Arrhythmogenic Manifestations of Chagas Disease: Perspectives From the Bench to Bedside.

Chagas cardiomyopathy caused by infection with the intracellular parasite Trypanosoma cruzi is the most common and severe expression of human Chagas disease. Heart failure, systemic and pulmonary thromboembolism, arrhythmia, and sudden cardiac death are the principal clinical manifestations of Chagas cardiomyopathy. Ventricular arrhythmias contribute significantly to morbidity and mortality and are the major cause of sudden cardiac death. Significant gaps still exist in the understanding of the pathogenesis mechanisms underlying the arrhythmogenic manifestations of Chagas cardiomyopathy. This article will review the data from experimental studies and translate those findings to draw hypotheses about clinical observations. Human- and animal-based studies at molecular, cellular, tissue, and organ levels suggest 5 main pillars of remodeling caused by the interaction of host and parasite: immunologic, electrical, autonomic, microvascular, and contractile. Integrating these 5 remodeling processes will bring insights into the current knowledge in the field, highlighting some key features for future management of this arrhythmogenic disease.

In vivo tebuconazole administration impairs heart electrical function and facilitates the occurrence of dobutamine-induced arrhythmias: involvement of reactive oxygen species.

Tebuconazole (TEB), a widely used pesticide in agriculture to combat fungal infections, is commonly detected in global food, potable water, groundwater, and human urine samples. Despite its known in vivo toxicity, its impact on heart function remains unclear. In a 28-day study on male Wistar rats (approximately 100 g), administering 10 mg/kg/day TEB or a vehicle (control) revealed no effect on body weight gain or heart weight, but an increase in the infarct area in TEB-treated animals. Notably, TEB induced time-dependent changes in in vivo electrocardiograms, particularly prolonging the QT interval after 28 days of administration. Isolated left ventricular cardiomyocytes exposed to TEB exhibited lengthened action potentials and reduced transient outward potassium current. TEB also increased reactive oxygen species (ROS) production in these cardiomyocytes, a phenomenon reversed by N-acetylcysteine (NAC). Furthermore, TEB-treated animals, when subjected to an in vivo dobutamine (Dob) and caffeine (Caf) challenge, displayed heightened susceptibility to severe arrhythmias, a phenotype prevented by NAC. In conclusion, TEB at the no observed adverse effect level (NOAEL) dose adversely affects heart electrical function, increases arrhythmic susceptibility, partially through ROS overproduction, and this phenotype is reversible by scavenging ROS with NAC.

Neurotoxicity of Pyrethroids in neurodegenerative diseases: From animals' models to humans' studies.

Neurodegenerative diseases are associated with diverse symptoms, both motor and mental. Genetic and environmental factors can trigger neurodegenerative diseases. Chemicals as pesticides are constantly used in agriculture and also domestically. In this regard, pyrethroids (PY), are a class of insecticides in which its main mechanism of action is through disruption of voltage-dependent sodium channels function in insects. However, in mammals, they can also induce oxidative stress and enzyme dysfunction. This review investigates the association between PY and neurodegenerative diseases as Alzheimer's, Huntington's, Parkinson's, Amyotrophic Lateral Sclerosis, and Autism in animal models and humans. Published works using specific and non-specific models for these diseases were selected. We showed a tendency toward the development and/or aggravating of these neurodegenerative diseases following exposure to PYs. In animal models, the biochemical mechanisms of the diseases and their interaction with the insecticides are more deeply investigated. Nonetheless, only a few studies considered the specific model for each type of disease to analyze the impacts of the exposure. The choice of a specific model during the research is an important step and our review highlights the knowledge gaps of PYs effects using these models reinforcing the importance of them during the design of the experiments.

Eugenol delays the onset of ouabain-induced ventricular cardiac arrhythmias in guinea pigs.

Eugenol is an aromatic compound used in the manufacture of medicines, perfumes, cosmetics and as an anaesthetic due to the ability of the drug to block the neuronal isoform of voltage-gated Na+ channels (NaV ). Some arrhythmias are associated with gain of function in the sodium current (INa ) found in cardiomyocytes, and antiarrhythmic sodium channel blockers are commonly used in the clinical practice. This study sought to elucidate the potential mechanisms of eugenol's protection in the arrhythmic model of ouabain-induced arrhythmias in guinea pig heart. Ex vivo arrhythmias were induced using 50 µM of ouabain. The antiarrhythmic properties of eugenol were evaluated in the ex vivo heart preparation and isolated ventricular cardiomyocytes. The compound's effects on cardiac sodium current and action potential using the patch-clamp technique were evaluated. In all, eugenol decreased the ex vivo cardiac arrhythmias induced by ouabain. Furthermore, eugenol showed concentration dependent effect upon peak INa , left-shifted the stationary inactivation curve and delayed the recovery from inactivation of the INa . All these aspects are considered to be antiarrhythmic. Our findings demonstrate that eugenol has antiarrhythmic activity, which may be partially explained by the ability of eugenol to change de biophysical properties of INa of cardiomyocytes.

