidelines on Cardio-Oncology, to prevent late cardiovascular complications. Monitoring of cancer patients must not stop during anticancer treatment but it must continue afterwards, depending on the patient's CV risk. CV toxicity risk should be reassessed 5 years after therapy to organize long-term follow-up. Considering late cardiotoxicity in CS, our review aims to evaluate the incidence of cardiovascular diseases in CS, their mechanisms, surveillance protocols, preventive strategies, diagnosis and treatment.

Immune-checkpoint myocarditis phenotype when using systematic troponin surveillance in cancer patients.

Abstract Background ICI myocarditis, often described as potentially fulminant in registries from specialized centers, lacks sufficient data on its subclinical forms. ESC 2022 guidelines advocate systematic troponin measurements for early detection, resulting in numerous suspected cases of ICI myocarditis. Additionally, many patients receive other potentially cardiotoxic cancer drugs (e.g.,anthracyclines), adding complexity to the management. Purpose To identify factors associated with ICI-myocarditis diagnosis in a single-center unselected cohort referred for suspicion of CV immune-related adverse events(irAEs). Patients underwent standardized evaluation, including serum testing, ECG, echocardiogram, cardiac MRI(CMRi) and multidisciplinary team assessment including cardiology and internal medicine specialists. Coronary imaging and myocardial biopsy (EMB) were performed as needed. ICI-myocarditis diagnosis was adjudicated by expert panel (FC, MM, SH) based on Dallas, ESC, and IC-OS criteria. Results We enrolled 175 consecutive patients from March 2022 to October 2023; 40 (22.89%) were male, with a median age of 61 (IQR 48.0-72.0) years. 92(52.57%) were referred for lone asymptomatic troponin raise, 101 (57.7%) had breast cancer; 142 (81.11%) received other cardiotoxic drugs. We identified 95 (61.71%) patients with CV irAEs at referral (8 had >=2 CV irAEs) with a total of 108 CV irAEs after follow up, including 72 (41.14%) with ICI-myocarditis at referall. No CV deaths occurred within 30 days from referall. Characteristics and univariate analysis are depicted in the Table1. Reasons for referral in the ICI-myocarditis population are depicted in the Figure1. On multivariate regression analysis, prior CV disease history (OR 6.232 [CI 1.204-32.260], p=0.029), EMB (OR 24.969 [5.139-121.32], p=<.0001), IC-OS criteria fulfilled (OR 43.108 [9.222-201.510], p=<.0001), and positive Lake Louise criteria on CMRi (OR 6.583[1.416-30.610], p=0.0163) were associated with myocarditis. In a sub-analysis group of breast cancer patients (largely receiving anthracyclines), ICI-myocarditis was associated on multivariate regression with abnormal ECG (OR 14.227 [CI 1.632-124.040], p=< 0.0163) and EMB (OR 8.508 [CI 2.718-26.638], p=0.0002). The same characteristics were significantly associated with other CV irAEs in the group of breast cancer patients. In the group of patients with cancers other than breast, factors significantly associated with ICI myocarditis diagnosis were: EMB (OR 70.656 [CI 5.970-836.161], 0.0007), and no arterial hypertension (OR 9.554 [CI 1.792-50.940], p= 0.0082). Conclusions The FAST TRACK study highlights the high proportion of patients fulfilling ICI-myocarditis criteria despite no or mild symptoms, mainly referred for troponin elevation. However, subclinical ICI-myocarditis appears to bear good prognosis. Endomyocardial biopsy is key to increase the diagnosis rate of ICI-myocarditis across different cancer populations when mildly symptomatic.Table 1Figure 1

Is there an association of anthracyclines with HFrEF and/or HFpEF in patients treated for breast cancer or lymphoma after 30 years of follow-up? Insights from the Rochester epidemiology project

