Introduction. In patients with obesity-associated type 2 diabetes, insulin and leptin resistance are linked to diabetic cardiomyopathy (DCM) development, a severe complication that increases the risk of heart failure. Impaired cardiomyocyte (CM) metabolic flexibility, characterized by increased fatty acid oxidation and decreased glucose utilization, is considered a major contributor to DCM.

Aims. This study aims to elucidate how loss-of-function of leptin receptor (LEPR) alters the metabolic properties of human CMs, thereby providing mechanistic insight into the pathogenesis of DCM.

Methods. Homozygous LEPR-deficient (LEPRΔ/Δ) human induced pluripotent stem cells (iPSCs) were generated using CRISPR/Cas9 technology. Both wild-type (WT) and LEPRΔ/Δ-iPSCs were differentiated into CMs and maintained for 60 days in either B27 medium (11 mM glucose, 700 nM insulin) or F2 medium (7 mM glucose, 50 nM insulin, 0.5% Albumax). Bulk RNA sequencing data analysis was used to identify genes associated with transcriptional remodelling under different metabolic conditions. The effect of acute leptin and/or insulin stimulation on molecular pathways, such as JAK2/STAT3 and PI3K/AKT, was investigated by Western blot. The metabolic phenotype was evaluated by Seahorse technology and reactive oxygen species (ROS) measurements.

Results. We demonstrate that F2 medium, which closely mimics physiological conditions for glucose, insulin, and fatty acids, was essential to investigate the role of LEPR in iPSC-CMs. Western blot analysis confirmed loss of LEPR signalling in LEPRΔ/Δ-iPSC-CMs, as demonstrated by abolished JAK2/STAT3 activation upon leptin stimulation in F2 medium when compared to WT-iPSC-CMs. Transcriptomic analysis revealed downregulation of the PI3K-AKT pathway in LEPRΔ/Δ-iPSC-CMs in F2 medium compared with WT-iPSC-CMs, consistent with preserved insulin sensitivity in WT-iPSC-CMs as demonstrated by increased AKT phosphorylation after acute insulin stimulation and impaired insulin signalling in LEPRΔ/Δ-iPSC-CMs. Additionally, WT-iPSC-CMs in F2 medium exhibited an adult CM-like metabolic phenotype, characterized by improved mitochondrial respiration and glycolytic function compared to the group cultured in B27 medium. Consistently, transcriptomic analysis revealed that F2 culture condition elicited upregulation of key metabolic genes involved in enhancing TCA cycle flux, NADPH production, mitochondrial oxidative capacity and induction of antioxidant and redox-regulatory genes, consistently with a reduction in ROS levels compared with B27 medium in both WT- and LEPRΔ/Δ-iPSC-CMs. In contrast, LEPRΔ/Δ-iPSC-CMs in F2 medium exhibited reduced mitochondrial function and downregulation of pathways critical for sarcomeric structure and contraction.

Conclusions. These findings identify that LEPR signalling plays a critical role in CM metabolic flexibility, providing mechanistic insight into the contribution of leptin resistance to the development of diabetic cardiomyopathy.