Effects of Sex on the Susceptibility for Atrial Fibrillation in Pigs with Ischemic Heart Failure.

Atrial fibrillation (AF) is the most prevalent arrhythmia, often caused by myocardial ischemia/infarction (MI). Men have a 1.5× higher prevalence of AF, whereas women show a higher risk for new onset AF after MI. However, the underlying mechanisms of how sex affects AF pathophysiology are largely unknown. In 72 pigs with/without ischemic heart failure (IHF) we investigated the impact of sex on ischemia-induced proarrhythmic atrial remodeling and the susceptibility for AF. Electrocardiogram (ECG) and electrophysiological studies were conducted to assess electrical remodeling; histological analyses were performed to assess atrial fibrosis in male and female pigs. IHF pigs of both sexes showed a significantly increased vulnerability for AF, but in male pigs more and longer episodes were observed. Unchanged conduction properties but enhanced left atrial fibrosis indicated structural rather than electrical remodeling underlying AF susceptibility. Sex differences were only observed in controls with female pigs showing an increased intrinsic heart rate, a prolonged QRS interval and a prolonged sinus node recovery time. In sum, susceptibility for AF is significantly increased both in male and female pigs with ischemic heart failure. Differences between males and females are moderate, including more and longer AF episodes in male pigs and sinus node dysfunction in female pigs.

Cardiac Macrophages and Their Effects on Arrhythmogenesis.

Cardiac electrophysiology is a complex system established by a plethora of inward and outward ion currents in cardiomyocytes generating and conducting electrical signals in the heart. However, not only cardiomyocytes but also other cell types can modulate the heart rhythm. Recently, cardiac macrophages were demonstrated as important players in both electrophysiology and arrhythmogenesis. Cardiac macrophages are a heterogeneous group of immune cells including resident macrophages derived from embryonic and fetal precursors and recruited macrophages derived from circulating monocytes from the bone marrow. Recent studies suggest antiarrhythmic as well as proarrhythmic effects of cardiac macrophages. The proposed mechanisms of how cardiac macrophages affect electrophysiology vary and include both direct and indirect interactions with other cardiac cells. In this review, we provide an overview of the different subsets of macrophages in the heart and their possible interactions with cardiomyocytes under both physiologic conditions and heart disease. Furthermore, we elucidate similarities and differences between human, murine and porcine cardiac macrophages, thus providing detailed information for researchers investigating cardiac macrophages in important animal species for electrophysiologic research. Finally, we discuss the pros and cons of mice and pigs to investigate the role of cardiac macrophages in arrhythmogenesis from a translational perspective.

Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node.

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by electrical impulses generated in sinoatrial node (SAN) and then conducted to ventricles via atrioventricular node (AVN). To further study the role of resident macrophages in cardiac conduction system, a proper isolation of resident macrophages from SAN and AVN is necessary, but it remains challenging. Here, we provide a protocol for the reliable microdissection of the SAN and AVN in murine hearts followed by the isolation and culture of resident macrophages. Both, SAN which is located at the junction of the crista terminalis with the superior vena cava, and AVN which is located at the apex of the triangle of Koch, are identified and microdissected. Correct location is confirmed by histologic analysis of the tissue performed with Masson's trichrome stain and by anti-HCN4. Microdissected tissues are then enzymatically digested to obtain single cell suspensions followed by the incubation with a specific panel of antibodies directed against cell-type specific surface markers. This allows to identify, count, or isolate different cell populations by fluorescent activated cell sorting. To differentiate cardiac resident macrophages from other immune cells in the myocardium, especially recruited monocyte-derived macrophages, a delicate devised gating strategy is needed. First, lymphoid lineage cells are detected and excluded from further analysis. Then, myeloid cells are identified with resident macrophages being determined by high expression of both CD45 and CD11b, and low expression of Ly6C. With cell sorting, isolated cardiac macrophages can then be cultivated in vitro over several days for further investigation. We, therefore, describe a protocol to isolate cardiac resident macrophages located within the cardiac conduction system. We discuss pitfalls in microdissecting and digesting SAN and AVN, and provide a gating strategy to reliably identify, count and sort cardiac macrophages by fluorescence-activated cell sorting.