Anna Llach

Atrial Fibrillation Is Associated With Increased Spontaneous Calcium Release From the Sarcoplasmic Reticulum in Human Atrial Myocytes

Background—Spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) can generate afterdepolarizations, and these have the potential to initiate arrhythmias. Therefore, an association may exist between spontaneous SR Ca2+ release and initiation of atrial fibrillation (AF), but this has not yet been reported. Methods and Results—Spontaneous Ca2+ release from the SR, manifested as Ca2+ sparks and Ca2+ waves, was recorded with confocal microscopy in atrial myocytes isolated from patients with and those without AF. In addition, the spontaneous inward current associated with Ca2+ waves was measured with the use of the perforated patch-clamp technique. The Ca2+ spark frequency was higher in 8 patients with AF than in 16 patients without (6.0±1.2 versus 2.8±0.8 sparks/mm per second, P<0.05). Similarly, the spontaneous Ca2+ wave frequency was greater in patients with AF (2.8±0.5 versus 1.1±0.3 waves/mm per second, P<0.01). The spontaneous inward current frequency was also higher in 10 patients with AF than in 13 patients without this arrhythmia (0.101±0.028 versus 0.031±0.007 per second, P<0.05, at a clamped potential of −80 mV). In contrast, both the Ca2+ released from the SR and the Na+-Ca2+ exchange rate induced by a rapid caffeine application were comparable in patients with and without AF. Conclusions—The observed increase in spontaneous Ca2+ release in patients with AF probably is due to an upregulation of the SR Ca2+ release channel activity, which may contribute to the development of AF.

Effect of aging on the pluripotential capacity of human CD105+ mesenchymal stem cells

Whether aging modifies mesenchymal stem cell (MSC) properties is unknown.

FGF-4 increases in vitro expansion rate of human adult bone marrow-derived mesenchymal stem cells.

Human bone marrow-derived mesenchymal stem cells (MSCs) exhibit limited in vitro growth. Fibroblast growth factors (FGFs) elicit a variety of biological responses, such as cell proliferation, differentiation and migration. FGF-4 represents one of the FGFs with the highest cell mitogenic activity. We studied the effect of FGF-4 on MSCs growth and pluripotency. MSCs duplication time (Td) was significantly reduced with FGF-4 compared to controls (2.2 ± 0.2 vs. 4.1 ± 0.2 days, respectively; p = 0.03) while BMP-2 and SCF-1 did not exert a significant growth effect. MSC expression of surface markers, differentiation into adipogenic and osteogenic lineages, and baseline expression of cardiomyogenic genes were unaffected by FGF-4. In summary, exogenous FGF-4 increases the rate at which MSC proliferate and has no significant effect on MSC pluripotency.

Abnormal calcium handling in atrial fibrillation is linked to up-regulation of adenosine A2A receptors

AIMS Atrial fibrillation (AF) is associated with abnormal sarcoplasmic reticulum (SR) calcium release, which is promoted by adenosine A(2A) receptor (A(2A)R) activation. Here, we tested the hypothesis that abnormal calcium release in AF is linked to A(2A)R remodelling. METHODS AND RESULTS Western blotting and quantitative real-time PCR were used to determine A(2A)R mRNA and protein levels in right atrial samples from patients with and without AF. Effects of A(2A)R activation on calcium handling were assessed with patch-clamp technique and confocal calcium imaging. A(2A)R mRNA levels and functional A(2A)Rs were moderately up-regulated in patients with atrial dilation and markedly up-regulated in those with AF. Accordingly, A(2A)R stimulation significantly increased ryanodine receptor phosphorylation in AF patients, and spontaneous calcium waves increased moderately in myocytes from patients with atrial dilation and strongly in patients with AF (2.2 ± 2.1 to 14.3 ± 8.8 min(-1), n = 6, P = 0.01). Moreover, the high baseline level of calcium waves in AF was reduced by A(2A)R antagonists (3.5 ± 2.0 to 1.3 ± 1.3 min(-1), n = 6, P = 0.007) or adenosine deaminase (1.7 ± 1.5 to 0.5 ± 0.6 min(-1), n = 10, P = 0.02) suggesting that A(2A)Rs are activated by endogenous adenosine. Indeed, intracellular perfusion with adenosine significantly increased the calcium wave frequency (1.1 ± 0.8 to 8.2 ± 3.3 min(-1), n = 8), whereas adenosine removal from the cytosol decreased it (2.1 ± 0.9 to 0.3 ± 0.3 min(-1), n = 8, P = 0.04). CONCLUSIONS Atrial fibrillation patients show increased A(2A)R expression that may account for the high baseline level of spontaneous SR calcium release seen in myocytes from these patients, and the ability of A(2A)R antagonists to reduce this abnormal calcium release points to the A(2A)R as a novel molecular target in AF.

Ageing is associated with deterioration of calcium homeostasis in isolated human right atrial myocytes.