Abstract Introduction Anthracyclines (ACs) have been identified as cardiotoxic drugs. They are associated with contractile dysfunction of the heart, which can manifest as heart failure. However, differentiating between HFrEF (left ventricular ejection fractions [LVEF] ≤40%) or HFpEF (LVEF >40%) has been overlooked. Purpose Using a retrospective cohort of patients with breast cancer (BC), Hodgkin (HL) or non-Hodgkin lymphoma (NHL) and controls from the Rochester Epidemiology Project, we evaluated the association of ACs use with risk of HFrEF and/or HFpEF. Methods 797 subjects with BC, HL or NHL diagnosed from 1988 to 2010 were identified, 658 treated with ACs (ACs group) and 139 not treated with ACs (no-ACs group). Controls (n=1526) were subjects without cancer. The median (Q1, Q3) follow up was 11.5 years (7.4, 16.7). Cox proportional hazards regression was used to compare the risk of HFpEF and HFrEF in the three cohorts, adjustment for sex, CV comorbidities (type 2 diabetes [T2D], hypertension [HTN] and dyslipidemia), radiotherapy (RT), and ACs cumulative dose was performed. Results Baseline characteristics are shown in table 1. HFpEF occurred in 29/1526 subjects in the control group, 3/139 in the no-ACs group and in 14/658 in the ACs group. Compared to the control group, there was not an increased risk of developing HFpEF in the no-ACs group (HR 1.67, CI 95%: 0.5 – 5.53, p = 0.39) or in the ACs group (HR 1.83, CI 95%: 0.95 – 3.52, p = 0.06) (Figure 1). Compared with no-ACs group, the ACs group had a similar risk of developing HFpEF (HR 1.09, CI 95%: 0.3 – 3.87, p = 0.88). RT was associated with a lower risk of HFpEF (HR 0.21, CI 95%: 0.06 – 0.75, p = 0.01). In the multivariable analysis, T2D (HR 6.91, CI 95%: 3.07-15.56, p = <0.0001) was associated with higher risk of HFpEF. A cumulative dose of ACs > 250 mg/m2 was not associated with increased risk of HFpEF. HFrEF occurred in 25/1526 subjects in the control group, in 4/139 in the no-ACs group and in 31/658 in the ACs group. Compared to the control group, the ACs group had a significant increase in the risk of HFrEF (HR 4.77, CI 95%: 2.76 – 8.22, p < 0.0001), the no-ACs group did not (HR 2.81, CI 95%: 0.97 – 8.12, p = 0.055) (figure 2). RT was associated with lower risk of HFrEF (HR 0.29, CI 95%: 0.13 – 0.65, p = 0.002). In the multivariable analysis, ACs (HR 4.23, CI 95%: 2.4-7.47, p = < 0.0001) being male (HR 2.45, CI 95%: 1.43 – 4.15, p < 0.0001), smoking history (HR 2, CI 95%: 1.16 – 3.44, p < 0.01), and HTN (HR 3.23, CI 95%: 1.9 – 5.61, p < 0.0001), were associated with higher risk of HFrEF. A cumulative dose of ACs > 250 mg/m2 was not associated with increased risk of HFrEF. Conclusion To our knowledge this is the first study to differentiate between HFpEF and HFrEF in this cohort. ACs were not associated with increased risk of HFpEF. However, ACs were associated with increased risk of HFrEF, but not with a specific dose. These results should be validated in other population cohorts.

Development of a cardiotoxicity evaluation system using a Heart-on-a-Chip Microdevice with aligned fiber device

Abstract Background and Purpose Cardiovascular cells differentiated from human induced pluripotent stem (iPS) cells, such as cardiomyocytes, are expected to be useful for evaluating cardiac pharmacology and toxicity in humans. In recent years, myocardial damage caused by anticancer drugs has begun to attract attention. Being able to evaluate human cardiotoxicity early in the development of novel drugs like anticancer agents confers advantages in terms of cost and development speed. The aim of this study was to develop a highly sensitive bioassay system that can evaluate the physiological function of the human heart, and to validate the efficacy of the system toward detection of cardiotoxicity of drugs. Methods We differentiated human iPS cells into cardiovascular cells (cardiomyocytes, endothelial cells and mural cells) and applied dynamic culture training to generate vascularized microtissues (VCM) with vascular network structures. By integrating this VCM with a microfluidic chip containing microchannels, we developed a heart-on-a-chip microdevice (HMD) specialized for evaluating cardiac function (Figure 1). Furthermore, through a collaborative study with the Stem Cell & Device Laboratory (SCAD), application of their aligned fiber device improved throughput and stability, allowing development of a SCAD-HMD capable of evaluating longer-term cardiotoxicity. Results SCAD-HMD can quantify physiological parameters of VCM such as ejection volume and force based on displacement of particles within the microchannels. Our results showed that SCAD-HMD not only exhibited pharmacological responses to representative circulatory stimulants, but also reproduced clinical events occurring in actual practice, demonstrating that the anticancer drug trastuzumab, commonly used for breast cancer treatment, causes myocardial disfunction when combined with doxorubicin. Conclusions This study demonstrated that SCAD-HMD can stably evaluate the toxicity of drugs on the human heart. In the future, establishing SCAD-HMD using iPS cells derived from individual patients and assessing responses to specific drugs may enable "individualized cardiotoxicity assessment systems" that can preemptively avoid cardiac complications associated with anticancer drugs and other pharmacological agents.

