Figure 1.
Effect of AA on PTP opening in isolated mouse liver mitochondria.
A, chemical structure of AA. B, D, Ca2+ retention capacity (CRC) either in phosphate (Pi) buffer (B) or in arsenate (Asi) buffer (D). Calcium Green-5N fluorescence is reported as arbitrary units on the y axis. As the probe does not permeate mitochondria, Ca2+ uptake into the organelles is displayed as a rapid decrease of the fluorescence spike after administration of every Ca2+ pulse (10 µM each). AA (red trace, 8 µM) or CsA (0.8 µM) act as pore inhibitors only in Pi buffer (B), as they increase the threshold Ca2+ concentration required to trigger the permeability transition, i.e. the number of spikes before a sudden and marked fluorescence increase occurs. Ub0 (25 µM) inhibits the pore also in Asi buffer, albeit to a lesser extent. C, inset of D, quantification of the effect of PTP inhibitors is displayed as the ratio between the CRC detected in the presence (CRC) and absence (CRC0) of the compound. Results are mean±SD of at least 4 experiments. In C and D, we analyzed whether each pharmacological treatment increased mitochondrial Ca2+ uptake when compared to control conditions (Ca2+ uptake in the absence of the drug), and found a significant difference (Student's t test analysis; *: p<0.01) between the CRC of mitochondria treated with either AA (at various concentrations), or CsA, or Ub0 and the CRC of untreated mitochondria, indicating that each of these treatments inhibits the PTP. In C, significant differences were also observed between the CRC of mitochondria treated with either Ub0/AA or Ub0/CsA and with Ub0 by itself (Student's t test analysis; #: p<0.01), indicating that the inhibitory effect of both AA and CsA on the PTP is additive with that of Ub0.
Figure 2.
Phe residues in position 6 and 9 of AA are required for PTP inhibition.
A, amino acid sequence of AA derivatives tested on the PTP. Amino acid changes are outlined in red. B, Ratio between the CRC detected in the presence (CRC) and absence (CRC0) of increasing concentrations of AA and derivatives. The CRC/CRC0 ratio is calculated as in Figure 1. Results are mean±SD of at least 4 experiments. C, Representative experiment showing the lack of effect on Ca2+ retention capacity (CRC) of the octa-Gly9 AA derivative. Traces are reported as in Fig. 1B.
Figure 3.
Effect of AA on respiration and PTP opening in mitochondria of wild-type or CyP-D knock-out mouse fibroblasts.
A, upper part: Western blot analysis assessing CyP-D expression in fibroblasts obtained from either wild-type or CyP-D knock-out mice; HKII was utilized to verify protein load; lower part: representative experiment showing oxygen consumption as assessed by polarography (see Methods) in mitochondria isolated from wild-type fibroblasts in the absence (a) or presence (b) of 8 µM AA. Experiments were started by adding 0.6 mg of mitochondria (Mito), followed by 100 µM ADP and 50 µM 2,4-dinitrophenol (DNP) (arrows). Each experiment was repeated at least three times. Oxygen consumption is reported as percentage of initial value on the y axis. B, bar graphs report the ratio between the CRC detected in the presence (CRC) and absence (CRC0) of increasing concentrations of AA, in mitochondria from either wild-type fibroblasts (left) or CyP-D knock-out fibroblasts (right). PTP inhibition by AA and CsA is not additive. Results are mean±SD of at least 4 experiments. We analyzed whether each pharmacological treatment increased mitochondrial Ca2+ uptake when compared to control conditions (Ca2+ uptake in the absence of the drug), and found a significant difference (Student's t test analysis; *: p<0.01) between the CRC of mitochondria treated with either AA (at various concentrations), or CsA, and the CRC of untreated mitochondria, indicating that each of these treatments inhibits the PTP.
Figure 4.
Effect of AA on PTP opening in wild-type or CyP-D knock-out mouse fibroblasts and on CyP-D isomerase activity.