The fungicide tebuconazole modulates the sodium current of human NaV1.5 channels expressed in HEK293 cells.

The fungicide Tebuconazole is a widely used pesticide in agriculture and may cause cardiotoxicity. In our present investigation the effect of Tebuconazole on the sodium current (INa) of human cardiac sodium channels (NaV1.5) was studied using a heterologous expression system and whole-cell patch-clamp techniques. Tebuconazole reduced the amplitude of the peak INa in a concentration- and voltage-dependent manner. At the holding potential of -120 mV the IC50 was estimated at 204.1 ± 34.3 µM, while at -80 mV the IC50 was 0.3 ± 0.1 µM. The effect of the fungicide is more pronounced at more depolarized potentials, indicating a state-dependent interaction. Tebuconazole caused a negative shift in the half-maximal inactivation voltage and delayed recovery from fast inactivation of INa. Also, it enhanced closed-state inactivation, exhibited use-dependent block in a voltage-dependent manner. Furthermore, Tebuconazole reduced the increase in late sodium current induced by the pyrethroid insecticide ß-Cyfluthrin. These results suggest that Tebuconazole can interact with NaV1.5 channels and modulate INa. The observed effects may lead to decreased cardiac excitability through reduced INa availability, which could be a new mechanism of cardiotoxicity to be attributed to the fungicide.

Experimental hypothyroidism induces cardiac arrhythmias and ranolazine reverts and prevents the phenotype.

AIMS: Hypothyroidism is associated with an increased risk of cardiovascular disease and enhanced susceptibility to arrhythmias. In our investigation, we evaluated the potential involvement of late sodium current (INa,late) in cardiac arrhythmias in an experimental murine model of hypothyroidism. MAIN METHODS: Male Swiss mice were treated with methimazole (0.1 % w/vol, during 21 days) to induce experimental hypothyroidism before ECG, action potential (AP) and intracellular Ca2+ dynamics were evaluated. Susceptibility to arrhythmia was measured in vitro and in vivo. KEY FINDINGS: The results revealed that hypothyroid animals presented ECG alterations (e.g. increased QTc) with the presence of spontaneous sustained ventricular tachycardia. These changes were associated with depolarized resting membrane potential in isolated cardiomyocytes and increased AP duration and dispersion at 90 % of the repolarization. Aberrant AP waveforms were related to increased Ca2+ sparks and out-of-pace Ca2+ waves. These changes were observed in a scenario of enhanced INa,late. Interestingly, ranolazine, a clinically used blocker of INa,late, restored the ECG alterations, reduced Ca2+ sparks and aberrant waves, decreased the in vitro events and the severity of arrhythmias observed in isolated cardiomyocytes from hypothyroid animals. Using the in vivo dobutamine + caffeine protocol, animals with hypothyroidism developed catecholaminergic bidirectional ventricular tachycardia, but pre-treatment with ranolazine prevented this. SIGNIFICANCE: We concluded that animals with hypothyroidism have increased susceptibility to developing arrhythmias and ranolazine, a clinically used blocker of INa,late, is able to correct the arrhythmic phenotype.

Altered heart cytokine profile and action potential modulation in cardiomyocytes from Mas-deficient mice.

