Background: Quantitative real-time RT-PCR (RT-qPCR) is a highly sensitive method for mRNA quantification, but requires invariant expression of the chosen reference gene(s). In pathological myocardium, there is limited information on suitable reference genes other than the commonly used Gapdh mRNA and 18S ribosomal RNA. Our aim was to evaluate and identify suitable reference genes in human failing myocardium, in rat and mouse post-myocardial infarction (post-MI) heart failure and across developmental stages in fetal and neonatal rat myocardium. Results: The abundance of Arbp, Rpl32, Rpl4, Tbp, Polr2a, Hprt1, Pgk1, Ppia and Gapdh mRNA and 18S ribosomal RNA in myocardial samples was quantified by RT-qPCR. The expression variability of these transcripts was evaluated by the geNorm and Normfinder algorithms and by a variance component analysis method. Biological variability was a greater contributor to sample variability than either repeated reverse transcription or PCR reactions. Conclusions: The most stable reference genes were Rpl32, Gapdh and Polr2a in mouse post-infarction heart failure, Polr2a, Rpl32 and Tbp in rat post-infarction heart failure and Rpl32 and Pgk1 in human heart failure (ischemic disease and cardiomyopathy). The overall most stable reference genes across all three species was Rpl32 and Polr2a. In rat myocardium, all reference genes tested showed substantial variation with developmental stage, with Rpl4 as was most stable among the tested genes.

Cardiac responsiveness to neurohumoral stimulation is altered in congestive heart failure (CHF). In chronic CHF, the left ventricle has become sensitive to serotonin because of appearance of G _s -coupled 5-HT ₄ receptors. Whether this also occurs in acute CHF is unknown. Serotonin responsiveness may develop gradually or represent an early response to the insult. Furthermore, serotonin receptor expression could vary with progression of the disease. Postinfarction CHF was induced in male Wistar rats by coronary artery ligation with nonligated sham-operated rats as control. Contractility was measured in left ventricular papillary muscles and mRNA quantified by real-time reverse-transcription PCR. Myosin light chain-2 phosphorylation was determined by charged gel electrophoresis and Western blotting. Ca ²⁺ transients in CHF were measured in field stimulated fluo-4-loaded cardiomyocytes. A novel 5-HT _2A receptor-mediated inotropic response was detected in acute failing ventricle, accompanied by increased 5-HT _2A mRNA levels. Functionally, this receptor dominated over 5-HT ₄ receptors that were also induced. The 5-HT _2A receptor-mediated inotropic response displayed a triphasic pattern, shaped by temporally different activation of Ca ²⁺ -calmodulin-dependent myosin light chain kinase, Rho-associated kinase and inhibitory protein kinase C, and was accompanied by increased myosin light chain-2 phosphorylation. Ca ²⁺ transients were slightly decreased by 5-HT _2A stimulation. The acute failing rat ventricle is, thus, dually regulated by serotonin through G _q -coupled 5-HT _2A receptors and G _s -coupled 5-HT ₄ receptors.

Cardiac contractility is regulated by several neural, hormonal, paracrine, and autocrine factors. Amongst these, signaling through β-adrenergic and serotonin receptors generates the second messenger cyclic AMP (cAMP), whereas activation of natriuretic peptide receptors and soluble guanylyl cyclases generates cyclic GMP (cGMP). Both cyclic nucleotides regulate cardiac contractility through several mechanisms. Phosphodiesterases (PDEs) are enzymes that degrade cAMP and cGMP and therefore determine the dynamics of their downstream effects. In addition, the intracellular localization of the different PDEs may contribute to regulation of compartmented signaling of cAMP and cGMP. In this review, we will focus on the role of PDEs in regulating contractility and evaluate changes in heart failure.

Human 5-hydroxytryptamine₇ (5-HT₇) receptors display characteristics shared with receptors believed to form a tight physical coupling with G protein in the absence of ligand. Some receptors apparently preassociated with G_i/o and G_q/11 are reported to inhibit the signaling of other similarly coupled G protein-coupled receptors by limiting their access to activate a common G protein pool. Therefore, we determined whether 5-HT₇ receptor expression was sufficient to limit signaling of endogenously expressed G_s-coupled receptors in human embryonic kidney (HEK) 293 cells. Using the ecdysone-inducible expression system, which allows for the titration of increasing receptor density in the same clonal cell line, we compared the effects of 5-HT_4(b) and 5-HT_7(a,b,d) receptor expression on adenylyl cyclase (AC) stimulation by the endogenous G_s-coupled β-adrenergic (βAR) and prostanoid EP (EPR) receptors. βAR- and EPR-stimulated AC activity was attenuated by 5-HT₇ receptor expression in both membrane preparations and intact HEK293 cells. βAR- and EPR-stimulated AC activity was unaffected by expression of the G_s-coupled 5-HT₄ receptor. The mechanism of this heterologous desensitization seems independent of protein kinase A activation, nor does it occur at the level of G protein activation because 1) βAR- and EPR-stimulated AC activity was not restored to control values when Gα_s was overexpressed; and 2) β₁AR and β₂AR activation of Gα_s was unaffected by the expression of 5-HT₇ receptors. In addition, overexpression of AC isoforms was unable to rescue βAR- and EPR-stimulated AC activity. Therefore, 5-HT₇ receptors probably limit access and/or impede activation of AC by βAR and EP receptors. Although the 5-HT₇ receptor may preassociate with G protein and/or AC, the mechanism of this heterologous desensitization remains elusive.