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Background and aims: Leptin resistance or leptin signalling deficiency are associated with increased risk of diabetic cardiomyopathy and heart failure, which is a leading cause of obesity- and diabetes type 2 (T2DM)-related morbidity and mortality. Various metabolic disturbances are involved in this pathogenesis, such as elevated glucose and fatty acid levels, insulin resistance and altered myocardial substrate utilization. Rodent models provided useful insights into the underlying molecular mechanisms of obese- and T2DM-associated cardiometabolic diseases, however, they cannot fully recapitulate the disease phenotype of obese or T2DM patients. The aims of this study were to study the effect of leptin receptor (LEPR) mutations on the leptin-mediated signalling pathways in human cardiomyocytes, and to investigate glucose and fatty acid metabolism in the heart under (patho)physiological conditions. Methods and results: To study the role of LEPR in human cardiomyocytes (CMs), human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were used as a model. In the first part of this study, LEPR expression and function was investigated in wild type (WT)-iPSC-CMs by PCR and Western Blot. LEPR protein expression was almost not detectable in iPSCs and during early cardiac differentiation stages, however mRNA LEPR expression was comparable in the different steps of cardiac development. Importantly, LEPR protein expression was observed in WT-iPSC-CMs at the maturation stages, indicating that LEPR plays an important role in matured CMs. Thanks to CRISPR/Cas9 technology, LEPR mutations were introduced into iPSCs. Among the several clones obtained, 1B2 LEPRΔ/Δ-iPSC line was fully characterized and showed normal capacity to differentiate into spontaneously beating CMs. Although the B27 medium represents a well-established medium to cultivate iPSC-CMs, it has limitations for studying CM metabolism due to its high concentration of insulin and glucose, but low concentration of fatty acids. Physiological medium condition (F2) including physiological range of glucose, insulin and fatty acids was found to be fundamental to study LEPR signalling pathway in iPSC-CMs. Western blot analysis showed functional LEPR downstream pathway activation in WT-iPSC-CMs, while the absence of LEPR function was demonstrated in LEPRΔ/Δ-iPSC-CMs cultured in F2 medium. Moreover, improved medium condition, offered by the F2 medium, ameliorates insulin sensitivity as result of increased insulin-dependent AKT phosphorylation in WT-iPSC-CMs, while loss of LEPR function was associated with downregulation of insulin pathway activation. Additionally, leptin direct effect was observed on the regulation of glucose metabolism in WT-iPSC-CMs by reducing glycolytic fluxes, which was not observed in LEPRΔ/Δ-iPSC-CMs, as measured by 13C-isotope-assisted glucose metabolic flux. These data indicate that the signalling interaction between insulin and leptin is important in regulation of glucose metabolism and is abolished in LEPRΔ/Δ-iPSC-CMs. The matured WT-iPSC-CMs in F2 medium display adult CM-like metabolic phenotype such as enhanced mitochondrial respiration and glycolytic function, as measured by Seahorse analyser, compared to the same group cultured in the B27 medium. The mutation generated in LEPRΔ/Δ-iPSC-CMs caused an “energy starvation” status which led to increased AMPK phosphorylation compared to the WT group in B27 medium, which was associated with lower mitochondrial oxygen consumption rate (OCR) linked basal respiration and ATP production. In the next part of this study, the long-term leptin treatment of iPSC-CMs under physiological medium conditions in the presence of physiological range of insulin, glucose, and fatty acids (F2+) influenced LEPR downstream pathway activation such as JAK2 and AMPK suggesting a leptin-dependent role in fatty acid uptake and oxidation in WT-iPSC-CMs. On the contrary, leptin did not affect JAK2 and AMPK activation in LEPRΔ/Δ-iPSC-CMs. Culturing of (WT)-iPSC-CMs in F2+ medium demonstrated no significant difference in mitochondrial oxygen consumption, while slightly lower glycolysis and glycolytic capacity was observed. However, a leptin effect on fatty acid and glucose metabolism was observed in LEPR∆/∆-iPSC-CMs, which is independent from LEPR downstream regulation. To study the effect of high leptin levels, a medium mimicking some of the diabetic hallmarks, such as high glucose, high insulin, and high leptin levels, was used. Metabolic flexibility was observed in WT-iPSC-CMs in F3+ medium as showed by no difference in mitochondrial function in WT-iPSC-CMs in the presence or absence of high leptin. In contrast, LEPRΔ/Δ-iPSC-CMs in F3+ medium demostrated higher OCR compared to F2 medium, which is accompanied by lower glycolysis and glycolytic capacity, indicating the incapability of LEPRΔ/Δ-iPSC-CMs to use glucose as energy source, as measured by Seahorse analysis. Conclusion and outlook: Taken together, this study demonstrates the importance of leptin and LEPR at the late stage of CM maturation and the fundamental role of metabolic medium condition including physiological range of glucose and fatty acid to study the role of leptin in iPSC-CMs. In addition, LEPRΔ/Δ-iPSC-CMs in diabetic condition (F3+) represent a suitable model to investigate leptin-dependent cardiac metabolism, resulting in increased mitochondrial oxygen consumption and decreased glycolytic function, resembling the condition known in obesity-related T2DM patients. Further studies should focus on the regulation of the metabolic switch between glucose and fatty acid utilization in the absence of a functional LEPR. Understanding the contribution of leptin/LEPR signalling in human CM metabolism will shed light on novel therapeutic approaches to treat diabetic cardiomyopathy.