Article Figures & Data
Figures
- Fig. 1.
The chemical structure of istaroxime and its metabolite PST3093 (A) and PKs in humans (B and C). (A) The main metabolic pathways of istaroxime are reduction of the carbonyl in position 6 catalyzed by carbonyl reductases and oxidative deamination of the primary amino group catalyzed by monoamine oxidases or tissue-bound semicarbazide-sensitive amine oxidase. (B) Istaroxime (N = 22) and PST3093 (N = 29) plasma levels evaluated over 6 hours of istaroxime infusion in HF patients at 1 μg/kg per minute and up to 12 hours after discontinuation of infusion (from HORIZON-HF study, #NCT00616161). (C) Statistics of Cmax, Tmax, AUClast, and T0.5; *P < 0.05 versus istaroxime (unpaired t test). Data are the mean ± S.E.M. ista, istaroxime; PST, PST3093.
- Fig. 2.
Modulation of Na+/K+ ATPase activity. (A) Inhibition of Na+/K+ ATPase activity by istaroxime and PST3093 in dog renal preparations (N = 3). (B) Concentration-response curves for INaK inhibition by PST3093 (n = 15) and istaroxime (modified from Torre et al. (2022); this work is available under a CC-BY-NC license) in rat LV myocytes; INaK recording under increasing concentrations of PST3093 and finally to ouabain (OUA as reference) is shown on the right. Data are the mean ± S.E.M.
- Fig. 3.
Modulation of SERCA2a ATPase activity in healthy and diseased (STZ) preparations. Effect of PST3093 (N = 5–11) (A and B) and istaroxime (N = 5–16) (C and D) on SERCA2a Vmax estimated from Ca2+-dose response curves in cardiac homogenates from healthy and diabetic (STZ) rats. Internal controls (ctr) are provided. Data are the mean ± S.E.M. *P < 0.05 versus ctr (RM one-way ANOVA plus post hoc Tukey’s multiple comparisons test or paired t test).
- Fig. 4.
Modulation of SERCA1 ATPase activity. Concentration dependency of PST3093 and istaroxime modulation of SERCA1 KdCa in guinea pig skeletal muscle microsomes containing SERCA1 alone (A and C) (N = 5–6) and after reconstitution with the PLN1-32 fragment (B and D) (N = 5–6). Data are the mean ± S.E.M. *P < 0.05 (RM or mixed-model one-way ANOVA plus post hoc Tukey’s multiple comparisons).
- Fig. 5.
Modulation of intracellular Ca2+ handling in field-stimulated myocytes from healthy and diseased (STZ) hearts. (A) Representative recordings of CaT triggered by steady-state electrical stimulation at 2 Hz in intact cells, followed after 20 seconds by caffeine-induced Ca2+ release measuring CaSR. PST3093 (1 µM) was tested in normal (healthy) and STZ myocytes. (B) Statistics for CaT amplitude (ampl), CaSR, Carest, and time for 50% CaT decay (t0.5). CTR N = 3 (n = 28 without PST3093; n = 31 with PST3093), STZ N = 4 (n = 28 without PST3093; n = 30 with PST3093). *P < 0.05 (one-way ANOVA plus post hoc Tukey’s multiple comparisons).
- Fig. 6.
Modulation of SR Ca2+ uptake under NCX inhibition in V-clamped myocytes from STZ hearts. (A) Disease (STZ) effect on SR Ca2+ loading in patch-clamped myocytes. SR Ca2+ loading by a train of V-clamp pulses was initiated after caffeine-induced SR depletion; NCX was blocked by Na+ substitution to identify SERCA2a-specific effects (see Methods and Supplemental Fig. 1); myocytes from healthy (N = 9; n = 32) and diseased hearts (N = 6; n = 31) are compared. (B) PST3093 effect in STZ myocytes (N = 4; without PST3093 n = 18, with PST3093 n = 19). Panels from left to right: CaT amplitude, ER gain (the ratio between CaT amplitude and Ca2+ influx through ICaL), and τ of CaT decay. *P < 0.05 for the “interaction factor” in RM two-way ANOVA, indicating a different steepness of curves.
- Fig. 7.
