Increased mitochondrial Ca2+ contributes to health decline with age and Duchene muscular dystrophy in C. elegans

Sarcopenia is a geriatric syndrome characterized by an age-related decline in skeletal muscle mass and strength. Here, we show that suppression of mitochondrial calcium uniporter (MCU)-mediated Ca2+ influx into mitochondria in the body wall muscles of the nematode Caenorhabditis elegans improved the sarcopenic phenotypes, blunting movement and mitochondrial structural and functional decline with age. We found that normally aged muscle cells exhibited elevated resting mitochondrial Ca2+ levels and increased mitophagy to eliminate damaged mitochondria. Similar to aging muscle, we found that suppressing MCU function in muscular dystrophy improved movement via reducing elevated resting mitochondrial Ca2+ levels. Taken together, our results reveal that elevated resting mitochondrial Ca2+ levels contribute to muscle decline with age and in muscular dystrophy. Further, modulation of MCU activity may act as a potent pharmacological target in various conditions involving muscle loss.


Introduction
functional declines with age 12, 13 . C. elegans sarcomeres and mitochondria are located in the monolayer 53 within the cell and can be easily observed alive under a microscope. Declines in mitochondrial network 54 structure, increased fragmentation, and reduced mitochondrial volume occur earlier than sarcomere 55 decline and correlate more strongly with reduction in movement, maximum velocity, and lifespan 14,15,16 . 56 In addition, several molecular systems such as the dystrophin complex and mitophagy, which is 57 controlled by PINK and PERKIN, are conserved in C. elegans 17,18,19 . Furthermore muscle deterioration 58 can be examined without the influence of muscle regeneration since C. elegans has no muscle stem cells. 59 Therefore, the C. elegans body wall muscle is a simple model useful to study (primary) sarcopenia and 60 other inherited muscular diseases. 61 In this study, we examined the role of mitochondrial Ca 2+ homeostasis in the context of aging and 62 sarcopenia using C. elegans. Initially, like past studies, we observed aberrant changes in mitochondria in 63 the body wall muscle of aged worms. We next confirmed elevated levels in resting mitochondrial Ca 2+ 64 with age. Either pharmacologic or genetic inhibition of MCU function was sufficient to prevent increases 65 in mitochondrial Ca 2+ and improve sarcopenic phenotypes. In addition, we found that Duchene muscular 66 dystrophy (DMD) worms also exhibit abnormally high cytosolic and mitochondrial Ca 2+ levels, and that 67 MCU inhibition was similarly sufficient to prevent increases in mitochondrial Ca 2+ and improve health. 68 The results in this study indicate that altered mitochondrial Ca 2+ homeostasis is associated with muscle 69 aging and dystrophy in C. elegans. These findings raise the possibility that mitochondrial Ca 2+ 70 homeostasis is associated with mammalian muscle aging and dystrophy and that it may be a potential 71 therapeutic target in them.

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Results and discussion 74 Age-related mitochondrial changes with Ca 2+ accumulation 75 In the body wall muscle on D4 adulthood of WT worms, the mitochondria contained aligned filamentous 76 structures, but these structures fragmented and shortened with increasing age (Figures 1A and B). Our 77 qualitative analysis showed that more than 70% of muscular cells in the D10 WT worms were classified 78 colocalized with the autophagosomal marker LGG-1::GFP, or with the lysosomal marker LMP-1::GFP in 105 D10 muscle cells (Figure 1E). Thus, mtGFP-negative mtGECO structures were trafficked into acidic 106 lysosomal compartments, which must have quenched the fluorescence of GFP. In a weakly acidic 107 environment, mtGECO can fluoresce but decrease fluorescence emission as the pH decreases 20 . In 108 addition, mtGECO can have Ca 2+ -dependent fluorescence changes under a weak acidic condition (pH 5.5) 109 judging from 20 . Therefore, we assumed that intense fluorescence intensities observed in the mtGECO 110 structures could reflect Ca 2+ accumulation. Taken together, these results suggest that the fragmented 111 mitochondrial network in aged C. elegans muscle cells contain fragments with elevated Ca 2+ levels, and 112 that these Ca 2+ -accumulated portions of mitochondria are eliminated by mitophagic pathways, 113 presumably to maintain mitochondrial function.  mcu-1(ju1154) was introduced into this recombinant (ATU2302; mcu-1(ju1154)), cytosolic Ca 2+ changes 122 associated with muscle contraction were observed, but synchronized Ca 2+ influx into the mitochondria 123 was largely lost (Figure 2A, Supporting Movie 2). These results indicate that when a large amount of Ca 2+ 124 flows into the muscle cytosol due to muscle contraction, Ca 2+ is also taken up in mitochondria via MCU-1. 125 Gibhardt et al (2016) report similar usefulness of LAR-GECO as a mitochondrial Ca 2+ sensor in the 126 mammalian muscular system 23 .

