Modulation of L-type Ca2+ current (ICa,L) by H+ ions in cardiac myocytes is controversial, with widely discrepant responses reported. The pH sensitivity of ICa,L was investigated (whole cell voltage clamp) while measuring intracellular Ca2+ (Ca2+i) or pHi (epifluorescence microscopy) in rabbit and guinea pig ventricular myocytes. Selectively reducing extracellular or intracellular pH (pHo 6.5 and pHi 6.7) had opposite effects on ICa,L gating, shifting the steady-state activation and inactivation curves to the right and left, respectively, along the voltage axis. At low pHo, this decreased ICa,L, whereas at low pHi, it increased ICa,L at clamp potentials negative to 0 mV, although the current decreased at more positive potentials. When Ca2+i was buffered with BAPTA, the stimulatory effect of low pHi was even more marked, with essentially no inhibition. We conclude that extracellular H+ ions inhibit whereas intracellular H+ ions can stimulate ICa,L. Low pHi and pHo effects on ICa,L were additive, tending to cancel when appropriately combined. They persisted after inhibition of calmodulin kinase II (with KN-93). Effects are consistent with H+ ion screening of fixed negative charge at the sarcolemma, with additional channel block by H+o and Ca2+i. Action potential duration (APD) was also strongly H+ sensitive, being shortened by low pHo, but lengthened by low pHi, caused mainly by H+-induced changes in late Ca2+ entry through the L-type Ca2+ channel. Kinetic analyses of pH-sensitive channel gating, when combined with whole cell modeling, successfully predicted the APD changes, plus many of the accompanying changes in Ca2+ signaling. We conclude that the pHi-versus-pHo control of ICa,L will exert a major influence on electrical and Ca2+-dependent signaling during acid–base disturbances in the heart.

AimsSpontaneous Ca2+ waves in cardiomyocytes are potentially arrhythmogenic. A powerful controller of Ca2+ waves is the cytoplasmic H+ concentration ([H+]i), which fluctuates spatially and temporally in conditions such as myocardial ischaemia/reperfusion. H+-control of Ca2+ waves is poorly understood. We have therefore investigated how [H+]i co-ordinates their initiation and frequency.Methods and resultsSpontaneous Ca2+ waves were imaged (fluo-3) in rat isolated ventricular myocytes, subjected to modest Ca2+-overload. Whole-cell intracellular acidosis (induced by acetate-superfusion) stimulated wave frequency. Pharmacologically blocking sarcolemmal Na+/H+ exchange (NHE1) prevented this stimulation, unveiling inhibition by H+. Acidosis also increased Ca2+ wave velocity. Restricting acidosis to one end of a myocyte, using a microfluidic device, inhibited Ca2+ waves in the acidic zone (consistent with ryanodine receptor inhibition), but stimulated wave emergence elsewhere in the cell. This remote stimulation was absent when NHE1 was selectively inhibited in the acidic zone. Remote stimulation depended on a locally evoked, NHE1-driven rise of [Na+]i that spread rapidly downstream.ConclusionAcidosis influences Ca2+ waves via inhibitory normalHnormali+ and stimulatory Nanormali+ signals (the latter facilitating intracellular Ca2+-loading through modulation of sarcolemmal Na+/Ca2+ exchange activity). During spatial [H+]i-heterogeneity, normalHnormali+-inhibition dominates in acidic regions, while rapid Nanormali+ diffusion stimulates waves in downstream, non-acidic regions. Local acidosis thus simultaneously inhibits and stimulates arrhythmogenic Ca2+-signalling in the same myocyte. If the principle of remote H+-stimulation of Ca2+ waves also applies in multicellular myocardium, it raises the possibility of electrical disturbances being driven remotely by adjacent ischaemic areas, which are known to be intensely acidic.

Timothy syndrome (TS) is an extremely rare human disorder resulting from a single point mutation (G406R) in the intracellular part of the S6 transmembrane segment of domain 1 of Cav1.2. This region is encoded in a mutually exclusive manner by exons 8/8a. The identical glycine to arginine mutation occurs in both the 8a and 8 human splice variants resulting in TS1 and TS2. Heterozygous expression of the TS2 defect is lethal to mice. However, the TS2 defect is tolerated when suppressed though transcriptional interference by the NEO promoter (TS2-NEO mouse). By crossing TS2-NEO with a cardiac-specific tamoxifen-inducible CRE we can upregulate expression of the mutant TS2 calcium channel. We extracted mRNA from cardiac left ventricle and probed with PCR primers to exon 7 and exon 9. Individual PCR products were incorporated into plasmid DNA and individual clones were isolated. This method detected exon 8a:8 ratios in normal mice as 0:58 and 1:56 in TS2-NEO mice suggesting very low level expression of the exon related to TS1. Only 1 in 56 TS2-NEO colonies expressed mutant channels in heart suggesting that the NEO cassette effectively supressed mutant exon 8 isoforms. TS2-NEO mice were crossed with a cardiac specific promoter regulating a CRE recombinase which can be induced with tamoxifen. When activated with tamoxifen (to remove the NEO cassette and suppression of the TS2 mutation) the ratio of mutant to non-mutant exon increased from 1:34 to 29:72. Removal of the NEO cassette induced a strong slowing of inactivation in isolated myocytes. Tamoxifen-induced removal of suppression of the TS2 mutant exon increased QT duration from 33.151.8 ms to 74.055.5 ms (n=5,p<0.01). These results demonstrate the critical role of the L-type calcium channel inactivation in repolarization.

No abstract