DiaPASEF proteotype analysis indicates changes in cell growth and metabolic switch induced by caspase‐9 inhibition in chondrogenic cells

Caspase‐9 is the major apical caspase responsible for triggering the intrinsic apoptotic pathway. Our previous study indicated that specific inhibition of caspase‐9 caused microscopically evident alterations in appearance of the primary chondrogenic cultures which cannot be explained by decrease in apoptosis. To describe a complex molecular background of this effect, proteomics analysis of control and caspase‐9 inhibitor‐treated chondrogenic cultures were performed. Proteins were extracted, identified and quantified using LC‐MS in both data dependent and data independent acquisition (DIA) mode. While directDIA analysis of diaPASEF data obtained using timsTOF Pro LC‐MS system revealed 7849 protein groups (Q‐value <0.01), a parallel analysis of iTRAQ‐2DLC‐MS3 and conventional DIA‐MS data identified only 5146 and 4098 protein groups, respectively, showing diaPASEF a superior method for the study. The detailed analysis of diaPASEF data disclosed 236/551 significantly down‐/up‐regulated protein groups after caspase‐9 inhibition, respectively (|log2FC|>0.58, Q value <0.05). Classification of downregulated proteins revealed changes in extracellular matrix organization, collagen metabolism, and muscle system processes. Moreover, deregulations suggest a switch from glycolytic to lipid based metabolism in the inhibited cells. No essential changes were found in the proteins involved in apoptosis. The data indicate new non‐apoptotic participation of caspases in chondrocyte homeostasis with potential applications in cartilage pathophysiology.

Caspases are produced within cells as inactive zymogens and become activated upon extrinsic (receptor mediated) or intrinsic (mitochondria) triggering. Due to this fact, modulations of caspase cascade are performed at the protein level. The most common approach is application of inhibitors such as fluoromethylketone (FMK) [16]. These synthetic inhibitors bind to the active site of caspase and irreversibly block its activity, but do not affect the presence or detectability of caspase protein. So far, general caspase FMK inhibition in chondrogenic micromass cultures pointed to engagement of caspases in osteogenic [14,17] and autophagic pathways [18]. Recently, general caspase inhibition in chondrocytic cultures led to modified expression of several osteoarthritis related genes [19]. In order to reveal impact of individual caspases on the expression profile in chondrocytes, specific inhibitors may be applied.
Caspase-9 is the major apical caspase of the intrinsic pathway initiated mostly from mitochondria. Activated caspase-9 was present in growth plate chondrocytes [14] and was also detected during chondrogenesis in vitro [20], where its inhibition affected gene expression of chondrogenesis-related genes.
In our investigation, specific inhibition of caspase-9 caused morphological changes in chondrogenic micromass cultures which could not be simply explained by modulation of apoptosis. To search for possible background of such changes, proteomics analysis using the DIA -Parallel Accumulation Serial Fragmentation (diaPASEF) approach on timsTOF Pro was designed to compare the profile subjected to FMK-caspase-9 inhibitor treatment and control cultures. The timsTOF Pro employs the Trapped Ion Mobility Spectrometer (TIMS) analyzer that allows separation of eluting precursor ions based on the ion mobility. This additional separation dimension decreases the complexity of MS/MS spectra, resulting in increased sensitivity of peptide identification [21]. The combination of PASEF with DIA mode further improves the quantification capabilities of the method as almost complete sampling of the precursor ions is achieved [21]. We moreover compared the diaPASEF method to widely used data-dependent acquisition (DDA) method with iTRAQ-8plex labeling together with conventional DIA method, both executed on orbitrap Lumos, and we demonstrate superiority of the diaPASEF in number of quantified peptides and protein groups. Using this approach, we uncovered changes of proteins involved in extracellular matrix organization, muscle system

