Istaroxime stimulates SERCA2a and accelerates calcium cycling in heart failure by relieving phospholamban inhibition

Abstract

Background and Purpose

Calcium handling is known to be deranged in heart failure. Interventions aimed at improving cell Ca²⁺ 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 Approach

To 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 Results

We showed that istaroxime enhances SERCA2a activity, Ca²⁺ uptake and the Ca²⁺-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 Ca²⁺ cycling.

Conclusions and Implications

Istaroxime 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 Ca²⁺ uptake into the SR by relieving the PLB inhibitory effect on SERCA2a in a cAMP/PKA independent way.

Introduction

Two overarching pathologic features underlie the clinical findings in heart failure (HF): (i) an inotropic abnormality resulting in diminished systolic emptying (systolic failure) and (ii) a compliance abnormality in which the ability of the ventricles to accept blood is impaired (diastolic dysfunction). Most of these functional alterations are associated with disturbances in excitation–contraction coupling underlined by alterations in Ca²⁺ handling (Bers, 2002). The pivotal role of sarcoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA2a) in Ca²⁺ homeostasis is well recognized (Minamisawa et al., 1999; Bers, 2006). It improves SR Ca²⁺ loading during diastole making more Ca²⁺ available during systole and reducing cytosolic Ca²⁺ favouring relaxation. Data consistently indicate decreased levels/activity of SERCA2a in the failing heart (Del Monte et al., 1999; Bers, 2006). Therefore, the impaired efflux of cytosolic Ca²⁺ sustained by defects in SR Ca²⁺ loading and release represents molecular target(s) where pharmacological intervention may result not only in inotropic, but also in lusitropic activity. Indeed, recent reports have shown that targeting SERCA2a by gene transfer represents a novel therapeutic approach to improve diastolic and systolic function in HF (Kawase et al., 2008), with the challenges associated to the application of gene therapy into clinical practice. Thus, molecules that enhance SERCA2a function, in a cAMP-independent manner, represent a highly promising hypothesis to improve luso-inotropic performance in HF.

Istaroxime is an original luso-inotropic agent endowed of a double mechanism of action, stimulating SERCA2a activity and inhibiting Na-K ATPase (Micheletti et al., 2002; 2007; Rocchetti et al., 2005; 2008). Istaroxime has two primary functional effects: improves cellular Ca²⁺ cycling, favouring cytosolic Ca²⁺ accumulation during systole (inotropism), as well as rapid SR Ca²⁺ sequestration during diastole, yielding a lusitropic response, without enhancing spontaneous Ca²⁺ efflux from SR (Rocchetti et al., 2005; Micheletti et al., 2007; Sabbah et al., 2007). This allows improved systolic and diastolic performance and efficiency of contraction (low oxygen consumption for any level of cardiac work), minimizing the risk of arrhythmias or ischemia (Rocchetti et al., 2003; Alemanni et al., 2011). Istaroxime mechanism has been investigated in cardiac SERCA2a-enriched preparations or in dog kidney purified Na-K ATPase (Micheletti et al., 2002), and in isolated cardiac myocytes (Micheletti et al., 2007). Istaroxime efficacy in stimulating SERCA2a ATPase activity has been demonstrated in healthy and failing guinea pig and human SR heart preparations (Micheletti et al., 2007). Notably, in failing preparations, where the SERCA2a activity is reduced compared with healthy hearts, istaroxime reactivates SERCA2a approaching normal levels (Micheletti et al., 2007). In isolated guinea pig (Rocchetti et al., 2005) and mouse cardiac myocytes (Alemanni et al., 2011), istaroxime stimulates twitch amplitude and relaxation by increasing Ca²⁺ transients and accelerating Ca²⁺ re-uptake into SR through SERCA2a.

Although the ability of istaroxime to stimulate SERCA2a ATPase activity has been clearly substantiated, it has not yet been clarified whether istaroxime directly accelerates the enzyme turnover or relieves phospholamban (PLB) inhibitory activity by displacing the interaction between SERCA2a and PLB. Here, we approached these questions by extending previous observations to dog, a species with a sensitivity to Na-K ATPase inhibition by cardiac glycosides and SR Ca²⁺ handling similar to humans, and where the therapeutic efficacy of istaroxime in chronic HF models has been clearly demonstrated (Adamson et al., 2003; Mattera et al., 2007; Sabbah et al., 2007). By applying different experimental set-ups, we demonstrated that (i) istaroxime stimulatory activity on SERCA2a translates in a Ca²⁺ uptake increase into cardiac SR vesicles; (ii) istaroxime exerts such activity only when PLB is co-expressed with SERCA2a; and (iii) the specific interaction of istaroxime with SERCA2a/PLB complex results in displacement of PLB from SERCA2a, independently from cAMP/PKA. Therefore, istaroxime represents the first example of a therapeutic small molecule able to stimulate SERCA2a ATPase activity and to enhance Ca²⁺ uptake into SR by relieving PLB inhibitory effect on SERCA2a, independently from cAMP/PKA.

Methods

Canine SERCA2a and PLB expression in Sf21 insect cells

The canine variant of SERCA2a and PLB cDNA were subcloned in the donor plasmid pFastBac (Invitrogen, Carlsbad, CA, USA), and recombinant baculoviruses were produced after transfection of Sf21 insect cells using the Bac-to-Bac system (Invitrogen). SERCA2a and PLB were expressed in Sf21 cells grown in suspension at 27°C in Sf-900 II SFM medium (Invitrogen) supplemented with 5% fetal bovine serum. Microsomes were isolated from insect cells harvested 72 h after infection.

Animal models

All the experiments were carried out according to the guidelines of the Prassis Institute for Animal Care, approved by the Italian Ministry of Health and complied with European Directive 86/609, and with the Italian Law (DL116, 27 January 1992). Animals were monitored by a veterinarian.

