Age-related declines in α-Klotho drive progenitor cell mitochondrial dysfunction and impaired muscle regeneration

While young muscle is capable of restoring the original architecture of damaged myofibers, aged muscle displays a markedly reduced regeneration. We show that expression of the “anti-aging” protein, α-Klotho, is up-regulated within young injured muscle as a result of transient Klotho promoter demethylation. However, epigenetic control of the Klotho promoter is lost with aging. Genetic inhibition of α-Klotho in vivo disrupted muscle progenitor cell (MPC) lineage progression and impaired myofiber regeneration, revealing a critical role for α-Klotho in the regenerative cascade. Genetic silencing of Klotho in young MPCs drove mitochondrial DNA (mtDNA) damage and decreased cellular bioenergetics. Conversely, supplementation with α-Klotho restored mtDNA integrity and bioenergetics of aged MPCs to youthful levels in vitro and enhanced functional regeneration of aged muscle in vivo in a temporally-dependent manner. These studies identify a role for α-Klotho in the regulation of MPC mitochondrial function and implicate α-Klotho declines as a driver of impaired muscle regeneration with age.

A ging is associated with impaired skeletal muscle regenerative capacity after an acute injury, resulting in declines in force-producing capacity. The impaired regenerative response of aged muscle is characterized by a shift from functional myofiber repair following injury to fibrotic deposition 1 . This increased fibrosis has been attributed to muscle stem (satellite) cell (MuSCs) dysfunction 1 .
In response to muscle injury, MuSCs become activated from a quiescent state to repair damaged myofibers 2,3 . While MuSC activation in young muscle restores the original architecture of the damaged myofibers, aging is associated with MuSC dysfunction, as evidenced by increased apoptosis 4 , decreased proliferation 5 , impairment of autophagy 6 , and a decreased resistance to stress 7 . Aged MuSCs also display a manifold increase in expression of aging-associated senescence markers, including p16 Ink4a and p21 Cip18 . Though aged MuSCs clearly display cell-autonomous deficits that contribute to an impaired regenerative response [8][9][10][11] , it was recently suggested that extrinsic changes in the muscle microenvironment may provide the initial geroconversion trigger in MuSCs 12 . Indeed, several studies have demonstrated that rejuvenation of the systemic muscle microenvironment largely restores the healing capacity of aged skeletal muscle 1,5,13,14 , leading to interest in the identification of circulating "anti-geronic" proteins and an improved mechanistic understanding by which such proteins may transpose a youthful regenerative phenotype onto aged skeletal muscle.
To this end, genetic studies have identified a powerful aging suppressor gene, Klotho, which encodes a membrane-bound and circulating hormonal protein in mice and humans 15,16 . Klotho deficiency results in the onset of numerous aging phenotypes, including decreased activity levels, gait disturbances, cognitive impairment, sarcopenia, as well as an impaired wound repair process 15,[17][18][19][20][21][22] . Three homologs of Klotho, α, β, and γ, have been identified (reviewed in ref. 23 ). In tissues such as the skin, small intestine, and kidney, declines in α-Klotho have been shown to promote cellular senescence 24 and stem cell dysfunction 17 .
In this study, we tested the hypothesis that age-related declines in α-Klotho drive dysfunctional muscle progenitor cell (MPC) mitochondrial bioenergetics, ultimately resulting in an impaired tissue regeneration. Our findings demonstrate that young skeletal muscle displays a robust increase in local α-Klotho expression following an acute muscle injury with transient demethylation of the Klotho promoter. However, aged muscle displays no change in Klotho promoter methylation and no increase in α-Klotho expression following injury. Levels of α-Klotho in MPCs derived from aged mice are decreased relative to those of young animals, and genetic knockdown of α-Klotho in young MPCs confers an aged phenotype with pathogenic mitochondrial ultrastructure, decreased mitochondrial bioenergetics, mitochondrial DNA damage, and increased senescence. Further supporting a role for α-Klotho in skeletal muscle vitality, mice heterozygously deficient for Klotho (Kl +/− ) have impaired MPC bioenergetics that is consistent with a defective regenerative response following injury, but the regenerative defect of Kl +/− mice is rescued at the cellular and organismal level when mitochondrial ultrastructure is restored through treatment with the mitochondria-targeted peptide, SS-31 25 . Finally, we demonstrate that systemic delivery of exogenous α-Klotho rejuvenates MPC bioenergetics and enhances functional myofiber regeneration in aged animals in a temporally dependent manner. Together, these findings reveal a role for α-Klotho in the regulation of MPC mitochondrial function and skeletal muscle regenerative capacity.

Results
Aged muscle displays a blunted α-Klotho response to injury.
