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

Abstract

Alterations in the buffering of intracellular Ca²⁺, 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 Ca²⁺ buffering profile in persAF remains obscure. Therefore, we aimed to investigate the intracellular buffering of Ca²⁺ and its potential arrhythmogenic role in persAF. Transmembrane Ca²⁺ fluxes (patch-clamp) and intracellular Ca²⁺ signaling (fluo-3-acetoxymethyl ester) were recorded simultaneously in myocytes from right atrial biopsies of sinus rhythm (Ctrl) 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 were measured on a Langendorff system. Cytosolic Ca²⁺ buffering was decreased in atrial myocytes of patients with persAF because of a depleted amount of Ca²⁺ buffers. In agreement, protein levels of selected Ca²⁺ binding myofilament proteins, including cTnC (cardiac troponin C), a major cytosolic Ca²⁺ buffer, were significantly lower in patients with persAF. Small interfering RNA (siRNA)-mediated knockdown of cTnC (si-cTNC) in atrial iPSC-CM phenocopied the reduced cytosolic Ca²⁺ buffering observed in persAF. Si-cTnC treated atrial iPSC-CM exhibited a higher predisposition to spontaneous Ca²⁺ release events and developed action potential alternans at low stimulation frequencies. Last, indirect reduction of cytosolic Ca²⁺ 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 Ca²⁺ buffering and atrial arrhythmogenesis. Our findings suggest that loss of myofilament proteins, particularly reduced cTnC protein levels, causes diminished cytosolic Ca²⁺ buffering in persAF, thereby potentiating the occurrence of spontaneous Ca²⁺ release events and atrial fibrillation susceptibility. Strategies targeting intracellular buffering may represent a promising therapeutic lead in persAF management.