Mechanism Of Mitochondrial IMS Protease HtrA2 In Regulating Myogenic Differentiation Of C2C12 Myoblast

Ageing,external injury and malignant tumours can cause muscle atrophy,which in turn leads to the development of muscle degenerative pathologies such as sarcopenia.Factors such as imbalance in protein homeostasis,inflammatory response and mitochondrial dysfunction are thought to be involved in the development of skeletal muscle degeneration.Myogenic differentiation is a prerequisite for maintaining muscle mass and function,and involves a series of differentiation steps from muscle satellite cells to myoblast to myotubes.A key step in this process is myogenic differentiation,during which myosin is abundantly expressed to support muscle contractile function,while mitochondrial oxidative phosphorylation（OXPHOS）-related gene expression is upregulated to accommodate the energy demands of myofibers,suggesting that myogenic differentiation requires the coordination of proteostasis and mitochondrial biosynthesis.Therefore,exploring the role of mitochondria in myogenic differentiation from the perspective of proteostasis may provide new clues for the prevention and treatment strategies of skeletal muscle degeneration.The mitochondrial intermembrane space（IMS）is an important buffer region between the cytoplasm and the mitochondrial matrix,and an important site for the assembly of the mitochondrial protein transport and electron transport chain（ETC）complex,and the maintenance of IMS proteostasis is essential for mitochondrial function.High temperature requirement factor A2（HtrA2）,a serine protease localized to the IMS,was previously thought to have a role in regulating apoptosis,but recent studies have revealed that HtrA2 can regulate mitochondrial proteostasis through enzymatic activity and molecular chaperone function.In our team’s study of motor neuron degeneration 2（mnd2）mice with a missense mutation at HtrA2-Ser276Cys,we found that the reduction of muscle mass and hypofunction,suggested that it was related to mitochondrial dysfunction caused by mito-nuclear protein imbalance,suggesting that HtrA2 could be a key factor to study mitochondrial protein homeostasis and myoblast differentiation.However,the mnd2 mice as an in vivo model is difficult to achieve for the study of myogenic differentiation.Therefore,we used C2C12 myoblasts,which have the potential to differentiate into myotubes,and UCF 101,a specific inhibitor of HtrA2 protease,in combination with RNAi biotechnologies to investigate the role of mitochondrial protein homeostasis in myogenic differentiation.Mitochondrial Unfold Protein Response(UPR^mt)plays a role in coordinating protein homeostasis and mitochondrial biosynthesis as a mode of nuclear-mitochondrial interaction regulation.It has been suggested that the UPR^mttranscription factors ATF5,CHOP,and p-e IF2αrestore mitochondrial function through selective expression of nuclear and mitochondrial genes.However,recent studies have found that sustained activation of UPR^mt leads to an increase in mis-mutated mt DNA and mitochondrial dysfunction.Some studies have found that UPR^mtpromotes hepatic stem cells differentiation and inhibits neuronal differentiation,suggesting that the effect of UPR^mt on cell differentiation may depend on restoration of proteostasis.Cells undergo continuous changes in their proteome during differentiation,therefore exploring the relationship between mitochondrial proteostasis and biosynthesis during adult myoblast differentiation from the perspective of UPR^mt can further elucidate the mechanisms underlying the role of nuclear-mitochondrial interaction regulation in muscle degenerative diseases.In this study,we propose to use the mitochondrial membrane gap protease HtrA2as an entry point,and use bioinformatics techniques to screen the signalling pathways and molecular targets related to HtrA2 in the differentiation of adult myoblasts,in combination with C2C12 cell differentiation experiments to further explore the role of UPR^mt induced by proteostasis imbalance in influencing the metabolic shift in the differentiation of adult myoblasts.The role of mitochondria in myoblast differentiation was further investigated in the context of the C2C12 cell differentiation assay.MethodsⅠ.Explore the relationship between HtrA2 and myogenic differentiation1.To investigate the relationship between HtrA2 enzyme activity deficiency and muscle atrophy,this study assessed the locomotor function of mnd2 mice by suspension assay,grip strength test and other assays,assessed the morphology of muscle fibres by HE staining of skeletal muscle fibres,and examined the transcript levels of myosin heavy chain gene Myh1/2/4/7 by RT-q PCR assay,and the expression of mitochondrial membrane gap protease and myogenic differentiation marker protein by Western Blot to detect the