To determine whether α-Klotho is upregulated locally in response to an acute muscle injury, we performed immunofluorescence analysis of α-Klotho in the skeletal muscle of young (4-6 months) and aged (22-24 months) male mice under conditions of homeostasis and following a cardiotoxin-induced injury. α-Klotho was virtually undetectable in healthy, uninjured muscle, regardless of age (Fig. 1a-e). In contrast, strong expression of α-Klotho was observed at the regenerating site of young muscle 14 days post injury (dpi) (Fig. 1c, e; confirmation of antibody specificity is presented in Supplementary Fig. 1). Aged muscle, however, displayed no appreciable increase in α-Klotho expression following an acute injury (Fig. 1d, e). Serum α-Klotho levels followed a similar expression pattern according to age and injury status (Fig. 1f). RT-qPCR findings revealed that Klotho transcript expression increases significantly at 3 and 7 dpi injury in the skeletal muscle of young mice (Fig. 1g). Despite the fact that α-Klotho protein is still detected in young muscle at 14 dpi (Fig. 1c, e), gene expression approached baseline levels at this later time point. On the other hand, aged counterparts display unaltered gene expression across all the time points tested (Fig. 1g). The α-Klotho response to injury was not unique to a cardiotoxin injury, as we found that young mice exposed to a severe contusion injury displayed a robust α-Klotho response at the protein level 14 days after injury ( Supplementary Fig. 2). Young female mice displayed a similar, yet blunted, increase in Klotho expression in response to injury, but, like males, the response is lost with aging (Supplementary Fig. 3).
Epigenetic silencing of Klotho contributes to the impaired regenerative potential of dystrophic skeletal muscle, and a differentially methylated region (DMR) of 110 nucleotides within the Klotho promoter region was identified in the muscles of aged mdx mice 26 . Therefore, we measured methylation levels of the DMR after injury in young and aged muscle. An acute injury to young muscle triggered demethylation of the DMR in the Klotho promoter 3 and 7 dpi (Fig. 1h). Injury-induced demethylation was, however, absent in the Klotho promoter within aged muscle (Fig. 1h).
To examine whether modifying enzymes for DNA methylation contribute to the observed methylation changes, we used a chromatin immunoprecipitation (ChIP) assay to measure the enrichment of DNA methyltransferases 3a (DNMT3a) in the Klotho promoter region 26 . There was a decrease of DNMT3a binding to the Klotho promoter in the injured young muscle that, at the timepoints evaluated, reached a nadir 3 days after injury and was increased by day 14 (Fig. 1i). In contrast, the injuryinduced decrease in DNMT3a binding was delayed and blunted in injured aged muscle (Fig. 1i). Consistent with the finding that dimethylated histone H3 lysine 9 (H3K9me2) bound to the Klotho promoter in dystrophic skeletal muscle represses Klotho 26 , H3K9me2 binding to the Klotho promoter region was decreased after injury only in young muscle (Fig. 1j). Taken together, these findings suggest that acute injury drives the reactivation of Klotho by reducing DNA methylation and H3K9 dimethylation in the promoter of young muscle, but that Klotho remains epigenetically repressed after injury in aged muscle.
Genetic inhibition of α-Klotho impairs muscle regeneration. To directly implicate a functional role for α-Klotho in skeletal muscle regeneration, we next evaluated the regenerative response to acute muscle injury in adult mice that are heterozygously deficient for Klotho (Kl +/− mice). As expected, Kl +/−   Fig. 1 α-Klotho is increased in young muscle after injury, but the response is lost with increased age. a−d Immunofluorescent imaging of α-Klotho and Factin in skeletal muscle from uninjured young (UIY; 4-6 months) and old mice (UIO; 22-24 months) as well as 14 days post injury (dpi) in young (YI) and old (OI) mice. Scale: 50 µm. e Quantification of α-Klotho across the four comparison groups, UIY, UIO, YI, and OI. Experimental cohorts were performed in duplicate, n = 3/group/cohort. ****p < 0.0001. f ELISA analysis of serum obtained from UIY, UIO, YI, and OI mice (n = 8-11/group; ****p < 0.0001). g RT-qPCR analysis of α-Klotho in young and old muscles at 0 (control), 3, 7, and 14 dpi (n = 4/age/timepoint). h MSPCR analysis of young and old muscle at 0 (control), 3, 7, and 14 dpi (n = 4/age/timepoint). i ChIP analysis of DNMT3a in young and old muscle at 0 (control), 3, 7, and 14 dpi (n = 3-4/age/ timepoint). j ChIP analysis of H3K9me2 in the Klotho promoter in young and old muscle at 0 (control), 3, 7, and 14 dpi (n = 4/age/timepoint) (*p < 0.05 compared to young, sex-matched uninjured muscles; #p < 0.05 indicates a significant difference between young and aged groups at the respective timepoint mice displayed a significantly decreased local α-Klotho expression at the site of injury (Fig. 2a, b). Kl +/− mice also displayed a decreased regenerative index, smaller myofiber cross-sectional area, and increased fibrosis when compared to age-and sex-matched wild-type counterparts ( Fig. 2a−e). These findings are consistent with a recent study of muscle regeneration in α-Klotho hypomorphs 27 . Together, the data demonstrate that α-Klotho is necessary for effective skeletal muscle regeneration after injury. Given our findings of increased Klotho expression within the injured muscles of young mice (Fig. 1c,g,e), we next evaluated the contribution of local α-Klotho in functional muscle regeneration. The tibialis anterior (TA) muscles of young mice were injected with GFP-tagged SMARTpool® lentiviral particles carrying shRNA to α-Klotho, whereas control counterparts were injected with an equal volume of the non-targeting control (NTC) lentivirus. After 4 weeks, TAs were injured via a local cardiotoxin injection, and regeneration was evaluated 2 weeks after injury. As expected, muscles treated with shRNA to α-Klotho displayed a significant decrease in α-Klotho expression at the site of injury ( Supplementary Fig. 1B, C). Circulating α-Klotho was also significantly decreased ( Supplementary Fig. 1D). This suggests that muscle-derived α-Klotho may contribute to the increased circulating levels observed in young mice after injury (Fig. 1f).
