Bioengineering of a human innervated cardiac muscle model

Cardiac autonomic neurons control cardiac contractility. Dysregulation of the autonomic nervous system can lead to sympathetic overdrive resulting in heart failure and an increased incidence of fatal arrhythmias. Here, we introduce innervated engineered human myocardium (iEHM), a novel model of neuro-cardiac junctions, constructed by fusion of a bioengineered neural organoid (BENO) patterned to autonomic nervous system and engineered human myocardium (EHM). Projections of sympathetic neurons into engineered human myocardium formed presynaptic terminals in close proximity to cardiomyocytes and an extensive vascular network co-developing in the tissues. Contractile responses to optogenetic stimulation of the accordingly engineered neuronal component demonstrated functionality of the engineered neuro-cardiac junctions in iEHM. This model will serve as a human surrogate system to delineate neuron and cardiac cell contribution to brain and heart diseases and is an important step towards engineering a human brain to heart axis in a dish. Overall design: Sympathetic neuronal organoids (SNO) were generated by culturing human induced pluripotent stem cells (hiPSC) in a 3D collagen matrix and step-wise addition of growth factors and small molecules to direct them toward an autonomic, predominantly sympathetic neuronal fate. The medium used for the SNO was based on neuron-specific Neurobasal-A medium. EHM were engineered by combining human foreskin fibroblasts and hiPSC-derived cardiomyocytes with bovine collagen and casting this mixture into a ring-shaped mold. EHM were matured for four weeks in an EHM-specific medium on auxotonic stretchers. After 40 days of culture, SNO were fused with EHM by placing the SNO in the center of the muscle ring to generate iEHM. From this day, iEHM were cultured in a mixture of neuron- and EHM-specific medium in a 1:1 ratio. At this time, SNO were transitioned to the same medium mixture to examine changes in the neuronal tissues unspecific neuro-cardiac interaction. At 6 weeks after fusion (SNO were 12 weeks old), one iEHM sample was snap-frozen, single nuclei were isolated, and RNA was sequenced as described.