Mitochondrial respiratory chain function promotes extracellular matrix integrity in cartilage

Abstract

Energy metabolism and extracellular matrix (ECM) function together orchestrate and maintain tissue organization, but crosstalk between these processes is poorly understood. Here, we used single-cell RNA-Seq (scRNA-Seq) analysis to uncover the importance of the mitochondrial respiratory chain for ECM homeostasis in mature cartilage. This tissue produces large amounts of a specialized ECM to promote skeletal growth during development and maintain mobility throughout life. A combined approach of high-resolution scRNA-Seq, mass spectrometry/matrisome analysis, and atomic force microscopy was applied to mutant mice with cartilage-specific inactivation of respiratory chain function. This genetic inhibition in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage, showing disorganized chondrocytes and increased deposition of ECM material. scRNA-Seq analysis identified a cell cluster–specific decrease in mitochondrial DNA–encoded respiratory chain genes and a unique regulation of ECM-related genes in nonarticular chondrocytes. These changes were associated with alterations in ECM composition, a shift in collagen/noncollagen protein content, and an increase of collagen crosslinking and ECM stiffness. These results demonstrate that mitochondrial respiratory chain dysfunction is a key factor that can promote ECM integrity and mechanostability in cartilage and presumably also in many other tissues. Energy metabolism and extracellular matrix (ECM) function together orchestrate and maintain tissue organization, but crosstalk between these processes is poorly understood. Here, we used single-cell RNA-Seq (scRNA-Seq) analysis to uncover the importance of the mitochondrial respiratory chain for ECM homeostasis in mature cartilage. This tissue produces large amounts of a specialized ECM to promote skeletal growth during development and maintain mobility throughout life. A combined approach of high-resolution scRNA-Seq, mass spectrometry/matrisome analysis, and atomic force microscopy was applied to mutant mice with cartilage-specific inactivation of respiratory chain function. This genetic inhibition in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage, showing disorganized chondrocytes and increased deposition of ECM material. scRNA-Seq analysis identified a cell cluster–specific decrease in mitochondrial DNA–encoded respiratory chain genes and a unique regulation of ECM-related genes in nonarticular chondrocytes. These changes were associated with alterations in ECM composition, a shift in collagen/noncollagen protein content, and an increase of collagen crosslinking and ECM stiffness. These results demonstrate that mitochondrial respiratory chain dysfunction is a key factor that can promote ECM integrity and mechanostability in cartilage and presumably also in many other tissues. A large amount of energy is required to drive skeletal growth and synthesize the structural components of the cartilage extracellular matrix (ECM) that withstand mechanical forces and maintain lifelong mobility. The mitochondrial respiratory chain (mtRC) is the major source of cellular energy, but the connection between respiratory activity and ECM homeostasis is still poorly understood (1Romani P. Valcarcel-Jimenez L. Frezza C. Dupont S. Crosstalk between mechanotransduction and metabolism.Nat. Rev. Mol. Cell Biol. 2021; 22: 22-38Crossref PubMed Scopus (51) Google Scholar). We recently induced the expression of the mitochondrial (mt) DNA Twinkle (TW) helicase mutant K320E (2Weiland D. Brachvogel B. Hornig-Do H.T. Neuhaus J.F.G. Holzer T. Tobin D.J. Niessen C.M. Wiesner R.J. Baris O.R. Imbalance of mitochondrial respiratory chain complexes in the epidermis induces severe skin inflammation.J. Invest. Dermatol. 2018; 138: 132-140Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 3Baris O.R. Ederer S. Neuhaus J.F. von Kleist-Retzow J.C. Wunderlich C.M. Pal M. Wunderlich F.T. Peeva V. Zsurka G. Kunz W.S. Hickethier T. Bunck A.C. Stockigt F. Schrickel J.W. Wiesner R.J. Mosaic deficiency in mitochondrial oxidative metabolism promotes cardiac arrhythmia during aging.Cell Metab. 