In addition, we found that both 5-HT2A and 5-HT2B receptor mRNAs were increased at the time of birth, but only the former mediated an inotropic response. The failing heart has been associated with a foetal genotype and the increased 5-HT4 gene expression and concomitant 5- HT4-mediated inotropic response to serotonin previously demonstrated in the ventricles of failing rat heart is also observed in late foetal cardiac development. Thus, the 5-HT4 mRNA expression level and the corresponding 5-HT4- mediated inotropic response accompany each other through the different developmental stages of the heart and in HF. The presence of robust 5-HT4 responses in the foetal/neonatal cardiac ventricle implies a role of the 5-HT4 receptor in cardiac development. The importance of their re-expression in failing ventricle has been evaluated in other studies. Other lines of evidence also suggest a functional role of the 5- HT4 receptor in the foetal heart. Offspring of female mice immunised with a peptide corresponding to the second extracellular loop of the human 5-HT4 receptor developed foetal and neonatal arrhythmia, as well as ataxia. However, the impact of this 5-HT4 receptor autoimmunity on cardiac function remains uncertain. Neonatal cardiomyocytes are an attractive model system to characterise several aspects of cardiac function at the cellular level. Importantly, since neonatal rat hearts express functional 5-HT4 receptors linked to inotropic effects, it should be further evaluated whether neonatal rat cardiomyocytes may serve as a useful model for cardiomyocytes from failing rat hearts as well as from the less SCH772984 supply available failing human hearts in studies of cardiac 5-HT4 receptor function. An even lower level of the 5-HT4 receptors in the ventricle and possible species differences complicates the ability to demonstrate the presence of cardiac 5-HT4 receptors with currently available methods. Although the ability to label new 5- HT4 receptor antagonists with even higher selectivity and affinity for the 5-HT4 receptor and lower non-specific binding might improve future binding assays, the density of 5-HT4 receptors in cardiac ventricle will remain low and likely near the limit of proper quantification. In a previous publication we have demonstrated similar slopes of standard curves used to determine 5-HT4 mRNA expression in hippocampus and cardiac left ventricle. The difference in crossing point were,8 Cp’s, representing approximately 250-fold difference in mRNA level between the hippocampus and heart. Hippocampus expresses about 120 fmol 5-HT4 receptors per mg protein. Assuming similar ratios between mRNA and protein, the correspondingproteinlevelofthe 5-HT4 receptorinthe heartcan be estimated to about 0.5 fmol per mg protein, which would be below the limit of detection with the radiolabeled ligands available. Since 5- HT4 receptor protein is clearly below the level of detection with available radioligand binding methods, the presence of the protein and its level of expression in this and previous studies had to be inferred from its functional effects on contractility. Since 5-HT4 receptor stimulation in previous studies was shown to produce a submaximal inotropic response compared to betaadrenoceptor stimulation, a reasonable correlation between receptor expression levels and the maximal 5-HT4-mediated inotropic response can be inferred, and we are therefore interpreting an increasing maximal 5-HT4-mediated inotropic response to reflect increasing 5-HT4 receptor protein levels.