Tatjana Holzer

Kristina Probst

Julia Etich

Markus Auler

Veronika S Georgieva

Björn Bluhm

Christian Frie

Juliane Heilig

Anja Niehoff

Julian Nüchel

Markus Plomann

Jens M Seeger

Hamid Kashkar

Olivier R Baris

Rudolf J Wiesner

PhD Bent Brachvogel
email: bent.brachvogel@uni-koeln.de

Respiratory chain inactivation links cartilage-mediated growth retardation to mitochondrial diseases

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Introduction

Patients suffering from mitochondrial damage causing respiratory chain (RC) dysfunction, both due to mtDNA mutations or defects in nuclear genes encoding mitochondrial proteins, are reported to often present with short stature, but the pathomechanism of the impaired skeletal growth remains unclear [START_REF] Koenig | Presentation and diagnosis of mitochondrial disorders in children[END_REF][START_REF] Wolny | Abnormal growth in mitochondrial disease[END_REF].

Skeletal growth is driven by the transformation of cartilage into bone tissue as a result of unidirectional cell proliferation within the growth plate cartilage. Chondrocytes are the only cells of the growth plate and it is current belief that these cells rely on anaerobic glycolysis to promote skeletal growth in the avascular, severely hypoxic growth plate [START_REF] Martin | Mitochondrial electron transport and glycolysis are coupled in articular cartilage[END_REF]. However, this hypothesis is in conflict with the observation that respiratory dysfunction in patients reduces skeletal growth. Recent ex vivo studies have also reported that mitochondrial dysfunction could act as a pathogenic factor in degenerative cartilage disease, but the in vivo evidence is missing [START_REF] Blanco | The role of mitochondria in osteoarthritis[END_REF].

A major experimental limitation is the lack of models to study RC-function in cartilage. Access to growth plate cartilage from patients with mitochondrial diseases is limited and genetic approaches to study RC-function in vivo failed because of embryonal lethality when genes essential for mitochondrial homeostasis were manipulated. Only recently genetic tools were developed to selectively inactivate the RC in mice [START_REF] Dogan | Modelling mitochondrial dysfunction in mice[END_REF].

The aim of this study was to use these novel genetic tools and determine if the RC-dysfunction is a major cause for growth retardation and degenerative cartilage disease in the presence of mitochondrial damage. In order to achieve this goal we first analyzed the RC activity during development. Interestingly, the RC is hardly active in growth plate cartilage in newborns, but growth plate RC activity markedly increases in juvenile mice, when secondary ossification centers are formed and vascular networks are established at the proximal and distal end of the growth plate. We then generated transgenic mice which have an inactivated RC only in cartilage using the cartilage-specific expression of a mtDNA helicase Twinkle mutant [START_REF] Baris | Mosaic Deficiency in Mitochondrial Oxidative Metabolism Promotes Cardiac Arrhythmia during Aging[END_REF][START_REF] Weiland | Imbalance of Mitochondrial Respiratory Chain Complexes in the Epidermis Induces Severe Skin Inflammation[END_REF]. Here, we show that these mice, as a consequence of the lack of RC activation after birth, develop postnatal growth retardation and growth plate cartilage degeneration caused by energy deficiency, altered metabolic signaling, destabilization of the hypertrophic ECM and increased chondrocyte death at the cartilage-bone junction.

These findings illustrate that glycolysis is sufficient to drive fetal cartilage growth and, in contrast to the current view, a metabolic switch from fetal glycolysis to respiration in growth plate cartilage after birth is essential to promote postnatal skeletal growth. Moreover, the results provide an explanation at the molecular level why loss of RC dysfunction in mitochondrial diseases can cause growth plate cartilage degeneration and impaired skeletal growth.

Results

It was early proposed that the metabolism in cartilage is entirely anaerobic [START_REF] Bywaters | Metabolism of Cartilage[END_REF], but to our knowledge RC activity was never studied in situ during growth plate cartilage development.

Hence, we applied cytochrome c oxidase (CYTOCOX; complex IV) activity staining to femoral sections of newborn, 13 days and one month old mice to determine the complex IV activity in growth plate cartilage.

In newborns, CYTOCOX staining was restricted to the lateral growth plate and the perichondrium (Figure 1A) close to laminin 1 positive blood vessels (Figure 1B, upper panel, arrowheads), while it was hardly detectable in chondrocytes in the center (for cellularity see DAPI staining, Figure 1B, lower panel). After formation of the secondary ossification center in 13 days old mice, CYTOCOX staining was mainly detected in proliferating, but not in prehypertrophic or hypertrophic chondrocytes. In one month old mice most cells of the growth plate were CYTOCOX positive but the strongest staining was still found in proliferating chondrocytes. At this stage a vascular plexus has developed at the apical and distal growth plate (Figure 1B,arrowheads). These results unexpectedly indicate that the cartilage metabolism is partially aerobic and that respiration is activated at later stages of postnatal development when blood vessels surround the growth plate to supply sufficient oxygen and/or nutrient levels.

We next generated transgenic mice expressing a patient-derived dominant-negative mutant of the Twinkle helicase in chondrocytes to inhibit the RC and thus determine its function in growth platemediated skeletal growth. We crossed R26-K320E-Twinkle loxP/+ mice [START_REF] Weiland | Imbalance of Mitochondrial Respiratory Chain Complexes in the Epidermis Induces Severe Skin Inflammation[END_REF] with mice expressing Cre recombinase driven by the Col2a1 promotor (Cre) [START_REF] Bluhm | miR-322 stabilizes MEK1 expression to inhibit RAF/MEK/ERK pathway activation in cartilage[END_REF][START_REF] Ovchinnikov | Col2a1-directed expression of Cre recombinase in differentiating chondrocytes in transgenic mice[END_REF] and induced the expression of the Twinkle mutant protein in cartilage during early embryonal development. In these mice (CreTW), a marked reduction of the multi-copy mtDNA molecules was found and consequently a decrease in index subunits of those RC complexes which contain essential mtDNA encoded subunits (complexes I, III and IV) was observed in cartilage from one month old mice (Figure 2A, B). CYTOCOX activity was absent in newborns and 1 month old CreTW mice (Figure 2C), which indicates that the RC was successfully inactivated in growth plate cartilage. The consequences for chondrocyte metabolism were then studied in detail using quantitative flow cytometry approaches. As expected, the mitochondrial membrane potential was decreased while mitochondrial mass was slightly but significantly increased in isolated and cultured chondrocytes from newborn and one month old CreTW mice (Figure 2D,E). The pH in the cell culture medium was decreased at both time points, indicating increased lactate production upon RC dysfunction (Figure 2F). More pronounced changes, e.g. significantly reduced ATP levels and reduced ROS levels, were found in chondrocytes isolated from one month old mice compared to newborns (Figure 2G, H), whereas NADH levels were increased at both time points (Figure 2I). Hence, mutant Twinkle expression in chondrocytes leads to a depletion of mtDNA, followed by a decrease in RC complexes containing mtDNA encoded subunits and reduced energy production in newborns and more pronounced in older CreTW mice.

Respiratory capacity of chondrocytes was then characterized by Seahorse XF96 analyzer. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were recorded over time and basal respiration, ATP production-coupled respiration and maximal and spare respiratory capacities were determined in chondrocytes isolated from newborns and one-month old animals (Figure 3). The data obtained showed that basal respiration and ATP production-coupled respiration were low in both Cre and CreTW chondrocytes isolated from newborns. The calculated maximal and spare respiratory capacities were however reduced in young CreTW chondrocytes compared to Cre chondrocytes clearly indicating the lack functional mitochondrial respiration in CreTW chondrocytes. Interestingly, basal respiration, ATP production and maximal/spare respiration capacity were increased in older Cre chondrocytes compared to Cre chondrocytes isolated from newborns (Figure 3A,B). The aerobic energy production was activated in older control Cre chondrocytes, while mitochondrial respiratory dysfunction slightly stimulated glycolysis in old CreTW chondrocytes (Figure 3C). These results clearly showed that chondrocytes at early ages mainly utilize anaerobic glycolysis to cover cellular energetic demands and do not rely on mitochondrial respiration-coupled energy production. In contrast, at later stages chondrocytes also use mitochondrial respiration and may be dependent on intact mitochondria.

Next we studied the consequences of the lack of RC activation for skeletal growth in CreTW mice.

