Chronology of cellular events related to mitochondrial burnout leading to cell death in Fuchs endothelial corneal dystrophy

Abstract

Fuchs endothelial corneal dystrophy (FECD) is a degenerative eye disease characterized by corneal endothelial cell (CEC) death and the formation of guttae, an abnormal thickening of CEC's basement membrane. At the tissue level, an oxidative stress causing mitochondrial damage and CEC death have been described to explain FECD pathogenesis. At the cellular level, our group has previously observed significant variability in the mitochondrial mass of FECD CECs. This led us to hypothesize that mitochondrial mass variability might play a key role in the chronology of events eventually leading to CEC death in FECD. We thus used different fluorescent markers to assess mitochondrial health and functionality as a function of mitochondrial mass in FECD corneal endothelial explants, namely, intra-mitochondrial calcium, mitochondrial membrane potential, oxidation level and apoptosis. This has led us to describe for the first time a sequence of events leading to what we referred to as a mitochondrial burnout, and which goes as follow. FECD CECs initially compensate for endothelial cell losses by incorporating mitochondrial calcium to help generating more ATP, but this leads to increased oxidation. CECs then resist the sustained need for more ATP by increasing their mitochondrial mass, mitochondrial calcium and mitochondrial membrane potential. At this stage, CECs reach their maximum capacity and start to cope with irreversible oxidative damage, which leads to mitochondrial burnout. This burnout is accompanied by a dissipation of the membrane potential and a release of mitochondrial calcium, which in turn leads to cell death by apoptosis.

Figure 1

Vicious cycle of FECD pathogenesis. The CEC Na+K+−ATPase pump, required to maintain corneal deturgescence, uses 30% of the cellular ATP, this high energetic demand is met by increasing the number of mitochondria and/or by increasing mitochondrial calcium intake. However, this leads to oxidative stress and/or the formation of mitochondria permeability transition pores [MPTP], resulting in a loss of mitochondrial membrane potential [↓Δψm] and elimination of altered mitochondria by mitophagy or induction of cell death by apoptosis. When FECD CEC die [↓ Cells], the amount of energy globally required to maintain corneal deturgescence remains the same, therefore increasing ATP demand on remaining cells, in turn promoting mitochondrial exhaustion. Although all cells are not affected to the same degree, each additional cell death feeds the vicious cycle.

Figure 2

Higher variability of mitochondrial mass in FECD explants. (A) A marker of mitochondrial mass (mitotracker) was used in healthy and FECD endothelial explants. The typical CEC loss and the enlargement of remaining CECs in the FECD explants (right) compared to the healthy explants (left) is well illustrated. (B) Quantification of the mitotracker signal showed no statistically significant difference in mitochondrial mass between healthy and FECD explants. However, FECD cells presented more variation (Coefficient of variation (CV) = 0.44) than the Healthy cells (CV = 0.17). Scale bar = 40 μm. Median and standard deviation are reported. Experiments were performed with 3 different explants from Healthy (1200 cells analyzed, N = 3) and 3 FECD patients (1176 cells analyzed, N = 3).

Figure 3

Cell death is linked to mitochondrial mass depletion in FECD CEC. (A) A marker of cell death by apoptosis (caspase-3/7 activity; green) was used in conjunction with the mitochondrial mass marker (mitotracker; red) in healthy and FECD explants. (B) Mitochondrial and caspase-3/7 signals were quantified in healthy (blue) and FECD (red) explants and plotted. Each dot represents a single cell. No caspase-3/7 signal was detected in healthy explants. Caspase-3/7 negative and positive cells composed 48% and 52% of the FECD cells, respectively. The median mitochondrial mass was higher in caspase-3/7 negative cells than in caspase-3 positive cells. FECD CECs with mitochondrial mass ranging between Caspase-3/7 positive and negative medians composed 46% of the FECD cellular population. These cells are referred to as containing a “Normal mitochondrial level”, which happened to be very similar to that of healthy cells. Twenty seven percent of the Caspase-3/7 negative FECD cells were above the median mitochondrial mass level and are therefore referred to as containing a “High mitochondrial level”. On the other hand, 27% of the caspase-3/7 positive FECD cells were below the median mitochondrial mass level and are therefore referred to as containing a “Low mitochondrial level”. Scale bar = 40 μm. Experiments were performed with 3 different explants from Healthy (900 cells analyzed, N = 3) and 3 FECD patients (846 cells analyzed, N = 3).

