Minimum Information about a Cardiac Electrophysiology Experiment (MICEE): standardised reporting for model reproducibility, interoperability, and data sharing.

Cardiac experimental electrophysiology is in need of a well-defined Minimum Information Standard for recording, annotating, and reporting experimental data. As a step towards establishing this, we present a draft standard, called Minimum Information about a Cardiac Electrophysiology Experiment (MICEE). The ultimate goal is to develop a useful tool for cardiac electrophysiologists which facilitates and improves dissemination of the minimum information necessary for reproduction of cardiac electrophysiology research, allowing for easier comparison and utilisation of findings by others. It is hoped that this will enhance the integration of individual results into experimental, computational, and conceptual models. In its present form, this draft is intended for assessment and development by the research community. We invite the reader to join this effort, and, if deemed productive, implement the Minimum Information about a Cardiac Electrophysiology Experiment standard in their own work.

Measurement and analysis of sarcomere length in rat cardiomyocytes in situ and in vitro.

Sarcomere length (SL) is an important determinant and indicator of cardiac mechanical function; however, techniques for measuring SL in living, intact tissue are limited. Here, we present a technique that uses two-photon microscopy to directly image striations of living cells in cardioplegic conditions, both in situ (Langendorff-perfused rat hearts and ventricular tissue slices, stained with the fluorescent marker di-4-ANEPPS) and in vitro (acutely isolated rat ventricular myocytes). Software was developed to extract SL from two-photon fluorescence image sets while accounting for measurement errors associated with motion artifact in raster-scanned images and uncertainty of the cell angle relative to the imaging plane. Monte-Carlo simulations were used to guide analysis of SL measurements by determining error bounds as a function of measurement path length. The mode of the distribution of SL measurements in resting Langendorff-perfused heart is 1.95 mum (n = 167 measurements from N = 11 hearts) after correction for tissue orientation, which was significantly greater than that in isolated cells (1.71 mum, n = 346, N = 9 isolations) or ventricular slice preparations (1.79 mum, n = 79, N = 3 hearts) under our experimental conditions. Furthermore, we find that edema in arrested Langendorff-perfused heart is associated with a mean SL increase; this occurs as a function of time ex vivo and correlates with tissue volume changes determined by magnetic resonance imaging. Our results highlight that the proposed method can be used to monitor SL in living cells and that different experimental models from the same species may display significantly different SL values under otherwise comparable conditions, which has implications for experiment design, as well as comparison and interpretation of data.

K(ATP) channel current increases in postinfarction remodeled cardiomyocytes.

Adenosintriphosphate-sensitive potassium channels (K(ATP) channels) are an important linkage between the metabolic state of a cell and electrophysiological membrane properties. In this study, K(ATP) channels were studied in myocytes of normal and remodeled myocardium of the rat. Myocardial infarction was induced by ligature of the left anterior descending artery. Remodeled myocytes were obtained from the hypertrophied posterior left ventricular wall and interventricular septum 3 months after infarction. The current through K(ATP) channels was measured in whole-cell and inside-out patches by using the patch-clamp technique. After myocardial infarction, the heart weight/body weight ratio was doubled and the myocytes were hypertrophied yielding a cell capacitance of 266+/-16 pF compared to 122+/-12 pF in control cells. The amount of Kir6.2 protein was indistinguishable in corresponding regions of control and remodeled hearts. The ATP sensitivity of K(ATP) channels in remodeled cells was significantly lower than in control cells (half maximum block at 115 micromol/l ATP in remodeled and at 71 mumol/l ATP in control cells). The maximum I (KATP) density induced by metabolic inhibition was higher in small remodeled (176+/-15 pA/pF) than in control cells (127+/-11 pA/pF), but was unchanged in large remodeled cells. Both, the higher I (KATP) density and the lower sensitivity of the K(ATP) channels to ATP suggest that remodeled cardiomyocytes develop an improved tolerance to ischemia by stabilizing the resting potential and decreasing excitability.

The beta1 subunit but not the beta2 subunit colocalizes with the human heart Na+ channel (hH1) already within the endoplasmic reticulum.

Voltage-dependent Na+ channels are heteromultimers consisting of a pore-forming a subunit and accessory b subunits. In order to provide more insight into the trafficking and assembly of the cardiac Na+ channel complex, we investigated the subcellular localization of the Na+ channel beta1 and beta2 subunits, both in the absence and presence of the human heart Na+ channel (hH1). We fused spectrally distinct variants of the green fluorescent protein (GFP) to hH1 and to the beta1 and beta2 subunit, and expressed the optically labeled b subunits separately or in combination with hH1 in HEK293 cells. In contrast to the predominant localization of hH1 channels within the endoplasmic reticulum (ER), both beta subunits were clearly targeted to the plasma membrane when expressing their cDNAs alone. Upon coexpression of the a subunit, the beta1 subunit was efficiently retained within the ER and found to be colocalized with hH1. In contrast to this, hH1 and the beta2 subunit were not colocalized, i.e., they were detected mainly within the ER and the plasma membrane, respectively. These results indicate that hH1 and the b2 subunit are transported separately to the plasma membrane whereas the hH1/beta1 complex occurs already within the ER, which possibly facilitates trafficking of the channel complex to the plasma membrane.

Amiloride derivatives are potent blockers of KATP channels.

In cardiomyocytes sarcolemmal KATP channels open massively when the cytosolic [ATP] drops into the range of tens of micromolar, as during acute ischemia. The diuretic drug amiloride and related derivatives are well established as drugs blocking the Na+/H+- and the Na+/Ca2+-exchange, protecting the ischemic heart. Herein, the blocking action of amiloride and its derivatives 2',4'-dichlorobenzamil (DCB) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) on KATP channels was tested. In inside-out patches of mouse cardiac myocytes, amiloride, DCB, and EIPA reversibly blocked the KATP channels with the IC50 values 102, 1.80, and 2.14 micromol/l (-80 mV), respectively. Similar IC50 values were obtained in recombinant channels when coexpressing the KIR6.2 subunit with one of the sulfonylurea receptors SUR1 and SUR2A. All three drugs also blocked currents generated by the C-terminus deletion mutant KIR6.2delta26 in the absence of SUR. Amiloride blocked outward currents more effectively than inward currents whereas the block by DCB and EIPA was voltage independent. In cardiomyocytes, also whole-cell IKATP was blocked by the three drugs. In conclusion, amiloride, EIPA, and DCB block the pore-forming KIR6.2 subunit of cardiac KATP channels with higher potency than the Na+/H+- and the Na+/Ca2+-exchange, precluding a specific block of the exchanges under ischemic conditions.

Non-selective voltage-activated cation channel in the human red blood cell membrane.

Using the patch-clamp technique, a non-selective voltage-activated Na+ and K+ channel in the human red blood cell membrane was found. The channel operates only at positive membrane potentials from about +30 mV (inside positive) onwards. For sodium and potassium ions, similar conductances of about 21 pS were determined. Together with the recently described K+(Na+)/H+ exchanger, this channel is responsible for the increase of residual K+ and Na+ fluxes across the human red blood cell membrane when the cells are suspended in low ionic strength medium.