No length alterations were observed in newborns, while in one month and, more prominently, in one year old CreTW mice a significant reduction in body length was detected (Figure 4A). Accordingly, CreTW femora and tibiae were significantly shorter in one month and one year old mice than in controls (Figure 4B,C). In addition, µCT measurements were performed on femora of one, two, three, four, six months and one year old mice. Quantification of the µCT measurements revealed that the trabecular bone mass at the distal femoral metaphysis is increased in one month old CreTW mice compared to the control mice, as indicated by a higher trabecular number (Tb.N) combined with a smaller trabecular separation (Tb.Sp).

However, in older CreTW mice the trabecular bone mass was decreased and CreTW mice have less trabeculae with a greater distance between these (Figure S1B). In comparison to controls, the growth plate was smaller in 4 months old mice, partially lost in older animals and completely closed in one year old CreTW animals (Figure 4D, arrowheads, Figure S1). Hematoxylin/eosin and alcian blue stained, inplane matched sections showed that directly after birth there were no differences in the organisation of the growth plate between Cre and CreTW mice (Figure 4E,F). At one month the growth plate was expanded and the prehypertrophic/ hypertrophic area was even enlarged, whereas at one year no cartilage remained in CreTW mice. Hence, lack of RC activation in chondrocytes severely disturbs growth plate organization, induces premature growth plate closure and inhibits skeletal growth in older animals mimicking the abnormal skeletal growth in patients with mitochondrial diseases.

To understand the link between lack of RC activation and reduced skeletal growth, chondrocyte proliferation and cell death were analyzed in cartilage of CreTW mice. Chondrocytes isolated from newborns showed similar proliferation rates in culture, whereas cell numbers were significantly reduced in CreTW chondrocytes taken from one month old animals after two and three days in culture (Figure 5A).

The proportion of apoptotic and necrotic cells was significantly increased in cultured chondrocytes from one month old CreTW mice, while no differences were detected in chondrocytes isolated from newborn mice (Figure 5B) as determined by AnxA5/PI staining and flow cytometry [START_REF] Rosenbaum | Identification of novel binding partners (annexins) for the cell death signal phosphatidylserine and definition of their recognition motif[END_REF]. In growth plate sections of newborn and one month old Cre and CreTW mice similar number of proliferating cell nuclear antigen (PCNA) + cells were detected (Figure 5C,graph). Only few terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) + dying cells were observed in newborn growth plates, whereas the numbers increased significantly in one month old CreTW mice compared to controls (Figure 5D, graph). Dying cells were only detected in the late hypertrophic zone, close to the cartilage-bone junction, but not in the proliferating zone. Therefore, lack of the RC activation does not impair proliferation of chondrocytes in vivo in older animals, but a higher number of CreTW chondrocytes are damaged along the differentiation process and undergo cell death in the late hypertrophic zone, where cartilage is replaced by bone.

To define the molecular pathways leading to impaired chondrocyte survival, isolated femoral growth plates from 12 days old Cre and CreTW mice were subjected to transcriptome analysis using established protocols (Belluoccio et al., 2010a;[START_REF] Bergmeier | Identification of a myofibroblast-specific expression signature in skin wounds[END_REF][START_REF] Bluhm | miR-322 stabilizes MEK1 expression to inhibit RAF/MEK/ERK pathway activation in cartilage[END_REF]. At this time point length differences between Cre and CreTW femora were already detected and still sufficient amounts of growth plate cartilage could be isolated. Bioinformatic analysis revealed that within the 181 entities, which were found differentially expressed in CreTW cartilage compared to control, many were described as mitochondrial genes. mRNA levels for several mtDNA-encoded subunits of the respiratory chain complexes, i. e. the NADH dehydrogenase complex (Ndx), cytochrome B (Cytb) and ATP-synthase 8 (Atp8) were significantly lowered in CreTW mice (Figure 6A). Lower RC mRNA levels will result in a decrease in RC protein complexes and therefore cause RC inactivation in growth plate cartilage of CreTW mice. On the other hand, genes encoding main players of the integrated stress response (ISR, [START_REF] Quiros | Mitonuclear communication in homeostasis and stress[END_REF]) were upregulated in CreTW mice (Figure 6B). The activating transcription factor 3 (Atf3), Atf5 and DNA-damage inducible transcript 3 (Ddit3-Chop) were twofold enhanced, whereas the growth differentiation factor 15 (Gdf15) and tribbles homolog 3 (Trib3) were strongly increased in CreTW mice (Figure 4B). Fibroblast growth factor 21 (Fgf21) mRNA was increased fourfold, however expressed at only very low absolute levels. In contrast, Atf4 was significantly expressed but not differentially regulated. ISR genes were further validated by quantitative PCR analysis (Figure 6C). Here, the expression of Fgf21, Gdf15, Ddit3 (Chop) and Trib3 was clearly increased in CreTW chondrocytes.

The integrated stress response can be induced by an unfolded protein response (UPR) in the endoplasmatic recticulum (ER). We therefore determined the processing of X-box binding protein 1 (Xbp1) mRNA, which is spliced to a shorter mRNA (sXbp1) during UPR. The RNA of growth plate cartilage from 12 days old mice and from cultured chondrocytes of one month old mice was isolated and the relative abundance of the Xbp1 transcripts was analyzed by semiquantitative PCR. The unprocessed Xbp1 transcript was detected in growth plate cartilage and in isolated chondrocytes of Cre and CreTW mice, but not the processed spliced form (Figure 6D). The results show that a Xbp1-dependent UPR does not occur in CreTW mice.

If the integrated stress response causes the phenotype, its central mediators of the integrated stress response should be activated. We therefore analyzed the phosphorylation of EIF2a and the expression levels of the stress response target gene asparagine synthetase (ASNS) (Lomelino et al., 2017;Quiros et al., 2017) in growth plate cartilage from 12 days old mice. The phosphorylation of EIF2a may have been increased in CreTW mice, but variations in the ratio of phosphorylated EIF2a/total EIF2a were detected (Figure 7A), and ASNS levels were significantly increased in lysates of CreTW mice compared to control. The results point to the induction of an integrated stress response in growth plate cartilage of CreTW mice. Energetic stress in old CreTW chondrocytes could also be sensed by AMPactivated protein kinase (AMPK). The phosphorylation of AMPK was determined in growth plate cartilage from 12 days old mice (Figure 7B). Here, variations in the phosphorylation of AMPK were detected irrespectively of the genotype. We next studied AMPK activation in chondrocytes isolated from older mice. Chondrocytes of one month old mice were cultured for three days in growth medium and total and phosphorylated AMPK levels were determined in extracts by immunoblot analysis (Figure 7D). A strong phosphorylation of AMPK was observed in cultured 1 month old CreTW chondrocytes compared to Cre control. Hence, AMPK senses energy depletion when ATP levels were reduced in older CreTW chondrocytes (see Figure 2G).

Respiration inactivation was described to repress HIF1A protein synthesis through AMPKdepending signaling pathways [START_REF] Hsu | Mitochondrial dysfunction represses HIF-1alpha protein synthesis through AMPK activation in human hepatoma HepG2 cells[END_REF][START_REF] Martinez-Reyes | TCA Cycle and Mitochondrial Membrane Potential Are Necessary for Diverse Biological Functions[END_REF] and to inhibit the autophagic flux [START_REF] Lopez De Figueroa | Autophagy activation and protection from mitochondrial dysfunction in human chondrocytes[END_REF]. As p62/SQSTM1 accumulates when autophagy is inhibited (Bjorkoy et al., 2009) we next determined HIF1A and p62/SQSTM1 protein levels in extracts of growth plate cartilage isolated from 12 days old Cre and CreTW mice (Figure 7B). Interestingly, full length HIF1A protein levels were decreased and p62/SQSTM1 levels were increased in lysates of the growth plate derived from 12 days old CreTW mice compared to control. A decrease of HIF1A and inhibition of autophagy can suppress ECM production [START_REF] Kang | Cartilage-Specific Autophagy Deficiency Promotes ER Stress and Impairs Chondrogenesis in PERK-ATF4-CHOP-Dependent Manner[END_REF][START_REF] Pfander | HIF-1alpha controls extracellular matrix synthesis by epiphyseal chondrocytes[END_REF]. We therefore studied aggrecan, collagen X and matrilin 3 levels in growth plate cartilage of 12 days old mice (Figure 7C). Here a significant reduction in the protein amounts of collagen X was detected in CreTW mice compared to control.

