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Several factors trigger apoptosis in cochlear hair cells. Previous studies have shown that mitochondria play key roles in apoptosis, but the role of mitochondrial deoxyribonucleic acid (mtDNA) copy number in the pathogenesis of hair cell apoptosis remains largely unknown. We used mouse cochlear hair cells and House Ear Institute-Organ of Corti 1 (HEI-OC1) cells to explore the relationship between mtDNA copy number and cell apoptosis. We found that the mtDNA copy number of hair cells was reduced relative to mitochondrial mass and hypothesized that increasing it might have a protective effect. We then increased the mtDNA copy number of the hair and HEI-OC1 cells by transfecting them with an adeno-associated virus (AAV) vector containing mitochondrial transcription factor A (
Disabling hearing loss affects more than 5% of the world's population (466 million individuals). It is estimated that by 2050, more than 900 million people will be affected by disabling hearing loss (
The mitochondrion plays an important role in cell apoptosis; it is also the main site of intracellular oxidative phosphorylation and adenosine triphosphate (ATP) synthesis (
A total of 50 newborn C57BL/6J mice (P1-P3), each weighing ~1.5–2.5 grams, were used in this experiment, and their gender selection was random. International Council for Laboratory Animal Science (ICLAS) Ethical Guideline (
HEI-OC1 is a widely used progenitor hair cell line derived from mouse auditory organs (
Total RNA and DNA were extracted with a DNA/RNA Isolation Kit (Qiagen). Reverse transcription was performed using a PrimeScript RT Reagent Kit with gDNA Eraser (Takara) per the manufacturer's protocols. The mitochondrial gene
qPCR was performed using the Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific, Inc.) using GoTaq qPCR Master Mix (Promega). We designed validated primers for each target messenger RNA (mRNA) or DNA:
An AAV vector containing human
The probe was prepared using the FISH Tag DNA Kit (Thermo Fisher Scientific, Inc.), and the spectrum of the intended probe was from 278 to 1435 bases (NCBI Reference Sequence: NC_005089.1) in mouse mtDNA.
The cochlear basal membranes or HEI-OC1 cells were cultured, collected at proper time points on slides, and then fixed with 4% polyoxymethylene and permeabilized with 0.5% Triton X-100. Then, the FISH procedure was conducted as previously described (
MitoTracker Green FM (Thermo Fisher Scientific, Inc.) was used to determine mitochondrial mass, and MitoSOX Red (Thermo Fisher Scientific, Inc.) was used to determine reactive oxygen species (ROS) levels. Cochlear basal membranes were grown in culture medium for a certain period of time, the culture medium was removed and the samples were washed with PBS. We then added a pre-warmed (37°C) solution containing MitoTracker Green FM or MitoSOX Red and the cells were incubated at 37°C for 20 min. After staining, the samples were washed in PBS and imaged (living-cell imaging) under a Leica SP8 confocal microscope (magnification, ×40; Leica Biosystems). To label hair cells, anti-myosin VIIA rabbit polyclonal antibody (ab3481, Abcam) was added and the cells were incubated for 8 h (4°C) at a dilution of 1:200 after cochlear basal membranes had been fixed with 4% polyoxymethylene and permeabilized with 0.5% Triton X-100. We washed the samples 3 times with PBS and incubated them for 2 h with fluorescence-conjugated goat anti-rabbit immunoglobulin G (IgG) secondary antibody (ab150077, Abcam) at room temperature (RT).
Since GFP expression was extremely low on the second day after
To measure mitochondrial permeability, we conducted FCM using a MitoProbe Transition Pore Assay kit (M34153, Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. For each sample, we prepared 3 aliquots: 1 contained calcein-acetoxymethyl (calcein AM) only; 1 contained calcein AM and CoCl2; and 1 contained calcein AM, CoCl2 and ionomycin. The samples were incubated at 37°C in the dark for 15 min. Detailed information can be found in the manufacturer's protocols.
To determine mitochondrial membrane potential (MMP) and analyze reactive oxygen species (ROS), the HEI-OC1 cells were cultured, trypsinized and collected; and then resuspended in a pre-warmed (37°C) solution containing MitoTracker Red CMXRos or MitoSOX Red (Thermo Fisher Scientific, Inc.) for 10 min. The HEI-OC1 cells were then washed with PBS; and they were analyzed by FCM. For apoptosis analysis, a TUNEL assay kit was utilized. First, the cells were collected and fixed with paraformaldehyde, and then centrifuged for 5 min at 300 × g and then the supernatant was discarded. Next, the cells were washed in PBS, pelleted by centrifugation and then added to 5 ml ice-cold 70% (v/v) ethanol. Finally, the cells were analyzed by FCM. A Dead Cell Apoptosis Kit with Annexin V-FITC and PI (Thermo Fisher Scientific, Inc.) was used. The cells were washed twice with cold PBS and then resuspended at a concentration of 1×106 cells/ml. We added Annexin V/PI, gently mixed it with the cells, incubated the mixture at RT in the dark and then analyzed the cells by FCM as soon as possible. All tests were repeated at least 3 times.
