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

BACKGROUND: 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 aim to investigate the intracellular buffering of calcium and its potential arrhythmogenic role in persAF. METHODS: Simultaneous transmembrane fluxes (patch-clamp) and intracellular Ca2+ 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 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 in induced pluripotent stem cell-derived cardiac myocytes (si-cTnC) phenocopied the reduced cytosolic Ca2+ buffering observed in persAF. Si-cTnC induced pluripotent stem cell-derived cardiac myocytes 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. CONCLUSIONS: 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 AF susceptibility. Strategies targeting intracellular buffering may represent a promising therapeutic lead in AF management.

ProteoPlex: stability optimization of macromolecular complexes by sparse-matrix screening of chemical space.

Molecular machines or macromolecular complexes are supramolecular assemblies of biomolecules with a variety of functions. Structure determination of these complexes in a purified state is often tedious owing to their compositional complexity and the associated relative structural instability. To improve the stability of macromolecular complexes in vitro, we present a generic method that optimizes the stability, homogeneity and solubility of macromolecular complexes by sparse-matrix screening of their thermal unfolding behavior in the presence of various buffers and small molecules. The method includes the automated analysis of thermal unfolding curves based on a biophysical unfolding model for complexes. We found that under stabilizing conditions, even large multicomponent complexes reveal an almost ideal two-state unfolding behavior. We envisage an improved biochemical understanding of purified macromolecules as well as a substantial boost in successful macromolecular complex structure determination by both X-ray crystallography and cryo-electron microscopy.

Efeitos da ivermectina (Ivomec® 1 por cento injetável) administrada a ratas Wistar durante o período de organogênese / Effects of the endectocid ivermectin (Ivomec(r) 1 percent injection) on pregnants females wistar rats

Avaliou-se a segurança da ivermectina (IVOMEC 1 por cento injetável), um fármaco muito utilizado, administrado a ratas prenhes, no início da fase organogênica, em dosagem até 60 vezes superior (12mg/kg) a utilizada para o tratamento de escabiose em caninos e felinos. Os resultados revelaram ausência de toxicidade sistêmica e reprodutiva, fundamentados na ausência de alterações no desenvolvimento ponderal, consumo de água e ração, absorções embrionárias, massa relativa e exame histopatológico dos órgãos das ratas prenhes, bem como na massa corporal, vitalidade dos fetos, número de fetos por fêmea e alterações macroscópicas externas. Conclui-se pela segurança da ivermectina, quando administrada, em dose única, no início da fase de organogênese de ratas prenhes.