Cardiac Corpuscles: A "New" Morphofunctional Entity? / Corpúsculos Cardíacos: ¿Una "Nueva" Entidad Morfofuncional?

SUMMARY: The existence of "transitional muscular structures" between subendocardial branches (Purkinje fibers) and ventricular working muscle fibers (WF) was first described by the German anatomist, Kurt Goerttler, in 1964. He designated them as "subendocardial nucleus organs." He supposed such fibers functioned as mechanoreceptors, controlling of the intensity of contraction of the ventricular musculature. Brazilian anatomist Ferraz de Carvalho described similar structures in 1993. A thorough literature search failed to identify any other research articles confirming or denying their existence. The objective of this work was to find such structures in subendocardial ventricular walls in human hearts. We collected fifteen formalin-preserved hearts from the Anatomy Department of São Paulo University and sectioned the apical portions on the right and left ventricles according to method used by Goerttler. We utilized conventional histology (light microscopy- LM), scanning electron microscopy (SEM), and a new preservation method called micro- plastination (MP). At the anterior wall of the right ventricle in the subendocardial region between the interventricular septum and moderator band, we found several bundles of fusiform and helicoidal fibers of similar histology to the WF. The bundles measured between 400 and 1150 µm in length and were separated from adjacent muscular fibers by thin collagen fiber, thus acting as a "pseudo capsule." Some structures seemed to be linked to PF and were appeared to be lymphatic and blood vessels and nerves. We called those structures "cardiac corpuscles" (CC). The observation of the previously "unknown" CC in this initial study confirmed the previous descriptions and its discovery may contribute to new perspectives in the study of cardiac muscle structure and function.

La existencia de "estructuras musculares de transición" entre los ramos subendocárdicos (fibras de Purkinje) y las fibras musculares ventriculares activas(FMV) fue descrita por primera vez por el anatomista alemán Kurt Goerttler en 1964, quien las denominó "órganos del núcleo subendocárdico". Supuso que tales fibras funcionaban como mecanoreceptores, controlando la intensidad de la contracción de la musculatura ventricular. El anatomista brasileño Ferraz de Carvalho describió estructuras similares en 1993. Una búsqueda bibliográfica exhaustiva no logró identificar ningún otro artículo de investigación que confirmara o negara su existencia. El objetivo de este trabajo fue encontrar dichas estructuras en las paredes ventriculares subendocárdicas de corazones humanos. Recolectamos 15 corazones conservados en formalina del Departamento de Anatomía de la Universidad de São Paulo y seccionamos las porciones apicales de los ventrículos derecho e izquierdo según el método utilizado por Goerttler. Utilizamos histología convencional (microscopía de luz-LM), microscopía electrónica de barrido (SEM) y un nuevo método de conservación llamado microplastinación (MP). En la pared anterior del ventrículo derecho en la región subendocárdica entre el tabique interventricular y la banda moderadora, encontramos varios haces de fibras fusiformes y helicoidales de histología similar a la FMV. Los haces medían entre 400 y 1150 µm de longitud y estaban separados de las fibras musculares adyacentes por una fina fibra de colágeno, actuando así como una "pseudocápsula". Algunas estructuras parecían estar vinculadas a la fibras de purkinje y parecían ser vasos linfáticos, sanguíneos y nerviosos. Llamamos a esas estructuras "corpúsculos cardíacos" (CC). La observación del CC previamente "desconocido" en este estudio inicial confirmó las descripciones anteriores y su descubrimiento puede contribuir a nuevas perspectivas en el estudio de la estructura y función del músculo cardíaco.

Partial and complete loss of myosin binding protein H-like cause cardiac conduction defects.

