The ABA/LANCL1-2 system in ROS regulation in cardiomyocytes and in skeletal muscle differentiation and thermogenesis: evaluation of potential LANCL2 agonists

Abscisic acid (ABA) is an evolutionarily conserved hormone that originated in some of the earliest living organisms, with modern ABA-producing cyanobacteria likely representing their descendants. Its emergence occurred prior to the divergence of the plant and animal kingdoms, and its fundamental role as a signaling molecule mediating cellular responses to environmental stress has been maintained throughout evolution. In mammals, beyond its anti-inflammatory and neuroprotective functions, ABA at nanomolar concentrations modulates the metabolic response to glucose availability by promoting glucose uptake in skeletal muscle and adipose tissue through an insulin-independent mechanism. Moreover, ABA enhances mitochondrial energy production and energy dissipation in both brown and white adipocytes via activation of its receptors LANCL1 and LANCL2. Chronic oral administration of ABA at microgram-per-kilogram body weight doses has been reported to improve glycemic control, lipid homeostasis, and morphometric parameters in borderline subjects for prediabetes and metabolic syndrome. In rat H9c2 cardiomyocytes the cross-kingdom stress hormone ABA and its mammalian receptors LANCL1 and LANCL2 modulate the cellular response of cardiomyocytes to hypoxia by activating the AMPK/PGC-1α axis. This activation enhances increasing NO generation, mitochondrial proton gradient, respiration, and improves cell vitality after hypoxia/reoxygenation. In addition, overexpression of LANCL1/2 in rat H9c2 cardiomyocytes markedly increased, whereas silencing decreased, mitochondrial number, OXPHOS complex I activity, proton gradient, glucose- and palmitate-driven respiration, uncoupling protein transcription, and expression of proteins involved in cytoskeletal, contractile, and electrical functions. These effects, together with LANCL1/2-dependent NO production, are mediated by the transcription factor ERRα, which acts upstream of the AMPK/PGC-1α axis and is transcriptionally regulated by the ABA/LANCL1/2 system. Based on this evidence, this thesis investigated the role of the ABA/LANCL1-2 system in Reactive Oxygen Species (ROS) metabolism in H9c2 cardiomyocytes overexpressing or silenced for LANCL1/2, with or without concomitant ERRα knockdown. Expression of enzymes involved in ROS production and scavenging was assessed by qRT-PCR and Western blot, while mitochondrial proton gradient and ROS levels were measured using specific fluorescent probes. LANCL1/2 overexpression decreased ROS-generating enzymes, increased ROS-scavenging enzymes, and reduced mitochondrial ROS, whereas opposite effects were observed in LANCL1/2-silenced cells. The knockdown of ERRα abrogated all beneficial effects on ROS turnover in LANCL1/2 overexpressing cells. Overall, these results indicate that the ABA/LANCL1-2 system regulates key aspects of cardiomyocyte physiology and ROS turnover via the ERRα/AMPK/PGC-1α axis, highlighting it as a potential target to enhance mitochondrial function and resistance to oxidative stress. Investigating molecules that specifically activate this system and its receptors represents a promising research avenue. To this end, biochemical assays in H9c2 cells and preliminary binding studies were conducted to assess the interaction of novel potential agonists with human recombinant LANCL2. Recent findings in rat H9c2 cardiomyocytes have demonstrated that the ABA/LANCL1-2 system plays a role in regulating cellular thermogenesis, as evidenced by higher heat production in LANCL1/2-overexpressing cells compared with double-silenced cells. These results are consistent with previous observations in adipocytes and myoblasts, which revealed an enhanced browning process accompanied by an increased basal metabolic rate, elevated substrate oxidation, and upregulated expression of uncoupling proteins. These proteins dissipate the mitochondrial proton gradient to generate heat instead of ATP, thereby contributing to cellular energy expenditure, mechanisms that are crucial for maintaining normal body weight and counteracting obesity. In this thesis the potential role of the ABA/LANCL1-2 system in regulating the expression of genes involved in skeletal muscle thermogenesis and differentiation was investigated, aiming to provide further insights into the physiological functions of skeletal muscle, a tissue that constitutes approximately 40% of total human body weight and, together with adipose tissue, represents the majority of mammalian cell mass, significantly contributes to body weight balance.