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Analysis of the spatio-temporal distribution of calcium sparks showed a preferential increase in sparks near the sarcolemma in atrial myocytes from patients with atrial fibrillation (AF), linked to higher ryanodine receptor (RyR2) phosphorylation at s2808 and lower calsequestrin-2 levels. Mathematical modeling, incorporating modulation of RyR2 gating, showed that only the observed combinations of RyR2 phosphorylation and calsequestrin-2 levels can account for the spatio-temporal distribution of sparks in patients with and without AF. Furthermore, we demonstrate that preferential calcium release near the sarcolemma is key to a higher incidence and amplitude of afterdepolarizations in atrial myocytes from patients with AF., Postprint (published version)

Analysis of the spatio-temporal distribution of calcium sparks showed a preferential increase in sparks near the sarcolemma in atrial myocytes from patients with atrial fibrillation (AF), linked to higher ryanodine receptor (RyR2) phosphorylation at s2808 and lower calsequestrin-2 levels. Mathematical modeling, incorporating modulation of RyR2 gating, showed that only the observed combinations of RyR2 phosphorylation and calsequestrin-2 levels can account for the spatio-temporal distribution of sparks in patients with and without AF. Furthermore, we demonstrate that preferential calcium release near the sarcolemma is key to a higher incidence and amplitude of afterdepolarizations in atrial myocytes from patients with AF.

Aims Atrial fibrillation (AF) has been associated with excessive spontaneous calcium release, linked to cyclic AMP (cAMP)-dependent phosphorylation of calcium regulatory proteins. Because ß-blockers are expected to attenuate cAMP-dependent signaling, we aimed to examine whether the treatment of patients with ß-blockers affected the incidence of spontaneous calcium release events or transient inward currents (ITI). Methods The impact of treatment with commonly used ß-blockers was analyzed in human atrial myocytes from 371 patients using patch-clamp technique, confocal calcium imaging or immunofluorescent labeling. Data were analyzed using multivariate regression analysis taking into account potentially confounding effects of relevant clinical factors Results The L-type calcium current (ICa) density was diminished significantly in patients with chronic but not paroxysmal AF and the treatment of patients with ß-blockers did not affect ICa density in any group. By contrast, the ITI frequency was elevated in patients with either paroxysmal or chronic AF that did not receive treatment, and ß-blocker treatment reduced the frequency to levels observed in patients without AF. Confocal calcium imaging showed that ß-blocker treatment also reduced the calcium spark frequency in patients with AF to levels observed in those without AF. Furthermore, phosphorylation of the ryanodine receptor (RyR2) at Ser-2808 and phospholamban at Ser-16 was significantly lower in patients with AF that received ß-blockers. Conclusion Together, our findings demonstrate that ß-blocker treatment may be of therapeutic utility to prevent spontaneous calcium release-induced atrial electrical activity; especially in patients with a history of paroxysmal AF displaying preserved ICa density., Peer Reviewed, Postprint (published version)

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), favouring 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 homoeostasis 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.

Altres ajuts: Fundació Marato TV3 [20152030/31]., 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. 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 (I ) 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 (I ) 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/µm 2) and sparks were wider (3.0 ± 0.3 vs. 2.2 ± 0.3 µm) and lasted longer (79 ± 6 vs. 55 ± 8 ms), favouring their fusion into calcium waves that triggers I s and afterdepolarizations. This was linked to higher ryanodine receptor phosphorylation at s2808 in women with AF, and inhibition of adenosine A or beta-adrenergic receptors that modulate s2808 phosphorylation was able to reduce the higher incidence of I in women with AF. Perturbations of the calcium homoeostasis in AF is sex-dependent, concurring with increased spontaneous SR calcium release-induced electrical activity in women but not in men, and with diminished I density in men only. This work was supported by grants from The Spanish Ministry of Science Innovation and Universities [

