Uncovering Istaroxime effects on Pulmonary Artery Smooth Muscle Cells

Smooth muscle cells (SMCs) exhibit plasticity, transitioning between contractile and proliferative phenotypes in response to vascular diseases. Changes correlate with altered expression of Ca2+ handling proteins and intracellular Ca2+ levels1. In cardiac preparations, istaroxime, known for inhibiting Na+/K+ ATPase and stimulating SERCA2a, is considered a promising luso-inotropic agent for acute heart failure treatment2. Moreover, it reduces prostate tumor cell migration by decreasing Store Operated Ca2+ entry (SOCE)3. This project investigates istaroxime effects on intracellular Ca2+ dynamics in rat pulmonary artery (PA) SMCs (rPASMCs), aiming to uncover its potential role in vascular other than cardiac diseases. rPASMCs were characterized for their α-smooth muscle actin (α-SMA) positivity using western blot and immunofluorescence analysis, thus ensuring cell culture purity. Effects on intracellular Ca2+ dynamics were assessed by using Fluo4-AM (single cell analysis) or Fura2-AM (cell population analysis) dye; in particular, SOCE was evaluated in isolated cells, while resting Ca2+ levels and ATP-induced sarcoplasmic reticulum (SR) Ca2+ release through a cell population analysis. Na+/K+ ATPase current (INaK) was evaluated in isolated V-clamped rPASMCs. Finally, rPASMCs proliferation was analyzed with CCK-8 kit. Isolated cells from PA were positive to α-SMA, thus confirming their SMC nature. Chronic treatment (48 hrs) with istaroxime (1µM) decreased resting intracellular Ca2+ levels, an effect opposite to that of CPA, a selective SERCA blocker. Given that the selective SERCA2a stimulator PST3093 (main istaroxime metabolite) did not affect resting Ca2+, we hypothesized a SERCA-independent effect of istaroxime on rPASMCs. The drug significantly reduced SOCE, a finding potentially explaining the drug effects on Ca2+ levels. ATP-induced SR Ca2+ release was instead not altered by the drug and the potency of INaK inhibition was comparable to that observed in cardiac preparations. Furthermore, rPASMCs proliferation was reduced in the presence of istaroxime. Overall, these findings suggest that istaroxime modulates intracellular rPASMCs Ca2+ dynamics and proliferation possibly via a mechanism involving SOCE inhibition. Consequently, istaroxime may represent a promising candidate for vascular diseases treatment.