Mitochondrial calcium uniporter is essential for hearing and hair cell preservation in congenic FVB/NJ mice

Mitochondrial Ca2+ regulates a wide range of cell processes, including morphogenesis, metabolism, excitotoxicity, and survival. In cochlear hair cells, the activation of mechano-electrical transduction and voltage-gated Ca2+ channels result in a large influx of Ca2+. The intracellular rise in Ca2+ is partly balanced by the mitochondria which rapidly uptakes Ca2+ via a highly selective channel comprised of the main pore-forming subunit, the mitochondrial Ca2+ uniporter (MCU), and associated regulatory proteins. MCU thus contributes to Ca2+ buffering, ensuring cytosolic homeostasis, and is posited to have a critical role in hair cell function and hearing. To test this hypothesis, Ca2+ homeostasis in hair cells and cochlear function were investigated in FVB/NJ mice carrying the knockout allele of Mcu (Mcu+/− or Mcu−/−). The Mcu knockout allele, which originated in C57BL/6 strain cosegregated along with Cdh23ahl allele to the FVB/NJ strain, due to the close proximity of these genes. Neither Mcu+/− nor Mcu−/− genotypes affected cochlear development, morphology, or Ca2+ homeostasis of auditory hair cells in the first two postnatal weeks. However, Mcu−/− mice displayed high-frequency hearing impairment as early as 3 weeks postnatal, which then progressed to profound hearing loss at all frequencies in about 6 months. In Mcu+/− mice, significantly elevated ABR thresholds were observed at 6 months and 9 months of age only at 32 kHz frequency. In three-month-old Mcu−/− mice, up to 18% of the outer hair cells and occasionally some inner hair cells were missing in the mid-cochlear region. In conclusion, mitochondrial Ca2+ uniporter is not required for the development of cochlea in mice, but is essential for hearing and hair cell preservation in congenic FVB/NJ mice.


Discussion
The uniporter protein MCU traversing the inner mitochondrial membrane is a highly selective Ca 2+ channel necessary for rapid mitochondrial Ca 2+ uptake in intact cells [32][33][34] . Initially, MCU alone was reported to be sufficient for mitochondrial Ca 2+ uptake 34 . However, further research has identified several other proteins that either act in concert with MCU or fine-tune its activity, thus forming a uniporter complex or uniplex. With pentameric MCU forming the main Ca 2+ permeant pore that is incumbent for rapid Ca 2+ uptake, the uniplex includes other protein components such as MCUb, EMRE, MICU1, MICU2, MICU3, MCUR1, and SLC25A23 35,36 . The gene encoding for MCU is well conserved in eukaryotes except for yeasts 34 ; in humans, it is expressed in all major tissues and organs 37 18 . However, the authors did not assess ABR thresholds in mice later than 7 weeks of age. In the present www.nature.com/scientificreports/ study, we recorded the ABR thresholds longitudinally in mice starting as early as 3 weeks to 12 months of age, in B6-CD1 as well as FVB/NJ background. C57BL/6J and CD1 strains are genetically predisposed to progressive hearing loss 19,20 , and are known to harbor the Cdh23 ahl allele in their genome 23,39 . The B6-CD1 background, despite being isogenic for Cdh23 ahl , may present with genetic variability and/or heterozygosity at innumerable loci; based on our results, this mixed background turned out to be unsuitable to study the hearing phenotype especially relating to MCU. Another logical assumption is the presence of genomic variants in the mixed background that may mask or compensate for the loss of Mcu. Supporting this notion, Mcu loss was shown to be protective to a certain degree against acoustic stress in the mixed B6-CD1 background, while in the CBA/J mice, siRNA or Ru360 mediated inhibition of MCU was protective against noise-induced OHC loss and permanent hearing impairment 18   www.nature.com/scientificreports/ is important to understand the spectrum of genotype-phenotype correlation or variation, and how that might relate to human subjects 41 .
