LANCL2 and ERRα Interaction and Functional Collaboration in Cardiomyocytes

Abscisic acid (ABA), a terpenoid phytohormone classically associated with plant stress responses, has emerged as an endogenous mammalian hormone with a conserved role in energy metabolism and immune regulation. In mammals, ABA modulates glucose homeostasis, mitochondrial function, and oxidative metabolism through its receptors, lanthionine synthetase C–like proteins LANCL1 and LANCL2. The orphan nuclear receptor estrogen-related receptor alpha (ERRα) is a master transcriptional regulator of mitochondrial biogenesis, oxidative phosphorylation, and metabolic flexibility in tissues with high energetic demand, including muscle and immune cells. Although both ABA/LANCL signaling and ERRα independently control cellular energy metabolism, their potential functional interplay has not been previously investigated. The objective of this doctoral research was to investigate the molecular and functional collaboration between the ABA/LANCL hormone–receptor system and ERRα signaling in cardiomyocytes and immune cells, with particular emphasis on nitric oxide (NO) and reactive oxygen species (ROS) metabolism, mitochondrial function, and transcriptional regulation. By integrating cellular, molecular, biochemical, and advanced imaging approaches, this study aimed to investigate the possible role of ERRα as a downstream effector of ABA/LANCL signaling and to explore the relevance of this axis in both metabolic and immune contexts. Using rat embryonic H9c2 cardiomyocytes as a cell model, ABA stimulation and genetic modulation of LANCL1/2 expression were combined with ERRα silencing or overexpression. ROS production was quantified using H₂DCFDA and mitochondrial superoxide–specific MitoSOX™ assays, while NO signaling was evaluated through analysis of endothelial nitric oxide synthase (eNOS) expression. Transcriptional and protein-level changes were assessed by qPCR and Western blotting. Confocal microscopy and FRET–FLIM imaging were employed to investigate the subcellular localization and physical proximity of LANCL2 and ERRα, providing evidence of their spatial and functional association. In parallel, recombinant human ERRα was expressed and purified in Escherichia coli and reintroduced by plasmid transfection into LANCL2-overexpressing cardiomyocytes to validate causality in signaling responses. The results demonstrate that the ABA/LANCL1–2 system regulates NO and ROS metabolism in cardiomyocytes through an ERRα-dependent mechanism. ABA stimulation enhanced eNOS transcription and NO production, while concomitantly limiting excessive ROS generation, thereby promoting a metabolically efficient and redox-balanced phenotype. ERRα silencing significantly blunted these effects, establishing ERRα as a critical mediator of ABA/LANCL signaling. Confocal and FRET–FLIM analyses revealed co-localization and close molecular interaction between LANCL2 and ERRα in H9c2 supporting the existence of a functional signaling hub linking membrane-associated ABA perception to nuclear transcriptional control of mitochondrial metabolism. To extend these findings to the immune system, LANCL2 expression was selectively silenced or increased in human CD14⁺ monocytes and peripheral blood lymphocytes (PBLs). These experiments confirmed the feasibility of modulating the ABA/LANCL axis in immune cells and provided initial evidence that LANCL2 expression levels influence cellular metabolic and signaling competence, consistent with the established role of ERRα in immune cell activation and metabolic reprogramming. Together, these data suggest that the ABA/LANCL–ERRα axis represents a conserved metabolic signaling pathway operative in both cardiomyocytes and immune cells. In conclusion, this thesis identifies ERRα as a novel downstream effector and functional partner of the ABA/LANCL hormone–receptor system, linking extracellular metabolic cues to transcriptional programs controlling mitochondrial function, redox homeostasis, and energy metabolism. These findings provide new mechanistic insight into insulin-independent, ABA-mediated metabolic regulation and highlight the ABA/LANCL–ERRα axis as a promising therapeutic target for metabolic disorders, cardiomyopathies, and immune-metabolic dysregulation. Future perspectives include in vivo validation of this signaling pathway, exploration of its role in blood glucose homeostatis, metabolic and inflammatory diseases, and the development of pharmacological or nutraceutical strategies aimed at selectively modulating ERRα activity downstream of ABA signaling.