Differential changes in cyclic adenosine 3′‐5′ monophosphate (cAMP) effectors and major Ca²⁺ handling proteins during diabetic cardiomyopathy

Figures

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  FIGURE 1. Hyperglycaemia is associated with cardiac remodelling in DCM. (A) Comparison of the fasting blood glucose levels (mg/dL) in CON (black diamonds; n = 14/13/14 rats) and STZ rats (white diamonds; n = 15/14/19 rats) at 4, 8 and 12 weeks after STZ or vehicle injection. (B) BW (g) in CON (black diamonds; n = 14/13/14 rats) and STZ rats (white diamonds; n = 15/14/19 rats) at 4, 8 and 12 weeks after STZ or vehicle injection. Statistical analysis was performed with a two‐way anova followed by a post hoc Tukey's multiple comparison test. Statistically significant differences between CON and STZ rats of the same age are indicated as ***, p < 0.001. Statistically significant differences between CON‐4 weeks and CON‐8 weeks or between STZ‐4 weeks and STZ‐8 weeks are indicated as ^^, p < 0.01; ^^^, p < 0.001. Statistically significant differences CON‐4 weeks and CON‐12 weeks or between STZ‐4 weeks and STZ‐12 weeks are indicated as &, p < 0.05 and &&&, p < 0.001. (C) HW (g) in CON (black diamonds; n = 14/13/14 rats) and STZ rats (white diamonds; n = 15/14/19 rats) at 4, 8 and 12 weeks after STZ or vehicle injection. (D) mRNA expression of ANF normalized to GAPDH measured in CON (black diamonds;…

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  FIGURE 2. Expression of Epac1 and Epac2 isoforms in hearts from control and diabetic rats at 4, 8 and 12 weeks. (A). Epac1 mRNA expression in CON (black diamonds; n = 6/6/6 rats) and STZ rats (white diamonds; n = 6/6/6 rats) at 4, 8 and 12 weeks after STZ or vehicle injection. (B) Epac2 mRNA expression in CON (black diamonds; CON: 6/9/6 rats) and STZ rats (white diamonds; STZ: 6/7/6 rats) at 4, 8 and 12 weeks after STZ or vehicle injection. mRNA expression of both isoforms was normalized to GAPDH in both STZ and aged‐matched CON rats. Two‐way anova test showed no significant interaction between STZ treatment and time; however, STZ treatment alone had a statistically significant effect on Epac1 mRNA expression (p < 0.0001). A two‐way anova followed by a post hoc Tukey's multiple comparison test was performed to assess the changes in the expression of Epac2 mRNA. Statistically significant differences between CON and STZ rats of the same age are indicated as *, p < 0.05. Statistically significant differences between STZ‐4 weeks and STZ‐12 weeks are indicated as &&, p < 0.01. Equal amounts of cardiac proteins from control (CON) and diabetic rats (STZ) were separated on SDS/PAGE and revealed…

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  FIGURE 3. Expression of PKA subunits and CaMKII in hearts from control and diabetic rats at 4, 8, and 12 weeks. Equal amounts of cardiac proteins from control (CON) and diabetic rats (STZ) were separated on SDS/PAGE and revealed with PKA RI‐, PKA RIIα‐, PKA Cα‐, CaMKII‐specific antibodies. Calsequestrin and actin were used as loading controls. (A) Representative blots for PKA RI, PKA RIIα, PKA Cα, CaMKII, calsequestrin and actin in CON and STZ at 4, 8 and 12 weeks are shown. (B–E) Quantification of all data obtained in several immunoblots from hearts of CON (black diamonds) and STZ (white diamonds) and represented as mean ± S.E.M. In (B–D), two‐way anova showed no significant interaction between STZ treatment and time. Additionally, neither STZ treatment alone nor the time had any statistically significant effect on the expression of PKA RI (CON: n = 6/6/6; STZ: n = 4/6/6 at 4, 8 and 12 weeks, respectively), PKA RIIα (CON: n = 6/6/6; STZ: n = 5/6/6 at 4, 8 and 12 weeks, respectively) and PKA Cα (CON: n = 6/6/6; STZ: n = 5/6/6 at 4, 8 and 12 weeks, respectively) between CON and STZ rats. In (E), two‐way anova followed by a post hoc Tukey's multiple comparison test was performed to assess…

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  FIGURE 4. Expression of the major actors of the cardiac ECC in hearts from control and diabetic rats at 4, 8, and 12 weeks. Total RNA was extracted from hearts of 5 to 8 control (CON, black diamonds) and diabetic rats (STZ, white diamonds) at each time point and analysed by Real‐time PCR for SERCA2a (A), PLB (B) and TnI (C). mRNA expression was normalized to GAPDH in both STZ and aged‐matched CON rats. Data represent the mean ± S.E.M. Statistical analysis was performed with two‐way anova that showed no significant interaction between STZ treatment and time for all proteins. However, STZ treatment had the same statistically significant effect on PLB (p < 0.05) across time.

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  FIGURE 5. Expression of TnI, PLB and SERCA2a proteins in hearts from control and diabetic rats at 4, 8, and 12 weeks. Equal amounts of cardiac proteins from control (CON) and diabetic rats (STZ) were separated on SDS/PAGE and revealed with p‐TnI (Ser23/24), TnI, p‐PLB (Ser16), p‐PLB (Thr17), PLB and SERCA2a‐specific antibodies. Calsequestrin and actin were used as loading controls. (A), Representative blots for p‐TnI (Ser23/24), TnI, p‐PLB (Ser16), p‐PLB (Thr17), PLB, SERCA2a, calsequestrin and actin in CON and STZ at 4, 8 and 12 weeks are shown. (B–G) Quantification of all data obtained in several immunoblots from hearts of 4 to 8 CON (black diamonds) and STZ (white diamonds) and represented as mean ± S.E.M. Statistical analysis was performed with two‐way anova tests that showed no significant interaction between STZ treatment and time for all proteins. However, STZ treatment alone had the same statistically significant effect on p‐PLB (Thr17)/Total PLB (p < 0.01) across time.

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  FIGURE 6. Summary of cAMP and Ca2+ signalling pathways remodelling during DCM. We have previously shown that 4 weeks after STZ injection, a modest increase in cAMP content, although not significant, resulted from a balance between β1‐AR receptors, PDE4B and PDE4D whose expression was upregulated in the diabetic heart. 37 At Weeks 8 and 12, β1‐AR receptors and cAMP content were restored while the expression of PDE3A was increased. 37 In the present study, Epac2 protein levels as well as the expression of PKA subunits (RI, RIIα and Cα) remained unchanged with the progression of DCM, whereas CaMKII increased at Week 12 in the diabetic myocardium. Moreover, no changes were observed in the protein levels of PLB, SERCA2a and TnI irrespective of the disease evolution. Importantly, PLB phosphorylation at threonine‐17 was increased in DCM, whereas phosphorylation of both PLB at serine‐16 and TnI at serine‐23/24 remained unchanged (see text for more details). EF, ejection fraction; FS, fraction shortening; EDV, end‐diastolic volume; ESV, end‐systolic volume; SV, stroke volume; β1‐AR, β1‐adrenergic receptor; AC, adenylyl cyclase; cAMP, cyclic adenosine 3′‐5′ monophosphate; PDE, phosphodiesterase;…