A premature truncation of MYBPHL in humans and a loss of Mybphl in mice is associated with dilated cardiomyopathy, atrial and ventricular arrhythmias, and atrial enlargement. MYBPHL encodes myosin binding protein H-like (MyBP-HL). Prior work in mice indirectly identified Mybphl expression in the atria and in small puncta throughout the ventricle. Because of its genetic association with human and mouse cardiac conduction system disease, we evaluated the anatomical localization of MyBP-HL and the consequences of loss of MyBP-HL on conduction system function. Immunofluorescence microscopy of normal adult mouse ventricles identified MyBP-HL-positive ventricular cardiomyocytes that co-localized with the ventricular conduction system marker contactin-2 near the atrioventricular node and in a subset of Purkinje fibers. Mybphl heterozygous ventricles had a marked reduction of MyBP-HL-positive cells compared to controls. Lightsheet microscopy of normal perinatal day 5 mouse hearts showed enrichment of MyBP-HL-positive cells within and immediately adjacent to the contactin-2-positive ventricular conduction system, but this association was not apparent in Mybphl heterozygous hearts. Surface telemetry of Mybphl-null mice revealed atrioventricular block and atrial bigeminy, while intracardiac pacing revealed a shorter atrial relative refractory period and atrial tachycardia. Calcium transient analysis of isolated Mybphl-null atrial cardiomyocytes demonstrated an increased heterogeneity of calcium release and faster rates of calcium release compared to wild type controls. Super-resolution microscopy of Mybphl heterozygous and homozygous null atrial cardiomyocytes showed ryanodine receptor disorganization compared to wild type controls. Abnormal calcium release, shorter atrial refractory period, and atrial dilation seen in Mybphl null, but not wild type control hearts, agree with the observed atrial arrhythmias, bigeminy, and atrial tachycardia, whereas the proximity of MyBP-HL-positive cells with the ventricular conduction system provides insight into how a predominantly atrial expressed gene contributes to ventricular arrhythmias and ventricular dysfunction.

In vitro ion channel profile and ex vivo cardiac electrophysiology properties of the R(-) and S(+) enantiomers of hydroxychloroquine.

Hydroxychloroquine (HCQ) is a derivative of the antimalaria drug chloroquine primarily prescribed for autoimmune diseases. Recent attempts to repurpose HCQ in the treatment of corona virus disease 2019 has raised concerns because of its propensity to prolong the QT-segment on the electrocardiogram, an effect associated with increased pro-arrhythmic risk. Since chirality can affect drug pharmacological properties, we have evaluated the functional effects of the R(-) and S(+) enantiomers of HCQ on six ion channels contributing to the cardiac action potential and on electrophysiological parameters of isolated Purkinje fibers. We found that R(-)HCQ and S(+)HCQ block human Kir2.1 and hERG potassium channels in the 1 µM-100 µM range with a 2-4 fold enantiomeric separation. NaV1.5 sodium currents and CaV1.2 calcium currents, as well as KV4.3 and KV7.1 potassium currents remained unaffected at up to 90 µM. In rabbit Purkinje fibers, R(-)HCQ prominently depolarized the membrane resting potential, inducing autogenic activity at 10 µM and 30 µM, while S(+)HCQ primarily increased the action potential duration, inducing occasional early afterdepolarization at these concentrations. These data suggest that both enantiomers of HCQ can alter cardiac tissue electrophysiology at concentrations above their plasmatic levels at therapeutic doses, and that chirality does not substantially influence their arrhythmogenic potential in vitro.

Muscarinic agonists inhibit the ATP-dependent potassium current and suppress the ventricle-Purkinje action potential dispersion.

Activation of the parasympathetic nervous system has been reported to have an antiarrhythmic role during ischemia-reperfusion injury by decreasing the arrhythmia triggers. Furthermore, it was reported that the parasympathetic neurotransmitter acetylcholine is able to modulate the ATP-dependent potassium current (I K-ATP), a crucial current activated during hypoxia. However, the possible significance of this current modulation in the antiarrhythmic mechanism is not fully clarified. Action potentials were measured using the conventional microelectrode technique from canine left ventricular papillary muscle and free-running Purkinje fibers, under normal and hypoxic conditions. Ionic currents were measured using the whole-cell configuration of the patch-clamp method. Acetylcholine at 5 µmol/L did not influence the action potential duration (APD) either in Purkinje fibers or in papillary muscle preparations. In contrast, it significantly lengthened the APD and suppressed the Purkinje-ventricle APD dispersion when it was administered after 5 µmol/L pinacidil application. Carbachol at 3 µmol/L reduced the pinacidil-activated I K-ATP under voltage-clamp conditions. Acetylcholine lengthened the ventricular action potential under simulated ischemia condition. In this study, we found that acetylcholine inhibits the I K-ATP and thus suppresses the ventricle-Purkinje APD dispersion. We conclude that parasympathetic tone may reduce the arrhythmogenic substrate exerting a complex antiarrhythmic mechanism during hypoxic conditions.