Accumulation of p62/SQSTM1 protein points to a reduction in the autophagic flux and to an accumulation of damaged mitochondria in older CreTW chondrocytes. Therefore, mitochondrial fragmentation in cultured chondrocytes from newborns or one month old Cre and CreTW mice was characterized. Mitochondrial membrane organization was visualized by transfection of a reporter construct encoding the yellow fluorescence protein linked to a mitochondrial targeting sequence (pOCT-YFP) [START_REF] Neuspiel | Cargo-selected transport from the mitochondria to peroxisomes is mediated by vesicular carriers[END_REF] or by immunofluorescence detection of the endogenous mitochondrial outer membrane protein TOM20 (Figure 8A). Mitochondria were elongated and formed an extended membrane network in young chondrocytes and in older Cre chondrocytes. In contrast, massive mitochondrial fragmentation was observed in older CreTW chondrocytes accompanied by a pronounced increase in vesicle-like structures irrespective of the detection assay. Quantification of mitochondrial fragmentation per cell (Figure 8B) confirmed the strong increase in older CreTW chondrocytes. The results demonstrate that loss of membrane potential inhibits the removal of damaged mitochondria by autophagy and induces severe mitochondrial fragmentation in older chondrocytes.

Mitochondrial damage and energy loss can be translated into cellular damage responses and cell death [START_REF] Quiros | Mitonuclear communication in homeostasis and stress[END_REF]. More important, damage responses in chondrocytes have been shown to destabilize the organization of the extracellular matrix (ECM), which then results in the expansion of the hypertrophic growth plate cartilage and reduced skeletal growth [START_REF] Rajpar | Targeted induction of endoplasmic reticulum stress induces cartilage pathology[END_REF][START_REF] Tsang | In vivo cellular adaptation to ER stress: survival strategies with double-edged consequences[END_REF].

We could detect a reduction in collagen X in cultured older CreTW chondrocytes and hypothesized that mitochondrial damage and energy loss in chondrocytes also disturbs hypertrophic ECM organization in situ. Hence, the composition of the ECM in growth plate of CreTW mice was studied by immunofluorescence analysis. In newborns the main components of the cartilage ECM, the fibrillar collagen II, the extrafibrillar proteoglycan aggrecan and the network forming collagen X were normally distributed within the growth plate of CreTW mice (Figure 9A, Figure S2AB). In one month old CreTW mice the collagen II + /aggrecan + growth plate area was enlarged, which shows that RC inactivation results in a transiently expanded growth plate (see also Figure 4E,F) accompanied by an enlargement of the cartilage ECM. Collagen X levels were strongly reduced (Figure 9A, Figure S2B) and metallopeptidase 13 (MMP13, Figure 9B, Figure S2B) levels were decreased in the hypertrophic growth plate of one month old CreTW mice compared to Cre control. The decrease in MMP13 was confirmed by immunoblot analysis of rib cage chondrocytes from one month old CreTW mice (Figure 9C). Collagen X and MMP13 are specifically expressed in hypertrophic growth plate cartilage to stabilize and remodel the transition zone between cartilage-bone. Loss of collagen X and MMP13 are linked to expanded growth plates [START_REF] Grskovic | Depletion of annexin A5, annexin A6, and collagen X causes no gross changes in matrix vesicle-mediated mineralization, but lack of collagen X affects hematopoiesis and the Th1/Th2 response[END_REF][START_REF] Inada | Critical roles for collagenase-3 (Mmp13) in development of growth plate cartilage and in endochondral ossification[END_REF]. Therefore, RC dysfunction in CreTW mice induces severe damage in the hypertrophic chondrocytes with transient increase in hypertrophy, destabilization of the ECM at the cartilage-bone junction and growth plate degeneration in older CreTW mice.

Collectively, these results provide a molecular scenario which explains why a RC defect impairs skeletal growth in CreTW mice and may cause short stature in many patients with mitochondrial diseases. Moreover, the results illustrate the importance of the metabolic switch from fetal glycolysis to RC activation in growth plate cartilage after birth to promote postnatal growth, which may apply to various other cellular systems that are challenged with the transition from fetal to postnatal life.

Discussion

Anaerobic glycolysis was considered the main energy source for skeletal growth due to the avascular nature of growth plate cartilage with consequently low oxygen supply, but surprisingly mitochondrial diseases and RC dysfunction often present with slow growth and short stature. We proposed that RC activity is important for cartilage-mediated skeletal growth, but experimental systems to define the role of the RC in vivo were missing and to our knowledge the activity of the RC in growth plate cartilage was never determined.

Blood vessel infiltration supports the activation of respiration in the postnatal growth plate cartilage

We now provide the first evidence that the RC is active in growth plate cartilage at late stages of postnatal development. In newborns, complex IV activity was restricted to cartilage of the lateral growth plate and the chondro-osseous junction, both in close vicinity to the vasculature, while no complex IV activity was detectable in the zone of proliferating chondrocytes. In contrast, complex IV is active in proliferating chondrocytes of 13 days old mice and, even more markedly, of one month old mice. At this stage of development, the growth plate cartilage normally decreases in size [START_REF] Sivaraj | Blood vessel formation and function in bone[END_REF] and blood vessels invade the distal end of the femur cartilage to form the secondary ossification center, providing oxygen and nutrients to the distal side of the growth plate. Therefore, the locally increased oxygen tension or nutrient availability in older mice may induce a metabolic switch from glycolytic energy production to respiration in these chondrocytes. This is in line with the lack of changes in skeletal growth, cartilage organization or ECM deposition in CreTW newborn mice, despite a similar degree of mtDNA depletion and reduction of mtDNA encoded RC subunits as in one month old CreTW mice. Hence, glycolysis is sufficient to promote cartilage -mediated skeletal growth in the hypoxic fetal and neonatal environment, but not in later postnatal stages with increasing energy demand, when the growth plate cartilage becomes less hypoxic. Here the activation of the respiratory chain is crucial for energy production and for the inhibition of premature growth plate cartilage differentiation and degradation as demonstrated by the later onset and progression of the cartilage phenotype in older CreTW mice.

Therefore, respiration is essential to provide energy for postnatal cartilage growth, but is also needed to control cartilage differentiation and prevent premature cartilage degeneration and its transformation into bone.

Respiration promotes growth plate differentiation and hypertrophic ECM formation in aging mice

In three to four month old CreTW mice growth plate cartilage is reduced in size and completely resorbed and transformed into bone in older CreTW mice, while still present in control animals. In mice the growth plates are not fully degraded [START_REF] Hirai | Parathyroid hormone/parathyroid hormone-related protein receptor signaling is required for maintenance of the growth plate in postnatal life[END_REF], in contrast to humans. The phenotype of CreTW mice shows that the activation of the RC is important to prevent the complete resorption of the growth plate. Presumably, RC activity provides sufficient energy to maintain the growth plate cartilage, but the detailed analysis of the organization of the growth plate also showed that the RC is needed to regulate hypertrophic chondrocyte differentiation, survival and ECM stabilization. RC function may be important for progenitor cells [START_REF] Mizuhashi | Resting zone of the growth plate houses a unique class of skeletal stem cells[END_REF] normally responsible for sustaining growth plate chondrocytes and growth plate-derived osteoblasts. These progenitors could also be targeted by Col2-Cre expression and may increasingly suffer from mtDNA depletion during aging in CreTW mice and give rise to chondrocytes (and osteoblast) with a higher load of mtDNA damage compared to younger chondrocytes. Progenitor-derived older CreTW chondrocytes may accumulate further mitochondrial damage during replication cycles within the stacks of the growth plate and then reach a critical threshold in hypertrophic chondrocytes. A previous study indicated that terminally differentiated chondrocytes may undergo a maturation-dependent loss of mitochondrial function and become sensitive to signals for programmed cell death [START_REF] Rajpurohit | Chondrocyte death is linked to development of a mitochondrial membrane permeability transition in the growth plate[END_REF]. This sensitivity may be increased in the presence of mtDNA damage to promote specifically the death of older hypertrophic CreTW chondrocytes and to destabilize the hypertrophic ECM.

In one month old CreTW mice we detected a transient enlargement of the area of the hypertrophic zone. This enlargement was accompanied by decreased amounts of important ECM-related proteins in this zone, e. g. collagen X and MMP13, while the amounts of main ECM components like collagen II or aggrecan were not affected by RC inactivation. Mitochondrial dysfunction can modulate MMP expression and inhibit MMP13 production in human chondrocytes cultures [START_REF] Cillero-Pastor | Mitochondrial respiratory chain dysfunction modulates metalloproteases -1, -3 and -13 in human normal chondrocytes in culture[END_REF] and the decrease in collagen X and MMP13 is indicative of a general reduction of the hypertrophic ECM and ECM-remodeling proteins in cartilage of CreTW mice. Collagen II and aggrecan molecules have an exceptionally long lifetime and slow turn over [START_REF] Sivan | Aggrecan turnover in human intervertebral disc as determined by the racemization of aspartic acid[END_REF][START_REF] Verzijl | Effect of collagen turnover on the accumulation of advanced glycation end products[END_REF] while collagen X and MMP13 are located in the chondroosseus junction where the ECM is remodeled and cartilage is continuously replaced by bone. Therefore, collagen X and MMP13 production in the hypertrophic growth plate cartilage could be more susceptible for RC dysfunction in one month old CreTW mice.

