Exchanger In Cardiomyocytes Research Articles

Functional coupling of α2-isoform Na+/K+-ATPase and Ca2+ extrusion through the Na+/Ca2+-exchanger in cardiomyocytes

Intracellular Na+ concentration ([Na+]i) is an important regulator of Ca2+ homeostasis in cardiomyocytes through its influence on the Na+/Ca2+-exchanger (NCX). [Na+]i is regulated by the Na+/K+-ATPase (NKA) of which two catalytic isoforms are expressed in cardiomyocytes, α1 and α2. Here we report that the NKA α2-isoform can modulate NCX mediated Ca2+ transport by establishing a shared microdomain of Na+ where [Na+]i can be different from that in the bulk cytosol. Ca2+ transients and the corresponding NCX currents were induced by [Ca2+]i jumps via UV-flash photolysis of caged Ca2+ (DM-nitrophen), while Ca2+ release and uptake by the sarcoplasmic reticulum was blocked pharmacologically. We determined that specific inhibition of 55% of the α2-isoform was obtained with 0.3μM ouabain, whereas 5μM ouabain blocked ≈95% of α2-isoform and in addition, ≈8% of the α1-isoform. Specific inhibition of the α2-isoform caused slowing of Ca2+ extrusion by the NCX. However, this NCX inhibition could not be explained by an increase in global [Na+]i as assessed by the Na+ indicator SBFI, but rather by Na+ microdomains built up by the NKA α2-isoform in the “fuzzy space”. The results suggest that regulation of NKA α2-isoform activity is crucial for the regulation of Ca2+ homeostasis in cardiomyocytes.

A novel class of sodium/calcium exchanger inhibitor: design, synthesis, and structure–activity relationships of 3,4-dihydro-2(1H)-quinazolinone derivatives

Design, synthesis, and structure–activity relationships of 3,4-dihydro-2(1H)-quinazolinone derivatives as inhibitors of the sodium/calcium (Na+/Ca2+) exchanger are discussed. These studies, based on a lead compound 9a, which was identified in our library, involved systematic modification of three regions and revealed that (1) the 3,4-dihydro-2(1H)-quinazolinone having a tertiary amino alkyl side chain at the 3-position is essential for activity, (2) a nonsubstituted phenyl ring is most suitable for high activity, and (3) introduction of a 4-substituted piperidine moiety enhanced the activity, in particular 4-benzylpiperidin-1-yl showed strong inhibitory activity. Based on these SAR studies, a structurally novel and highly potent inhibitor against the Na+/Ca2+ exchanger, 12g (SM-15811), was discovered. In particular, SM-15811 directly inhibited the Na+-dependent Ca2+ influx via the Na+/Ca2+ exchanger in cardiomyocytes with a high potency. The activity was almost two orders more potent than the lead compound 9a and SM-15811 exerted a protective effect against myocardial ischemic reperfusion injury. These Na+/Ca2+ inhibitors could have a therapeutic potential for the treatment of ischemic reperfusion injury.

Discovery of a novel potent Na+/Ca2+ exchanger inhibitor: design, synthesis and structure-activity relationships of 3,4-dihydro-2(1H)-quinazolinone derivatives.

Design, synthesis and structure-activity relationships for 3,4-dihydro-2(1H)-quinazolinone derivatives with the inhibitory activities of the Na(+)/Ca(2+) exchanger are discussed. These studies based on lead compound 1a lead to the discovery of a structurally novel and highly potent inhibitor against the Na(+)/Ca(2+) exchanger 4f (SM-15811), which directly inhibited the Na(+)-dependent Ca(2+) influx via the Na(+)/Ca(2+) exchanger in cardiomyocytes with a high potency.

Alpha-cardiac actin (ACTC) binds to the band 3 (AE1) cardiac isoform.

