Beyond the Heart: Istaroxime, a Novel Candidate against Vascular Smooth Muscle Cell Remodeling

Pulmonary arterial hypertension (PAH) is a progressive and life-threatening vascular disorder characterized by increased pulmonary vascular resistance, structural remodeling of the pulmonary arteries, right ventricular overload, and ultimately, heart failure. A hallmark of PAH pathogenesis is the maladaptive phenotypic transition of pulmonary artery smooth muscle cells (PASMCs) from a contractile to a proliferative state. This process is driven by disrupted intracellular Ca²⁺ homeostasis, impaired sarcoplasmic reticulum (SR) function, and activation of Ca²⁺-dependent signaling pathways, particularly the calcineurin–NFAT axis. Despite advances in therapy, current treatments remain largely symptomatic and fail to reverse vascular remodeling, highlighting the need for innovative pharmacological strategies targeting the underlying cellular mechanisms. Istaroxime is a luso-inotropic compound developed for the treatment of acute heart failure, acting through a dual mechanism that combines stimulation of the SR Ca²⁺-ATPase isoform 2a (SERCA2a) with inhibition of the Na⁺/K⁺-ATPase. Istaroxime thus improves cardiac contractility and relaxation, stimulating Ca²⁺ reuptake into the SR and preventing cytosolic Ca²⁺ overload. Given the central role of Ca²⁺ dysregulation in PASMCs proliferation and vascular remodeling, istaroxime may offer therapeutic benefits beyond cardiac disease, representing a promising candidate for the treatment of PAH. This doctoral project investigated the potential role of istaroxime as a modulator of pulmonary vascular remodeling, focusing on its effects on Ca²⁺ homeostasis, SR functionality, and prevention of maladaptive remodeling. Fistly, PASMCs were treated with istaroxime (1 µM), both acutely and chronically (48 hours) to evaluate broader effects on SR performance and Ca²⁺-dependent pathways. A pathological context was then reproduced in vitro by using angiotensin II (AngII), a well-established inducer of Ca²⁺ dysregulation and proliferation, and comparative experiments were performed pre-treating or not PASMCs with istaroxime. Results showed that prolonged exposure to istaroxime reduced cytosolic Ca2+ and PASMCs proliferation, mainly through SERCA2 stimulation. Furthermore, istaroxime prevented AngII–induced PASMCs proliferation, cytosolic Ca2+ overload, ROS induction and calcineurin-NFAT-dependent signaling. Overall, this work identifies a novel role for istaroxime in counteracting pulmonary vascular remodeling, mainly favoring SR Ca²⁺ confinement and limiting intracellular Ca2+-dependent pathways associated to pathological remodeling processes. These findings extend the therapeutic relevance of istaroxime beyond heart failure and provide mechanistic insights supporting its repurposing as a promising pharmacological strategy for the treatment of PAH and related vascular disorders. In addition to this doctoral project, a six-month research stay was undertaken at King’s College London under the supervision of Dr Giancarlo Forte. This experience offered the opportunity to expand the research framework by integrating complementary experimental approaches. During this stay, the work progressed from a two-dimensional culture model of human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) to a three-dimensional engineered heart tissue (EHT) system. The 3D model was functionally characterized through the assessment of contractile force and beating rate over time. Furthermore, fluorimetric analysis of electrical activity and Ca²⁺ transients, together with molecular analyses, revealed a higher degree of structural and functional maturation in the EHT constructs. This training provided a deeper understanding of molecular and cellular mechanisms, bridging 2D culture studies with more physiologically relevant 3D tissue models.