Characteristics and time course of acute and chronic myocardial lesion formation after electroporation ablation in the porcine model.

INTRODUCTION: Electroporation ablation creates deep and wide myocardial lesions. No data are available on time course and characteristics of acute lesion formation. METHODS: For the acute phase of myocardial lesion development, seven pigs were investigated. Single 200 J applications were delivered at four different epicardial right ventricular sites using a linear suction device, yielding a total of 28 lesions. Timing of applications was designed to yield lesions at seven time points: 0, 10, 20, 30, 40, 50, and 60 min, with four lesions per time point. After killing, lesion characteristics were histologically investigated. For the chronic phase of myocardial lesion development, tissue samples were used from previously conducted studies where tissue was obtained at 3 weeks and 3 months after electroporation ablation. RESULTS: Acute myocardial lesions induce a necrosis pattern with contraction band necrosis and interstitial edema, immediately present after electroporation ablation. No further histological changes such as hemorrhage or influx of inflammatory cells occurred in the first hour. After 3 weeks, the lesions consisted of sharply demarcated loose connective tissue that further developed to more fibrotic scar tissue after 3 months without additional changes. Within the scar tissue, arteries and nerves were unaffected. CONCLUSION: Electroporation ablation immediately induces contraction band necrosis and edema without additional tissue changes in the first hour. After 3 weeks, a sharply demarked scar has been developed that remains stable during follow-up of 3 months. This is highly relevant for clinical application of electroporation ablation in terms of the electrophysiological endpoint and waiting period after ablation.

High-frequency irreversible electroporation for cardiac ablation using an asymmetrical waveform.

BACKGROUND: Irreversible electroporation (IRE) using direct current (DC) is an effective method for the ablation of cardiac tissue. A major drawback of the use of DC-IRE, however, are two problems: requirement of general anesthesia due to severe muscle contractions and the formation of bubbles containing gaseous products from electrolysis. The use of high-frequency alternating current (HF-IRE) is expected to solve both problems, because HF-IRE produces little to no muscle spasms and does not cause electrolysis. METHODS: In the present study, we introduce a novel asymmetric, high-frequency (aHF) waveform for HF-IRE and present the results of a first, small, animal study to test its efficacy. RESULTS: The data of the experiments suggest that the aHF waveform creates significantly deeper lesions than a symmetric HF waveform of the same energy and frequency (p = 0.003). CONCLUSION: We therefore conclude that the use of the aHF enhances the feasibility of the HF-IRE method.

Neonatal rat cardiomyocytes as an in vitro model for circadian rhythms in the heart.

Circadian rhythms are biorhythms with a 24-hour period that are regulated by molecular clocks. Several clinical and animal models have been developed to analyze the role of these rhythms in cardiovascular physiology, disease and therapy, but a convenient in vitro model that mimics both molecular and functional circadian effects of the heart is not available. Therefore, we established a neonatal rat cardiomyocyte model that recapitulates in vivo circadian rhythmicity, as measured by anti-phasic oscillatory mRNA expression of two core clock genes, Bmal1 and Per2 and that shows functional dependence on the clock as indicated by an oscillating response in apoptosis induced by doxorubicin, hydroperoxide or hypoxia. In addition, perturbation of the cardiac clock by the use of several compounds including Resveratrol and Ex-527 was found to result in loss of functional rhythmicity. This indicates that neonatal rat cardiomyocytes are a good model to investigate the cardiac circadian clock as well as a system that allows for fast and easy preclinical testing of the influence of compounds on circadian rhythmicity that might have crucial effects on cardiac health.

Circadian rhythms in cell maturation.

Circadian rhythms are of major importance in mammalian physiology and disease. In this review, we give an overview of the present knowledge on origination of circadian rhythms. We discuss the development of both master and peripheral clocks and compare the origination of circadian rhythms in utero and in vitro.

Minimal coronary artery damage by myocardial electroporation ablation.