The renin-angiotensin system (RAS) is a key hormonal system. In recent years, the functional analysis of the novel axis of the RAS (ACE2/Ang-(1-7)/Mas receptor) revealed that its activation can become protective against several pathologies, including cardiovascular diseases. Mas knockout mice (Mas-KO) represent an important tool for new investigations. Indeed, extensive biological research has focused on investigating the functional implications of Mas receptor deletion. However, although the Mas receptor was identified in neonatal cardiomyocytes and also in adult ventricular myocytes, only few reports have explored the Ang-(1-7)/Mas signaling directly in cardiomyocytes to date. This study investigated the implication of Mas receptor knockout to the cytokine profile, energy metabolism, and electrical properties of mice-isolated cardiomyocytes. Here, we demonstrated that Mas-KO mice have modulation in some cytokines, such as G-CSF, IL-6, IL-10, and VEGF in the left ventricle. This model also presents increased mitochondrial number in cardiomyocytes and a reduction in the myocyte diameter. Finally, Mas-KO cardiomyocytes have altered action potential modulation after diazoxide challenge. Such electrical finding was different from the data showed for the TGR(A1-7)3292 (TGR) model, which overexpresses Ang-(1-7) in the plasma by 4.5, used by us as a control. Collectively, our findings exemplify the importance of understanding the ACE2/Ang-(1-7)/Mas pathway in cardiomyocytes and heart tissue. The Mas-KO mice model can be considered an important tool for new RAS investigations.

Impact of IFN-γ Deficiency on the Cardiomyocyte Function in the First Stage of Experimental Chagas Disease.

Chagas disease (CD) is caused by the parasitic protozoan T. cruzi. The progression of CD in ~30% of patients results in Chagasic Cardiomyopathy (CCM). Currently, it is known that the inflammatory system plays a significant role in the CCM. Interferon-gamma (IFN-γ) is the major cytokine involved in parasitemia control but has also been linked to CCM. The L-type calcium current (ICa,L) is crucial in the excitation/contraction coupling in cardiomyocytes. Thus, we compared ICa,L and the mechanical properties of cardiomyocytes isolated from infected wild type (WT) and IFN-γ(-/-) mice in the first stage of T. cruzi infection. Using the patch clamp technique, we demonstrated that the infection attenuated ICa,L in isolated cardiomyocytes from the right and left ventricles of WT mice at 15 days post-infection (dpi), which was not observed in the IFN-γ(-/-) cardiomyocytes. However, ICa,L was attenuated between 26 and 30 dpi in both experimental groups. Interestingly, the same profile was observed in the context of the mechanical properties of isolated cardiomyocytes from both experimental groups. Simultaneously, we tracked the mortality and MCP-1, TNF-α, IL-12, IL-6, and IL-10 serum levels in the infected groups. Importantly, the IFN-γ(-/-) and WT mice presented similar parasitemia and serum inflammatory markers at 10 dpi, indicating that the modifications in the cardiomyocyte functions observed at 15 dpi were directly associated with IFN-γ(-/-) deficiency. Thus, we showed that IFN-γ plays a crucial role in the electromechanical remodeling of cardiomyocytes during experimental T. cruzi infection in mice.

The insecticide ß-Cyfluthrin induces acute arrhythmic cardiotoxicity through interaction with NaV1.5 and ranolazine reverses the phenotype.

ß-Cyfluthrin, a class II Pyrethroid, is an insecticide used worldwide in agriculture, horticulture (field and protected crops), viticulture, and domestic applications. ß-Cyfluthrin may impair the function of biological systems; however, little information is available about its potential cardiotoxic effect. Here, we explored the acute toxicity of ß-Cyfluthrin in isolated heart preparations and its cellular basis, using isolated cardiomyocytes. Moreover, ß-Cyfluthrin effects on the sodium current, especially late sodium current (INa-L), were investigated using human embryonic kidney cells (HEK-293) cells transiently expressing human NaV1.5 channels. We report that ß-Cyfluthrin raised INa-L in a dose-dependent manner. ß-Cyfluthrin prolonged the repolarization of the action potential (AP) and triggered oscillations on its duration. Cardiomyocytes contraction and calcium dynamics were disrupted by the pesticide with a marked incidence of non-electronic-stimulated contractions. The antiarrhythmic drug Ranolazine was able to reverse most of the phenotypes observed in isolated cells. Lastly, ventricular premature beats (VPBs) and long QT intervals were found during ß-Cyfluthrin exposure, and Ranolazine was able to attenuate them. Overall, we demonstrated that ß-Cyfluthrin can cause significant cardiac alterations and Ranolazine ameliorated the phenotype. Understanding the insecticides' impacts upon electromechanical properties of the heart is important for the development of therapeutic approaches to treat cases of pesticides intoxication.

The fungicide Tebuconazole induces electromechanical cardiotoxicity in murine heart and human cardiomyocytes derived from induced pluripotent stem cells.