The hanging‐heart chip: A portable microfluidic device for high‐throughput generation of contractile embryonic stem cell‐derived cardiac spheroids

AbstractStem cell‐derived cardiac spheroids are promising models for cardiac research and drug testing. However, generating contracting cardiac spheroids remains challenging because of the laborious experimental procedure. Here, we present a microfluidic hanging‐heart chip (HH‐chip) that uses a microchannel and flow‐driven system to facilitate cell loading and culture medium replacement operations to reduce the laborious manual handling involved in the generation of a large quantity of cardiac spheroids. The effectiveness of the HH‐chip was demonstrated by simultaneously forming 50 mouse embryonic stem cell‐derived embryonic bodies, which sequentially differentiated into 90% beating cardiac spheroids within 15 days of culture on the chip. A comparison of our HH‐chip method with traditional hanging‐drop and low‐attachment plate methods revealed that the HH‐chip could generate higher contracting proportions of cardiac spheroids with higher expression of cardiac markers. Additionally, we verified that the contraction frequencies of the cardiac spheroids generated from the HH‐chip were sensitive to cardiotoxic drugs. Overall, our results suggest that the microfluidic hanging drop chip‐based approach is a high‐throughput and highly efficient method for generating contracting mouse embryonic stem cell‐derived cardiac spheroids for cardiac toxicity and drug testing applications.

Extracorporeal membrane oxygenation in cardiovascular medication poisoning. A German-wide retrospective study

Medication poisoning, resulting from the ingestion of cardiotoxic drugs, presents a significant health issue. The mortality rate remains high for patients with myocardial dysfunction refractory to conventional treatments. Venoarterial Extracorporeal Membrane Oxygenation (V-A ECMO) provides temporary support, potentially enhancing patient outcomes. This study aims to assess the efficacy of V-A ECMO in treating cardiovascular failure induced by cardiovascular medication poisoning. We utilized inpatient data from all hospitalisations in Germany from 2007 to 2022 due to cardiovascular medication poisoning treated with V-A ECMO. Patient characteristics, comorbidities, complications and application of ECMO were described descriptively and analysed for statistical significance between survivors and non-survivors. Overall, 49 patients received V-A ECMO for cardiovascular medication poisoning, with a survival rate of 63.6%. The most ingested medications were calcium-channel blockers (38.8%) and beta-adrenoceptor antagonists (34.7%). Half of non-survivors received in-hospital CPR, compared to 12.9% of survivors. Early ECMO implantation (within 24 h of admission) was common (83.7%) but did not significantly impact survival rates. A substantial number of patients presented with multiple substances ingested. V-A ECMO represents a viable option for patients experiencing cardiac failure due to medication poisoning. A structured implementation of V-A ECMO for cardiovascular medication poisoning could lead to higher survival rates.

Ferroptosis-induced Cardiotoxicity and Antitumor Drugs.

The induction of regulated cell death ferroptosis in tumors is emerging as an intriguing strategy for cancer treatment. Numerous antitumor drugs (e.g., doxorubicin, etoposide, tyrosine kinase inhibitors, trastuzumab, arsenic trioxide, 5-fluorouracil) induce ferroptosis. Although this mechanism of action is interesting for fighting tumors, the clinical use of drugs that induce ferroptosis is hampered by cardiotoxicity. Besides in cancer cells, ferroptosis induced by chemotherapeutics can occur in cardiomyocytes, and this feature represents an important drawback of antitumor therapy. This inconvenience has been tackled by developing less or no cardiotoxic antitumor drugs or by discovering cardioprotective agents (e.g., berberine, propofol, fisetin, salidroside, melatonin, epigallocatechin- 3gallate, resveratrol) to use in combination with conventional chemotherapeutics. This review briefly summarizes the molecular mechanisms of ferroptosis and describes the ferroptosis dependent mechanisms responsible for cardiac toxicity developed by cancer- suffering patients following the administration of some chemotherapeutics. Additionally, the pharmacological strategies very recently proposed for potentially preventing this inconvenience are considered.

Multiscale mapping of transcriptomic signatures for cardiotoxic drugs

Drug-induced gene expression profiles can identify potential mechanisms of toxicity. We focus on obtaining signatures for cardiotoxicity of FDA-approved tyrosine kinase inhibitors (TKIs) in human induced-pluripotent-stem-cell-derived cardiomyocytes, using bulk transcriptomic profiles. We use singular value decomposition to identify drug-selective patterns across cell lines obtained from multiple healthy human subjects. Cellular pathways affected by cardiotoxic TKIs include energy metabolism, contractile, and extracellular matrix dynamics. Projecting these pathways to published single cell expression profiles indicates that TKI responses can be evoked in both cardiomyocytes and fibroblasts. Integration of transcriptomic outlier analysis with whole genomic sequencing of our six cell lines enables us to correctly reidentify a genomic variant causally linked to anthracycline-induced cardiotoxicity and predict genomic variants potentially associated with TKI-induced cardiotoxicity. We conclude that mRNA expression profiles when integrated with publicly available genomic, pathway, and single cell transcriptomic datasets, provide multiscale signatures for cardiotoxicity that could be used for drug development and patient stratification.