Statement of significance of the study
Emerging non-apoptotic functions of caspases are one of the hot topics because of their significance in several pathologies. Such novel aspects have been recently reported in the bone and cartilage, for example, with respect to osteoarthritis. The role of individual caspases, however, remains to be specified. Inhibition of caspase-9, a key apical caspase, in chondrogenic cultures resulted in clear morphological alterations which cannot be explained by modification of apoptosis. To clarify the molecular mechanisms associated with caspase-9 inhibition, we applied three LC-MS/MS-based proteomics strategies, including diaPASEF, iTRAQ-2DLC-MS3, and conventional DIA, and demonstrated superior performance of the diaPASEF approach allowing deep quantification of the proteome.
Our results provide a complex insight into the background of the effect of capsase-9 inhibition and point to significant changes in extracellular matrix organization, particularly collagen metabolic processes. Additionally, a switch from glycolytic to lipid metabolism in chondrocytes treated by caspase-9 inhibitor was indicated. Caspase inhibitors and collagen-based treatment are considered in anti-osteoarthritic therapies and metabolic pathways in osteoarthritis become also intensely investigated. processes and in glycolytic and lipid metabolism after the caspase-9 inhibition.

Statistical analysis
The Pearson correlation analysis and visualization of the correlation plots and was performed using GraphPad Prism (version 9.4.1).

Gene set enrichment analysis
Gene set enrichment analysis (GSEA) in GSEA Java desktop application [23] version 4.1.0 was conducted using the list of all quantified proteins pre-ranked according to the negative log2 of the q value and the sign of the log2 fold change to identify enriched pathways, with a priori defined pathways from the GSKB mouse database [24]. Minimal size of a gene set was adjusted to 2, otherwise default settings were used.
The GSEA analysis was performed also using the WEB-based GEne SeT AnaLysis Toolkit (WebGestalt, http://www.webgestalt.org/) [25] Organism of interest was set to Mus musculus and Method of interest to GSEA. Analysis was performed against Gene Ontology Biological Process (GOBP) database with minimum number of genes for a category set to 3 and with FDR significance level 0.05.

Enrichment analysis of molecular pathways
UniProt ID lists of proteins that were either statistically (q value

Caspase-9 activity assay
Caspase-9 activity was measured by luminescence using Caspase-Glo® 9 Assay (Promega). Cell suspension (approximately 200,000 cells) was mixed with 100 μL Caspase-Glo 9 Reagent containing Caspase-Glo 9 Substrate. Cells were incubated for 2 h in the dark at 37 • C. For each sample (n = 6), caspase-9 activity was measured in parallel in the control group and the group to which the caspase-9 inhibitor was added. Luminescence was measured by Synergy HT Microplate Reader (BioTek). The statistical significance of the difference was estimated by paired t-test.

Staining of micromass cultures and proliferation detection
Micromass cultures grown on culture glass were fixed by 4% PFA.
Alcian blue and eosin staining was used for visualisation of micromass structure and differentitation.

3.2
Inhibition of caspase-9 affects structure of micromass cultures

Caspase-9 inhibition affects protein abundance in micromass cultures
To identify proteins that could be associated with caspase-9 inhibition  Table S3). Further, we re-analyzed the   Figure 2A,B). However, 2494 protein groups and 52,301 peptides were quantified exclusively by the diaPASEF method (Figure 2A,B).
Comparison of quantities of protein groups specific for diaPASEF dataset and protein groups quantified also by iTRAQ 2DLC-MS3 and conventional DIA clearly show the ability of diaPASEF to analyze low abundant proteins elusive for the other two methods, due to the increased sensitivity by ion mobility spectrometry ( Figure 2C). We next assessed the level of correlation between quantification by diaPASEF, Our results clearly indicate that diaPASEF method offers a superior proteome coverage compared to the iTRAQ and conventional DIA based approaches using the Orbitrap instrument. Moreover, the caspase-9 protein, whose inhibition is object of this study, was quantified only by the diaPASEF method and is missing in the iTRAQ-2DLC-MS3 and conventional DIA datasets. Thus, we proceeded with diaPASEF results for further evaluating the caspase-9 inhibition in micromass cells.
Although protein levels of caspase-9 were not influenced by the inhibitor (log2FC = 0.23, q value = 0.046) according to our criteria, effects of the caspase-9 inhibitor treatment of the cells were observed through significant (q value <0.05) upregulation (log2FC >0.58) and downregulation (log2 FC <-0.58) of 551 and 236 proteins, respectively.
The heatmap ( Figure 3A) visualizes precise clustering of individual biological replicates according to the caspase-9 inhibition condition.
These proteins could be negatively associated with caspase-9 functions. The most significantly downregulated proteins after caspase-9 inhibition represent proteins Acp5, Hbb-y, Ca9, Trim72, Atp6v0d2, Tnnc1, Mmp9, Tnni1, Tnnt1, and Myh7 (Table 1). These results represent a panel of proteins that could be associated with caspase-9 activity. All significantly downregulated and upregulated proteins are listed in Table S5.
On the other hand, from the 7 caspases quantified in our experiment, only caspase-12 showed decreased protein levels, abundances of other caspases were not affected by the caspase-9 inhibition (Table 2).
Thus, we did not identify any compensatory mechanism based on elevated expression of other caspases after inhibition of caspase-9 activity in the micromass cells.

