Distinct PKA Signaling in Cytosolic and Mitochondrial Compartments in Electrically Paced Atrial Myocytes

Figure 1. Mean PKA activity in the cytosol. (A) Representative example of cytosolic PKA activity (AKAR4 emission Y/C) in a single atrial cell in response to adenylyl cyclase (AC) activation by 10 µM forskolin (FSK) followed by 6 µM H-89. (B) Mean PKA activity in the cytosol in response to FSK and H-89 (n = 7, 3 rabbits). (C) Representative example of cytosolic PKA activity (AKAR4 emission Y/C) in a single atrial cell in response to AC activation by 10 and 20 µM forskolin (FSK) and 100 µM 3-isobutyl-1-methylxanthine (IBMX). (D) Mean PKA activity in the cytosol in response to FSK and IBMX (n = 5. 3 rabbits). Data are presented as mean ± SEM.

Figure 2. The effect of a PKA inhibitor on mean PKA activity in the cytosol and the ability to be electrically stimulated. (A) Representative example of cytosolic PKA activity (AKAR4 emission Y/C) in a single atrial cell in response to increasing concentrations of H-89 (0.6, 1, 6 µM). (B) Mean PKA activity in the cytosol in response to increasing concentrations of H-89 (n = 13, 4 rabbits). (C) Percentage of atrial cell contraction at 1, 2, and 3 Hz, in the absence vs. presence of H-89 (0.6, 1, 6 µM) (n = 19, 3 rabbits). Data are presented as mean ± SEM. * p < 0.05 compared to baseline.

Figure 3. The effect of a Ca2+ chelator on mean PKA activity in the cytosol. (A) Representative example of cytosolic PKA activity (AKAR4 emission Y/C) in a single atrial cell treated with BAPTA-AM (10 µM). (B) Mean PKA activity in the cytosol of atrial cells treated with BAPTA-AM followed by 10 µM forskolin (FSK) and 6 µM H-89 (n = 16, 5 rabbits). Data are presented as mean ± SEM. * p < 0.05 compared to control conditions.

Figure 4. Mean PKA activity in the mitochondrial compartments. (A) Representative example of PKA activity in the outer mitochondrial membrane (OMM-AKAR4 emission Y/C) in response to adenylyl cyclase (AC) activation by 10 µM forskolin (FSK) and 6 µM H-89. (B) Mean PKA activity in the OMM in response to FSK and H-89 (n = 7, 3 rabbits). (C) Representative example of mitochondrial matrix PKA activity (MM-AKAR4 emission Y/C) in response to AC activation by 10 µM forskolin (FSK) and 6 µM H-89. (D) Mean PKA activity in MM in response to FSK and H-89 (n = 12, 3 rabbits) and 6 µM H-89. Data are presented as mean ± SEM. * p < 0.05 compared to cytosol.

Figure 5. PKA activity in response to electrical stimulation. Representative example of (A) cytosolic PKA activity (AKAR4 emission Y/C), (B) outer mitochondrial membrane PKA activity (OMM-AKAR4 emission Y/C), and (C) mitochondrial matrix PKA activity (MM-AKAR4 emission Y/C) in response to electrical stimulation (1, 2, or 3 Hz) followed by adenylyl cyclase (AC) activation by 10 µM forskolin (FSK) and PKA inhibition by 6 µM H-89. (D) Mean PKA activity in the cytosol (n = 7, 3 rabbits), outer mitochondrial membrane (n = 7, 3 rabbits), and mitochondrial matrix (n = 12, 3 rabbits) in response to electrical stimulation (1, 2, or 3 Hz) followed by 10 µM FSK and 6 µM H-89. Data are presented as mean ± SEM.

Figure 6. Mean PKA activity in response to electrical stimulation in the presence of isoproterenol. Representative example of (A) cytosolic PKA activity (AKAR4 emission Y/C), (B) outer mitochondrial membrane PKA activity (OMM-AKAR4 emission Y/C), and (C) mitochondrial matrix PKA activity (MM-AKAR4 emission Y/C) in response to electrical stimulation (1, 2 or 3 Hz) followed by adenylyl cyclase (AC) activation by 10 µM forskolin (FSK) and 6 µM PKA inhibition by H-89. (D) Mean PKA activity in the cytosol (n = 10, 3 rabbits), outer mitochondrial membrane (n = 7, 3 rabbits), and mitochondrial matrix (n = 7, 3 rabbits) in response to electrical stimulation (1, 2 or 3 Hz) (E) followed by 10 µM FSK and 6 µM H-89. Data are presented as mean ± SEM. * p < 0.05 compared to 1 Hz.

Figure 7. Representative example of cytosolic Ca2+ levels (A) in the absence and (B) presence of isoproterenol and electrical stimulation at 1 Hz. Representative example of cytosolic Ca2+ levels (C) in the absence and (D) presence of isoproterenol and electrical stimulation at 3 Hz.

Figure 8. Summary of PKA activity in the cytosol under different interventions (electrical stimulation and drug perturbations). ISO—isoproterenol; FSK—forskolin. Data are presented as mean ± SEM.