Tebuconazole (TEB) is an important fungicide that belongs to the triazole family. It is widely used in agriculture and its use has experienced a tremendous increase in the last decade. The long-term exposure of humans to this pesticide is a real threat because it is stable in water and soil. The association between long-term exposure to TEB and damage of several biological systems, including hepatotoxicity and cardiotoxicity is evident, however, acute toxicological studies to reveal the toxicity of TEB are limited. This research paper addressed the acute exposure of TEB in murine hearts, cardiomyocytes, and human cardiomyocytes derived from an induced pluripotent stem cell (hiPSC-CMs), spelling out TEB's impact on electromechanical properties of the cardiac tissue. In ex vivo experiments, TEB dose dependently, caused significant electrocardiogram (ECG) remodeling with prolonged PR and QTc interval duration. The TEB was also able to change the action potential waveform in murine cardiomyocytes and hiPSC-CMs. These effects were associated with the ability of the compound to block the L-type calcium current (IC50 = 33.2 ± 7.4 µmol.l-1) and total outward potassium current (IC50 = 5.7 ± 1.5 µmol.l-1). TEB also increased the sodium/calcium exchanger current in its forward and reverse modes. Additionally, sarcomere shortening and calcium transient in isolated cardiomyocytes were enhanced when cells were exposed to TEB at 30 µmol.l-1. Combined, our results demonstrated that acute TEB exposure affects the cardiomyocyte's electro-contractile properties and triggers the appearance of ECG abnormalities.

Diminazene Aceturate, an angiotensin converting enzyme 2 (ACE2) activator, promotes cardioprotection in ischemia/reperfusion-induced cardiac injury.

This study aimed to investigate whether the Diminazene Aceturate (DIZE), an angiotensin-converting enzyme 2 (ACE2) activator, can revert cardiac dysfunction in ischemia reperfusion-induced (I/R) injury in animals and examine the mechanism underlying this effect. Wistar rats systemically received DIZE (1 mg/kg) for thirty days. Cardiac function in isolated rat hearts was evaluated using the Langendorff technique. After I/R, ventricular non-I/R and I/R samples were used to evaluate ATP levels. Mitochondrial function was assessed using cardiac permeabilized fibers and isolated cardiac mitochondria. Cardiac cellular electrophysiology was evaluated using the patch clamp technique. DIZE protected the heart after I/R from arrhythmia and cardiac dysfunction by preserving ATP levels, independently of any change in coronary flow and heart rate. DIZE improved mitochondrial function, increasing the capacity for generating ATP and reducing proton leak without changing the specific citrate synthase activity. The activation of the ACE2 remodeled cardiac electrical profiles, shortening the cardiac action potential duration at 90 % repolarization. Additionally, cardiomyocytes from DIZE-treated animals exhibited reduced sensibility to diazoxide (KATP agonist) and a higher KATP current compared to the controls. DIZE was able to improve mitochondrial function and modulate cardiac electrical variables with a cardio-protective profile, resulting in direct myocardial cell protection from I/R injury.

Inhibition of calcium/calmodulin (Ca2+ /CaM)-Calcium/calmodulin-dependent protein kinase II (CaMKII) axis reduces in vitro and ex vivo arrhythmias in experimental Chagas disease.

Chagasic cardiomyopathy (CCC) is one of the main causes of heart failure and sudden death in Latin America. To date, there is no available medication to prevent or reverse the onset of cardiac symptoms. CCC occurs in a scenario of disrupted calcium dynamics and enhanced oxidative stress, which combined, may favor the hyper activation of calcium/calmodulin (Ca2+ /CaM)-calcium/calmodulin-dependent protein kinase II (CaMKII) (Ca2+ /CaM-CaMKII) pathway, which is fundamental for heart physiology and it is implicated in other cardiac diseases. Here, we evaluated the association between Ca2+ /CaM-CaMKII in the electro-mechanical (dys)function of the heart in the early stage of chronic experimental Trypanosoma cruzi infection. We observed that in vitro and ex vivo inhibition of Ca2+ /CaM-CaMKII reversed the arrhythmic profile of isolated hearts and isolated left-ventricles cardiomyocytes. The benefits of the limited Ca2+ /CaM-CaMKII activation to cardiomyocytes' electrical properties are partially related to the restoration of Ca2+ dynamics in a damaged cellular environment created after T. cruzi infection. Moreover, Ca2+ /CaM-CaMKII inhibition prevented the onset of arrhythmic contractions on isolated heart preparations of chagasic mice and restored the responsiveness to the increase in the left-ventricle pre-load. Taken together, our data provide the first experimental evidence for the potential of targeting Ca2+ /CaM-CaMKII pathway as a novel therapeutic target to treat CCC.