Modulation of electrical activity in guinea pig myocytes. The effect of 100 nM PST3093 was tested on AP parameters and their steady-state rate dependency in guinea pig myocytes (N = 5). (A) representative APs recorded at 0.5, 1, 2, and 4 Hz in control (left) and with 100 nM PST3093 (right). (B) Effect on the rate dependency of diastolic potential (Ediast) and AP duration (APD50, APD90) (n ≥ 24 without PST3093; n ≥ 22 with PST3093). (C) Effect on the τ of APD90 adaptation following a step change in rate (n ≥ 22 without PST3093; n ≥ 16 with PST3093). (D) Effect on the rate dependency of APD90 STV (n ≥ 24 without PST3093; n ≥ 20 with PST3093). (E) Effect on the correlation between STV of APD90 and APD90 values; data from 1, 2, and 4 Hz were pooled. *P < 0.05 for the “interaction factor” of RM two-way ANOVA. The effect of PST3093 at a higher concentration (1 µM) is reported in Supplemental Fig. 3.
- Fig. 8.
Disease (STZ) effects on in vivo echocardiographic parameters. Echocardiographic parameters are compared between healthy rats (N = 18) and 8 weeks after STZ treatment (N = 20). Top row, global function parameters; mid row, systolic function parameters; bottom row, diastolic function parameters: *P < 0.05 (unpaired t test).
- Fig. 9.
PST3093 effects on in vivo echocardiographic parameters in diseased (STZ) rats. PST3093 was intravenously infused (0.22 mg.kg−1.min−1) in rats 8 weeks after STZ treatment. Echocardiographic parameters were measured before and at 15 and 30 minutes during drug infusion. Rows and symbols as in Fig. 8; N = 10 rats. Data are the mean ± S.E.M. *P < 0.05 (RM one-way ANOVA plus post hoc Tukey’s multiple comparisons). Effects of PST3093 on all echocardiographic parameters at 15 minutes of infusion in STZ rats in comparison with istaroxime and digoxin are reported in Table 2.
Tables
Evaluation Experimental Model Scope Pharmacokinetics in humans. Human blood samples from the clinical HORIZON-HF study on istaroxime (Gheorghiade et al., 2008). To assess compatibility of pharmacokinetics with chronic usage. Effects on SERCA2a and Na+/K+ ATPase activities in cell-free preparations (enzymatic assays). Tissue homogenates/microsomes from: dog kidney (Na+/K+ ATPase); rat and guinea pig ventricle (SERCA2a); guinea pig skeletal muscle (SERCA1). To assess efficacy and selectivity in modulating the ATPase proteins relevant to the ino-lusitropic effect.
Demonstration of PLN-SERCA2a interaction as the molecular target.In vitro effects for ligands potentially accounting for off-target actions. High-throughput in vitro ligation to a panel of 50 molecular ligands. To screen for a wide range of interactors potentially involved in off-target effects. Effects on Na+/K+ ATPase current and intracellular Ca2+ dynamics in healthy and diseased myocytes. Ventricular myocytes isolated from healthy and diseased rats with known SERCA2a dysfunction (STZ-diabetic rats). To assess in isolated cells efficacy in restoring the SR function in diseased cardiac myocytes. Effects on electrical activity of healthy myocytes. Ventricular myocytes isolated from guinea pigs, a species with human-like repolarization. To test for potential off-target effects of proarrhythmic relevance. Effects on in vivo hemodynamics of diseased hearts. Healthy and STZ-diabetic rats; echocardiographic evaluation before and during drug infusion. To assess efficacy in reversing disease-induced hemodynamic abnormalities in vivo. In vivo acute toxicity. Healthy mice; evaluation of LD50. To assess drug toxicity in vivo. - TABLE 2
Effects of PST3093 (0.22 mg.kg−1.min−1), istaroxime (0.22 mg.kg−1.min−1), and digoxin (0.11 mg.kg−1.min−1) on echo indexes of STZ rats at 15 minutes of infusion Data are the mean ± S.E.M.