Histological analysis revealed that knockdown of α-Klotho expression resulted in a decreased number of regenerating fibers, a decrease in the percentage of myofiber area/total area, and increased adiposity ( Fig. 2f−j). There was, however, no difference in the fibrosis across groups (Fig. 2k). The crosssectional area of regenerating (centrally nucleated) fibers in muscles treated with shRNA to α-Klotho was also significantly smaller than NTC controls (Fig. 2l), further confirming a defective regenerative response. Unexpectedly, we also observed the presence of a number of large, nonregenerating myofibers at the injury site of muscles treated with shRNA to α-Klotho (Fig. 2f). To evaluate the structural integrity of these myofibers, we performed second harmonic generation (SHG) imaging, which allows for three-dimensional visualization of myofiber structure and organization. Consistent with histological findings, SHG analysis revealed that muscles treated with shRNA to α-Klotho contain a decreased number of centrally nucleated fibers (Fig. 2m, n). This decreased evidence of active regeneration was concomitant with pathologic myofiber architecture and integrity ( Fig. 2m and Supplementary Movies 1 and 2). Most importantly, the impaired regenerative response and disrupted myofiber structure was concomitant with a decreased functional recovery after injury (Fig. 2o).
α-Klotho is expressed by MuSCs and their progeny. The fact that α-Klotho expression in young muscle is elevated at 3 days after injury (Fig. 1g)-a time point that corresponds with MuSC activation-led us to next investigate whether MuSCs express α-Klotho and whether α-Klotho is necessary for the MuSC response to injury. We accessed RNAseq data from a recent study 28 , which is stored on the Gene Expression Omnibus (GEO) publicly accessible database. Analysis of archived data revealed a ten-fold increase in Klotho expression of freshly sorted MuSCs as compared to whole muscle lysates (Fig. 3a). Structured illumination microscopy (SIM) confirmed robust α-Klotho in MPCs isolated from young mice (Fig. 3b, c). MPCs were cultured for no more than three passages prior to analysis and were confirmed to be >90% MyoD + . MPCs isolated from aged muscle, however, displayed a markedly decreased α-Klotho protein expression (Fig. 3b, c). We also evaluated α-Klotho expression in MuSCs isolated by fluorescence-activated cell sorting 29 . As observed in MPCs, we found that young MuSCs displayed a robust α-Klotho expression, but that α-Klotho expression was decreased in aged MuSCs ( Supplementary Fig. 4).
Tissues previously shown to express high levels of α-Klotho, such as the kidney, contain a membrane-bound form of α-Klotho (~120 kDa), which functions as an obligate co-receptor for fibroblast growth factor-23 (FGF23) 30 . However, upon proteolytic cleavage of the extracellular domain, α-Klotho is released from the cells, where it functions as a humoral factor 15 . To determine whether MPCs secrete α-Klotho, we performed ELISA of the conditioned media from MPCs isolated from young and old muscle. Conditioned media derived from young MPCs contained significantly more α-Klotho than the conditioned media from aged MPCs (Fig. 3d), suggesting that MuSCs secrete α-Klotho but that secretion declines with age.