2015; 21: 667-677Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) specifically in growth plate cartilage by Col2a1-directed expression of Cre recombinase (Cre; (4Ovchinnikov D.A. Deng J.M. Ogunrinu G. Behringer R.R. Col2a1-directed expression of Cre recombinase in differentiating chondrocytes in transgenic mice.Genesis. 2000; 26: 145-146Crossref PubMed Scopus (294) Google Scholar, 5Bluhm B. Ehlen H.W.A. Holzer T. Georgieva V.S. Heilig J. Pitzler L. Etich J. Bortecen T. Frie C. Probst K. Niehoff A. Belluoccio D. Van den Bergen J. Brachvogel B. miR-322 stabilizes MEK1 expression to inhibit RAF/MEK/ERK pathway activation in cartilage.Development. 2017; 144: 3562-3577Crossref PubMed Scopus (20) Google Scholar)) to generate mice with impaired mtRC function in chondrocytes (CreTW) (Fig. 1A). These mice developed postnatal growth retardation and a chondrodysplasia-like phenotype caused by disturbed metabolic signaling and destabilization of the cartilage-to-bone junction during postnatal development (6Holzer T. Probst K. Etich J. Auler M. Georgieva V.S. Bluhm B. Frie C. Heilig J. Niehoff A. Nuchel J. Plomann M. Seeger J.M. Kashkar H. Baris O.R. Wiesner R.J. et al.Respiratory chain inactivation links cartilage-mediated growth retardation to mitochondrial diseases.J. Cell Biol. 2019; 218: 1853-1870Crossref PubMed Scopus (1) Google Scholar). Bulk RNA transcriptome profiling showed that lack of respiration was translated into a general integrated stress response, but the individual load of mtRC dysfunction in chondrocyte subpopulations and the impact on ECM homeostasis remained unclear. Single-cell RNA-Seq (scRNA-Seq) has evolved as a method to provide a high-resolution map of cellular transcriptomes with the promise of improving the understanding of individual cell function and dysfunction (7Eberwine J. Sul J.Y. Bartfai T. Kim J. The promise of single-cell sequencing.Nat. Methods. 2014; 11: 25-27Crossref PubMed Scopus (160) Google Scholar). In joint biology, scRNA-Seq was used to study cellular heterogeneity and lineage specification during development. Tissue-specific cell clusters of hind limb development were identified, distinct transcriptome signatures of joint progenitor cells were described (8Kelly N.H. Huynh N.P.T. Guilak F. Single cell RNA-sequencing reveals cellular heterogeneity and trajectories of lineage specification during murine embryonic limb development.Matrix Biol. 2020; 89: 1-10Crossref PubMed Scopus (14) Google Scholar, 9Feng C. Chan W.C.W. Lam Y. Wang X. Chen P. Niu B. Ng V.C.W. Yeo J.C. Stricker S. Cheah K.S.E. Koch M. S. Ng Chan D. and progenitor cells in the lineage Cell 2019; Full Text Full Text PDF PubMed Scopus Google and of the major for joint were Kim Wang H. S. S. P. A cell of joint 2020; PubMed Scopus Google Scholar). scRNA-Seq analysis also the expression of genes in growth plate cartilage J. H. Wang J. S. J. K. Y. H. Y. G. of development single-cell Full Text Full Text PDF PubMed Scopus Google Scholar) by profiling D. Etich J. S. Frie C. J. Ehlen H. S. F. von K. J.F. Brachvogel B. of growth plate chondrocytes the and of cells of a PubMed Scopus Google Scholar). This to ECM-related genetic in chondrocyte subpopulations with impaired mtRC but to scRNA-Seq was applied to high-resolution transcriptome analysis of mutant mice with a cartilage The of study was to the individual cellular load of mtRC dysfunction and the impact on ECM homeostasis and cartilage a combined approach of high-resolution scRNA-Seq, mass spectrometry/matrisome analysis, and atomic force microscopy in mutant mice with cartilage-specific inactivation of mtRC function. The mouse is a unique source of cartilage to cell for scRNA-Seq We to mutant mice with increased mtRC deficiency and mtRC activity and cartilage in the of 1-month-old Cre and activity activity in and growth plate cartilage of 1-month-old Cre with the the junction to the In of