Collagen X forms hexagonal networks [START_REF] Kwan | Macromolecular organization of chicken type X collagen in vitro[END_REF] and interacts with collagen II fibrils [START_REF] Poole | Immunoelectron microscopic studies of type X collagen in endochondral ossification[END_REF][START_REF] Schmid | Hypertrophic cartilage matrix. Type X collagen, supramolecular assembly, and calcification[END_REF] and proteoglycans [START_REF] Chen | Domains of type X collagen: alteration of cartilage matrix by fibril association and proteoglycan accumulation[END_REF] to stabilize the hypertrophic growth plate and the cartilage-bone junction, whereas MMP13 degrades various substrates to remodel the cartilage ECM. The lack of MMP13 or collagen X in mice results in expanded growth plates [START_REF] Grskovic | Depletion of annexin A5, annexin A6, and collagen X causes no gross changes in matrix vesicle-mediated mineralization, but lack of collagen X affects hematopoiesis and the Th1/Th2 response[END_REF][START_REF] Inada | Critical roles for collagenase-3 (Mmp13) in development of growth plate cartilage and in endochondral ossification[END_REF]) like in our mice and mutations in collagen X are often associated with short stature and waddling gait in humans [START_REF] Bateman | Mutations of COL10A1 in Schmid metaphyseal chondrodysplasia[END_REF][START_REF] Chan | Phenotypic and biochemical consequences of collagen X mutations in mice and humans[END_REF]. Interestingly, increased hypertrophic differentiation and decreased collagen X or MMP13 expression are also believed to be induced by stress responses in cartilage [START_REF] Cameron | Transcriptional profiling of chondrodysplasia growth plate cartilage reveals adaptive ER-stress networks that allow survival but disrupt hypertrophy[END_REF][START_REF] Tsang | In vivo cellular adaptation to ER stress: survival strategies with double-edged consequences[END_REF][START_REF] Yan | mTORC1 regulates PTHrP to coordinate chondrocyte growth, proliferation and differentiation[END_REF]. Hence, collagen X and MMP13 are required to maintain the integrity of the ECM and the growth plate cartilage at the chondro-osseous junction. We now show that RC inactivation induces a similar damage response in chondrocytes to destabilize the growth plate cartilage and consequently impair skeletal growth.

RC inactivation provokes a cellular stress response

Lack of RC activation has to be translated into cellular responses in CreTW mice. The transcriptome analysis of growth plate cartilage indicates that a form of mito-nuclear integrated stress response (ISR) is induced in these chondrocytes. A mitochondrial unfolded protein response (UPRmt) has been mainly described in C. elegans, which is commonly induced as a response to mitochondrial stress signals in order to activate a compensatory gene expression program [START_REF] Quiros | Mitonuclear communication in homeostasis and stress[END_REF][START_REF] Shpilka | The mitochondrial UPR: mechanisms, physiological functions and implications in ageing[END_REF]. The mammalian counterpart is driven by activating transcription factors (ATFs; [START_REF] Khan | mTORC1 Regulates Mitochondrial Integrated Stress Response and Mitochondrial Myopathy Progression[END_REF][START_REF] Shpilka | The mitochondrial UPR: mechanisms, physiological functions and implications in ageing[END_REF]). Interestingly, we could detect an increased expression of Atf3 which facilitates cell cycle exit and terminal differentiation of chondrocytes [START_REF] James | The transcription factor ATF3 is upregulated during chondrocyte differentiation and represses cyclin D1 and A gene transcription[END_REF], modulates Mmp13 expression in chondrocytes [START_REF] Chan | Cytokine-induced MMP13 Expression in Human Chondrocytes Is Dependent on Activating Transcription Factor 3 (ATF3) Regulation[END_REF] and can act as an adaptive response gene with proapoptotic functions [START_REF] Thompson | ATF3 transcription factor and its emerging roles in immunity and cancer[END_REF]. The expression of Ddit3/Chop and Atf5 is also increased in CreTW cartilage and DDIT3/CHOP protein has been reported before to mediate stressinduced apoptosis in cartilage [START_REF] Cameron | Transcriptional profiling of chondrodysplasia growth plate cartilage reveals adaptive ER-stress networks that allow survival but disrupt hypertrophy[END_REF] and directly induce Atf5 expression to potentiate DDIT3/CHOP-dependent apoptosis [START_REF] Teske | CHOP induces activating transcription factor 5 (ATF5) to trigger apoptosis in response to perturbations in protein homeostasis[END_REF]. ATF5 is capable of activating the integrated stress response in nematodes and presumably in mammals [START_REF] Melber | UPR(mt) regulation and output: a stress response mediated by mitochondrial-nuclear communication[END_REF]. It harbors a mitochondrial targeting sequence potentially allowing it to immediately translate mitochondrial damage into cellular responses [START_REF] Fiorese | The Transcription Factor ATF5 Mediates a Mammalian Mitochondrial UPR[END_REF]. These three transcriptional regulators may act in concert to induce the expression of damage-response genes, e.g. ASNS, GDF15 and FGF21, but the molecular interactions remain to be determined. The secreted proteins GDF15 and FGF21 are described as mitokines produced in response to mitochondrial damage [START_REF] Kim | Autophagy deficiency leads to protection from obesity and insulin resistance by inducing Fgf21 as a mitokine[END_REF][START_REF] Suomalainen | FGF-21 as a biomarker for muscle-manifesting mitochondrial respiratory chain deficiencies: a diagnostic study[END_REF] and interestingly both are upregulated in chondrocytes of CreTW mice. FGF21 directly acts on chondrocytes of the growth plate to inhibit collagen X expression and to mediate growth hormone insensitivity in hypertrophic chondrocytes [START_REF] Wang | GDF15 is a heart-derived hormone that regulates body growth[END_REF][START_REF] Wu | Fibroblast growth factor 21 (FGF21) inhibits chondrocyte function and growth hormone action directly at the growth plate[END_REF], which could contribute to reduced collagen X protein levels in one month old mice. At later stages FGF21 as well as GDF15 may act cooperatively to inhibit cartilage-mediated skeletal growth as both are known to exert effects on systemic energy homeostasis and growth hormone mediated body growth [START_REF] Chung | Growth differentiation factor 15 is a myomitokine governing systemic energy homeostasis[END_REF].

Patients with mitochondrial diseases often present with increased GFD15 and FGF21 serum levels [START_REF] Davis | A comparison of current serum biomarkers as diagnostic indicators of mitochondrial diseases[END_REF][START_REF] Ji | Growth Differentiation Factor 15 Is a Novel Diagnostic Biomarker of Mitochondrial Diseases[END_REF][START_REF] Lehtonen | FGF21 is a biomarker for mitochondrial translation and mtDNA maintenance disorders[END_REF][START_REF] Montero | GDF-15 Is Elevated in Children with Mitochondrial Diseases and Is Induced by Mitochondrial Dysfunction[END_REF][START_REF] Yatsuga | Growth differentiation factor 15 as a useful biomarker for mitochondrial disorders[END_REF] although clinical studies correlating growth retardation with these factors are needed. These mitokines could be produced locally in growth plate cartilage in response to RC dysfunction to inhibit cartilage-mediated skeletal growth, but also to contribute to systemic metabolic effects on body growth in these patients.