Immunohistochemical analysis of young muscle three days after injury revealed that 96.7% of MyoD + cells express α-Klotho (Fig. 3e). MyoD is a marker of activated MuSCs and is required for myogenic lineage progression 31 . We accessed and analyzed MuSCs RNAseq data housed on the GEO repository 28 and found that activated MuSCs display significantly increased Klotho expression when compared to quiescent MuSC counterparts that were isolated from 1% PFA-perfused mice (Fig. 3f). These findings suggest that α-Klotho expression is increased in the transition from MuSC quiescence to activation. To investigate this more directly, we compared α-Klotho expression in freshly sorted (quiescent) MuSCs and MuSCs that were maintained in culture for 3 days, thereby promoting activation in vitro 32 . Activated MuSCs displayed a 4.5-fold increase in α-Klotho, when compared to quiescent counterparts (Fig. 3g, h). We also isolated MuSCs from uninjured young muscle and young muscle 3 dpi, yielding a population of quiescent and activated MuSCs, respectively ( Supplementary Fig. 4C). As observed when MuSCs were activated in vitro, there was a significant increase in α-Klotho expression in MuSCs that were activated in vivo (Fig. 3j, k). As a comparison, fibroadipogenic progenitor cells (FAPs), which also play a critical role in the skeletal muscle regenerative cascade 33 , displayed no change in α-Klotho over 3 days of activation in culture, nor was α-Klotho expression increased when FAPS were isolated from acutely injured muscle ( Fig. 3g-k, Supplementary Fig. 4D). The conditioned media from activated FAPs also contained significantly less α-Klotho when compared to the conditioned media from activated MuSCs (Fig. 3i). Therefore, at least some of the α-Klotho protein detected in muscle after injury could come from MPCs themselves, although other neighboring cell populations may also express and secrete α-Klotho in response to an acute injury. Indeed, it was recently demonstrated that α-Klotho derived from macrophages promotes muscle regeneration 34 .
We next asked whether α-Klotho is necessary for normal MuSC lineage progression. MPCs isolated from the skeletal muscle of Kl +/− mice displayed a small, but significant, decrease in the percentage of MyoD + cells when compared to age-matched wild-type counterparts. There was, however, no difference in Pax7 expression across groups ( Fig. 3l-n). These findings are consistent with a previous report demonstrating that α-Klotho hypomorphs display a decreased number of MyoD + cells as compared to control counterparts 27 . In vivo, lentiviral shRNA inhibition of α-Klotho resulted in a decreased MyoD expression at the site of injury (Fig. 3o, p). Taken together, these data suggest that a loss of α-Klotho disrupts MuSC lineage progression.  multiple processes associated with inhibition of senescence 24 . Senescence is actively repressed in young MuSCs, but this capacity declines with aging, resulting in a failure of MuSCs to activate and proliferate in response to injury 35 . Therefore, we investigated whether the decreased MuSC activation in α-Klotho-deficient muscles may be attributed to cellular senescence. To confirm an inhibitory role for α-Klotho in myogenic cell senescence, we used small interference RNA (siRNA) inhibition, which resulted in ã 3-fold decrease in α-Klotho ( Supplementary Fig. 1G, H). As expected, knockdown of α-Klotho induced a senescent phenotype in young MPCs, as evidenced by the percentage of senescence associated (SA)-βgal-positive cells, increased cytosolic HMGB1  Given that senescent cells are known to have higher levels of DNA damage 36 , we used LXRepair multiplex technology 37,38 to evaluate the DNA base excision repair (BER) enzyme activities of OGG1 and APE1, which work on two common oxidative DNA lesions, 8-oxodG and abasic sites, respectively. When compared to scrambled siRNA-treated young MPCs, there was no significant decline in BER in the nucleus of cells treated with siRNA to α-Klotho ( Supplementary Fig. 5G, H). However, just 48 h of α-Klotho supplementation to siRNA-treated MPCs from young animals dramatically stimulated the activity of these BER enzymes ( Supplementary Fig. 5G, H). These data suggest that α-Klotho's role in MPC senescence may be attributed to induction of key enzymes in the BER pathway, which is rapid and can occur even within a couple of hours 39 .
While α-Klotho's role in cellular senescence has been demonstrated in multiple systems, the mechanisms underlying this role are incompletely understood. Cellular senescence is associated with altered mitochondrial morphology and dysregulated bioenergetics that can lead to aging and aging-associated pathologies 40,41 . Thus, regulation of MPC senescence by α-Klotho raised the intriguing possibility that α-Klotho may regulate MPC mitochondrial structure and function. Using an antibody against the mitochondrial membrane protein, Tom20, we observed no difference in mitochondrial morphology, as determined by sphericity, the number of mitochondria per cell, or mitochondrial volume according to age or α-Klotho levels ( Supplementary Fig. 6A, C-F). This was further supported qualitatively using STimulation Emission Depletion (STED) microscopy to visualize the mitochondrial network (Supplementary Fig. 6B). These findings suggest that a loss of α-Klotho does not affect mitochondrial morphology or mass. However, detailed analysis by transmission electron microscopy (TEM) revealed a striking alteration of ultrastructural integrity of the mitochondrial cristae and endoplasmic reticulum in addition to lipid droplet accumulation when young MPCs were treated with siRNA to α-Klotho (Fig. 4a).
