Effect of neuronal innervation on the properties of hPSC-derived cardiomyocytes

Abstract

The sympathetic division of the autonomic nervous system is known as the sympathetic nervous system (SNS) and is especially important for the involuntary control of heart rhythm. Specifically, ionotropic (force), chronotropic (rate) and dromotropic (conduction velocity) response in the heart is affected through autonomic innervation. The heart-brain axis has been studied extensively in vivo. Given this knowledge, conventionally immature human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) were used in coculture with sympathetic neurons (SN) isolated from neonatal rat pups, to model the neuro-cardiac junction (NCJ), and better understand this cell-cell interaction in vitro. IMR90 stem cells were differentiated in-house into hPSC-CM. The structural relationship was first investigated through confocal imaging and microscopy (SICM). The functional relationship was investigated using FRET microscopy, which measures changes in the important secondary messenger molecule (cAMP) of the CM. Contraction and calcium handling were investigated in cells paced at 1 Hz in a novel attempt, using the CytoCypher. Immunostaining showed that SN express a prominent marker of neuroexocytosis. They also fire action potentials in response to nicotine, indicating functional intercellular communication in coculture. Neurite sprouting increased over time but with no significant difference in the morphology of the NCJ. HPSC-CM in coculture became elongated, as shown by reduced cell circularity index. Functional investigation into this relationship found that cAMP levels in response to nicotine are significantly higher in cocultured CM compared to monocultures. Moreover, contraction and calcium transient amplitude was significantly larger in coculture and at baseline, prior to stimulation of SN with nicotine. Interestingly, the action potential dynamics of the IMR90 hPSC-CM was matured in coculture, with larger amplitude and greater sodium channel activity. This thesis highlights a critical role for the structural and functional development of hPSC-CM towards a more physiological condition when cocultured with SN in vitro.