Male Beagle dogs (Harlan, Monza, Italy; 14–18 kg) were used for obtaining cardiac tissues for SERCA2a-enriched SR preparations. Healthy (n = 8) and failing hearts (n = 6) were utilized. Chronic HF was induced in dogs in the General Pharmacology Department of Sigma-Tau, Rome, Italy, as described (Sabbah et al., 1991; 2007; Adamson et al., 2003). Chronic left ventricle (LV) systolic and diastolic dysfunction and HF were induced by multiple intracoronary micro-embolizations with polystyrene latex microspheres (45–90 μm; Polysciences, Warrington, PA, USA), resulting in reduced LV ejection fraction (<35%), increased LV end-diastolic pressure and LV end-diastolic volume, decreased cardiac output, reduced maximum rate of LV pressure rise (+dP/dt) and pressure decay (−dP/dt), and increased pulmonary artery wedge pressure and systemic vascular resistance, as reported (Sabbah et al., 1991; 2007; Adamson et al., 2003). Anaesthetized dogs (Zoletil 100®; Virbac, Milan, Italy) were killed with an intravenous lethal dose of Tanax® (Intervet, Milan, Italy). Hearts were excised, LV was isolated, infarcted tissue was discarded and viable myocardium was divided in portions and frozen.

Male rabbits (New Zealand White, Charles River, Italy; 3 kg, n = 10) were used for SERCA1-enriched SR preparations. Anaesthetized animals (ketamine, 50 mg·kg⁻¹ intramuscularly) were killed, and fast-twitch hind leg muscles were excised and frozen.

SR vesicle isolation

Dog LV tissues were used for SERCA2a-enriched SR preparations while rabbit muscles for vesicles containing SERCA1. Tissues were homogenized, as described (Nediani et al., 1996), in 10 mM NaHCO₃, pH 7, 1 mM PMSF, 10 μg·mL⁻¹ aprotinin and leupeptin, and centrifuged at 12 000× g. Contractile proteins were extracted with 0.6 M KCl, 30 mM histidine, pH 7 and centrifuged at 100 000× g. Pellets were reconstituted with 300 mM sucrose, 30 mM histidine, pH 7.4 and stored in aliquots. Ten dog cardiac preparations were made both from healthy and failing heart tissues and used for SERCA2a ATPase activity, Ca²⁺ uptake and electrical measurements. Two preparations of skeletal microsomes were obtained and used for current measurements.

Microsomes were isolated from Sf21 cells expressing canine SERCA2a and PLB, as described (Mahaney et al., 2000). Sf21 cells were washed with 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄, pH 7.4, centrifuged at 1500 r.p.m. and resuspended in 10 mM NaHCO₃, 0.5 mM pefabloc, 0.4 μM aprotinin, 10 μM leupeptin, 18 μM bestatin, 7.5 μM pepstatin A, and 7 μM E-64. After homogenization, samples were centrifuged at 9000 r.p.m. and supernatants were centrifuged at 26 000 r.p.m. Pellets were resuspended in 250 mM sucrose, 30 mM histidine, pH 7.4 and stored in aliquots.

SERCA2a ATPase activity measurement

SERCA2a activity was measured in microsomes as ³²P-ATP hydrolysis, as described (Micheletti et al., 2007) and identified as 10 μM cyclopiazonic acid (CPA)-inhibited fraction (Seidler et al., 1989). Istaroxime (0.0001–100 nM) was preincubated with microsomes for 5 min at 4°C. Ca²⁺ activation curves were fitted to sigmoidal curves, and maximum velocity (V_max) and Ca²⁺ affinity [Kd(Ca²⁺) ] determined.

To evaluate the PKA dependence of istaroxime stimulatory effect on SERCA2a, staurosporin (10–1000 nM) was added without or with 100 nM istaroxime during the preincubation and SERCA2a activity measured.

Ca²⁺ uptake measurements

SERCA2a-mediated Ca²⁺ uptake into dog cardiac SR vesicles was measured by two independent methods: (i) using ⁴⁵Ca as a tracer at steady state after 10 min of incubation and (ii) by a stopped-flow method for 60 s to measure the initial fast phase of Ca²⁺ uptake.

• SR vesicles were preincubated for 10 min at 4°C, without or with 50 nM istaroxime, in 100 mM KCl, 5 mM MgCl₂, 50 mM 3-(N-morpholino) propane sulfonic acid (MOPS), 5 mM Na-oxalate, 1 mM EGTA, pH 7, free Ca²⁺ concentrations (range 0.1–3 μM) and 0.14 μCi of ⁴⁵CaCl₂ (21.7 mCi·mg⁻¹; Perkin Elmer, Monza, Italy), as described (Nediani et al., 1996). Mg-ATP (5 mM) was added and Ca²⁺ uptake continued for 10 min at 37°C and stopped by automatic filtration. Filters were washed with 150 mM NaCl and radioactivity measured. SERCA2a-dependent Ca²⁺ uptake was identified as 10 μM CPA-inhibited fraction.

• Cardiac SR vesicles were suspended in 100 mM KCl, 1 mM MgCl₂, 50 μM arsenazo III, 5 mM Na-azide, 20 mM MOPS, pH 7.4 and 0.19 or 2 μM free Ca²⁺ concentrations, as described (Tocchetti et al., 2007) without or with 100 nM istaroxime and incubated for 15 min. Vesicles were mixed with 1 mM Na₂ ATP in a manually operated stopped-flow apparatus (Applied Photophysic Ltd., Leatherhead, Surrey, UK). Ca²⁺ concentration changes were monitored at 0.1 s intervals for 60 s by a single-beam UV-VIS spectrophotometer (AVIV model 14DS; Lakewood, NJ, USA) with monochromator setting at 650 nm. The kinetic parameters for Ca²⁺ uptake were evaluated by fitting stopped-flow signals using non-linear regression analysis.

Electrical measurements

Charge movements were measured by adsorbing microsomes from dog LV containing SERCA2a, or rabbit skeletal muscle containing SERCA1, onto an alkanethiol/phospholipid bilayer anchored to a gold electrode [solid supported membrane (SSM); Pintschovius and Fendler, 1999; Tadini-Buoninsegni et al., 2004]. Once adsorbed, microsomes were activated by rapid injection of ATP. If the ATP concentration jump induces charge displacement within the protein, a current transient can be recorded (Tadini-Buoninsegni et al., 2006; 2008a). We precise that SSM technique detects pre-steady state current transients within the first catalytic cycle and is not sensitive to stationary currents following the first cycle (Tadini-Buoninsegni et al., 2006; 2008a; 2010). In ATP concentration jump experiments, the buffer solution contained 150 mM choline chloride, 25 mM histidine, pH 7, 0.25 mM EGTA, 1 mM MgCl₂, 1 mM DTT and 0.25 mM CaCl₂ (10 μM free Ca²⁺). SERCA2a and SERCA1 activation was obtained by addition of 100 μM ATP. To investigate istaroxime effect on charge movements, 100 nM istaroxime was added and incubated for 3 min. ATP-induced current transients without and with istaroxime were compared. To prevent Ca²⁺ accumulation into microsomes, 1 μM calcium ionophore A23187 (calcimycin) was used.

