Tissue Engineered Innervated Cardiac Units: A Three-Dimensional Platform Recapitulating the Spatial Organization of Cardiac Sympathetic Innervation

Autonomic innervation governs cardiac development, functional maturation, and electrophysiological integration, yet in vitro models rarely reproduce the spatial organization through which sympathetic neurons reach the heart. We first established planar neuro-cardiac cocultures of intact neonatal rat superior cervical ganglia (SCG) and embryonic rat cardiac aggregates. Sympathetic axons were tyrosine hydroxylase (TH) positive, penetrated the cardiac aggregates, and increased the spontaneous contraction rate. RT-qPCR on these planar cocultures showed that innervated cardiac tissue upregulated contractile, gap-junction, and adrenergic transcripts (Actc1, Actn2, Myh6, Adrb1, Gja5/Cx40, Cx43, Cx45) relative to both freshly isolated tissue and cardiac-only cultures, without re-expression of the fetal isoform Myh7. We then developed 3D Tissue Engineered Innervated Cardiac Units (TE-ICUs), in which SCG and cardiac aggregates occupy opposite ends of a 3–10 mm methacrylated hyaluronic acid microcolumn. Sympathetic axons in isolation projected up to ~6 mm and released ~170 nM norepinephrine upon electrical stimulation. Within TE-ICUs, fasciculated TH+ tracts bridged the microcolumn and penetrated the cardiac unit, while cardiac aggregates self-organized into chamber-like compartments; the contraction rate was significantly higher than that of cardiac-only controls at 8 days in vitro. TE-ICUs provide an anatomically inspired platform for studying neuro-cardiac development and engineering innervated cardiac tissue.