Haemodynamic Forces: Emerging Markers of Ventricular Remodelling in Multiple Myeloma Cardiovascular Baseline Risk Assessment.

Multiple myeloma (MM) affects a population with a high prevalence of cardiovascular (CV) disease. These patients benefit from an accurate CV risk evaluation in order to choose the safest drug regimen. Haemodynamic forces (HDFs) analysis allows for the earlier detection of myocardial damage compared with standard markers; the role played by MM in HDFs alteration, with or without the influence of hypertension, is yet to be studied. Therefore, we aimed to identify differences in HDFs analysis in patients with MM, hypertension or both versus normotensive non-oncologic subjects. A total of 173 patients (MM hypertensive patients, MMHT; MM normotensive patients, MMNT; non-oncologic hypertensive patients, CoHT; and non-oncologic normotensive patients, CoNT) underwent transthoracic echocardiography for HDFs analysis and pulse wave velocity (PWV) assessment. Hypertensive patients (MMHT, CoHT) showed decreased ejection fraction (EF), global longitudinal strain (GLS) and HDFs values compared with CoNT, whereas ventricular mass (LVMi) and PWV increased. MMNT displayed a significant reduction in systolic HDFs (p < 0.006) and systolic ejection HDFs (p < 0.008) compared with CoNT, without significant change in EF, GLS, LVMi or PWV. In conclusion, MM leads to ventricular remodelling regardless of hypertension; HDFs application for MM patients could help detect early myocardial damage, especially in patients receiving cardiotoxic drugs.

Zebrafish as a rapid model system for early cardiotoxicity assessment of drugs

ObjectiveThere are some problems in the evaluation of drug cardiotoxicity in zebrafish, such as unsystematic indicators and weak sensitivity. This study aims to explore the advantages and disadvantages of various evaluation methods in rapid cardiotoxicity assessment, and to identify and establish representative and highly sensitive evaluation indicators. MethodsFour typical cardiotoxic drugs (Doxorubicin, 5-Fluorouracil, Ibuprofen and Lidocaine) with different concentrations were selected to act on zebrafish embryos. The death, cardiac malformation, heart rate, blood flow and sinus venosus-bulbus arteriosus (SV-BA) distance of zebrafish in each group were observed and recorded by stereo microscopy. The behavioral changes of zebrafish after drug treatment were counted and analyzed using a zebrafish behavior recorder. Adult zebrafish were used to screen the cardiotoxic expressed genes, and the spatiotemporal expression stability and concentration-dose dependence of the specifically highly expressed genes were studied. ResultsAll the 4 drugs can reduce the heart rate and blood flow velocity of zebrafish embryos to varying degrees, increase the SV-BA distance of zebrafish embryos, reduce the activity behavior of zebrafish embryos, and have different degrees of inhibitory effects on the cardiac function of zebrafish. Among the selected 12 genes expressed only in the heart, one and only CMLC1 showed high specific expression in the heart of zebrafish. However, doxorubicin did not affect the expression of CMLC1 gene in a concentration-dose dependent manner, and the other three drugs could significantly induce the up-regulation of CMLC1 gene expression. ConclusionEthology is a faster and more sensitive method than the traditional cardiotoxicity evaluation indicators (heart rate, blood flow velocity, and SV-BA distance). Zebrafish have a small number of highly heart-specific expressed genes and are not suitable for assessing cardiotoxicity based solely on heart-specific expression of circulating mRNA.

Semisupervised Learning to Boost hERG, Nav1.5, and Cav1.2 Cardiac Ion Channel Toxicity Prediction by Mining a Large Unlabeled Small Molecule Data Set.