In light of the loss of mitochondrial ultrastructure resulting from decreased α-Klotho expression, we next evaluated whether loss of α-Klotho drives MPC mitochondrial dysfunction. Indeed, recent reports have shown a critical role for mitochondria in MuSC function 42,43 . The bioenergetic profiles of young and aged MPCs were studied using a Seahorse XF e 96 Flux analyzer, which quantifies the oxygen consumption rate (OCR), a measure of oxidative phosphorylation. OCR was measured again after sequential injection of oligomycin, FCCP, 2-deoxyglucose (2-DG), and rotenone. These data demonstrate that, when normalized to total number of cells, MPCs from older animals display dramatically decreased levels of basal OCR as compared to young counterparts ( Fig. 4b; Supplementary Fig. 7A, B). A similar ageassociated deficit in the OCR of freshly isolated and cultured MuSCs was recently reported 44 . When young MPCs were treated with siRNA to α-Klotho, basal OCR was reduced to~25% values of scramble-treated counterparts (Fig. 4c, Supplementary Fig. 7A, B). MPCs isolated from uninjured Kl +/− mice display a similarly blunted bioenergetic profile (Fig. 4d). Though there was no appreciable decrease in reserve capacity in aged MPCs, young MPCs treated with siRNA to α-Klotho and MPCs isolated from Kl +/− mice both showed a~25% decrease in the reserve capacity ( Fig. 4e-g; Supplementary Fig. 8A). Reserve capacity represents the spare bioenergetic capacity, is calculated as the difference between the basal and maximal OCR, and indicates the ability of a cell to respond to stress 45 . Taken together, these data support the hypothesis that age-related declines in α-Klotho drive impaired MPC mitochondrial bioenergetics.
One factor that could contribute to decreased mitochondrial function with decreased α-Klotho is oxidative damage, which could manifest itself as mtDNA damage 46 . Therefore, we next examined mtDNA integrity. Using a qPCR-based assay that we have successfully applied to different cell types including mouse tissue and human peripheral lymphocytes 47-49 , we evaluated mtDNA damage in MPCs isolated from young or aged mice. The method used is based on the principle that a wide variety of types of DNA damage have the propensity to block DNA polymerase progression 46 . Therefore, this assay detects numerous kinds of base DNA damage or DNA repair intermediates such as abasic sites, as well as single and double DNA strand breaks.
There was no difference in steady-state mtDNA copy number across groups (Supplementary Fig. 7C, D), consistent with immunocytochemical analysis showing no difference in mitochondrial mass (Supplementary Fig. 6C). However, we found that MPCs isolated from aged mice displayed higher levels of mtDNA damage as compared to young counterparts (Fig. 4h). Accordingly, young MPCs receiving siRNA to α-Klotho and MPCs isolated from Kl +/− mice displayed increased numbers of mtDNA lesions when compared to scramble-treated and wildtype controls, respectively (Fig. 4i, j).
Cardiolipin is an anionic phospholipid that is confined almost exclusively to the inner mitochondrial membrane where it is synthesized. It is required for the mitochondrial respiratory supercomplex assembly and activity, the disruption of which enhances the generation of mitochondrial ROS 25 . We found that cardiolipin content is depleted in MPCs isolated from Kl +/− mice, as compared to wild-type counterparts (Fig. 5c, d). However, treatment with SS-31, a mitochondrially targeted peptide that mitigates cardiolipin peroxidation 25 , restored Kl +/− MPC ROS generation, mtDNA damage and cellular bioenergetics to levels comparable to wild-type counterparts (Fig. 5a-i,  Supplementary Figure 8). Administration of SS-31 to Kl +/− mice also resulted in an increased number of MyoD + cells at the site of injury, an enhanced regenerative index, and an increased myofiber cross-sectional area (Fig. 5j-n). Accordingly, Kl +/− mice treated with SS-31 display an increased strength recovery after injury that parallels that of wild-type counterparts (Fig. 5o). Of note, treatment of wild-type mice with SS-31 did not alter strength recovery after injury when compared to saline-injected counterparts. Taken together, these findings suggest that the defect in MPC mitochondrial bioenergetics and the accumulation of mtDNA lesions accompanying a loss of α-Klotho are mediated by disruption of mitochondrial ultrastructure, ultimately resulting in dysfunctional muscle regeneration. h, i RT-qPCR based analysis of mtDNA damage in young, old, young + scramble and young + siRNA MPCs. j RT-qPCR analysis of mtDNA damage in MPCs isolated from wild-type and Kl +/− mice (*p < 0.05, **p < 0.01, ****p < 0.0001, one-tailed Student's t test). Data represented as mean ± SEM mitochondrial function in aged MPCs. We found that when MPCs isolated from aged skeletal muscle were cultured in the presence of recombinant α-Klotho for 48 h, the aged mitochondrial phenotype was improved, as determined by decreased mtDNA damage, increased OCR, and increased reserve capacity (Fig. 6a-c).
