Regulation of Cardiac L-Type Ca2+ Current in Na+-Ca2+ Exchanger Knockout Mice: Functional Coupling of the Ca2+ Channel and the Na+-Ca2+ Exchanger

Abstract

L-type Ca2+ current (I(Ca)) is reduced in myocytes from cardiac-specific Na+-Ca2+ exchanger (NCX) knockout (KO) mice. This is an important adaptation to prevent Ca2+ overload in the absence of NCX. However, Ca2+ channel expression is unchanged, suggesting that regulatory processes reduce I(Ca). We tested the hypothesis that an elevation in local Ca2+ reduces I(Ca) in KO myocytes. In patch-clamped myocytes from NCX KO mice, peak I(Ca) was reduced by 50%, and inactivation kinetics were accelerated as compared to wild-type (WT) myocytes. To assess the effects of cytosolic Ca2+ concentration on I(Ca), we used Ba2+ instead of Ca2+ as the charge carrier and simultaneously depleted sarcoplasmic reticular Ca2+ with thapsigargin and ryanodine. Under these conditions, we observed no significant difference in Ba2+ current between WT and KO myocytes. Also, dialysis with the fast Ca2+ chelator BAPTA eliminated differences in both I(Ca) amplitude and decay kinetics between KO and WT myocytes. We conclude that, in NCX KO myocytes, Ca2+-dependent inactivation of I(Ca) reduces I(Ca) amplitude and accelerates current decay kinetics. We hypothesize that the elevated subsarcolemmal Ca2+ that results from the absence of NCX activity inactivates some L-type Ca2+ channels. Modulation of subsarcolemmal Ca2+ by the Na+-Ca2+ exchanger may be an important regulator of excitation-contraction coupling.

Suppression of Early and Late Afterdepolarizations by Heterozygous Knockout of the Na + /Ca 2+ Exchanger in a Murine Model

“…These findings are in line with previous studies in other murine models with altered NCX expression. 16,17 These observations support the hypothesis that reduced Ca 2+ extrusion capacity by reduced NCX activity promotes Ca 2+ -dependent I Ca inactivation by accumulation of Ca 2+ in the dyadic cleft. Thus, next to the genetic suppression of NCX inward current, this direct functional interplay between NCX and I Ca will also contribute to the observed reduction of AP duration and the suppression of EAD in hetKO.…”

“…Indeed, this has been observed in homozygous knockout mice 16,25 and-conversely-a slowed inactivation was observed in homozygous overexpressor mice. 5,17 In our study, we also observed a trend toward accelerated I Ca inactivation in heterozygous KO mice, however, other than in homozygous KO mice, this trend did not reach statistical significance.…”

“…The cells (WT: n=15; hetKO: n=17) were dialyzed via the pipette solution with BAPTA (10 mmol/L) similar to previously reported studies. 16,17 Under these conditions, there was no significant difference in I Ca amplitude (maximum I Ca amplitude; in pA/pF: WT, 9.7±0.6; hetKO, 9.1±0.8; P=0.57 hetKO n.s. Figure 4.…”

“…[16][17][18] We therefore measured I Ca in voltage clamped cardiomyocytes in ruptured patch configuration. Cells were clamped at −40 mV to inhibit I Na and then depolarized by square wave pulses (400 ms) ranging from −30 to +40 mV ( Figure 7A To test, whether enhanced Ca 2+ -dependent inactivation of I Ca is responsible for the observed reduction of I Ca in hetKO, we repeated the measurements of I Ca in the presence of the Ca 2+ buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA; Figure 7C).…”

“…This observation is supported by previous findings in a model with homozygous (complete) knockout of NCX. 16,25 Further evidence for this adaptive mechanism has been obtained in a model with increased NCX expression in which-complementary to NCX knockout mice-I Ca is increased, 5,17 thereby preventing cellular Ca 2+ loss because of increased Ca 2+ removal by increased NCX activity.…”