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.

Figure 1

Urocortin 2 (Ucn2) elicits positive inotropic and lusitropic effects in mouse ventricular myocytes. (A) Fractional shortening (FS, % of resting cell length, RCL) 0, 5, 10, 15 and 20 min after application of 100 nM Ucn2. Asterisks indicate two extra shortenings not elicited by the electrical stimulation. (B) Direct comparison of individual shortenings from (A) after 0 (a) and 20 (b) min of Ucn2 exposure. (C) Time course of FS (normalized to the initial control) in untreated control myocytes (Ctrl, n = 5 myocytes from three mouse hearts) and in myocytes challenged with 100 nM Ucn2 (n = 10 myocytes from five mouse hearts). **P < 0.01 versus 0 min Ucn2; ##P < 0.01 versus Ucn2; tested by two-way anova for repeated measures, followed by Tukey's range test. (D) Maximal velocities of shortening and relengthening in untreated control cells (Ctrl, n = 5 myocytes from three mouse hearts) and in myocytes treated with 100 nM Ucn2 in the absence (Ucn2, n = 10 myocytes from five mouse hearts) and presence of 10 nM antisauvagine-30 (+ASV, n = 6 myocytes from five mouse hearts). **P < 0.01 and *P < 0.05 versus Ctrl; #P < 0.05 versus Ucn2; tested by one-way anova followed by Tukey's range test. (E) Concentration-response curve of the Ucn2-induced increase in FS. Values were obtained from 3–9 ventricular myocytes from 3–5 mouse hearts. Line is a fit of the Hill equation to the Ucn2 data yielding an EC₅₀ of 19 nM Ucn2 and a Hill coefficient of 3.2. In the presence of 10 nM antisauvagine-30 (+ASV, n = 6 myocytes from five mouse hearts), the Ucn2 effect was blocked. #P < 0.05 versus Ucn2 alone, tested by unpaired Student's t-test.

Figure 2

The positive inotropic and lusitropic effects of urocortin 2 (Ucn2) are Ca²⁺-dependent. (A) Original recordings of [Ca²⁺]_i transients, measured as normalized Fluo-4 fluorescence, F/F₀ (top) and fractional shortening (FS), measured as % of resting cell length (bottom) 0, 5, 10 and 15 min after application of 100 nM Ucn2. (B) Systolic [Ca²⁺]_i and kinetics of [Ca²⁺]_i transients and (C) FS and shortening kinetics in untreated control myocytes (Ctrl, n = 10 myocytes from eight mouse hearts) and in myocytes exposed to 100 nM Ucn2 (n = 19 myocytes from 10 mouse hearts). [Ca²⁺]_i transient decay and relaxation were quantified by the time constant τ of fluorescence decrease and the time required from peak shortening to 50% of relengthening (RT50), respectively, *P < 0.05 versus 0 min Ucn2; **P < 0.01 versus 0 min Ucn2, tested by paired Student's t-test.

Figure 4

Urocortin 2 (Ucn2) increases [Ca²⁺]_i transients in Indo-1-loaded myocytes in an H89-sensitive manner. (A) Systolic (left) and diastolic (right) [Ca²⁺]_i, measured as the ratio F405/F485 of the Indo-1 fluorescence signal in myocytes left untreated (Ctrl, n = 13 myocytes from three mouse hearts), treated with 100 nM Ucn2 (n = 15 myocytes from four mouse hearts), treated with 2 µM H89 (H89, n = 7 myocytes from three mouse hearts), or treated with 100 nM Ucn2 in the presence of 2 µM H89 (+Ucn2, n = 10 myocytes from three mouse hearts). (B) Amplitude, that is systolic minus diastolic Indo-1 fluorescence ratio, of the [Ca²⁺]_i transients in the same myocytes. **P < 0.01 versus Ctrl, H89, and +Ucn2, tested by one-way anova followed by Tukey's range test.