These encouraging in vitro findings led us to next probe whether α-Klotho supplementation may enhance skeletal muscle regeneration in vivo. To do this, α-Klotho was administered to aged mice via osmotic pump. Osmotic pumps were implanted three days prior to injury and were maintained for 14 dpi. At the dose tested, we observed a significant increase in local α-Klotho within the injured muscle areas (Fig. 6d, e). Systemic administration of α-Klotho in aged muscle resulted in an increased number of regenerating fibers after injury, as determined both by histology and SHG imaging (Fig. 6d-h). These findings were consistent with an approximately 3.5-fold increase in the number of MyoD + cells at the site of injury in animals that received supplementation with α-Klotho (Fig. 6i, j). However, osmotic pump delivery of α-Klotho yielded no significant increase in myofiber cross-sectional area or total muscle area when compared to saline counterparts. Given that osmotic pump administration delivers α-Klotho continuously, we next tested the hypothesis that the timing of α-Klotho administration may be critical for functional tissue regeneration. To do this, we performed daily intraperitoneal injections of recombinant α-Klotho to aged mice either from 1-3 dpi or from 3-5 dpi (i.e. the timepoints at which we found Klotho to be highly expressed in young muscle (Fig. 1g)). Similar to our findings using osmotic pumps, α-Klotho delivery both from 1-3 dpi and 3-5 dpi resulted in an increased number of regenerating fibers when compared to vehicle controls (Fig. 6k, l). Both treatment groups also displayed an increased myofiber area, though the magnitude of the improvement was greater in mice receiving α-Klotho over 3-5 dpi (Fig. 6m). Importantly, only animals receiving α-Klotho over 3-5 dpi displayed an improved functional recovery (Fig. 6n, o, Supplementary Table 1).
Taken together, the data suggest that α-Klotho is required for an adequate regenerative response to an acute injury, and that supplementation with α-Klotho via the circulation promotes MuSC commitment and myofiber regeneration in aged mice when administered at the appropriate timepoint. These findings implicate declines in this longevity protein as a contributor to a defective muscle regenerative response with aging and raise the possibility of systemic administration of α-Klotho as a therapeutic approach to promote the healing of aged skeletal muscle after injury.

Discussion
Though aged muscle typically demonstrates a shift from functional myofiber repair following injury, to a quick-fix default towards fibrosis 5 , these age-related declines are reversible. Heterochronic parabiosis, in which circulatory systems of young and old animals are surgically joined, has clearly shown that systemic factors in young animals have a positive impact on the health of older animals 1,5,13,51 . Here, we tested the hypothesis that α-Klotho, a circulating hormone and paracrine factor associated with the attenuation of tissue aging is critical for effective MPC function and muscle regeneration after injury. We found that α-Klotho is highly upregulated at both the mRNA and protein level locally within the acutely injured muscles of young skeletal muscles. However, the response is markedly attenuated in aged muscles. At the cellular level, we uncovered a role for α-Klotho in the maintenance of mitochondrial ultrastructure, mtDNA damage, and MPC bioenergetics (Fig. 7). Though skeletal muscle α-Klotho expression under conditions of homeostasis is low 15 , the data indicate that expression of α-Klotho within injured muscle is required for functional regeneration. As an alternative, systemic supplementation with α-Klotho at physiologically relevant time points may promote muscle regeneration in aged mice.
Previous studies have demonstrated that decreased α-Klotho levels impair the wound healing response of the skin and small intestine through regulation of stem cell function 17 . In dystrophic muscle, α-Klotho levels are epigenetically repressed at the level of the Klotho promoter via DNA methylation and histone modification 26 . Our observation that Klotho is silenced in normal muscle but poised to lose the repressive marks following an injury presents a novel mechanism for regulating the contribution of local Klotho expression to regeneration after acute injury. It is intriguing that failure to derepress the Klotho promoter in the injury response contributes to the deficiency of aged muscle regeneration. This may have implications for mechanisms of degeneration or failed regeneration in other tissues where α-Klotho has been demonstrated to decline with age.
