cAMP Compartmentalisation in Human Myometrial Cells

Abstract

Preterm birth is the leading cause of childhood mortality and morbidity. A better understanding of the processes that drive the onset of human labour is essential to reduce the adverse perinatal outcomes associated with dysfunctional labour. Beta-mimetics, which activate the myometrial cyclic adenosine monophosphate (cAMP) system, successfully delay preterm labour, suggesting a key role for cAMP in the control of myometrial contractility; however, the mechanisms underpinning this regulation are incompletely understood. Here we used genetically encoded cAMP reporters to investigate cAMP signalling in human myometrial smooth muscle cells at the subcellular level. We found significant differences in the dynamics of the cAMP response in the cytosol and at the plasmalemma upon stimulation with catecholamines or prostaglandins, indicating compartment-specific handling of cAMP signals. Our analysis uncovered significant disparities in the amplitude, kinetics, and regulation of cAMP signals in primary myometrial cells obtained from pregnant donors compared with a myometrial cell line and found marked response variability between donors. We also found that in vitro passaging of primary myometrial cells had a profound impact on cAMP signalling. Our findings highlight the importance of cell model choice and culture conditions when studying cAMP signalling in myometrial cells and we provide new insights into the spatial and temporal dynamics of cAMP in the human myometrium.

Keywords: cAMP; hTERT-HM cells; myometrium; phosphodiesterases; pregnancy; signalling compartmentalisation.

Figure 1

Localisation of EPAC-S^H187 and AKAP79-CUTie sensors in HPMCs. YFP emission (shown in greyscale) and corresponding representative schematics demonstrating the localisation of EPAC-S^H187 sensor (A) and AKAP79-CUTie sensor (B) in the HPMCs. For FRET measurements, grey values were averaged within regions of interest after subtraction of grey values averaged within the background region.

Figure 2

Comparison of the cAMP peak response in the hTERT-HM cells and HPMCs in the cytoplasm and plasmalemma following isoproterenol or PGE2 stimulation. FRET responses in hTERT-HM cells or HPMCs expressing the cytosolic (blue) Epac-S^H187 sensor to (A) ISO treatment or (B) prostaglandin treatment. FRET responses in hTERT-HM cells or HPMCs expressing the plasmalemma (pink) AKAP79-CUTie sensor to (C) ISO treatment or (D) prostaglandin treatment. For hTERT-HM, each data point (circles) represents one cell (measurements from at least three independent cultures). For HPMCs, each data point (squares) indicates averaged measurements from individual donors; see Table S4 for cell numbers per donor. Data are expressed as changes relative to maximal FRET change at sensor saturation and show mean ± SEM; data normality was tested using the Shapiro–Wilk test followed by a Mann–Whitney test, * = 0.05 < p < 0.01, ** = 0.01 < p < 0.001, *** = 0.001 < p < 0.0001.

Figure 3

Comparison of the cAMP response to IBMX in the hTERT-HM cells and HPMCs in the cytoplasm and plasmalemma following agonist application. FRET responses to IBMX (100 μM) applied following ISO or PGE2 treatment of hTERT-HM cells (A) or HPMCs (B) expressing the Epac-S^H187 sensor (blue) or AKAP79-CUTie sensor (pink). For hTERT-HM, each data point (circles) represents one cell (measurements from at least three independent cultures). For HPMCs, each data point (squares) indicates averaged measurements from individual donors; see Table S4 for cell numbers per donor. Values were calculated as the FRET change on application of IBMX relative to maximal FRET change at saturation and are presented as mean ± SEM; normality was tested using Shapiro–Wilk test followed by a Mann–Whitney test, *** = 0.001 < p < 0.0001, **** = p < 0.0001.

Figure 4

Kinetics of cAMP changes measured in the cytosol of hTERT-HM cells and HPMCs in response to ISO or PGE2 application. FRET-change kinetics recorded in individual cells expressing the cytosolic FRET sensor Epac-S^H187. (A) hTERT-HM cells (n = 12 cells) treated with 1 nM ISO. (B) HPMCs (n = 11 cells, 3 donors) treated with1 nM ISO. (C) HPMCs (n = 26 cells, 7 donors) treated with 1 μM ISO. (D) hTERT-HM cells (n = 25 cells) treated with 1 μM PGE2. (E) HPMCs (n = 12 cells, 4 donors) treated with 1 μM PGE2. (F) HPMCs (n = 28 cells, 6 donors) treated with 30 nM PGE2. Same colour lines indicate cells from the same patient.