Figure 3

KT5720 and KN93 abolish the positive inotropic effect of urocortin 2 (Ucn2). (A) Original recordings of Fluo-4 fluorescence (i.e. [Ca²⁺]_i transients; top) and cell shortenings (bottom) of myocytes treated with 100 nM Ucn2 in the absence (Ucn2) and presence of 1 µM KT5720 (KT + Ucn2) or 1 µM KN93 (KN + Ucn2). Recordings were obtained after 0 and 15 min of Ucn2 exposure and have been normalized to the amplitude at 0 min. Average values of systolic [Ca²⁺]_i (B) and fractional shortening (FS) (C) obtained after 0 and 15 min of Ucn2 exposure in the absence (Ucn2, red, n = 19) or presence of 1 µM KT5720 (KT + Ucn2, n = 14) or 1 µM KN93 (KN + Ucn2, n = 6) or in time-matched controls (Ctrl, n = 10). Results from a total of 15 mouse hearts. **P < 0.01 versus 0 min Ucn2, tested by paired Student's t-test.

Figure 5

Urocortin 2 (Ucn2) increases phospholamban (PLN) phosphorylation at serine-16 in a time- and concentration-dependent manner. Original immunoblots of phosphorylated (P-PLN Ser-16) and total PLN following exposure to Ucn2 for various times (A) or at various concentrations (B). Average values of the ratio of P-PLN to total PLN from n = 3–8 independent experiments (i.e. mouse hearts) are shown in (C) and (D) respectively. Results with H89 (2 µM) were obtained from an additional series of experiments (n = 3 mouse hearts). *P < 0.05 versus 0 min Ucn2 (C) or Ctrl (D) respectively. #P < 0.05 versus 100 nM Ucn2 alone, tested by one-way anova followed by Tukey's range test.

Figure 6

Urocortin 2 (Ucn2) elicits arrhythmogenic events. (A) [Ca²⁺]_i signals (top), that is raw Fluo-4 fluorescence in arbitrary units (a.u.), fractional shortening (FS) (middle), and the electrical stimulation (bottom) following application of 100 nM Ucn2. [Ca²⁺]_i signals and shortening are rhythmic in the beginning and become progressively more irregular, that is arrhythmogenic, after 5–15 min Ucn2 exposure. The first extra [Ca²⁺]_i transient and shortening not caused by the electrical stimulation is marked by the asterisks. (B) Enlarged traces corresponding to the shaded areas a and b in (A). Extra [Ca²⁺]_i transients and shortenings are marked by the asterisks. (C) Arrhythmogenic events in untreated control myocytes (Ctrl, n = 18) and in myocytes treated with 100 nM Ucn2 in the absence (Ucn2, n = 20) and presence, respectively, of 1 µM KT5720 (+KT, n = 14) or 1 µM KN93 (+KN, n = 6). Results from a total of 17 mouse hearts. **P < 0.01 versus 0 min Ucn2; ##P < 0.01 and #P < 0.05 versus Ucn2; tested by two-way anova of repeated measures followed by Tukey's range test. (D) Cumulative number of arrhythmogenic events within the 15 min observation period in the same myocytes shown in (C). **P < 0.01 versus Ctrl, +KT and +KN, tested by one-way anova followed by Tukey's range test.

Figure 7

Urocortin 2 (Ucn2) elicits a positive inotropic effect and spontaneous contractions in human atrial trabeculae. (A) Tracings of twitch force of a human atrial trabecula before and during exposure to 300 nM Ucn2. Ucn2 increased twitch force by 15%. (B) Average values of developed force, normalized to the pre-Ucn2 control (=100%), in Ucn2-treated trabeculae (n = 10 trabeculae from seven atria) and in untreated, time-matched control trabeculae (Ctrl; n = 12 trabeculae from nine atria). **, P < 0.01 versus Ctrl, tested by unpaired Student's t-test. (C) Original chart recording of isometric contractions of an atrial trabecula before (left) and during (right) exposure to 100 nM Ucn2. Force recording is shown at two time scales. At the faster time scale, individual twitches are visible. Black circles indicate electrical stimulations. Asterisks mark spontaneous contractions not elicited by the electrical stimulation.