Background/Aims: Nesfatin-1 (NF-1), an anorexic nucleobindin-2 (NUCB2)-derived hypothalamic peptide, acts as a peripheral cardiac modulator and it can induce negative inotropic effects. However, the mechanisms underlying these effects in cardiomyocytes remain unclear. Methods: Using patch clamp, protein kinase assays, and western blot analysis, we studied the effect of NF-1 on L-type Ca2+ currents (/(Ca,L)) and to explore the regulatory mechanisms of this effect in adult ventricular myocytes. Results: NF-1 reversibly decreased /(Ca,L) in a dose-dependent manner. This effect was mediated by melanocortin 4 receptor (MC4-R) and was associated with a hyperpolarizing shift in the voltage-dependence of inactivation. Dialysis of cells with GDP-beta-S or anti-G(beta) antibody as well as pertussis toxin pretreatment abolished the inhibitory effects of NF-1 on /(Ca,L). Protein kinase C (PKC) antagonists abolished NF-1-induced responses, whereas inhibition of PKA activity or intracellular application of the fast Ca2+-chelator BAPTA elicited no such effects. Application of NF-1 increased membrane abundance of PKC theta isoform (PKC theta), and PKC theta inhibition abolished the decrease in /(Ca,L) induced by NF-1. Conclusion: These data suggest that NF-1 suppresses L-type Ca2+ channels via the MC4-R that couples sequentially to the beta gamma subunits of G(i/o)-protein and the novel PKC theta isoform in adult ventricular myocytes. Copyright (C) 2015 S. Karger AG, Basel