Aims Atrial fibrillation (AF) has been associated with intracellular calcium disturbances in human atrial myocytes, but little is known about the potential influence of sex and we here aimed to address this issue. Methods and Results Alterations in calcium regulatory mechanisms were assessed in human atrial myocytes from patients without AF or with long-standing persistent or permanent AF. Patch-clamp measurements revealed that L-type calcium current (ICa) density was significantly smaller in males with than without AF (-1.15±0.37 vs. -2.06±0.29 pA/pF) but not in females with AF (-1.88±0.40 vs. -2.21±0.0.30 pA/pF). In contrast, transient inward currents (ITi) were more frequent in females with than without AF (1.92±0.36 vs. 1.10±0.19 events/min) but not in males with AF. Moreover, confocal calcium imaging showed that females with AF had more calcium spark sites than those without AF (9.8±1.8 vs. 2.2±1.9 sites/µm2) and sparks were wider (3.0±0.3 vs. 2.2±0.3 µm) and lasted longer (79±6 vs. 55±8 ms), favoring their fusion into calcium waves that triggers ITIs and afterdepolarizations. This was linked to higher ryanodine receptor phosphorylation at s2808 in women with AF, and inhibition of adenosine A2A or beta-adrenergic receptors that modulate s2808 phosphorylation was able to reduce the higher incidence of ITI in women with AF. Conclusion Perturbations of the calcium homeostasis in AF is sex-dependent, concurring with increased spontaneous SR calcium release-induced electrical activity in women but not in men, and with diminished ICa density in men only. Translational Perspective Statistical analysis taking into account confounding effects of concurrent disease, risk factors and treatments revealed differential sex-dependent alterations of the calcium homeostasis in AF. The analysis suggests that suppression of calcium release-induced membrane depolarizations with adenosine receptor antagonists may be efficient in women with AF only while therapies aiming to restore L-type calcium current may be more efficient in males with AF.

Cardiac ryanodine receptors (RyR2s) are Ca 2þ release channels clustering in the sarcoplasmic reticulum membrane. These clusters are believed to be the elementary units of Ca 2þ release. The distribution of these Ca 2þ release units plays a critical role in determining the spatio-temporal profile and stability of sarcoplasmic reticulum Ca 2þ release. RyR2 clusters located in the interior of cardiomyocytes are arranged in highly ordered arrays. However, little is known about the distribution and function of RyR2 clusters in the periphery of cardiomyocytes. Here, we used a knock-in mouse model expressing a green fluorescence protein (GFP)-tagged RyR2 to localize RyR2 clusters in live ventricular myocytes by virtue of their GFP fluorescence. Confocal imaging and total internal reflection fluorescence microscopy was employed to determine and compare the distribution of GFP-RyR2 in the interior and periphery of isolated live ventricular myocytes and in intact hearts. We found tightly ordered arrays of GFP-RyR2 clusters in the interior, as previously described. In contrast, irregular distribution of GFP-RyR2 clusters was observed in the periphery. Time-lapse total internal reflection fluorescence imaging revealed dynamic movements of GFP-RyR2 clusters in the periphery, which were affected by external Ca 2þ and RyR2 activator (caffeine) and inhibitor (tetracaine), but little detectable movement of GFP-RyR2 clusters in the interior. Furthermore, simultaneous Ca 2þ -and GFP-imaging demonstrated that peripheral RyR2 clusters with an irregular distribution pattern are functional with a Ca 2þ release profile similar to that in the interior. These results indicate that the distribution of RyR2 clusters in the periphery of live ventricular myocytes is irregular and dynamic, which is different from that of RyR2 clusters in the interior.

Aims It is unknown how β‐adrenergic stimulation affects calcium dynamics in individual RyR2 clusters and leads to the induction of spontaneous calcium waves. To address this, we analysed spontaneous calcium release events in green fluorescent protein (GFP)‐tagged RyR2 clusters. Methods Cardiomyocytes from mice with GFP‐tagged RyR2 or human right atrial tissue were subjected to immunofluorescent labelling or confocal calcium imaging. Results Spontaneous calcium release from single RyR2 clusters induced 91.4% ± 2.0% of all calcium sparks while 8.0% ± 1.6% were caused by release from two neighbouring clusters. Sparks with two RyR2 clusters had 40% bigger amplitude, were 26% wider, and lasted 35% longer at half maximum. Consequently, the spark mass was larger in two‐ than one‐cluster sparks with a median and interquartile range for the cumulative distribution of 15.7 ± 20.1 vs 7.6 ± 5.7 a.u. (P < .01). β2‐adrenergic stimulation increased RyR2 phosphorylation at s2809 and s2815, tripled the fraction of two‐ and three‐cluster sparks, and significantly increased the spark mass. Interestingly, the amplitude and mass of the calcium released from a RyR2 cluster were proportional to the SR calcium load, but the firing rate was not. The spark mass was also higher in 33 patients with atrial fibrillation than in 36 without (22.9 ± 23.4 a.u. vs 10.7 ± 10.9; P = .015). Conclusions Most sparks are caused by activation of a single RyR2 cluster at baseline while β‐adrenergic stimulation doubles the mass and the number of clusters per spark. This mimics the shift in the cumulative spark mass distribution observed in myocytes from patients with atrial fibrillation.

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