Predicting drug toxicity is a critical aspect of ensuring patient safety during the drug design process. Although conventional machine learning techniques have shown some success in this field, the scarcity of annotated toxicity data poses a significant challenge in enhancing models' performance. In this study, we explore the potential of leveraging large unlabeled small molecule data sets using semisupervised learning to improve drug cardiotoxicity predictive performance across three cardiac ion channel targets: the voltage-gated potassium channel (hERG), the voltage-gated sodium channel (Nav1.5), and the voltage-gated calcium channel (Cav1.2). We extensively mined the ChEMBL database, comprising approximately 2 million small molecules, and then employed semisupervised learning to construct robust classification models for this purpose. We achieved a performance boost on highly diverse (i.e., structurally dissimilar) test data sets across all three targets. Using our built models, we screened the whole ChEMBL database and a large set of FDA-approved drugs, identifying several compounds with potential cardiac ion channel activity. To ensure broad accessibility and usability for both technical and nontechnical users, we developed a cross-platform graphical user interface that allows users to make predictions and gain insights into the cardiotoxicity of drugs and other small molecules. The software is made available as open source under the permissive MIT license at https://github.com/issararab/CToxPred2.

Abstract We025: Human Engineered Heart Tissues Demonstrate Clinically-Relevant Disease indicators of Duchenne Muscular Dystrophy

Accurate in vitro modeling of healthy and disease conditions is crucial for assessing the safety and efficacy of novel therapeutics before clinical trials. For cardiac diseases, direct evaluation of muscle contraction is a reliable indicator of overall tissue function, however, this often necessitates expensive and low-throughput non-human in vivo models. Moreover, animal models may not accurately predict human responses to compound exposure. Here, we have developed a turnkey platform for facile fabrication of 3D engineered heart tissues (EHTs) that enables label-free, push-button measurements of contractility. The system allows for customized stimulation protocols to individual constructs, while simultaneously measuring contractility across 24 tissues in parallel with minimal user input. This approach enables stratification of muscle phenotypes and facilitates compound safety and efficacy screening. We present a 3D model of Duchenne muscular dystrophy that displays functional deficits consistent with in vivo disease indicators, including reduced contractile force and slowed relaxation kinetics, though beat rate remains unchanged. We show both acute and chronic effects of cardiotoxic drugs, as well as positive inotropic responses to external calcium and Isoproterenol. This highlights the utility of using EHTs to identify toxicity and derisk drug development and clinical testing outcomes. Spontaneously beating EHTs were also subjected to in situ stretching, leading to increased levels of cardiac troponin I (cTnI) in the tissue medium, indicating stretch induced injury. These data demonstrate a first-and-only commercial platform that integrates individual, fully-customizable, well-based control of electrical stimulation across a 24-well plate of tissues to pace muscle constructs, model force-frequency, and enable exercise regimens or damage protocols. Heart organoids were stretched under load presenting a challenge for therapeutics targeting injury prevention in the myocardium. Stimulation is coupled with automated assessment of 3D muscle contraction, providing an inclusive, medium-throughput platform for studying muscle-related disorders, advancing therapeutic discovery using a modality-agnostic biosystem.

Advancing Cardiovascular Drug Screening Using Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as a promising tool for studying cardiac physiology and drug responses. However, their use is largely limited by an immature phenotype and lack of high-throughput analytical methodology. In this study, we developed a high-throughput testing platform utilizing hPSC-CMs to assess the cardiotoxicity and effectiveness of drugs. Following an optimized differentiation and maturation protocol, hPSC-CMs exhibited mature CM morphology, phenotype, and functionality, making them suitable for drug testing applications. We monitored intracellular calcium dynamics using calcium imaging techniques to measure spontaneous calcium oscillations in hPSC-CMs in the presence or absence of test compounds. For the cardiotoxicity test, hPSC-CMs were treated with various compounds, and calcium flux was measured to evaluate their effects on calcium dynamics. We found that cardiotoxic drugs withdrawn due to adverse drug reactions, including encainide, mibefradil, and cetirizine, exhibited toxicity in hPSC-CMs but not in HEK293-hERG cells. Additionally, in the effectiveness test, hPSC-CMs were exposed to ATX-II, a sodium current inducer for mimicking long QT syndrome type 3, followed by exposure to test compounds. The observed changes in calcium dynamics following drug exposure demonstrated the utility of hPSC-CMs as a versatile model system for assessing both cardiotoxicity and drug efficacy. Overall, our findings highlight the potential of hPSC-CMs in advancing drug discovery and development, which offer a physiologically relevant platform for the preclinical screening of novel therapeutics.

Experimentally validated modeling of dynamic drug-hERG channel interactions reproducing the binding mechanisms and its importance in action potential duration.

