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.

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 K ATP 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.

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-β1 (TGF-β1)-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-β1-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-β1-stimulation. Illumination of cardiac bodies prepared with Archaerhodopsin expressing fibroblasts led to hyperpolarization of cardiomyocytes and complete block of spontaneous beating after TGF-β1-stimulation. Effects of light were significantly smaller without TGF-β1-stimulation. Conclusion Transforming growth factor-β1-stimulation leads to increased hetero-cellular coupling and optogenetic hyperpolarization of fibroblasts reduces TGF-β1 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.

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Background Cardiac inflammation driven by the Toll-Like-Receptor 4 (TLR4) is correlated to increased risk of arrhythmia. Cardiac arrhythmogenesis and the formation of re-entry tachycardia is highly dependent on conduction velocity (CV) and action potential (AP) duration (APD). As TLR4 induced APD shortening has been shown, we analyze in this study for the first time the TLR4 effect on conductance disturbances in a LPS induced septic mouse model. Methods Systemic activation of TLR4 in mice was achieved by intraperitoneal LPS injection 3.5 hours prior to experiments. In vivo electrophysiological investigation (EPI) was performed using an octapolar transvenous catheter placed to the right heart. Ex vivo experiments were performed on Langendorff-perfused hearts. AP propagation was measured by optical voltage mapping (OVM) with voltage sensitive dye Di-4-ANEPPS. For AP analysis including RMP, intracellular electrical recordings were performed using sharp microelectrodes. Wildtype mice after LPS injection were compared to wildtype mice after vehicle (NaCl) injection or ubiquitous TLR4 knockout (TLR4−/−) mice with LPS or vehicle application. Results In vivo EPI showed a tendency to more atrial fibrillation after LPS injection (+LPS 5/6, +NaCl 2/6, p=0.2). Ventricular stimulation evoked ventricular tachycardia in every LPS treated WT mouse but less in controls (+LPS 6/6, +NaCl 1/6, p=0.01). OVM measured decreased CV in both atria and ventricle after LPS treatment (atria: +LPS: 43.1±3.1cm/s, n=5; +NaCl: 72.6±9.8cm/s, n=10, p=0.04; ventricle: +LPS: 50.2±2.2cm/s, n=6; +NaCl: 67.7±5.0cm/s, n=10, p=0.02). In analysis of AP in atria upstroke velocity was slightly decreased (max.dV/dt: +LPS: 123.1±4.8V/s, n=22; +NaCl: 158.5±5.3V/s, n=39, p=0.04) but highly reduced in ventricle (max.dV/dt: +LPS: 91.8±3.6V/s, n=27; +NaCl: 140.7±6.3V/s, n=35, p<0.0001). RMP in atria was depolarised after LPS injection (+LPS: −70.1±1.9mV, n=22; +NaCl: −81.1±1.2mV, n=39, p=0.004) explaining decreased upstroke velocity and CV slowing in atria by voltage-dependent Na+ channel inactivation. Ventricular RMP was unaffected by LPS injection (+LPS: −75.1±1.1mV, n=44; +NaCl: −76.3±0.9mV, n=55, p=0.83). Therefore the Na+ currents were measured in isolated ventricular cardiomyocytes using whole cell patch clamp revealing the maximum Na+ current density lowered after LPS treatment (+LPS: −20.6±1.7 pA/pF, n=16, +NaCl 27.1±2.6 pA/pF, n=10, p=0.03). LPS did not affect EPI, CV, upstroke velocity or current density in TLR4−/− mice. Conclusion Herein we report for the first time impaired cardiac depolarisation and conduction after short term activation of TLR4 in vivo. Pro arrhythmogenic mechanisms differ in atria and ventricle: Increased atrial RMP inactivates Na+ current leading to reduction of CV. Ventricular slow CV is caused by reduced current density of Na+ channels. This different TLR4 effect might be important for novel antiarrhythmic and antiinflammatory applications. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): GEROK-grant, University Bonn