Journeying down the long and winding road: the whole picture of volume-activated Cl- channel activation in cardiac myocytes

Patch-clamp studies of anion channels show a bewildering variety in their properties and regulation mechanisms.2 Amongst them, the anion channels activated by hypo-osmotic cell swelling (ICl,swell) have drawn attention because of their wide expression in virtually every vertebrate cell(s), including cardiac myocytes, and their critical roles in cell volume regulation.2–4 Cells need to regulate their volume in the face of several external and internal challenges. For this purpose, cells are endowed with various ion channels and transporters that become activated upon cell swelling or shrinkage. ICl,swell are also called by different names like volume-regulated anion channels and volume-stimulated osmolyte and anion channels, implicating their permeability to a wide variety of anionic molecules including organic substances and ATP4−. In spite of their importance and ubiquitous expression, their molecular nature is still the subject of controversy and remains unclear.2,3 One of the key issues in ICl,swell is the activation mechanism, i.e. how is the hypo-osmotic stress transduced into anion channel activation? A direct mechanical gating is unlikely with regard to the considerable time lag between hypotonic challenge and steady-state activation of ICl,swell. The list of suggested mechanisms or critical factors supported by experimental evidence include reduced ionic activity, G protein activation, lipoxygenase metabolites, MAP kinase and tyrosine kinase pathways, and reactive oxygen species generated from NADPH oxidase (NOX).2–4 Such a perplexing picture definitely indicates that multiple mechanisms are involved and knotted to activate ICl,swell, depending on the tissue and cell types. In cardiac myocytes, ICl,swell is supposed to regulate the duration of the action potential due to the negative equilibrium potential of Cl−in vivo.4,5. Interestingly, the tonic activation of ICl,swell in isotonic condition was found by Baumgarten and coworkers6 in cardiac myocytes isolated from the over-paced heart failure model. The same group has made continued efforts to reveal the signalling pathways of ICl,swell under both isotonic and hypotonic conditions.7–10 Their previous studies have revealed another entrance (i.e. integrin receptor), short cuts (angiotensin-II and EGF receptors), and common paths (NOX and H2O2) leading to ICl,swell. Evidently, the road seems to be lengthy, with many winding turns. Through this paper, the authors have connected the gaps between the paths previously found by themselves and other groups. In summary, swelling of ventricular myocytes releases angiotensin II, and the autocrine/paracrine stimulation of angiotensin-II receptor (AT1R) triggers transactivation of EGFR that subsequently activates the downstream pathways of PI-3K, NOX2, and H2O2 generation.1 Thus, we seem to have the guide map from hypotonic swelling to Cl− channel activation, at least in ventricular myocytes. Readers who are familiar with the previous work of Baumgarten's group could have expected that the stimuli from hypo-osmotic swelling and the stretch via integrin receptor converge on a common pathway downstream of AT1R activation. However, as argued by the authors, the osmotic swelling and integrin stretch seem to use different pathways (as well), because the pharmacological inhibition of Src family kinases with PP2 has the opposite effect on the final activation of ICl,swell, depending on the types of stimuli.1,7–11 The role of tyrosine phosphorylation signalling in the activation of ICl,swell in cardiac myocytes has been previously suggested, but depending on the tissues and species it remains controversial.4 The differential responses to Src kinase inhibitors need further investigation. Another question is how the different types of stimulation could commonly release angiotensin II in the ventricular myocytes. Although the local renin-angiotensin system and its physiological role have been extensively proposed, the actual amount of secreted angiotensin might be low. Moreover, the direct activation of AT1R by mechanical stress has been recently suggested.12 The conclusion of the study and the signalling mechanism deduced by Ren et al.1 is convincingly supported by much evidence. However, readers might be puzzled by the complexity of the signalling cascade starting from the osmotic swelling. Why should it be so long? Certainly, not every cell would release angiotensins or similar ligands upon cell swelling, whereas all types of cells show ICl,swell. In cells other than ventricular myocytes, other mechanisms are required to provide other pathways or a shortcut to ICl,swell. In addition, it would be intriguing to examine whether those shortcuts might also converge on the common denominator, namely NOX and H2O2, suggested by the authors1 and other researchers.13 NOX and their products (H2O2) are believed to play key roles in the pathophysiology of cardiovascular diseases, and the upregulation of NOX in remodelled and failing heart has been reported.14 In this respect, the present study by Ren et al.1 again explains the tonic activation of ICl,swell in cardiac remodelling and suggests the plausible mechanism, i.e. NOX. Then, what would be the actual role of ICl,swell in the pathophysiology of heart failure? Is it pros or cons? The effect of ICl,swell activation on cardiac rhythm and excitability would be highly variable, depending on the cytoplasmic Cl− activity and other settings. The impact would be even greater under ischaemic conditions where cell swelling is expected. However, as has been recently demonstrated by A. Noma's beautiful simulation model and experiments, the interaction of Na+/K+–ATPase inhibition and Cl− conductance (in this case, CFTR) revealed more obscure results.15 The chronic activation of ICl,swell and its impact on cardiac excitability remains to be examined in vivo as well as in silico. In addition to the electrophysiological effect, the long-term changes in Cl− conductance and cell volume would affect multiple aspects of cell biology.2,3 The work by Ren et al.1 would be a very helpful map. Nonetheless, the road beyond that would be still far away from the goal and have many surprises.