AIMS: Radiofrequency catheter ablation is a successful treatment for cardiac arrhythmias, but may lead to major complications such as permanent coronary damage. Irreversible electroporation (IRE) is a new non-thermal ablation modality, but its effect on coronary arteries is still unknown. METHODS AND RESULTS: In a porcine model, epicardial IRE lesions were created at the base of the left ventricle in four hearts (group A) and directly on the left anterior descending artery (LAD) in five hearts (group B). After 3 weeks, coronary arteries inside IRE lesions and in apparently undamaged myocardium next to the lesions were (immuno-)histologically studied. Two untreated hearts served as controls. Coronary damage was defined as intimal hyperplasia. Left anterior descending artery angiograms were obtained before ablation, directly after ablation, and before termination in group B. In group A, 103 arterial branches were studied. Of these, 5 of 56 arterial branches inside lesions and 1 of 47 outside lesions showed intimal hyperplasia, but all had <50% area stenosis. Targeted LADs (group B) did not reveal intimal hyperplasia and angiograms showed no signs of stenosis. Expression of connective tissue growth factor was observed in the scar tissue, but not in the fibrotic tissue directly around the arteries, confirming that the arteries are indeed spared from tissue damage and remodelling. CONCLUSION: Coronary arteries remain free of clinically relevant damage 3 weeks after epicardial IRE ablation, even amid very large myocardial lesions. This suggests that IRE ablation can be applied safely near or even on coronary arteries. With IRE ablation, arterial blood flow does not appear to affect lesion formation.

The circadian clock remains intact, but with dampened hormonal output in heart failure.

BACKGROUND: Circadian (24-h) rhythms are important regulators in physiology and disease, but systemic disease may disrupt circadian rhythmicity. Heart failure (HF) is a systemic disease affecting hormonal regulation. We investigate whether HF affects the rhythmic expression of melatonin and cortisol, main endocrine products of the central clock, and cardiac-specific troponin in patients. We corroborate the functionality of the peripheral clock directly in the organs of translational models, inaccessible in human participants. METHODS: We included 46 HF patients (71.7% male, median age of 60 years, NYHA class II (32.6%) or III (67.4%), ischemic cardiomyopathy (43.5%), comorbidities: diabetes 21.7%, atrial fibrillation 30.4%), and 24 matched controls. Blood was collected at seven time-points during a 24-h period (totalling 320 HF and 167 control samples) for melatonin, cortisol, and cardiac troponin T (cTnT) measurements after which circadian rhythms were assessed through cosinor analyses, both on the individual and the group level. Next, we analysed peripheral circadian clock functionality using cosinor analysis in male animal HF models: nocturnal mice and diurnal zebrafish, based on expression of core clock genes in heart, kidneys, and liver, every 4 h during a 24-h period in a light/darkness synchronised environment. FINDINGS: Melatonin and cortisol concentrations followed a physiological 24-h pattern in both patients and controls. For melatonin, acrophase occurred during the night for both groups, with significantly decreased amplitude (median 5.2 vs 8.8, P = 0.0001) and circadian variation ([maximum]/[minimum]) in heart failure patients. For cortisol, mesor showed a significant increase for HF patients (mean 331.9 vs 275.1, P = 0.017) with a difference of 56.8 (95% CI 10.3-103.3) again resulting in a relatively lower variation: median 3.9 vs 6.3 (P = 0.0058). A nocturnal blood pressure dip was absent in 77.8% of HF patients. Clock gene expression profiles (Bmal, Clock, Per, Cry) were similar and with expected phase relations in animal HF models and controls, demonstrating preserved peripheral clock functionality in HF. Furthermore, oscillations in diurnal zebrafish were expectedly in opposite phases to those of nocturnal mice. Concordantly, cTnT concentrations in HF patients revealed significant circadian oscillations. INTERPRETATION: Central clock output is dampened in HF patients while the molecular peripheral clock, as confirmed in animal models, remains intact. This emphasises the importance of taking timing into account in research and therapy for HF, setting the stage for another dimension of diagnostic, prognostic and therapeutic approaches. FUNDING: Hartstichting.

Circadian Dependence of the Acute Immune Response to Myocardial Infarction.