The impaired tissue healing previously observed in α-Klotho knockout mice is associated with decreased cellular proliferation 17,52 , decreased resistance to stress 52,53 , fibrosis formation 26 , and increased senescence 24 . Mitochondrial dysfunction is a hallmark of the aged tissue phenotype 54 , and, increasingly, mitochondria are emerging as a nexus that modulates cell-wide signaling pathways governing these cellular processes. Among these, Wnt/TGFβ, insulin growth factor (IGF)-1, and fibroblast growth factor (FGF) signaling, for example, have been shown to be responsive to signals emanating from the mitochondria, predominantly through ROS production [55][56][57][58] . It is noteworthy that these pathways have been implicated both in the antiaging effects of α-Klotho 17,30,59 and in age-related declines in muscle regeneration 1,6,9,60,61 . A recent report demonstrated that α-Klotho inhibits Wnt signaling in aged MuSCs 27 . However, a direct role of α-Klotho on cellular mitochondrial function has remained poorly understood. While an impaired mitochondrial morphology with decreased α-Klotho levels has been previously demonstrated in α-Klotho knockout mice 62 , it is difficult to discern whether the mitochondrial defects are a primary result of α-Klotho deficiency, or if they represent a secondary consequence of cumulative tissue dysfunction in these progeroid mice.  5 Mitochondrial structure and function are impaired in Kl +/− mice, but are rescued with SS-31. a, b Representative TEM images and analysis of damaged mitochondria of wild-type (WT), Kl +/− and Kl +/− + SS-31 groups (10-20 images were analyzed to quantify >100 mitochondria/group; ****p < 0.0001, one-way ANOVA with Tukey's post-hoc test). Scale: 500 nm. c, d Representative immunofluorescent images and quantification of cardiolipin content, by Nonyl Acridine Orange staining (NAO, green) in WT, Kl +/− and Kl +/− + SS-31 MPCs (*p < 0.05, one-way ANOVA with Tukey's post-hoc test). Scale: 50 µm. e, f Representative immunofluorescent images from live imaging and quantification of ROS as determined by MitoSox staining (red) on live cells from WT, Kl +/− and Kl +/− + SS-31 groups (****p < 0.0001, one-way ANOVA with Tukey's post-hoc test). Scale: 50 µm. g RT-qPCR-based analysis of mtDNA damage on WT, Kl +/− and Kl +/− + SS-31 MPCs (n = 3/group; *p < 0.05, one-way ANOVA with Tukey's post-hoc test). h, i Seahorse analysis of basal OCR and reserve capacity of WT, Kl +/− and Kl +/− + SS-31 MPCs (n = 4-6/group; *p < 0.05, one-way ANOVA with Tukey's post-hoc test). j, k Quantification of MyoD + cells at the site of injury of TA muscles from WT, Kl +/− and Kl +/− + SS-31 groups (n = 3-6/group; ***p < 0.001, one-way ANOVA with Tukey's post-hoc test). Scale: 25 µm. l, m Representative SHG images and analysis of the percentage of centrally nucleated fibers from WT, Kl +/− and Kl +/− + SS-31 mice at 14 dpi (n = 3-4/group; *p < 0.05, **p < 0.01, one-way ANOVA with Tukey's post-hoc test). Scale: 50 µm. n Quantification of myofiber cross-sectional area from WT, Kl +/− and Kl +/− + SS-31 groups (n = 3-4/group; *p < 0.05, **p < 0.01, one-way ANOVA with Tukey's post-hoc test). o Hang impulse (calculated as hanging time × mouse weight) at 14 dpi, represented as a fold-change from 1 dpi score during the wire hang test (n = 3-6/group; *p < 0.05, ***p < 0.001, two-way ANOVA with Tukey's post-hoc test). Each mouse was used as its own control in order to account for baseline variability (shown in Supplementary Figure 8). Data represented as mean ± SEM It is plausible that the main actions of α-Klotho and FGF23 on phosphorous homeostasis that are lost in aging and in Kl +/− mice promotes mitochondrial hyperpolarization and subsequent ROS production due to increased phosphate levels 63 . The studies presented here employed gain-and loss-of-function paradigms to demonstrate a direct role for α-Klotho in the maintenance of MPC mitochondrial morphology, genome integrity, and bioenergetics. Decreased levels of α-Klotho in MPCs and skeletal muscle were associated with vacuolated mitochondria possessing compromised cristae structure. This loss of architecture may lead to reorganization and oxidation of cardiolipin that, in turn, converts cytochrome c into an oxidant-generating peroxidase 25 . The reversal of the aged or Kl +/− mitochondrial phenotype with SS-31 supports this mechanism, as SS-31 binding to cardiolipin protects the structure of mitochondrial cristae, prevents cardiolipin interactions with cytochrome c, and promotes oxidative phosphorylation 25 . SS-31 administration has also been shown to rejuvenate aged skeletal muscle mitochondrial energetics and performance 64 . It was recently shown that Consistent with the induction of an aged phenotype when α-Klotho expression is inhibited in young MPCs in vitro, depletion of α-Klotho in vivo impairs myofiber regeneration and drives fibrosis after an acute injury event. α-Klotho has been shown to regulate fibrosis formation under conditions of renal pathology, skin lesions, and muscular dystrophy 17,26,65 . The in vivo data suggest that, with acute injury, α-Klotho plays an important role in regulating physiological muscle regenerative cascade. It appears that only a portion of this role is attributed to α-Klotho within the muscle itself as local inhibition of α-Klotho results in an attenuated regenerative defect when compared to whole body knockdown. The fact that administration of α-Klotho enhanced MuSC lineage progression and myofiber regeneration in aged skeletal muscle suggests that supplementation via the circulation may be an effective means to compensate for the regenerative deficit observed in aged animals.