For all experiments, values for the normal controls were set to 1, except for the experiments of flow cytometry. Therefore, most of our histograms show relatively quantified (RQ) values. We made cross-sectional reconstruction after confocal microscope scanning in all our hair cell imaging except for the apoptosis experiments. Al fluorescence quantification was performed with Adobe Photoshop CC (Adobe Systems Inc.). The targeted area (hair cell) was selected and the average brightness of each cell in gray mode was calculated. At least 50 cells in each group were selected. We repeated this procedure at least 3 times for all our experiments. Statistical analyses were conducted with SigmaPlot software version 12.3 (Systat Software, Inc.). A 2-tailed t test was used to compare values between groups. Data are presented as mean ± standard deviation (SD). The difference was considered significant when P<0.05.
Mitochondria are the powerhouses of cells. As auditory receptors and mechano-electrical transducers, hair cells need a large number of mitochondria, which we confirmed in this experiment. After culturing mouse cochlear basal membranes
In the apoptosis induction experiment, we added neomycin into the medium at a final concentration of 1 mM on the second day after
To verify the previous results on hair cells, we repeated the experiment on HEI-OC1 cells. After transfecting them with
In this experiment, the protection mechanisms of mtDNA copy number in HEI-OC1 cells were explored. As previously mentioned, mtDNA copy number peaked at the third day after
Then, we analyzed the mitochondrial transmembrane potential of HEI-OC1 cells by FCM using MitoTracker Red CMXRos at the same time point after DDP treatment. Mitochondrial transmembrane potential was higher in the
Mitochondria have their own genetic material, mtDNA, which encodes 13 proteins, 22 transfer RNAs (tRNAs), and 2 ribosomal RNAs (rRNAs), which are involved in maintaining the mitochondrial function. mtDNA copy number varies by cell (
In the present study, we measured the mitochondrial mass, ROS level, and mtDNA copy number in newborn mouse cochlear hair cells. It was found that the mitochondrial mass of hair cells was significantly higher than that of the supporting cells, and the same was true for their ROS level. These results were anticipated as hair cells require a great deal of energy from mitochondria to sustain their normal function, and the levels of by-product ROS increase during the ATP production process. Then, the mtDNA copy number in hair cells was then assessed and it was higher than that of the supporting cells, but to a smaller degree than was true for mitochondrial mass, meaning that mtDNA copy number was reduced relative to mitochondrial mass in hair cells. This result was not what we had expected. We think the reduced mtDNA copy may have influenced the normal mitochondrial function to a certain extent, giving rise to a high level of ROS and leading to the fragility of hair cells. Therefore, increased mtDNA copy number may have a protective effect on hair cells.
The regulation of intracellular mtDNA copy number is complicated. The catalytic subunit of DNA polymerase γ and its processivity factor (both encoded by the
In the apoptosis induction experiment, we used neomycin, which is an aminoglycoside antibiotic and can specifically damage hair cells in the cochlear basal membrane but does not damage the surrounding supporting cells (
To further confirm the protective effect of mtDNA copy number, we repeated these experiments using HEI-OC1 cells. The results were the same as for hair cells. The mtDNA copy number was increased and remained higher for approximately 1 week after
Increased mtDNA copy number may exert its protective effect by enhancing the mitochondrial function of the cell. However, the detailed mechanisms are not clear. When the mtDNA copy number was increased in HEI-OC1 cells, we analyzed the transcription of mtDNA genes (
Mitochondrial DNA is compacted into nucleoids with numerous nucleoid-associated proteins, each nucleoid containing just 1 copy of mtDNA (
The mitochondrial permeability transition pore (mPTP) is formed in the inner membrane of the mitochondrion, and it plays an important role in the physiological regulation of Ca2+ and in ROS homeostasis. The opening of the mPTP initiates the production and release of ROS, which damages mitochondrial and nuclear DNA, proteins, and phospholipids (
We analyzed the mitochondrial permeability of the HEI-OC1 cells by FCM using a mitochondrial-permeability transition pore assay kit and found that permeability was decreased in the
In summary, we found that the mtDNA copy number of cochlear hair cells was reduced relative to mitochondrial mass, which may contribute to the fragility of these cells. The mtDNA copy number can be increased
Not applicable.
