Abstract
Heart development is one of the earliest developmental events, and its pumping action is directly linked to the intensity of development of other organs. Heart contractions mediate the circulation of the nutrients and signalling molecules to the focal points of developing embryos. In the present study, we used in vivo, ex vivo, in vitro, and in silico methods for chick embryo model to characterize and identify molecular targets under the influence of ectopic nitric oxide in reference to cardiogenesis. Spermine NONOate (SpNO) treatment of 10 μM increased the percentage of chick embryos having beating heart at 40th h of incubation by 2.2-fold (p < 0.001). In an ex vivo chick embryo culture, SpNO increased the percentage of embryos having beats by 1.56-fold (p < 0.05) compared with control after 2 h of treatment. Total body weight of SpNO-treated chick embryos at the Hamburger and Hamilton (HH) stage 29 was increased by 1.22-fold (p < 0.005). Cardiac field potential (FP) recordings of chick embryo at HH29 showed 2.5-fold (p < 0.001) increased in the amplitude, 3.2-fold (p < 0.001) increased in frequency of SpNO-treated embryos over that of the control group, whereas FP duration was unaffected. In cultured cardiac progenitors cells (CPCs), SpNO treatment decreased apoptosis and cell death by twofold (p < 0.001) and 1.7-fold (p < 0.001), respectively. Transcriptome analysis of chick embryonic heart isolated from HH15 stage pre-treated with SpNO at HH8 stage showed upregulation of genes involved in heart morphogenesis, heart contraction, cardiac cell development, calcium signalling, structure, and development whereas downregulated genes were enriched under the terms extracellular matrix, wnt pathway, and BMP pathway. The key upstream molecules predicted to be activated were p38 MAPK, MEF2C, TBX5, and GATA4 while KDM5α, DNMT3A, and HNF1α were predicted to be inhibited. This study suggests that the ectopic nitric oxide modulates the onset of cardiac development.
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Acknowledgements
We are thankful to SciGenom Labs (Kochi, India) for the transcriptome sequencing. P.K. is thankful for the RUBICON programme, Erasmus MC for IPA® software support.
Funding
This work was supported by a grant from University Grant Commission Faculty Recharge Programme (UGC-FRP), Government of India, to S.C. P.K. received financial support from University Grant Commission-Junior Research Fellowship (UGC-JRF) programme, Government of India. L.S. received financial support from Department of Biotechnology-Junior Research Fellowship (DBT-SRF) programme, Government of India. Pr.K. received financial support from Lady Tata Memorial Trust-Senior Research Fellowship (LTMT-SRF), India.
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P.K., A.G., and Pr.K. performed the experiments. P.K., Pr.K., and L.K. prepared the manuscript. P.K., A.G., Pr.K., and L.K. analyzed the data. K.S. supervised the electrophysiology experiments. S.C. supervised the experiments and approved the manuscript. All authors reviewed the manuscript.
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The authors declare that the research was conducted with prior approval from the Institutional Biosafety and Ethical Committee (IBEC) of the AU-KBC Research Centre as per Annexure I; Project 02 (14-11-2011).
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Kumar, P., Ghosh, A., Sundaresan, L. et al. Ectopic release of nitric oxide modulates the onset of cardiac development in avian model. In Vitro Cell.Dev.Biol.-Animal 56, 593–603 (2020). https://doi.org/10.1007/s11626-020-00495-w
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DOI: https://doi.org/10.1007/s11626-020-00495-w