Presented at the Neonatal Society 2011 Spring Meeting.
Tranter MH1, Hyde MJ2, Clarke L3, Kemp PR4, Harding SE1, Lyon AR1
1 Dept. of Cardiac Medicine, National Heart and Lung Institute, Imperial College, London, UK
2 Dept. of Neonatal Medicine, Section of Paediatrics, Imperial College, London, UK
3 Section of Comparative Medicine, Imperial College, London, UK
4 Dept. of Molecular Medicine, National Heart and Lung Institute, Imperial College, London, UK
Background: It has previously been reported that vaginal delivery (VD) leads to a change in hepatic gene expression compared with caesarean section (CS) at 7 days post partum in piglets (1). It has been hypothesised that the “stress” hormonal milieu of normal labour and VD is an important priming event for normal development of hepatic metabolism, the immune system, and the transition from the fetal to the neonatal phenotype. Regulation of calcium cycling is maintained by inhibitory action of phospholamban (PLB) on the sarcoplasmic reticulum protein SERCA. Through phosphorylation of the monomer of PLB (which is the active form, as opposed to the relatively inactive pentamer (2)) at the serine-16 residue this inhibition is lifted and the SERCA protein activity is increased; myocardial beta-adrenoreceptor activation by catecholamines increases protein kinase A activity and PLB-serine-16 phosphorylation. We studied whether the stress at birth causes changes in the levels of PLB expression and phosphorylation as a reflection of cardiac stimulation by catecholamines.
Methods: Piglets were euthanized at birth, following either CS delivery (day 112 of gestation; n=4) or VD (day 114; n=4). Further groups of piglets, born by CS (day 112; n=4) and by VD (day 114; n=4), underwent bilateral jugular catheterisation (for blood sampling purposes) before being fed milk replacement formula ad libitum; on day 7 post-partum they were euthanized. Hearts from these animals were collected and frozen in liquid nitrogen. These piglets have been previously described in the study by Hyde et al (1); all studies received Home Office approval. Apical myocardial samples from the hearts were homogenised under reducing conditions and protein content quantified using a modification of the Lowry method.These proteins were separated on a 10% acrylamide gel (140V for 1 hour). After transfer to a PVDF membrane and blocking, blots were probed for PLB (total (PLB), serine-16 phosphorylated phospholamban (PLB-P)), and calsequestrin with detection by chemiluminesence using a peroxidase conjugated secondary antibody. The images were quantified and the values for PLB and PLB-P normalised to that for calsequestrin for the same sample; the PLB-P/PLB ratio was calculated from these normalised values. Plasma triiodothyronine (T3) concentrations were measured at day 7 post-partum using a radio-immuno assay (MP Biomedicals T3 RIA Kit, MP Biomedicals, UK).
Results: At birth the myocardial monomeric PLB-P/PLB ratio was significantly greater in the VD samples (n=4) compared with the CS samples (n=4) (VD=0.86±0.08; CS=0.47±0.08 (p<0.05)). The levels of monomeric, pentameric or total PLB levels were not significantly different at this time point. At 7 days post-partum however there was a significant increase in the levels of monomeric PLB in the VD samples (n=4) compared with the CS samples (n=4) (VD=16.2±2.32; CS=4.31±0.44 (p<0.05)). There was also a statistically significant increase in total PLB levels in the VD samples (n=4) compared with the CS samples (n=4) at 7 days post-partum (VD=24.2±4.43; CS=7.17±0.63 (p=0.03)). Also in the 7 days post-partum group there is a significantly higher T3 serum concentration in VD piglets compared with CS piglets (VD=1.62±0.37; CS=0.62±0.16 nmol.L-1 (p<0.05)).
Conclusion: Neonatal hearts have higher levels of phosphorylated PLB following VD compared with CS, suggesting higher sympathetic drive and beta-adrenoreceptor activation, consistent with a ‘high stress’ birth compared with that of the ‘low stress’ CS birth. These changes are associated with increased protein expression of PLB, and an increase in levels of the active monomeric PLB fraction, at 7 days post-partum. Increased T3 levels are known to control SERCA activity by up-regulating SERCA protein and down-regulating PLB (3, 4). However, T3 was raised at 7 days post-partum and this therefore does not provide an explanation for the changes in PLB seen.
Corresponding author: firstname.lastname@example.org
1. Hyde MJ, Griffin JL, Herrera E, Byrnes CD, Clarke L, Kemp PR. Clin Sci 2010; 118:47-59
2. Autry JM, Jones LR. J Biol Chem 1997; 272: 15872-15880
3. Kiss E, Jakab G, Kranias EG, Edes I. Circ Res 1994; 75:245-251
4. Trivieri MG, Oudit GY, Sah R, Kerfant BG, Sun H et al. PNAS 2006; 103:6043-6048