Although there was no significant difference in the cAMP levels after non selective PDE inhibition (ISO and IBMX treatment) seen in the summary of FRET experiments
conducted (Figure 4-6), it was important to look closer at the contribution that PDEs may play in the control of cAMP level. This was done by taking the final response detected upon addition of ISO + IBMX treatment in these cells and by subtracting the ISO response (Figure 4-8A) to get a clearer idea of the PDEs contribution in determining cAMP levels in the PKA-RI and PKA-RII compartments. In the PKA-RI compartment there was a
significantly greater increase in cAMP intensity compared with the PKA-RII compartment, indicating there was a significantly greater contribution of PDEs acting in the PKA-RI compartment compared with the PKA-RII compartment. To explore whether or not this was due to a higher basal level of PDEs activity in the PKA-RI compartment, ARVM transduced with the targeted sensors were challenged with 100 μM IBMX alone (Figure 4-8B). This treatment was found to induce a cAMP increase which was comparable in the two locations. These results suggest that it is only after β-adrenoceptor stimulation and cAMP generation that this disparity in the PDE activity in the two compartments can be identified. One possible explanation for this is that a number of PDE, including PDE3 and PDE4, have PKA phosphorylation sites (Omori and Kotera 2007) and therefore activation of PKA by ISO stimulation leads to the modification of PDE activity in a compartment specific manner.
Figure 4-8. The contribution of phosphodiesterases (PDEs) in ARVM transduced with RI_epac or RII_epac.
(A) Myocytes are stimulated first with 100 nM Isoproterenol, then IBMX (100 μM). The contribution of the IBMX response alone is shown here. (B) Myocytes are treated with IBMX without Isoproterenol. IBMX after ISO: RI_epac n =26; RII_epac n = 27. IBMX alone: RI_epac: n =5; RII_epac n = 6. Error bars represent SEM. Two tailed; paired t-test, *** p<0.001.
In order to identify which family of phosphodiesterases may be controlling each
subcellular location which could possibly account for the higher PDE activity in the PKA- RI compartment; myocytes were first pre-treated with a selective PDE inhibitor before stimulation of cAMP production with 100 nM ISO. Among the cAMP-hydrolysing PDEs expressed in the heart, selective inhibitors for PDE2, PDE3 and PDE4 but not for PDE1 and PDE8 are commercially available, so the analysis focused on the former. In the experiments investigating the effect of PDE2, ARVM were incubated for 10 minutes with 50 nM of the PDE2 inhibitor Bay 60 7550 at 37˚C before imaging. Cells were then
challenged as before with ISO, IBMX and then finally with Forskolin to achieve the maximal response. Under basal conditions (Figure 4-9A), there was a comparable level of cAMP in each condition. Upon ISO stimulation (Figure 4-9B), there was a greater cAMP increase in PKA-RII compartment than the PKA-RI compartment in control myocytes. Pre-treatment of the myocytes with Bay 60-7550 resulted in a significantly greater increase in cAMP in both compartments compared to controls. There was a 2 fold increase in the amount of cAMP generated in the PKA-RI compartment and a 1.3 fold increase in the PKA-RII compartment upon PDE2 inhibition. In addition, PDE2 inhibition abolished the difference between the PKA-RI and PKA-RII compartments. Maximal FRET responses
were achieved by saturating the sensors with 100 μM IBMX and 25 μM forskolin (RI_epac control: ΔR/R0 max =11.042 ±0.413, n =9; RII_epac control: ΔR/R0 max = 11.170 ± 0.465
n = 8; RI_epac + Bay 60-7550: ΔR/R0 max =10.287 ±0.285, n =8; RII_epac + Bay 60-
7550: ΔR/R0 max = 10.118± 0.433 n = 5). These results indicate that although PDE2 is
present in both the PKA-RI and PKA-RII compartments in the adult myocytes, after β-AR stimulation PDE2 activity appears to higher in the PKA-RI compartment compared to the PKA-RII compartment.
Figure 4-9. Summary of FRET change upon the selective inhibition of PDE2.
