title: Targeting cardiac arrhythmia by enhancing mitochondrial calcium uptake
Creators: name: Schredelseker, Johann
Publisher: name: Ludwig-Maximilians-Universität München
Year: 2020
Subjects: subject: 600
subject: 610
subject: FOS: Medical and Health Sciences

description: Cardiovascular diseases remain the number one cause for morbidity and mortality worldwide with an estimated half of cardiovascular disease-related deaths being attributed to cardiac arrhythmia. Despite this enormous importance for public health, existing antiarrhythmic drugs are still far from being ideal as they display perilous side effects and can not be administered over extended time periods. It is thus a major endeavor of cardiovascular research to identify novel safer drug targets and therapeutic strategies for the treatment of cardiac arrhythmia. Since cardiac rhythmicity is directly dependent on a tight regulation of intracellular Ca2+ and cardiac arrhythmia is often associated with disturbances in Ca2+ homeostasis, we used an unbiased approach to identify novel regulators of cardiac Ca2+ handling and modulators thereof. A library of newly synthesized, organic-like compounds was screened for their ability to restore rhythmic cardiac contractions in a zebrafish model for cardiac fibrillation. From this screen we identified the small ester compound efsevin, which binds to the voltage-dependent anion channel 2 (VDAC2) in the outer mitochondrial membrane. We demonstrated that treatment with efsevin enhances mitochondrial Ca2+ uptake and thereby prevents propagation of spontaneous intracellular Ca2+ release events in cardiomyocytes, the triggers for ectopic excitations and arrhythmia. Since this work presented a novel role for VDAC2 in cardiomyocytes we analyzed the structure of VDAC2 by crystallography to identify elements that promote specificity of this isoform over VDAC1 and VDAC3. Though we could not detect large structural differences, we identified moieties that interact with regulatory proteins, which differ between the isoforms, and could thus explain the distinct role of VDAC2 in cardiomyocytes. We then used the crystal structure of VDAC2 to identify the binding site of efsevin by computational modeling and identified a binding pocket located between the wall of the VDAC2 pore and the pore-lining α helix, that was previously suggested to promote channel gating. In planar lipid bilayers we demonstrated that efsevin promotes gating of the channel from an anion-selective high conductance state into a more cation-selective low conductance state, thereby explaining the enhanced mitochondrial Ca2+ uptake induced by efsevin. To analyze the translational potential of efsevin, we tested efsevin in experimental models for the human cardiac arrhythmia catecholaminergic polymorphic ventricular tachycardia (CPVT). Efsevin reduced spontaneous diastolic Ca2+ signals and action potentials in cardiomyocytes isolated from CPVT mice and significantly reduced episodes of ventricular tachycardia in in vivo. Furthermore, efsevin reduced spontaneous, diastolic Ca2+ signals in induced pluripotent stem cell derived cardiomyocytes from a CPVT patient. Because efsevin lacks several features essential for druggability like e.g. a nanomolar affinity to the target and key pharmacokinetic properties like oral bioavailability, we then screened a library of clinically approved compounds for additional mitochondrial Ca2+ uptake enhancers. We identified increased uptake of Ca2+ into mitochondria of cardiomyocytes upon treatment with either the cholesterol uptake inhibitor ezetimbe or disulfiram, used for the treatment of alcohol abuse. Both were active at significantly lower concentrations compared to efsevin and showed efficacy experimental models for cardiac arrhythmia. Taken together, this thesis (i) establishes the outer mitochondrial membrane as a regulated barrier for Ca2+, (ii) establishes mitochondrial Ca2+ uptake as a novel regulator of cardiac rhythmicity and (iii) provides a novel candidate structure and lead substances for the development of a treatment for human cardiac arrhythmia.