Background and ObjectiveAssessment of drug cardiotoxicity is critical in the development of new compounds and modeling of drug-binding dynamics to hERG can improve early cardiotoxicity assessment. We previously developed a methodology to generate Markovian models reproducing preferential state-dependent binding properties, trapping dynamics and the onset of IKr block using simple voltage clamp protocols. Here, we test this methodology with real IKr blockers and investigate the impact of drug dynamics on action potential prolongation. MethodsExperiments were performed on HEK cells stably transfected with hERG and using the Nanion SyncroPatch 384i. Three protocols, P-80, P0 and P 40, were applied to obtain the experimental data from the drugs and the Markovian models were generated using our pipeline. The corresponding static models were also generated and a modified version of the O´Hara-Rudy action potential model was used to simulate the action potential duration. ResultsThe experimental Hill plots and the onset of IKr block of ten compounds were obtained using our voltage clamp protocols and the models generated successfully mimicked these experimental data, unlike the CiPA dynamic models. Marked differences in APD prolongation were observed when drug effects were simulated using the dynamic models and the static models. ConclusionsThese new dynamic models of ten well-known IKr blockers constitute a validation of our methodology to model dynamic drug–hERG channel interactions and highlight the importance of state-dependent binding, trapping dynamics and the time-course of IKr block to assess drug effects even at the steady-state.

CRISPR/Cas9-mediated knock-in of a fluorescent reporter into the target locus of interest in human pluripotent stem cells

The method presented herein is associated with the Lab Resource article titled “Generation of αMHC-EGFP knock-in in human pluripotent stem cell line, SNUe003-A-3, using CRISPR/CAS9-based gene targeting” [1]. The cardiac muscle-specific protein, α-myosin heavy chain (αMHC), is encoded by the human MYH6 gene, which is expressed in both the atria and ventricles during embryonic development and is predominantly expressed in the atria after birth [2]. Herein, the methods used to achieve CRISPR/SpCas9-mediated introduction of an EGFP reporter into αMHC, the target locus in human pluripotent stem cells (hPSCs) for cardiac lineage tracing and clinical cell sorting are described. The CRISPR-Cas9 system enables efficient replacement of the stop codon in the last exon of αMHC with a 2A non-joining peptide (T2A)-EGFP cassette. First, hPSCs are transfected with the donor construct and Cas9/sgRNA plasmids via electroporation and selected with neomycin for approximately 3 weeks. Thereafter, the established cell line exhibits typical characteristics of human embryonic stem cells (hESCs). When these cells differentiate into cardiomyocytes, the expression of EGFP is confirmed using confocal microscopy, flow cytometry analysis, and immunostaining.•The line enables monitoring of cell maturation events during human cardiac development.•The line is a valuable platform for cardiotoxicity tests and drug screening.•This method has already been employed in two original studies, as previously reported for reporter cell line generation using CRISPR/Cas9.

Cardiotoxicity of HER2-Targeted Drugs When Combined with Other Drugs: A Systematic and Meta-analysis of Randomized Controlled Trials.

The development and use of HER2-targeted drugs has improved the prognosis of HER2-positive cancer patients. However, in addition to improved survival rates, treatment-induced adverse events and nontumor-related deaths have increased. We sought to assess the incidence of cardiovascular adverse events when HER2-targeted drugs are combined with other drugs. We systematically searched the literature on the cardiotoxicity of anti-HER2 drugs in electronic databases, including PubMed, Web of Science, Cochrane Library, OVID and CNKI, from their inception to April 2022. The Cochrane Collaboration's tool for assessing risk of bias and the Jadad scale were used to evaluate the risk of bias and quality of the studies, respectively. For each included trial, we calculated the incidence of cardiovascular adverse effects (CAEs) and 95% confidence intervals (95% CIs) and performed a meta-analysis using a random effects model (REM). The meta-analysis was performed using R 4.2.1. We included 41 randomized clinical trials (RCTs) in the meta-analysis, consisting of 56 groups and 31,934 patients. The meta-analysis revealed the following: (1) The incidence of cardiotoxicity in groups given monoclonal antibody treatment was 14% for single therapy (95% CI: 2-34%) and 10%, 11%, and 12% for adjuvant therapy combined with combined therapy (95% CI: 6-13%), chemotherapy (95% CI: 8-13%) and endocrine therapy (95% CI: 7-18%), respectively. However, in the groups treated with the antibody‒drug conjugates (ADCs), the percentage of patients treated with the combination therapy was 1% (95% CI: 0-2%) and 5% (95% CI: 4-7%), respectively, with a significant difference (P < 0.01). The heterogeneity among the included studies was significant (I2 = 94%, p < 0.01). (2) When monoclonal antibodies were combined with chemotherapy, the incidence of cardiotoxicity under anthracycline-containing therapy (10.3%) was significantly greater than that under nonanthracycline-containing therapy (8.8%). (3) Significant differences were found between subgroups, except for the endocrine group versus some others, although this difference might result from the different inclusion criteria of the original trials. (1) When anti-HER2 drugs are administered in combination with anthracycline-containing chemotherapy, the incidence of cardiotoxicity is greater than with other drugs. (2) Safety benefits can be achieved by replacing traditional monoclonal antibodies with ADCs. The comprehensive use of these drugs necessitates collaboration between oncologists and cardiologists.