Charge measurements were performed by the SURFE²R^One device (Nanion Technologies, Munich, Germany). The temperature was maintained at 23°C.

To verify the reproducibility of the current transients on the same SSM, each measurement was repeated six times and then averaged to improve signal-to-noise ratio. Standard deviations did not exceed 5%. Each set of measurements was usually reproduced using four to six different gold sensors.

Co-immunoprecipitation of SERCA2a with PLB

Dog cardiac SR vesicles were incubated as described for SERCA2a activity with Ca²⁺ (0.1-1-5 μM) and without or with istaroxime (1-10-100 nM). The samples were then mixed with 40 mM Hepes-NaOH, pH 7.5, 300 mM NaCl, 2 mM EDTA, 4 mM PMSF, 1% Tween 20 (Sigma-Aldrich, Milan, Italy) and centrifuged, as described (Asahi et al., 1999). The supernatant was mixed with G-Sepharose/PLB monoclonal antibody (clone A1, specific epitope between PLB residues 7–16), rotated and centrifuged. The pellet was washed three times with 20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA and 0.5% Tween 20 (Sigma-Aldrich), treated with sample buffer, centrifuged and the supernatant used for immunoblotting.

Western blot

Samples were separated by SDS-PAGE (4–12% Bis-Tris Criterion BIO-RAD gels, Milan, Italy), blotted and incubated with primary antibodies, followed by incubation with secondary antibodies labelled with fluorescent markers and quantified by Odyssey Infrared Imaging System (LI-COR, Milan, Italy). Antibodies: anti-SERCA2 (N-19) and anti-Thr¹⁷ PLB (Santa Cruz Biotechnology, Dallas, TX, USA); anti-PLB, clone A1 (Upstate Millipore, Milan, Italy); anti-Ser¹⁶ PLB (Millipore); anti-actin (Sigma-Aldrich).

Statistical analysis

Data are mean ± SEM. Free Ca²⁺ concentrations were calculated with WinMAXC program (Stanford University, Stanford, CA, USA). Ca²⁺ dose–response curves were calculated by a sigmoidal curve fitting software (Synergy Software KaleidaGraph 3.6; Reading, PA, USA), obtaining SERCA2a Kd(Ca²⁺) and V_max. Statistical analysis was performed by one-way anova and paired or unpaired t-test analysis, as specified. P < 0.05 was considered statistically significant.

Chemicals

Istaroxime {PST2744: [E,Z]-3-[(2-aminoethoxy)imino]-androstane-6,17-dione hydrochloride} was synthesized and developed at Prassis Research Institute and Sigma-Tau Pharmaceutical Company (Micheletti et al., 2002).

Results

Istaroxime stimulates SERCA2a ATPase activity and Ca²⁺ uptake into cardiac SR vesicles

We compared the kinetics of SERCA2a activity and its expression in cardiac SR vesicles from dog healthy and failing LV. Cardiac preparations from healthy dogs showed significantly higher V_max and Kd(Ca²⁺) (Figure 1A, Supporting Information Table S1) than dogs with failing hearts (Figure 1B, Supporting Information Table S2). The reduced SERCA2a ATPase activity of failing heart vesicles was associated with a decreased protein expression of SERCA2a (−20%) and monomeric (−21%), but not pentameric, PLB and a lower Ser¹⁶ phosphorylation of monomeric (−64%) and pentameric PLB (−48%), as measured by immunoblotting in two different preparations of healthy and failing hearts (Figure 1D). Actin did not differ between the two samples (not shown).

Figure 1.

Effect of istaroxime on SERCA2a ATPase activity and Ca²⁺ uptake into dog cardiac SR vesicles. (A) Ca²⁺ activation curves of SERCA2a ATPase activity were measured as CPA insensitive component in cardiac SR microsomes from healthy dogs in the absence (control, open circles) and presence of 100 nM istaroxime (closed circles; n = 22 independent experiments of Ca²⁺ activation curves). The kinetic parameters, maximum velocity (V_max) and calcium affinity [Kd(Ca²⁺)] of the Ca²⁺ activation curves were determined by a sigmoidal fitting. The data are reported in the text. (B) Ca²⁺ activation curves of SERCA2a ATPase activity were measured in dog failing heart microsomes in the absence (control, open circles) and presence of 1 nM istaroxime (closed circles; n = 20 experiments). (C) Ca²⁺ activation curves of SERCA2a ATPase activity were measured in cardiac SR microsomes from healthy dogs in the absence (control, open circles) and presence of 100 nM digoxin (closed circles; n = 6 experiments). (D) Western blot analysis for SERCA2a and monomeric (m) and pentameric (p) un-phosphorylated and Ser¹⁶ phosphorylated (pSer¹⁶) PLB in one representative microsome preparation from dog healthy and failing hearts (10 μg protein/lane). Standard molecular weights are indicated on the left. (E) SERCA2a-dependent Ca²⁺ uptake into cardiac SR vesicles from healthy dogs was measured using ⁴⁵Ca as a tracer in the absence (control, open circles) and presence (closed circles) of 50 nM istaroxime (n = 11 experiments of Ca²⁺ activation curves). (F) Representative stopped-flow recordings of active Ca²⁺ uptake into cardiac SR vesicles from healthy dogs monitored at 650 nm at 0.19 μM free Ca²⁺ in the absence (control) and presence of 100 nM istaroxime. The curves were fitted to a biexponential equation. Kinetic parameters of Ca²⁺ uptake monitored at 0.19 μM (n = 8 time course experiments) and 2 μM free Ca²⁺ (n = 8 time course experiments) are reported in Table 3.

