Impaired Intracellular Calcium Buffering Contributes to the Arrhythmogenic Substrate in Atrial Myocytes From Patients With Atrial Fibrillation

Abstract

BACKGROUND: Alterations in the buffering of intracellular Ca 2+ , for which myofilament proteins play a key role, have been shown to promote cardiac arrhythmia. It is interesting that although studies report atrial myofibrillar degradation in patients with persistent atrial fibrillation (persAF), the intracellular Ca 2+ buffering profile in persAF remains obscure. Therefore, we aim to investigate the intracellular buffering of calcium and its potential arrhythmogenic role in persAF. METHODS: Simultaneous transmembrane fluxes (patch-clamp) and intracellular Ca 2+ signaling (fluo-3-acetoxymethyl ester) were recorded in myocytes from right atrial biopsies of sinus rhythm (control) and patients with persAF, alongside human atrial subtype induced pluripotent stem cell–derived cardiac myocytes (iPSC-CMs). Protein levels were quantified by immunoblotting of human atrial tissue and induced pluripotent stem cell–derived cardiac myocytes. Mouse whole heart and atrial electrophysiology was measured on a Langendorff system. RESULTS: Cytosolic Ca 2+ buffering was decreased in atrial myocytes of patients with persAF because of a depleted amount of Ca 2+ buffers. In agreement, protein levels of selected Ca 2+ binding myofilament proteins, including cTnC (cardiac troponin C), a major cytosolic Ca 2+ buffer, were significantly lower in patients with persAF. Small interfering RNA (siRNA)–mediated knockdown of cTnC in induced pluripotent stem cell–derived cardiac myocytes (si-cTnC) phenocopied the reduced cytosolic Ca 2+ buffering observed in persAF. Si-cTnC induced pluripotent stem cell–derived cardiac myocytes exhibited a higher predisposition to spontaneous Ca 2+ release events and developed action potential alternans at low stimulation frequencies. Last, indirect reduction of cytosolic Ca 2+ buffering using blebbistatin in an ex vivo mouse whole heart model increased vulnerability to tachypacing-induced atrial arrhythmia, validating the direct mechanistic link between impaired cytosolic Ca 2+ buffering and atrial arrhythmogenesis. CONCLUSIONS: Our findings suggest that loss of myofilament proteins, particularly reduced cTnC protein levels, causes diminished cytosolic Ca 2+ buffering in persAF, thereby potentiating the occurrence of spontaneous Ca 2+ release events and AF susceptibility. Strategies targeting intracellular buffering may represent a promising therapeutic lead in AF management.