Background: Trifunctional lipids are a multifunctional tool to display the interaction between lipids and proteins. By incorporating specific protein-binding groups, these chemically modified lipids enable targeted investigation of these interactions and contribute to a deeper understanding of the underlying mechanisms. In this study, we focus on the biological functions of sphingolipids, especially on sphingosine-1-phosphate (S1P) and its precursor sphingosine. In the cardiovascular system, dysregulation of sphingolipid metabolism has been associated with metabolic stress, mitochondrial dysfunction and cardiac remodeling, contributing to the development of cardiomyopathies and heart failure. Understanding how sphingolipids interact with cardiac proteins is therefore essential to uncover novel molecular mechanism underlying cardiac metabolism.
Aim: To identify sphingolipid-binding proteins (SBPs) in cardiomyocytes and to investigate their physiological functions and potential roles in the development of cardiac diseases.
Methods: Adult cardiomyoctes were isolated from wildtype mice using standard Langendorff perfusion system with enzymatic digestion. After isolation, cells were treated with photoclickable sphingosine (pac sph) for 1 hour, followed by UV irradiation for 5 minutes to induce crosslinking. The resulting protein lysates were subjected to a biotin click reaction using biotin azide, CuSO₄, Tris(benzyltriazolylmethyl)amine (TBTA), and ascorbic acid, followed by protein precipitation and bead-based elution. The resulting eluates were used for either proteomic analysis or Western blotting to identify and characterize sphingolipid-interacting proteins. In parallel, sphingolipid analysis of cardiomyocytes was performed using LC-MS. RNA sequencing was conducted on hearts from wildtype and diet-induced obesity (DIO) mice.
Results: Cardiomyocytes isolated from DIO mice exhibited altered sphingolipid levels compared to controls. Targeted proteomic analysis in cardiomyocytes from wildtype mice identified numerous sphingolipid-binding proteins, which are involved in metabolic processes, calcium homeostasis, and the maintenance of cardiac function. KEGG pathway analysis revealed that these proteins are associated with diabetic, hypertrophic, and dilated cardiomyopathy, as well as cardiac muscle contraction. Comparison with RNA sequencing data from DIO hearts showed an overlap with sphingolipid-interacting proteins that were upregulated under metabolic stress. Overall, these findings indicate that sphingolipid-binding proteins play a critical role in coordinating mitochondrial function, calcium handling, and cellular stress responses, which are essential for sustaining efficient contraction and cardiomyocyte survival under both physiological and pathological conditions.
Conclusion: Sphingolipid-binding proteins are closely associated with metabolic regulation, calcium handling, and mitochondrial function in cardiomyocytes, highlighting their potential role in cardiac physiology and disease. These findings underscore lipid–protein interactions as promising targets for future therapeutic investigation.