The effect of istaroxime (0.0001–100 nM) on SERCA2a-mediated Ca²⁺ activation curves was then measured and compared in dog healthy and failing cardiac SR vesicles. The compound significantly increased SERCA2a V_max in both preparations, starting from 0.1 nM (Tables 1 and 2). The maximum effect on SERCA2a V_max was achieved at 100 nM in healthy (+28%, P < 0.01; Figure 1A, Table 1) and at 1 nM (+34%, P < 0.01) in failing cardiac SR vesicles (Figure 1B, Table 2). Istaroxime effect on Ca²⁺ activation curves in dog healthy and failing cardiac SR vesicles was also expressed as percentage increase versus the respective control at all the free Ca²⁺ concentrations, showing that it was statistically significant in the low (0.3–0.5 μM) and high range of Ca²⁺ (1–3 μM) in both cardiac preparations (Supporting Information Tables S1 and S2). Even though the kinetic analysis of Ca²⁺ activation curves did not detect a statistically significant effect of istaroxime on SERCA2a Kd(Ca²⁺) in healthy and failing heart vesicles, these findings imply that istaroxime may exert its stimulatory activity also at low Ca²⁺ concentrations, as previously demonstrated in guinea pig SR vesicles where istaroxime significantly reduced SERCA2a Kd(Ca²⁺) (Micheletti et al., 2007).

Table 1.

Effect of istaroxime on the kinetic parameters of the Ca²⁺-dependent activity curves in SR microsomes from healthy dog hearts

Healthy	Kd(Ca2+), μM	Vmax, μmol·min−1·mg−1
Control	0.651 ± 0.073	1.013 ± 0.054
0.0001 nM istaroxime	0.667 ± 0.091	1.079 ± 0.07
0.001 nM istaroxime	0.675 ± 0.074	1.135 ± 0.071
0.01 nM istaroxime	0.753 ± 0.093	1.164 ± 0.059
0.1 nM istaroxime	0.715 ± 0.079	1.220 ± 0.068*
1 nM istaroxime	0.731 ± 0.076	1.244 ± 0.065*
10 nM istaroxime	0.713 ± 0.086	1.281 ± 0.077**
100 nM istaroxime	0.703 ± 0.09	1.293 ± 0.078**

Data are mean ± SEM of 22 independent experiments of Ca activation curves in the absence (control) and presence of increasing concentrations of istaroxime. The statistical significance among control and istaroxime was measured by one-way anova.

^*P < 0.05,

^**P < 0.01 istaroxime versus control.

Table 2.

Effect of istaroxime on the kinetic parameters of the Ca²⁺-dependent activity curves in SR microsomes from failing dog hearts

Failing	Kd(Ca2+), μM	Vmax, μmol·min−1·mg−1
Control	0.376 ± 0.024$$	0.736 ± 0.044$$
0.0001 nM istaroxime	0.436 ± 0.053	0.787 ± 0.052
0.001 nM istaroxime	0.458 ± 0.037	0.827 ± 0.055
0.01 nM istaroxime	0.413 ± 0.033	0.821 ± 0.046
0.1 nM istaroxime	0.482 ± 0.04	0.95 ± 0.053*
1 nM istaroxime	0.439 ± 0.036	0.987 ± 0.056**
10 nM istaroxime	0.41 ± 0.04	0.953 ± 0.055*
100 nM istaroxime	0.436 ± 0.05	0.872 ± 0.056

Data are mean ± SEM of 20 independent experiments of Ca activation curves in the absence (control) and presence of increasing concentrations of istaroxime. The statistical significance among control and istaroxime was measured by one-way anova. The statistical significance of Kd(Ca²⁺) and V_max between failing and healthy control was measured by t-test analysis.

^*P < 0.05,

^**P < 0.01 istaroxime versus control;

^$$P < 0.01 failing versus healthy.

Digoxin (100 nM), a reference compound known as a selective Na-K ATPase inhibitor (Katz et al., 2010), with an IC₅₀ of 0.45 μM on dog renal Na-K ATPase (Micheletti et al., 2002), failed to stimulate SERCA2a activity and did not affect SERCA2a Kd in SR vesicles from healthy dogs (n = 6 experiments; Figure 1C).

To verify whether the stimulatory effect of istaroxime on SERCA2a V_max activity was paralleled by a direct effect on Ca²⁺ movements, SERCA2a-dependent Ca²⁺ uptake was measured into cardiac SR microsomes from healthy dogs with two different methodologies: (i) using ⁴⁵Ca as a tracer over a period of incubation of 10 min; and (ii) by a stopped-flow method using arsenazo III to monitor the fast phase of Ca²⁺ removal from the extravesicular compartment over 60 s of incubation.

• SERCA2a-mediated ⁴⁵Ca uptake into cardiac SR vesicles was fitted by a sigmoidal curve (Figure 1E). Istaroxime (50 nM) significantly increased ⁴⁵Ca uptake V_max (+22%, n = 11 experiments, P < 0.05) without affecting Kd (control 700 ± 4 nM, + istaroxime 715 ± 29, n = 11 experiments; Figure 1E).

• SERCA2a-dependent time course of Ca²⁺ uptake into cardiac SR vesicles measured by a stopped-flow method was fitted to a biphasic curve (Figure 1F), with a very fast (k1 from 0.41 to 0.55·s⁻¹) and a slower (k2 from 0.066 to 0.091·s⁻¹) component (Table 3). Incubation with 100 nM istaroxime significantly increased Ca²⁺ uptake mediated by the fast and slow uptake phases measured both in the range of low (0.19 μM, n = 8 experiments) and high (2 μM, n = 8 experiments) free Ca²⁺ concentrations (Figure 1F, Table 3).

Table 3.