3D Bioprinting the Cardiac Purkinje System Using Human Adipogenic Mesenchymal Stem Cell Derived Purkinje Cells.

PURPOSE: The objective of this study was to reprogram human adipogenic mesenchymal stem cells (hADMSCs) to form Purkinje cells and to use the reprogrammed Purkinje cells to bioprint Purkinje networks. METHODS: hADMSCs were reprogrammed to form Purkinje cells using a multi-step process using transcription factors ETS2 and MESP1 to first form cardiac progenitor stem cells followed by SHOX2 and TBX3 to form Purkinje cells. A novel bioprinting method was developed based on Pluronic acid as the sacrificial material and type I collagen as the structural material. The reprogrammed Purkinje cells were used in conjunction with the novel bioprinting method to bioprint Purkinje networks. Printed constructs were evaluated for retention of functional protein connexin 40 (Cx40) and ability to undergo membrane potential changes in response to physiologic stimulus. RESULTS: hADMSCs were successfully reprogrammed to form Purkinje cells based on the expression pattern of IRX3, IRX5, SEMA and SCN10. Reprogrammed purkinje cells were incorporated into a collagen type-1 bioink and the left ventricular Purkinje network was printed using anatomical images of the bovine Purkinje system as reference. Optimization studies demonstrated that 1.8 mg/mL type-I collagen at a seeding density of 300,000 cells per 200 µL resulted in the most functional bioprinted Purkinje networks. Furthermore, bioprinted Purkinje networks formed continuous syncytium, retained expression of vital functional gap junction protein Cx40 post-print, and exhibited membrane potential changes in response to electric stimulation and acetylcholine evaluated by DiBAC4(5), an electrically responsive dye. CONCLUSION: Based on the results of this study, hADMSCs were successfully reprogrammed to form Purkinje cells and bioprinted to form Purkinje networks.

Electrophysiological mechanisms of vandetanib-induced cardiotoxicity: Comparison of action potentials in rabbit Purkinje fibers and pluripotent stem cell-derived cardiomyocytes.

Vandetanib, a multi-kinase inhibitor used for the treatment of various cancers, has been reported to induce several adverse cardiac effects. However, the underlying mechanisms of vandetanib-induced cardiotoxicity are unclear. This study aimed to investigate the mechanism of vandetanib-induced cardiotoxicity using intracellular electrophysiological recordings on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), rabbit Purkinje fibers, and HEK293 cells transiently expressing human ether-a-go-go-related gene (hERG; the rapidly activating delayed rectifier K+ channel, IKr), KCNQ1/KCNE1 (the slowly activating delayed rectifier K+ current, IKs), KCNJ2 (the inwardly rectifying K+ current, IK1) or SCN5A (the inward Na+ current, INa). Purkinje fiber assays and ion channel studies showed that vandetanib at concentrations of 1 and 3 µM inhibited the hERG currents and prolonged the action potential duration. Alanine scanning and in silico hERG docking studies demonstrated that Y652 and F656 in the hERG S6 domain play critical roles in vandetanib binding. In hiPSC-CMs, vandetanib markedly reduced the maximum rate of depolarization during the AP upstroke. Ion channel studies revealed that hiPSC-CMs were more sensitive to inhibition of the INa by vandetanib than in a heterogeneously expressed HEK293 cell model, consistent with the changes in the AP parameters of hiPSC-CMs. The subclasses of Class I antiarrhythmic drugs inhibited INa currents in a dose-dependent manner in hiPSC-CMs and SCN5A-encoded HEK293 cells. The inhibitory potency of vandetanib for INa was much higher in hiPSC-CMs (IC50: 2.72 µM) than in HEK293 cells (IC50: 36.63 µM). These data suggest that AP and INa assays using hiPSC-CMs are useful electrophysiological models for prediction of drug-induced cardiotoxicity.

