Connexin hemichannels in atrial fibrillation: orphaned and irrelevant?

This editorial refers to ‘RyR2 regulates Cx43 hemichannel intracellular Ca2+-dependent activation in cardiomyocytes’ by A. Lissoni et al. , pp. 123–136. Compared to the pivotal role of gap junctions in cardiac cellular electrophysiology, there is only limited knowledge about the role of connexin hemichannels in the heart, with even lesser known of their relevance in cardiac arrhythmias. 1 Connexins are well known to form intercellular channels, so-called gap junctions, which connect cells chemically and electrically. In the healthy heart, gap junctions between cardiomyocytes are usually formed by connexin 43 (Cx43). Six of these Cx43 proteins, located at the intercalated discs, form so-called hemichannels that connect to hemichannels of adjacent myocytes. Given their central role in electrical impulse propagation throughout the myocardium, alterations in gap junction activity likely contributes to the pathophysiology of ventricular and atrial arrhythmias. 1, 2 Nevertheless, in the sarcolemma of cardiomyocytes, connexins may also form orphaned hemichannels which do not couple with hemichannels of adjacent cells. In contrast to gap junctions, these connexin hemichannels are closed under resting conditions. Their opening, which has been studied mainly in isolated ventricular cardiomyocytes and expression systems, occurs primarily at positive membrane potentials in response to metabolic inhibition, low extracellular Ca2+ or an increase in intracellular Ca2+ concentration. 3 In their present manuscript, Lissoni et al. 4 observed a characteristic pattern of spiking inward currents in isolated mouse ventricular myocytes, which occurs in response to the application of caffeine. Based on the characteristic conductance and the absence of these currents in Cx43 knockout mice, the authors attributed these spikes to single-channel openings of Cx43 hemichannels. With great enthusiasm, we read about the careful evaluation of these phenomena, since the method employed by the authors in detecting Cx43 hemichannels is well established in our lab for cellular electrophysiology and Ca2+ homeostasis interaction studies. 5, 6 Caffeine is widely used to induce depletion of sarcoplasmic reticulum (SR) Ca2+ content by activation of ryanodine receptor channels (cardiac type 2, RyR2) within the SR. While holding the membrane potential at −80 mV using the conventional patch-clamp technique, the released Ca2+ is removed from the cytosol via Na+/Ca2+ exchanger (NCX), which brings 3 Na+ ions into the cell per extruded Ca2+ ion. This ion exchange is an electrogenic process that induces a depolarizing inward current. In the representative recordings from Lissoni et al. 4 this current is overlayed with single-channel openings attributed to Cx43 hemichannel openings. Indeed, when reanalysing experiments we recently performed in mouse atrial myocytes, all cells showed the characteristic spiking behaviour in response to caffeine (Figure 1A). Next, we wondered if this behaviour manifests in human atrial myocytes. We screened all our experiments performed during the last year in isolated human atrial myocytes obtained from sinus rhythm patients undergoing cardiac surgery for coronary bypass grafting. Surprisingly, we found clear hemichannel openings in about 15% of atrial myocytes (Figure 1B). We obviously missed this behaviour in previous analysis, probably due to the low occurrence rate or misinterpretation of these inward spikes for electrical noise. The exemplary experiments shown in Figure 1A clearly demonstrate that connexin hemichannel activity with characteristic single-channel conductance is also observed in atrial myocytes from human and mice. Occurrence of unitary current events mediated by RyR2 activation in atrial myocytes from mice and humans. (A) Representative recordings showing events of unitary openings provoked by application of caffeine on myocytes from mouse (left) and patients with sinus rhythm (human, right) held at −80 mV. Magnified grey portion (insert) was obtained by the exclusion of the inward depolarizing current, predominated by NCX. L0 represents baseline current levels while L1 and L2 depict levels of single and double channel openings. (B) Prevalence of unitary current events in atrial myocytes obtained from mice and humans. n/N, number of myocytes/mouse or patient. Based on the insights provided by the detailed analysis of Lissoni et al. and our preliminary observations in isolated atrial myocytes, the following questions forced themselves into our minds: The principal atrial connexins are Cx43 and Cx40. While Cx43 is expressed ubiquitously in the heart, Cx40 is expressed in the atria, but not in the ventricles. 1 Given the central role of gap junctions in determining conduction velocity in atrial tissue, and genetic studies that suggest an association between connexin gene variants and atrial fibrillation (AF), it is not surprising that a wide range of studies investigated the role of connexin remodelling in AF pathophysiology (see comprehensive reviews by Leybaert et al. 1 and Kato et al. 2). Nevertheless, results on total atrial expression of connexions in both AF patients and animal models show remarkable variations even within the same model. 2 In contrast, connexin redistribution from cell-ends to lateral margins appears to be a more consistent finding in both animal models and human AF. The resulting reduction in connexin density at the intercalated discs is likely to contribute to AF promoting conduction abnormalities. 1 On a different note, the laterally redistributed connexin channels do not assemble into gap junction channels. 7 One may speculate that these channels form functional hemichannels that may also contribute to AF pathophysiology. When open, connexin hemichannels are supposed to conduct unspecific ion currents. They could essentially function to mediate K+ efflux and Na+ influx, thereby leading to Na+ overload in atrial myocytes from AF patients. In fact, cytosolic Na+ accumulation has been suggested to contribute to AF pathophysiology. 8 The increased cytosolic Na+ load inhibits Ca2+ extrusion by NCX, thereby promoting intracellular Ca2+ overload. The latter facilitates the occurrence of spontaneous Ca2+ release events (SCaEs) from the SR, which may give rise to arrhythmogenic delayed afterdepolarizations (DADs). 5 Accordingly, inhibition of Cx43 hemichannels has been demonstrated to normalize accumulation of intracellular Ca2+ which mitigates increased frequency of SCaEs from the SR (sparks) in ventricular myocytes from a mouse model of arrhythmogenic right ventricular cardiomyopathy. 9 Whether a similar mechanism may play a role in atrial myocytes from AF patients is highly speculative. However, a recent screening study identified the Cx43 hemichannel blocker carbenoxolone as a robust suppressor of abnormal electrophysiological phenotypes in both a human embryonic stem cell model and a zebrafish model of myosin light chain 4 (MYL4) mutation-associated AF. 10 Taken together, it is well justified to hypothesize that connexin hemichannels play an important role in AF pathophysiology. Therefore, selective hemichannel blocking peptides such as GAP19, L2, or RyRHCIp may act as lead structures for the development of novel antiarrhythmic strategies. 1, 4 Nevertheless, substantial further research is clearly necessary to answer this question. The opinions expressed in this article are not necessarily those of the Editors of Cardiovascular Research or of the European Society of Cardiology. All available data are incorporated into this article. Conflict of interest: none declared. This work was supported by German Research Foundation (DFG; Clinician Scientist Program In Vascular Medicine (PRIME), MA 2186/14-1 to P. T. ; VO1568/3-1, IRTG1816, and SFB1002 project A13 to N. V. ), German Research Foundation under Germany's Excellence Strategy (EXC 2067/1- 390729940), German Centre for Cardiovascular Research (DZHK SE181 to N. . V. ), and the Else-Kröner-Fresenius Foundation (EKFS 2016A20 to N. V. ).