Effect of istaroxime on the kinetic parameters for Ca²⁺ uptake into cardiac SR vesiscles from healthy dogs by a stopped-flow method

Free Ca2+ 0.19 μM	k1 (s−1)	A1	k2 (s−1)	A2
Control N = 8	0.4117 ± 0.0379	0.003308 ± 0.00046	0.066 ± 0.0096	0.00474 ± 0.00022
+ Istar N = 8	0.5854 ± 0.0351*	0.003483 ± 0.00023	0.0841 ± 0.0067*	0.00376 ± 0.00012

Free Ca2+ 2 μM	k1 (s−1)	A1	k2 (s−1)	A2
Control N = 8	0.5523 ± 0.0449	0.00282 ± 0.000158	0.0911 ± 0.0060	0.00357 ± 0.00019
+ Istar N = 8	0.7348 ± 0.0036*	0.00341 ± 0.000258	0.1187 ± 0.0103*	0.00407 ± 0.00017

Data are mean ± SEM of absorbance at 650 nm measured in the absence (control) and presence of 100 nM istaroxime at 0.19 and 2 μM free Ca²⁺ concentrations. N, number of replicates of time course experiments; k1, rate constant for fast phase of Ca²⁺ uptake; k2, rate constant for slow phase of Ca²⁺ uptake; A1, amplitude of fast phase of Ca²⁺ uptake; A2, amplitude of slow phase of Ca²⁺ uptake. The statistical significance between control and istaroxime was measured by t-test analysis.

^*P < 0.05 istaroxime versus control.

These data demonstrate that chronic HF in dog is characterized by an impaired activity and expression of SERCA2a, as already reported for human HF (Hasenfuss, 1998). In this setting, istaroxime displays its stimulatory effect on SERCA2a ATPase activity at lower concentrations in SR vesicles from failing than from healthy hearts from dogs and this effect translates into a fast Ca²⁺ uptake into native cardiac SR vesicles, in line with previous findings obtained in guinea pig and human cardiac preparations (Rocchetti et al., 2005; Micheletti et al., 2007).

Istaroxime increases the ATP-induced charge movements within a single catalytic cycle of SERCA2a

To confirm that the stimulatory effect of istaroxime on SERCA2a activity translates into a functional effect on Ca²⁺ movement, we applied an experimental method to measure charge transfer within the first catalytic cycle of SERCA2a across the SR membrane (Tadini-Buoninsegni et al., 2006; 2008b; Bartolommei et al., 2011; Lewis et al., 2012). Native SR SERCA2a from dog healthy hearts was characterized by pre-steady state charge measurements on an SSM. As shown in Figure 2A (dashed line), a current transient was observed after ATP addition in the presence of 10 μM free Ca²⁺ (n = 6 measurements). In analogous experiments with native SR SERCA1 from rabbit skeletal muscle, the charge obtained by numerical integration of the ATP-induced current transient was attributed to an electrogenic event corresponding to vectorial displacement of bound Ca²⁺ at the end of the first cycle following ATP utilization.

Figure 2.

Charge measurements in dog cardiac SR vesicles. (A) Current transients after 100 μM ATP jumps on cardiac SR microsomes from healthy dogs in the presence of 10 μM free Ca²⁺ (dashed line), in the absence of Ca²⁺ (1 mM Mg²⁺ only, solid line) and in the presence of 100 nM TG (dotted line). Current transients are the average of six measurements on the same SSM (see Methods). Inset: Normalized charges related to ATP jumps in the presence of Ca²⁺ (white column), in the absence of Ca²⁺ (grey column) and in the presence of Ca²⁺ and 100 nM TG (black column). The charges were normalized to the maximum charge attained in the presence of 10 μM free Ca²⁺. Normalized charges are mean ± SEM of six independent experiments. (B,C) Current transients after 100 μM ATP jumps on cardiac SR microsomes (SERCA2a/PLB) from healthy dogs incubated with (B) 10 μM free Ca²⁺ or (C) without Ca²⁺. Charge measurements were performed in the absence (solid lines) or presence of 100 nM istaroxime (dashed lines). Current transients are the average of six consecutive measurements on the same SSM. (D) Current transients after 100 μM ATP jumps on rabbit skeletal muscle SERCA1 in the absence (solid line) or presence of 100 nM istaroxime (dashed line). The curves are the average of six measurements on the same SSM. (E) Normalized charges related to ATP jumps on cardiac SR microsomes (SERCA2a/PLB) from healthy dogs and on rabbit skeletal muscle SR vesicles (SERCA1) in the absence (white and black columns) or presence of 100 nM istaroxime (grey columns). The charges were normalized to the charge attained in the presence of 10 μM free Ca²⁺ (white columns). Normalized charges are mean ± SEM of four independent experiments. The statistical significance between control and istaroxime was measured by t-test analysis. *P < 0.05 istaroxime versus control.

We then performed an ATP jump in the absence of Ca²⁺ and presence of 1 mM Mg²⁺ and we observed a reduced current transient (n = 6 measurements; Figure 2A, solid line), whose related charge was approximately 50% of that obtained in the presence of Ca²⁺ (Figure 2A, inset). The total charge measured in the presence of Ca²⁺ was inhibited ∼50% by thapsigargin (TG; n = 6 measurements; Figure 2A, inset), a specific SR Ca²⁺-ATPase inhibitor (Sagara and Inesi, 1991). Therefore, the TG-insensitive current peak (Figure 2A, dotted line), which corresponds to the current transient measured in the presence of Mg²⁺ alone (Figure 2A, solid line), may be presumably related to other ATPases present in cardiac SR vesicles.

Subsequently, we investigated the effects of istaroxime on charge movements generated by SERCA2a in canine SR vesicles. We found that 100 nM istaroxime, added to the same SSM prepared for control measurements, increased the peak current obtained after the ATP jump in the presence of Ca²⁺ (Figure 2B). In particular, the addition of istaroxime produced an ∼20% increase of the total charge measured in dog SR vesicles in the presence of Ca²⁺ (n = 4 experiments, P < 0.05; Figure 2E) in agreement with the effects obtained with biochemical measurements of SERCA2a activity and Ca²⁺ uptake. Istaroxime had no effect on the TG-insensitive and Ca²⁺-independent current signal (Figure 2C, E) and on charge movements generated by SERCA1 from rabbit skeletal muscle (n = 4 experiments; Figure 2D, E).

These results indicate that istaroxime enhances Ca²⁺ translocation through SERCA2a/PLB-enriched vesicles, but not in skeletal SERCA1 vesicles devoid of PLB.

