The effects on the L-type Ca 2+ current

Experimental data on mouse ventricular myocytes shows that the application of 1 µM

isoproterenol, which stimulates both β1-ARs and β2-ARs, robustly increases the magnitude of the

L-type Ca2+ current by about 2 folds41,56,57,58. Stimulation of β1-ARs alone with 1 µM isoproterenol

and a block of β2-ARs by 50 nM ICI-118551 results in a similar increase in the magnitude of ICaL41.

Further, application of 1 µM of isoproterenol with β1-ARs blocked with 0.3 µM of CGP-20712A41

or application of PTX without stimulation of β1-ARs or β2-ARs43,58,59 does not show any effect on

ICaL, demonstrating silence of the β2-adrenergic signaling system under these experimental

conditions. However, the small effects of β2-ARs can be revealed by stimulating β2-ARs with 1

µM isoproterenol and additional application of PTX58 or with inhibition of PDE3 and PDE441. Figure 3.7 shows simulations of these experimental findings. Application of PTX does not

Figure 3.6 Compartmentalization of cAMP dynamics in mouse ventricular myocytes. Panel A: Experimental data on normalized cAMP concentration in mouse ventricular myocytes in caveolar and extracaveolar compartments upon selective stimulation of β1-ARs and β2-ARs are shown by black bars34;

the corresponding simulation data is shown by gray bars. Panel B: Experimental data on normalized cAMP concentration in mouse ventricular myocytes (whole cell concentrations) upon stimulation of both β1-ARs and β2-ARs, β1-ARs alone, or β2-ARs alone are shown by black bars34; the corresponding

control and long-dashed line for PTX in Fig. 3.7A). Stimulation of both β1-ARs and β2-ARs by 1

µM isoproterenol increases the magnitude of ICaL by 1.9 folds (compare solid line for control and

small-dashed line for 1 µM isoproterenol in Fig. 3.7A and Fig. 3.8C for the experimental data).

An additional application of PTX to 1 µM isoproterenol slightly increases the amplitude of ICaL to

reflect the effects of β2-ARs (dotted line in Fig. 3.7A). Finally, the magnitude of ICaL shows a larger

increase upon application of 1 µM isoproterenol with inhibition of PDE3 and PDE4 (dashed-dotted

line in Fig. 3.7A). To reveal the contribution of β1-ARs and β2-ARs to the total effects, we

performed simulations with the same protocols, but with inhibition of β2-ARs or β1-ARs. The

results are shown in Figs. 3.7, B and C). The effects of stimulation of β1-ARs with 1 µM

isoproterenol and a block of β2-ARs are very similar to those when both β1-ARs and β2-ARs are

intact (Fig. 3.7B). The only difference is that no effects of PTX are observed.

In contrast, only small increases in the magnitudes of ICaL are observed upon stimulation of β2-

ARs when β1-ARs are blocked under different experimental conditions (Fig. 3.7C). The

application of PTX did not change the magnitude of ICaL (compare solid line for control and long-

dashed line for PTX in Fig. 3.7C). Stimulation of β2-ARs when β1-ARs are blocked with 1 µM

isoproterenol leads to a tiny increase in ICaL (3% increase), which virtually cannot be detected in

the experiments (compare solid line for control and small-dashed line for 1 µM isoproterenol in

Fig. 3.7C). Application of 1 µM isoproterenol in the presence of PTX results in a larger increase

in ICaL (12% increase, dotted line in Fig. 3.7C). Even larger effects of stimulation of β2-ARs are

obtained upon application of 1 µM isoproterenol and inhibition of PDE3 and PDE4 (22% increase,

Figure 3.7 The effects of stimulation of β1-ARs and β2-ARs on the L-type Ca2+ current. Simulated current-

voltage relationships obtained upon the stimulation of both β1-ARs and β2-ARs (Panel A), β1-ARs alone

(Panel B), and β2-ARs alone (Panel C). Currents are obtained by voltage pulses from −70 mV to +50 mV

(in 10-mV increments) from a holding potential of −80 mV and without Ca2+_{-induced Ca}2+ _{release to}

account for heavy buffering conditions. Simulations were performed for several physiological conditions: control (solid lines), inhibition of Gi without isoproterenol (long dashed lines, they are almost invisible in

figures due to very tiny effects), after application of 1 μM isoproterenol (medium dashed lines), after application of 1 μM isoproterenol with inhibition of Gi (dotted lines), or after application of 1 μM

isoproterenol with inhibition of PDE3 and PDE4 (dash-dotted lines).

Comparisons of the simulated and experimental data under different experimental conditions

is shown in Fig. 3.8. No effect is observed in simulations and in the experiments43,59 _upon

application of 1 µM isoproterenol without and with PTX in simulations and in the experiment by

Xu et al.58 _{(Fig. 3.8B). The statistically significant effects of stimulation of β}

2-ARs alone

(37%±13% increase) is observed upon application of 1 µM isoproterenol and inhibition of PDE3

and PDE4 (by cilostamide and rolipram, respectively), which is close to our simulated effect under

similar experimental conditions (third set of bars in Fig. 3.8C). However, as expected, stimulation

of β1-ARs alone with 1 µM isoproterenol and inhibition of PDE3 and PDE4 results in a much

larger increase in ICaL (two last bars in Fig. 3.8C), which was also predicted by our simulations.

Thus, our model for the first time provides non-contradictive simulated data on the effects of

β1-ARs and β2-ARs on the L-type Ca2+ current in mouse ventricular myocytes. Simulations show

that the effects of β2-ARs on the L-type Ca2+ current can be revealed by stimulation with

isoproterenol with additional inhibition of Gi or PDE3 and PDE4.