Circadian rhythms influence the recruitment of immune cells and the onset of inflammation, which is pivotal in the response to ischemic cardiac injury after a myocardial infarction (MI). The hyperacute immune response that occurs within the first few hours after a MI has not yet been elucidated. Therefore, we characterized the immune response and myocardial damage 3 hours after a MI occurs over a full twenty-four-hour period to investigate the role of the circadian rhythms in this response. MI was induced at Zeitgeber Time (ZT) 2, 8, 14, and 20 by permanent ligation of the left anterior descending coronary artery. Three hours after surgery, animals were terminated and blood and hearts collected to assess the immunological status and cardiac damage. Blood leukocyte numbers varied throughout the day, peaking during the rest-phase (ZT2 and 8). Extravasation of leukocytes was more pronounced during the active-phase (ZT14 and 20) and was associated with greater chemokine release to the blood and expression of adhesion molecules in the heart. Damage to the heart, measured by Troponin-I plasma levels, was elevated during this time frame. Clock gene oscillations remained intact in both MI-induced and sham-operated mice hearts, which could explain the circadian influence of the hyperacute inflammatory response after a MI. These findings are in line with the clinical observation that patients who experience a MI early in the morning (i.e., early active phase) have worse clinical outcomes. This study provides further insight on the immune response occurring shortly after an MI, which may contribute to the development of novel and optimization of current therapeutic approaches.

Circadian rhythms in ischaemic heart disease: key aspects for preclinical and translational research: position paper of the ESC working group on cellular biology of the heart.

Circadian rhythms are internal regulatory processes controlled by molecular clocks present in essentially every mammalian organ that temporally regulate major physiological functions. In the cardiovascular system, the circadian clock governs heart rate, blood pressure, cardiac metabolism, contractility, and coagulation. Recent experimental and clinical studies highlight the possible importance of circadian rhythms in the pathophysiology, outcome, or treatment success of cardiovascular disease, including ischaemic heart disease. Disturbances in circadian rhythms are associated with increased cardiovascular risk and worsen outcome. Therefore, it is important to consider circadian rhythms as a key research parameter to better understand cardiac physiology/pathology, and to improve the chances of translation and efficacy of cardiac therapies, including those for ischaemic heart disease. The aim of this Position Paper by the European Society of Cardiology Working Group Cellular Biology of the Heart is to highlight key aspects of circadian rhythms to consider for improvement of preclinical and translational studies related to ischaemic heart disease and cardioprotection. Applying these considerations to future studies may increase the potential for better translation of new treatments into successful clinical outcomes.

Prognostic biomarker soluble ST2 exhibits diurnal variation in chronic heart failure patients.

AIM: Soluble suppression of tumorigenicity-2 (sST2) is a strong prognostic biomarker in heart failure. The emerging understanding of circadian biology in cardiovascular disease may lead to novel applications in prognosis and diagnosis and may provide insight into mechanistic aspects of the disease-biomarker interaction. So far, it is unknown whether sST2 exhibits a diurnal rhythm. Repeated measurements of sST2 may aid in clinical decision making. The goal of this study was to investigate whether sST2 exhibits diurnal variation in patients with heart failure with reduced ejection fraction (HFrEF) and in control subjects, thereby enhancing its diagnostic and prognostic values. METHODS AND RESULTS: The study comprised 32 subjects: 16 HFrEF patients and 16 controls. Blood was collected at seven subsequent time points during a 24 h time period. sST2, N-terminal pro-B-type natriuretic peptide (NT-proBNP), melatonin, and cortisol were measured from serum. Peak values of sST2 clustered at daytime (modal value: 5 p.m.) in 87.6% of all subjects (81.3% of patients, P = 0.021; 93.8% of controls, P = 0.001), and minimum concentrations at night-time (modal value: 5 a.m.) in 84.4% (87.5% of patients, P = 0.004 81.3% of controls, P = 0.021). A cosinor analysis of mean normalized sST2 values revealed significant cosine shaped 24 h oscillations of patients (P = 0.026) and controls (P = 0.037). NT-proBNP in contrast did not show a diurnal rhythm, while melatonin and cortisol patterns were intact in all subjects. CONCLUSIONS: sST2 exhibits a diurnal rhythm with lower values in the morning than in the late afternoon. This new insight could lead to refinement of its diagnostic and prognostic values through specified and consistent sampling times with repeated measurements. For example, by measuring sST2 during the afternoon, when levels are at their highest, false negatives on prognosis prediction could be avoided.