(A)Summary of basal cAMP levels in ARVM in the presence or absence of PDE2 inhibitor Bay 60-7550 (50 nM). RI_epac control n =9; RII_epac control n = 9; RI_epac + Bay 60-7550 n =18; RII_epac + Bay 60-7550 n = 12. (B) Summary of FRET change induced by 100 nM isoproterenol in the presence or absence of Bay 60-7550. RI_epac control: ΔR/R0 =3.47 ± 0.152 %, n =8; RII_epac control: ΔR/R0 = 5.871 ± 0.405 %, n = 16; RI_epac + Bay 60-7550: ΔR/R0 =7.616 ± 0.528 %, n =10; RII_epac + Bay 60-7550: ΔR/R0 = 7.633 ± 0.427 %, n = 9 Statistical significance calculated by two way ANOVA with Bonferroni’s post-test, *** p<0.001.
To explore the local contribution on the control of cAMP levels of the PDE3 family of phosphodiesterases, adult myocytes were pre-incubated with 10 μM cilostamide, a
selective PDE3 inhibitor. Results similar to PDE2 inhibition at basal levels of cAMP were obtained with cilostamide, and there was no significant differences between the groups measured (Figure 4-10A). As a control again the amount of cAMP generated in the two compartments upon β-adrenergic stimulation was measured, before comparing these
results with ARVM which had been pre treated with cilostamide and stimulated with ISO (Figure 4-10B). It was found that PDE3 inhibition only had an effect on the level of cAMP generated in the PKA-RI compartment, with a 1.4 fold increase in cAMP production. The maximal FRET response was measured again in the presence of this selective inhibitor (figure not shown) and it was found that PDE3 inhibition had no effect on the maximal FRET change at saturation (RI_epac control: ΔR/R0 max =10.281 ±0.536, n =7; RII_epac
control: ΔR/R0 max = 10.264 ± 0.363 n = 10; RI_epac + cilostamide: ΔR/R0 max =9.051
±0.449, n =10; RII_epac + cilostamide: ΔR/R0 max = 9.198± 0.550 n = 7). These results
indicate that PDE3 controls a pool of cAMP which is functionally coupled to the PKA-RI compartment alone.
Figure 4-10. Summary of FRET change upon the selective inhibition of PDE3.
(A) Summary of basal cAMP levels in ARVM in the presence or absence of PDE3 inhibitor cilostamide (10 μM). RI_epac control n =7; RII_epac control n = 10; RI_epac + cilostamide n =9; RII_epac + cilostamide n = 7. (B) Summary of FRET change induced by 100 nM
isoproterenol in the presence or absence of cilostamide. RI_epac control: ΔR/R0 =4.402 ± 0.276 %, n =8; RII_epac control: ΔR/R0 = 5.634 ± 0.276 %, n = 11; RI_epac + cilostamide: ΔR/R0 =6.23 ± 0.541 %, n =11; RII_epac + cilostamide: ΔR/R0 = 5.884 ± 0.834 %, n = 8.
Statistical significance calculated by two way ANOVA with Bonferroni’s post-test, *p<0.05, ** p<0.01.
The PDE4 family is expressed at high levels in the heart (Mongillo et al. 2004) and therefore it is important to investigate the role of this family in compartmentalised cAMP
signalling. This was achieved by selectively inhibiting all PDE4 isoforms by pre treating the cells for 10 minutes with 10 μM rolipram. As with the other selective inhibitors, rolipram incubation had no effect on basal cAMP levels in adult myocytes (Figure 4-11A). Upon addition of 100 nM ISO in control myocytes (Figure 4-11B) there was a higher generation of cAMP in the PKA-RII compartment compared to the PKA-RI compartment. In myocytes which had been treated with rolipram before imaging there was an increased level of cAMP in both compartments compared with control cells. Results were similar to those found in PDE2 with a larger increase in the PKA-RI compartment (1.8 fold increase) than recorded in the PKA-RII compartment (1.3 fold increase). Again this indicates that although PDE4 isoforms are found in both locations, their effect is predominant in the PKA-RI compartment. The maximal FRET response was measured in the presence of rolipram (figure not shown) and it was found that PDE4 inhibition which had no
significant effect on the maximal FRET change at saturation (RI_epac control: ΔR/R0 max
=10.281 ±0.536, n =7; RII_epac control: ΔR/R0 max = 10.264 ± 0.363 n = 10; RI_epac +
rolipram: ΔR/R0 max = 8.697 ± 0.753, n =7; RII_epac + rolipram: ΔR/R0 max = 10.522 ±
0.801 n = 7).
