Giedrius Kanaporis

Gap junction permeability: selectivity for anionic and cationic probes

Gap junction channels formed by different connexins exhibit specific permeability to a variety of larger solutes including second messengers, polypeptides, and small interfering RNAs. Here, we report the permeability of homotypic connexin26 (Cx26), Cx40, Cx43, and Cx45 gap junction channels stably expressed in HeLa cells to solutes with different size and net charge. Channel permeability was determined using simultaneous measurements of junctional conductance and the cell-cell flux of a fluorescent probe. All four connexins allowed passage of both cationic and anionic probes, but the transfer rates were connexin dependent. The negatively charged probes [Lucifer yellow (LY; median axial diameter 9.9 Å, charge -2), carboxyfluorescein (CF; 8.2 Å; -2), and Alexa Fluor350 (AF350, 5.4 Å; -1)] exhibited the following permeability order: Cx43 > Cx45 > Cx26 > Cx40. In contrast, for the positively charged species permeability, the orders were as follows: Cx26 ≈ Cx43 ≈ Cx40 ≈ Cx45 for N,N,N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl) amino] ethanaminium (NBD-m-TMA; 5.5 Å, +1) and Cx26 ≥ Cx43 ≈ Cx40 > Cx45 for ethidium bromide (10.3 Å, +1). Comparison of probe permeability relative to K(+) revealed that Cx43 and Cx45 exhibited similar permeability for NBD-m-TMA and AF350, indicating weak charge selectivity. However, lesser transfer of CF and LY through Cx45 relative to Cx43 channels suggests stronger size-dependent discrimination of solute. The permeability of NBD-m-TMA for Cx40 and Cx26 channels was approximately three times higher than to anionic AF350 despite the fact that both have similar minor diameters, suggesting charge selectivity. In conclusion, these results confirm that channels formed from individual connexins can discriminate for solutes based on size and charge, suggesting that channel selectivity may be a key factor in cell signaling.

Coupling an HCN2‐expressing cell to a myocyte creates a two‐cell pacing unit

We examined whether coupling of a ventricular myocyte to a non‐myocyte cell expressing HCN2 could create a two‐cell syncytium capable of generating sustained pacing. Three non‐myocyte cell types were transfected with the mHCN2 gene and used as sources of mHCN2‐induced currents. They were human mesenchymal stem cells and HEK293 cells, both of which express connexin43 (Cx43), and HeLa cells transfected with Cx43. Cell–cell coupling between heterologous pairs increased with time in co‐culture, and hyperpolarization of the myocyte induced HCN2 currents, indicating current transfer from the mHCN2‐expressing cell to the myocyte via gap junctions. The magnitude of the HCN2 currents recorded in myocytes increased with increasing junctional conductance. Once a critical level of electrical cell–cell coupling between myocytes and mHCN2 transfected cells was exceeded spontaneous action potentials were generated at frequencies of ∼0.6 to 1.7 Hz (1.09 ± 0.05 Hz). Addition of carbenoxolone (200 μm), a gap junction channel blocker, to the media stopped spontaneous activity in heterologous cell pairs. Carbenoxolone washout restored activity. Blockade of HCN2 currents by 100 μm 9‐amino‐1,2,3,4‐tetrahydroacridine (THA) stopped spontaneous activity and subsequent washout restored it. Neither THA nor carbenoxolone affected electrically stimulated action potentials in isolated single myocytes. In summary, the inward current evoked in the genetically engineered (HCN2‐expressing) cell was delivered to the cardiac myocyte via gap junctions and generated action potentials such that the cell pair could function as a pacemaker unit. This finding lays the groundwork for understanding cell‐based biological pacemakers in vivo once an understanding of delivery and target cell geometry is defined.

The Mechanisms of Calcium Cycling and Action Potential Dynamics in Cardiac Alternans

RATIONALE Alternans is a risk factor for cardiac arrhythmia, including atrial fibrillation. At the cellular level alternans manifests as beat-to-beat alternations in contraction, action potential duration (APD), and magnitude of the Ca(2+) transient (CaT). Electromechanical and CaT alternans are highly correlated, however, it has remained controversial whether the primary cause of alternans is a disturbance of cellular Ca(2+) signaling or electrical membrane properties. OBJECTIVE To determine whether a primary failure of intracellular Ca(2+) regulation or disturbances in membrane potential and AP regulation are responsible for the occurrence of alternans in atrial myocytes. METHODS AND RESULTS Pacing-induced APD and CaT alternans were studied in single rabbit atrial and ventricular myocytes using combined [Ca(2+)]i and electrophysiological measurements. In current-clamp experiments, APD and CaT alternans strongly correlated in time and magnitude. CaT alternans was observed without alternation in L-type Ca(2+) current, however, elimination of intracellular Ca(2+) release abolished APD alternans, indicating that [Ca(2+)]i dynamics have a profound effect on the occurrence of CaT alternans. Trains of 2 distinctive voltage commands in form of APs recorded during large and small alternans CaTs were applied to voltage-clamped cells. CaT alternans was observed with and without alternation in the voltage command shape. During alternans AP-clamp large CaTs coincided with both long and short AP waveforms, indicating that CaT alternans develop irrespective of AP dynamics. CONCLUSIONS The primary mechanism underlying alternans in atrial cells, similarly to ventricular cells, resides in a disturbance of Ca(2+) signaling, whereas APD alternans are a secondary consequence, mediated by Ca(2+)-dependent AP modulation.

