Functional Adult Myocardium in the Absence of Na ⁺ -Ca ²⁺ Exchange

Abstract

Abstract-The excitation-contraction coupling cycle in cardiac muscle is initiated by an influx of Ca 2ϩ through voltage-dependent Ca 2ϩ channels. Ca 2ϩ influx induces a release of Ca 2ϩ from the sarcoplasmic reticulum and myocyte contraction. To maintain Ca 2ϩ homeostasis, Ca 2ϩ entry is balanced by efflux mediated by the sarcolemmal Na ϩ -Ca 2ϩ exchanger. In the absence of Na ϩ -Ca 2ϩ exchange, it would be expected that cardiac myocytes would overload with Ca 2ϩ . Using Cre/loxP technology, we generated mice with a cardiac-specific knockout of the Na ϩ -Ca 2ϩ exchanger, NCX1. The exchanger is completely ablated in 80% to 90% of the cardiomyocytes as determined by immunoblot, immunofluorescence, and exchange function. Surprisingly, the NCX1 knockout mice live to adulthood with only modestly reduced cardiac function as assessed by echocardiography. At 7.5 weeks of age, measures of contractility are decreased by 20% to 30%. We detect no adaptation of the myocardium to the absence of the Na ϩ -Ca 2ϩ exchanger as measured by both immunoblots and microarray analysis. Ca 2ϩ transients of isolated myocytes from knockout mice display normal magnitudes and relaxation kinetics and normal responses to isoproterenol. Under voltage clamp conditions, the current through L-type Ca 2ϩ channels is reduced by 50%, although the number of channels is unchanged. An abbreviated action potential may further reduce Ca 2ϩ influx. Rather than upregulate other Ca 2ϩ efflux mechanisms, the myocardium appears to functionally adapt to the absence of the Na ϩ -Ca 2ϩ exchanger by limiting Ca 2ϩ influx. With our current understanding of excitation-contraction coupling, it is almost inconceivable that myocardium could survive in the absence of Na ϩ -Ca 2ϩ exchange activity. Without a vigorous Ca 2ϩ efflux mechanism, cardiac myocytes should Ca 2ϩ overload, leading to a nonfunctional myocardium. Four laboratories, including ours, have reported that global knockout of the Na ϩ -Ca 2ϩ exchanger, NCX1, is embryonic lethal. 3-6 The result is not surprising and the lethality has been attributed to a cardiac phenotype. 3,5 One study, however, indicated that the lethality had an extracardiac origin. 7 Unexpectedly, heart tubes from NCX knockout embryos, dissected at day 9.5, had almost normal Ca 2ϩ transients in response to electrical stimulation. 5 homeostasis. The heart tubes were operating under low stress conditions (1 Hz stimulation at 26°C), however, and did not tolerate interventions (eg, increased stimulation rate) that increased the need for Ca 2ϩ efflux. Additionally, day 9.5 NCX knockout embryos are within 1 day of death and other adaptations of the myocardium have occurred. 8 Thus, embryonic heart tubes are a difficult preparation and not ideal for analysis of NCX ablation.We have now generated, using Cre/loxP technology, mice with a cardiac-specific knockout of NCX1. The exchanger is completely ablated in at least 80% of the myocytes. Strikingly, these mice survive to adulthood with diminished, yet surprisingly good, cardiac function. We...

Mechanism of shortened action potential duration in Na+-Ca2+ exchanger knockout mice

“…The model was downloaded from the CellML web site (www.cellml.org) and was run on a Pentium-based personal computer. Changes in peak current and inactivation kinetics were modeled in the voltage-clamp mode for INCX, ICa, and Ito until we achieved similarity with changes observed in vitro in this and previous studies (13,27,28). The modified currents were then introduced into the model's AP pacing mode, and data were obtained after AP kinetics reached steady state during 1-Hz pacing.…”

“…NCX cardiac-specific KO mice were generated using Cre/Lox technology, as previously described (13). NCX KO mice used for this study were between 8 and 12 wk of age and did not display any gross pathology or signs of heart failure.…”

“…Ten to twenty percent of the myocytes isolated from KO hearts have a WT phenotype (13). To identify and exclude such cells from this study, myocytes used from KO mice were tested for INCX either during rapid application of caffeine (27) or during SR Ca 2ϩ release induced by a brief depolarization (28).…”

“…NCX is therefore viewed as an essential component of cardiac excitation-contraction coupling (ECC), and functional myocardium in the absence of NCX would seem impossible. Nevertheless, cardiac-specific NCX knockout (KO) mice are viable to adulthood with almost normal cardiac performance (13,26). We reported previously (27) that there are two fundamental mechanisms of adaptation to the absence of NCX: 1) a decrease in L-type Ca 2ϩ current (I Ca ) with an increased ECC gain and 2) a shortened and more rapidly repolarizing action potential (AP) that further reduces Ca 2ϩ influx.…”

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

“…[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).…”