Figure 5

Kinetics of cAMP changes measured at the plasmalemma of hTERT-HM cells and HPMCs in response to ISO or PGE2 treatment. FRET-change kinetics recorded in individual cells expressing the plasmalemma-targeted sensor AKAP79-CUTie. (A) hTERT-HM cells (n = 6 cells) treated with 1 nM ISO. (B) HPMCs (n = 9 cells, 3 donors) treated with 1 nM ISO. (C) HPMCs (n = 29 cells, 7 donors) treated with 1 μM ISO. (D) hTERT-HM cells (n = 9 cells) treated with 1 μM PGE2. (E) HPMCs (n = 9 cells, 3 donors) treated with 1 μM PGE2. (F) HPMCs (n = 26 cells, 8 donors) treated with 30 nM PGE2. Same colour lines indicate cells from the same patient.

Figure 6

cAMP responses to ISO or PGE2 in HPMCs at passage 0 (P0) to passage 5 (P5) in the cytosol and plasmalemma. FRET responses to ISO (1 µM) in HPMCs expressing (A) Epac-S^H187 (blue) sensor or (B) AKAP79-CUTie (pink) sensor and at different passages after isolation, as indicated. FRET responses to PGE2 (30 nM) in HPMCs expressing (C) Epac-S^H187 (blue) sensor or (D) AKAP79-CUTie (pink) sensor and at different passages after isolation. Each data point (square) is the average of n = 3–5 cells per donor. Normality was tested using the Shapiro–Wilk test. For non-normally distributed data, a Kruskal–Wallis test followed by a Dunn’s multiple comparisons test was used, ** = 0.01 < p < 0.001, *** = 0.001 < p < 0.0001.

Figure 7

cAMP increase on inhibition of PDEs in HPMCs at passage 0 (P0) to passage 5 (P5) in the cytosol and plasmalemma and pre-treated with ISO or PGE2. FRET responses to IBMX (100 µM) measured in HPMCs pre-treated with ISO (1 µM) and expressing (A) Epac-S^H187 (blue) sensor or (B) AKAP79-CUTie (pink) sensor and at different passages after isolation, as indicated. FRET change in response to IBMX (100 µM) measured in HPMCs pre-treated with PGE2 (30 nM) and expressing (C) Epac-S^H187 (blue) sensor or (D) AKAP79-CUTie (pink) sensor and at different passages after isolation, as indicated. Each data point (square) is the average of n = 3–5 cells per donor. Normality was tested using the Shapiro–Wilk test. For non-normally distributed data, a Kruskal–Wallis test followed by a Dunn’s multiple comparisons test was used, * = 0.05 < p < 0.01, ** = 0.01 < p < 0.001.

Figure 8

PDE4B, ADRB2 and PTGER2 mRNA expression and PDE4B, β2-AR and EP2 receptor protein levels in term no labour HPMCs from P0 to P4. HPMCs were cultured to 80% confluence at P0 and passaged to P4. At each passage, RNA was extracted and synthesised to cDNA for subsequent qPCR for PDE4B (A), ADRB2 (D) and PTGER2 (G). Protein was also extracted and analysed by western blotting at each passage. Densitometric analysis for PDE4B (B) with representative blot (C) β2-AR (E) with representative blot (F) and EP2 receptor (H) with representative blot (I). Data were normalised to GAPDH and show mean ± SEM. Normality was tested using the Shapiro–Wilk test. For mRNA, data were analysed using a one-way ANOVA followed by a Turkey’s multiple comparisons test. For protein, data were analysed using a paired t-test. * = 0.05 < p < 0.01. Each data point or protein band indicates HPMCs from individual donors. mRNA (circles) [n = 5–6]; protein p0 (circles) [n = 7]; protein p4 (squares) [n = 6].

Figure 9

cAMP responses to ISO or PGE2 in the cytosol and at the plasmalemma of HPMCs from individual donors. Individual donor FRET response to ISO (1 µM) in HPMCs expressing Epac-S^H187 (blue) sensor (A) or AKAP79-CUTie (pink) sensor (B). Individual donor FRET response to PGE2 (30 nM) in HPMCs expressing Epac-S^H187 sensor (C) or AKAP79-CUTie sensor (D). For HPMCs, each data point (squares) indicates averaged measurements from individual donors; see Table S4 for cell numbers per donor. Data are expressed as changes relative to maximal FRET change at sensor saturation and show mean ± SEM.