Background Delayed afterdepolarizations (DADs) carried by Na+-Ca2+-exchange current (INCX) in response to sarcoplasmic reticulum (SR) Ca2+-leak can promote atrial fibrillation (AF). The mechanisms leading to DADs in AF-patients have not been defined. Methods and Results Protein levels (Western-blot), membrane-currents and action-potentials (patch-clamp), and [Ca2+]i (Fluo-3) were measured in right-atrial samples from 77 sinus-rhythm (Ctl) and 69 chronic-AF (cAF) patients. Diastolic [Ca2+]i and SR-Ca2+-content (integrated INCX during caffeine-induced-Ca2+-transient [cCaT]) were unchanged, whereas diastolic SR Ca2+-leak, estimated by blocking RyR2 with tetracaine, was ~50% higher in cAF vs. Ctl. Single-channel recordings from atrial RyR2 reconstituted into lipid-bilayers revealed enhanced open-probability in cAF-samples, providing a molecular basis for increased SR Ca2+-leak. Calmodulin-expression (+60%), CaMKII-autophosphorylation at Thr287 (+40%) and RyR2-phosphorylation at Ser2808 (PKA/CaMKII-site, +236%) and Ser2814 (CaMKII-site, +77%) were increased in cAF. The selective CaMKII-blocker KN-93 decreased SR Ca2+-leak, the frequency of spontaneous Ca2+-release events and RyR2 open-probability in cAF, whereas PKA-inhibition with H-89 was ineffective. Knock-in mice with constitutively-phosphorylated RyR2 at Ser2814 showed a higher incidence of Ca2+-sparks and increased susceptibility to pacing-induced AF vs. controls. The relationship between [Ca2+]i and INCX-density revealed INCX-upregulation in cAF. Spontaneous Ca2+-release events accompanied by inward INCX-currents and DADs/triggered-activity occurred more often and the sensitivity of resting membrane voltage to elevated [Ca2+]i (diastolic [Ca2+]i–voltage coupling gain) was higher in cAF vs. Ctl. Conclusions Enhanced SR Ca2+-leak through CaMKII-hyperphosphorylated RyR2, in combination with larger INCX for a given SR Ca2+-release and increased diastolic [Ca2+]i–voltage coupling gain, cause AF-promoting atrial DADs/triggered-activity in cAF patients.

Background Advancing structural and functional maturation of stem cell-derived cardiomyocytes remains a key challenge for applications in disease modelling, drug screening, and heart repair. Here, we sought to advance cardiomyocyte maturation in engineered human myocardium (EHM) towards an adult phenotype under defined conditions. Methods We systematically investigated cell composition, matrix and media conditions to generate EHM from embryonic and induced pluripotent stem cell-derived cardiomyocytes and fibroblasts with organotypic functionality under serum-free conditions. We employed morphological, functional, and transcriptome analyses to benchmark maturation of EHM. Results EHM demonstrated important structural and functional properties of postnatal myocardium, including: (1) rod-shaped cardiomyocytes with M-bands assembled as a functional syncytium; (2) systolic twitch forces at a similar level as observed in bona fide postnatal myocardium; (3) a positive force-frequency-response; (4) inotropic responses to β-adrenergic stimulation mediated via canonical β1- and β2-adrenoceptor signaling pathways; and (5) evidence for advanced molecular maturation by transcriptome profiling. EHM responded to chronic catecholamine toxicity with contractile dysfunction, cardiomyocyte hypertrophy, cardiomyocyte death, and NT-proBNP release; all are classical hallmarks of heart failure. Additionally, we demonstrate scalability of EHM according to anticipated clinical demands for cardiac repair. Conclusions We provide proof-of-concept for a universally applicable technology for the engineering of macro-scale human myocardium for disease modelling and heart repair from embryonic and induced pluripotent stem cell-derived cardiomyocytes under defined, serum-free conditions.

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