SERCA stimulatory effect of istaroxime depends on the presence of PLB

Previous data indicate that istaroxime exerts its stimulatory effect on native cardiac SR vesicles expressing SERCA2a/PLB, but not on skeletal muscle vesicles expressing SERCA1 alone. This implies that istaroxime displays its activity by specifically interacting with and/or displacing PLB from SERCA2a/PLB complex. To further investigate this aspect, canine SERCA2a was overexpressed alone, or with PLB, in Sf21 cells. Ca²⁺-dependent activation curves on microsomes from Sf21 cells expressing SERCA2a alone (Figure 3A) were analysed by a sigmoidal fitting (Figure 3B) and showed a significantly (P < 0.001) higher Ca²⁺ affinity (Kd 280 ± 24 nM, n = 6 experiments) and V_max (0.71 ± 0.022 μmol·min⁻¹·mg⁻¹, n = 6 experiments) as compared with cells co-expressing SERCA2a with PLB (Figure 3C; Kd 451 ± 24 nM, V_max 0.24 ± 0.03 μmol·min⁻¹·mg⁻¹, n = 19 experiments; Figure 3D). As shown by immunoblotting, SERCA2a protein expression of these two samples was comparable (∼20% of total proteins; Figure 3A, C), indicating that the difference in V_max was due to the presence of PLB. In Sf21 cells expressing canine SERCA2a alone, istaroxime did not modify either the V_max or Kd (Figure 3B). Conversely, in Sf21 cells co-expressing canine SERCA2a and PLB, istaroxime (0.0001–100 nM) significantly enhanced SERCA2a V_max, but not Kd, starting from 0.01 nM (Supporting Information Table S3). Figure 3D shows the effect of 100 nM istaroxime.

Figure 3.

Effect of istaroxime on canine SERCA2a co-expressed with PLB in Spodoptera frugiperda (Sf21) insect cells. (A,B) Canine SERCA2a was overexpressed without PLB in Sf21 cells and microsomes were prepared. (A) Western blot analysis of Sf21 microsomes expressing canine SERCA2a alone. Molecular weights are reported on the right. (B) Ca²⁺-dependent activity curves in Sf21 microsomes expressing SERCA2a without PLB were measured in the absence (control, open circles) and presence (closed circles) of 100 nM istaroxime and analysed by a sigmoidal fitting. Data are mean ± SEM of six experiments of Ca²⁺-dependent activation curves. Kinetic parameters (V_max, Kd) are shown in the text. (C,D) Microsomal preparations of Sf21 cells expressing canine SERCA2a with PLB. (C) Western blot analysis of Sf21 microsomes indicating the presence of SERCA2a and monomeric (m) and pentameric (p) PLB. Molecular weights are reported on the right. (D) Ca²⁺-dependent activity curves in Sf21 microsomes expressing SERCA2a/PLB were measured in the absence (control, open circles) and presence (closed circles) of 100 nM istaroxime and analysed by a sigmoidal fitting. Data are mean ± SEM of 19 experiments of Ca²⁺-dependent activation curves. Kinetic parameters (V_max, Kd) are shown in the text.

These data further indicate that istaroxime increased the maximum rate of SERCA2a ATPase activity, but did not change the Kd, in microsomial preparations from Sf21 cells only when canine SERCA2a was co-expressed with PLB (Figure 3D), as found in native dog cardiac SR preparations (Figure 1A). Conversely, the compound does not affect SERCA1 (Figure 2D, E) or SERCA2a when expressed without PLB (Figure 3B).

Istaroxime activates SERCA2a in a cAMP/PKA independent way

All the previous finding converges towards the hypothesis that istaroxime activates SERCA2a by favouring its de-inhibition via PLB detachment. Under physiological conditions, SERCA2a inhibition by PLB is relieved through the phosphorylation of PLB by PKA at Ser¹⁶ and by calmodulin-kinase at Thr¹⁷ (Asahi et al., 1999; Karim et al., 2006; Traaseth et al., 2006; Bidwell et al., 2011; James et al., 2012), leading to a partial dissociation of PLB cytoplasmic domain from SERCA2a and SERCA2a functional activation. To verify whether the stimulation of SERCA2a activity by istaroxime might be mediated through PKA activation, rather than on a direct PLB displacement from SERCA2a/PLB complex, Ca²⁺-dependent SERCA2a ATPase activity was measured in cardiac SR vesicles from healthy dogs at increasing concentrations of the PKA inhibitor staurosporin (10-100-1000 nM) in the absence and presence of 100 nM istaroxime. At the highest concentration, staurosporin modestly decreased SERCA2a activity, although the trend was not statistically significant. At any dose, staurosporin affected neither istaroxime-mediated stimulation of SERCA2a V_max, which remained persistently activated (range from +24 to +27%, n = 4 experiments, P < 0.05; Figure 4A), nor Kd (control 710 ± 70 nM, + istaroxime 690 ± 40 nM, n = 4 experiments). Accordingly, the immunoblotting of dog SR vesicles incubated with 100 nM istaroxime did not show any change of Ser¹⁶ phosphorylation of the monomeric and pentameric PLB, compared with the control (Figure 4B).

Figure 4.

Effect of PKA inhibition by staurosporin on istaroxime-induced activation of SERCA2a. (A) Ca²⁺-dependent activation curves of SERCA2a ATPase activity were measured in SR microsomes from healthy dogs in the absence and presence of 100 nM istaroxime. The effect of addition of PKA inhibitor, staurosporin, at 10, 100, 1000 nM was evaluated. Kinetic parameters (V_max, Kd) have been determined by fitting sigmoidal curves to the data and are reported in the text. The figure shows the V_max of the Ca²⁺ activation curves. Data are mean ± SEM of four experiments of Ca²⁺-dependent activation curves. The statistical significance between control and istaroxime was measured by paired t-test. *P < 0.05 istaroxime versus control. (B) Effect of istaroxime on PLB Ser¹⁶ phosphorylation. Dog cardiac microsomes were incubated in the absence (control) and presence of 100 nM istaroxime as indicated in SERCA2a ATPase activity and analysed by Western blotting for SERCA2a and PLB Ser¹⁶ phosphorylation (pSer¹⁶) of its monomeric (m) and pentameric (p) form. The immunoblotting is one representative of three independent experiments.

These results suggest that istaroxime stimulates SERCA2a activity by a cAMP/PKA independent mechanism.