activity was in the and growth as by (Fig. These changes in mice were associated with the expansion of the central area of the cartilage and disturbed chondrocyte as by (Fig. The results that distinct chondrocyte subpopulations a of mtRC to changes in cartilage of scRNA-Seq analysis was applied to changes in individual chondrocyte subpopulations of mice with the load of mtRC cartilage of 1-month-old Cre and mice were by and DNA and the were and expression was used to and expression (Fig. of cells were and a of genes cell was identified in Cre and (Fig. unique were for the of and cell clusters were identified by of the transcriptome (Fig. and expression analysis was used to cell genes and to individual cell clusters (Fig. of cells and and of cells to the pathway were but the of cells were chondrocytes. we identified of and chondrocytes as as a unique of chondrocytes (Fig. the of cells in unique cartilage was increased in the of cells in the and was (Fig. mtRC deficiency the cellular of cartilage in of cell expression of cell genes was used to individual cell expression of individual cells the was identified, and subpopulations of and unique chondrocytes with and cells were the cell the of Cre and mice in unique expression analysis of the chondrocyte subpopulations was to genes that chondrocyte of mice in to impaired mtRC dysfunction of cells analysis genes between Cre and genes were in chondrocytes (Fig. of the genes were used to expression between chondrocyte subpopulations in Cre and chain chain and were in chondrocyte subpopulations Cre of these genes was in chondrocytes of and the was in the and unique subpopulations (Fig. and genes were in nonarticular chondrocytes but in chondrocytes. genes to the ECM were identified (Fig. A and and and were in The results that mtRC dysfunction the expression of and ECM-related genes in nonarticular chondrocyte the of expression for ECM and deposition in cartilage, we protein and of and of and were increased in of cartilage with as by analysis (Fig. In microscopy showed that was in the and growth plate cartilage of Cre and mice (Fig. and in the central area of the cartilage in A of extracellular was the of the growth plate cartilage in Cre and area was into the central area of mice (Fig. and collagen and ECM C. Niehoff A. D. L. M. A. collagen in cartilage of Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, S. J.C. promotes matrix homeostasis by with collagen and and collagen 2018; PubMed Scopus Google and a increased of and into the ECM of mice the cartilage ECM the for ECM composition, we the of cartilage 1-month-old Cre and mice V.S. Etich J. Bluhm B. M. Frie C. F. J. Brachvogel B. of the has on extracellular matrix protein in J. Mol. 2020; 21: Scopus Google Scholar). In were between cartilage Cre and mice (Fig. A and analysis showed that and the and were was the the (Fig. that ECM alterations a of mtRC deficiency in cartilage. we the changes in ECM in by analysis to the V. Etich J. Pitzler L. Frie C. Koch M. M. G. H. Brachvogel B. of a expression in skin Biol. 2018; PubMed Scopus Google Scholar, J. Koch M. F. M. Brachvogel B. expression profiling of the extracellular matrix in of activation for skin J. Mol. 2019; Scopus Google Scholar). the ECM-related were associated to the and to the (Fig. and the were in cartilage of mice but (Fig. the collagen and were but in analysis, we of collagen and increased of by analysis (Fig. In with the the of the protein and the protein was In were identified, and analysis showed that these in ECM collagen and crosslinking of collagen (Fig. and a distinct protein in collagen and we collagen that in mature cartilage in collagen PubMed Scopus Google Scholar) and the of the and Here, we a increase in the mature in cartilage of mice with but of the (Fig. The increase in was associated with a decrease in collagen and an increase in (Fig. The results that crosslinking in collagen is increased in mtRC cartilage, the and mechanostability of the ECM in cartilage. in of the ECM were by of the of the were in the and of the growth