The fact that the Cdh23 ahl allele is linked to the Mcu knockout allele may partly explain the accelerated hearing loss observed in the FVB/NJ double homozygous mice. However, we argue that this hearing impairment is a direct result of loss of Mcu based on the following observations: (i) The elevation in ABR threshold in Mcu −/− mice is noticed as early as 3 weeks, a timeline when the effect of Cdh23 ahl allele is insignificant on hearing in several inbred mouse strains 22,[42][43][44] . Additionally, the rapid ABR threshold elevation at 8 kHz and 16 kHz in FVB/NJ Mcu −/− mice at 1 month does not correspond to Cdh23 ahl -induced phenotype. (ii) Moreover, Cdh23 ahl homozygosity is shown to be necessary in the C57BL/6 mice, but not by itself sufficient to account for the hearing loss 44 ; also the C57BL/6J-derived Cdh23 ahl allele had little effect on hearing loss in the CBA/CaJ background 44 . (iii) The  [49][50][51][52][53][54] , voltage-sensitive Ca 2+ channels at the basolateral surface of IHCs, as well as additional Ca 2+ release from internal stores [55][56][57][58][59] . This excess Ca 2+ in the HCs is attenuated by several molecules such as the endogenous Ca 2+ buffering proteins, Ca 2+ ATPase (PMCA) pumps that extrude Ca 2+ , and organelles including mitochondria and endoplasmic reticulum that serve as Ca 2+   17,64 . Debate continues on whether these additional channels allow rapid Ca 2+ uptake or a gradual accumulation to sustain mitochondrial Ca 2+ homeostasis in the absence of MCU 65 . The ability of model organisms, specifically selective strains of mice, to survive without any overt phenotype in the absence of MCU supports the idea that MCU is either dispensable or is functionally compensated 17,66-71 . Nonetheless, the crucial role of MCU in several contexts, including acute energy demand, strenuous exercise, pathological conditions, tissue bioenergetics, and longevity cannot be disregarded 11 49 . The integrity of stereocilia in HCs is dependent on chronic Ca 2+ influx via MET channels 74 , and without MCU to handle excess Ca 2+ , the stereocilia may degenerate. In addition, the presence of Cdh23 ahl allele disrupts the normal exon splicing of Cdh23 and causes in-frame exon-skipping 46 . Because Cadherin 23 is a component of the tip links of hair cell stereocilia along with Protocadherin 15 75,76 , the presence of Cdh23 ahl variant may affect the preservation of stereocilia function contributing to HC degeneration 42,44 . However, the lack of correlation in the timeline corresponding to the onset of hearing loss and the degeneration of HCs in the FVB/NJ Mcu −/− mice indicates that the latter is a secondary manifestation of the hearing deficit. Genetic mouse models carrying mutated Otof, Ocm, or Vglut3 genes [77][78][79] are good examples to show impairment of hearing function ahead of visible hair cell or stereocilia damage. Alternatively, when compared against ABR, a quantitative technique, SEM imaging may fail to capture small structural changes in hair cells that commensurate with a decline in hearing. Degeneration of stereociliary bundles of OHCs and, less frequently, IHCs in older mice, initiating at the base of the cochlea and gradually progressing to the apex, is a distinctive characteristic of the Cdh23 ahl allele 42,48,80 . In contrast, the HC and stereocilia degeneration observed in FVB/NJ Mcu −/− mice at 12 weeks of age, predominantly along the mid-frequency region of the cochlea, might be the outcome of Mcu loss. A burgeoning question is why do only a middle turn fraction of HCs and their stereocilia degenerate in Mcu −/− mice? While further research is warranted, the scientific community is starting to appreciate the tissuespecific or cell-type-specific functions of MCU 81 . In summary, the present study provides convincing evidence that Mcu is essential for the preservation of hearing and stereocilia maintenance in congenic FVB/NJ mice. Understanding the precise mechanism associated with the loss of Mcu and the primary site of dysfunction that lead to the observed phenotype is important. Since the FVB/NJ Mcu mutant mice used in this study are constitutive knockouts, it is difficult to ascertain the initial site of the genesis of hearing loss in this model. It is also possible that the observed phenotype in Mcu mutant mice is a synergistic effect of mitochondria-rich cell types in the cochlea (hair cells, stria vascularis, and spiral ganglion neurons) and the Cdh23 ahl allele. Future studies involving cell-type-specific conditional knockout mice will be necessary to dissect the mechanism of hearing loss induced by the lack of MCU. DNA isolation and genotyping. Ear punched tissues of weaned mice were collected in sterile Eppendorf tubes and were processed for DNA isolation. Briefly, the tissues were lysed in SNET Lysis buffer, hair and other debris were removed by centrifugation, and the remaining supernatant was mixed with an equal volume of Isopropanol. DNA was pelleted by centrifugation, washed once with 70% Ethanol, and air-dried at RT for 15 min. The DNA was dissolved in 40 µL 0.5× TE buffer at 50 °C for 1 h and then 2 µL of each of the sample was used for PCR genotyping with DreamTaq Green PCR Master Mix (Thermo Fischer Scientific), and the following primers:

Animals. The
Mcu WT Fwd: 5′-GGA GTT AAG TCA TGA GCT GC-3′. The primers were synthesized by Integrated DNA Technologies and were used at a final concentration of 200 nmol/PCR reaction. The total reaction volume of the PCR was 20 µL, and the conditions were initial denaturation at 94 °C for 5 min., followed by 35 cycles of 94 °C for 15 s, 48 °C for 15 s, and 72 °C for 1 min, followed by a final extension at 72 °C for 10 min and 4 °C hold. The wild type allele of Mcu produced amplicons of 311 bp while the KO allele amplicons were 176 bp in length. The PCR products were resolved in a 1.5% Agarose Gel run in 1X TAE at 100 V, 400 mA for 35 min. Bullseye DNA SafeStain (MidSci) was used to visualize the DNA.

Scientific Reports
We developed a PCR-RFLP (Restriction Fragment Length Polymorphism) method for genotyping the Cdh23 c.753A/G polymorphism. A segment of exon 7 spanning the single nucleotide variant of interest of Cdh23 was amplified using the following primers: Cdh23.E7-Fwd 5′-AAG CTG TGT CAT TAT GTG TGG TAC -3′.
Cdh23.E7-Rev 5′-TAC TGT AGC ACC ATC AGG CTC-3′. PCR was set up to a total volume of 20 µL, with 100 nmol final concentration of each of the primers, 100 ng of genomic DNA and 1× DreamTaq Green PCR Master Mix. The PCR conditions were 95 °C for 2 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, a final extension of 72 °C for 2 min, and 10 °C hold. 10 µL of the PCR products were run in 1.5% agarose gel to verify amplification, and the remaining 10 µL was subjected to restriction digestion using 6 units of BsrI restriction enzyme (New England Biolabs) in 1× NEB 3.1 buffer totaling to a final volume of 25 µL. The digestion was performed at 65 °C for 1 h, followed by inactivation at 80 °C for 20 min. and 10 °C hold. Following digestion, the products were resolved in a 1.5% agarose gel with undigested controls and 100 bp DNA ladder. The 534 bp amplicons with Cdh23 c.753G allele remain undigested with BsrI, while the Cdh23 c.753A allele produces two fragments of 350 bp and 184 bp upon BsrI digestion. Validation was carried out by sequencing the PCR amplicons to verify restriction digestion.
Auditory brainstem response. The mice were weighed, and anesthetized with an Intra-peritoneal (IP) injection of a cocktail containing ketamine (20 mg/mL) and xylazine (1.75 mg/mL) at a dose of 0.1 mL/20 g. The depth of anaesthesia was tested by the toe-pinch response, eyes lubricated by application of PURALUBE ® VET Ointment, and the mice were placed in a sound-isolated chamber. The body temperature was maintained at 37 °C through a temperature-controlled heating pad. Subdermal electrodes were inserted at the vertex of the skull (positive), the mastoid region under the left ear (ground), and the mastoid region under the right ear (negative). Pure tone bursts were delivered through earphones using a computer-aided evoked potential system (Intelligent Hearing Systems, the Smart-EP software 3.30, Miami, FL, USA). Thresholds were determined for the 8 kHz, 16 kHz, and 32 kHz frequencies by escalating or deescalating the intensity in 10-dB steps starting at 70 dB, and then in 5-dB steps near-threshold until no organized responses were detected. For each stimulus, 768 sweeps were averaged for a response. Thresholds were defined as the lowest stimulus level where an organized response is observed. The recordings were performed blindly without knowledge of the genotype, and later the recorded values were matched with the genotype and grouped for statistical analyses.
