Decreased expression and activity of cAMP phosphodiesterases in cardiac hypertrophy and its impact on beta-adrenergic cAMP signals

Abstract

Rationale: Multiple cyclic nucleotide phosphodiesterases (PDEs) degrade cAMP in cardiomyocytes but the role of PDEs in controlling cAMP signaling during pathological cardiac hypertrophy is poorly defined.

Objective: Evaluate the beta-adrenergic regulation of cardiac contractility and characterize the changes in cardiomyocyte cAMP signals and cAMP-PDE expression and activity following cardiac hypertrophy.

Methods and results: Cardiac hypertrophy was induced in rats by thoracic aortic banding over a time period of 5 weeks and was confirmed by anatomic measurements and echocardiography. Ex vivo myocardial function was evaluated in Langendorff-perfused hearts. Engineered cyclic nucleotide-gated (CNG) channels were expressed in single cardiomyocytes to monitor subsarcolemmal cAMP using whole-cell patch-clamp recordings of the associated CNG current (I(CNG)). PDE variant activity and protein level were determined in purified cardiomyocytes. Aortic stenosis rats exhibited a 67% increase in heart weight compared to sham-operated animals. The inotropic response to maximal beta-adrenergic stimulation was reduced by approximately 54% in isolated hypertrophied hearts, along with a approximately 32% decrease in subsarcolemmal cAMP levels in hypertrophied myocytes. Total cAMP hydrolytic activity as well as PDE3 and PDE4 activities were reduced in hypertrophied myocytes, because of a reduction of PDE3A, PDE4A, and PDE4B, whereas PDE4D was unchanged. Regulation of beta-adrenergic cAMP signals by PDEs was blunted in hypertrophied myocytes, as demonstrated by the diminished effects of IBMX (100 micromol/L) and of both the PDE3 inhibitor cilostamide (1 micromol/L) and the PDE4 inhibitor Ro 201724 (10 micromol/L).

Conclusions: Beta-adrenergic desensitization is accompanied by a reduction in cAMP-PDE and an altered modulation of beta-adrenergic cAMP signals in cardiac hypertrophy.

Figure 1. β-AR responses in whole hearts and isolated ventricular myocytes from sham and CH rats

(A) CRC to ISO on LV +dP/dt_max used as an index of cardiac contractility in 5 normal hearts and 7 hypertrophied hearts. (B) Comparison of the effect of ISO (100 nmol/L) on I_CNG density in sham (black bars) and hypertrophied (CH, white bars) cardiomyocytes using the low and high affinity CNG channels mutants: E583M CNGA2 and C460W/E583M CNGA2, respectively. I_CNG was measured every 8 s at -50 mV from a holding potential of 0 mV. The number of cells tested from 2 normal hearts and 3 hypertrophied hearts for E583M CNGA2 and from 18 normal hearts and 19 hypertrophied hearts for E583M/C460W CNGA2 is indicated in brackets above the bars. Statistically significant differences in I_CNG density between sham-operated and CH cardiomyocytes are indicated as *, p<0.05. (C) CRC to ISO on I_CNG density in cardiomyocytes from 16 hypertrophied hearts (CH, dash line) and 13 normal hearts (Sham, continuous line) using C460W/E583M CNGA2. Cells were first superfused with control external Ringer solution and then exposed to increasing concentrations of ISO. Data represent the mean ± S.E.M. of the number of cardiomyocytes indicated near each data point. (D) Effect of the forskolin analog L-858051 (100 μmol/L) on I_CNG density in sham (black bars) and hypertrophied (CH, white bars) cardiomyocytes using C460W/E583M CNGA2. Data represent the mean ± S.E.M. of the number of cells (from 23 normal hearts and 21 hypertrophied hearts) indicated near the bars.

Figure 2. Regulation of β-AR cAMP signals by PDEs in sham and hypertrophied cardiomyocytes

Subsarcolemmal cAMP signals were recorded by measuring I_CNG in sham (A) and CH (B) cardiomyocytes expressing E583M CNGA2. Cells were superfused with control external Ringer solution and then challenged with ISO (100 nmol/L) in the absence or presence of IBMX (100 μmol/L) for the periods indicated with horizontal lines. The individual I_CNG traces shown on top were recorded at the times indicated by the corresponding letters on the graph below. (C) Summary of the responses shown in A and B. Data are expressed as mean ± S.E.M. The numbers above the bars represent cardiomyocytes isolated from 2 normal hearts and 3 hypertrophied hearts. Asterisks and dollars signs indicate statistically significant differences (paired t-test) resulting from the treatment with IBMX or statistically significant differences (unpaired t-test) between hypertrophied (CH) and sham cardiomyocytes, respectively, and are indicated as: ^$, p<0.05; **, p<0.01; ***, p<0.001.

Figure 3. Regulation of β-AR cAMP signals by PDE3 and PDE4 in sham and hypertrophied cardiomyocytes

Subsarcolemmal cAMP signals were recorded by measuring I_CNG in sham (A) and CH (B) cardiomyocytes expressing C460W/E583M CNGA2. Cells were superfused with control external Ringer solution and then challenged with different drugs for the periods indicated with horizontal lines. The individual I_CNG traces shown on top were recorded at the times indicated by the corresponding letters on the graph below. (C) and (D), Summary of the responses shown in A and B, represented either in absolute current density (C) or in percentage increase over the ISO response (D). All data are expressed as mean ± S.E.M. The numbers above the bars represent cardiomyocytes isolated from 2 normal and 5 hypertrophied hearts. Asterisks and dollars signs indicate statistically significant differences (paired t-test) resulting from the treatment with PDE inhibitors or statistically significant differences (unpaired t-test) between CH and sham cardiomyocytes, respectively, and are indicated as: ^$, p<0.05; **^{; $$}, p<0.01; ***, p<0.001.

Figure 4. Expression of PDE3 and PDE4 proteins in sham and hypertrophied cardiomyocytes

Equal amounts of proteins from ventricular myocytes isolated from CH (n=4) and sham-operated (n=6) rat hearts were separated on SDS/PAGE and revealed with PDE subtype-specific antibodies. Whole brain (PDE4A, PDE4B and PDE4B) and total heart extracts (PDE3A) of wild type and the respective PDE KO mice were loaded as immunoblot controls (IB ctr.). Calsequestrin (Calseq) was used as a loading control. (A) Shown are representative blots. (B) Quantification of all data obtained in several immunoblots from 6 sham-operated samples and 4 CH samples.

Figure 5. Pattern of cAMP-PDE activities in sham and hypertrophied cardiomyocytes

(A) PDE activity in sham and CH cardiomyocytes using 1 μmol/L cAMP as substrate. PDE3 and PDE4 activities are defined as the fraction of the total PDE activity inhibited by CIL (1 μmol/L) or rolipram (10 μmol/L), respectively. (B) Detergent extracts from sham and CH cardiomyocytes were immunoprecipitated with PDE4 subtype-specific antibodies as described in Materials and Methods. The PDE activities recovered in the IP pellets are reported. The number of samples analyzed for each group is shown in brackets above the bars. All data show the mean ± S.E.M. Statistically significant differences in PDE activity between CH and sham-operated rats are indicated as: *, p<0.05; **, p<0.01; ***, p<0.001.