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SERCA (sarco(endo)plasmic reticulum Ca 2ϩ -ATPase) is a well characterized cation transport ATPase (1-6) that is obtained with vesicular fragments of sarcoplasmic reticulum (SR).2 Two Ca 2ϩ are transported from the medium into the vesicles, whereas one ATP is utilized. ATPase activation requires binding of two Ca 2ϩ per enzyme molecule (E 1 ⅐2Ca 2ϩ ) followed by ATP utilization and formation of a phosphoenzyme intermediate (E 1 -P). The free energy derived from ATP is utilized by the phosphoenzyme for a conformational transition (E 1 -P to E 2 -P) that favors translocation and release of the bound Ca 2ϩ against its concentration gradient. The cycle is completed by hydrolytic cleavage of E 2 -P. Ca 2ϩ /H ϩ countertransport and electrogenicity were noted (7-9) with native SR vesicles, but most useful information was obtained with reconstituted proteoliposomes (10, 11) that are not leaky to H ϩ or other electrolytes. It was then possible to demonstrate that, at neutral pH, the stoichiometry of Ca 2ϩ

BACKGROUND AND PURPOSECalcium handling is known to be deranged in heart failure. Interventions aimed at improving cell Ca 2+ cycling may represent a promising approach to heart failure therapy. Istaroxime is a new luso-inotropic compound that stimulates cardiac contractility and relaxation in healthy and failing animal models and in patients with acute heart failure (AHF) syndrome. Istaroxime is a Na-K ATPase inhibitor with the unique property of increasing sarcoplasmic reticulum (SR) SERCA2a activity as shown in heart microsomes from humans and guinea pigs. The present study addressed the molecular mechanism by which istaroxime increases SERCA2a activity. EXPERIMENTAL APPROACHTo study the effect of istaroxime on SERCA2a-phospholamban (PLB) complex, we applied different methodologies in native dog healthy and failing heart preparations and heterologous canine SERCA2a/PLB co-expressed in Spodoptera frugiperda (Sf21) insect cells. KEY RESULTSWe showed that istaroxime enhances SERCA2a activity, Ca 2+ uptake and the Ca 2+ -dependent charge movements into dog healthy and failing cardiac SR vesicles. Although not directly demonstrated, the most probable explanation of these activities is the displacement of PLB from SERCA2a.E2 conformation, independently from cAMP/PKA. We propose that this displacement may favour the SERCA2a conformational transition from E2 to E1, thus resulting in the acceleration of Ca 2+ cycling. CONCLUSIONS AND IMPLICATIONSIstaroxime represents the first example of a small molecule that exerts a luso-inotropic effect in the failing human heart through the stimulation of SERCA2a ATPase activity and the enhancement of Ca 2+ uptake into the SR by relieving the PLB inhibitory effect on SERCA2a in a cAMP/PKA independent way. LINKED ARTICLE AbbreviationsAHF, acute heart failure; CPA, cyclopiazonic acid; +dP/dt, rate of LV pressure rise; −dP/dt, rate of LV pressure decay; LV, left ventricle; PLB, phospholamban; SERCA, sarcoplasmic reticulum Ca 2+

Cisplatin, carboplatin, and oxaliplatin are widely used anticancer drugs. Their efficacy is strongly reduced by development of cell resistance, a phenomenon not entirely understood, with contribution of drug detoxification, defective accumulation, and efflux from the cell. Down-regulation of CTR1, responsible for Cu uptake by the cell, and up-regulation of the Cu-ATPases, ATP7A and ATP7B, which accept Cu from the cytosolic chaperone Atox1 and transfer the metal ion into the secretory pathway where it is incorporated into cuproenzymes, have been associated to augmented drug resistance. To gain information on translocation of Pt drugs by human Cu-ATPases, we performed electrical measurements on COS-1 cell microsomal fraction, enriched with recombinant ATP7A, ATP7B, and selected mutants, adsorbed on a solid supported membrane (SSM). The experimental results demonstrate that Pt drugs activate Cu-ATPases and undergo ATP-dependent translocation with a mechanism identical to that of Cu. We then used NMR spectroscopy and ESI-MS to determine the binding mode of these drugs to the first N-terminal metal binding domain of ATP7A (Mnk1).

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