Fig 1.
Ca2+ sparks and waves in permeabilized myocytes from WT, RyR2-R4496C and Casq2-/- mice under control (VEH) conditions.
A. Averaged spark frequency, full duration at half maximun (FDHM), spark mass and spark-mediated leak measured for each one of the groups studied. ***P<0.001, n = 35–60 cells, N = 2–3 mice/group. Results expressed as mean±SEM. B. Representative line-scans (LS) showing sparks registered in permeabilized WT, RyR2-R4496C, and Casq2-/- myocytes in internal solution (IS) containing Fluo 4 pentapotassium salt ([EGTA]IS = 0.4 mM, estimated free [Ca2+]IS = 0.04 μM (Maxchelator)). C. Averaged amplitude, frequency and propagation speed of Ca2+ waves measured in WT, RyR2-R4496C and Casq2-/- permeabilized myocytes. *P<0.05, **P<0.01. n = 30–60 cells, N = 2 mice/group. D. Representative LS of Ca2+ waves from permeabilized WT, RyR2-4496C mutant cells and Casq2-/- myocytes in internal solution containing Fluo-4 pentapotassium salt ([EGTA]IS = 0.05 mM, estimated free [Ca2+]IS = 3.9 μM (Maxchelator)).
Fig 2.
Effects of flecainide (FLEC) on Ca2+ wave parameters from permeabilized WT, RyR2-R4496C, and Casq2-/- myocytes.
A. Representative LS (left side of the panel) obtained for WT, RyR2-R4496C, and Casq2-/- myocytes, and their corresponding averaged space LS (right side of the same panel) under control condition (VEH) and in the presence of flecainide (FLEC) 10 μM. B. Concentration-response curves (CRCs) for wave incidence, amplitude, frequency, and propagation speed as a function of the concentration of FLEC (in μM) in WT, RyR2-4496C mutant cells, and Casq2-/- myocytes. Results are expressed as mean±SE for each concentration and each parameter. For each group studied: n = 20 cells, N = 3 mice/ condition tested.
Fig 3.
Comparison of Ca2+ spark frequency in WT, caffeine (CAFF) treated WT and Casq2-/- myocytes.
A. Average spark frequency, N = 25–70 cells/group, ***P<0.001. B. Representative LS showing sparks registered in permeabilized WT myocytes, CAFF-treated permeabilized WT myocytes, and Casq2-/- myocytes in internal solution (IS) containing Fluo 4 pentapotassium salt ([EGTA]IS = 0.4 mM, nominal [Ca2+]IS = 0.06 mM, estimated free [Ca2+]IS = 0.04 μM (Maxchelator)).
Fig 4.
Caffeine increases potency of flecainide (FLEC) against Ca2+ waves.
A. Representative LS of Ca2+ waves in permeabilized WT myocytes, WT cells sensitized with 150 μM caffeine (WT+CAFF 150μM) and Casq2-/- myocytes under control condition (VEH) and in the presence of FLEC 10 μM. B. CRCs for wave parameters (incidence, amplitude, frequency, and propagation speed) as a function of the concentration of FLEC (in μM) in WT, WT+CAFF 150μM, and Casq2-/- cells. FLEC effect on Ca2+ wave parameters is dose-dependent, with a strongest effect on Ca2+ waves in Casq2-/- myocytes. FLEC effects on Ca2+ waves in WT cells are partially restored by sensitizing the cells with CAFF 150 μM. Mean±SE for each concentration and each parameter. For each group: n = 20 cells, N = 3 mice/condition tested.
Fig 5.
Caffeine increases potency of R-propafenone (RPROP) against Ca2+ waves.
A. Representative line-scans of Ca2+ waves in permeabilized WT myocytes and Casq2-/- myocytes under control condition (VEH) and in the presence of RPROP 10 μM. B. CRCs for wave parameters (incidence, amplitude, frequency, and propagation speed) as a function of the concentration of RPROP (in μM) in WT and Casq2-/- cells. Similarly to FLEC, the effect of RPROP on the Ca2+ waves is dose-dependent, with a strongest effect on Casq2-/- myocytes. Results expressed as mean±SE for each concentration and each parameter. For each group: n = 20–25 cells, N = 2 mice/condition tested.
Table 1.
Potency (IC50, expressed in μM) and efficacy (defined as maximum drug effect measured at 100 μM) of all the drugs tested in the different mouse groups.
Fig 6.
Caffeine has no effect on potency of tetracaine (TET) against Ca2+ waves.
A. Representative LS of Ca2+ waves in permeabilized WT myocytes, WT+CAFF 150μM, and Casq2-/- myocytes under control condition (VEH) and in the presence of tetracaine (TET) 25 μM. B. CRCs for wave parameters (incidence, amplitude, frequency, and propagation speed) as a function of TET concentration (in μM) in WT, WT+CAFF 150μM, and Casq2-/- cells. Mean±SEM is depicted for each concentration and each parameter. For each group: n = 20–25 cells, N = 2–3 mice/ condition tested.
Fig 7.
Effect of RPROP and TET on Ca2+ sparks and waves in permeabilized rabbit ventricular myocytes.
A. Bar representation (left side of the panel) of averaged Ca2+ spark parameters (amplitude, frequency and mass) measured in permeabilized rabbit ventricular myocytes under control conditions (VEH) and in the presence of RPROP 10 μM and TET 50 μM. **P<0.01. n = 30–40 cells, N = 3 rabbits/condition tested. B Representative LS for each condition. C CRCs for RPROP and TET for Ca2+ wave parameters. n = 30 cells, N = 3 rabbits/condition tested. D. Representative LS for VEH, RPROP 25 μM and TET 25 μM.
Table 2.
Potency (IC50, expressed in μM) and efficacy (defined as maximum drug effect measured at 100 μM) of RPROP and TET in permeabilized rabbit ventricular myocytes.