The potential of microRNAs (miRNA) as non-invasive diagnostic, prognostic, and predictive biomarkers, as well as therapeutic targets, has recently been recognized. Previous studies have highlighted the importance of consistency in the methodology used, but to our knowledge, no study has described the methodology of sample preparation and storage systematically with respect to miRNAs as blood biomarkers. The aim of this study was to investigate the stability of miRNAs in blood under various relevant clinical and research conditions: different collection tubes, storage at different temperatures, physical disturbance, as well as serial freeze-thaw cycles. Blood samples were collected from 12 healthy donors into different collection tubes containing anticoagulants, including EDTA, citrate and lithium-heparin, as well as into serum collection tubes. MiRNA stability was evaluated by measuring expression changes of miR-1, miR-21 and miR-29b at different conditions: varying processing time of whole blood (up to 72 hours (h)), long-term storage (9 months at -80°C), physical disturbance (1 and 8 h), as well as in a series of freeze/thaw cycles (1 and 4 times). Different collection tubes revealed comparable concentrations of miR-1, miR-21 and miR-29b. Tubes with lithium-heparin were found unsuitable for miRNA quantification. MiRNA levels were stable for at least 24 h at room temperature in whole blood, while separated fractions did show alterations within 24 h. There were significant changes in the miR-21 and miR-29b levels after 72 h incubation of whole blood at room temperature (p<0.01 for both). Both miR-1 and miR-21 showed decreased levels after physical disturbance for 8 h in separated plasma and miR-1 in serum whole blood, while after 1 h of disturbance no changes were observed. Storage of samples at -80°C extended the miRNA stability remarkably, however, miRNA levels in long-term stored (9 months) whole blood samples were significantly changed, which is in contrast to the plasma samples, where miR-21 or miR-29b levels were found to be stable. Repetitive (n = 4) freeze-thaw cycles resulted in a significant reduction of miRNA concentration both in plasma and serum samples. This study highlights the importance of proper and systematic sample collection and preparation when measuring circulating miRNAs, e.g., in context of clinical trials. We demonstrated that the type of collection tubes, preparation, handling and storage of samples should be standardized to avoid confounding variables influencing the results.

The voltage-gated KCNQ1 (KvLQT1, Kv7.1) potassium channel plays a crucial role in shaping the cardiac action potential as well as in controlling the water and salt homeostasis in several epithelial tissues. KCNQ1 channels in these tissues are tightly regulated by auxiliary proteins and accessory factors, capable of modulating the properties of the channel complexes. This paper reviews the current knowledge about the KCNQ1 channel with a major focus on interacting proteins and physiological functions.

Small-conductance calcium-activated potassium (SK) channels are expressed in the heart of various species, including humans. The aim of the present study was to address whether SK channels play a functional role in human atria. Quantitative real-time PCR analyses showed higher transcript levels of SK2 and SK3 than that of the SK1 subtype in human atrial tissue. SK2 and SK3 were reduced in chronic atrial fibrillation (AF) compared with sinus rhythm (SR) patients. Immunohistochemistry using confocal microscopy revealed widespread expression of SK2 in atrial myocytes. Two SK channel inhibitors (NS8593 and ICAGEN) were tested in heterologous expression systems revealing ICAGEN as being highly selective for SK channels, while NS8593 showed less selectivity for these channels. In isolated atrial myocytes from SR patients, both inhibitors decreased inwardly rectifying K⁺ currents by ∼15% and prolonged action potential duration (APD), but no effect was observed in myocytes from AF patients. In trabeculae muscle strips from right atrial appendages of SR patients, both compounds increased APD and effective refractory period, and depolarized the resting membrane potential, while only NS8593 induced these effects in tissue from AF patients. SK channel inhibition did not alter any electrophysiological parameter in human interventricular septum tissue. SK channels are present in human atria where they participate in repolarization. SK2 and SK3 were down-regulated and had reduced functional importance in chronic AF. As SK current was not found to contribute substantially to the ventricular AP, pharmacological inhibition of SK channels may be a putative atrial-selective target for future antiarrhythmic drug therapy.

Background—: Recently, evidence has emerged that small-conductance Ca ²⁺ -activated K ⁺ (SK) channels are predominantly expressed in the atria in a number of species including human. In rat, guinea pig, and rabbit ex vivo and in vivo models of atrial fibrillation (AF), we used 3 different SK channel inhibitors, UCL1684, N -(pyridin-2-yl)-4-(pyridin-2-yl)thiazol-2-amine (ICA), and NS8593, to assess the hypothesis that pharmacological inhibition of SK channels is antiarrhythmic. Methods and Results—: In isolated, perfused guinea pig hearts, AF could be induced in all control hearts (n=7) with a combination of 1 μmol/L acetylcholine combined with electric stimulation. Pretreatment with 3 μmol/L NS8593, which had no effect on QT interval, prolonged the atrial effective refractory period by 37.1±7.7% ( P <0.001) and prevented acetylcholine-induced AF ( P <0.001, n=7). After AF induction, perfusion with NS8593 (10 μmol/L), UCL1684 (1 μmol/L), or ICA (1 μmol/L) terminated AF in all hearts, comparable to 10 μmol/L amiodarone. In isolated, perfused rat hearts, AF was induced with electric stimulation; 10 μmol/L NS8593 terminated AF and prevented reinduction of AF in all hearts (n=6, P <0.001). In all hearts, AF could be reinduced after washing. In isolated, perfused rabbit hearts, AF was induced with 10 μmol/L acetylcholine and burst pacing; 10 μmol/L NS8593 terminated AF and prevented reinduction of AF in all hearts (n=6, P <0.001). After washing, AF could be reinduced in 75% of the hearts (n=4, P =0.06). In an in vivo rat model of acute AF induced by burst pacing, injection of 5 mg/kg of either NS8593 or amiodarone shortened AF duration significantly to (23.2±20.0%, P <0.001, n=5, and 26.2±17.9%, P <0.001, n=5, respectively) as compared with injection of vehicle (96.3±33.2%, n=5). Conclusions—: Inhibition of SK channels prolongs atrial effective refractory period without affecting QT interval and prevents and terminates AF ex vivo and in vivo, thus offering a promising new therapeutic opportunity in the treatment of AF.