Multiple factors recognize mitochondrial dysfunction and it is well described that mtDNA damage and respiration failure to increased intracellular pO 2 , destabilized HIF1A levels and impaired cell survival [START_REF] Doege | Inhibition of mitochondrial respiration elevates oxygen concentration but leaves regulation of hypoxia-inducible factor (HIF) intact[END_REF][START_REF] Martinez-Reyes | TCA Cycle and Mitochondrial Membrane Potential Are Necessary for Diverse Biological Functions[END_REF]. Interestingly, we could indeed observe reduced HIF1A in extracts of growth plate from 12 days old CreTW mice. HIF1A was originally described to activate Sox9 in prechondrogenic mesenchymal cells and maintain differentiation and ECM production in response to hypoxia [START_REF] Amarilio | HIF1alpha regulation of Sox9 is necessary to maintain differentiation of hypoxic prechondrogenic cells during early skeletogenesis[END_REF] during early embryonic development. However, the same group demonstrated that in differentiated chondrocytes HIF1A directly controls collagen hydroxylation and secretion during later embryonal development independently of Sox9 [START_REF] Bentovim | HIF1alpha is a central regulator of collagen hydroxylation and secretion under hypoxia during bone development[END_REF]. Hif1A directly drives prolyl-4-hydroxylase expression, a central stabilizer of collagen triple helix formation [START_REF] Wagner | Coexpression of alpha and beta subunits of prolyl 4-hydroxylase stabilizes the triple helix of recombinant human type X collagen[END_REF] and lack of HIF1a leads to reduced collagen hydroxylation, arrested ECM secretion and ER stress in differentiated growth plate chondrocytes in vivo [START_REF] Bentovim | HIF1alpha is a central regulator of collagen hydroxylation and secretion under hypoxia during bone development[END_REF]. This is in line with the decrease of collagen X levels at the chondro-osseous junction and the occurrences of cellular stress in the growth plate of older CreTW mice. Moreover, it was shown that chondrocyte metabolism and HIF1a signaling control collagen modification and matrix resistance to protease digestion [START_REF] Stegen | HIF-1alpha metabolically controls collagen synthesis and modification in chondrocytes[END_REF]. Hence, HIF1a can exert important effects on ECM homeostasis and the chondrocyte phenotype in the differentiated growth plate cartilage. Considering that terminal differentiated chondrocytes have a unique sensitivity to mitochondrial function [START_REF] Rajpurohit | Chondrocyte death is linked to development of a mitochondrial membrane permeability transition in the growth plate[END_REF], an increase in intracellular pO 2 levels in older CreTW mice could decrease HIF1a levels to directly impair collagen hydroxylation and secretion, and chondrocyte survival at the chondro-osseous junction.

Diminished respiration can also inhibit ROS production, which is necessary for the activation of HIF1A [START_REF] Martinez-Reyes | TCA Cycle and Mitochondrial Membrane Potential Are Necessary for Diverse Biological Functions[END_REF] and the induction of autophagy [START_REF] Marambio | Glucose deprivation causes oxidative stress and stimulates aggresome formation and autophagy in cultured cardiac myocytes[END_REF][START_REF] Wang | 2-Deoxy-D-glucose treatment of endothelial cells induces autophagy by reactive oxygen species-mediated activation of the AMP-activated protein kinase[END_REF]. We could also observe reduced ROS levels in extracts of growth plate from 12 days old CreTW mice and detect the accumulation of fragmented mitochondria and the reduction in the autophagic flux in older CreTW chondrocytes. Autophagy was previously described to promote chondrocyte differentiation, survival [START_REF] Vuppalapati | Targeted Deletion of Autophagy Genes Atg5 or Atg7 in the Chondrocytes Promotes Caspase-Dependent Cell Death and Leads to Mild Growth Retardation[END_REF] and ECM production [START_REF] Pfander | HIF-1alpha controls extracellular matrix synthesis by epiphyseal chondrocytes[END_REF] and is likely to contribute to cell death and ECM dysregulation in CreTW mice.

The consequences of respiratory chain inactivation in mitochondria of growth plate cartilage may not necessarily be explained by the simplistic view of a single key mechanism or pathway, but in our view a unique damage response is evoked due to energy depletion in older CreTW mice. Energy depletion is first sensed in hypertrophic chondrocytes based on their unique sensitivity to respiration dysfunction. High pO2 and reduced ROS levels can decrease HIF1a levels to impair hydroxylation of collagens and ECM secretion. In addition, inhibition of autophagy can induce a stress response to impair ECM homeostasis and chondrocyte differentiation and the combination of these factors destabilizes the ECM, promotes chondrocyte death, growth plate degradation and closure in older CreTW animals.

In conclusion, this study demonstrates to our knowledge for the first time that, in contrast to the current view, postnatal cartilage development requires RC activity in chondrocytes and indicates that RC activation protects from premature growth plate closure and growth retardation. Glycolysis is sufficient to drive fetal cartilage development, but cannot compensate for the lack of RC activation during postnatal skeletal growth in CreTW mice and most likely in humans with mitochondrial diseases and RC dysfunction. Mechanistically, the phenotype is caused by a defect in the energy production, which disturbs metabolic signaling and chondrocyte hypertrophy, destabilizes the ECM and finally induces cell death. Hence, the metabolic switch in cartilage from anaerobic glycolysis to RC activation is crucial to drive postnatal skeletal development, while RC dysfunction accelerates premature growth plate closure and induces mitochondria-related skeletal pathologies. In general, this study illustrates the importance of metabolic adaptation for the coordination of postnatal cartilage development and homeostasis.

Materials and Methods

Animals

Previously established R26-K320E-TWINKLE loxP/+ C57BL/6J mice were crossed with Col2a1-Cre C57BL/6N mice [START_REF] Bluhm | miR-322 stabilizes MEK1 expression to inhibit RAF/MEK/ERK pathway activation in cartilage[END_REF][START_REF] Ovchinnikov | Col2a1-directed expression of Cre recombinase in differentiating chondrocytes in transgenic mice[END_REF] to induce cartilage-specific Tw K320E expression in heterozygous Col2a1-Cre-R26-K320E-TWINKLE loxP/+ mice (TW). Col2a1-Cre C57BL/6 mice of the same litter (Cre) were used as controls. For neonatal assessment of skeletal development newborns of both sexes were analyzed. All experiments were performed in accordance with the guidelines of the German animal protection law. Institutional review board: "Landesamt für Natur, Umweltund Verbraucherschutz NRW".

Histology and immunohistochemistry

Microtome sections (5µm) of decalcified right femora from mice were used for morphological and immunohistological analyses. The organization of the growth plate was assessed by hematoxylin/eosin/alcian blue staining and the distribution of ECM proteins was determined by immunofluorescence microscopy. To detect collagen II and X, sections were pretreated with 0.025% pepsin for 15min at 37°C, 5mg/ml hyaluronidase (Sigma) for 30min at 37°C and 10µg/ml Proteinase K (Sigma) for 10min at 50°C. To study aggrecan distribution, sections were preincubated with 10µg/ml Proteinase K (Sigma) for 10min at 50°C and to localize the laminin γ1 chain, sections were incubated with 0.005% Trypsin for 10min at 37°C. The following primary antibodies were used: mouse-anti-collagen II (1:500, Calbiochem), mouse-anti-collagen X (1:100, hybridoma clone X53; [START_REF] Girkontaite | Immunolocalization of type X collagen in normal fetal and adult osteoarthritic cartilage with monoclonal antibodies[END_REF]), rabbit-anti-aggrecan (1:100, Millipore/Chemicon), rabbit-anti-laminin γ1 chain (1:2.000, [START_REF] Tunggal | Expression of the nidogen-binding site of the laminin gamma1 chain disturbs basement membrane formation and maintenance in F9 embryoid bodies[END_REF]). Corresponding secondary antibodies coupled to Cy3 (1:800, Jackson ImmunoResearch) were applied. To determine the distribution of MMP13 and PCNA, immunohistochemical stainings were performed. Sections were pretreated with citrate buffer (pH 6.0) for 1 hour at 60°C. Primary antibodies against MMP13 (1:500, Abcam) or PCNA (1:200, Abcam) were applied and appropriate secondary antibodies labeled with horseradish peroxidase (DAKO) were used for the detection (Zytomed Systems).

Sections were analyzed using a Nikon Eclipse TE2000-U Microscope.

mtDNA copy number

DNA of femoral head cartilage was extracted using phenol-chloroform extraction and the relative mtDNA content was analyzed by quantitative PCR [START_REF] Baris | The mitochondrial electron transport chain is dispensable for proliferation and differentiation of epidermal progenitor cells[END_REF]. The Ct values of the mtDNA encoded Nd2 and the nuclear DNA encoded Pecam were determined using the QuantiTect SYBR Green PCR assay (Qiagen). Pecam amplification was used for normalization and the fold change was calculated with delta delta CT method [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF].

