Subcellular mechanisms underlying the low cardiotoxicity of istaroxime

RACIOPPI, MARÍA FLORENCIA; BURGOS, JUAN IGNACIO; MORELL, MALENA; PETROFF, MARTÍN VILA; GONANO, LUIS ALBERTO

Congreso; Reunión anual de la Sociedad Argentina de Hipertensión Arterial (SAHA); 2021

INTRODUCTION Pharmacological blockade of Na+/K+ ATPase (NKA) with cardiac glycosides (digitalis) has extensively been used as a therapy for patients suffering heart failure given its inotropic effect. However, the use of digitalis like digoxin was strongly reduced because there is no evidence of reduction of mortality in clinical trials, and because of its toxicity potential that includes life-threatening arrhythmias 1. Istaroxime is the first-in-class original luso-inotropic agent, shown to be effective and safe in patients2. Structurally it is a steroideal compound non related with glycosides, which has a dual effect that combines NKA blocking capacity with a potentiation of Ca2+ uptake into the sarcoplasmic reticulum (SR) mediated by the SR Ca2+ ATPase isoform 2a (SERCA2a).This peculiar combination makes istaroxime an inotropic-lusitropic agent, and seems to confer istaroxime a better safety profile compared to other NKA inhibitors, with a low risk of Ca2+ triggered arrhythmias reported 3.We have previously shown that low therapeutic concentrations of digitalis can promote Ca2+-calmodulin dependent kinase II (CaMKII) activation and apoptotic cardiomyocyte death4. Interestingly, istaroxime has been shown to have an antiproliferative capacity in tumoral cell lines5, and similar to other NKA inhibitors, is proposed as an antitumoral drug6. However, its impact on myocyte viability and apoptosis has not been previously reported. Our aim was to test if therapeutic concentrations of istaroxime have a significant impact on adult cardiomyocyte viability.The currently accepted mechanism for the lusitropic effect and the low arrhythmogenicity of istaroxime is its capacity to dissociate the SERCA from its inhibitory protein Phospholamban (PLB)7, but if this is the exclusive mechanism supporting istaroxime´s improved safety has not been fully explored. In this context, an additional aim of this work is to gain insight into these mechanistic aspects comparing the impact exerted by istaroxime and ouabain in cardiomyocytes lacking PLB (PLB-KO myocytes).OBJECTIVES To assess if therapeutic concentrations of istaroxime have a significant impact on adult cardiomyocyte viability and to gain insight into the mechanisms that explain the different safety profile exerted by istaroxime and classic NKA inhibitors.METHODS Rat cardiac myocytes isolation and culture: Male Wistar rat cardiac ventricular myocytes were isolated by enzymatic digestion of Langendorff perfused hearts. For culture, isolated cells were re-suspended in M199 medium and then plated into culture dishes for 1 h to allow cell attachment. After this period, the culture media was changed for a fresh one in the presence of the drugs. After 24 h of culture at 37 ° C, the cells were photographed and evaluated morphologically, being classified as viable or nonviable according to their length-to-width ratio (> 3 were considered viable). Caspase-3 activity positive cells were quantified as a marker of apoptosis activation.Cell shortening measurements: Ventricular cardiomyocytes were paced at 1 Hz and superfused with Hepes-buffer in the presence of ouabain 2 µM or istaroxime 10 µM. Cell shortening was registered by video edge-detection.Detection of spontaneous Ca2+ release: Ca2+ spark and wave frequency were measured by confocal microscopy in Fluo-4 loaded myocytes after incubation for 1 hour with the indicated drugs. Inmunodetection by Western blot analysis: Homogenates were prepared from the ventricular cells isolated from the Langendorff-perfused hearts. These cells were also incubated for 1 hour with the drugs previously mentioned. CAMKII activity (p-CAMKII), phosphorylation of CAMKII substrate threonine-17 (Thr17) of phospholamban (PLN) and the apoptotic index BAX/BCL-2 were quantified by western blot.Statistical analysis: Statistical analysis was performed by ANOVA or T Test followed by post hoc-test. Data are expressed as means ± SEM. Differences were considered significant at p ≤ 0.05. RESULTS In cardiomyocytes paced at 1 Hz and perfused with Hepes-buffer in the presence of ouabain 2 µM or istaroxime 10 µM, both drugs promoted a similar increase in cell shortening after 5 minutes. Therefore, we continued our experiments with these equi-inotropic concentrations. After 24 hours of culture at 37 ° C in the presence and absence of 2 µM ouabain and 10 and 20 µM istaroxime, , we count the following percentages of cell viability: Control without treatment 52 ± 2.5%; Ouabain 2 µM 33 ± 3%; Istaroxime 10 and 20 µM 46 ± 3 and 42 ± 6% respectively (Fig 1). We observed a significant reduction in cell viability accompanied by an increase in caspase-3 activity in ouabain-treated cells. Interestingly, 10 µM and even 20 µM istaroxime did not significantly affect cell viability and caspase-3 activity compared to control without drugs (n=5 cultures per group. p