cAMP‐ and Ca²⁺/calmodulin‐dependent protein kinases mediate inotropic, lusitropic and arrhythmogenic effects of urocortin 2 in mouse ventricular myocytes

Abstract

BACKGROUND AND PURPOSE Urocortin 2 is beneficial in heart failure, but the underlying cellular mechanisms are not completely understood. Here we have characterized the functional effects of urocortin 2 on mouse cardiomyocytes and elucidated the underlying signalling pathways and mechanisms. EXPERIMENTAL APPROACH Mouse ventricular myocytes were field‐stimulated at 0.5 Hz at room temperature. Fractional shortening and [Ca2+]i transients were measured by an edge detection and epifluorescence system respectively. Western blots were carried out on myocyte extracts with antibodies against total phospholamban (PLN) and PLN phosphorylated at serine‐16. KEY RESULTS Urocortin 2 elicited time‐ and concentration‐dependent positive inotropic and lusitropic effects (EC50: 19 nM) that were abolished by antisauvagine‐30 (10 nM, n= 6), a specific antagonist of corticotrophin releasing factor (CRF) CRF2 receptors. Urocortin 2 (100 nM) increased the amplitude and decreased the time constant of decay of the underlying [Ca2+]i transients. Urocortin 2 also increased PLN phosphorylation at serine‐16. H89 (2 µM) or KT5720 (1 µM), two inhibitors of protein kinase A (PKA), as well as KN93 (1 µM), an inhibitor of Ca2+/calmodulin‐dependent protein kinase II (CaMKII), suppressed the urocortin 2 effects on shortening and [Ca2+]i transients. In addition, urocortin 2 also elicited arrhythmogenic events consisting of extra cell shortenings and extra [Ca2+]i increases in diastole. Urocortin 2‐induced arrhythmogenic events were significantly reduced in cells pretreated with KT5720 or KN93. CONCLUSIONS AND IMPLICATIONS Urocortin 2 enhanced contractility in mouse ventricular myocytes via activation of CRF2 receptors in a cAMP/PKA‐ and Ca2+/CaMKII‐dependent manner. This enhancement was accompanied by Ca2+‐dependent arrhythmogenic effects mediated by PKA and CaMKII.

Urocortin 2 in cardiovascular health and disease

“…Another signaling pathway induced by CRH-R2 activation is Ca 2+ -calmodulin-CaMKII, which, along with PKA, is important for intracellular Ca 2+ homeostasis given that they have the ability to phosphorylate key Ca 2+ -regulating proteins like the L-type Ca 2+ channel, the ryanodine receptor and PLB [27]. The activation of these proteins leads ultimately to increased Ca 2+ influx, sarcoplasmic reticulum (SR) Ca 2+ content and accelerated [Ca 2+ ] i transients [27,28].…”

Intravenous Adeno-Associated Virus Serotype 8 Encoding Urocortin-2 Provides Sustained Augmentation of Left Ventricular Function in Mice

“…UCn2 peptide infusion increases contractile function independent of loading conditions in normal mice, indicating a direct cardiac effect (Bale et al 2004). The mechanisms for inotropic effects have not been defined, but studies suggest beneficial effects on Ca 2 + handling (Yang et al, 2011). The assumption is that the beneficial direct cardiac effects of UCn2 occur via activation of CRFR2 on cardiac myocytes.…”

Interactions of Enhanced Urocortin 2 and Mineralocorticoid Receptor Antagonism in Experimental Heart Failure

“…When CA was given in combination with Ucn2, the resulting hemodynamic responses were largely comparable with those produced by Ucn2 alone with marked improvements in CO in association with substantial reductions (a halving) in peripheral resistance and LAP-effects observed previously in this model of HF. [12][13][14][15] Although the Ucn2-induced increase in CO is presumably in part a consequence of the large falls in peripheral resistance, with the peptide shown to directly reduce vascular tone, 9 Ucn2 also exhibits potent inotropic activity, 10,44 as evidenced here by the concurrent rise in dP/dt max . The reductions in LAP are probably secondary to the rise in CO, although the peptide is reported to have lusitropic 10,11 and venodilator 45 activity, which may also have contributed.…”

“…[12][13][14][15] Although the Ucn2-induced increase in CO is presumably in part a consequence of the large falls in peripheral resistance, with the peptide shown to directly reduce vascular tone, 9 Ucn2 also exhibits potent inotropic activity, 10,44 as evidenced here by the concurrent rise in dP/dt max . The reductions in LAP are probably secondary to the rise in CO, although the peptide is reported to have lusitropic 10,11 and venodilator 45 activity, which may also have contributed. Importantly, not only were the beneficial hemodynamic actions of Ucn2 not inhibited by concurrent MR antagonism, but the combination therapy negated the fall in blood pressure seen with CA alone for most of the infusion period and well on into the postinfusion follow-up.…”

“…Ucn2 is reported to bind with high affinity to the G-protein-coupled CRF receptor subtype CRF 2 , 8 which is localized in diverse tissues throughout the brain and periphery (with strong expression demonstrated in the heart and vasculature). 9 This receptor is thought to mediate the majority of actions seen with systemically administered peptide, including direct vasodilation and positive cardiac chronotropic and lusitropic activity 9,10 -effects which have identified Ucn2 as a potential therapeutic agent in heart disease. Several studies have shown that Ucn2 improves cardiovascular and renal function in experimental HF, in association with suppression of a number of vasoconstrictor/volume-retaining hormone systems (including the RAAS).…”