Aims Ageing-related cardiac disorders such as heart failure and atrial fibrillation often present with intracellular calcium homeostasis dysfunction. However, knowledge of the intrinsic effects of ageing on cellular calcium handling in the human heart is sparse. Therefore, this study aimed to analyse how ageing affects key mechanisms that regulate intracellular calcium in human atrial myocytes. Methods and results Whole membrane currents and intracellular calcium transients were measured in isolated human right atrial myocytes from 80 patients with normal left atrial dimensions and no history of atrial fibrillation. Patients were categorized as young (<55 years, n = 21), middle aged (55–74 years, n = 42), and old (≥75 years, n = 17). Protein levels were determined by western blot. Ageing was associated with the following electrophysiological changes: (i) a 3.2-fold decrease in the calcium transient (P < 0.01); (ii) reduction of the L-type calcium current (ICa) amplitude (2.4 ± 0.3 pA/pF vs. 1.4 ± 0.2 pA/pF, P < 0.01); (iii) lower levels of L-type calcium channel alpha-subunit (P < 0.05); (iv) lower rates of both fast (14.5 ± 0.9 ms vs. 20.9 ± 1.9, P < 0.01) and slow (73 ± 3 vs. 120 ± 12 ms, P < 0.001) ICa inactivation; and (v) a decrease in the sarcoplasmic reticulum calcium content (10.1 ± 0.8 vs. 6.4 ± 0.6 amol/pF, P < 0.005) associated with a significant decrease in both SERCA2 (P < 0.05) and calsequestrin-2 (P < 0.05) protein levels. In contrast, ageing did not affect spontaneous sarcoplasmic reticulum calcium release. Conclusion Ageing is associated with depression of SR calcium content, L-type calcium current, and calcium transient amplitude that may favour a progressive decline in right atrial contractile function with age.

Low Density Lipoproteins Promote Unstable Calcium Handling Accompanied by Reduced SERCA2 and Connexin-40 Expression in Cardiomyocytes

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 µg LDL/mL maximally reduced the calcium transient amplitude by 43% from 0.30±0.04 to 0.17±0.02 (p<0.05). Moreover, LDL-cholesterol 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 µg LDL/mL (p<0.05) and SR calcium loading was reduced by 38±6% (p<0.001). By contrast, HDL-cholesterol had no significant effect on SERCA expression or SR calcium loading. LDL-cholesterol also slowed the conduction velocity of the calcium signal from 3.2+0.2 mm/s without LDL to 1.7±0.1 mm/s with 500 µg LDL/mL (p<0.05). This coincided with a reduction in Cx40 expression (by 44±3%; p<0.05 for mRNA and by 79±2%; p<0.05 for Cx40 protein at 200 µg/ml LDL) whereas the Cx-43 expression did not significantly change. In conclusion, LDL-cholesterol 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.

Effect of beta-adrenergic stimulation on the relationship between membrane potential, intracellular [Ca2+] and sarcoplasmic reticulum Ca2+ uptake in rainbow trout atrial myocytes.

SUMMARY Long depolarizations cause a steady tonic contraction and induce sarcoplasmic reticulum (SR) Ca2+-uptake in trout atrial myocytes. Simultaneous measurements of cytosolic [Ca2+] ([Ca2+]i) and whole membrane current showed an elevated [Ca2+]i throughout the depolarization. Rapid caffeine (Caf) applications at –80 mV before and after a long depolarization were used to determine SR Ca2+ loading and its dependency on membrane potential and [Ca2+]i during depolarization. Following a 10 s depolarization, the maximal SR Ca2+ load was 597 μmol l–1 and loading was half-maximal at –12 mV. Theβ -adrenergic agonist isoproterenol (ISO) did not affect the maximal SR Ca2+ loading but shifted the potential for half-maximal loading by– 26 mV. Following a 3 s depolarization, the maximal SR Ca2+ uptake rate (V̇max) was 418μ mol l–1 s–1 in control conditions. ISO did not affect V̇max, but significantly lowered the average free Ca2+ transient during the depolarization and shifted the K0.5 for the relationship between SR Ca2+ uptake and [Ca2+]i from 1.27 in control to 0.8 μmol l–1 with ISO. Following repetitive 200 ms depolarizations, ISO increased the l-type Ca2+ current (ICa) amplitude by 91±29% and the peak Ca2+ transient by 41±10%, and decreased the half life of the Ca2+ transient from 151±12 to 111±6 ms. Using the relationship between [Ca2+]i and SR Ca2+ uptake to calculate the total SR Ca2+ uptake during a Ca2+ transient elicited by a 200 ms depolarization, a significant increase in the SR Ca2+ uptake from 37±6 μmol l–1 in control to 68±4 μmol l–1 with ISO was seen. When normalized to the total Ca2+ transport the contribution of the SR was not significantly different in the absence (35±6%) or presence of ISO (41±4%). Exposure of cells to ISO and low extracellular [Ca2+] increased ICa by 67±40% (N=5) but significantly reduced SR Ca2+ uptake at membrane potentials above –30 mV. Together, these results suggest that (i) ISO has a stimulatory effect on the SR Ca2+ pump that may contribute to the faster decay of the Ca2+ transient, and (ii) the relative contribution of the SR to the Ca2+ removal during relaxation is not altered by ISO in trout atrial myocytes.