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

Abstract

Aims: Despite massive efforts, we remain far behind in our attempts to identify effective therapies to treat heart failure with preserved ejection fraction (HFpEF). Diastolic function is critically regulated by sarcoplasmic/endoplasmic reticulum (SR) calcium ATPase 2a (SERCA2a), which forms a functional cardiomyocyte (CM) microdomain where 3',5'-cyclic adenosine monophosphate (cAMP) produced upon β-adrenergic receptor (β-AR) stimulation leads to phospholamban (PLN) phosphorylation and facilitated Ca2+ re-uptake.

Methods and results: To visualize real-time cAMP dynamics in the direct vicinity of SERCA2a in healthy and diseased myocytes, we generated a novel mouse model on the leprdb background that stably expresses the Epac1-PLN Förster resonance energy transfer biosensor. Mice homozygous for the leprdb mutation (db/db) developed obesity and type 2 diabetes and presented with a HFpEF phenotype, evident by mild left ventricular hypertrophy and elevated left atria filling pressures. Live cell imaging uncovered a substantial β2-AR subtype stimulated cAMP response within the PLN/SERCA2a microdomain of db/db but not healthy control (db/+) CMs, which was accompanied by increased PLN phosphorylation and accelerated calcium re-uptake. Importantly, db/db CMs also exhibited a desensitization of β1-AR stimulated cAMP pools within the PLN/SERCA2a microdomain, which was accompanied by a blunted lusitropic effect, suggesting that the increased β2-AR control is an intrinsic compensatory mechanism to maintain PLN/SERCA2a-mediated calcium dynamics and cardiac relaxation. Mechanistically, this was due to a local loss of cAMP-degrading phosphodiesterase 4 associated specifically with the PLN/SERCA2a complex.

Conclusion: These newly identified alterations of cAMP dynamics at the subcellular level in HFpEF should provide mechanistic understanding of microdomain remodelling and pave the way towards new therapies.

Keywords: 3′, 5′-Cyclic adenosine monophosphate; Förster resonance energy transfer; Heart failure with preserved ejection fraction; Obesity; Phosphodiesterase; Phospholamban; Sarcoplasmic/endoplasmic reticulum calcium ATPase 2a; Type 2 diabetes.

Graphical Abstract

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, L, N, and O) was confirmed by Kolmogorov–Smirnov test (P > 0.1); in (Q), normal distribution was tested by Shapiro–Wilk test, P values were P = 0.0777, P = 0.6468, P = 0.0357, P = 0.0069, P = 0.0447, P = 0.4695, P = 0.1930, and P = 0.0594, respectively. Significance at *P < 0.05. Normally distributed data in (A–F, K, L, and O) analysed by unpaired t-test and in (N) by mixed ANOVA followed by Wald χ  ² test and Sidak multiple comparisons test for data not normally distributed; (Q) analysis performed by Kruskal–Wallis ANOVA followed by Dunn’s multiple comparison test. *P < 0.05 vs. db/+ same condition.

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) quantification, and for β₂-AR stimulation (70 nM ISO + 100 nM CGP20712A) in (P) db/+ (N = 4 mice/72 cells) and (Q) db/db (N = 4 mice/56 cells) CMs and (R) quantification. Normal distribution was tested by Kolmogorov–Smirnov test (P > 0.1) in all data except (C) where db/+, P = 0.0771 and db/db, P = 0.0448 and (I) where db/+, P = 0.0132. Data in (C, F, and I) were then analysed by Kruskal–Wallis ANOVA followed by Dunn’s multiple comparison test. For data in (L, O, and R), comparisons of baseline and stimulated within the same group were analysed by paired t-test, and comparisons of baselines and stimulated between different groups were analysed by unpaired t-test. *P < 0.05 and ***P < 0.001 vs. db/+ same condition. #P < 0.05 vs. baseline same group.

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 all data except in (C) where β₂-AR db/+, P = 0.0777 and β₂-AR + Roli db/db, P = 0.0016. Normally distributed data in (B and D) analysed mixed ANOVA followed by Wald χ  ² test and Sidak multiple comparisons test. Data not normally distributed in (C) analysed by mixed ANOVA followed by Kruskal–Wallis test and Dunn’s multiple comparisons test. *P < 0.05 vs. db/+ same condition; #P < 0.05 and ###P < 0.001 vs. β₂-AR stimulation same group; †P < 0.05 in db/+ compared to db/+ at basal; and ‡P < 0.05 in db/+ compared to db/+ post β₂-AR stimulation.

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.