The impact of oxidative stress in zebrafish cardiac looping.

ARMESTO, RUBINA; HERRERA, IDALIA; BINOLFI, ANDRES; LOMBARDO, VERÓNICA A.

Rosario

Congreso; L Reunión Anual de la Sociedad Argentina de Biofísica.; 2022

Zebrafish (Danio rerio) is a small vertebrate used as a model to study heart development and human cardiac diseases(1). Cardiac looping is a highly conserved phase of heart morphogenesis in vertebrates in which the heart tube loops to give an “S” shape heart(3). Alterations in this process are associated with congenital heart defects(4). An increase of reactive oxygen species produce the constitutive activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) associated with cardiac diseases. This activation requires the oxidation of methionine residues within the regulatory region of the enzyme and is reverted by Methionine Sulfoxide Reductase A (MSRA)(5). The aim of this work is to analyze how oxidative stress affects cardiac looping and how it modulates the expression and regulatory interaction of CamKII and MSRA in cardiac tissue. Thus, we used a zebrafish reporter line [Tg(myl7:eGFP)twu34](6), that expresses GFP protein within the myocardium, to analyze the morphogenesis of embryonic hearts under normal and oxidative stress conditions. We observed that oxidative treatment interfere with the normal cardiac looping in a dose-dependent manner, and we measured the looping extension by two previously reported parameters(7). To analyze the expression levels of CamKII and MSRA by quantitative polymerase chain reaction (qPCR), we first performed an in silico analysis of zebrafish camk2 and msra genes and found seven isoforms for camk2 (α, β1, β2, δ1, δ2, γ1, γ2), each of them generating splicing variants, and one gene for msra, with two splice variants. Then, we carried out qPCR and immunodetection assays to study their expression levels during cardiac looping morphogenesis in both cardiac tissue and whole embryos. Interestingly, we observed a 30-times increase of camk2d2 transcript level. Altogether, these findings contribute to understand the role of oxidative regulation during normal and abnormal cardiac development. 1) Leong, et al., Acta Physiol., 2010, 199, 257-76. 2) Bakkers, 2011. 3) Ramsdell, A.F., Left-right asymmetry and congenital cardiac defects: getting to the heart of the matter in vertebrate left-right axis determination. Dev Biol, 2005. 288(1): p. 1-20. 4) Schleich, et al, Arch. Cardiovasc. Dis., 2013, 106, 612-23. 5) Erickson, et al., Cell, 2008, 133, 462-74. 6) Huang et al., Dev Dyn., 2003, 228 (1), 30-40. 7) Lombardo et al., Development 2019, 146 (22).