Figure 1.
Effect of FCCP on mitochondrial membrane potential (ΔΨm) and mPTP opening in isolated mouse ventricular myocytes.
A. TMRM fluorescence was monitored as an indicator of Δψm. FCCP (1 and 10 µM) was perfused as indicated by the horizontal bar. Two snapshots of TMRM fluorescence (control and ~2.5 min after the treatment with 10 µM FCCP) are shown. The fluorescence in the presence of 30 µM FCCP was set as 100% dissipation in each cell. B. Calcein fluorescence intensity was monitored as an indicator of mPTP opening. B-a. Two snapshots of calcein fluorescence at baseline (0 min) and 6 min after the treatment with FCCP (1 and 10 µM) and FCCP (1 µM) + CsA (1 µM). B-b. A representative recording of calcein fluorescence showing the rate of fluorescence decline in the presence of 1 and 10 µM FCCP. B-c. Summary data showing the calcein fluorescence decline in the presence of FCCP (100 nM, 1 and 10 µM) and CsA + FCCP as indicated. The fluorescence in the presence of 30 µM FCCP was set as 0% in each cell. *p < 0.05 vs. Control; #p < 0.05 vs. FCCP (1 µM).
Figure 2.
Effect of FCCP on High [Ca2+]o-induced Cai2+ waves.
A. FCCP (100nM) induced spontaneous CaWs (indicated by arrows) under normal excitation contraction coupling (ECC). The cells were paced by field stimulation at 0.5 Hz in the presence of 1 mM Ca2+ concentration under control (ctl) condition and ~ 5 min after perfusion with 100 nM FCCP. B. A Cai2+ fluorescence trace recorded from a mouse ventricular myocytes. The cell was first perfused with the normal Tyrode's solution (1 mM Ca2+) and then with a high Ca2+ Tyrode's solution (4 mM Ca2+). Cai2+ waves (CaWs) were consistently observed in the presence of high external Ca2+ (Cao2+; 4 mM). Spontaneous Ca2+ CaW were eliminated by Tetracaine (2 mM). C-a. A representative Cai2+ fluorescence trace showing the dose-dependent effects of FCCP on the SCWs. C-b. Effect of FCCP on CaW frequency in a dose-dependent and biphasic manner. C-c. Summary data showing the effect of FCCP on basal Cai2+. ∗p < 0.05,∗∗p < 0.01 vs. control (n = 6). D. Cyclosporin A (CsA), a mPTP inhibitor, significantly counteracted the effects of FCCP (100nM) on SCWs. A representative trace (D-a) and summarized data for CaW frequency (D-b) and basal [Cai2+] level (D-c) are shown. **p < 0.01 vs. Control; #P<0.05, ##p < 0.01 vs. FCCP, n = 8.
Figure 3.
Action potentials (APs) were triggered by FCCP-enhanced Cai2+ waves.
A. A line-scan image along the long axis of the cell (A-a), whole-cell Ca fluorescence intensity (A-b), and membrane potential (A-c), were recorded simultaneously from a ventricular myocyte. CaWs (W) and sub-theshold depolarizations (SD) were exacerbated by FCCP (100 nM) so that triggered APs/activities (TA) and Ca transients (T) were induced. B. Summary data showing that FCCP (100 nM) markedly increased the incidence of TAs (**p < 0.01 vs. control, n = 4).
Figure 4.
Elimination of SCWs by high dose FCCP is due to the reduction of SR Cai2+ level via Ca2+ extrusion by sNCX and less SR Ca2+ uptake by SERCA.
A. SR Ca2+ content was assessed by rapid application of 10 mM caffeine in the absence and presence of 1µM FCCP. FCCP significantly reduced SR Ca2+ concentration, presumably via Ca2+ efflux by Na+-Ca2+ exchanger (NCX) (**p < 0.01 vs. control, n = 5). B. Suppressing NCX restored SCWs in the myocytes pretreated with 1µM FCCP, while no SCWs were observed when SERCA2a was inhibited with thapsigargin (TG, 1µM). C. High-dose FCCP-inhibited CaWs were restored by elevating [Ca2+]o from 4 mM to 8 mM. D. Lack of CaW inhibition by high-dose FCCP (1 µM) in the presence of Oligomycin (Oligo 1 µM). The expanded trace in the inset shows a fast Cai2+ oscillation status. E. Mag-fluo-4 fluorescence (evaluation of reciprocal changes of [ATP]i) traces recorded in mouse ventricular myocytes treated with (E-a) 100 nM FCCP or (E-b) 1 µM FCCP. The baseline value (i.e. before perfusion of FCCP) was normalized to 1, as indicated by the leftward-facing arrow in each panel. The F/F0 level at 6 min after FCCP treatment (as indicated by the rightward-facing arrow) was compared between different groups. E-c. Summary data showing the intensity of the fluorescence (F/F0) recorded 6 min after the treatment (**p < 0.01 vs. Ctl).
