Dual role of miR-1 in the development and function of sinoatrial cells.

miR-1, the most abundant miRNA in the heart, modulates expression of several transcription factors and ion channels. Conditions affecting the heart rate, such as endurance training and cardiac diseases, show a concomitant miR-1 up- or down-regulation. Here, we investigated the role of miR-1 overexpression in the development and function of sinoatrial (SAN) cells using murine embryonic stem cells (mESC). We generated mESCs either overexpressing miR-1 and EGFP (miR1OE) or EGFP only (EM). SAN-like cells were selected from differentiating mESC using the CD166 marker. Gene expression and electrophysiological analysis were carried out on both early mES-derived cardiac progenitors and SAN-like cells and on beating neonatal rat ventricular cardiomyocytes (NRVC) over-expressing miR-1. miR1OE cells increased significantly the proportion of CD166+ SAN precursors compared to EM cells (23% vs 12%) and the levels of the transcription factors TBX5 and TBX18, both involved in SAN development. miR1OE SAN-like cells were bradycardic (1,3 vs 2 Hz) compared to EM cells. In agreement with data on native SAN cells, EM SAN-like cardiomyocytes show two populations of cells expressing either slow- or fast-activating If currents; miR1OE SAN-like cells instead have only fast-activating If with a significantly reduced conductance. Western Blot and immunofluorescence analysis showed a reduced HCN4 signal in miR-1OE vs EM CD166+ precursors. Together these data point out to a specific down-regulation of the slow-activating HCN4 subunit by miR-1. Importantly, the rate and If alterations were independent of the developmental effects of miR-1, being similar in NRVC transiently overexpressing miR-1. In conclusion, we demonstrated a dual role of miR-1, during development it controls the proper development of sinoatrial-precursor, while in mature SAN-like cells it modulates the HCN4 pacemaker channel translation and thus the beating rate.

Striatin knock out induces a gain of function of INa and impaired Ca2+ handling in mESC-derived cardiomyocytes.

AIM: Striatin (Strn) is a scaffold protein expressed in cardiomyocytes (CMs) and alteration of its expression are described in various cardiac diseases. However, the alteration underlying its pathogenicity have been poorly investigated. METHODS: We studied the role(s) of cardiac Strn gene (STRN) by comparing the functional properties of CMs, generated from Strn-KO and isogenic WT mouse embryonic stem cell lines. RESULTS: The spontaneous beating rate of Strn-KO CMs was faster than WT cells, and this correlated with a larger fast INa conductance and no changes in If. Paced (2-8 Hz) Strn-KO CMs showed prolonged action potential (AP) duration in comparison with WT CMs and this was not associated with changes in ICaL and IKr. Motion video tracking analysis highlighted an altered contraction in Strn-KO CMs; this was associated with a global increase in intracellular Ca2+, caused by an enhanced late Na+ current density (INaL) and a reduced Na+/Ca2+ exchanger (NCX) activity and expression. Immunofluorescence analysis confirmed the higher Na+ channel expression and a more dynamic microtubule network in Strn-KO CMs than in WT. Indeed, incubation of Strn-KO CMs with the microtubule stabilizer taxol, induced a rescue (downregulation) of INa conductance toward WT levels. CONCLUSION: Loss of STRN alters CMs electrical and contractile profiles and affects cell functionality by a disarrangement of Strn-related multi-protein complexes. This leads to impaired microtubules dynamics and Na+ channels trafficking to the plasma membrane, causing a global Na+ and Ca2+ enhancement.

Cardiac mesoangioblasts are committed, self-renewable progenitors, associated with small vessels of juvenile mouse ventricle.

Different cardiac stem/progenitor cells have been recently identified in the post-natal heart. We describe here the identification, clonal expansion and characterization of self-renewing progenitors that differ from those previously described for high spontaneous cardiac differentiation. Unique coexpression of endothelial and pericyte markers identify these cells as cardiac mesoangioblasts and allow prospective isolation and clonal expansion from the juvenile mouse ventricle. Cardiac mesoangioblasts express many cardiac transcription factors and spontaneously differentiate into beating cardiomyocytes that assemble mature sarcomeres and express typical cardiac ion channels. Cells similarly isolated from the atrium do not spontaneously differentiate. When injected into the ventricle after coronary artery ligation, cardiac mesoangioblasts efficiently generate new myocardium in the peripheral area of the necrotic zone, as they do when grafted in the embryonic chick heart. These data identify cardiac mesoangioblasts as committed progenitors, downstream of earlier stem/progenitor cells and suitable for the cell therapy of a subset of juvenile cardiac diseases.

HCN2 overexpression in newborn and adult ventricular myocytes: distinct effects on gating and excitability.

