TABLE 1

Synopsis of the studies

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.