Istaroxime stimulates SERCA2a activity by dissociating SERCA2a/PLB complex

Because istaroxime appears to stimulate SERCA2a activity independently from cAMP/PKA and only when SERCA2a/PLB complex is expressed, we finally investigated whether istaroxime may induce a physical disruption of SERCA2a/PLB interaction in co-immunoprecipitation experiments from cardiac SR vesicles of healthy dogs. Using a PLB monoclonal antibody, specific for an epitope between PLB residues 7–16, we verified that increasing free Ca²⁺ concentrations from 0.1 to 5 μM decreased the amount of SERCA2a co-immunoprecipitated with PLB (−35%, n = 6 experiments, P < 0.01; Figure 5A), but did not completely abolish SERCA2a/PLB interaction, as already published (Asahi et al., 1999). Then, we tested the effect of 100 nM istaroxime at 0.1-1-5 μM free Ca²⁺ concentrations. The compound significantly reduced the amount of SERCA2a co-immunoprecipitated with PLB at the free Ca²⁺ concentration of 0.1 μM (−37%, n = 6 experiments, P < 0.01), but not at 1 and 5 μM (Figure 5A). The concentration-dependent effect of 1-10-100 nM istaroxime on SERCA2a co-immunoprecipitation was evaluated at 0.1 μM free Ca²⁺ concentration (Figure 5B). Istaroxime significantly reduced SERCA2a co-immunoprecipitation in a concentration-dependent manner by 22% (n = 12, P < 0.05), 40% (n = 20, P < 0.01) and 43% (n = 16, P < 0.01), respectively, at 1-10-100 nM istaroxime, compared with controls without compound (n = 29; Figure 5B). The reduction of co-immunoprecipitation obtained with 100 nM istaroxime at 0.1 μM Ca²⁺ (Figure 5B) was comparable with that obtained in the presence of 5 μM Ca²⁺ alone (Figure 5A).

Figure 5.

Co-immunoprecipitation of SERCA2a with PLB. (A) Effect of Ca²⁺ on the interaction between SERCA2a and PLB. Dog cardiac microsomes were incubated with increasing free Ca²⁺ concentrations (0.1-1-5 μM) in the absence and presence of 100 nM istaroxime. Co-immunoprecipitation of SERCA2a with PLB was initiated by addition of anti-PLB antibody (clone A1, epitope between PLB residues 6–17). The amount of SERCA2a in the co-immunoprecipitated sample was revealed by Western blot by anti-SERCA2a antibody. Data are mean ± SEM of six independent co-immunoprecipitation experiments. The statistical significance among control and istaroxime concentrations was measured by one-way anova. **P < 0.01 plus versus minus istaroxime; ^$$P < 0.01 5 μM free Ca²⁺ versus 0.1 μM free Ca²⁺. (B) Concentration-dependent effect of istaroxime on the interaction between SERCA2a and PLB at 0.1 μM free Ca²⁺. Dog cardiac microsomes were incubated with istaroxime (1-10-100 nM). Co-immunoprecipitation of SERCA2a with PLB was performed by addition of anti-PLB antibody. SERCA2a in the co-immunoprecipitated sample was revealed by Western blot by anti-SERCA2a antibody. Data are mean ± SEM; control n = 29; istaroxime: 1 nM, n = 12; 10 nM, n = 20; 100 nM, n = 16; n represents the number of co-immunoprecipitation experiments. The statistical significance among control and istaroxime concentrations was measured by one-way anova. *P < 0.05, **P < 0.01 istaroxime versus control.

Collectively, these findings indicate that istaroxime activates SERCA2a by a partial disruption (−43%) of the physical interaction between SERCA2a and PLB, which occurs through a cAMP/PKA independent mechanism.

Discussion and conclusions

The present study furnishes consistent data on the molecular mechanism of istaroxime, a new luso-inotropic agent under clinical development for the treatment of acute heart failure (AHF), shown to be highly effective and safe both in animal models and AHF patients (Sabbah et al., 2007; Gheorghiade et al., 2008; Shah et al., 2009).

By applying different methodologies, we showed that istaroxime, at nanomolar concentrations, stimulates SERCA2a activity and Ca²⁺ uptake through a direct interaction with SERCA2a/PLB complex, independently from cAMP/PKA and PLB phosphorylation. Istaroxime significantly stimulated the maximum rate of SERCA2a ATPase activity both in dog healthy and failing heart vesicles. More interestingly, in the latter, where SERCA2a ATPase activity was significantly reduced as compared with healthy preparations, istaroxime re-establishes a SERCA2a activity close to that of healthy preparations. Furthermore, istaroxime exerts its maximum activation in SR vesicles from dog failing hearts at lower concentrations (1 nM) than in healthy heart SR vesicles (100 nM), suggesting a higher potency of the drug in the altered cardiac failing condition. The stimulatory effect of istaroxime in dog healthy and failing heart vesicles was statistically significant both in the low (0.3–0.5 μM) and high (1–3 μM) Ca²⁺ concentration range. This confirms previous published data in human and guinea pig SR preparations (Rocchetti et al., 2005; Micheletti et al., 2007) and indicates that istaroxime can be specifically effective in the physiological range of low diastolic Ca²⁺ concentrations resulting in improvement of lusitropy in HF. Similarly, the compound enhanced SERCA2a-dependent Ca²⁺ uptake into SR cardiac vesicles from healthy dogs as measured by (i) ⁴⁵Ca as a tracer at steady state; (ii) a stopped-flow method, permitting the time-course evaluation for the fast and slow uptake kinetics within 60 s; and (iii) measurements of SERCA2a-generated current transients on a SSM, related to vectorial displacement of bound Ca²⁺ upon ATP utilization within a single catalytic cycle.

Several experimental evidences have indicated that istaroxime-mediated SERCA2a stimulation appears to be dependent on the presence of PLB, the physiological SERCA2a regulatory protein. Indeed, istaroxime did not affect SERCA ATPase activity and SERCA-generated charge movements using skeletal SERCA1, devoid of PLB, or when canine SERCA2a was expressed in Sf21 cells without PLB. However, istaroxime enhancement of SERCA2a activity observed in native dog SR preparations was reproduced in microsomes from Sf21 cells co-expressing heterologous recombinant canine SERCA2a and PLB.