Circadian rhythms and the molecular clock in cardiovascular biology and disease.

The Earth turns on its axis every 24 h; almost all life on the planet has a mechanism - circadian rhythmicity - to anticipate the daily changes caused by this rotation. The molecular clocks that control circadian rhythms are being revealed as important regulators of physiology and disease. In humans, circadian rhythms have been studied extensively in the cardiovascular system. Many cardiovascular functions, such as endothelial function, thrombus formation, blood pressure and heart rate, are now known to be regulated by the circadian clock. Additionally, the onset of acute myocardial infarction, stroke, arrhythmias and other adverse cardiovascular events show circadian rhythmicity. In this Review, we summarize the role of the circadian clock in all major cardiovascular cell types and organs. Second, we discuss the role of circadian rhythms in cardiovascular physiology and disease. Finally, we postulate how circadian rhythms can serve as a therapeutic target by exploiting or altering molecular time to improve existing therapies and develop novel ones.

[Reduced consciousness levels caused by hyperammonaemia]. / Bewustzijnsdaling door hyperammoniëmie.

Hyperammonaemia is an important cause of lethargy. In this article, we describe a lesser-known but potential fatal cause of hyperammonaemia. A 27-year-old woman presented with lethargy caused by hyperammonaemia. She was treated with the emergency regime that is used to treat hyperammonaemia in urea cycle defects. Although this effectively lowered the ammonia levels, the clinical situation of the patient initially deteriorated and she was transferred to the Intensive Care Unit and intubated. Urine culture identified Proteus mirabilis, a urea-splitting bacterium that caused the hyperammonaemia. Prompt and adequate treatment with antibiotics and adequate drainage of urine was started and she completely recovered. Although every patient can get hyperammonaemia caused by urinary tract infection with urea-splitting bacteria, patients with structural bladder abnormalities are at greater risk. Lethargy can be the only presenting symptom. When recognized early, it is quite treatable and has a good prognosis.

Analysis of 24-h Rhythm in Ventricular Repolarization Identifies QT Diurnality As a Novel Clinical Parameter Associated with Previous Ventricular Arrhythmias in Heart Failure Patients.

Introduction: Cardiac repolarization abnormalities are among the major causes of ventricular arrhythmias and sudden cardiac death. In humans, cardiac repolarization duration has a 24-h rhythm. Animal studies show that this rhythm is regulated by 24-h rhythms in ion channel function and that disruption of this rhythm leads to ventricular arrhythmias. We hypothesized that 24-h rhythms in QT duration can be used as a predictor for sudden cardiac death and are associated with ventricular arrhythmias. Secondly, we assessed a possible mechanistic explanation by studying the putative role of hERG channel dysfunction. Materials and Methods: In 2 retrospective studies, measures of the 24-h variation in the QT and QTc intervals (QT and QTc diurnality, QTd and QTcd, respectively) have been derived from Holter analyses and compared between groups: 1) 39 post-infarct patients with systolic heart failure (CHF: EF < 35%), of which 14 with, and 25 without a history of ventricular arrhythmias and 2) five patients with proven (LQTS2) and 16 with potential (Sotalol-induced) hERG channel dysfunction vs. 22 controls. Results: QTd was two-fold higher in CHF patients with a history of ventricular arrhythmias (38 ± 15 ms) compared to CHF patients without VT (16 ± 9 ms, p = 0.001). QTd was significantly increased in LQT2 patients (43 ± 24 ms) or those treated with Sotalol (30 ± 10 ms) compared to controls (21 ± 8 ms, p < 0.05 for both). Discussion: QT diurnality presents a novel clinical parameter of repolarization that can be derived from Holter registrations and may be useful for identification of patients at risk for ventricular arrhythmias.