Two-Dimensional Deep Learning Frameworks for Drug-Induced Cardiotoxicity Detection.

The identification of drug-induced cardiotoxicity remains a pressing challenge with far-reaching clinical and economic ramifications, often leading to patient harm and resource-intensive drug recalls. Current methodologies, including in vivo and in vitro models, have severe limitations in accurate identification of cardiotoxic substances. Pioneering a paradigm shift from these conventional techniques, our study presents two deep learning-based frameworks, STFT-CNN and SST-CNN, to assess cardiotoxicity with markedly improved accuracy and reliability. Leveraging the power of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) as a more human-relevant cell model, we record mechanical beating signals through impedance measurements. These temporal signals were converted into enriched two-dimensional representations through advanced transformation techniques, specifically short-time Fourier transform (STFT) and synchro-squeezing transform (SST). These transformed data are fed into the proposed frameworks for comprehensive analysis, including drug type classification, concentration classification, cardiotoxicity classification, and new drug identification. Compared to traditional models like recurrent neural network (RNN) and 1-dimensional convolutional neural network (1D-CNN), SST-CNN delivered an impressive test accuracy of 98.55% in drug type classification and 99% in distinguishing cardiotoxic and noncardiotoxic drugs. Its feasibility is further highlighted with a stellar 98.5% average accuracy for classification of various concentrations, and the superiority of our proposed frameworks underscores their promise in revolutionizing drug safety assessments. With a potential for scalability, they represent a major leap in drug safety assessments, offering a pathway to more robust, efficient, and human-relevant cardiotoxicity evaluations.

Effect of fructose on anthracycline and HER-2 blocking agent mediated cardiotoxicity through NLRP3 and MyD88 mediated pathways: New roles of sweeteners in cardio-oncology.

e24020 Background: Combinatorial therapy based on anthracyclines and HER-2 blocking agents increases the risk of cardiomyopathy in women with breast cancer. Fructose is a monosaccharide associated with high risk of diabetes, non-alcoholic fatty liver disease, atherosclerosis and visceral obesity. A high fructose diet is a potential cardiometabolic risk factor and may increase cardiac risk in women with breast cancer treated with cardiotoxic drugs. In this study, we highlighted the role of sweeteners fructose in the worsening of cardiotoxicity induced by combinatorial therapy anthracycline-HER2-blocking agents. Methods: Human cardiomyocytes were pre-exposed with doxorubicin combined to trastuzumab (at 300 and 1000 nM) for 48 and 72h alone or combined to fructose at 0.25, 1 and 2.5 mM. After the incubation period, we performed the following tests: determination of cell viability, through mitochondrial dehydrogenase activity, study of lipid peroxidation (quantifying cellular Malondialdehyde and 4-hydroxynonenal), intracellular Ca2+ homeostasis. Moreover, pro-inflammatory studied were also performed (activation of NLRP3 inflammasome; expression of peroxisome proliferator-activated receptor-α; transcriptional activation of p65/NF-κB and secretion of cytokines involved in cardiotoxicity (Interleukins 1β, 8, 6, CXCL-12). Results: High fructose increases significantly cell mortality of cardiomyocytes exposed to doxorubicin and trastuzumab through the activation of NLRP-3 and Myd-88 expression. Intracellular Ca++ levels were also increased of 4 times compared to non-fructose exposed cardiomyocytes; MDA and 4-HNA levels were increased of 48,3 and 51,2 % compared to non-fructose exposed cells (p&lt;0.001). Fructose increases IL-1, IL-6 and CXCL-12 expression in cardiomyocytes in a concentration dependent manner, compared to doxorubicin-trastuzumab group (p&lt;0.05). Conclusions: Data of the present study indicate that fructose induces a pro-inflammatory phenotype in human cardiac cells exposed to anticancer cardiotoxic drugs, providing even more data on the harmful role of fructose as a key player of cardiotoxic phenomena.

