Istaroxime, a potential anticancer drug in prostate cancer, exerts beneficial functional effects in healthy and diseased human myocardium

The current gold standard for prostate cancer treatment is androgen deprivation therapy and antiandrogenic agents. However, adverse cardiovascular events including heart failure can limit therapeutic use. Istaroxime, which combines Na+-K+-ATPase (NKA) inhibition with sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) stimulation, has recently shown promising anti-neoplastic effects in prostate cancer (PC) models and may also improve cardiac function. Considering the promising anticancer effects of istaroxime, we aimed to assess its functional effects on human myocardium. Results Istaroxime and strophanthidin elicited dose-dependent positive inotropic effects with a decline in developed force at supraphysiological concentrations in human atrial, nonfailing, and failing ventricular (ToF) myocardium. Diastolic force and RT50% did not change after exposure to both drugs. The maximal developed force in our in-vitro model of heart failure (ToF) was significantly higher after istaroxime administration. Such a difference did not occur in atrial or nonfailing ventricular trabeculae and was not applicable to the diastolic force. Materials and Methods Human atrial and ventricular trabeculae were isolated from nonfailing hearts and hearts of infants with tetralogy of Fallot (ToF), which were used as an in-vitro model of heart failure. The samples were electrically stimulated and treated with increasing concentrations of istaroxime and strophanthidin (10 nM–1 μM). Systolic and diastolic force development and relaxation parameters (RT50%) were analyzed. Conclusions Combined NKA inhibition/SERCA2a stimulation increases contractility in atrial, nonfailing, and failing myocardium. Considering that heart failure is a potential side effect of current PC treatments, especially in elderly patients, istaroxime might combine beneficial cardiac and anti-cancer properties.

, respectively (forcefrequency relationship, 0.5 to 3 Hz; FFR). The same was true for istaroxime (0.1 µM). Moreover, with respect to diastolic force, both substances showed no difference versus baseline in the force frequency relationship. Supplementary Figure 3 shows the effect of both substances (in increasing concentrations) on the RT50% (= time from peak tension to 50% relaxation) (A) and the effect of each substance at 0.1 µM is further investigated versus baseline using different pacing frequencies (B and C). Overall, strophanthidin and istaroxime reveal similar functional effects in human atrial myocardium.

Force frequency relationship -non-failing ventricle
Using different pacing frequencies, we further investigated changes in developed and diastolic force in response to strophanthidin (Supplementary Figure Figure 3D shows changes of RT50% with administration of either substance in increasing concentrations. Further investigation of each substance at 0.1µM using different pacing frequencies (Supplementary Figure 3E-3F) revealed no significant difference compared to baseline. Thus, strophanthidin and istaroxime reveal similar functional effects in human ventricular myocardium.

Comprehensive force frequency relationship analysis
In order to identify potential differences between strophanthidin and istaroxime, we took a closer look focusing only on the maximum developed force values presented in the previous FFR figures (S1/S2). We analyzed the maximal developed force recorded after incubation with 0.1 µM istaroxime and 0.1 µM strophanthidin, respectively, regardless of the pacing frequency. Supplementary Figure 4 shows box plots of the maximal developed force, and the corresponding diastolic force and RT50% values, irrespective of the pacing frequency. This representation allows for a better comparison between strophanthidin and istaroxime, and for detection of differences that might otherwise be overlooked. However, analysis of both atrial (Supplementary Figure

Trabeculae preparation
With the help of a stereomicroscope, trabeculae were carefully dissected, transferred to an organ bath and fixed on hooks, stretched to optimal length in tyrode's solution with a Ca 2+ concentration of 2.5 mM and electrically stimulated. Trabeculae were maintained at 37°C. Developed force, diastolic force and RT50% were recorded using a force-transducer (Scientific Instruments, Germany). Tracings were recorded on a thermorecorder and stored digitally for offline analysis.

Statistical analysis
For each separate group (atrium, nonfailing ventricle, failing ventricle (ToF)) we performed a repeated measures analysis of variances (rmANOVA) with all data rank transformed to test the effects on the muscle force parameters of the concentration level (baseline, 0.01, 0.03, 0.1, 0.3, 0.5, 1) as within subjects factor and the two groups (istaroxime and strophanthidin) as between subjects factor. We defined contrasts to test which concentration level differs significantly from baseline in total and within each group. Beyond the main between subject effect of a group difference, we defined contrasts to test at which concentration level the two groups differ significantly and whether there is a significant interaction of group*concentration between two adjacent concentration levels. P-values for contrasts were corrected for multiple comparisons according to Bonferroni. Differences in maximum developed force and differences in diastolic force and RT50% at the concentration level at which the muscle strips attained maximum developed force were assessed with the Wilcoxon rank sum test. P-values less than 0.05 were considered statistically significant. All computations were done using IBM SPSS Statistics (