expression of IMS protease and muscle differentiation marker proteins to evaluate the degenerative phenotype of skeletal muscle in mnd2mice.2,To investigate the possible mechanism of muscle atrophy in mice due to the absence of HtrA2 enzyme activity,GO enrichment analysis was performed on the m RNA sequencing dataset of mouse muscle degeneration to preliminarily explore the possible mechanisms by which HtrA2 affects muscle atrophy.3.To investigate whether the absence of HtrA2 enzyme activity affected myogenic differentiation,a C2C12 myogenic differentiation model was constructed using the HtrA2 inhibitor UCF 101（20μM）and RNAi technology.The effect of HtrA2 on differentiation of myogenic cells was investigated by light microscopic observation of cell morphology,immunofluorescence assessment of Fusion Index（FI）,and RT-q PCR and Western Blot techniques to detect the transcription and expression levels of Myosin heavy chain and Myogenin,the markers of differentiation.Ⅱ.Investigating the role of HtrA2 on energy metabolism in myogenic differentiation1.In order to explore the mechanism by which the absence of HtrA2 enzyme activity leads to impaired myogenic differentiation,KEGG pathway enrichment analysis was performed on a high-throughput dataset of myogenic differentiation m RNA.2.Based on the results obtained from the enrichment analysis,HtrA2 may be associated with oxidative phosphorylation function during myogenic differentiation,energy metabolism indicators such as ATP content,lactate secretion,glucose uptake and cellular oxygen consumption rate during C2C12 cell differentiation were examined to further investigate the role of mitochondrial oxidative phosphorylation function in myogenic differentiation.Ⅲ.Exploring the mechanism of HtrA2 affects myogenic differentiation1,To further investigate the specific mechanisms by which HtrA2 affects mitochondrial oxidative phosphorylation during differentiation of adult myoblasts,the expression of ETC subunits encoded by mt DNA and n DNA was examined,and the levels of mitochondrial biosynthesis and related signalling pathways were assessed by flow cytometry,RT-q PCR,and Western Blot techniques.2,To investigate whether HtrA2 is involved in the mechanism of nuclear-mitochondrial interaction regulation through activation of UPR^mt,the expression and transcript levels of UPR^mt-related proteins during C2C12 differentiation were examined by Western Blot and RT-q PCR assay.The String database was also used to construct a PPI protein interaction network to initially explore the possible mechanisms by which mitochondrial protein homeostasis affects mitochondrial biosynthesis through UPR^mt.ResultⅠ.Deficiency of HtrA2 enzyme activity leads to the impairement of myogenic differentiation1.The weight of gastrocnemius and gastrocnemius muscle in mnd2 pure mice were significantly lower than those in wild-type and heterozygous mice;the results of kinematic experiments showed that the grip strength of mnd2 pure mice was significantly reduced and the resting time of hanging tail was significantly prolonged;the results of morphological experiments showed that the cross-sectional HE stained sections of gastrocnemius muscle in mnd2 pure mice showed that the muscle fiber gap was enlarged and the muscle fibers tended to be more fragmented.The quantitative morphological analysis showed that the CSA of the gastrocnemius muscle was significantly reduced compared to that of wild-type and heterozygous mice;RT-q PCR results showed that the m RNA transcript level of Myh2,7 gene was significantly reduced in mnd2 pure gastrocnemius muscle,suggesting that the content of atrophic muscle fibres type I and IIA in mnd2 pure mice was reduced.These results suggest that mnd2 pure mice have muscle atrophy and reduced muscle function.2.GO enrichment analysis showed that the genes affected by HtrA2 in the degenerated muscle tissues were mainly located in the cytochrome complex and mitochondrial respiratory chain complex;in terms of biological processes,they may be related to the assembly of mitochondrial respiratory chain complex and tricarboxylic acid cycle,suggesting that in the pathogenesis of muscle atrophy The western blot of myogenin and myosin genes in gastrocnemius muscle of mnd2 pure mice showed that the expression levels of IMS proteases OMA1,YME1L1 and HtrA2were elevated.The expression levels of Myogenin and Myosin were significantly reduced,suggesting that the muscle degenerative lesions in mnd2 mice may be related to the impairement of muscle differentiation.3.C2C12 cell differentiation assay showed that UCF 101（20μM）and HtrA2-sh RNA treated C2C12 cells formed less myotubes and had shorter myotubes compared to the CON group under light microscopy;in the immunofluorescence assay,the myofusion index of C2C12 cells with HtrA2 enzyme activity deficiency was found to be significantly lower in D3 and D5 than that of the normal differentiation group.The transcription and expression levels of myosin and myogenin,the markers of differentiation in C2C12 cells with HtrA2 enzyme activity deficiency,were also significantly lower than those in the CON group at the same time,suggesting that HtrA2 enzyme activity deficiency inhibits differentiation of C2C12 myogenic cells.