High resolution 3-Dimensional imaging of the human cardiac conduction system from microanatomy to mathematical modeling.

Cardiac arrhythmias and conduction disturbances are accompanied by structural remodelling of the specialised cardiomyocytes known collectively as the cardiac conduction system. Here, using contrast enhanced micro-computed tomography, we present, in attitudinally appropriate fashion, the first 3-dimensional representations of the cardiac conduction system within the intact human heart. We show that cardiomyocyte orientation can be extracted from these datasets at spatial resolutions approaching the single cell. These data show that commonly accepted anatomical representations are oversimplified. We have incorporated the high-resolution anatomical data into mathematical simulations of cardiac electrical depolarisation. The data presented should have multidisciplinary impact. Since the rate of depolarisation is dictated by cardiac microstructure, and the precise orientation of the cardiomyocytes, our data should improve the fidelity of mathematical models. By showing the precise 3-dimensional relationships between the cardiac conduction system and surrounding structures, we provide new insights relevant to valvar replacement surgery and ablation therapies. We also offer a practical method for investigation of remodelling in disease, and thus, virtual pathology and archiving. Such data presented as 3D images or 3D printed models, will inform discussions between medical teams and their patients, and aid the education of medical and surgical trainees.

Morphological study of the atrioventricular conduction system and Purkinje fibers in yak.

We studied the morphology of the atrioventricular conduction system (AVCS) and Purkinje fibers of the yak. Light and transmission electron microscopy were used to study the histological features of AVCS. The distributional characteristics of the His-bundle, the left bundle branch (LBB), right bundle branch (RBB), and Purkinje fiber network of yak hearts were examined using gross dissection, ink injection, and ABS casting. The results showed that the atrioventricular node (AVN) of yak located in the right side of interatrial septum and had a flattened ovoid shape. The AVN of yak is composed of the slender, interweaving cells formed almost entirely of the transitional cells (T-cells). The His-bundle extended from the AVN, and split into left LBB and RBB at the crest of the interventricular septum. The LBB descended along the left side of interventricular septum. At approximately the upper 1/3 of the interventricular septum, the LBB typically divided into three branches. The RBB ran under the endocardium of the right side of interventricular septum, and extended to the base of septal papillary muscle, passed into the moderator band, crossed the right ventricular cavity to reach the base of anterior papillary muscle, and divided into four fascicles under the subendocardial layer. The Purkinje fibers in the ventricle formed a complex spatial network. The distributional and cellular component characteristics of the AVCS and Purkinje fibers ensured normal cardiac function.

A new electrophysiologic observation in patients with idiopathic left ventricular tachycardia.

BACKGROUND: In patients with idiopathic left ventricular tachycardia (ILVT), the arrhythmogenic substrate is poorly understood. OBJECTIVE: The purpose of this study was to elucidate the ILVT characteristics and outcome of radiofrequency catheter ablation in patients with ILVT. METHODS: Twenty-four patients with ILVT and 15 patients with left accessory pathways (control) underwent high-density mapping of the left His-Purkinje system during sinus rhythm (SR) using 3-dimensional electroanatomic mapping. RESULTS: Fragmented antegrade Purkinje potential (FAP) was represented at the left ventricular septum slightly inferoposterior to the left posterior fascicle (LPF) in 23 patients with ILVT. In control subjects, no FAPs could be recorded at the same region, FAPs were identified at the proximal portion of the LPF (4 patients) and at the distal LPF (1 patient). The finding of any FAPs in ILVT patients was significantly higher than that in control patients (23/24 vs 5/15, P < .01). Radiofrequency ablation at the area of FAP resulted in successful ablation in 23 patients with ILVT. No ILVT recurred during follow-up of 16.3 ± 7.2 months. CONCLUSION: In patients with ILVT, FAP located at the left ventricular septum slightly inferoposterior to the LPF is a novel finding using 3-dimensional electroanatomic mapping. The FAP may represent an arrhythmogenic substrate in ILVT and may be used for guiding successful ablation.