Caspase-9 inhibition significantly affects biological pathways in primary chondrogenic cultures
To cover the biological pathways affected by caspase-9 inhibition, identified proteins were used to perform several in silico enrichment and  (Table S6).
For Reactome analysis, the cut-off used for the comparison was q value <0.05 and (|log2FC|) >0.58. Networks displaying impacted pathways are shown in Figure 4A,B. In the case of upregulated proteins after caspase-9 inhibition, the affected reactome pathways included platelet degranulation, neutrophil degranulation or formation of cornified envelope ( Figure 4A). The impact on the extracellular matrix organization, gluconeogenesis, and muscle contraction was apparent in downregulated proteins ( Figure 4B).

DISCUSSION
Increasing body of recent studies provides evidence about yet unexpected contribution of the apoptotic caspases to proliferation and differentiation of cells [15,33]. Recently, caspases were even assigned as a key switch between life and death [34].
Micromass cultures prepared from limb buds (autopodium) allow for work with population of chondrogenic progenitor cells and represent a traditional and well established model to investigate endochondral ossification in vitro resembling cell interactions in vivo [35].
As such, the micromass complex includes not only chondrogenic precursors, chondroblasts and chondrocytes, but also of fibroblasts, tenoblasts and myoblasts organized in concentric nodular structure upon cultivation [32]. Specific FMK-based caspase-9 inhibitor can effectively reduce caspase-9 activity in this type of cells, as confirmed by luminescence results.
The presented proteomics analysis was designed to search for the molecular background of changed formation within micromass cultures after caspase-9 inhibition which cannot be explained by decreased apoptosis as would be expected based on the traditional pro-apoptotic function of caspase-9. We applied a recent timsTOF technology [36]. offering increased sensitivity based on the implemented ion mobility separation dimension. We performed the analyzes in diaPASEF mode combining high reproducibility of DIA and sensitivity of TIMS [21]. The additional TIMS precursor separation dimension in the diaPASEF surpassed the iTRAQ-MS3 based and conventional DIA strategies on Orbitrap Lumos in the proteome coverage in our study.
To our knowledge, this is one of the first applications of diaPASEF in a comparative biological experiment.
The proteomics analysis revealed molecules with multiple roles which may be related to the presence of several cell types in micromass culture. Caspase-9 inhibition significantly affected proteins associ-F I G U R E 4 Pathways significantly enriched with up-regulated and down-regulated protein groups in micromass cells after the caspase-9 inhibitor treatment. Significantly enriched Reactome pathways [28] (p < 0.01) and related significantly up-regulated protein groups (q value <0.05, log2FC >0.58) in micromass cells after 6 days of caspase-9 inhibition (A). Significantly enriched Reactome pathways [28] (p < 0.01) and related significantly down-regulated protein groups (q value <0.05, log2FC<-0.58) in micromass cells after 6 days of caspase-9 inhibition (B).
ated with cell proliferation, tumorigenic processes, and metabolism.
Altered cell proliferation after caspase-9 inhibition was subsequently confirmed by BrdU incorporation assay.
In vivo, caspase-9 is required for proper prenatal development, mainly due to its apoptotic functions [37,38]. Caspase-9 deficiency is perinatally lethal and includes brain malformations caused by insufficient apoptotic removal. Caspase-9 inhibited micromass cultures showed significant phenotypical changes even after three days of treatment and more pronounced after 6 days. Inhibition strongly affected outer parts of micromass spots whereas chondrogenic dif-ferentiation in central parts was affected less. Based on these observations, several possible explanations of caspase-9 effect are offered and supported by proteomics. Caspase-9 thus can play a role in proliferation, metabolism or migration of low differentiated micromass cells.
Interestingly, the most downregulated protein was tartrateresistant acid phosphatase type 5 (PPA5). This marker of bone resorption is highly expressed in osteoclasts and chondroclasts, but PPA5 activity was also found in osteoathritic chondrocytes and as such, it was proposed as a diagnostic factor [39,40]. The level of PPA5 was decreased after general caspase inhibition [39], in agreement with our findings which further specify the impact of caspase-9 selective inhibition.