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Intriguingly, the mcu-1 mutant was shown to suppress mitochondrial fragmentation and severe 128 loss of mitochondrial mass, as well as age-related Ca 2+ accumulation in mitochondria (Figure 2B, C and 129 D). As a result, the proportion of muscle cells displaying progressing mitophagy with age also decreased 130 ( Figure 2B and E). In the control wild-type (WT) with aceIs1 and ccIs4251 transgenes, approximately 131 half of the population had died by 10-day-old adulthood (D10), with surviving individuals displaying 132 significantly reduced motor activity (mean velocity) and oxygen consumption rate (OCR) activity 133 compared to 1-day-old adulthood (D1) (Figures 2F, G and H). These age-related declines were 134 significantly reduced in the mcu-1(ju1154) null mutation in the same background. In addition, in the 135 muscle on D10 adults treated with mcu-1 RNAi, less than 20% muscular cells were 'fragmented', 136 [Ca 2+ ] mito did not significantly increase until 10-days old, and mitophagy activity in muscle cells was 137 decreased (Supplementary Figure S3). The life span was also extended in the mcu-1 mutant; the 138 difference in life expectancy between the mutant and control was 3.2 days (control, 12.5 ± 0.2 days; mcu-139 1(ju1154), 15.7 ± 0.4 days; p < 0.01) ( Figure 2F). A similar result was observed between wild-type N2 140 and the mutant strain CZ19982 without the transgenic reporters; the original mcu-1(ju1154) mutation 141 extended life span (N2, 14.7 ± 0.5 days; CZ19982 mcu-1(ju1154), 17.6 ± 0.8 days; p < 0.05).

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Recently, it has been reported that the highly conserved ryanodine receptor (RyR), UNC-68 in C. 143 elegans, is oxidized with age which results in age-dependent 'leaky' channels 24 Figure S4). The full-width half-maximum (FWHM) 25   Having established that genetic ablations of mcu-1 were sufficient to improve age-related muscle 153 mitochondrial changes, we examined whether pharmacological inhibition of MCU-1 could similarly 154 improve muscle health with age. Ru360, a specific mitochondrial calcium uptake inhibitor 26,27 , was used 155 to inhibit mitochondrial Ca 2+ influx. As this compound is significantly restricted in intact mammalian systems due to its poor cell permeability 26,27 , we first evaluated the penetration of Ru360 into intact C. 157 elegans continuously cultured at a final concentration of 10 μM Ru360 from egg to adulthood. In adults 158 of ATU2301 treated with Ru360, synchronized Ca 2+ influx into the mitochondria was significantly lost 159 ( Figure 3A), as it was the mcu-1 null mutant (Figure 2A), indicating that Ru360 is permeable to C. 160 elegans muscle cells. As expected, Ru360 treatment prevented age-related changes in the body wall 161 muscles of C. elegans including upregulated [Ca 2+ ] mito , mitochondrial fragmentation and formation of 162 mtGECO-structures ( Figures 3B-E). Furthermore, Ru360 improved mobility with age ( Figure 3F). These 163 results confirm that inhibition of MCU-1 mediated Ca 2+ influx attenuates age-dependent changes in 164 mitochondrial morphology and Ca 2+ levels, suppress the emergence of Ca 2+ -accumulated structures, and 165 ultimately attenuates movement decline with age. The mcu-1 mutant, mcu-1 RNAi, and MCU-1 inhibitor 166 studies combine in demonstrating that C. elegans muscle decline with age is normally driven by excessive 167 Ca 2+ influx into mitochondria and not merely elevated cytosolic Ca 2+ levels due to leaky RyR channels 24 .  After immobilization with polystyrene microspheres, cytosolic Ca 2+ live imaging was performed on 175 single muscle cells of D4 animals ( Figure 4A). The FWHM duration of the dys-1(eg33) mutant, was 176 significantly (23.0 ± 13.5 sec) longer than that of the wild type (8.0 ± 0.8 sec), suggesting that muscle 177 rigidity occurred ( Figure 4A and B). FWHM broadened in dys-1(eg33) D4 animals and was similar to 178 WT aged animals. In addition, [Ca 2+ ] mito also maintained higher levels in mutant muscle cells ( Figure 4A).