Echo Parameters Istaroxime PST3093 Digoxin Before At 15 Min Before At 15 Min Before At 15 Min Morphometric Parameters IVSTd (mm) 1.91 ± 0.09 1.97 ± 0.16 1.89 ± 0.09 1.86 ± 0.11 1.79 ± 0.07 1.81 ± 0.04 PWTd (mm) 1.6 ± 0.07 1.68 ± 0.09 1.46 ± 0.05 1.47 ± 0.07 1.71 ± 0.08 1.66 ± 0.1 LVEDD (mm) 7.25 ± 0.15 7.1 ± 0.2 7.22 ± 0.17 7.57 ± 0.17 6.76 ± 0.15 6.67 ± 0.15 IVSTs (mm) 2.4 ± 0.1 2.45 ± 0.12 2.16 ± 0.11 2.31 ± 0.13 2.25 ± 0.14 2.31 ± 0.1 PWTs (mm) 2.33 ± 0.16 2.42 ± 0.15 2.43 ± 0.12 2.62 ± 0.14 2.47 ± 0.16 2.68 ± 0.14 LVESD (mm) 3.82 ± 0.21 3.8 ± 0.2 3.89 ± 0.18 3.66 ± 0.15 3.53 ± 0.17 3.14 ± 0.14a Systolic Parameters FS (%) 47.21 ± 2.4 46.72 ± 2.1 46.2 ± 2.02 51.8 ± 1.89a 47.62 ± 1.9 53.09 ± 1.8a s′ (mm/s) 23.37 ± 0.9 23.05 ± 0.7 22.0 ± 0.72 23.4 ± 0.5a 23.5 ± 0.9 26 ± 1.5a EF (%) 83 ± 2 82 ± 62 82 ± 1.9 87 ± 1.5a 84 ± 1.5 88 ± 1.3a Diastolic Parameters E (mm/s) 0.91 ± 0.04 0.94 ± 0.04 0.91 ± 0.06 1.05 ± 0.07a 0.831 ± 0.03 0.924 ± 0.06 A (mm/s) 0.66 ± 0.04 0.78 ± 0.05a 0.79 ± 0.08 0.87 ± 0.07 0.59 ± 0.05 0.79 ± 0.05a E/A 1.39 ± 0.06 1.22 ± 0.08 1.18 ± 0.05 1.22 ± 0.04 1.48 ± 0.11 1.2 ± 0.06a DT (ms) 46.62 ± 4.1 34.12 ± 2.5a 48.5 ± 3.8 40.2 ± 3.0 53.4 ± 3.8 44.1 ± 3.2a DT/E 51.91 ± 4.9 36.66 ± 3.3a 56.7 ± 6.5 40.39 ± 4.8a 65.21 ± 5.3 50.18 ± 5.4a E/DT 21.28 ± 3.2 28.98 ± 2.8a 20.61 ± 3.0 27.51 ± 2.6a 16.53 ± 1.7 22.66 ± 3.0 e′ (mm/s) 21.23 ± 0.99 24.73 ± 0.61a 22.94 ± 1.0 27.01 ± 1.1a 20.9 ± 0.5 23.8 ± 0.9a a′ (mm/s) 27.61 ± 1.9 31.35 ± 1.5a 28.72 ± 2.2 31.06 ± 1.5 25.9 ± 1.9 29.8 ± 1.9 e′/a′ 0.78 ± 0.04 0.79 ± 0.03 0.83 ± 0.06 0.88 ± 0.04 0.85 ± 0.06 0.82 ± 0.04 E/e′ 43.6 ± 2.6 38.29 ± 1.6a 39.5 ± 1.7 38.61 ± 1.5 39.64 ± 0.9 38.95 ± 2.3 Cardiac Function HR (bpm) 266.2 ± 9.8 316.2 ± 7.5a 270 ± 17 271 ± 10 236 ± 12 257 ± 11 SV (ml) 0.71 ± 0.04 0.67 ± 0.05 0.702 ± 0.05 0.847 ± 0.06a 0.589 ± 0.03 0.601 ± 0.04 CO (ml/min) 188.8 ± 10.5 211.2 ± 13.2a 187.7 ± 13.7 231.7 ± 20.2a 138.5 ± 11.2 155.6 ± 14.4 N 8 8 10 10 10 10 aP < 0.05 versus before drug infusion (paired t test).
Data Supplement
- Supplemental Data -
Supplemental Methods
Supplemental Figure S1. Protocol to evaluate intracellular Ca2+ dynamics in patch-clamped cells under Na+ free condition.
Supplemental Figure S2. Analytical characterization of PST3093.
Supplemental Figure S3. Effects of 1 μM PST3093 on electrical activity in guinea-pig myocytes.
Supplemental Table S1. Effect of PST3093 and istaroxime on SERCA2a kinetic parameters in cardiac preparations from healthy guinea-pigs.
Supplemental Table S2. Effect of PST3093 (10 μM) on the panel of molecular targets (Eurofins, Taiwan).
Supplemental Table S3. Characterization of the STZ diabetic rat model.
Supplemental Table S4. Echocardiographic parameters in healthy and STZ diabetic rats.
Supplemental References.
- Supplemental Data -