Cataracts are caused by alterations of a critical N-terminal positive charge in connexin50.

PURPOSE To elucidate the basis of the autosomal dominant congenital nuclear cataracts caused by the connexin50 mutant, CX50R23T, by determining its cellular distribution and functional behavior and the consequences of substituting other amino acids for arginine-23. METHODS Connexin50 (CX50) mutants were generated by PCR and transfected into HeLa or N2a cells. Expressed CX50 protein was detected by immunoblot analysis and localized by immunofluorescence. Intercellular communication was assessed by microinjection of neurobiotin or by double whole-cell patch-clamp recording. RESULTS HeLa cells stably transfected with CX50R23T or wild-type CX50 produced immunoreactive CX50 bands of identical electrophoretic mobility. Whereas HeLa cells stably expressing CX50 contained abundant gap junction plaques, CX50R23T localized predominantly in the cytoplasm. HeLa cells expressing wild-type CX50 showed large gap junctional conductances and extensive transfer of neurobiotin, but those expressing CX50R23T did not show significant intercellular communication by either assay. Moreover, CX50R23T inhibited the function of coexpressed wild-type CX50. Three CX50R23 substitution mutants (CX50R23K, CX50R23L, and CX50R23W) formed gap junction plaques, whereas two mutant substitutions with negatively charged residues (CX50R23D, CX50R23E) did not form detectable plaques. Only the mutant with a positive charge substitution (CX50R23K) allowed neurobiotin transfer at levels similar to those of wild-type CX50; none of the other mutants induced transfer. CONCLUSIONS These results suggest that replacement of amino acid 23 in CX50 by any residue that is not positively charged would lead to cataract formation.

Membrane potential determines calcium alternans through modulation of SR Ca(2+) load and L-type Ca(2+) current.

Alternans is a risk factor for cardiac arrhythmia, including atrial fibrillation. At the cellular level alternans is observed as beat-to-beat alternations in contraction, action potential (AP) morphology and magnitude of the Ca2+ transient (CaT). It is widely accepted that the bi-directional interplay between membrane voltage and Ca2+ is crucial for the development of alternans, however recently the attention has shifted to instabilities in cellular Ca2+ handling, while the role of AP alternation remains poorly understood. This study provides new insights how beat- to-beat alternation in AP morphology affects occurrence of CaT alternans in atrial myocytes. Pacing-induced AP and CaT alternans were studied in rabbit atrial myocytes using combined Ca2+ imaging and electrophysiological measurements. To determine the role of AP morphology for the generation of CaT alternans, trains of two voltage commands in form of APs recorded during large and small alternans CaTs were applied to voltage-clamped cells. APs of longer duration (as observed during small amplitude alternans CaT) and especially beat-to-beat alternations in AP morphology (AP alternans) reduced the pacing frequency threshold and increased the degree of CaT alternans. AP morphology contributes to the development of CaT alternans by two mechanisms. First, the AP waveform observed during small alternans CaTs coincided with higher end-diastolic sarcoplasmic reticulum Ca2+ levels ([Ca2+]SR), and AP alternans resulted in beat-to-beat alternations in end-diastolic [Ca2+]SR. Second, L-type Ca2+ current was significantly affected by AP morphology, where the AP waveform observed during large CaT elicited L-type Ca2+ currents of higher magnitude and faster kinetics, resulting in more efficient triggering of SR Ca2+ release. In conclusion, alternation in AP morphology plays a significant role in the development and stabilization of atrial alternans. The demonstration that CaT alternans can be controlled or even prevented by modulating AP morphology has important ramifications for arrhythmia prevention and therapy strategies.

Photo-Control of Excitation Waves in Cardiomyocyte Tissue Culture

Azobenzene photoswitches were recently reported to control the activity of neural cells and heart beat in leeches. Here, we report photocontrol of excitation of cultured cardiomyocytes that have been made light sensitive by using the addition of azobenzene trimethylammonium bromide (AzoTAB). The trans-isomer of AzoTAB reversibly suppresses spontaneous activity and propagation of excitation waves, whereas the cis-isomer has no detectable effect on the electrical properties of cardiomyocytes. Photoisomerization of AzoTAB was achieved by switching the illumination wavelength, λ, from ~440 nm (trans-isomer) to ~350 nm (cis-isomer). Simultaneous irradiation at two wavelengths with properly chosen intensities allowed for dynamic control of the cis-isomer/trans-isomer ratio and the level of excitability from normal to fully unexcitable. Experiments were conducted by using AzoTAB-treated confluent monolayers of neonatal rat cardiomyocytes. Excitation waves were monitored by using the Ca2+-sensitive fluorescent dye Fluo-4. By projecting two-wavelength illumination patterns onto otherwise uniform cell layers, we were able to create excitable networks with the desired topology, dimensions, and functional properties. The present article discusses potential applications of this technique for the analysis of complex patterns of electrical excitation and cardiac arrhythmias.

