Exploring Istaroxime Unknown Effects on Pulmonary Artery Smooth Muscle Cells

Smooth muscle cells (SMCs) demonstrate versatility, transitioning between contractile and proliferative states in response to vascular conditions. These changes are associated with alterations in the expression of Ca2+ handling proteins and intracellular Ca2+ levels. Istaroxime, able to inhibit Na+/K+ ATPase and stimulate SERCA2a, is considered a promising agent for acute heart failure treatment. Moreover, istaroxime inhibits prostate cancer cells migration associated to decreased Store Operated Calcium Entry (SOCE). This study investigates the effects of istaroxime on intracellular Ca2+ dynamics and proliferation of rat pulmonary artery (rPA) SMCs, aiming to uncover its potential role in vascular diseases beyond the cardiac ones. rPASMCs were characterized for α-smooth muscle actin (α-SMA) positivity using western blot and immunofluorescence analysis, ensuring the purity of the cell culture. Drug effects on intracellular Ca2+ dynamics were evaluated using Fluo4-AM for single-cell analysis and Fura2-AM for cell population analysis. Na+/K+ ATPase current (INaK) was assessed as K+out activated current in isolated voltage-clamped rPASMCs (hp -40 mV). Finally, rPASMC proliferation was quantified using the CCK-8 kit. rPASMCs treated for 48 hours with istaroxime (1µM) showed decreased resting Ca2+ levels; opposite effects were observed with the selective SERCA blocker, CPA. Since the selective SERCA2a stimulator PST3093 (the main istaroxime metabolite) did not affect resting Ca2+ levels, a SERCA-independent effect of istaroxime was hypothesized. The drug significantly reduced SOCE, while ATP-induced SR Ca2+ release was unaffected by the drug; moreover, the potency of Na+/K+ ATPase inhibition was comparable to that observed in cardiac preparations. Finally, istaroxime reduced rPASMC proliferation. Overall, these results suggest that istaroxime modulates intracellular Ca2+ dynamics and proliferation of rPASMCs through a SERCA-independent mechanism, possibly through SOCE inhibition. Therefore, istaroxime might represent a new candidate for vascular diseases treatment.