In situ detection assays

The DeadEnd TM Fluorometric TUNEL assay (Promega) was applied to paraffin sections (5 µm) as previously described [START_REF] Bluhm | miR-322 stabilizes MEK1 expression to inhibit RAF/MEK/ERK pathway activation in cartilage[END_REF]. Briefly, sections were digested with 20µg/ml Proteinase K for 8min at room temperature and then incubated with 4% paraformaldehyde for 10min. The rTdT labeling reaction was performed for one hour at 37°C. Nuclei were detected with DAPI and then sections were embedded in Mowiol. The number of TUNEL positive cells was quantified by ImageJ software.

Deparaffinized sections (5µm) were permeabilized with 0.2% Triton X-100 in TBS for 10min, washed with TBS for 10min and incubated with the BCIP/NBT staining solution (86µg/ml BCIP, 162µg/ml NBT, 5mM MgCl 2 in 10% polyvinylalcohol/0.1M Tris (pH 9.0)/0.1M NaCl at 30°C until staining was visible. Sections were extensively washed with H 2 O, stained with 0.001% Fast Green FCF for 5min and embedded in gelatin. To visualize complex IV activity in situ, cryotome sections (10µm) of right mouse femora were prepared using Kawamoto's film method [START_REF] Kawamoto | Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, whole-animals, insects and plants[END_REF] and stained for complex IV (CYTOCOX) activity as previously described [START_REF] Sciacco | Cytochemistry and immunocytochemistry of mitochondria in tissue sections[END_REF].

Cell culture

To analyze sufficient cell numbers in downstream experiments rib cage growth plates from newborn and one month old mice were incubated in collagenase II solution (450U/ml, Biochrom) for 6 hours at 37°C. Femoral growth plates could not be used due to the insufficient yield in cell numbers, especially from one month old mice. To obtain adequate numbers of cells, rib cage derived chondrocytes from 2-3 animals of both genders were pooled unless otherwise stated (Figure 7B) and cultured in DMEM-F12 supplemented with 10% FCS (Biochrom), penicillin (100U/ml, Biochrom), streptomycin (100µg/ml, Biochrom), ascorbate (44µg/ml, Sigma) and L-ascorbate-2-phosphate (130µg/ml, Sigma).

After 24h chondrocytes were detached with collagenase II in DMEM-F12 (2mg/ml, Biochrom), centrifuged and resuspended in medium. Cells were cultured for another three days and then used for experiments.

The pH of the cell culture supernatant was determined using a Seven-Easy-pH-Meter (Mettler, Toledo).

Mitochondrial mass, membrane potential, ATP production, MitoSOX and NADH

In order to assess mitochondrial mass, chondrocytes were detached with collagenase II, washed with cell culture medium and stained with 50nM MitoTracker deep red dye in cell culture medium for 30min at 37°C. Cells were washed and resuspended in 5% FCS/PBS containing propidium iodide. The mean fluorescence intensity of the MitoTracker deep red signal (APC-A; 660 ± 20nm) was determined using a FACS Canto II flow cytometer (Becton Dickinson). To evaluate the mitochondrial membrane potential, chondrocytes were resuspended in cell culture medium containing 5µM JC-1 and then incubated for 15min at 37°C. Cells were washed with 5% FCS/PBS, resuspended in 1µM SYTOX Blue (Thermo Fischer Scientific)/ 5% FCS/PBS and after 15min incubation on ice they were analyzed by flow cytometry. The ratio of the mean PE-A (mitochondrial JC-1 + ) and the mean FITC signal (cytoplasmic JC-1 + ) from SYTOX Blue -viable chondrocytes was calculated. The ATP content of chondrocytes was studied using the ATP Bioluminescence Assay Kit CLS (Sigma). Briefly, 9 x 10 4 chondrocytes were cultured in a 12-well plate for three days, detached with collagenase II, washed with PBS and resuspended in 100µl PBS. The protein content of 4.5x10 4 cells was determined with the BC assay (Interchim). The remaining 4.5x10 4 cells were dissolved in 450µl of hot 100mM Tris/ 4mM EDTA (pH 7.8) and incubated for two min at 100°C. The luciferase reagent was added and the ATP-dependent production of a bioluminescence signal was determined (Infinite M1000, Tecan) and normalized to the protein amount. Reactive oxygen levels of chondrocytes were analyzed by the MitoSOX Red staining assay. Chondrocytes were incubated for 15min at 37°C in 250 µl of 5 µM MitoSOX Red (Thermo Fisher Scientific) in DMEM w/o phenol red and mice). The image data were segmented using a threshold (dependent on the age of the mice) of 19% (1 and 2 months), 21 % (3 months) and 22% (4 months, 6 months and 1 year) of the maximum grayscale value. Trabecular parameters included bone volume fraction (BV/TV, 1), connectivity density (Conn-Dens, 1/mm 3 ), trabecular number (Tb.N, 1/mm), trabecular thickness (Tb.Th, mm), and trabecular separation (Tb.Sp, mm). Statistical analysis (Mann-Whitney U test) was performed using IBM®SPSS® Statistics (Version 21, IBM Deutschland GmbH, Ehningen, Germany). For whole bone overview pictures the image data were segmented using a threshold of 21% (1 month), 24% (2 months), 26% (4 months) and 27% (3 and 6 months, 1 year) of the maximum grayscale value.

Cell viability

Proliferation of chondrocytes was analyzed using the neutral red assay [START_REF] Repetto | Neutral red uptake assay for the estimation of cell viability/cytotoxicity[END_REF]. 7.5

x 10 3 chondrocytes were cultured in 96-well plates for one, two or three days. Neutral red dye solution (0.5mg/ml in 0.9% NaCl) was added and incubated for 2 hours at 37°C. Cells were washed twice with PBS and then the dye was extracted with 0.05M Na 2 HPO 4 in 50% ethanol overnight at -20°C.

Absorbance was measured at 540nm (Infinite M1000, Tecan). Apoptosis was studied by Annexin A5 (ANXA5) staining as described [START_REF] Rosenbaum | Identification of novel binding partners (annexins) for the cell death signal phosphatidylserine and definition of their recognition motif[END_REF]. Briefly, chondrocytes were detached with collagenase II, washed and stained with ANXA5-Alexa 647 and propidium iodide (PI). The number of ANXA5-Alexa647 -/PI -viable, ANXA5-Alexa647 + /PI -apoptotic and Anxa5-Alexa647 + /PI + dead cells was determined by flow cytometry (FACS Canto II, Becton Dickinson).

Gene expression analysis

The RNA from femoral growth plates of 12 days old Cre and CreTW littermate mice or cultured chondrocytes was isolated by phenol-chloroform extraction and RNA integrity was confirmed by microcapillary electrophoresis (2100 Bioanalyzer, Agilent). For microarray analysis, 100ng RNA of growth plate cartilage from four Cre and CreTW mice, respectively, were amplified, labeled and hybridized to a 8x60K whole genome mRNA microarray using the Agilent protocol. Microarrays were scanned (Agilent G2595C scanner), data extracted and analyzed by GeneSpring V14.9 software (Agilent). mRNAs were selected according to their differential expression, to their similar expression levels in all four microarrays of a given genotype and to their statistical significance.

Quantitative PCR (qPCR)

RNA was reversely transcribed into cDNA with the Omniscript RT assay (Qiagen) and used for sqPCR or mRNA SYBR®Green-(Gdf15, Qiagen) or probe-based qPCR assays (Fgf21, Ddit3, Trib3, Roche, Invitrogen) (Belluoccio et al., 2010a). The expression was normalized to Mapk7, constantly expressed in the proliferative, prehypertrophic and hypertrophic zone of growth plate cartilage (Belluoccio et al., 2010b;[START_REF] Etich | MiR-26a modulates extracellular matrix homeostasis in cartilage[END_REF]. The fold change was calculated with the delta-delta C(T) method [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF]. The following primers were used: Actin (fw 5'-gacgaggcccagagcaagag-3'; rev 5'-ctagagcaacatagcacagc-3'), Ddit3 (fw 5'-atctcatccccaggaaacg-3'; rev 5'-atgtgcgtgtgacctctgtt-3'), Fgf21 (fw 5'-aagacactgaagcccacctg-3'; rev 5'-gaatgacccctggcttcaa-3'), Gdf15 (fw 5'-gagaggactcgaactcagaac-3'; rev 5'-gaccccaatctcacctctg-3') [START_REF] Wang | GDF15 is a heart-derived hormone that regulates body growth[END_REF], Mapk7 (fw 5'-ggttcatctcagacccacctt-3'; rev 5'-ctgttatggctcggtggtg-3'), Trib3 (fw 5'-gctatcgagccctgcact-3'; rev 5´-acatgctggtgggtaggc-3'), Xbp1 (fw 5´-gatcctgacgaggttccaga-3´; rev 5´-acagggtccaacttgtccag-3´).