Ⅱ.HtrA2 enzyme activity deficiency inhibits oxidative phosphorylation during myogenic differentiation1.The results of KEGG pathway enrichment analysis showed that HtrA2 may be involved in oxidative phosphorylation function during myogenic cell differentiation.2.The results of the energy metabolism-related indexes showed that C2C12 cells in the UCF 101（20μM）treated group had higher lactate production in differentiation D3 and D5,while the ATP content and cellular oxygen consumption rate were lower than those in the CON group during the same period,while there was no significant difference in glucose uptake,suggesting that the deficiency of HtrA2 enzyme activity inhibited the shift of energy metabolism from glycolysis to oxidative phosphorylation.Ⅲ,Investigating the mechanism of HtrA2 affects myogenic differentiation1,Western Blot assay revealed that the expression levels of mt DNA-encoded ETC subunits ND1,CYTB and ATP6A1 were reduced during the differentiation of C2C12 cells in the UCF 101（20μM）treated group,while there was no significant change in n DNA-encoded subunits,suggesting the inhibition of mitochondria-encoded gene expression and the occurrence of nuclear-mitochondrial protein imbalance.Flow cytometry analysis of the number of mitochondria stained with Mito Tracker Green showed that the number of mitochondria in the UCF 101（20μM）treated group of C2C12 cells at differentiation D3 and D5 was significantly reduced compared to the control group during the same period.RT-q PCR analysis of the DNA copy numbers of mitochondrial genes NADH1,CYTB and COXII showed that the UCF 101（20μM）treatment group showed a decrease in the number of mitochondrial DNA copies in differentiated D3 and D5 mitochondria,suggesting that the level of mitochondrial biosynthesis was inhibited.In addition,Western Blot showed that the expression levels of VDAC1 and NRF2 were significantly reduced in the UCF 101（20μM）treated group,and the expression levels of mitochondrial transcription complex subunits POLRMT and TFAM were significantly reduced,suggesting that mitochondrial biosynthesis and gene transcription functions were impaired.2.Western Blot and RT-q PCR results showed that the expression levels of UPR^mt-related proteins LONP1,HSP10,HSP60,CHOP and ATF5 were significantly increased in the HtrA2 inhibitor and RNAi-treated groups during differentiation D1and D3,and the early phosphorylation level of e IF2αwas significantly up-regulated,suggesting that the deficiency of HtrA2 enzyme activity during In the HtrA2-centered protein interactions regulatory network,various UPR^mt-related proteins such as Trap1（HSP75）,Lonp1,Hspd1,Yme1L1,Hspa1l,ATF5 and HtrA2 have protein interactions with HtrA2,suggesting that HtrA2 may be involved in UPR^mt during myogenic cell differentiation through directly or indirectly involved in the regulation of UPR^mt.Conclusion1.Mnd2 mice exhibit muscle atrophy and reduced muscle function compared to wild-type mice,suggesting that HtrA2 plays an important role in the maintenance of muscle mass and function.2.The results of C2C12 myogenic differentiation assays and bioinformatics analysis suggest that HtrA2 enzyme activity deficiency leads to impaired myogenic differentiation of myoblast cells.3.HtrA2 enzyme activity deficiency leads to abnormal mitochondrial IMS protease expression in skeletal muscle of mnd2 mice and C2C12 myoblasts,suggesting that imbalance in mitochondrial protein homeostasis may be a cause of impaired myoblast differentiation.4.HtrA2 deficiency inhibited OXPHOS function in C2C12 myoblast,leading to a reduction in the content of the mt DNA-encoded ETC complex subunits and inhibition of the nuclear regulatory mitochondrial biosynthesis signaling pathway,suggesting that imbalance in mitochondrial protein homeostasis caused by HtrA2deficiency could affect myogenic differentiation by inhibiting mitochondrial biosynthesis.5.Upregulation of UPR^mt-related protein expression by imbalance in mitochondrial proteostasis caused by inhibition of HtrA2 enzyme activity during myogenic differentiation.The PPI suggests that HtrA2 may be involved in nuclear-mitochondrial cross-talk through direct or indirect activation of UPR^mt.In summary,this study provides a new direction for targeting mitochondria to prevent and treat muscle atrophy,and provides a new research idea to explore mitochondrial proteostasis as a counter-regulatory nuclear signaling pathway during cell differentiation.