Phosphorylation of phospholamban and troponin I through 5-HT4 receptors in the isolated human atrium

Our objective was to investigate the role of phosphodiesterase (PDE)3 and PDE4 and cGMP in the control of cAMP metabolism and of phosphorylation of troponin I (TnI) and phospholamban (PLB) when 5-HT 4 receptors are activated in pig left atrium. Electrically paced porcine left atrial muscles, mounted in organ baths, received stimulators of particulate guanylyl cyclase (pGC) or soluble guanylyl cyclase (sGC) and/or specific PDE inhibitors followed by 5-HT or the 5-HT 4 receptor agonist prucalopride. Muscles were freezeclamped at different moments of exposure to measure phosphorylation of the cAMP/protein kinase A targets TnI and PLB by immunoblotting and cAMP levels by enzyme immunoassay. Corresponding with the functional results, 5-HT only transiently increased cAMP content, but caused a less quickly declining phosphorylation of PLB and did not significantly change TnI phosphorylation. Under combined PDE3 and PDE4 inhibition, the 5-HT-induced increase in cAMP levels and PLB phosphorylation was enhanced and sustained, and TnI phosphorylation was now also increased. Responses to prucalopride per se and the influence thereupon of PDE3 and PDE4 inhibition were similar except that responses were generally smaller. Stimulation of pGC together with PDE4 inhibition increased 5-HT-induced PLB phosphorylation compared to 5-HT alone, consistent with functional responses. sGC stimulation hastened the fade of inotropic responses to 5-HT, while cAMP levels were not altered. PDE3 and PDE4 control the cAMP response to 5-HT 4 receptor activation, causing a dampening of downstream signalling. Stimulation of pGC is able to enhance inotropic responses to 5-HT by increasing cAMP levels, while sGC stimulation decreases contraction to 5-HT cAMP independently.

Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

Section: Western Blot Analysismentioning

confidence: 99%

mentioning

confidence: 99%

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Influence of phosphodiesterases and cGMP on cAMP generation and on phosphorylation of phospholamban and troponin I by 5-HT4 receptor activation in porcine left atrium

Weninger

Maeyer

Lefebvre

2013

“…Correspondingly, Gergs et al (2009) provided evidence for the PKA-dependent phosphorylation of PLB at Ser16 and of troponin I in human atrium. In addition, they also demonstrated phosphorylation of PLB at Thr17, not a substrate for PKA but a substrate for calcium calmodulin kinase II (CaMKII).…”

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confidence: 90%

Surprises from a cardiac 5-HT4 TG mouse: spontaneous atrial arrhythmias by endogenous 5-HT of atrial origin? Different mechanism of arrhythmias through 5-HT4 receptors and β-adrenoceptors?