Intractable Electrical Storm After Coronary Artery Bypass Grafting Originating in Abnormal Purkinje Fibers.

Electrical storm is a rare but critical complication following revascularization in patients with ischemic heart disease. We report the case of a 67-year-old man who developed drug refractory intractable electrical storm after emergent coronary artery bypass grafting for ischemic cardiomyopathy. The electrical storm was successfully eliminated by percutaneous endocardial radiofrequency catheter ablation targeting the abnormal Purkinje-related triggering ventricular premature contractions in a low-voltage zone.

Catheter ablation targeting Purkinje potentials controlled ventricular fibrillation in a patient with a malignant lymphoma occurring in the ventricular septum.

Malignant lymphoma is known to cause various types of arrhythmia, including ventricular fibrillation. However, radiofrequency catheter ablation of ventricular fibrillation associated with malignant lymphoma has never been reported. We describe the case of a 53-year-old man with refractory ventricular fibrillation that was associated with malignant lymphoma. Electrophysiological testing revealed that a Purkinje potential appeared before ventricular contraction at the tumour site. We successfully treated the ventricular fibrillation with radiofrequency catheter ablation, using the Purkinje potential as an indicator. Physicians should consider this treatment if ventricular fibrillation cannot be controlled using other strategies.

Purkinje fibers after myocardial ischemia-reperfusion.

The purpose of this study was to evaluate the effects of ischemia-reperfusion on Purkinje fibers, comparing them with the adjacent cardiomyocytes. In a model of heterotopic heart transplantation in pigs, the donor heart was subjected to 2 hours of ischemia (n=9), preserved in cold saline, and subjected to 24 hours of ischemia with preservation in Wisconsin solution, alone (n=6), or with an additive consisting of calcium (n=4), Nicorandil (n=6) or Trolox (n=7). After 2 hours of reperfusion, we evaluated the recovery of cardiac electrical activity and took samples of ventricular myocardium for morphological study. The prolonged ischemia significantly affected atrial automaticity and A-V conduction in all the groups subjected to 24 hours of ischemia, as compared to 2 hours. There were no significant differences among the groups that underwent prolonged ischemia. Changes in the electrical activity did not correlate with the morphological changes. In the Purkinje fibers, ischemia-reperfusion produced a marked decrease in the glycogen content in all the groups. In the gap junctions the immunolabeling of connexin-43 decreased significantly, adopting a dispersed distribution, and staining the sarcolemma adjacent to the connective tissue. These changes were less marked in the group preserved exclusively with Wisconsin solution, despite the prolonged ischemia. The addition of other substances did not improve the altered morphology. In all the groups, the injury appeared to be more prominent in the Purkinje fibers than in the neighboring cardiomyocytes, indicating the greater susceptibility of the former to ischemia-reperfusion injury.

Cardiovascular safety pharmacology profile of etamicastat, a novel peripheral selective dopamine-ß-hydroxylase inhibitor.

Etamicastat, a peripheral reversible dopamine-ß-hydroxylase inhibitor, blocked the hERG current amplitude with an IC50 value of 44.0µg/ml in HEK 293 cells. At 0.3 and 3µg/ml, etamicastat had no effects on the action potential (AP) in male dog Purkinje fibers. At 30µg/ml, etamicastat significantly affected resting membrane potential (+4%), AP amplitude (-4%), AP duration at 60% (-14%) and AP duration at 90% (+5%) repolarization, and AP triangulation (+79%). In the telemetered conscious male dog, etamicastat (up to 20mg/kg) had no effects on arterial blood pressure, heart rate and the PR interval. At 10 and 20mg/kg, the QTc interval was slightly prolonged (8-9% max, P<0.05). No arrhythmia or other changes in the morphology of the ECG were observed. The maximum observed plasma concentrations (Cmax) of etamicastat (i.e. 3h post-administration) were 1.4 and 3.7µg/ml at 10 and 20mg/kg, respectively. No deleterious effects, including ECG disturbance were observed in male and female dogs dosed by gavage with etamicastat (up to 20mg/kg/day) for 28 days. Mean plasma Cmax etamicastat levels ranged between 2.4 and 6.3µg/ml on Day 1 and Day 28 of treatment, respectively. It is concluded that the blockade of the delayed rectifier potassium channels by etamicastat together with the QTc interval prolongation observed in conscious dogs can be considered as modest with respect to the measured plasmatic concentrations. These findings suggest that etamicastat is not likely to prolong the QT interval at therapeutic doses (~0.2µg/ml).