The in the the and the (Fig. The ECM of 1-month-old cartilage mice showed a general to increase in of the collagen in the was in the nonarticular and of growth plate cartilage. was in the cartilage and the distinct for Cre and Here, the in mice was by in mice with (Fig. ECM and processes L. C. T. M. T. H. J. K. to and function of Mol. 2020; PubMed Scopus Google Scholar, A. of on the structural and of collagen for 2015; PubMed Scopus Google and we that the ECM in mice a of into collagen and We used microscopy to study the in the of cartilage. were in the ECM of chondrocytes in Cre and mice (Fig. in Cre the chondrocytes were by and these were in In mtRC dysfunction in cartilage induces a in growth plate cartilage that the between and ECM crosslinking and and in the The mtRC is a major for postnatal cartilage growth and but the of mtRC for ECM homeostasis in mature cartilage has Here, we by scRNA-Seq analysis that impaired mtRC to a cell and an mechanostability in cartilage. The of the transcriptome the single-cell in a cell is but was for the joint and growth plate cartilage (8Kelly N.H. Huynh N.P.T. Guilak F. Single cell RNA-sequencing reveals cellular heterogeneity and trajectories of lineage specification during murine embryonic limb development.Matrix Biol. 2020; 89: 1-10Crossref PubMed Scopus (14) Google Scholar, 9Feng C. Chan W.C.W. Lam Y. Wang X. Chen P. Niu B. Ng V.C.W. Yeo J.C. Stricker S. Cheah K.S.E. Koch M. S. Ng Chan D. and progenitor cells in the lineage Cell 2019; Full Text Full Text PDF PubMed Scopus Google Scholar, Kim Wang H. S. S. P. A cell of joint 2020; PubMed Scopus Google Scholar). Here, we applied scRNA-Seq analysis to mature cartilage 1-month-old helicase K320E transgenic cell were and cell were identified in the cartilage of Cre that the has on the of the of and cartilage the a of cells to the cartilage was were the cell that cartilage-specific ECM for and This to the described to collagen and to in tissue and M. P. J. K. H. D. J. S. of collagen by A in PubMed Scopus Google Scholar). the chondrocyte and subpopulations identified by expression The of joint Biol. 2014; PubMed Scopus Google Scholar) and Belluoccio D. L. J.F. protein is in and cartilage and in Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) of cartilage, and were in cartilage, but expression is to a of This the in expression of and in cartilage, is in the of cartilage P. D. A cartilage protein in the of cartilage with Biol. Full Text Full Text PDF PubMed Scopus Google is in the and of cartilage The of joint Biol. 2014; PubMed Scopus Google Scholar, Y. M. P. M. Cell and of cartilage and to in mouse Biol. 2017; PubMed Scopus Google Scholar). is by chondrocytes D. Etich J. S. Frie C. J. Ehlen H. S. F. von K. J.F. Brachvogel B. of growth plate chondrocytes the and of cells of a PubMed Scopus Google Scholar) and was used to the the scRNA-Seq a unique of nonarticular was and to the and disorganized central of in mice in was in a of chondrocytes a to mature growth plate J. Brachvogel B. M. F. Niehoff A. deficiency to and of murine an of and 2020; Full Text Full Text PDF PubMed Scopus Google presumably cells of the and was also by scRNA-Seq The results that the is in to mtRC dysfunction with clusters of cells and in cartilage. These results the area in the was (6Holzer T. Probst K. Etich J. Auler M. Georgieva V.S. Bluhm B. Frie C. Heilig J. Niehoff A. Nuchel J. Plomann M. Seeger J.M. Kashkar H. Baris O.R. Wiesner R.J. et al.Respiratory chain inactivation links cartilage-mediated growth retardation to mitochondrial diseases.J. Cell Biol. 2019; 218: 1853-1870Crossref PubMed Scopus (1) Google Scholar). the and in development. In the in the joint with in the but with of the cartilage M. G. J.M. development of the and and in PubMed Scopus Google Scholar). In a in the of the and can provide and to of the growth cells in the of the in a unique to mtRC dysfunction with the joint scRNA-Seq analysis is to these cells and unique response, and we an increase in cell of the unique in This that scRNA-Seq analysis also on the cellular of tissue to scRNA-Seq analysis provide as a combined to individual transcriptomes and cell to study and changes in We a of of mtRC complexes in mice with the decrease in expression was in and unique chondrocyte to in of expression between chondrocyte subpopulations in K320E expression is by the (4Ovchinnikov D.A. Deng J.M. Ogunrinu G. Behringer R.R. Col2a1-directed expression of Cre recombinase in differentiating chondrocytes in transgenic mice.Genesis. 