Atrial-Selective Approaches for the Treatment of Atrial Fibrillation

Atrial-selective pharmacologic approaches represent promising novel therapeutic options for the treatment of atrial fibrillation (AF). Medical treatment for AF is still more widely applied than interventional therapies but is hampered by several important weaknesses. Besides limited clinical efficacy (cardioversion success and sinus-rhythm maintenance), side effects like ventricular proarrhythmia and negative inotropy are important limitations to present class I and III drug therapy. Although no statistically significant detrimental survival consequences have been documented in trials, constitutional adverse effects might also limit applicability. Cardiac targets for novel atrial-selective antiarrhythmic compounds have been identified, and a large-scale search for safe and effective medications has begun. Several ionic currents (IKACh, IKur) and connexins (Cx-40) are potential targets, because atrial-selective expression makes them attractive in terms of reduced ventricular side-effect liability. Data on most agents are still experimental, but some clinical findings are available. Atrial fibrillation generates a specifically remodeled atrial milieu for which other therapeutic interventions might be effective. Some drugs show frequency-dependent action, whereas others target structurally remodeled atria. This review focuses on potential atrial-selective compounds, summarizing mechanisms of action in vitro and in vivo. It also mentions favorable interventions on the milieu in terms of conventional (such as antifibrotic effects of angiotensin-system antagonism) and innovative gene-therapy approaches that might add to future AF therapeutic options

N-Terminal Arginines Modulate Plasma-Membrane Localization of Kv7.1/KCNE1 Channel Complexes

BACKGROUND AND OBJECTIVE: The slow delayed rectifier current (I(Ks)) is important for cardiac action potential termination. The underlying channel is composed of Kv7.1 α-subunits and KCNE1 β-subunits. While most evidence suggests a role of KCNE1 transmembrane domain and C-terminus for the interaction, the N-terminal KCNE1 polymorphism 38G is associated with reduced I(Ks) and atrial fibrillation (a human arrhythmia). Structure-function relationship of the KCNE1 N-terminus for I(Ks) modulation is poorly understood and was subject of this study. METHODS: We studied N-terminal KCNE1 constructs disrupting structurally important positively charged amino-acids (arginines) at positions 32, 33, 36 as well as KCNE1 constructs that modify position 38 including an N-terminal truncation mutation. Experimental procedures included molecular cloning, patch-clamp recording, protein biochemistry, real-time-PCR and confocal microscopy. RESULTS: All KCNE1 constructs physically interacted with Kv7.1. I(Ks) resulting from co-expression of Kv7.1 with non-atrial fibrillation '38S' was greater than with any other construct. Ionic currents resulting from co-transfection of a KCNE1 mutant with arginine substitutions ('38G-3xA') were comparable to currents evoked from cells transfected with an N-terminally truncated KCNE1-construct ('Δ1-38'). Western-blots from plasma-membrane preparations and confocal images consistently showed a greater amount of Kv7.1 protein at the plasma-membrane in cells co-transfected with the non-atrial fibrillation KCNE1-38S than with any other construct. CONCLUSIONS: The results of our study indicate that N-terminal arginines in positions 32, 33, 36 of KCNE1 are important for reconstitution of I(Ks). Furthermore, our results hint towards a role of these N-terminal amino-acids in membrane representation of the delayed rectifier channel complex

<資料>新西蘭,加奈陀,印度の中央銀行設立計畫

<p><b>Panels A–C,</b> representative images obtained from confocal microscopy of transiently transfected HEK cells. R835Q mutant channels do not appear differently distributed in comparison to WT KCNH2. <b>D</b>, Immunoblots using anti-erg1 (2, 5 µg/mL) of crude membrane extracts from heterologous expression in HEK cells, indicating equal protein expression level. Illustrated below are endoplasmic reticulum and plasma membrane fraction with respective markers of equal protein loading (calnexin for endoplasmic reticulum, spectrin for plasma membranes). Exemplary Western blots of preparations at physiological temperature (37°C) and 40°C (to simulate febrile illness of the index patient’s brother) are shown. No differences were observed in Kv11.1-WT or Kv11.1-R835Q plasma membrane representation of the two proteins under the two conditions. ER: endoplasmic reticulum fraction; PM: plasma-membrane fraction; WT: wild type; NT: non-transfected cells.</p

mRNA expression levels.

<p>Mean±SEM results of quantitative real-time PCR for mRNA expression of KCNE1 (<b>A</b>) and Kv7.1 (<b>B</b>) from HEK cells transfected with Kv7.1 and the various KCNE1 constructs (n = 5 transfections for each panel). Data were normalized to ß-actin expression. There were no differences in mRNA transcription that could account for changes in membrane currents.</p

Biophysical characteristics.

<p><b>A</b>, illustrates half-activation voltages (V₅₀) of currents resulting from expression of Kv7.1 with respective constructs. Mean±SEM V₅₀ values were similar between constructs ‘38S’: 5.8±3.7 mV, ‘38G’: −2.7±4.1 mV, ‘Δ1-38’: 6.0±4.1 mV, ‘linker’: 1.0±2.5 mV, ‘38S-3xA’: 3.4±8.6 mV, ‘38G-3xA’: 8.7±6.8 mV; <i>P</i> = n.s vs. ‘38S’. Lines shown are Boltzmann fits to mean data (obtained with the formula: A = A₀/(1+exp[(V₅₀-V)/S])). <b>B</b> shows results of mono-exponential fits (y = A^(−t/τ)+C) to activating currents with time-constants plotted over a test potential of 0 mV. <b>C</b> shows results of mono-exponential fits to deactivating ionic currents. Currents obtained from co-transfection of Kv7.1 with ‘linker’ and ‘38S-3xA’ deactivated more slowly than currents obtained with the remainder of the constructs (<i>P</i><0.05 vs. ‘38S’). TP – test potential.</p

Primers for KCNE1 mutagenesis.

<p>Mutation containing sequences in italic, underlined.</p

Primers for quantitation of Kv7.1 and KCNE1 mRNA.

<p>F – forward primer, R – reverse primer.</p

Electrophysiological properties.

<p><b>A–F</b>, representative ionic currents elicited by whole-cell patch-clamp with the protocol shown in inset A. <b>G</b>, current-voltage relations of mean±SEM depolarization induced activating step-current densities from cells transfected with Kv7.1 plus various KCNE1 constructs. <b>H,</b> mean±SEM current-voltage relationships of repolarization induced tail-current densities. P-values are shown for currents recorded from cells transfected with Kv7.1+’38S’ vs. all other constructs. The remainder of constructs did not lead to significantly different current sizes compared with ‘38G’ (<i>P</i> = n.s.). TP – test potential.</p

Immunofluorescent studies of membrane representation.

<p>Confocal images of HEK cells transfected with Kv7.1 and flag-tagged KCNE1 constructs. The top row localizes Kv7.1 (red) within cells. The middle row shows the distribution of KCNE1 (green), bottom panels illustrate the plasma-membrane marker pan-cadherin (blue). Bars represent 10 µm. The respective bar graphs below represent the mean±SEM data of Kv7.1 and KCNE1 correlation with plasma-membrane marker pan-cadherin. White bars represent Kv7.1/pan-cadherin ratio, hatched bars are KCNE1/pan-cadherin ratio. Results are from 4-9 experiments each. Cad – cadherin, other abbreviations as above.</p