Calcium‐activated chloride current determines action potential morphology during calcium alternans in atrial myocytes

Cardiac alternans – periodic beat‐to‐beat alternations in contraction, action potential (AP) morphology or cytosolic calcium transient (CaT) amplitude – is a high risk indicator for cardiac arrhythmias and sudden cardiac death. However, it remains an unresolved issue whether beat‐to‐beat alternations in intracellular Ca2+ ([Ca2+]i) or AP morphology are the primary cause of pro‐arrhythmic alternans. Here we show that in atria AP alternans occurs secondary to CaT alternans. CaT alternans leads to complex beat‐to‐beat changes in Ca2+‐regulated ion currents that determine alternans of AP morphology. We report the novel finding that alternans of AP morphology is largely sustained by the activity of Ca2+‐activated Cl− channels (CaCCs). Suppression of the CaCCs significantly reduces AP alternans, while CaT alternans remains unaffected. The demonstration of a major role of CaCCs in the development of AP alternans opens new possibilities for atrial alternans and arrhythmia prevention.

Optical mapping at increased illumination intensities

Abstract. Voltage-sensitive fluorescent dyes have become a major tool in cardiac and neuro-electrophysiology. Achieving high signal-to-noise ratios requires increased illumination intensities, which may cause photobleaching and phototoxicity. The optimal range of illumination intensities varies for different dyes and must be evaluated individually. We evaluate two dyes: di-4-ANBDQBS (excitation 660 nm) and di-4-ANEPPS (excitation 532 nm) in the guinea pig heart. The light intensity varies from 0.1 to 5  mW/mm2, with the upper limit at 5 to 10 times above values reported in the literature. The duration of illumination was 60 s, which in guinea pigs corresponds to 300 beats at a normal heart rate. Within the identified duration and intensity range, neither dye shows significant photobleaching or detectable phototoxic effects. However, light absorption at higher intensities causes noticeable tissue heating, which affects the electrophysiological parameters. The most pronounced effect is a shortening of the action potential duration, which, in the case of 532-nm excitation, can reach ∼30%. At 660-nm excitation, the effect is ∼10%. These findings may have important implications for the design of optical mapping protocols in biomedical applications.

Ca2+-activated chloride channel activity during Ca2+ alternans in ventricular myocytes

ABSTRACT Cardiac alternans, defined beat-to-beat alternations in contraction, action potential (AP) morphology or cytosolic Ca transient (CaT) amplitude, is a high risk indicator for cardiac arrhythmias. We investigated mechanisms of cardiac alternans in single rabbit ventricular myocytes. CaTs were monitored simultaneously with membrane currents or APs recorded with the patch clamp technique. A strong correlation between beat-to-beat alternations of AP morphology and CaT alternans was observed. During CaT alternans application of voltage clamp protocols in form of pre-recorded APs revealed a prominent Ca2+-dependent membrane current consisting of a large outward component coinciding with AP phases 1 and 2, followed by an inward current during AP repolarization. Approximately 85% of the initial outward current was blocked by Cl− channel blocker DIDS or lowering external Cl− concentration identifying it as a Ca2+-activated Cl− current (ICaCC). The data suggest that ICaCC plays a critical role in shaping beat-to-beat alternations in AP morphology during alternans.

Metabolic inhibition reduces cardiac L-type Ca2+ channel current due to acidification caused by ATP hydrolysis

Metabolic stress evoked by myocardial ischemia leads to impairment of cardiac excitation and contractility. We studied the mechanisms by which metabolic inhibition affects the activity of L-type Ca2+ channels (LTCCs) in frog ventricular myocytes. Metabolic inhibition induced by the protonophore FCCP (as well as by 2,4- dinitrophenol, sodium azide or antimycin A) resulted in a dose-dependent reduction of LTCC current (ICa,L) which was more pronounced during β-adrenergic stimulation with isoprenaline. ICa,L was still reduced by metabolic inhibition even in the presence of 3 mM intracellular ATP, or when the cell was dialysed with cAMP or ATP-γ-S to induce irreversible thiophosphorylation of LTCCs, indicating that reduction in ICa,L is not due to ATP depletion and/or reduced phosphorylation of the channels. However, the effect of metabolic inhibition on ICa,L was strongly attenuated when the mitochondrial F1F0-ATP-synthase was blocked by oligomycin or when the cells were dialysed with the non-hydrolysable ATP analogue AMP-PCP. Moreover, increasing the intracellular pH buffering capacity or intracellular dialysis of the myocytes with an alkaline solution strongly attenuated the inhibitory effect of FCCP on ICa,L. Thus, our data demonstrate that metabolic inhibition leads to excessive ATP hydrolysis by the mitochondrial F1F0-ATP-synthase operating in the reverse mode and this results in intracellular acidosis causing the suppression of ICa,L. Limiting ATP break-down by F1F0-ATP-synthase and the consecutive development of intracellular acidosis might thus represent a potential therapeutic approach for maintaining a normal cardiac function during ischemia.