New recellularizable cardio-matrix scaffold as a base for alternative in vitro models for the cardiotoxicity risk prediction

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): SID 227944/22 DOR 2331157/23 Background Cardiotoxicity is the occurrence of cardiac dysfunction induced by pathophysiological stimuli as a result of toxic effects. A considerable number of drugs, including chemotherapics, antibiotics antidepressants but also antiarrhythmics, could cause an alteration of cardiac physiology, and then cardiac damage. These include disturbances in ventricular repolarization and QT interval, arrhythmias, bradycardia, tachycardia, decreases in left ventricular ejection fraction, and congestive heart failure. Precinical evaluation of drug cardiotoxicity has used animal models, which tend to be expensive, have lo throughput, and have limitations as not ever faithfully reflect the human pathophysiology, while current two-dimensional cellular models revealed to be not sufficient and specific. Purpose The aim of this study is to develop a myocardial tissue-like scaffold, in order to realize an efficient and easy-replicable in vitro model for drug cardiotoxicity prediction. This must be able to mimic the morphological, cellular, and electrophysiological complexity of the intact adult human myocardium. Material and Methods Starting from fresh porcine hearts, cardiac specimens were isolated from left ventricles using a scalpel, sectioned with a vibratome and punched with a biopsy puncher obtaining myocardial patches. Thus, samples were decellularized through a novel serial decellularization treatment based on osmotic shock and detergents. The decellularization procedure exploited a new automated, dynamic perfusion device for a standardized and optimized removal of cardiac cells and non-ECM proteins. These samples were analysed for decellularization effectiveness by DNA quantification, histology and immunofluorescence staining to evaluate cell removal and extracellular matrix integrity. Moreover, they were tested for cytocompatibility using human mesenchymal stem cells and cardiac progenitors, also derived from induced pluripotent stem cells. Cellular adhesion, behaviour, vitality, proliferation, and possible apoptotic effects were tested, too. Results In order to advance a robust bioelectronics model, an automated platform was developed to generate a three-dimensional, bioengineered replica of human myocardial tissue. With respect to the common decellularization protocol characterized by mild agitation, results based on the automated, dynamic treatments have shown a superior ability to obtain acellular, biocompatible scaffolds in terms of cell elements’ removal and extracellular matrix preservation, as well as time- and cost-effectiveness. Indeed, these decellularized scaffolds allow cell penetration, adhesion and survival. Conclusions Further experiments will be focused on evaluating functional bioengineered myocardial tissues with electrophysiological and molecular analysis to evaluate this new model's throughput capacity. Finally, the validation of these bioelectronic platforms will be performed with drugs with known cardiotoxic effects.

FRUCTOSE EXACERBATES ANTHRACYCLINE AND HER–2 BLOCKING AGENT MEDIATED CARDIOTOXICITY THROUGH NLRP3 AND MYD88 MEDIATED PATHWAYS: NEW ROLES OF SWEETENERS IN CARDIO–ONCOLOGY

Abstract Background Combinatorial therapy based on anthracyclines and HER–2 blocking agents increases the risk of cardiomyopathy in women with breast cancer. Fructose is a monosaccharide associated with high risk of diabetes, non–alcoholic fatty liver disease, atherosclerosis and visceral obesity. A high fructose diet is a potential cardiometabolic risk factor and may increase cardiac risk in women with breast cancer treated with cardiotoxic drugs. Purpose In this study, we highlighted the role of sweeteners fructose in the worsening of cardiotoxicity induced by combinatorial therapy anthracycline–HER2–blocking agents. Methods Human cardiomyocytes were pre–exposed with doxorubicin combined to trastuzumab (at 300 and 1000 nM) for 48 and 72h alone or combined to fructose at 0.25, 1 and 2.5 mM. After the incubation period, we performed the following tests: determination of cell viability, through mitochondrial dehydrogenase activity, study of lipid peroxidation (quantifying cellular Malondialdehyde and 4–hydroxynonenal), intracellular Ca2+ homeostasis. Moreover, pro–inflammatory studied were also performed (activation of NLRP3 inflammasome; expression of peroxisome proliferator–activated receptor–α; transcriptional activation of p65/NF–κB and secretion of cytokines involved in cardiotoxicity (Interleukins 1β, 8, 6, CXCL–12). Results High fructose increases significantly cell mortality of cardiomyocytes exposed to doxorubicin and trastuzumab thorugh the activation of NLRP–3 and Myd–88 expression. Intracellular Ca++ levels were also increased of 4 times compared to non–fructose exposed cardiomyocytes; MDA and 4–HNA levels were increased of 48,3 and 51,2 % compared to non–fructose exposed cells (p&amp;lt;0.001). Fructose increases IL–1, IL–6 and CXCL–12 expression in cardiomyocytes in a concentration dependent manner, compard to doxorubicin–trastuzumab group (p&amp;lt;0.05). Conclusion Data of the present study indicate that fructose induces a pro–inflammatory phenotype in human cardiac cells exposed to anticancer cardiotoxic drugs, providing even more data on the harmful role of fructose as a key player of cardiotoxic phenomena.