RNA extraction and quantitative PCR. The mice cochleae were quickly dissected and isolated in icecold PBS, transferred to RNAlater™ solution (Thermo Fischer Scientific), and stored at 4 °C for a day. The next day, cochleae were transferred to ice-cold PBS to give a quick wash, and TRIzol (Invitrogen, Eugene, OR, USA) was used for total RNA extraction following the manufacturer's recommendation and instructions. The Super-Script IV First-Strand Synthesis System (Invitrogen) was utilized to synthesize cDNA from 2 µg of total RNA and used subsequently at a final concentration of 10 ng/reaction in qPCR. Quantitative PCR was performed using the PowerUp SYBR Green Master Mix (Thermo Fischer Scientific, A25742) on an ABI StepOnePlus™ Real-Time PCR System (Thermo Fischer Scientific). Details of the primers are provided in Table 1. The cycle reaction was performed as follows: 50 °C for 2 min, 95 °C for 2 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min, and finally a dissociation curve of 95 °C for 15 s and 60 °C for 15 s. The gene expression was calculated relative to the housekeeping genes Hprt and PolR2f and then analyzed using the 2 −ΔΔCT method 82 .
The organ of Corti explants, imaging and analysis. Organ of Corti explants were obtained as described previously [24][25][26] . Organs were dissected at postnatal days 3-4 (P3-P4). Briefly, the sensory epithelium was separated first from the lateral wall and then from the modiolus. The most basal "hook" region was cut out and the remaining organ was placed into a glass-bottom Petri dish (FluoroDish, WPI) and cultured in DMEM medium supplemented with 7% fetal bovine serum (Invitrogen, Carlsbad, CA) at 37 °C and 10% CO 2 . The organ of Corti explants were kept in vitro from one to five days before the experiments. The equivalent age (age of dissection plus days in vitro) of the specimens reported in this study was P5-P9. To prevent contamination, 10 µg/mL of ampicillin (Calbiochem, La Jolla, CA) was added to the medium. FM1-43 (Invitrogen) was used to evaluate mechanotransduction and to count functional hair cells. FM1-43 was dissolved in DMSO to obtain a stock solution with a concentration of 1 mM. Immediately before the experiment, the stock solution was diluted to 5 μM in Ca 2+ -free HBSS. Then, all specimens were briefly incubated in FM1-43 for 30 s at room temperature and then carefully rinsed in standard HBSS. MitoSOX Red superoxide indicator (Invitrogen), as well as calcium indicators Fluo-2 LeakRes AM (TEFlabs) and Rhod-2 AM (Biotium), were used according to manufacturer provided protocol. The raw images were gathered using an upright Olympus BX51WI microscope equipped with a 100 × 1 NA objective and a Grasshopper3 CMOS camera (FLIR), using manufacturer provided software. Images were subjected to fluorescence measurements using ImageJ (NIH). A region of interest was used to obtain measurements from the hair cells (I cell ) and an area without cells (I background) in the same image. Fluorescence intensity (I load ) for the hair cells were normalized (I load = I cell − I background ). After removing the stapes, and opening the round and oval window, the cochleae were immersed in the fixative for 2 h and then stored at 4 °C in 1/10th fixative diluted with buffer until collected for further preparation. The cochleae were removed, then washed with PBS, and placed in 5% EDTA, pH 7.4 at 4 °C for three days to decalcify. Following decalcification, samples were dissected and organs of Corti were removed and divided into apical, middle, and basal parts. The specimens were then dehydrated in a gradient ethanol series, critical-point dried using CO 2 , mounted on an SEM stub, and sputter-coated with 10 nm palladium. Cochlear epithelia were viewed and imaged with a high-resolution scanning electron microscope (FEI Helios NanoLab 650, Germany) housed at Swagelok Center for Surface Analysis of Materials (SCSAM), CWRU.
Statistical analysis. All statistical analyses were performed using Microsoft Excel and GraphPad Prism 7. Data are reported as mean ± SEM unless noted otherwise. Two way ANOVA analyses with post hoc pairwise comparisons using Bonferroni adjustments or Kruskal-Wallis test with Dunn's multiple comparisons were used depending on data distribution. Comparisons between two groups were tested by Student's t-test. P values < 0.05 were considered significant.
Received: 8 December 2020; Accepted: 13 April 2021 www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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