TLR4 pathway induces proarrhythmogenic slow conduction by impaired depolarisation
Authors: Funken M , Knappe V , Beiert T , Nickenig G , Sasse P , Schrickel J W 
Published: October 2022

Abstract

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&lt;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