Immunoblotting

Femoral growth plate, femur head cartilage or cultured cells were taken up in RIPA buffer containing 5% NP-40, 0.25%, Triton-X-100, 2.5%, 750mM NaCl, 100mM Tris-HCl (pH 7.4) and cOmplete™ Protease Inhibitor (for metabolic signaling) and disrupted by sonication (Branson). Protein concentration was determined using the BC Protein Assay (Interchim) according to the manufacturer's recommendations and equal amounts were resolved on SDS-polyacrylamide gels. After transfer onto a nitrocellulose membrane (Whatman), blots were incubated for 30 to 60min at RT with 5% milk powder or 5% BSA in TBS-0.1% Tween20 and then a primary antibody cocktail was added. Corresponding secondary antibodies coupled to horseradish peroxidase (Dako) were applied and antibody binding was visualized by enhanced chemoluminescence. Membranes were incubated in 0.2M NaOH for 2min, rinsed with water and TBS-0.1% Tween20 and after blocking reused for additional immunoblot experiments. The following antibodies were used: Actin (1:2000, Millipore), aggrecan (1:1000, Millipore), p-/t-AMPK (1:1000, CST), ASNS (1:500, Biorbyt), collagen X (1:500, [START_REF] Girkontaite | Immunolocalization of type X collagen in normal fetal and adult osteoarthritic cartilage with monoclonal antibodies[END_REF], p-/t-EIF2A (1:1000, CST), HIF1A (1:500, Genetex), SQSTM1 (1:1000, MBL), Matrilin3 (1:2000[START_REF] Klatt | Molecular structure and tissue distribution of matrilin-3, a filament-forming extracellular matrix protein expressed during skeletal development[END_REF], MMP13

(1:1000, Abcam), Respiratory chain complexes (1:500, Abcam). For quantification band intensities were analyzed using ImageJ software (NIH).

Mitochondrial fragmentation

For immunofluorescence detection of the outer mitochondrial membrane protein TOM20 chondrocytes were cultured for 24h on glass coverslips in a 24-well plate, fixed with 4% PFA/PBS for 10min at RT, permeabilized with 0.5% NP-40/PBS for 10min and incubated with 1% FCS/PBS for 30min at RT. The TOM20 antibody (Santa Cruz) was added and after one hour at RT cells were washed three times with blocking buffer and incubated with appropriate Alexa-Fluor-conjugated secondary antibodies and DAPI for additional 45min at RT. In addition, chondrocytes were transfected with 1µg plasmid DNA (pOCT-YFP) [START_REF] Neuspiel | Cargo-selected transport from the mitochondria to peroxisomes is mediated by vesicular carriers[END_REF] per well using XtremeGENE HP (Roche, Mannheim, Germany) according to the manufacturer's protocol in a 1:2 ratio (DNA:XtremeGENE HP) to fluorescently label mitochondria (MitoYFP). 24h-post transfection cells were fixed in 4% PFA / PBS for 10min at RT. After mounting (Dako, Hamburg, Germany) images were acquired using a Leica TCS SP5 microscope controlled with LAS AF Software (Leica, Wetzlar, Germany).

Statistical analysis

Statistical significance was determined for n≥3 using the unpaired two-tails method of the student's t-test or Mann-Whitney U-test (Figure 4D). P-values < 0.05 (*) were considered to be significant and p-values < 0.01 (**) as highly significant. Data are shown as mean ± standard deviation. For relative changes one sample t-test was applied.

Online supplemental material

Figure S1 demonstrates changes in femur organization and bone formation in 1, 2, 3, 4, 6 months and one year old Cre and CreTW mice using µCT analysis. In Figure S2 the distribution of collagen II, aggrecan, collagen X and MMP13 protein was determined in sections from three individual newborns and/or from one month old Cre and CreTW mice using immunostainings. Table S1

Figures legends

Figure 1 Characterization of respiratory chain activity in the developing growth plate cartilage (A) Femoral growth plate sections from one day, 13 days and one month old mice were stained for CYTOCOX activity (brown) (Pproliferative, PHprehypertrophic, Hhypertrophic chondrocytes, BMbone marrow, SOCsecondary ossification center). (B) Laminin γ1 chain immunostaining was used to visualize the vascular network in the proximity of the growth plate cartilage and DAPI to detect nuclei. Arrowheads show the blood vessels close to the growth plate cartilage. Bars: (A) 200µm, (B) 100µm. The brightness of the red signal for laminin γ1 staining and the blue signal for DAPI was adjusted for visualization. The Seahorse XF Cell Mito Stress assay was used to determine the bioenergetics profile in isolated chondrocytes from one day (n=2 Cre, n=3 CreTW) and one month old Cre and CreTW mice (n=3). (A) The oxygen consumption rate (OCR) over time was studied and the (B) Basal respiration, ATP production-coupled respiration and maximal and spare respiratory capacities were determined by adding the ATP synthase inhibitor oligomycin A (2µM), the uncoupler of the respiratory chain FCCP (1µM) and the inhibitors of complex III and I antimycin A (2.5µM) and rotenone (0.5µM), respectively [START_REF] Coutelle | Embelin inhibits endothelial mitochondrial respiration and impairs neoangiogenesis during tumor growth and wound healing[END_REF]. (C) The OCR and extracellular acidification rate ECAR were plotted to obtain the energetic profile of the chondrocytes. * p < 0.05, ** p < 0.01. The transcriptome of femoral growth plate cartilage (dashed line) isolated from 12 days old Cre and CreTW mice was compared using whole genome mRNA arrays. The growth plates of four individual animals per genotype were analyzed and (A) the corresponding expression intensity plots and Venn diagrams of differentially expressed genes are shown (fold-change ≥2, P<0.05, Cre versus CreTW), mitochondrial genes as annotated in the array (ochre) and genes of the integrated stress response are given (purple). (B) Cluster analysis -integrated stress response genes. Entities not differentially expressed or below expression threshold are displayed in desaturated colors. For some genes results of multiple probes are shown. FC = fold change (C) The expression of selected genes was validated by quantitative PCR using cDNA from growth plate cartialge of 12 days old mice. Quantitative data are mean ± SD (n= 3 animals per genotype), * p < 0.05, ** p < 0.01. (D) The induction of an unfolded protein response was studied by semiquantitative PCR using cDNA from isolated primary rib cage chondrocytes of one month old Cre and CreTW mice or femoral growth plate cartilage isolated from 12 days old mice. The expression of Xbp1 and spliced sXbp1 mRNA was determined. Tunicamycin-treated chondrocytes were used as control.

Figure 7 Regulation of metabolic sensitive pathways and ECM proteins

Extracts of Cre (n=6) and CreTW (n=5) growth plates were analyzed for the activation of the (A) integrated stress response (p-EIF2A, t-EIF2A, ASNS), (B) the metabolic response (p-AMPK, t-AMPK, HIF1A, p62/SQSTM1) and (C) for ECM proteins (ACAN, COL10A1, MATN3 by immunoblotting. Actin was used as loading control. * As all proteins shown in A and C were tested on a single blot the same actin control is included in both panels. (D) The activation of AMPK was determined in extracts of cultured chondrocytes from individual isolations of 1 month old growth plate cartilage of Cre and CreTW mice (n=6). Image J analysis was used for quantification and fold changes in protein activation or expression between Cre and CreTW are shown (graphs). p-phosphorylated protein, t-total protein. Quantitative data are mean ± SD, * p < 0.05, ** p < 0.01.

Figure 8 Mitochondrial membrane organization in Cre and CreTW chondrocytes

Cultured chondrocytes from one day or one month old Cre and CreTW mice were transfected with a MitoYFP reporter gene construct or immunostained with a TOM20 specific antibody to visualize mitochondrial membrane organization. Quantification of the tubular and fragmented mitochondria (n≥3 per genotype) is given as mean ± SD, * p < 0.05, ** p < 0.01. The brightness and contrast of the fluorescence signal was adjusted to enable visualization. Bar: 10µm.