Methods
Animals. C57BL/6 young (4-6 months) and old (22-24 months) mice were received from the Jackson Laboratories or NIA rodent colony. Kl +/− mice were obtained from MMRRC, UC Davis and were genotyped prior to inclusion in the studies. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Animals were ear-tagged, randomly assigned to intervention group, and compared to age-matched littermate controls whenever possible. Mice were evaluated prior to inclusion in the study, and animals with obvious health problems were eliminated. Animal experiments were repeated across a minimum of two separate cohorts of the experimental groups. All primary endpoints were prospectively selected prior to analyses and investigators performing endpoint analysis were blinded to the experimental group whenever possible.
Histological analysis of muscle regeneration. Wild-type male C57BL/6 young, Kl +/− mice, or old mice received injuries to bilateral TA muscles via an intramuscular injection of cardiotoxin (10 µL of 1 mg/mL). Fourteen days following the injury, TAs were harvested for histological analysis of α-Klotho, fibrosis (Sirius red), degenerating myofibers (IgG), and myofiber regeneration (laminin). SHG imaging was performed on isolated TA muscles treated with an NTC or lentiviral knockdown of α-Klotho, as well as pump-administered animals in order to visualize collagen and myofibers within the muscle 14 dpi, as we previously described 66 .
ELISA. The levels of α-Klotho protein were measured by a colorimetric sandwich enzyme immunoassay (SEH757Mu, Cloud-Clone Corp), according to the manufacturer's instructions. Each sample was measured in duplicate.
Hanging-wire test. Strength endurance was tested using the hang-wire test, as in ref. 68 . The Hang Impulse (HI) score was calculated as body weight (grams) × time hung (seconds). Male mice were used for all testing using C57Bl/6 mice. Wild-type and Kl +/− were females for testing.
Epigenetic regulation of α-Klotho. At baseline, 3, 7, and 14 days after injury, TAs were snap frozen using liquid nitrogen for gene expression, methylation-specific PCR (MSPCR), and ChIP analysis, essentially as previously described 69 .
MPC bioenergetics and mitochondrial DNA damage. OCR and extracellular acidification rate were measured in real time using a Seahorse XFe96 Extracellular Flux Analyzer (Billerica, MA) as previously described 45 . The basal OCR was measured by averaging the OCR values before treating the cells with oligomycin. Total reserve capacity was calculated by the differences of OCR between treatment with FCCP and 2DG and basal values. Mitochondrial DNA damage was quantified as previously described 47,49 .
SS-31 administration. Isotonic saline or SS-31 (3 mg/kg dissolved in saline at 0.3 mg/mL) was administered daily via an i.p. injection to wild-type and Kl +/− animals for the entire duration of injury 64 . For in vitro experiments, 100 nM of SS-31 was administered to MPCs isolated from Kl +/− animals for 48 h. Dosing was based on studies demonstrating the effectiveness in a mouse model of chronic cardiomyopathy 70 as well as in vitro dose ranging studies performed in C2C12s to evaluate inhibition of stress-induced mitochondrial membrane hyperpolarization and ROS generation.
In vivo lentiviral knockdown of α-Klotho. In vivo α-Klotho knockdown was done using lentiviral vectors for a SMARTpool of 2.0×10 5 TU/TA or 3.82×10 6 TU/TA shRNA to α-Klotho per TA muscle. Given that there was no significant difference in the local α-Klotho expression between the two treatment groups, samples across the two treatment groups were pooled for analysis. Control animals received equal volumes of empty lentiviral vector. Knockdown was maintained for 3 weeks, after which time bilateral TAs were injured. Histology or SHG imaging was performed 14 days after injury.
Supplementation of α-Klotho in vivo. Mini osmotic pumps containing either saline or α-Klotho (324 pg/mL in saline vehicle) were inserted subcutaneously into aged mice. After 2 days, bilateral TA muscles were injured by intramuscular CTX injection (as above). Osmotic pumps remained implanted until euthanasia 14 dpi. Isotonic saline or α-Klotho (10 µg/kg body weight) was administered to aged animals via daily intraperitoenal injections from 1-3 dpi or 3-5 dpi. The TAs were then harvested 14 dpi and preserved for histology or SHG analysis. Blood serum was also collected to evaluate circulating α-Klotho levels via ELISA. The activity of α-Klotho was confirmed as previously described 71 .

Data availability
The data supporting the results of this paper can be made available by the corresponding author upon reasonable request.