The present study was supported by grants from the National Science Foundation for Young Scientists of China (no. 81500788), the National Key R&D Program of China (nos. 2017YFA0103900, 2016YFC0905200) and the National Natural Science Foundation of China (nos. 81620108005, 81470687, 81830029, 81570913).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
HM, DM and HY performed the experiments, collected the data and wrote the manuscript; SS performed the PCR analysis and collected the data. YC and YZ performed the transfection experiments and collected the data. RC performed the flow cytometry analysis and collected the data. HL designed the research and gave final approval of the version to be published. HL and SS directed the research and provided supporting materials.
Experimental protocols and procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Affiliated Eye and ENT Hospital, Fudan University (Shanghai, China).
Not applicable.
The authors declare that they have no competing interests.
cisplatin
House Ear Institute-Organ of Corti 1
mitochondrial deoxyribonucleic acid
mitochondrial permeability transition pore
reactive oxygen species
mitochondrial transcription factor A
mitochondrial membrane potential
flow cytometry
TdT-mediated dUTP nick-end labeling
fluorescein isothiocyanate
propidium iodide
Measurement of mitochondrial mass, mtDNA and ROS in newborn mouse cochlear hair cells. (A) Mitochondria were marked in cochlear basal membranes using MitoTracker Green (cross-sectional reconstruction). The mitochondrial mass of hair cells was significantly higher than that of the supporting cells (a, marked hair cells; b, mitochondrial staining; c, nucleus staining; d, the merged image). (B) FISH of mtDNA in cochlear basal membrane, in which the intensity of red fluorescence is indicative of mtDNA copy number (cross-sectional reconstruction). The mtDNA copy number of hair cells was higher than that of the supporting cells, but the difference in mtDNA copy number between hair cells and supporting cells was obviously smaller than the difference in mitochondrial mass (a, marked hair cells; b, mtDNA staining; c, nucleus staining; d, the merged image). (C) The ROS level was measured using MitoSOX Red (cross-sectional reconstruction). The ROS level in hair cells was higher than that in the supporting cells (a, marked hair cells; b, ROS staining; c, nucleus staining; d, the merged image). (D) Quantitative analysis of fluorescence intensity in supporting cells and hair cells in A (1±0.198; 44.177±6.881; t=14.024; n=5; P=0.000000649). (E) Quantitative analysis of fluorescence intensity in supporting cells and hair cells in B (1±0.110; 1.252±0.091; t=4.083; n=6; P=0.00274). (F) Quantitative analysis of fluorescence intensity in supporting cells and hair cells in C (1±0.210; 1.443±0.040; t=4.6422; n=5; P=0.00166). (G) Quantitative analysis of fluorescence intensity in supporting cells and hair cells in A and B (1±0.232; 0.027±0.004; t=9.366; n=5; P=0.0000138). Fluorescence intensity of mtDNA was divided by that of mitochondrial mass. Scale bars, 20 µm. **P<0.01, ***P<0.001. ROS, reactive oxygen species; RQ, relatively quantified values.
Increased mtDNA copy number has a protective effect on hair cells. (A) FISH of mtDNA in control cochlear basal membrane (a, marked hair cells; b, transfected cells; c, mtDNA staining; d, nucleus staining; e, the merged image), in which the intensity of red fluorescence is indicative of the mtDNA copy number, and the green fluorescence is indicative of the transfected cell (white arrow; cross-sectional reconstruction). (B) FISH of mtDNA in cochlear basal membrane on the second day after TFAM transfection (cross-sectional reconstruction) (a, marked hair cells; b, transfected cells; c, mtDNA staining; d, nucleus staining; e, the merged image). The outer two hair cells were transfected, and the mtDNA copy number was higher (white arrows). (C) We treated the control hair cells with neomycin (a, marked hair cells; b, nucleus staining; c, apoptotic cells; d, the merged image). Many outer hair cells were lost or apoptotic (apoptotic cells are labeled red). (D) We treated hair cells in the TFAM-transfected group with neomycin for the same length of time (a, marked hair cells; b, nucleus staining; c, apoptotic cells; d, the merged image). Few hair cells were lost. (E) Quantitative analysis of red fluorescence intensity in hair cells in A and B (1±0.220; 1.392±0.147; t=4.44; n=9; P=0.000412). (F) qPCR of mtDNA copy number. Cochlear basal membrane mtDNA copy number was increased after TFAM transfection (1±0.084; 1.649±0.206; t=5.052; n=3; P=0.00722). (G) Quantitation of C and D (1±0.127; 0.694±0.071; t=3.638; n=3; P=0.022). Apoptosis rate of TFAM-transfected hair cells was lower than that of the control. Scale bars, 20 µm. *P<0.05, **P<0.01, ***P<0.01. TFAM, mitochondrial transcription factor A; RQ, relatively quantified values.