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Intriguingly, Ru360 treatment not only suppressed Ca 2+ influx into mitochondria, but also significantly 180 improved the broadening of FWHM (11.6 ± 7.7 sec) in the dys-1 mutants (Figure 4 A and B). We also 181 found that fragmentation of mitochondria and Ca 2+ accumulation in mitochondria in the muscle cells of 182 dystrophin mutant worms were decreased by Ru360 treatment (Figures 4C and D).

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In both mammalian cells and C. elegans body wall muscle cells, intracellular acidification is 184 caused by mitochondrial fragmentation and the pH drops from 7.5 to about 7.0 28 . Although measured 185 with different pH indicators, we obtained similar results with wild-type pH 7.4 versus pH 6.1 in the dys-1 186 mutant and pH 6.7 in Ru360 treated dys-1 mutants ( Figure 4E). In addition, we monitored Ca 2+ level in 187 the cytosol of the muscle cells on D2 of adulthood after anesthesia with sodium azide. Compared with the 188 WT counterparts, the dystrophin mutants had significantly higher cytosolic Ca 2+ levels, and surprisingly,

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Ru360 treatment decreased cytosolic Ca 2+ level in dystrophin mutants (Figure 4F   In MCU knockout mice, skeletal muscle showed altered phosphorylation and activity of pyruvate 197 dehydrogenase, significantly impairing the ability to perform strenuous work 29 . Rizzuto's group also 198 shows that MCU silencing causes muscle atrophy 10 . In contrast, our present study shows that MCU 199 inhibition (genetic ablation of mcu-1 and Ru360 treatment) ameliorated muscular function decline with 200 age and DMD in C. elegans. Recently, MCU-1 inhibitors, which are also ruthenium compounds with is reduced 32 , is highly similar to the aging of body wall muscle cells in adult C. elegans. All together 209 suggest that controlling MCU function can be the potential target for diagnosis of sarcopenia even in the Here we show that the blockage of MCU-1function by genetic or pharmacological modulation improves 214 health in both aging C. elegans and in C. elegans with muscular dystrophy. These observations suggest 215 that loss of calcium homeostasis is an early event in muscle aging that can be mitigated against by 216 improving mitochondrial calcium buffering capacity. Suppression of mitochondrial Ca 2+ influx prevented 217 the formation of Ca 2+ -accumulated structures in body wall muscle cells. These age associated Ca 2+ -218 accumulations appear to normally be removed via mitophagy. These results suggest that RyR, which 219 causes increased Ca 2+ with age, MCU-1, which facilitates increase mitochondrial Ca 2+ with age, and 220 mitophagy, which maintains mitochondrial homeostasis, are part of a coordinated system that fails to 221 maintain muscle health with age and which may be targeted for improved muscle health.          338 We followed standard procedures for C. elegans strain maintenance 33 . All strains were cultured on 339 nematode growth medium (NGM) plates with OP50 as a food source at 20°C. The worms were 340 synchronized by egg laying for 3h.

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Imaging of mitochondrial morphology, mitochondrial Ca 2+ , cytosolic Ca 2+ and nuclear morphology. 343 Mitochondrial morphology and mitochondrial Ca 2+ in body wall muscle cells were observed by assaying