Ventricular pacemaker current (I(f)) shows distinct voltage dependence as a function of age, activating outside the physiological range in normal adult ventricle, but less negatively in neonatal ventricle. However, heterologously expressed HCN2 and HCN4, the putative molecular correlates of ventricular I(f), exhibit only a modest difference in activation voltage. We therefore prepared an adenoviral construct (AdHCN2) of HCN2, the dominant ventricular isoform at either age, and used it to infect neonatal and adult rat ventricular myocytes to investigate the role of maturation on current gating. The expressed current exhibited an 18-mV difference in activation (V(1/2) -95.9+/-1.9 in adult; -77.6+/-1.6 mV in neonate), comparable to the 22-mV difference between native I(f) in adult and neonatal cultures (V(1/2) -98.7 versus -77.0 mV). This did not result from developmental differences in basal cAMP, because saturating cAMP in the pipette caused an equivalent positive shift in both preparations. In the neonate, AdHCN2 caused a significant increase in spontaneous rate compared with control (88+/-5 versus 48+/-4 bpm). In adult, where HCN2 activates more negatively, the effect was evident only during anodal excitation, requiring significantly less stimulus energy than control (2149+/-266 versus 3140+/-279 mV. ms). Thus, ventricular maturational state influences the voltage dependence of expressed HCN2, resulting in distinct physiological impact of expressed channels in neonate and adult myocytes. The full text of this article is available at http://www.circresaha.org.

Effects of dronedarone on acetylcholine-activated current in rabbit SAN cells.

1. The effect of the antiarrhythmic drug dronedarone on the Acetylcholine-activated K(+) current (I(K(ACh))) was investigated in single cells isolated from sinoatrial node (SAN) tissue of rabbit hearts. 2. Externally perfused dronedarone (0.001 - 1 microM) caused a potent, voltage independent block of I(K(ACh)). Fitting of the dose response curve of I(K(ACh)) block yielded an IC(50) value of 63 nM, a value over one order of magnitude lower than those reported for dronedarone block of other cardiac currents. 3. I(K(ACh)) block was not due to an inhibitory action of dronedarone on the muscarinic M2 receptor activation, since the drug was effective on I(K(ACh)) constitutively activated by intracellular perfusion with GTP-gammaS. 4. External cell perfusion with dronedarone inhibited the activity of I(K(ACh)) channels recorded from cell-attached patches by reducing the channel open probability (from 0.56 to 0.11) without modification of the single-channel conductance. 5. These data suggest that dronedarone blocks I(K(ACh)) channels either by disrupting the G-protein-mediated activation or by a direct inhibitory interaction with the channel protein.

Analysis and improvements of fringe jump corrections by electronics on the JET tokamak far infrared interferometer.

For the Tore Supra interferometer phase measurements, an electronics had been developed electronics using field programmable gate array processors. The embedded algorithm can correct the fringe jumps. For comparison, the electronics ran at JET during the 2009 campaign. The first analysis concluded that the electronics was not correcting all the fringe jumps. An analysis of the failures led to improvements in the algorithm, which was tested during the rest of the campaign. In this article, we evaluate the increases in the performance. From the analysis of the remaining faults, further improvements are discussed for designing future boards that are foreseen for JET using the second wavelength and the Cotton­Mouton effect information.

Fast sweeping reflectometry upgrade on Tore Supra.

In order to study the temporal dynamics of turbulence, the sweep time of our reflectometry has been shortened from 20 to 2 µs with 1 µs dead time. Detailed technical aspects of the upgrade are given, namely, about the stability of the ramp generation, the detection setup, and the fast acquisition module. A review of studies (velocity measurement of the turbulence, modifications of the wavenumber spectrum, radial mapping of correlation time, etc.) offered by such improvements is presented.

Fringe jump analysis and electronic corrections for the Tore Supra far infrared interferometer.

On the Tore Supra tokamak, the ten-channel far infrared interferometer consists of a double color (119 and 195 microm) system with two detectors for each channel to measure the plasma density. The phase measurement is obtained by combining a 100 kHz shifted reference beam with the probing beam that has crossed the plasma. The achieved precision--a few percent of a fringe--is very good compared with the expected variations due to plasma, which are on the order of several fringes. However, the counting of the fringe variations can be affected when the signal is perturbed by electromagnetic interferences or when it deviates in the presence of strong plasma refraction changes occurring during ICRH breakdowns, pellet injections, or disruptions. This induces a strong decrease in the reliability of the measurement, which is an important concern when the diagnostic is used for density control. We describe in this paper the renewing of the electronics that has been achieved to reduce and correct the number of the so-called fringe jumps. A new zero crossing method for phase measurement is used, together with a field programmable gate array semiconductor integration, to measure the phase and activate the algorithm of corrections every 10 micros. Comparisons between a numerical oscilloscope analysis and the corrected acquired data in the case of laboratory amplitude modulation tests and in the case of real plasma perturbations are also discussed.