Measurement of the transcription of TFAM and the mtDNA copy number of cochlear hair cells at the fifth day after AAV-TFAM-GFP transfection. (A) qPCR of the mRNA levels of TFAM. At the first, second and fifth day, we extracted the RNA of both control and TFAM-transfected cochlear basal membranes and performed PCR analysis. We found the mRNA levels were statistically higher in the TFAM-transfected group (1±0.159/43.615±5.386, 1±0.259/137.804±12.377, 1±0.131/69.955±6.770; t=13.698, 19.14, 17.638; n=3, 3, 3; P=0.000165, 0.0000439, 0.0000607). (B) qPCR of mtDNA copy number. At the fifth day, we extracted the DNA of both control and TFAM-transfected cochlear basal membranes and performed PCR analysis. No statistically significant differences were found (1±0.091; 1.149±0.180; t=1.282; n=3; P=0.269). At the fifth day, the transfected hair cells had bright-green fluorescence, but its mtDNA copy number (red fluorescence) was not higher than those of the other 2 outer hair cells: (C) control group and (D) TFAM group (cross-sectional reconstruction; a, marked hair cells; b, transfected cells; c, mtDNA staining; d, nucleus staining; e, the merged image). Scale bar, 20 µm. ***P<0.01. TFAM, mitochondrial transcription factor A; RQ, relatively quantified values.
Increased mtDNA copy number has a protective effect on HEI-OC1 cells. (A) Expression levels of TFAM RNA in HEI-OC1 cells. The expression was significantly increased, peaked at the second day (1±0.059; 67.804±5.377; t=21.516; n=3; P=0.0000276) and then decreased. (B) The mtDNA copy number of HEI-OC1 cells as analyzed by qPCR. The number was increased gradually after TFAM transfection, peaked at the third day (1±0.199; 4.152±0.808; t=6.556; n=3; P=0.0028), and then fell back. (C) Apoptosis analysis by FCM. We induced apoptosis in HEI-OC1 cells with DDP, and then analyzed the results using TUNEL. I, normal cells; II, apoptotic cells. The apoptosis rate decreased obviously after TFAM transfection. (D) Apoptosis analysis by FCM. We induced apoptosis in HEI-OC1 cells with DDP, and then analyzed the results using Annexin V/PI staining. The apoptosis rate decreased obviously after TFAM transfection (the value of each quadrant was a percentage). (E and F) Quantification of C and D. The results were consistent with that for hair cells (0.587±0.035, 0.515±0.013; 0.46±0.034, 0.274±0.006; t=4.797, 16.067; n=4, 3; P=0.00301, 0.0000878). *P<0.05, **P<0.01, ***P<0.001. TFAM, mitochondrial transcription factor A; RQ, relatively quantified values; DDP, cisplatin.
mtDNA copy number affects mitochondrial permeability, MMP, and ROS in HEI-OC1 cells. (A) mPTP assay by FCM; (a) Fluorescent calcein was present in the cytosol as well as the mitochondria, resulting in a bright signal. (b) In the presence of CoCl2, only calcein in the mitochondria emitted a signal and reflected cell mitochondrial permeability; (c) When ionomycin, a calcium ionophore, and CoCl2 were added to the cells at the same time, fluorescence signals were largely abolished. (B) Mitochondrial permeability of HEI-OC1 cells, analyzed by FCM. Fluorescence was obviously stronger in the TFAM group (1±0.078; 2.144±0.125; t=7.772; n=3; P=0.00148). (C) Mitochondrial transmembrane potential analyzed by FCM. This was higher in the TFAM group, but not to a statistically significant degree (1±0.053; 1.323±0.264; t=2.082, n=3, P=0.106). (D) ROS level analyzed by FCM. The level was lower in the TFAM group (1±0.024; 0.594±0.034; t=9.766; n=3; P=0.000616). **P<0.01, ***P<0.001. MMP, mitochondrial membrane potential; ROS, reactive oxygen species; TFAM, mitochondrial transcription factor A; mPTP, mitochondrial permeability transition pore; RQ, relatively quantified values.