Figure 2

 2 Figure 2 Characterization of mitochondrial function in chondrocytes of CreTW mice(A) Relative mtDNA copy number was determined by quantitative PCR analysis (n=3 biological samples per group). (B) Immunoblot analysis of respiratory chain complexes I to V in femoral head cartilage of one day and one month old Cre and CreTW mice. (C) Femoral growth plate (GP) sections were stained for CYTOCOX in one day and one month old CreTW mice. Flow cytometric assessment of (D) mitochondrial membrane potential and (E) mitochondrial mass in cultured primary chondrocytes from one day and one month old Cre and CreTW mice using JC-1 and MitoTracker Deep Red staining, respectively. (F) The pH of the cell culture supernatant was analyzed. (G) A bioluminescence assay was used to determine the ATP content in isolated chondrocytes cultured for three days in DMEM-F12 medium. (H) Relative ROS (Mitosox) changes were assessed by flow cytometry analysis and (I) relative NADH levels were determined by MTS measurements. Quantitative data are mean ± SD (n = 3 mice per group), * p < 0.05, ** p < 0.01. No standard deviation is given for control when calculating relative changes of independent experiments.

Figure 3

 3 Figure 3 Analysis of the energy production in Cre and CreTW chondrocytes.The Seahorse XF Cell Mito Stress assay was used to determine the bioenergetics profile in isolated chondrocytes from one day (n=2 Cre, n=3 CreTW) and one month old Cre and CreTW mice (n=3). (A) The oxygen consumption rate (OCR) over time was studied and the (B) Basal respiration, ATP production-coupled respiration and maximal and spare respiratory capacities were determined by adding the ATP synthase inhibitor oligomycin A (2µM), the uncoupler of the respiratory chain FCCP (1µM) and the inhibitors of complex III and I antimycin A (2.5µM) and rotenone (0.5µM), respectively[START_REF] Coutelle | Embelin inhibits endothelial mitochondrial respiration and impairs neoangiogenesis during tumor growth and wound healing[END_REF]. (C) The OCR and extracellular acidification rate ECAR were plotted to obtain the energetic profile of the chondrocytes. * p < 0.05, ** p < 0.01.

Figure 4

 4 Figure 4 Analysis of the skeletal phenotype in mice with cartilage-specific respiratory chain inactivation (A) Quantification of the body length (nose to tail) in one day, one month and one year old mice (n≥10 mice per genotype and time point). (B) Representative femora and tibiae (C) of one month and one year old mice. (D) µCT pictures of the femur of one month, three months and one year old mice. The localization of the growth plate is indicated (red arrowheads). Bars: 1mm. (E) In-plane matched paraffin sections from Cre and CreTW femora of one day, one month, three months and one year old mice were stained with hematoxylin (nuclei, purple), eosin (cytoplasm, pink) and alcian blue (proteoglycans, blue). The proliferative (P), prehypertrophic (PH), hypertrophic (H) zones and the growth plate are marked. Black arrowheads indicate loss of growth plate cartilage in one year old CreTW mice. (F) Quantification of the total area of growth plate cartilage and of the hypertrophic (H)-zone in femoral growth plate cartilage from one day and one month old Cre and CreTW mice. Quantitative data are mean ±SD, (n ≥ 5 animals per genotype and time point were analyzed), ** p < 0.01.

Figure 5

 5 Figure 5 Characterization of proliferation and cell death in Cre and CreTW chondrocytes (A) Cell numbers were determined as the incorporation of neutral red into the cytoplasm of viable cells over time in culture. (B) Cell viability was characterized by studying the proportion of AnxA5-Alexa647 -/PI -viable, AnxA5-Alexa647 + /PI -apoptotic and AnxA5-Alexa647 + /PI + necrotic cells by flow cytometry after 3 days in culture. Quantitative data are mean ±SD (n = 3 mice per group), * p < 0.05, ** p < 0.01. (C) The number of proliferating PCNA + and (D) apoptotic TUNEL + cells was determined on paraffin sections from growth plate cartilage of one day and one month old Cre and CreTW mice. DAPI

Figure 6

 6 Figure 6 Determination of transcriptome changes in the growth plate cartilage and in cultured chondrocytes of CreTW miceThe transcriptome of femoral growth plate cartilage (dashed line) isolated from 12 days old Cre and CreTW mice was compared using whole genome mRNA arrays. The growth plates of four individual animals per genotype were analyzed and (A) the corresponding expression intensity plots and Venn diagrams of differentially expressed genes are shown (fold-change ≥2, P<0.05, Cre versus CreTW), mitochondrial genes as annotated in the array (ochre) and genes of the integrated stress response are given (purple). (B) Cluster analysis -integrated stress response genes. Entities not differentially expressed or below expression threshold are displayed in desaturated colors. For some genes results of multiple probes are shown. FC = fold change (C) The expression of selected genes was validated by quantitative PCR using cDNA from growth plate cartialge of 12 days old mice. Quantitative data are mean ± SD (n= 3 animals per genotype), * p < 0.05, ** p < 0.01. (D) The induction of an unfolded protein response was studied by semiquantitative PCR using cDNA from isolated primary rib cage chondrocytes of one month old Cre and CreTW mice or femoral growth plate cartilage isolated from 12 days old mice. The expression of Xbp1 and spliced sXbp1 mRNA was determined. Tunicamycin-treated chondrocytes were used as control.

Figure 9

 9 Figure 9 Consequences of respiratory chain inactivation for ECM organization in CreTW mice (A) Distribution of collagen II, aggrecan and collagen X protein was analyzed by immunofluorescence staining. (B) Immunohistochemical staining was used to localize MMP13 protein in one month old growth plate. Pictures are representative for n≥3 per genotype and time point. Bars: 200µm. (C) Immunoblot analysis of MMP13 and actin (ACTA1) in cultured chondrocytes of one month old Cre and CreTW mice.

  reports trabecular parameters of bone volume fraction (BV/TV and connectivity density (Conn-Dens, 1/mm 3 ).

	Author contributions
	TH	-designing research studies, conducting experiments, acquiring data, analyzing data and writing
		the manuscript
	KP	-conducting experiments, acquiring data, analyzing data
	JE	-conducting experiments, acquiring data, analyzing data
	MA	-conducting experiments, acquiring data, analyzing data
	VG	-conducting experiments, acquiring data
	BB	-conducting experiments, acquiring data
	CF	-conducting experiments, acquiring data
	JH	-conducting experiments, acquiring data
	AN	-analyzing data, writing the manuscript
	JN	-conducting experiments, acquiring data
	MP	-analyzing the data, writing the manuscript
	JS	-conducting experiments, acquiring data
	HK	-analyzing the data, writing the manuscript
	HK	-conducting experiments, acquiring data
	RW	-designing research studies, analyzing data, providing reagents, writing the manuscript
	BB	-designing research studies, conducting experiments, acquiring data, analyzing data and writing
		the manuscript

Acknowledgements

This work was funded by grants from the Deutsche Forschungsgemeinschaft (BR 2304/9-1, BR2304/12-1, FOR2722 to B.B; Wi 889/6-3 to R.J.W, CRC1218 to H.K). The authors declare no competing financial interests.

the mean fluorescence intensity was determined by flow cytometry. Reducing equivalents were analyzed using the CellTiter 96® AQ ueous One Solution (Promega) according to the manufacturer's specification.

Cells were cultured for two, 24, 48 and 72 hours, substrate was added and production of colored formazan product was detected at 492nm using a 96-well plate reader (Sunrise, Tecan).

Bioenergetic profiling

For bioenergetics profiling of chondrocytes, 10.000 cells/well were seeded on Seahorse 96-well plates and analysed according to Agilent protocols (MitoStress Kit) with a Seahorse XF96 analyzer.

Parameters of metabolic function depicted as bar charts in the figures were calculated according to Agilent protocols [START_REF] Coutelle | Embelin inhibits endothelial mitochondrial respiration and impairs neoangiogenesis during tumor growth and wound healing[END_REF]. Basal respiration: (Last rate measurement before oligomycin

µCT analysis

A high-resolution µCT scanner (µCT 35, Scanco Medical AG) was used to determine the microarchitecture of the left femora. Isolated bones were scanned with an isotropic voxel size of 7x7x7 µm using 70 kVp tube voltage, 114 µA tube current and 400 ms integration time. Image noise was removed by preprocessing of the grayscale data of the raw CT images using a 3D Gaussian filter algorithm and separation of mineralized tissue from the soft tissue by a global thresholding procedure [START_REF] Stauber | Micro-computed tomography: a method for the non-destructive evaluation of the three-dimensional structure of biological specimens[END_REF]. The segmentation steps were applied with support=1, sigma=0.8. For assessment of trabecular bone, 142 (Cre mice, 1mm) or 107 (CreTW mice, 0.75 mm) slices were evaluated at the femoral metaphysis 1 mm (Cre mice) or 0.75 mm (CreTW mice) below the growth plate which included only secondary spongiosa (dependent on the different femur length of the Cre and CreTW