Notably, MMP9 was found among the top ten downregulated proteins. Regulation of Mmp9 gene expression by caspase-9 was recently described [20] [41]. Troponin displayed downregulation at the level of all 3 components (troponin C, T, I) after caspase-9 inhibition. Troponin-T was shown to cause high levels of apoptosis in aging cells [42] and thus could be associated with the apoptotic function of caspase-9. Troponin I was reported to inhibit angiogenesis in cartilage [43] and is also able to reduce proliferation in cancer cells [44]. Notably, troponin belongs to factors being discussed in emerging roles beyond the canonical ones [45]. These findings are likely to be associated with myoblastic cells present within the micromass cultures [32] which are affected by the caspase-9 inhibition. The changes are also in agreement with previous reports showing caspase-9 to affect muscle differentiation [46].
Additionally, caspase-9 inhibited cultures showed significant decrease in many other muscle-associated proteins, for example Actin alpha, Alpha actinin-2, Calponin, or Desmin. Desmin prolonged proliferation during myogenesis in myoblasts [47] and calponin in osteoblast lineage cells lead to decreased proliferation and bone mass [48]. Myoblast differentiation is also regulated by palmdelphin [49], which was decreased after caspase-9 inhibition. Alpha actinin proteins bind actin filaments and associate with a number of cytoskeletal and signaling molecules [50]. Deregulation of cytoskeleton thus should be one mechanism of caspase-9 inhibition effect in treated cultures.
Among the top 30 upregulated proteins in caspase-9 inhibited group, several were associated with osteogenesis. One of them was osteoactivin (Gpnmb). Osteoactivin is important osteogenic regulator involved in differentiation of osteoblast and mineralization, but is highly expressed also in hypertrophic chondrocytes of growth plate [51,52]. Others included, for example, Phex or Scarb1. Scarb1 is important for osteoblastic differentiation as shown in deficient animals [53].
Phex in abundantly expressed by osteoblasts and is involved in mineralization [54]. Recently, changes of Phex expression on RNA level were detected after caspases inhibition in osteoblastic cells [55]. Caspase-9 was previously shown to be a molecule with osteogenic potential since its inhibition caused altered expression of genes which may impact chondrogenic differentiation (Bmp4, Bmp7, Sp7, Gli1), mineral deposition (Alp, Itgam) or the remodeling of the extracellular matrix (Col1a2, Mmp9) [20].
Along with alterations in the osteogenic factors, a switch from glycolytic to lipid based metabolism was suggested by the GSEA. In cartilage, changes of lipid metabolism can lead to sever disorders such as osteoarthritis [56]. The impact of caspases in metabolism captures increasing interest in possible therapeutic applications since they apparently play roles in the pathogenesis of metabolic diseases such as obesity, the nonalcoholic steatohepatisis (NASH) and even more severe liver diseases [57]. Application of caspase inhibitors is thus a challenging approach [58]. However, one of the remaining open questions is which specific caspases have non-apoptotic roles in metabolism and related disorders [57]. The presented proteomics results indicate caspase-9 as yet another caspase participating in metabolism regulations, at least in association with chondrogenesis and endochondral osteogenesis. Despite caspase-1 and caspase-8 are of major interest in recent metabolic research, the irreversible selective inhibitor including caspase-9 (GS-9450) entered the phase-II clinical trial in patients with NASH [59]. Regional differences in chondrocyte metabolism in osteoarthritis were reported earlier [60] and caspase-9 could be one of the contributing factors.
In conclusion, inhibition of caspase-9 caused altered structural appearance of the chondrogenic micromass cultures. The proteomics analysis revealed that the changes are likely to be associated particularly with non-canonical roles of caspase-9. These novel functions included impact on proteins participating in metabolism which would thus support the novel insight into caspases as a switch between life and death [34]. Additionally, inhibition of caspase-9 modified levels of proteins having roles in cell collagen metabolic processes which would further explain the micromass morphology after caspase-9 inhibition.