SCA1+ Cells from the Heart Possess a Molecular Circadian Clock and Display Circadian Oscillations in Cellular Functions.

Stem cell antigen 1-positive (SCA1+) cells (SPCs) have been investigated in cell-based cardiac repair and pharmacological research, although improved cardiac function after injection has been variable and the mode of action remains unclear. Circadian (24-hr) rhythms are biorhythms regulated by molecular clocks that play an important role in (patho)physiology. Here, we describe (1) the presence of a molecular circadian clock in SPCs and (2) circadian rhythmicity in SPC function. We isolated SPCs from human fetal heart and found that these cells possess a molecular clock based on typical oscillations in core clock components BMAL1 and CRY1. Functional analyses revealed that circadian rhythmicity also governs SPC proliferation, stress tolerance, and growth factor release, with large differences between peaks and troughs. We conclude that SPCs contain a circadian molecular clock that controls crucial cellular functions. Taking circadian rhythms into account may improve reproducibility and outcome of research and therapies using SPCs.

Pulmonary vein stenosis after catheter ablation: electroporation versus radiofrequency.

BACKGROUND: Radiofrequency ablation inside pulmonary vein (PV) ostia can cause PV stenosis. A novel alternative method of ablation is irreversible electroporation, but the long-term response of PVs to electroporation ablation is unknown. METHODS AND RESULTS: In ten 6-month-old pigs (60-75 kg), the response of PVs to circular electroporation and radiofrequency ablation was compared. Ten consecutive, nonarcing, electroporation applications of 200 J were delivered 5 to 10 mm inside 1 of the 2 main PVs, using a custom-deflectable, 18-mm circular decapolar catheter. Inside the other PV, circular radiofrequency ablation was performed using 30 W radiofrequency applications via an irrigated 4-mm ablation catheter. PV angiograms were made before ablation, immediately after ablation, and after 3-month survival. PV diameters and heart size were measured. With electroporation ablation, PV ostial diameter decreased 11±10% directly after ablation, but had increased 19±11% after 3 months. With radiofrequency ablation, PV ostial diameter decreased 23±15% directly after ablation and remained 7±17% smaller after 3 months compared with preablation diameter despite a 21±7% increase in heart size during aging from 6 to 9 months. CONCLUSIONS: In this porcine model, multiple circumferential 200-J electroporation applications inside the PV ostia do not affect PV diameter at 3-month follow-up. Radiofrequency ablation inside PV ostia causes considerable PV stenosis directly after ablation, which persists after 3 months.

Stem cells for cardiac repair: an introduction.

Cardiovascular disease is a major cause of morbidity and mortality throughout the world. Most cardiovascular diseases, such as ischemic heart disease and cardiomyopathy, are associated with loss of functional cardiomyocytes. Unfortunately, the heart has a limited regenerative capacity and is not able to replace these cardiomyocytes once lost. In recent years, stem cells have been put forward as a potential source for cardiac regeneration. Pre-clinical studies that use stem cell-derived cardiac cells show promising results. The mechanisms, though, are not well understood, results have been variable, sometimes transient in the long term, and often without a mechanistic explanation. There are still several major hurdles to be taken. Stem cell-derived cardiac cells should resemble original cardiac cell types and be able to integrate in the damaged heart. Integration requires administration of stem cell-derived cardiac cells at the right time using the right mode of delivery. Once delivered, transplanted cells need vascularization, electrophysiological coupling with the injured heart, and prevention of immunological rejection. Finally, stem cell therapy needs to be safe, reproducible, and affordable. In this review, we will give an introduction to the principles of stem cell based cardiac repair.

[A man with atypical appendicitis]. / Een man met atypische appendicitis.

A 43-year-old man presented with acute left-sided middle and lower abdominal pain. He was diagnosed with 'left-sided acute appendicitis with non-rotation of the colon'. This is a rare and usually asymptomatic congenital anomaly.