Remodelling of cAMP dynamics within the SERCA2a microdomain in heart failure with preserved ejection fraction caused by obesity and type 2 diabetes

Figure 1. HFpEF phenotype and ‘snapshot’ of β-AR subtype signalling. (A) Left ventricular posterior wall during diastole (LVPWd), (B) left ventricle mass (LV mass), (C) ejection fraction % (EF %), (D) E/A ratio, (E) E/e′, and (F) isovolumic relaxation time (IVRT). Representative transthoracic echocardiography images, (G) short-axis view of the left ventricle in M-mode, (H) pulsed-waved Doppler, and (I) tissue Doppler from four-chamber view. (J) Representative images of picrosirius red, scale bar 40 µm, and (K) quantification (N = 5 mice/group). (L) Fold change in Col1a1 mRNA expression (N = 6 mice/group). (M) Representative images and (N) quantification of the protein expression of total PLN, SERCA2a, RyR, cTnI, PDE2A, PDE3A, PDE4B, and PDE4D, normalized to calsequestrin (Calsq; N = 6–15 hearts/group). (O) Quantification of the PLN/SERCA2a ratio from the data shown in (N). (P) Representative blots and (Q) quantification of Ser16 PLN phosphorylation at both submaximal (3 nM ISO + 50 nM ICI118551) and maximal (100 nM ISO + 50 nM ICI118551) selective β1-AR stimulation and of maximal selective β2-AR stimulation (100 nM ISO + 100 nM CGP20712A; N = 4 mice/group). Normal distribution in (A–F, K,…

Figure 2. Real-time monitoring of cAMP levels in the SERCA2a microdomain post selective β-adrenergic stimulation. Representative cAMP FRET traces obtained from selective β-AR stimulation in db/+ and db/db CMs, as well as quantification of the % change in FRET as compared to maximum response induced by 10 µM forskolin and 100 µM IBMX. Representative traces for submaximal β1-AR stimulation (1 nM ISO in the presence of 50 nM ICI118551) in (A) db/+ (N = 17 mice/45 cells) and (B) db/db CMs (N = 15 mice/32 cells) quantified in (C), for maximal β1-AR stimulation (100 nM ISO in the presence of 50 nM ICI118551) in (D) db/+ (N = 8 mice/26 cells) and (E) db/db (N = 5 mice/17 cells) CMs quantified in (F), and for β2-AR stimulation (100 nM ISO in the presence of 100 nM CGP20712A) in (G) db/+ (N = 10 mice/17 cells) and (H) db/db (N = 4 mice/13 cells) CMs quantified in (I). T90 calcium re-uptake with submaximal β1-AR stimulation (0.3 nM ISO + 50 nM ICI118551) in (J) db/+ (N = 3 mice/61 cells) and (K) db/db (N = 3 mice/69 cells) CMs and (L) quantification, with maximal β1-AR stimulation (1 nM ISO + 50 nM ICI118551) in (M) db/+ (N = 4 mice/62 cells) and (N) db/db (N = 3 mice/29 cells) CMs and (O)…

Figure 3. Role of PDEs in cAMP regulation after selective β-AR subtype stimulations. Representative cAMP FRET traces of selective PDE2 (100 nM BAY 60-7550, Bay), PDE3 (10 μM Cilo), and PDE4 inhibition (10 μM Roli) inhibition post selective β1-AR (100 nM ISO in the presence of 50 nM ICI118551) stimulation in (A–C) db/+ (Bay, N = 4 mice/12 cells; Cilo, N = 7 mice/16 cells; Roli, N = 7 mice/13 cells) and (D–F) db/db (Bay, N = 8 mice/14 cells; Cilo, N = 7 mice/15 cells; Roli, N = 9 mice/14 cells) CMs. Representative FRET traces of selective PDE2, PDE3, and PDE4 inhibition post selective β2-AR stimulation (100 nM ISO in the presence of 100 nM CGP20712A) in (H–I) db/+ (Bay, N = 6 mice/13 cells; Cilo, N = 5 mice/13 cells; Roli, N = 8 mice/12 cells) and (J–L) db/db (Bay, N = 8 mice/16 cells; Cilo, N = 8 mice/15 cells; Roli, N = 8 mice/15 cells) CMs. (M–N) Quantification of FRET responses as % FRET change to IBMX 100 µM. Normal distribution was confirmed by Kolmogorov–Smirnov test (P > 0.1) followed by (M, N) analysis by nested ANOVA. *P < 0.05 vs. db/+ same condition.

Figure 4. Loss of PDE4 association with PLN. (A) Quantification of T90 calcium reuptake with 10 µM Roli treatment post selective β2-AR stimulation (70 nM ISO + 100 nM CGP20712A) in db/+ (N = 3 mice/38 cells) and db/db (N = 3 mice/40 cells) CMs. Representative fluorescence traces are shown in Supplementary material online, Figure S6D and E. (B) Representative blots and quantification of Ser16 phosphorylated PLN/total PLN in control and PDE4B overexpressing (OE) CMs under basal conditions and post selective β2-AR stimulation (100 nM ISO + 100 nM CGP20712A) without 10 µM Roli (N = 3 mice/group). pPLN and total PLN intensities were first normalized on Calsq before calculating the pPLN/PLN ratio. (C) Co-IP performed with PLN pull down, representative blots for PDE3A, PDE4B, and PDE4D (F1 being the first flow through when beads washed, F5 the final flow through when beads washed, and Elutant, the eluted protein samples bound to the beads). The right-hand side shows negative controls performed under the same conditions using an unrelated antibody for IP. (D) Quantification of Co-IP normalized to input (N = 5 mice/group). Normal distribution was confirmed by Kolmogorov–Smirnov test (P > 0.1) in…

Figure 5. Summary diagrams of cAMP dynamics within the SERCA2a microdomain. In (A) healthy db/+ CMs, cAMP pools are generated by β1-AR signalling with PDE4B and PDE4D associating with PLN and locally controlling β2-AR/cAMP effects in this microdomain. In (B) db/db CMs, β1-AR-induced cAMP pools and PLN phosphorylation are desensitized and β2-AR signalling is enhanced, due to a loss of PDE4 coupling with the β2-AR and association with PLN.