However, on comparison of the ISO stimulated ARVM pre-treated with rolipram and the saturating response or ARVM pre-treated with rolipram, it was found that there was no significant difference in the PKA-RI compartment. This suggests that the ISO + rolipram response saturated the response in this sensor. No valid conclusions can be drawn for the PDE4 activity in the PKA-RI compartment of ARVM.
Figure 4-11. Summary of FRET change upon the selective inhibition of PDE4.
(A) Summary of basal cAMP levels in ARVM in the presence or absence of PDE4 inhibitor rolipram (10 μM). RI_epac control n =7; RII_epac control n = 10; RI_epac rolipram n =9; RII_epac rolipram n = 7. (B) Summary of FRET change induced by 100 nM isoproterenol in the presence or absence of rolipram. RI_epac control: ΔR/R0 =4.152 ± 0.239 %, n =10;
RII_epac control: ΔR/R0 = 5.838 ± 0.299 %, n = 13; RI_epac rolipram: ΔR/R0 =7.512 ± 0.68 %, n =7; RII_epac rolipram: ΔR/R0 = 7.733 ± 0.643 %, n = 7. Statistical significance calculated by two way ANOVA with Bonferroni’s post-test, *p<0.05, **p<0.01; *** p<0.001.
The above figures show that selective inhibition of each family has no effect on basal cAMP, nor does IBMX treatment at basal level. This could be due to compensation by other PDE families controlling the compartment which have not been inhibited (i.e PDE8 which is IBMX insensitive). It is only after β-AR stimulation that any differences in PDE activity are observed. However, Mika and colleagues have shown that in ARVM selective inhibition of PDE2, 3 and 4 under basal conditions does have an effect on cAMP and ECC. The authors show that selective inhibition of PDEs results in an increase in sarcomere shortening and the calcium transient at basal levels as well as after the addition of 1 nM ISO (Mika et al. 2013). Measurements were recorded in isolated myocytes loaded with 5 µM Fura-2 AM at stimulated at a frequency of 0.5 Hz before determining changes in sarcomere length and Fura-2 ratio at 512 nm using an IonOptix system. Although this study and Mika et al. investigate the impact of selective PDE inhibition in ARVM and utilise the same inhibitors at similar concentrations, this thesis studies the impact of PDEs in specific compartments deep within the cardiomyocyte whereas Mika and co-workers focus on the functional effects these inhibitors have on ECC and do not record the levels of cAMP. It is possible that there may be an effect on global cAMP which could impact ECC
in the cell, however no changes recorded by FRET in either compartment. It has been shown that more than one PDE family controls the PKA-RI and PKA-RII compartments, therefore it is possible that one family may try to compensate for another when its activity is impaired to maintain the correct level of cAMP required for normal function.
Figure 4-12 provides a summary of the above 3 experiments investigating the PDEs that are responsible for controlling the cAMP signalling in the PKA-RI and PKA-RII
subcellular compartments. At the PKA-RI location, PDE2, PDE3 and PDE4 all seem to play a role in the control of cAMP signalling. PDE3 appears to have no effect in the PKA- RII compartment where control of cAMP hydrolysis appears to be mediated solely by PDE2 and PDE4.
Figure 4-12. Summary of experiments recording effect of selective PDE inhibition on ISO stimulation.
ARVM were transduced with adenovirus containing a targeted FRET-based sensor and preincubated for 10 minutes with the selective PDE inhibitor. A. Effect of selective PDE inhibition upon ISO stimulation (100 nM) in the PKA-RI compartment. B. Effect of selective PDE inhibition upon ISO stimulation (100 nM) in the PKA-RII compartment. Statistical significance calculated by one way ANOVA with Dunnett’s post-test, * p<0.05, *** p<0.001.