Chemical modification of methionine residues of the phosphatidylcholine transfer protein from bovine liver. A spin-label study.

The role of methionine residues in the interaction of the phosphatidylcholine transfer protein from bovine liver with phospholipid vesicles was investigated by specific modification of these residues with iodoacetamide. The modified protein was digested with cyanogen bromide in order to determine which methionine residues had become resistant to this cleavage. Automated Edman degradation on the digest indicated that after 72 h of reaction, Met-1 was modified for 80%, Met-73 for 50%, Met-109 for 20%, whilst Met-173 and Met-203 were found to be unmodified. This distinct modification did not result in any loss of phosphatidylcholine transfer activity. The interaction of the phosphatidylcholine transfer protein with phospholipid vesicles was investigated by making use of electron spin resonance spectroscopy. The interaction of unmodified protein with vesicles composed of phosphatidylcholine/phosphatidic acid/spin-labeled phosphatidylethanolamine (79:16:5, mol%) or composed of phosphatidylserine/spin-labeled phosphatidylethanolamine (95:5, mol%), gave an increase of about 50% in the rotation correlation time. A similar increase was observed with the modified protein. This interaction was further investigated by labeling Met-1 and Met-73 in the transfer protein with iodoacetamidoproxyl spin-label. Spin-labeling did not inactivate the transfer protein. In addition, the electron spin resonance spectra of the spin-labeled protein were not affected upon addition of vesicles composed of phosphatidylcholine/phosphatidic acid (80:20, mol%). These experiments strongly suggest that Met-1 and Met-73 are not part of the site that interacts with the membrane.

Kinetic and ionic properties of the human HCN2 pacemaker channel.

Human cDNA coding for the hyperpolarization-activated "pacemaker" channel HCN2 was expressed in Phoenix cells and yielded an inward current (IhHCN2) activated on hyperpolarization. The average IhHCN2 was half-activated at -83.1 mV and its kinetics could be described by second-order Hodgkin-Huxley gating. The time constant curve was bell-shaped and peaked at -82.2 mV. With 115 mM external Na+ and 30 mM external K+, IhHCN2 reversed at -17.1 mV, and had a mean conductance of 5.6 nS. Reducing the external K+ or Na+ concentration led to a concentration-dependent reduction of the IhHCN2 conductance and to a hyperpolarizing shift of reversal potential. External Cs+ ions (5 mM) blocked IhHCN2 in a voltage-dependent way according to a Woodhull-type block model, at an electrical distance of 0.66 from the external membrane surface, and with a dissociation constant of 15 mM at 0 mV. Increasing cytoplasmic cAMP using forskolin increased IhHCN2 by shifting the current activation curve to more positive voltages (11.7 mV). Exposure of the intracellular side of inside-out macro-patches to cAMP led to a depolarizing shift of the channel open probability curve (15.2 mV with 10 microM cAMP). These results indicate that although hHCN2 channels share several properties with native cardiac f-channels, differences also exist in permeability and block properties, suggesting that native channels may not be composed simply of homomeric constructs.

Action of internal pronase on the f-channel kinetics in the rabbit SA node.

1. The hyperpolarization-activated If current was recorded in inside-out macropatches from sino-atrial (SA) node myocytes during exposure of their intracellular side to pronase, in an attempt to verify if cytoplasmic f-channel domains are involved in both voltage- and cAMP-dependent gating. 2. Superfusion with pronase caused a quick, dramatic acceleration of channel opening upon hyperpolarization and slowing, rapidly progressing into full blockade, of channel closing upon depolarization; these changes persisted after wash off of pronase and were irreversible, indicating proteolytic cleavage of channel regions which contribute to gating. 3. If recorded from patches normally responding to cAMP became totally insensitive to cAMP following pronase treatment, indicating partial or total removal of channel regions involved in the cAMP-dependent activation. 4. The fully activated I-V relationship was not modified by pronase, indicating that internal proteolysis did not affect the f-channel conductance. 5. The changes in If kinetics induced by pronase were due to a large depolarizing shift of the f-channel open probability curve (56.5 +/- 1.1 mV, n = 7). 6. These results are consistent with the hypothesis that cytoplasmic f-channel regions are implicated in dual voltage- and cAMP-dependent gating; also, since pronase does not abolish hyperpolarization-activated opening, an intrinsic voltage-dependent gating mechanism must exist which is inaccessible to proteolytic cleavage. A model scheme able to account for these data thus includes an intrinsic gating mechanism operating at depolarized voltages, and a blocking mechanism coupled to cAMP binding to the channel.