Kaumann

2013

5-HT 4 receptors of human myocardium mediate cardiostimulant effects of 5-hydroxytryptamine (5-HT, serotonin) mainly through the G s protein→cAMP→PKA pathway. Several 5-HT 4 receptor splice variants are expressed in human heart but functional differences have not yet been detected (Reviewed in Kaumann and Levy 2006). 5-HT does not only increase myocardial force and hasten relaxation Sanders and Kaumann 1992;Brattelid et al. 2004), but can also elicit arrhythmic contractions in atrial trabeculae (Kaumann and Sanders 1994) and myocytes (Sanders et al. 1995) as well as in ventricular trabeculae (Brattelid et al. 2004). It has been proposed that 5-HT can initiate atrial fibrillation (Kaumann 1994) and facilitate arrhythmias in patients with ischaemic heart disease (Brattelid et al. 2004). Chronic treatment of patients with β-blockers potentiates the inotropic effects of 5-HT in atrial trabeculae and myocytes (Sanders et al. 1995). 5-HT increases the L-type Ca 2+ current (I Ca,L )in human atrial myocytes in a PKA-dependent manner (Ouadid et al. 1992). Chronic treatment of patients with β-blockers potentiates the effects of 5-HT on I Ca,L and arrhythmic activity of atrial myocytes (Pau et al. 2003). 5-HT causes also cardiostimulation in porcine heart (Kaumann 1990;Parker et al. 1994;Brattelid et al. 2004), but not in other non-primate species, at least under physiological conditions. Arrhythmias, probably mediated through 5-HT 4 receptors, have also been reported in porcine atrium (Rahme et al. 1999). Rat ventricular 5-HT 4 receptor expression and function is induced in rats with ischaemic heart failure but not in normal rats (Qvigstad et al. 2005), plausibly by reactivating a phenotype resembling late foetal development (Brattelid et al. 2012).Gergs et al. (2010) overexpressed human 5-HT 4a receptors in the heart of mice that normally do not express functional cardiac 5-HT 4 receptors. In these transgenic mice 5-HT produced sinoatrial tachycardia, increases in ventricular I Ca,L and Ca 2+ transients, positive inotropic and lusitropic effects in ventricle, associated with phosphorylation of phospholamban (PLB) at Ser16 in ventricular myocytes and Thr 17 in perfused working hearts. These features resemble the pharmacology of 5-HT 4 receptors in human heart. 5-HT, but hardly isoprenaline, also caused arrhythmic Ca 2+ transients in venricular myocytes from TG hearts. Interesting, in isolated hearts from these TG hearts Gergs et al. (2010) demonstrated episodes of spontaneous polymorphic atrial arrhythmias in the absence of exogenous 5-HT that resemble shortlasting episodes of atrial fibrillation.In an additional report of Gergs et al. (2013) in the present issue of Naunyn-Schmiedeberg's Archives of Pharmacology, the authors studied the effects of 5-HT on paced left atria and spontaneously beating right atria from their TG mouse. As expected, 5-HT produced sinoatrial tachycardia and increases in left atrial force, left atrial arrhythmias, phosphorylation of PLB at Ser16 and arrhythmias, which were antagonised by 5-HT 4 re...

Agents increasing cyclic GMP amplify 5-HT4-elicited positive inotropic response in failing rat cardiac ventricle

Afzal

Qvigstad

Aronsen

et al. 2011

Activation of 5-HT4 receptors in failing ventricles elicits a cAMP-dependent positive inotropic response which is mainly limited by the cGMP-inhibitable phosphodiesterase (PDE) 3. However, PDE4 plays an additional role which is demasked by PDE3 inhibition. The objective of this study was to evaluate the effect of cGMP generated by particulate and soluble guanylyl cyclase (GC) on the 5-HT4-mediated inotropic response. Extensive myocardial infarctions were induced by coronary artery ligation in Wistar rats, exhibiting heart failure 6 weeks after surgery. Contractility was measured in left ventricular preparations. Cyclic GMP was measured by EIA. In ventricular preparations, ANP or BNP displayed no impact on 5-HT4-mediated inotropic response. However, CNP increased the 5-HT4-mediated inotropic response as well as the β1-adrenoceptor (β1-AR)-mediated response to a similar extent as PDE3 inhibition by cilostamide. Pretreatment with cilostamide eliminated the effect of CNP. Inhibition of nitric oxide (NO) synthase and soluble GC by l-NAME and ODQ, respectively, attenuated the 5-HT4-mediated inotropic response, whereas the NO donor Sin-1 increased this response. The effects were absent during PDE3 inhibition, suggesting cGMP-dependent inhibition of PDE3. However, in contrast to the effects on the 5-HT4 response, Sin-1 inhibited whereas l-NAME and ODQ enhanced the β1-AR-mediated inotropic response. cGMP generated both by particulate (NPR-B) and soluble GC increases the 5-HT4-mediated inotropic response in failing hearts, probably through inhibition of PDE3. β1-AR and 5-HT4 receptor signalling are subject to opposite regulatory control by cGMP generated by soluble GC in failing hearts. Thus, cGMP from different sources is functionally compartmented, giving differential regulation of different Gs-coupled receptors.