Figure 3. Cav3 modulates cAMP levels and its spatial distribution after β₂AR stimulation.

A) Expression of the dominant negative Cav3DN construct results in an increase in intracellular cAMP stimulated by β₂AR. Cells were transduced with either LacZ (control) or Cav3DN together with the Epac2-camps sensor adenovirus (all at MOI 500) and stimulated as described in Figure 2C and D. Data are means ± SE (n=12–13 cells). * - Difference from control is significant at P<0.05.

B) SICM/FRET analysis of β₂AR distribution in Cav3DN expressing cells. β₂AR was locally stimulated with the scanning pipette in either single T-tubules or in cell crests located between the Z-lines. The experiment was performed exactly as previously described [34]. β₂AR-cAMP signals can be detected only upon local receptor stimulation in the T-tubules, suggesting that β₂AR localization is unaltered in Cav3DN expressing cardiomyocytes. Data are presented as means±SE (n=5–8 cells). ** - Difference is significant at P<0.01.

C) Local β₂AR stimulation in single T-tubules (white arrow denotes the position of the SICM pipette) of control (LacZ expressing) cardiomyocytes results in a highly localized cAMP signal which is measurable only in the subcellular region adjacent to the pipette. Data in the graph show YFP/CFP ratios in different color-labeled regions across the cell cytosol. Representative experiment, n=>6 cells

D) Local β₂AR stimulation in single T-tubules of Cav3DN expressing cardiomyocytes results in a far-reaching cAMP gradient which diffuses across the entire cell. Data are presented as in C. Representative experiment, n=>8 cells.

E) Local T-tubular β₂AR stimulation in failing cardiomyocytes leads to a similar far-reaching cAMP gradient. Representative experiment, n=10 cells.