Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

Engineering of a human sympathetically innervated cardiac muscle model

L. V. Schneider1, G. Bao1, N. Liaw1, O. Jensen2, K. A. Schmoll1, J. Brockmöller2, W.-H. Zimmermann1, M. P. Zafeiriou1, für die Studiengruppe: AG31

1Institut für Pharmakologie und Toxikologie, Universitätsmedizin Göttingen, Göttingen; 2Institut für Klinische Pharmakologie, Univeritätsmedizin Göttingen, Göttingen;

The interaction between heart and brain via the autonomous nervous system (ANS) is of fundamental importance for cardiac homeostasis and its dysregulation contributes to heart failure. Here, we aimed to engineer an autonomically innervated human heart muscle from induced pluripotent stem cells (iPSC) to emulate the complex heart-brain interplay in health and disease. To construct innervated Engineered Human Myocardium (iEHM), we fused sympathetic neuronal organoid (SNO) with engineered human myocardium (EHM). For optogenetic control of iEHM, SNO were engineered from genetically modified iPSC with a red-light activatable channelrhodopsin (Chrimson). SNOs are comprised of 55±1% PHOX2B-positive autonomic neuron progenitors (n=9 tissues, N=3 independent differentiations) and present robust transcript as well as protein expression of SN-marker dopamine-beta-hydroxylase and tyrosine hydroxylase (DBH 7123±981-fold and TH 3.86±0.52-fold to undifferentiated iPSC) while showing low expression of other peripheral or cortical neuronal markers (MNX1 and PAX6, respectively). Liquid chromatography-mass spectrometry further confirmed SN-properties such as high norepinephrine (60.4±6.2 pmol/mg tissue) and low parasympathetic or pre-ganglionic neurotransmitter acetylcholine (18.4±4.9 pmol/mg tissue) abundance. Wholemount immunofluorescence analysis of iEHM revealed axons from the SNO extending towards cardiomyocytes. iEHM developed 0.46±0.04 mN (n=20, N=1) maximum force of contraction, 2-fold higher than EHM (0.26±0.04 mN, n=6, N=1) when cultured under the same conditions, suggesting a contractility-modulating activity of the engineered SN in iEHM. Light stimulation of optogenetic iEHM evoked a clear positive chronotropic response (24±4% in iEHM, n=40, N=3 vs 1±3% in EHM, n=16, N=3). Since SNO contained low levels of cholinergic neurons, we tested the effect of muscarinic receptor blocker atropine (1 nM). No significant differences were identified in the beating rate of iEHM (5.21±0.1%), suggesting that the cholinergic neurons may represent pre-ganglionic sympathetic neurons. Notably, in the presence of atropine, addition of the autonomic neuron stimulant nicotinic acid (30 µM) resulted in a statistically significant increase in beating rate in 19 out of 27 iEHM (38±13% to baseline, n=19, N=5). Further evidence for the development of a functional sympathetic nerve system in iEHM stemmed from experiments, in which the nicotinic acid-induced chronotropic response was blunted (-13±11 % to baseline, n=19, N=5) by beta adrenoreceptor-blocker (propranolol 10 µM). In conclusion, sympathetically innervated human heart muscle (iEHM) can be developed in vitro for future applications in disease modelling and drug screening.

https://dgk.org/kongress_programme/jt2022/aP790.html