Mapping and ablating ventricular premature contractions that trigger ventricular fibrillation: trigger elimination and substrate modification.

Ventricular fibrillation (VF) is a malignant arrhythmia, usually initiated by a ventricular premature contraction (VPC) during the vulnerable period of cardiac repolarization. Ablation therapy for VF has been described and increasingly reported. Targets for VF triggers are VPC preceded Purkinje potentials or the right ventricular outflow tract (RVOT) in structurally normal hearts, and VPC triggers preceded by Purkinje potentials in ischemic cardiomyopathy. The most important issue before the ablation session is the recording of the 12-lead electrocardiogram (ECG) of the triggering event, which can prove invaluable in regionalizing the origin of the triggering VPC for more detailed mapping. In cases where the VPC is not spontaneous or inducible, ablation may be performed by pacemapping. During the session, mapping should be focused on the earliest activation and determining the earliest potential is the key to a successful ablation. However, a modification of the Purkinje network might be applied when the earliest site cannot be determined or is located close to the His-bundle. Furthermore, the electrical isolation of the pulmonary artery (PA) can suppress RVOT type polymorphic ventricular tachycardia in some patients with rapid triggers from the PA. Suppression of VF can be achieved by not only the elimination of triggering VPCs, but also by substrate modification of possible reentry circuits in the Purkinje network, or between the PA and RVOT. Further studies are needed to evaluate the precise mechanisms of this arrhythmia.

Congenital multifocal increase of Purkinje fibres in a calf with cardiac conduction delay.

A female 4-month-old Holstein-Friesian calf was presented in heart failure. Microscopical examination of samples of the cardiac wall taken at necropsy examination revealed numerous aggregates of Purkinje fibres, particularly in the perivascular areas. Some Purkinje fibres were stained strongly with phosphotungstic acid haematoxylin and immunohistochemically were shown to express alpha smooth muscle actin, indicating an embryonic-like Purkinje fibre phenotype. A diagnosis of congenital multifocal increase of Purkinje fibres was made. The histological features of this case resemble multifocal cardiac Purkinje cell tumour of the heart in man.

Limitations of animal electrical cardiac safety models.

INTRODUCTION: Human electrical safety standards are based almost exclusively on animal studies and there is an unjustified assumption that ventricular fibrillation (VF) thresholds in animals are the same as those in humans. METHODS AND RESULTS: We analyzed differences between animals and humans in cardiac stimulation. A broad literature survey revealed that swine are a fragile electrophysiologic research species and have a dense intramural Purkinje fiber network, which is not found in some other species, including humans. Anesthesia agents have to be chosen carefully as swine are prone to malignant hyperthermia. Cardiac stimulation thresholds depend on weight and capture rates. Thus, the animal weight has to be representative of the weight of human subjects. Studies have shown significant ECG differences between humans and other species, including swine and canine. At least one study suggested that rabbit hearts tend to develop VF in a manner more similar to that seen in humans. CONCLUSION: Animal studies can play a role in conservatively evaluating cardiac safety. However, while still abiding by the precautionary principle, animal study design has to take into account the significant anatomical and electrophysiological differences between humans and other mammals. Data from multiple animal models may offer broader perspectives. If attempts are made to extrapolate animal results to humans then appropriate numerical correction factors should be applied, such as some of those discussed in this article.