Ischemic heart disease (IHD) is one of the leading causes of mortality worldwide. Preserving functionality and preventing arrhythmias of the heart are key principles in the management of patients with IHD. Levosimendan, a unique calcium (Ca²⁺) enhancer with inotropic activity, has been introduced into clinical usage for heart failure treatment. Human-induced pluripotent cell-derived cardiomyocytes (hiPSC-CMs) offer an opportunity to better understand the pathophysiological mechanisms of the disease as well as to serve as a platform for drug screening. Here, we developed an in vitro IHD model using hiPSC-CMs in hypoxic conditions and defined the effects of the subsequent hypoxic stress on CMs functionality. Furthermore, the effect of levosimendan on hiPSC-CMs functionality was evaluated during and after hypoxic stress. The morphology, contractile, Ca²⁺-handling, and gene expression properties of hiPSC-CMs were investigated in response to hypoxia. Hypoxia resulted in significant cardiac arrhythmia and decreased Ca²⁺ transient amplitude. In addition, disorganization of sarcomere structure was observed after hypoxia induction. Interestingly, levosimendan presented significant antiarrhythmic properties, as the arrhythmia was abolished or markedly reduced with levosimendan treatment either during or after the hypoxic stress. Moreover, levosimendan presented significant protection from the sarcomere alterations induced by hypoxia. In conclusion, this chip model appears to be a suitable preclinical representation of IHD. With this hypoxia platform, detailed knowledge of the disease pathophysiology can be obtained. The antiarrhythmic effect of levosimendan was clearly observed, suggesting a possible new clinical use for the drug.

Acute hypoxia is a major contributor to cardiomyocyte damage and dysfunction in ischemic heart disease, and the effective therapeutic strategies remain limited. Levosimendan, a calcium sensitizer with both inotropic and vasodilatory effects, along with its active metabolite OR-1896, is utilized in the treatment of acute heart failure. In this study, we investigated the cardioprotective and antiarrhythmic effects of levosimendan and its metabolite OR-1896 under hypoxic conditions using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). hiPSC-CMs were exposed to acute hypoxia and treated with levosimendan or its metabolite OR-1896. Structural integrity was assessed via immunostaining and electron microscopy imaging. Calcium transient abnormalities were evaluated using live-cell imaging. Hypoxia-induced injury was further assessed by measuring cardiac biomarkers and gene expression profiling of hypoxia-associated pathways. Hypoxia induced significant structural damage, including sarcomere disorganization, mitochondrial cristae fragmentation, and nuclear shrinkage, accompanied by increased release of cardiac biomarkers. Hypoxia also upregulated genes associated with the hypoxia response, oxidative stress, and apoptosis, while disrupting calcium handling and increasing arrhythmic events. Treatment with levosimendan and its metabolite OR-1896 preserved cellular structure, reduced biomarker release, and stabilized calcium transients, significantly reducing hypoxia-induced arrhythmogenesis. Both compounds also modulated gene expression, downregulating hypoxia-responsive and oxidative stress markers, and inhibiting apoptotic pathways. Notably, the metabolite OR-1896 exhibited protective effects comparable to or even greater than those of levosimendan. This study provides the first comprehensive evidence of the cardioprotective and antiarrhythmic properties of levosimendan’s metabolite, demonstrating its ability to reduce hypoxia-induced cellular injury and correct abnormal Ca²⁺ transients. These findings highlight the therapeutic potential of levosimendan and its clinically significant long-acting metabolite, OR-1896, in the treatment of cardiac ischemia.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer a powerful platform for disease modeling, drug discovery, and regenerative therapies. However, their clinical and research utility remains limited by their immature, fetal-like phenotype. In the human heart, postnatal metabolic maturation involves a critical switch from glycolysis to fatty acid β-oxidation, enabling efficient ATP production via oxidative phosphorylation. In this study, we investigated whether mimicking this metabolic shift in vitro by culturing hiPSC-CMs in a fatty acid-based maturation medium (FAM) could enhance their structural and functional development compared to a conventional glucose-based medium (GLM). hiPSC-CMs cultured in FAM for two weeks exhibited significant improvements in morphological, metabolic, and mechanical maturation markers. Morphologically, FAM-cultured CMs exhibited enhanced sarcomeric organization, increased cellular alignment, and a more elongated and rod-like shape, which are characteristics typically associated with mature CMs. Ultrastructural analysis further confirmed improved maturation, revealing more organized sarcomeres and densely packed mitochondria compared to GLM-cultured CMs. Metabolically, FAM-cultured CMs demonstrated a clear shift from glycolytic to oxidative metabolism, as evidenced by higher mitochondrial membrane potential, increased oxidative phosphorylation capacity, elevated ATP production, and reduced glycolytic activity. These metabolic adaptations indicate a more adult-like energy profile, consistent with enhanced fatty acid β-oxidation. Mechanically, FAM-cultured cardiomyocytes exhibited enhanced functional maturity, as evidenced by faster calcium transients and greater contraction amplitude, indicating improvements in specific electrophysiological properties. In conclusion, fatty acid supplementation effectively promotes the structural, metabolic, and mechanical maturation of hiPSC-CMs, resulting in a more adult-like phenotype. This strategy provides a robust and straightforward approach to enhance the physiological relevance of hiPSC-CMs for preclinical applications in disease modeling, drug testing, and regenerative medicine.