An endo-lysosomal Ca2+ store in cardiomyocytes controlled by OCaR proteins determines fatal tachyarrhythmias

R. Ottenheijm (Heidelberg)¹, M. Berlin (Heidelberg)¹, J. Londono (Heidelberg)¹, M. V. Malz (Heidelberg)¹, S. bhunia (Heidelberg)², A. Kraft (Heidelberg)³, W. Bildl (Freiburg)⁴, V. Tsvilovskyy (Heidelberg)⁵, S. Meyer (Heidelberg)⁶, K. Hennis (München)⁷, T. Tavhelidse-Suck (Heidelberg)⁸, A. Cornean (Heidelberg)⁹, F. Leuschner (Heidelberg)⁶, M. Biel (München)⁷, A. Hansen (Hamburg)¹⁰, C. Wahl-Schott (Planegg-Martinsried)¹¹, C. Grimm (München)¹², U. Schulte (Freiburg)¹³, M. Freichel (Heidelberg)^1
1Universitätsklinikum Heidelberg Pharmakologisches Institut Heidelberg, Deutschland; ²Pharmakologisches Institut Heidelberg, Deutschland; ³Universität Heidelberg Pharmakologisches Institut Heidelberg, Deutschland; ⁴Institute of Physiology II, Molecular Physiology Freiburg, Deutschland; ⁵Pharmakologisches Institut Universität Heidelebrg Heidelberg, Deutschland; ⁶Universitätsklinikum Heidelberg Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie Heidelberg, Deutschland; ⁷Ludwig-Maximilians-Universität München Center for Drug Research, Department of Pharmacy München, Deutschland; ⁸Universtität Heidelberg Heidelberg, Deutschland; ⁹Universität Heidelberg Centre for Organismal Studies Heidelberg, Deutschland; ¹⁰Universitätsklinikum Hamburg-Eppendorf Institut für Experimentelle Pharmakologie und Toxikologie Hamburg, Deutschland; ¹¹LMU München Biomedizinisches Zentrum, Institut für Kardiovaskuläre Physiologie und Pathophysiologie Planegg-Martinsried, Deutschland; ¹²Walther-Straub-Institut für Pharmakologie und Toxikologie, Ludwig-Maximilian-Universität München München, Deutschland; ¹³University of Freiburg Institute of Physiology Freiburg, Deutschland

We identified OCaR1 (Organellar Ca²⁺ Regulator), a membrane protein that determines NAADP-mediated Ca²⁺-release from acidic organelles in pancreatic acinar cells, playing a critical role in pancreatitis (Tsvilovskyy, Ottenheijm,et al., J.Clin. Invest. 134: 7, 2024). In cardiomyocytes, NAADP is generated upon catecholamine stimulation and evokes Ca²⁺-release from acidic stores via TPCs. This increase in Ca²⁺ at the lysosomal-SR junction contributes to ventricular arrhythmias and pathological cardiac remodelling. OCaR2, a protein of the same protein family as OCaR1, is expressed in cardiomyocytes and determines the role for Ca²⁺-homeostasis in cardiomyocytes and Ca²⁺-dependent arrhythmias and remodelling in vivo.

We identified that OCaR2 is located in endosomes and lysosomes in ventricular cardiomyocytes using co-localization analysis as well as organellar proteomics. In resting OCaR2-deficient cardiomyocytes, isoproterenol (ISO), NAADP, and Forskolin evoked intracellular Ca²⁺-oscillations that were not triggered in control cells. Ca²⁺-depletion of acidic stores by Bafilomycin A1 abolished the ISO-evoked Ca²⁺-oscillations. Additionally, inhibiting NAADP suppressed the Ca²⁺-signals in OCaR2-deficient cardiomyocytes. Inactivating TPCs significantly reduced ISO-evoked Ca²⁺-transients. Furthermore, ECG recordings in cardiomyocytes specific OCaR2-deficient mice (OCaR2^CM-KO) revealed an increase of ventricular extrasystoles and sudden cardiac death, which was prevented by inactivating TPCs. Moreover, ischemia and reperfusion enhanced ISO-evoked arrhythmogenesis and development of pathological remodelling in OCaR2-deficient mice.

To develop a therapeutic strategy for this pathophysiological phenotype in OCaR2^CM-KO requires high-precision gene-editing tools capable of targeted DNA modifications. Cytosine base editing (CBE) enables precise C-to-T transition mutations without inducing double-strand DNA breaks, offering significant potential for gene therapy. We investigate ventricular arrhythmias and pathological cardiac remodeling driven in OCaR2^CM-KO. We aim to deliver optimized (mini)CBE candidates via cardiomyocyte-specific AAVs to establish a specific, stable gene therapy strategy against catecholamine-evoked arrhythmia. This integrative approach, combining high-precision base editing with mechanistic insights into cardiomyocyte calcium signaling, has the potential to advance therapeutic gene editing and address current limitations in CVD treatment.