The use of three-dimensional (3D) cultures may induce cardiac progenitor cells to synthesize theirown extracellular matrix (ECM) and sarcomeric proteins to initiate cardiac differentiation. 3Dcultures grown on synthetic scaffolds may favour the implantation and survival of stem cells for celltherapy when pharmacological therapies are not efficient in curing cardiovascular diseases and whenorgan transplantation remains the only treatment able to rescue the patient’s life. Silk fibroin-basedscaffolds may be used to increase cell affinity to biomaterials and may be chemically modified toimprove cell adhesion. In the present study, porous, partially orientated and electrospun nanometricnets were used. Cardiac progenitor cells isolated from adult rats were seeded by capillarity in the 3Dstructures and cultured inside inserts for 21 days. Under this condition, the cells expressed a highlevel of sarcomeric and cardiac proteins and synthesized a great quantity of ECM. In particular,partially orientated scaffolds induced the synthesis of titin, which is a fundamental protein insarcomere assembly.