Selective optogenetic stimulation of fibroblasts enables quantification of hetero-cellular coupling to cardiomyocytes in a three-dimensional model of heart tissue.

AIMS: Besides providing mechanical stability, fibroblasts in the heart could modulate the electrical properties of cardiomyocytes. Here, we aim to develop a three-dimensional hetero-cellular model to analyse the electric interaction between fibroblasts and human cardiomyocytes in vitro using selective optogenetic de- or hyperpolarization of fibroblasts. METHODS AND RESULTS: NIH3T3 cell lines expressing the light-sensitive ion channel Channelrhodopsin2 or the light-induced proton pump Archaerhodopsin were generated for optogenetic depolarization or hyperpolarization, respectively, and characterized by patch clamp. Cardiac bodies consisting of 50% fibroblasts and 50% human pluripotent stem cell-derived cardiomyocytes were analysed by video microscopy and membrane potential was measured with sharp electrodes. Myofibroblast activation in cardiac bodies was enhanced by transforming growth factor-ß1 (TGF-ß1)-stimulation. Connexin-43 expression was analysed by qPCR and fluorescence recovery after photobleaching. Illumination of Channelrhodopsin2 or Archaerhodopsin expressing fibroblasts induced inward currents and depolarization or outward currents and hyperpolarization. Transforming growth factor-ß1-stimulation elevated connexin-43 expression and increased cell-cell coupling between fibroblasts as well as increased basal beating frequency and cardiomyocyte resting membrane potential in cardiac bodies. Illumination of cardiac bodies generated with Channelrhodopsin2 fibroblasts accelerated spontaneous beating, especially after TGF-ß1-stimulation. Illumination of cardiac bodies prepared with Archaerhodopsin expressing fibroblasts led to hyperpolarization of cardiomyocytes and complete block of spontaneous beating after TGF-ß1-stimulation. Effects of light were significantly smaller without TGF-ß1-stimulation. CONCLUSION: Transforming growth factor-ß1-stimulation leads to increased hetero-cellular coupling and optogenetic hyperpolarization of fibroblasts reduces TGF-ß1 induced effects on cardiomyocyte spontaneous activity. Optogenetic membrane potential manipulation selectively in fibroblasts in a new hetero-cellular cardiac body model allows direct quantification of fibroblast-cardiomyocyte coupling in vitro.

Comparison of Arctic Front Advance Pro and POLARx cryoballoons for ablation therapy of atrial fibrillation: an intraprocedural analysis.

INTRODUCTION: Cryoballoon (CB) ablation has become a popular method for pulmonary vein isolation (PVI) in atrial fibrillation (AF) treatment. This study aimed to compare the intraprocedural ablation characteristics of two cryoballoons, Arctic Front Advance Pro™ (AFA-Pro, Medtronic) and POLARx™ (Boston Scientific). METHODS AND RESULTS: In this retrospective single-center study, 230 symptomatic paroxysmal or persistent AF patients underwent CB ablation with either AFA-Pro or POLARx. Propensity-score matching resulted in two cohorts of 114 patients each. Baseline and procedural characteristics were comparable between both CBs. POLARx achieved lower minimal temperatures (e.g., left superior pulmonary vein, LSPV: AFA-Pro - 49.0 °C vs. POLARx - 59.5 °C) and lower temperatures at time-to-isolation (TTI). Additionally, POLARx reached lower temperatures faster, as evidenced by lower temperatures after 40 and 60 s, and a larger mean temperature change between 20 and 40 s. POLARx also had a greater area under the curve below 0 °C and a longer thawing phase. Both CBs achieved comparable high rates of final PV-isolation. TTI, minimal esophagus temperature, and first-pass isolation rates were similar between groups. Periprocedural complications, including phrenic nerve injuries, were comparable. Troponin levels in the left atrium were elevated with both systems. Values and change in troponin were numerically higher in the POLARx group (delta troponin: AFA-Pro 36.3 (26.4, 125.4) ng/L vs. POLARx 104.9 (49.5, 122.2) ng/L), p = 0.077). CONCLUSION: AFA-Pro and POLARx are both highly effective and safe CB systems for PVI. POLARx exhibited significant faster and lower freezing characteristics, and numerically higher troponin levels might indicate greater myocardial injury. However, these differences did not translate into improved performance, procedural efficiency, or safety.

Optogenetic Termination of Cardiac Arrhythmia: Mechanistic Enlightenment and Therapeutic Application?

Optogenetic methods enable selective de- and hyperpolarization of cardiomyocytes expressing light-sensitive proteins within the myocardium. By using light, this technology provides very high spatial and temporal precision, which is in clear contrast to electrical stimulation. In addition, cardiomyocyte-specific expression would allow pain-free stimulation. In light of these intrinsic technical advantages, optogenetic methods provide an intriguing opportunity to understand and improve current strategies to terminate cardiac arrhythmia as well as for possible pain-free arrhythmia termination in patients in the future. In this review, we give a concise introduction to optogenetic stimulation of cardiomyocytes and the whole heart and summarize the recent progress on optogenetic defibrillation and cardioversion to terminate cardiac arrhythmia. Toward this aim, we specifically focus on the different mechanisms of optogenetic arrhythmia termination and how these might influence the prerequisites for success. Furthermore, we critically discuss the clinical perspectives and potential patient populations, which might benefit from optogenetic defibrillation devices.

Optogenetic Hyperpolarization of Cardiomyocytes Terminates Ventricular Arrhythmia.

Cardiac defibrillation to terminate lethal ventricular arrhythmia (VA) is currently performed by applying high energy electrical shocks. In cardiac tissue, electrical shocks induce simultaneously de- and hyperpolarized areas and only depolarized areas are considered to be responsible for VA termination. Because electrical shocks do not allow proper control over spatial extent and level of membrane potential changes, the effects of hyperpolarization have not been explored in the intact heart. In contrast, optogenetic methods allow cell type-selective induction of de- and hyperpolarization with unprecedented temporal and spatial control. To investigate effects of cardiomyocyte hyperpolarization on VA termination, we generated a mouse line with cardiomyocyte-specific expression of the light-driven proton pump ArchT. Isolated cardiomyocytes showed light-induced outward currents and hyperpolarization. Free-running VA were evoked by electrical stimulation of explanted hearts perfused with low K+ and the KATP channel opener Pinacidil. Optogenetic hyperpolarization was induced by epicardial illumination, which terminated VA with an average efficacy of ∼55%. This value was significantly higher compared to control hearts without illumination or ArchT expression (p = 0.0007). Intracellular recordings with sharp electrodes within the intact heart revealed hyperpolarization and faster action potential upstroke upon illumination, which should fasten conduction. However, conduction speed was lower during illumination suggesting enhanced electrical sink by hyperpolarization underlying VA termination. Thus, selective hyperpolarization in cardiomyocytes is able to terminate VA with a completely new mechanism of increased electrical sink. These novel insights could improve our mechanistic understanding and treatment strategies of VA termination.