UC San Diego

The bioactive molecule sphingosine-1-phosphate (S1P) activates five recognized G-protein coupled receptors (S1P₁₋₅) to affect various tissues, including myocardium. Activation of these receptors and the ability to affect functional responses including differentiation, migration, contraction, and ionic currents in isolated cardiac fibroblasts (CFb) and myocytes was investigated. CFb are essential components of myocardium, and study of their cell signaling and physiology is required for a number of emerging disciplines. However, in order to conduct meaningful studies on CFb, methods for selective, reproducible cell isolations are necessary. A protocol was therefore developed that significantly reduced resident macrophage levels in CFb isolates and utilized more CFb- specific markers, instead of, or in addition to, more commonly used cytoskeletal markers. Primary isolated, purified mouse CFb express predominantly S1P₁₋₃; however, the relative levels of these receptor subtypes are modulated with time and by culture conditions. Co-culture with macrophages altered CFb S1P receptor levels relative to controls. Further investigations using known macrophage -secreted factors (e.g. TFG[beta]1, TNF[alpha], and PDGF- BB) all altered S1P receptor subtypes. Low serum concentrations increased S1P₂, which was diminished using a Rho-associated protein kinase inhibitor. Similarly, elevated S1P₃ levels by PDGF-BB were diminished using a Rac1 inhibitor. S1P₂ and S1P₃ (which activate Rho and Rac pathways, respectively) regulate cell migration, and CFb isolated from S1P₃-null mice had reduced migration. These results highlight the importance of demonstrating CFb culture purity in functional studies of S1P and present conditions that modulate S1P receptor expression in CFb. CFb were characterized using novel patch-clamping technology platforms to assess potassium channel currents and the effect of S1P on CFb electrophysiology. Both Kv and Kir currents were identified by conventional and patch -on-a-chip platforms; however, chip platforms were more likely to activate mechanosensitive ion channels. S1P strongly activated both inward and outward currents in adult rat CFb. Lastly, mouse ventricular myoycte contractility was investigated experimentally and through mathematical modeling to characterize signaling pathways involved in S1P-regulated negative inotropy. S1P reduced cell shortening via S1P₁ and S1P₃. In particular, S1P₁ reduced L-type calcium channel activity and activated an acetylcholine-like potassium current to alter action potential duration and intracellular calcium concentrations