Myocardium expression of connexin 43, SERCA2a, and myosin heavy chain isoforms are preserved in nitrofen-induced congenital diaphragmatic hernia rat model

Abstract

Background Previous morphological studies had produced controversial results with regard to heart development in congenital diaphragmatic hernia (CDH), whereas a few publications investigated cardiac function and myocardial maturation. Myocardium maturation is associated with age-dependent increasing of gene expression of gap junction protein connexin 43 (Cx43), adenosine triphosphatase of the sarcoplasmic reticulum (SERCA2a), as well as switching of myosin heavy chains (MHCs) from β to α isoforms. Our aim was to evaluate myocardium maturity in nitrofen-induced CDH rat model. Methods Fetuses from dated pregnant Sprague-Dawley rats were assigned to 3 experimental groups: control, nitrofen (exposed to nitrofen, without CDH), and CDH (exposed to nitrofen, with CDH). Myocardial samples collected from left ventricle free wall were processed to (i) quantification of messenger RNA (mRNA) of Cx43, SERCA2a, α and β MHC isoforms, as well as β-actin (housekeeping gene); and (ii) separation of MHC isoforms ( α and β isoforms) by sodium dodecyl sulfate polyacrylamide gel electrophoresis silver stained. Results We demonstrated that there is no difference in myocardial gene expression of Cx43 (control, 1.0 ± 0.1; nitrofen, 1.1 ± 0.2; CDH, 1.3 ± 0.2) and SERCA2a (control, 1.0 ± 0.1; nitrofen, 0.9 ± 0.1; CDH, 1.0 ± 0.2). Myocardial gene expressions of α and β mRNA of MHC isoforms were slightly decreased both in nitrofen and CDH fetuses when compared with control fetuses, but evaluation of the α-to- β ratios of MHC isoforms at protein level revealed no significant differences between CDH and control (control, 16.9 ± 2.5; CDH, 17.0 ± 2.0). Conclusions Myocardial quantification of Cx43 and SERCA2a mRNA, as well as the expression pattern of MHC isoforms at protein levels, was similar in all studied groups. We predict, therefore, that acute heart failure commonly observed in CDH infants might be attributed predominantly to cardiac overload secondary to severe pulmonary hypertension rather than to myocardial immaturity.