Multiple cytosolic and transmembrane interaction sites between PLB and SERCA2a have been reported (Chen et al., 2006). The physical interaction and the functional inhibition of SERCA2a by PLB are reversed by PLB phosphorylation or high Ca²⁺ (Karim et al., 2006; Traaseth et al., 2006; Bidwell et al., 2011; James et al., 2012; Winther et al., 2013). PLB phosphorylation seems to induce only a partial physical dissociation of PLB cytoplasmic domain from SERCA2a (Asahi et al., 1999; Karim et al., 2006) or an order-to-disorder conformational transition of PLB cytoplasmic domain (Traaseth et al., 2006). Membrane lipids further modulate this transition (Gustavsson et al., 2011). However, it has not been uniformly demonstrated whether the relief of PLB inhibition by Ca²⁺ induces, or not, a complete dissociation of PLB from SERCA2a (Asahi et al., 1999; Bidwell et al., 2011).

Although the data reported here do not allow to fully elucidate the molecular mechanism of istaroxime, it is reasonable to assume that it involves targeting within a pocket generated by SERCA2a/PLB protein–protein interaction or a direct binding to one or more sites on PLB. As a result of this interaction, it may be expected that istaroxime favours the dissociation of PLB from SERCA2a leading to the relief of SERCA2a inhibition. The effects of istaroxime and high Ca²⁺ on the dynamics of SERCA2a/PLB interaction were studied in co-immunoprecipitation experiments from cardiac SR preparations of healthy dogs. It was shown that at low Ca²⁺ (0.1 μM), known to stabilize SERCA2a.E2 conformation, istaroxime maximally reduced (−43%, P < 0.01) the amount of SERCA2a co-immunoprecipitated with PLB. Analogously, saturating Ca²⁺ (5 μM) reduced the co-immunoprecipitation of SERCA2a with PLB (−35%, P < 0.01), confirming Asahi's results (Asahi et al., 1999). Although a direct demonstration is lacking, the present data let us to speculate that istaroxime preferentially binds to SERCA2a.E2, the conformation that is stabilized by low Ca²⁺ and that is bound and inhibited by unphosphorylated PLB, favouring SERCA2a.E2 to E1 conformational transition that results in the relief of PLB inhibition and in the acceleration of Ca²⁺ cycling.

Indeed, cross-linking and molecular modelling studies led to different proposals for PLB and SERCA interaction (Toyoshima et al., 2003; Chen et al., 2006). One model predicts that during SERCA.E2 to E1. Ca²⁺ conformational transition, SERCA and the N-terminal helix of PLB cytoplasmic domain undergo changes that alter the cross-linking of PLB with SERCA (Toyoshima et al., 2003; Zamoon et al., 2005). Alternatively, it has been proposed that in the presence of Ca²⁺, the binding of PLB to SERCA2a transmembrane domain may induce a conformational change, so that the helical structure extending above the Ca²⁺ binding cavity in SERCA.E2 is converted to an extended conformation in E1 state (Traaseth et al., 2006). The key point of divergence of the two models is whether PLB dissociates from SERCA during systole or remains bound as a subunit of Ca²⁺ pump.

The present data with istaroxime seem to indicate that the compound dissociates the physical interaction between SERCA2a and PLB. The same was observed in the presence of high Ca²⁺. However, an alternative interpretation is possible. To reconcile the two opposite theories with the results obtained here in the co-immunoprecipitation experiments, it can be postulated that istaroxime, rather than simply displacing PLB from SERCA2a, may induce an ‘ordered’ conformation conversion of PLB cytoplasmic domain that becomes stabilized by the interaction with the membrane surface lipids, as demonstrated (Gustavsson et al., 2011). This may make the site less accessible to the anti-PLB antibody. The result of this conformational change would be a reduced amount of SERCA2a/PLB complex in the co-immunoprecipitate. A similar reduction of SERCA2a/PLB complex in the co-immunoprecipitate was observed in the presence of high Ca²⁺. The question of whether the molecular mechanism and the conformational changes of SERCA2a/PLB complex through which istaroxime exerts its effect are similar to those mediated by high Ca²⁺ remain to be elucidated.

Several reports indicate that an altered cAMP/PKA signalling can contribute to chronic cardiac negative remodelling and failure (Mann and Bristow, 2005). Therapies favouring PLB phosphorylation (isoproterenol, phosphodiesterase inhibitors) have been developed to increase cardiac contractility in HF (Mann and Bristow, 2005). Although they result effective in rescuing the patients from the acute syndrome in the short term, their long-term effects have been questioned both in terms of efficacy and safety considerations (Packer et al., 1991; Cuffe et al., 2002; Mann and Bristow, 2005). A new agent able to improve cardiac function by activating SERCA2a in a cAMP/PKA independent way is therefore highly desirable. Istaroxime that stimulates SERCA2a, independently from cAMP-PKA, represents to our knowledge the first and unique example of a new class of luso-inotropic agent able to improve cardiac contraction–relaxation cycle in failing hearts (Sabbah et al., 2007; Gheorghiade et al., 2008; Shah et al., 2009) through a specific reduction of SERCA2a/PLB protein–protein interaction.

Glossary

AHF

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 Ca²⁺ ATPase

Sf21

Spodoptera frugiperda

SR

sarcoplasmic reticulum

SSM

solid supported membrane

TG

thapsigargin

Conflicts of interest

At the time of the experiments, MF, PB, IM, MGT, CR and PF were employees of Prassis Sigma-Tau Research Institute, Settimo Milanese, Milan, Italy. All the other authors state no conflict of interests.

Supporting information

Additional Supporting Information may be found in the online version of this article at the publisher's web-site:

Table S1 Effect of istaroxime at increasing free Ca²⁺ concentrations on SERCA2a ATPase activity of SR microsomes from dog healthy hearts.

Table S2 Effect of istaroxime at increasing free Ca²⁺ concentrations on SERCA2a ATPase activity of SR microsomes from dog failing hearts.

Table S3 Effect of istaroxime on SERCA2a ATPase activity of SR microsomes from Sf21 cells co-expressing canine SERCA2a and PLB.