Sympathetic neurons regulate cardiomyocyte maturation

Authors

• WJ Kowalski
• IH Garcia-Pak
• W Li
• H Uosaki
• E Tampakakis
• J Zou
• Y Lin
• K Patterson
• C Kwon
• Y-S Mukouyama

Abstract

Sympathetic neurons invade the heart at mid-gestation and vastly expand, regulating local cardiomyocyte function. Embryos devoid of innervation succumb to sudden death, between E13.5-E15.5 in mice. Our previous studies of embryos lacking autonomic nerves showed normal embryo features, despite cessation of heartbeat and blood coagulation. These results suggested lethal cardiac arrhythmia as a probable cause of death in innervation-null mutants. Despite this requirement for innervation, its role in maintaining cardiac function has been underexplored. In this study, we investigated the potential for sympathetic neurons (SNs) to influence cardiomyocyte (CM) maturation. Using an in vitro model, we co-cultured phenotypically immature CMs derived from human induced pluripotent stem cells with embryonic mouse SNs for 30 days. We found that co-culture with SNs significantly up-regulated expression of multiple genes important for CM maturation, encoding structural components, ion channels, and calcium machinery. CMs in co-culture with SNs had greater density of connexin-43 and improved sarcomere organization, hallmarks of a more mature phenotype. Calcium imaging additionally showed larger transient amplitudes in co-cultured CMs carrying a GCaMP6 reporter. Sarcomere spacing and relaxation time, however, were not improved. While intrinsic calcium release in response to caffeine stimulus was reduced, co-culture with SNs resulted in a more uniform response, which may be important for anti-arrhythmic protection. In further experiments, we found that additional, combined co-culture with endothelial and epicardial cells did not alter CM maturation beyond the effects of SNs. In total, our results demonstrate that sympathetic neurons have a significant and complex role in regulating CM development.

Scientific Focus Area: Developmental Biology

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