Sarcolemmal Ca 2+ -entry through L-type Ca 2+ channels controls the profile of Ca 2+ -activated Cl − current in canine ventricular myocytes

Abstract

Highlights• Ca 2+ -entry via I Ca,L is essential for the activation of I Cl(Ca) • I Cl(Ca) can be activated even in the absence of CICR• TMEM16A and Bestrophin-3 are expressed on human left ventricular muscle• TMEM16A and Bestrophin-3 co-localize with each other and with Ca v 1.2 channels (I Cl(Ca) ) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of I Cl(Ca) during an actual ventricular action potential (AP), however, is poorly understood. We aimed to study the profile of I Cl(Ca) systematically under physiological conditions (normal Ca 2+ cycling and AP voltage-clamp) as well as in conditions designed to change [Ca 2+ ] i . The expression of TMEM16A and/or Bestrophin-3 in canine and human left ventricular myocytes was examined. The possible spatial distribution of these proteins and their co-localization with Ca v 1.2 was also studied. The profile of I Cl(Ca), identified as a 9-anthracene carboxylic acid-sensitive current under AP voltageclamp conditions, contained an early fast outward and a late inward component, overlapping early and terminal repolarizations, respectively. Both components were moderately reduced by ryanodine, while fully abolished by BAPTA, but not EGTA. [Ca 2+ ] i was monitored using Fura-2-AM. Setting [Ca 2+ ] i to the systolic level measured in the bulk cytoplasm (1.1 µM) decreased I Cl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of I Cl(Ca) . Ca 2+ -entry through L-type Ca 2+ channels was essential for activation of I Cl(Ca) . TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Ca v 1.2 channels, when assessed using immunolabeling and confocal microscopy in both canine myocytes and human ventricular myocardium. Activation of I Cl(Ca) in canine ventricular cells requires Ca 2+ -entry through neighboring L-type Ca 2+ channels and is only augmented by SR Ca 2+ -release. Substantial activation of I Cl(Ca) requires high Ca 2+ in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA.

Ca2+-activated Cl− current is antiarrhythmic by reducing both spatial and temporal heterogeneity of cardiac repolarization

“…I Cl(Ca) started to activate at −20 mV and had its highest value at +60 mV in all studied cell types (Fig. 3B, C) similarly to our earlier studies [28,31]. …”

“…Previously we have confirmed the expression of TMEM16A and Bestrophin-3 on both canine and human isolated left ventricular cardiomyocytes [28]. It was also shown that these two proteins co-localize with each other and Ca v 1.2 suggesting a direct control of I Cl(Ca) by Ca 2+ entry through L-type Ca 2+ channels (LTCCs) in canine ventricular myocytes [28]. Although a previous publication found no evidence for the existence of I Cl(Ca) in healthy human ventricular myocytes [29] but this was not confirmed later.…”

“…I 9-AC could be used to explore the contribution of I Cl(Ca) to the AP in closely physiological conditions (without the application of intracellular Ca 2+ buffering during APVC) [28]. Regional differences, however, were not studied previously with APVC despite some earlier studies where either a “typical” AP was applied as the command signal during perforated patch recording using nonspecific blockers to dissect I Cl(Ca) [35] or 10 mmol/L EGTA was applied in the pipette solution interfering with physiological Ca 2+ homeostasis [18].…”

“…ISO is well known to increase [Ca 2+ ] i and therefore most probably [Ca 2+ ] cleft as well. This leads to a larger I Cl(Ca) [28] which upon its blockade by 9-AC leads to a greater EAD incidence (Fig. 6B).…”

“…Moreover, TMEM16A is expressed in murine ventricle and it was confirmed to be responsible for I Cl(Ca) in ventricular myocytes [27]. Previously we have confirmed the expression of TMEM16A and Bestrophin-3 on both canine and human isolated left ventricular cardiomyocytes [28]. It was also shown that these two proteins co-localize with each other and Ca v 1.2 suggesting a direct control of I Cl(Ca) by Ca 2+ entry through L-type Ca 2+ channels (LTCCs) in canine ventricular myocytes [28].…”