Inhibition of adenylyl cyclase 1 by ST034307 inhibits IP3-evoked changes in sino-atrial node beat rate

Figures

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  FIGURE 1. 1 µM ST034307 inhibits changes in chronotropy in intact mouse right atria with intact SAN but not inotropy in left atria. (A) Dose response curves to show the change in beating rate on cumulative addition of PE to spontaneously beating mouse right atria preparations under control conditions (circles, n = 15) and in the presence of 1 µM ST034307 (squares, n = 12). (B) Dose response curves to show the change in tension generated on cumulative addition of PE to mouse left atria preparations under control conditions (circles, n = 5) and in the presence of 1 µM ST034307 (squares, n = 7). Dose-response curves in (A,B) (solid lines) were fitted using log(agonist) vs. response (three-parameter model) using Graphpad Prism v9. Asterisks indicate significance level for effect of ST034307 compared to control at individual concentrations as determined using 2-way repeated measures ANOVA followed by Šídák’s multiple comparisons test. Data are represented as mean ± SEM; ns = not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

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  FIGURE 2. IP3R2 is expressed in close proximity with AC1 in guinea pig SAN myocytes. (A) Representative example of a fixed, isolated guinea pig SAN myocyte immunolabelled for (i) IP3R2 (magenta), (ii) AC1 (cyan), (iii) co-immunolabelled for IP3R2 (magenta) and AC1 (cyan). Example brightfield image shown in (iv). (B) Intensity plot to show staining intensity along the white line shown in (A). R-value calculated from n = 7 cells. (C–E) Intensity surface plots showing the distribution of staining of IP3R2 [magenta, (D)] and AC1 [cyan, (E)] for the regions of cell shown in (C). Scale bars representing 10 µm are indicated in (A) and 2 µm in (C). For the purposes of presentation only, red and green channels have been represented as magenta and cyan, respectively.

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  FIGURE 3. 1 μM ST034307 does not alter the effect of PE on Ca2+ transients in isolated guinea pig atrial myocytes. (A,B): Representative average Ca2+ transients recoded from atrial myocytes during baseline recording (black trace) and following addition of PE (grey trace) in the absence (B) or presence (C) of 1 μM ST034307. (C): Effect of 10 μM PE on the Ca2+ transient amplitude recorded from isolated guinea pig atrial cells (n = 6; N = 3). (D): Effect of 1 μM ST034307 on the basal Ca2+ transient amplitude before addition of PE (PE, n = 18; ST, n = 15; N = 3). (E): Effect of 10 μM PE on the Ca2+ transient amplitude of cells in the presence of 1 μM ST034307 (n = 15; N = 3). (F,G): Effect of 10 μM PE on the 90–10% decay time (F) and 10–90% rise time (G) of Ca2+ transients recorded from cells perfused with PSS in the absence (black bars) or presence (grey bars) of 1 μM ST034307 before and after addition of 10 μM PE. All experiments were carried out at 35 ± 2°C and recordings were made 5 min following the start of each solution perfusion. Data are represented as mean ± SEM. Data in C and E were analysed using paired t-test. Data in D were analysed using unpaired t-test. Data in F and G were…

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  FIGURE 4. 1 µM ST034307 inhibits the basal spontaneous beating rate and response to PE of isolated guinea pig SAN cells. (A) Brightfield image to show example of an isolated guinea pig SAN cell as indicated by black arrow. Scale bar represents 10 µm. (B) Effect of 1 µM ST034307 on the basal SAN cell Ca2+ transient amplitude before addition of PE. (C,D) Representative 4 s recordings of changes in fluorescence (expressed as (F-F0)/F0) from spontaneously beating guinea pig SAN cells loaded with Fluo-5F-AM in PSS (C) or in PSS +1 µM ST034307 (D). Black and red/blue traces represent recordings before and 5 min after addition of 10 μM PE, respectively. Traces have been aligned according to the peak of the first transient. (E) Effect of 10 μM PE on the spontaneous beating rate of isolated SAN cells in the presence (squares) and absence (circles) of 1 µM ST034307. (F) Effect of 10 μM PE on the Ca2+ transient amplitude recorded from isolated SAN cells in the presence (squares) an absence (circles) of 1 µM ST034307. Time in E and F represents time following addition of PE to the perfusion solution with 0s representing levels in absence of PE. All experiments were carried out at 35 ± 2°C. Data are…