Eugenol interacts with cardiac sodium channel and reduces heart excitability and arrhythmias.

AIMS: Eugenol is a natural compound found in the essential oils of many aromatic plants. The compound is used as a local anesthetic because of its inhibitory effect on the voltage-gated Na+ channels (Nav), which are expressed in the nociceptive neurons. Eugenol has shown wide range of activities in the cardiovascular system; most of these activities are attributed to the modulation of voltage-sensitive Ca2+ channels. However, its action on Nav1.5, the main subtype of Nav expressed in the mammalian myocardium, is unknown. The interaction of eugenol with Nav1.5 could also contribute to its antiarrhythmic properties in vitro and ex vivo. We investigated the compound's effect on sodium current (INa) and its possible cardiac antiarrhythmic activity. METHODS: The effect of eugenol on cardiac contractility was investigated using isolated atrium from guinea pig (for isometric force measurements). The compound's effect on INa was evaluated using human embryonic cell transiently expressing human Nav1.5 and patch-clamp technique. KEY FINDINGS: Eugenol caused negative inotropic and chronotropic effects in the atria. In the ex vivo arrhythmia model, eugenol decreased atrial pacing disturbance induced by ouabain. Eugenol reduced the INa in a concentration-dependent manner. Furthermore, the compound left-shifted the stationary inactivation curve, delayed recovery from inactivation of the INa, and preferentially blocked the channel in the inactivated state. Importantly, eugenol was able to attenuate the late sodium current. All these aspects are considered to be antiarrhythmic. SIGNIFICANCE: Overall, our findings demonstrate that eugenol has antiarrhythmic activity due, at least in part, to its interaction with Nav1.5.

A novel substrate for arrhythmias in Chagas disease.

BACKGROUND: Chagas disease (CD) is a neglected disease that induces heart failure and arrhythmias in approximately 30% of patients during the chronic phase of the disease. Despite major efforts to understand the cellular pathophysiology of CD there are still relevant open questions to be addressed. In the present investigation we aimed to evaluate the contribution of the Na+/Ca2+ exchanger (NCX) in the electrical remodeling of isolated cardiomyocytes from an experimental murine model of chronic CD. METHODOLOGY/PRINCIPAL FINDINGS: Male C57BL/6 mice were infected with Colombian strain of Trypanosoma cruzi. Experiments were conducted in isolated left ventricular cardiomyocytes from mice 180-200 days post-infection and with age-matched controls. Whole-cell patch-clamp technique was used to measure cellular excitability and Real-time PCR for parasite detection. In current-clamp experiments, we found that action potential (AP) repolarization was prolonged in cardiomyocytes from chagasic mice paced at 0.2 and 1 Hz. After-depolarizations, both subthreshold and with spontaneous APs events, were more evident in the chronic phase of experimental CD. In voltage-clamp experiments, pause-induced spontaneous activity with the presence of diastolic transient inward current was enhanced in chagasic cardiomyocytes. AP waveform disturbances and diastolic transient inward current were largely attenuated in chagasic cardiomyocytes exposed to Ni2+ or SEA0400. CONCLUSIONS/SIGNIFICANCE: The present study is the first to describe NCX as a cellular arrhythmogenic substrate in chagasic cardiomyocytes. Our data suggest that NCX could be relevant to further understanding of arrhythmogenesis in the chronic phase of experimental CD and blocking NCX may be a new therapeutic strategy to treat arrhythmias in this condition.

Ethnic-Related Sodium Voltage-Gated Channel α Subunit 5 Polymorphisms Shape the In Vitro Pharmacological Action of Amiodarone upon Nav1.5.