2000; 26: 145-146Crossref PubMed Scopus (294) Google and of the collagen were in nonarticular cartilage. K320E expression in nonarticular cartilage, to a and impaired mtRC expression with cartilage. In cell (2Weiland D. Brachvogel B. Hornig-Do H.T. Neuhaus J.F.G. Holzer T. Tobin D.J. Niessen C.M. Wiesner R.J. Baris O.R. Imbalance of mitochondrial respiratory chain complexes in the epidermis induces severe skin inflammation.J. Invest. Dermatol. 2018; 138: 132-140Abstract Full Text Full Text PDF PubMed Scopus (13) Google but chondrocytes by cell expansion and cell Y. M. P. M. Cell and of cartilage and to in mouse Biol. 2017; PubMed Scopus Google Scholar). a in nonarticular This is by that mtRC inhibition was in nonarticular cartilage with cartilage, and nonarticular subpopulations in The of study that the severe in cartilage of genes were This is a of the scRNA-Seq analysis with transcriptome amounts of but cell and of RNA in a cell to RNA-Seq T. J.C. in single-cell 2015; PubMed Scopus Google Scholar). The of genes can also and cells We a of genes cell by scRNA-Seq of cartilage. scRNA-Seq analysis has a of with RNA scRNA-Seq unique on expression study that analysis of expression with the load of mtRC in chondrocyte subpopulations and with in changes in cartilage. scRNA-Seq analysis can the and of mtRC deficiency in cartilage and presumably also in other tissues. scRNA-Seq analysis, we identified the ECM as a central of the to mtRC deficiency in nonarticular cartilage of Here, scRNA-Seq analysis a cellular to individual cellular but a combined approach by and mass spectrometry/matrisome analysis was to and the results of the scRNA-Seq mass analysis on that in ECM collagen and crosslinking of collagen but by scRNA-Seq We of to provide a on ECM changes in to mtRC and in scRNA-Seq and mass analysis and to the mitochondrial is to induced by PubMed Scopus Google Scholar) and presumably mitochondrial dysfunction S. M. D. S. G. 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G. understanding of the Biol. 2014; PubMed Scopus Google Scholar). The was to chondrocyte and in cartilage K. P. Koch M. C. von K. B. J. in cartilage tissue induced by A. PubMed Scopus Google Scholar). and increased deposition in the ECM of mice provide to and of cartilage in mtRC deposition is associated with the expansion of the chondrocytes in central of in mice and the increase in and of the ECM also a of changes in the of chondrocyte of ECM also to ECM in to mtRC dysfunction in cartilage. ECM changes by chondrocytes to cell and and we the of cell cells and cells in cartilage of 1-month-old Cre and Here, we increased of chondrocytes the that to (Fig. These in with results that cell is induced the junction of the but changes in cell were in the area of cartilage. increased of changes in cell of chondrocytes. In and described to cell and Chen J. 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Cell Biol. 2021; 22: 22-38Crossref PubMed Scopus (51) Google and we a combined approach of high-resolution scRNA-Seq, mass and analysis to that ECM homeostasis in mature growth plate cartilage We demonstrate that mtRC induces an to increased collagen crosslinking and matrix stiffness. scRNA-Seq identified mtRC deficiency as a major for impaired ECM integrity and mechanostability in cartilage in mice were with mice to generate mice (CreTW) mutant helicase in cartilage and mice B. Ehlen H.W.A. Holzer T. Georgieva V.S. Heilig J. Pitzler L. Etich J. Bortecen T. Frie C. Probst K. Niehoff A. Belluoccio D. Van den Bergen J. 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