Aims: Single nucleotide polymorphisms on chromosome 4q25 have been associated with risk of atrial fibrillation (AF) but the exiguous knowledge of the mechanistic links between these risk variants and underlying electrophysiological alterations hampers their clinical utility. Here, we here tested the hypothesis that 4q25 risk variants cause alterations in the intracellular calcium homeostasis that predispose to spontaneous electrical activity. Methods and results: Western blotting, confocal calcium imaging, and patch-clamp techniques were used to identify mechanisms linking the 4q25 risk variants rs2200733T and rs13143308T to defects in the calcium homeostasis in human atrial myocytes. Our findings revealed that the rs13143308T variant was more frequent in patients with AF and that myocytes from carriers of this variant had a significantly higher density of calcium sparks (14.1±4.5 vs. 3.1±1.3 events/min, p¿=¿0.02), frequency of transient inward (ITI) currents (1.33±0.24 vs. 0.26±0.09 events/min, p¿<¿0.001) and incidence of spontaneous membrane depolarizations (1.22±0.26 vs. 0.56±0.17 events/min, p¿=¿0.001) than myocytes from patients with the normal rs13143308G variant. These alterations were linked to higher sarcoplasmic reticulum calcium loading (10.2±1.4 vs. 7.3±0.5amol/pF, p¿=¿0.01), SERCA2 expression (1.37±0.13 fold, p¿=¿0.03) and RyR2 phosphorylation at s2808 (0.67±0.08 vs. 0.47±0.03, p¿=¿0.01) but not at s2814 (0.28±0.14 vs. 0.31±0.14, p¿=¿0.61) in patients carrying the rs13143308T risk variant. Furthermore, the presence of a risk variant or AF independently increased the ITI frequency and the increase in the ITI frequency observed in carriers of the risk variants was exacerbated in those with AF. By contrast, the presence of a risk variant did not affect the amplitude or properties of the L-type calcium current in patients with or without AF. Conclusions: We here identify the 4q25 variant rs13143308T as a genetic risk marker for AF, specifically associated w

Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic disease caused by defective prelamin A processing, leading to nuclear lamina alterations, severe cardiovascular pathology, and premature death. Prelamin A alterations also occur in physiological aging. It remains unknown how defective prelamin A processing affects the cardiac rhythm. We show age-dependent cardiac repolarization abnormalities in HGPS patients that are also present in the Zmpste24-/- mouse model of HGPS. Challenge of Zmpste24-/- mice with the ß-adrenergic agonist isoproterenol did not trigger ventricular arrhythmia but caused bradycardia-related premature ventricular complexes and slow-rate polymorphic ventricular rhythms during recovery. Patch-clamping in Zmpste24-/- cardiomyocytes revealed prolonged calcium-transient duration and reduced sarcoplasmic reticulum calcium loading and release, consistent with the absence of isoproterenol-induced ventricular arrhythmia. Zmpste24-/- progeroid mice also developed severe fibrosis-unrelated bradycardia and PQ interval and QRS complex prolongation. These conduction defects were accompanied by overt mislocalization of the gap junction protein connexin43 (Cx43). Remarkably, Cx43 mislocalization was also evident in autopsied left ventricle tissue from HGPS patients, suggesting intercellular connectivity alterations at late stages of the disease. The similarities between HGPS patients and progeroid mice reported here strongly suggest that defective cardiac repolarization and cardiomyocyte connectivity are important abnormalities in the HGPS pathogenesis that increase the risk of arrhythmia and premature death., Peer Reviewed, Postprint (published version)

The damaging effects of high plasma levels of cholesterol in the cardiovascular system are widely known, but little attention has been paid to direct effects on cardiomyocyte function. We therefore aimed at testing the hypothesis that Low Density Lipoprotein (LDL) cholesterol affects calcium dynamics and signal propagation in cultured atrial myocytes. For this purpose, mRNA and protein expression levels were determined by real time PCR and western blot analysis, respectively, and intracellular calcium was visualized in fluo-4 loaded atrial HL-1 myocyte cultures subjected to field stimulation. At low stimulation frequencies all cultures had uniform calcium transients at all tested LDL concentrations. However, 500 mg LDL/mL maximally reduced the calcium transient amplitude by 43% from 0.3060.04 to 0.1760.02 (p,0.05). Moreover, LDLcholesterol dose-dependently increased the fraction of alternating and irregular beat-to-beat responses observed when the stimulation interval was shortened. This effect was linked to a concurrent reduction in SERCA2, RyR2, IP3RI and IP3RII mRNA levels. SERCA2 protein levels were also reduced by 43% at 200 mg LDL/mL (p,0.05) and SR calcium loading was reduced by 3866% (p,0.001). By contrast, HDL-cholesterol had no significant effect on SERCA expression or SR calcium loading. LDLcholesterol also slowed the conduction velocity of the calcium signal from 3.2+0.2 mm/s without LDL to 1.760.1 mm/s with 500 mg LDL/mL (p,0.05). This coincided with a reduction in Cx40 expression (by 4463%; p,0.05 for mRNA and by 7962%; p,0.05 for Cx40 protein at 200 mg/ml LDL) whereas the Cx-43 expression did not significantly change. In conclusion, LDLcholesterol destabilizes calcium handling in cultured atrial myocytes subjected to rapid pacing by reducing SERCA2 and Cx40 expression and by slowing the conduction velocity of the calcium signal., Peer Reviewed, Postprint (published version)