The band 3 protein is the major integral protein present in the erythrocyte membrane. Two tissue-specific isoforms are also expressed in kidney alpha intercalated cells and in cardiomyocytes. It has been suggested that the cardiac isoform predominantly mediates the anion exchange in cardiomyocytes, but the role of the cytoplasmic domain of the band 3 (CDB3) protein in the cardiac tissue is unknown. In order to characterize novel associations of the CDB3 in the cardiac tissue, we performed the two-hybrid assay, using a bait comprising the region from leu 258 to leu 311 of the erythrocyte band 3, which must also be present in the cardiac isoform. The assay revealed two clones containing the C-terminal region of the alpha-cardiac actin. Immunoprecipitation of whole rat heart using an anti-actin antibody, immunoblotted with anti-human band 3, showed that actin binds to band 3 which was confirmed in the reverse assay. The confocal microscopy showed band 3 in the intercalated discs. Thus, besides the in vivo physical interaction in the Saccharomyces cerevisiae cell, we demonstrated using immunopreciptation that there is a physical association of band 3 with alpha-cardiac actin in cardiomyocyte, and we suggest that the binding occur "in situ," in the intercalated disc, a site of cell-cell contact and attachment of the sarcomere to the plasma membrane.

BDM drives protein dephosphorylation and inhibits adenine nucleotide exchange in cardiomyocytes.

Contractile dysfunction plays a key role in injury sustained by ischemic myocardium at reperfusion, whereas interventions that impede hypercontracture enhance recovery. In permeabilized adult rat cardiomyocytes, the negative inotrope 2,3-butanedione monoxime (BDM; 10-50 mM) inhibited rigor at low MgATP concentration but stimulated net ATP hydrolysis. Hydrolysis was attenuated by H-7, kaempferol, chelerythrine, and genistein. Evidently BDM opposed phosphorylation of both serine/threonine and tyrosine kinase target proteins, either directly or by enhancing protein phosphatase activity, in a futile cycle of ATP hydrolysis independent of cross-bridge cycling. Although 20 mM BDM did not affect the onset of rigor contracture in permeabilized cells at low MgATP, in intact cells exposed to the metabolic inhibitors cyanide and 2-deoxyglucose rigor onset was accelerated, indicating that BDM increases ATP depletion in quiescent cardiomyocytes. Conversely, in cells exposed to the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone, BDM delayed the onset of contracture and hence ATP depletion, consistent with an inhibition of adenine nucleotide movement across the mitochondrial inner membrane. Such effects will limit the value of BDM as a cardioprotective agent at physiological temperature.

A study of the mechanism of action of befol on Ca2+ metabolism in cardiomyocytes using a FURA-2 fluorescent probe

The dynamics of the Ca-response of cardiomyocytes is studied and the efficiency of befol, verapamil, and amiodarone is compared using various experimental models of stimulation of [Ca2+]i. Befol (1–5 μM) is shown to inhibit the caffeine-and strophanthin G-induced rise of [Ca2+]i. Unlike verapamil and amiodarone, befol exhibits no Ca-blocking activity in modeled K-depolarization. It is concluded that the cardiotropic effect of befol is mediated through its primary action on Na+/Ca2+ exchange in cardiomyocytes, while the cardioplegic effect of verapamil and amiodarone is due to their ability to block the slow Ca2+ inward current.

Diamide: Positive inotropic effect in isolated atria and inhibition of Na+/Ca2+ exchange in cardiomyocytes

The influence of frequency of stimulation and external calcium on the positive inotropic response of guinea-pig left atria to diamide and the inhibitory action on Na+/Ca2+ exchange activity of rat cardiomyocytes by this oxidant of sulphhydryl groups have been investigated. Diamide (50-500 microM) induces a concentration-dependent positive inotropic effect which is more pronounced when atria are driven at 1.0 Hz rather than at 0.5 and 0.1 Hz, and are bathed in 2.72 mM rather than in 1.36 mM external calcium. A decrease in the positive inotropic effect at 35 degrees C with respect to 29 degrees C is also observed. In addition, diamide in positive inotropic concentrations (100-300 microM) significantly reduces Na+/Ca2+ exchange activity and cytoplasmic glutathione levels in adult rat cardiomyocytes. The thiol reducing agent dithiothreitol either reverses or prevents diamide effects both in isolated atria and cardiomyocytes, suggesting that the actions of diamide are correlated to its property to oxidize sulphhydryl groups to disulphides. In view of the functional importance of Na+/Ca2+ exchange in myocardial contractility, it is proposed that diamide may increase the heart force of contraction by an inhibition of the sarcolemmal Na+/Ca2+ exchange activity.