Figure 5.
Effect of FCCP on basal [Cai2+] level (mitochondrial Ca2+ efflux).
A. Effects of FCCP (1 µM) on CaWs and basal Cai2+ level under control condition (4 mM Cao2+). B. Effects of FCCP on basal [Cai2+] level in a Cao2+-free condition. C. Effects of FCCP on basal Cai2+ level in Cao2+-free, and SR-Ca depleted condition. D. Summary data showing the effect of 1 µM FCCP on basal Cai2+ (∗∗p < 0.01 vs. control, n =11).
Figure 6.
Effect of mCU Ca2+ flux on CaWs.
A. Kaempferol, a mitochondrial uniporter (mCU) opener, significantly attenuated CaWs induced by 100nM FCCP. A representative trace (A-a) and summarized data for CaW frequency (A-b) and basal [Cai2+] level (c) are shown (**p < 0.01 vs. control; #p < 0.05 vs. FCCP, n = 5). B. The effect of Ru360, a mCU blocker, on CaW frequency and basal [Cai2+] level in the presence of 50 nM FCCP. A representative trace (B-a) and summarized data (B-b & c) are shown. #p < 0.05 vs. FCCP (n = 5). C. The effect of Ru360 on CaW in the absence of FCCP. Note a persistent and fast oscillating status after Ru360 treatment.
Figure 7.
Metabolic inhibition exhibited less direct effect on CaWs.
A. The effect of antimycin A (an electron transport chain inhibitor disrupting ΔΨm) on CaW frequency and basal [Cai2+] level. Representative traces (5 and 10 µM antimycin A) (A-a) and summarized data (A-b & c) are shown. ∗∗p < 0.01 vs. control (n=6). B. The effects of oligomycin alone (B-a) and in the presence (B-b) of high-dose antimycine A (10 µM) on CaWs. B-b & c. Summary data showing the CaW frequencies under each condition. (n = 5 for each, ∗∗p < 0.01 vs. control). C. the same as A, except for Iodoacetic acid (IAA, a glycolytic inhibitor, n = 7) was tested. IAA exerted no significant effects on CaWs or basal [Cai2+] (p > 0.05).
Figure 8.
Less involvement of ROS in CaW facilitation by FCCP.
A. Mitosox Red fluorescence (evaluation of mitochondrial superoxide level) traces recorded in mouse ventricular myocytes treated with (A-a) 100 nM FCCP or (A-b) 1 µM FCCP. The baseline value (i.e. before perfusion of FCCP) was normalized to 1, as indicated by the leftward-facing arrow in each panel. The F/F0 level at 6 min after FCCP treatment (as indicated by the rightward-facing arrow) was compared between different groups. (A-c) Summary data showing the intensity of the fluorescence (F/F0) recorded 6 min after the treatment (**p < 0.01 vs. Ctl). B. Effect of MnTMPyP (10 µM) on spontaneous CaWs. Oligomycin was applied to exclude potential influences of cellular metabolic status. FCCP (100 nM) promoted CaWs either in the absence (B-a) or the presence (B-b) of MnTMPyP. (A-c). Summary data showing the CaW frequencies under differential treatments. The cell number for measurement in each group is indicated (** p < 0.01).
Figure 9.
Schematic illustration of Ca2+ waves modulation by mitochondrial Ca2+ fluxes.
Mitochondrial Ca2+ is released into the cytoplasm via the permeability transition pore (mPTP) and up-taken by the mitochondrial calcium uniporter (mCU). An increase in local cytosolic [Ca2+] prompts additional uptake of Ca2+ by the SERCA pump, and triggers a greater release of Ca2+ by the ryanodine receptor (RyR), thus generating/enhancing CaWs. Either inhibition of mPTP by CsA or activation of mCU by kaempferol suppresses CaWs.