Calcium release from the sarcoplasmic reticulum (SR) plays a central role in the regulation of cardiac contraction and rhythm in mammals and humans but its role is controversial in teleosts. Since the zebrafish is an emerging model for studies of cardiovascular function and regeneration we here sought to determine if basic features of SR calcium release are phylogenetically conserved. Confocal calcium imaging was used to detect spontaneous calcium release (calcium sparks and waves) from the SR. Calcium sparks were detected in 16 of 38 trout atrial myocytes and 6 of 15 ventricular cells. The spark amplitude was 1.4560.03 times the baseline fluorescence and the time to half maximal decay of sparks was 2763 ms. Spark frequency was 0.88 sparks mm21 min21 while calcium waves were 8.5 times less frequent. Inhibition of SR calcium uptake reduced the calcium transient (F/F0) from 1.7760.17 to 1.1260.18 (p=0.002) and abolished calcium sparks and waves. Moreover, elevation of extracellular calcium from 2 to 10 mM promoted early and delayed afterdepolarizations (from 0.660.3 min21 to 8.162.0 min21, p = 0.001), demonstrating the ability of SR calcium release to induce afterdepolarizations in the trout heart. Calcium sparks of similar width and duration were also observed in zebrafish ventricular myocytes. In conclusion, this is the first study to consistently report calcium sparks in teleosts and demonstrate that the basic features of calcium release through the ryanodine receptor are conserved, suggesting that teleost cardiac myocytes is a relevant model to study the functional impact of abnormal SR function., Peer Reviewed, Postprint (published version)

Calcium release from the sarcoplasmic reticulum (SR) plays a central role in the regulation of cardiac contraction and rhythm in mammals and humans but its role is controversial in teleosts. Since the zebrafish is an emerging model for studies of cardiovascular function and regeneration we here sought to determine if basic features of SR calcium release are phylogenetically conserved. Confocal calcium imaging was used to detect spontaneous calcium release (calcium sparks and waves) from the SR. Calcium sparks were detected in 16 of 38 trout atrial myocytes and 6 of 15 ventricular cells. The spark amplitude was 1.45±0.03 times the baseline fluorescence and the time to half maximal decay of sparks was 27±3 ms. Spark frequency was 0.88 sparks µm−1 min−1 while calcium waves were 8.5 times less frequent. Inhibition of SR calcium uptake reduced the calcium transient (F/F0) from 1.77±0.17 to 1.12±0.18 (p = 0.002) and abolished calcium sparks and waves. Moreover, elevation of extracellular calcium from 2 to 10 mM promoted early and delayed afterdepolarizations (from 0.6±0.3 min−1 to 8.1±2.0 min−1, p = 0.001), demonstrating the ability of SR calcium release to induce afterdepolarizations in the trout heart. Calcium sparks of similar width and duration were also observed in zebrafish ventricular myocytes. In conclusion, this is the first study to consistently report calcium sparks in teleosts and demonstrate that the basic features of calcium release through the ryanodine receptor are conserved, suggesting that teleost cardiac myocytes is a relevant model to study the functional impact of abnormal SR function.

Gilthead seabream is of high commercíal interest and has become an important aquacultural resource in the Mediterranean countries. Now, the aim of improving the quality of reared products in order to meet markets preferences of conformity with wild standards is a new priority, The aim of this study is the use of morphometrics and meristics for the quantification of the differences in quality assessment and on their causative factors between and within sea bream reared in different fish farms and wild standards. Reared S.auratus show higher body depth, caudal peduncle depth, last dorsal spine, anal fin rays number and position of first anal spine respect to dorsal, and lower head depth, snout length, preorbital length, predorsal length, dorsal fin base, pectoral fin length, ventral fin length, caudal peduncle length, scales berween lateral line and dorsal, pectoral fin rays number and largest dorsal fin spine than wild sea bream. Apart of this measures, Cupimar hatchery facility has fish with lower head length, interorbital length and distance between pelvic and pectoral fins than wild sea bream; Maresa hatchery físhes have lower anal fin base and higher head length than wild standards, Blanes Peix sea cages show lower head length, orbital diameter and interorbital length than wild standards; and Aquadelt fishes show lower head length, orbital diameter, interorbital length, distance berween pelvic and pectoral fins and preanal length than wild sea bream and a non significant difference in last dorsal spine.

We have developed an automatic method for the analysis and identification of dynamical regimes in intracellular calcium patterns from confocal calcium images. The method allows the identification of different dynamical patterns such as spatially concordant and discordant alternans, irregular behavior or phase-locking regimes such as period doubling or halving. The method can be applied to the analysis of different cardiac pathologies related to anomalies at the cellular level such as ventricular reentrant arrhythmias., Peer Reviewed, Postprint (published version)