Abstract Body (Do not enter title and authors here): Introduction: Cardiac sympathetic innervation plays important roles in regulation of heart rate, contractility, and conduction, as well as postnatal maturation and injury response. However, few physiologically relevant in vitro models of functionally innervated myocardium have been reported to date. Therefore, we sought to engineer a 3D tissue-engineered model of human innervated myocardium to enable functional and pharmacological studies of cardiac innervation.
Methods: We first developed a protocol to differentiate functional sympathetic neurons (SNs) from human induced pluripotent stem cells (hiPSCs) using a PHOX2B::eGFP reporter line, followed by characterization by qPCR, immunostaining, and Ca²⁺ imaging. Compartmentalized model of innervated engineered cardiac tissues (ECTs) was fabricated using 6-wk old hiPSC-SNs and 3-wk old hiPSC-cardiomyocytes (CMs) transduced with MHCK7-gCaMP6. After 4-6 weeks of culture, structural and functional characterization was performed using immunostaining, force testing, optical mapping, Ca²⁺ imaging, and pharmacological tests.
Results: hiPSC-SNs expressed canonical transcription factors (Phox2b, Ascl1, Hand2) and functional enzymes (TH, DBH, AChRs), and robustly responded to presynaptic nicotine and electrical stimulation. After 4 weeks of coculture, SN axons were evident in ECT cross-sections (0.23% area) and whole-tissue mounts (4.44% area), indicating successful axon ingrowth. Compared to aneural ECTs, SN-innervated ECTs displayed similar contractile forces and conduction velocities, and 1.32-fold higher Ca²⁺ transient amplitudes. Additionally, the spontaneous beating rate and beat-to-beat variability in innervated ECTs was significantly higher than in aneural ECTs (2.1-fold and 2.85-fold, respectively). Upon the addition of 100µM nicotine, the spontaneous beating rate of innervated tissues increased significantly compared to aneural ECTs (1.61-fold), while beat-to-beat variability was unchanged relative to control.
Conclusion: Collectively, our results demonstrate the successful generation of an in vitro model of functionally innervated human myocardium, which will enable studies of pathological SN remodeling after myocardial injury and diseases of the heart-brain axis.