Figure 4

Oxidative stress correlates with an increase in mitochondrial mass in FECD cells. (A) A marker of oxidative stress (CM-H₂DCFDA; green) was measured in conjunction with mitochondrial mass marker (mitotracker; red) in healthy and FECD. (B) Quantification of the oxidative stress marker showed no detectable level of oxidation in healthy endothelial explants, but a significantly higher amounts of oxidation in CECs of FECD explants. (C) CM-H₂DCFDA and mitotracker signal levels were plotted and each dot represents a single CEC (blue for healthy cells and red for FECD). A strong positive correlation was found between the mitochondrial mass and the oxidative stress level (R² = 0.514; p < 0.0001). (D) Quantification of CM-H₂DCFDA in FECD CEC populations according to their mitotracker level, i.e. low, normal and high mitochondrial level categories as depicted in Fig. 3. The significant difference in oxidation level between low and normal, as well as between normal and high categories confirms that a higher level of mitochondrial mass is associated with a higher level of oxidative stress, and vice versa. Scale bar = 40 μm. Experiments were performed with 3 different explants from Healthy (600 cells analyzed, N = 3) and 3 FECD patients (594 cells analyzed, N = 3). *p < 0.01;**p < 0.001;***p < 0.0001.

Figure 5

Higher mitochondrial calcium is found in FECD cells. (A) A marker of intra-mitochondrial calcium (Rhod-2; red) was measured in conjunction with mitochondrial mass marker (mitotracker; green) in healthy and FECD explants. Rhod-2 labelling allows the observation that CECs were much larger in the FECD than in the healthy explant and numerous guttae (star) are seen, disorganizing the architecture of the endothelial mosaic. (B) Quantification of intra-mitochondrial calcium showed a significantly higher levels of calcium in the FECD CECs, with no detectable level in healthy CECs. (C) Rhod-2 and mitotracker signal levels were plotted, each dot representing a single CEC (blue for healthy cells and red for FECD). A weak correlation was observed (R² = 0.020; p < 0.001). (D) Quantification of Rhod-2 in FECD CEC populations according to their mitotracker mass, i.e. low, normal and high mitochondrial level categories as depicted in Fig. 3, revealed that only the category with a high mitochondrial mass had a calcium level significantly higher than that of the other two categories. Scale bar = 40 μm. Experiments were performed with 3 different explants from Healthy (800 cells analyzed, N = 3) and 4 FECD patients (710 cells analyzed, N = 4). *p < 0.01 and **p < 0.001.

Figure 6

Mitochondrial membrane potential (ΔΨm) is lower in FECD and correlates with an increase in mitochondrial mass. (A) A marker of ΔΨm (JC-1; red) was measured in conjunction with mitochondrial mass marker (mitotracker; blue) in FECD and in healthy explants as controls. (B) Quantification of ΔΨm showed a high level of membrane potential in healthy cells, indicating of a normal capacity to generate ATP. On the other hand, a clear qualitative decrease in ΔΨm in FECD CEC, indicating a lower capacity to produce ATP in these cells. (C) Signal of ΔΨm and mitotracker mass were plotted, each dot representing a single CEC (blue for healthy cells and red for FECD). A weak positive correlation was found between mitochondrial mass and ΔΨm in healthy cells (R² = 0.235; P < 0.0001) while that correlation was negligible in FECD cells (R² = 0.038; p < 0.0001). (D) Distribution of the ΔΨm values of FECD CECs according to their mitotracker mass category (low, normal and high) as depicted in Fig. 3, revealed that ΔΨm was lowest in the “Low” and highest in the “High” mitochondrial level cell populations. Scale bar = 40 μm. Experiments were performed with 3 different explants from Healthy (1176 cells analyzed, N = 3) and 3 FECD patients (1200 cells analyzed, N = 3). *p < 0.01; **p < 0.001;***p < 0.0001.

Figure 7

Theory of mitochondrial burnout leading to CEC death in FECD. Theoretical series of events related to the burnout stages leading to FECD CEC cell death. The relative level of each markers investigated in this study is plotted against a timeline. (1) Compensation stage: Normal mitochondrial mass, high mitochondrial calcium and high oxidative stress. No sign of apoptosis. FECD CEC in this stage are compensating for the loss of surrounding cells by incorporating mitochondrial calcium to help providing more ATP. The consequence is an increase in oxidative stress which leads to a lower ΔΨm. (2) Resistance stage: Further increase in mitochondrial mass, mitochondrial calcium, oxidative stress and membrane potential. FECD CEC in this stage are resisting the sustained need for more ATP caused by depletion of surrounding cells but they have reached their maximum capacity. The increase in mitochondrial mass leads to an increase in oxidative stress. (3) Burnout stage: Apoptotic cells with normal mitochondrial mass, lower ΔΨm, oxidation, and mitochondrial calcium. Mitochondrial permeability transition pore are most likely opening, which leads to a loss in ΔΨm and mitochondrial calcium. This leads to apoptosis of FECD CEC. (4) Death stage: Apoptotic CEC FECD with low mitochondrial mass, low oxidation stress and depleted ΔΨm. Alteration to mitochondria leads to a loss in mitochondrial mass through mitophagy and their loss of ΔΨm through the opening of the mitochondrial permeability transition pore.