IP₃-mediated Ca²⁺ release regulates atrial Ca²⁺ transients and pacemaker function by stimulation of adenylyl cyclases

Figures

• 

  Figure 1.. IP3R2 colocalizes with AC8 in guinea pig atrial myocytes. A–C: representative examples of fixed, isolated guinea pig atrial myocytes labeled for IP3R1 (A), IP3R2 (B), IP3R3 (C), and negative controls (D). E: representative example of a fixed, isolated guinea pig atrial myocyte co-immunolabeled for IP3R2 (red) and AC8 (green). F: digital zoom of the area indicated on image D. G: intensity plot to show staining intensity along the line shown in E. H: representative example of a fixed, isolated guinea pig atrial myocyte co-immunolabeled for IP3R2 (red) and AC1 (green). I: digital zoom of the area indicated on image G. J: intensity plot to show staining intensity along the line shown in H. IP3R1, inositol trisphosphate receptor type 1; IP3R2, inositol trisphosphate receptor type 2; IP3R3, inositol trisphosphate receptor type 3.

• 

  Figure 2.. The direct actions of IP3 in guinea pig atrial myocytes require adenylyl cyclases and PKA. Ai: montage to show the upstroke of a control Ca2+ transient in PSS illustrating the classical U-shaped activation pattern. Numbers indicate time in ms from start of calcium transient upstroke. Aii: pseudolinescan image generated across the line drawn in Ai to show the U-shaped calcium transient seen in guinea pig atrial myocytes. Vertical scale bar indicates 5 µm. B: summary data to show cellular responses to IP3 photorelease (0.5 µmol/L caged-IP3/PM loaded for 1 h) in isolated guinea pig atrial myocytes under control conditions (n = 16) and in the presence of 1 µmol/L H89 (n = 9) to inhibit PKA, 3 µmol/L MDL (n = 6) to inhibit adenylyl cyclases, 10 µmol/L ODQ (n = 10) to inhibit soluble guanylyl cyclase, and 100 µmol/L L-NAME (n = 4) to inhibit nitric oxide synthase. *significant difference in comparison to IP3 photorelease alone (P < 0.05, ANOVA with Dunnett’s post hoc test). C: representative Ca2+ transient response to photorelease of IP3 in PSS under control conditions and at indicated timepoints after photorelease. D–G: representative Ca2+ transients under control conditions and 120 s…

• 

  Figure 3.. The positive chronotropic effect of PE requires function of adenylyl cyclases and a proposed scheme for regulation of intracellular calcium via IP3 signaling. A: dose-response curves to show the change in beating rate on cumulative addition of PE to spontaneously beating murine right atrial preparations under control conditions (n = 10) and in the presence of either 2-APB (IP3R inhibitor, 2.5 µmol/L, n = 7), MDL (AC inhibitor, 1 µmol/L, n = 5), ST034307 (AC1 inhibitor, 1 µmol/L, n = 8), H89 (PKA inhibitor, 1 µmol/L, n = 5), U73122 (PLC inhibitor, 5 µmol/L, n = 5), L-NAME (NOS inhibitor, 100 µmol/L, n = 6), or ODQ (sGC inhibitor, 30 µmol/L, n = 5). Dose-response curves (solid lines) were fit with log(agonist) versus response (three-parameter model) using GraphPad Prism 8. *significant reduction in maximum response of the fitted curve by ANOVA in comparison to PE. B: comparison of beating rate change in spontaneously beating murine atrial preparations from stable beating in PSS on addition of the inhibitors used in A, prior to stimulation by PE. Control indicates addition of β-blockade only as a time control (1 µmol/L metoprolol, n = 10). Other conditions are β-blockade plus…