Transcriptional pathways and potential therapeutic targets in the regulation of Ncx1 expression in cardiac hypertrophy and failure.

Changes in cardiac gene expression contribute to the progression of heart failure by affecting cardiomyocyte growth, function, and survival. The Na(+)-Ca(2+) exchanger gene (Ncx1) is upregulated in hypertrophy and is often found elevated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. Several transcriptional pathways mediate Ncx1 expression in pathological cardiac remodeling. Both α-adrenergic receptor (α-AR) and ß-adrenergic receptor (ß-AR) signaling can play a role in the regulation of calcium homeostasis in the cardiomyocyte, but chronic activation in periods of cardiac stress contributes to heart failure by mechanisms which include Ncx1 upregulation. Our studies have even demonstrated that NCX1 can directly act as a regulator of "activity-dependent signal transduction" mediating changes in its own expression. Finally, we present evidence that histone deacetylases (HDACs) and histone acetyltransferases (HATs) act as master regulators of Ncx1 expression. We show that many of the transcription factors regulating Ncx1 expression are important in cardiac development and also in the regulation of many other genes in the so-called fetal gene program, which are activated by pathological stimuli. Importantly, studies have revealed that the transcriptional network regulating Ncx1 expression is also mediating many of the other changes in genetic remodeling contributing to the development of cardiac dysfunction and revealed potential therapeutic targets for the treatment of hypertrophy and failure.

New modes of exchanger regulation: physiological implications.

Exchange activity is regulated principally by cytosolic Na+, Ca2+, and PIP2. However, the properties of these modes of regulation that have emerged from excised patch studies appear to be poorly suited to regulating exchange activity on a beat-to-beat basis. Here we summarize recent findings from our lab indicating that (a) allosteric activation by Ca2+ exhibits hysteresis, (b) elevated concentrations of cytosolic Na+ induce a mode of activity that no longer requires regulatory Ca2+ activation, and (c) the requirement for PIP2 is reduced or eliminated after allosteric Ca2+ activation. Our results suggest that exchange activity in cardiac myocytes may be regulated by the time-integral of Ca2+ transients occurring over multiple beats.

Sodium-dependent inactivation of sodium/calcium exchange in transfected Chinese hamster ovary cells.

High concentrations of cytosolic Na(+) ions induce the time-dependent formation of an inactive state of the Na(+)/Ca(2+) exchanger (NCX), a process known as Na(+)-dependent inactivation. NCX activity was measured as Ca(2+) uptake in fura 2-loaded Chinese hamster ovary (CHO) cells expressing the wild-type (WT) NCX or mutants that are hypersensitive (F223E) or resistant (K229Q) to Na(+)-dependent inactivation. As expected, 1) Na(+)-dependent inactivation was promoted by high cytosolic Na(+) concentration, 2) the F223E mutant was more susceptible than the WT exchanger to inactivation, whereas the K229Q mutant was resistant, and 3) inactivation was enhanced by cytosolic acidification. However, in contrast to expectations from excised patch studies, 1) the WT exchanger was resistant to Na(+)-dependent inactivation unless cytosolic pH was reduced, 2) reducing cellular phosphatidylinositol-4,5-bisphosphate levels did not induce Na(+)-dependent inactivation in the WT exchanger, 3) Na(+)-dependent inactivation did not increase the half-maximal cytosolic Ca(2+) concentration for allosteric Ca(2+) activation, 4) Na(+)-dependent inactivation was not reversed by high cytosolic Ca(2+) concentrations, and 5) Na(+)-dependent inactivation was partially, but transiently, reversed by an increase in extracellular Ca(2+) concentration. Thus Na(+)-dependent inactivation of NCX expressed in CHO cells differs in several respects from the inactivation process measured in excised patches. The refractoriness of the WT exchanger to Na(+)-dependent inactivation suggests that this type of inactivation is unlikely to be a strong regulator of exchange activity under physiological conditions but would probably act to inhibit NCX-mediated Ca(2+) influx during ischemia.

Sodium-calcium exchange does not require allosteric calcium activation at high cytosolic sodium concentrations.

The activity of the cardiac Na(+)-Ca(2+) exchanger (NCX1.1) is allosterically regulated by Ca(2+), which binds to two acidic regions in the cytosolically disposed central hydrophilic domain of the NCX protein. A mutation in one of the regulatory Ca(2+) binding regions (D447V) increases the half-activation constant (K(h)) for allosteric Ca(2+) activation from approximately 0.3 to > 1.8 microm. Chinese hamster ovary cells expressing the D447V exchanger showed little or no activity under physiological ionic conditions unless cytosolic [Ca(2+)] was elevated to > 1 microm. However, when cytosolic [Na(+)] was increased to 20 mm or more (using ouabain-induced inhibition of the Na(+),K(+)-ATPase or the ionophore gramicidin), cells expressing the D447V mutant rapidly accumulated Ca(2+) or Ba(2+) when the reverse (Ca(2+) influx) mode of NCX activity was initiated, although initial cytosolic [Ca(2+)] was < 100 nm. Importantly, the time course of Ca(2+) uptake did not display the lag phase that reflects allosteric Ca(2+) activation of NCX activity in the wild-type NCX1.1; indeed, at elevated [Na(+)], the D447V mutant behaved similarly to the constitutively active deletion mutant Delta(241-680), which lacks the regulatory Ca(2+) binding sites. In cells expressing wild-type NCX1.1, increasing concentrations of cytosolic Na(+) led to a progressive shortening of the lag phase for Ca(2+) uptake. The effects of elevated [Na(+)] developed rapidly and were fully reversible. The activity of the D447V mutant was markedly inhibited when phosphatidylinositol 4,5-bisphosphate (PIP2) levels were reduced. We conclude that when PIP2 levels are high, elevated cytosolic [Na(+)] induces a mode of exchange activity that does not require allosteric Ca(2+) activation.

Calcium-dependent regulation of calcium efflux by the cardiac sodium/calcium exchanger.

Allosteric regulation by cytosolic Ca2+ of Na(+)/Ca2+ exchange activity in the Ca2+ efflux mode has received little attention because it has been technically difficult to distinguish between the roles of Ca2+ as allosteric activator and transport substrate. In this study, we used transfected Chinese hamster ovary cells to compare the Ca2+ efflux activities in nontransfected cells and in cells expressing either the wild-type exchanger or a mutant, Delta(241-680), that operates constitutively; i.e., its activity does not require allosteric Ca2+ activation. Expression of the wild-type exchanger did not significantly lower the cytosolic Ca2+ concentration ([Ca2+](i)) compared with nontransfected cells. During Ca2+ entry through store-operated Ca2+ channels, Ca2+ efflux by the wild-type exchanger became evident only after [Ca2+](i) approached 100-200 nM. A subsequent decline in [Ca2+](i) was observed, suggesting that the activation process was time dependent. In contrast, Ca2+ efflux activity was evident under all experimental conditions in cells expressing the constitutive exchanger mutant. After transient exposure to elevated [Ca2+](i), the wild-type exchanger behaved similarly to the constitutive mutant for tens of seconds after [Ca2+](i) had returned to resting levels. We conclude that Ca2+ efflux activity by the wild-type exchanger is allosterically activated by Ca2+, perhaps in a time-dependent manner, and that the activated state is briefly retained after the return of [Ca2+](i) to resting levels.