A, B, PTP opening of digitonized wild-type (A) or CyP-D knock-out (B) fibroblasts was measured with the whole-cell CRC assay. Experiments were started by the addition of digitonized cells (not shown) followed by pulses of Ca2+ (5 µM each) and are plotted as in Figure 1B. The use of AA is shown with a red trace. Alamethicin (Alm, 1 µM) was added at the end of each measurement to fully release Ca2+ from intracellular stores. C, the CRC/CRC0 ratio calculated as in Figure 1C indicates that both AA and CsA display an inhibitory effect on the PTP in wild-type, but not in CyP-D knock-out fibroblasts. Results are mean±SD of at least 4 experiments. We analyzed whether each pharmacological treatment increased mitochondrial Ca2+ uptake when compared to control conditions (Ca2+ uptake in the absence of the drug), and found a significant difference (Student's t test analysis; *: p<0.01) between the CRC of mitochondria treated with either AA or CsA, and the CRC of untreated mitochondria, indicating that each of these treatments inhibits the PTP in wild-type fibroblasts. D, isomerase activity assay (see Materials and Methods): after addition of the substrate peptide N-succinyl-Ala-Ala-cis-trans-Pro-Phe-p-nitroanilide (arrow), chimotrypsin cleaves the trans-isomeric form, causing a rise in absorbance. AA (8 µM) and CsA (8 µM) act as inhibitors of the enzymatic activity of CyP-D.
Figure 5.
Effect of AA on mitochondrial potential of HeLa cells.
A, bar graphs report the ratio between the CRC detected in the presence (CRC) and absence (CRC0) of increasing concentrations of AA in mitochondria from HeLa cells. PTP inhibition by AA and CsA is not additive. Results are mean±SD of at least 4 experiments. We analyzed whether, in mitochondria from HeLa cells, each pharmacological treatment increased Ca2+ uptake when compared to control conditions (Ca2+ uptake in the absence of the drug), and found a significant difference (Student's t test analysis; *: p<0.01) between the CRC of mitochondria treated with either AA (at various concentrations), or CsA, and the CRC of untreated mitochondria, indicating that each of these treatments inhibits the PTP. B,C cytofluorimetric analysis of mitochondrial depolarization. HeLa cells were incubated with the TMRM probe and treated for 1 hour with the reported concentrations of either clotrimazole (CTM) or TAT-HK peptide. An unrelated TAT-linked peptide (TAT-Ctr) is used as a negative control. Before the addition of either CTM or TAT-peptides, cells were preincubated for 30 minutes with CsA or AA, or with the CsA analogue cyclosporin H (in the dubbed Ctr conditions), to exclude for changes in TMRM signal unrelated to mitochondrial potential (see Methods). Bar graphs in B display the percentage of cells with depolarized mitochondria (mean±SD, n = 4). We analyzed whether each pharmacological treatment increased the percentage of HeLa cells with depolarized mitochondria when compared to control conditions (i.e., percentage of cells with depolarized mitochondria in the absence of the drug), and found a significant difference (Student's t test analysis; #: p<0.01) between cells treated with either CTM or TAT-HK, and untreated cells, indicating that each of these compounds damages mitochondria. A similar pair wise comparison allowed to establish that pretreatment with either AA or CsA significantly decreased the percentage of cells with mitochondria depolarized by CTM (Student's t test analysis; *: p<0.01) or by TAT-HK (Student's t test analysis; **: p<0.01). Histograms in C report a representative experiment, where cells with polarized and depolarized mitochondria are indicated in red and grey, respectively.
Figure 6.
Effect of AA on HeLa cell viability.
A, cytofluorimetric analysis of cell death induction in HeLa cells treated for 1 hour with TAT-HK. Where indicated, cells were preincubated for 30 minutes with CsA or AA. TAT-Ctr was used as a negative control. Cells were double stained with Annexin V-FITC and propidium iodide (PI), to respectively evaluate apoptosis induction as phosphatidylserine exposure on the cell surface (increased Annexin V-FITC staining) and cell death as plasma membrane permeabilization (increased PI staining). We considered Annexin V+ as early/intermediate apoptotic cells; PI+ as necrotic cells; double stained as late apoptotic cells. Bar graphs display the percentage of each cell population (mean±SD, n = 4; double positive cells are added to both bars). The number of PI+ cells was significantly increased by TAT-HK treatment (Student's t test analysis; #: p<0.01 when comparing untreated and TAT-HK treated HeLa cells). Moreover, HeLa cell pretreatment with either AA or CsA significantly protected from TAT-HK induced death (Student's t test analysis; *: p<0.01 when comparing TAT-HK treated HeLa cells pretreated or not with either AA or CsA). B, histograms reporting a representative experiment, where viable cells are indicated in red, PI+- and Annexin V+-cells are indicated in grey and green, respectively.