Nav1.5-derived Na+ current (INa) exerts a pivotal role in the depolarization phase of cardiomyocytes' action potential, and, therefore, changes in INa can contribute to fatal arrhythmias. Nav1.5 displays naturally occurring ethnicity-related polymorphisms, which might alter the functioning and pharmacology of the channel. Some studies have shown how single-nucleotide polymorphism can change the response to antiarrhythmic drugs. Investigations on the role of Nav1.5 in arrhythmogenesis associated with its functional polymorphisms are currently growing as well as the possible variability in the antiarrhythmic pharmacotherapy among ethnic groups. The influence of the ethnicity-related polymorphisms (S524Y, S1103Y, R1193Q, V1951L) on the responsiveness, selectivity, and pharmacological efficacy of the clinically used antiarrhythmic amiodarone (AMIO) is not completely known. Our objectives were to analyze biophysical and pharmacological aspects of four ethnicity-related polymorphisms before and after exposure to AMIO. Polymorphisms caused reduced AMIO potency compared with wild type (WT), which can vary by up to 4× between them. AMIO shifted the voltage dependency for current inactivation without significant effect in voltage-dependent activation to a similar extent in WT and polymorphisms. The recovery from inactivation was altered between the polymorphisms when compared with WT. Finally, the use dependency of AMIO differed between studied groups, especially at a more depolarized cell membrane. Thus, our work may guide future studies focusing on the efficiency of AMIO in treating different arrhythmias and establish more individualized guidelines for its use depending on the Nav1.5 polymorphism after validating our findings using in vivo studies. SIGNIFICANCE STATEMENT: Sodium voltage-gated channel α subunit 5 (SCN5A) gene encodes the α subunit of Nav1.5, the main cardiac voltage-gated Na+ channel. Interestingly, ethnicity-related polymorphisms are found in SCN5A. Amiodarone is used in clinical practice, and some of its effects are attributed to interaction with Nav1.5. Important, amiodarone efficacy is variable among patients. Here we show that ethnicity-related SCN5A polymorphisms lead to altered Nav1.5-amiodarone interaction, which may be the cause for the variable efficacy observed in clinical usage of amiodarone.

Cardiac electrical remodeling and neurodegenerative diseases association.

Cardiac impairment contributes significantly to the mortality associated with several neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD), primarily recognized as brain pathologies. These diseases may be caused by aggregation of a misfolded protein, most often, in the brain, although new evidence also reveals peripheral abnormalities. After characterization of the cardiac involvement in neurodegenerative diseases, several studies concentrated on elucidating the cause of the impaired cardiac function. However, most of the current knowledge is focused on the mechanical aspects of the heart rather than the electrical disturbances. The main objective of this review is to summarize the most recent advances in the elucidation of cardiac electrical remodeling in the neurodegenerative environment. We aimed to determine a crosstalk between the heart and the brain in three neurodegenerative conditions: AD, PD, and HD. We found that the most studies demonstrated important alterations in the electrocardiogram (ECG) of patients with neurodegeneration and in animal models of the conditions. We also showed that little is described when considering excitability disruptions in cardiomyocytes, for example, action potential impairments. It is a matter of contention whether central nervous system abnormalities or the peripheral ones increase the risk of heart diseases in patients with neurodegenerative conditions. To determine this notion, there is a need for new heart studies focusing specifically on the cardiac electrophysiology (e.g., ECG and cardiomyocyte excitability). This review could serve as an important guide in designing novel accurate approaches targeting the heart in neuronal conditions.

NOTCH1 is critical for fibroblast-mediated induction of cardiomyocyte specialization into ventricular conduction system-like cells in vitro.

Cardiac fibroblasts are present throughout the myocardium and are enriched in the microenvironment surrounding the ventricular conduction system (VCS). Several forms of arrhythmias are linked to VCS abnormalities, but it is still unclear whether VCS malformations are cardiomyocyte autonomous or could be linked to crosstalk between different cell types. We reasoned that fibroblasts influence cardiomyocyte specialization in VCS cells. We developed 2D and 3D culture models of neonatal rat cardiac cells to assess the influence of cardiac fibroblasts on cardiomyocytes. Cardiomyocytes adjacent to cardiac fibroblasts showed a two-fold increase in expression of VCS markers (NAV1.5 and CONTACTIN 2) and calcium transient duration, displaying a Purkinje-like profile. Fibroblast-conditioned media (fCM) was sufficient to activate VCS-related genes (Irx3, Scn5a, Connexin 40) and to induce action potential prolongation, a hallmark of Purkinge phenotype. fCM-mediated response seemed to be spatially-dependent as cardiomyocyte organoids treated with fCM had increased expression of connexin 40 and NAV1.5 primarily on its outer surface. Finally, NOTCH1 activation in both cardiomyocytes and fibroblasts was required for connexin 40 up-regulation (a proxy of VCS phenotype). Altogether, we provide evidence that cardiac fibroblasts influence cardiomyocyte specialization into VCS-like cells via NOTCH1 signaling in vitro.

Correction: Reactive oxygen species and nitric oxide imbalances lead to in vivo and in vitro arrhythmogenic phenotype in acute phase of experimental Chagas disease.

[This corrects the article DOI: 10.1371/journal.ppat.1008379.].