Exploring The Role Of Mitonuclear Imbalance In Sarcopenia Based On The Differential Expression Of NRF-1/NRF-2 In Skeletal Muscle Of Mnd2 Mouse

Background:One of the most significant changes of the body during aging is the progressive decline in skeletal muscle mass and function,which eventually progressed into sarcopenia.Imbalance among skeletal muscle protein synthesis and breakdown,muscle cell apoptosis,and mitochondrial dysfunction is associated with the occurrence and development of sarcopenia.Due to the key role of mitochondria in intracellular energy supply and redox homeostasis,mitochondrial dysfunction is considered to be the core mechanism of sarcopenia.Disorders of mitochondrial biogenesis lead to abnormal oxidative phosphorylation,which is the main cause of mitochondrial dysfunction.Mitochondria are double-genome organelles,the respiratory chain is encoded by both the nucleus and mitochondrial DNA.Presently,it is believed that mitochondrial functions such as oxidative phosphorylation and mitochondrial division and fusion require the coordination of the nuclear and mitochondrial genomes.Function of oxidative phosphorylation is believed to be associated with mitonuclear communication.Thus,exploring the involvement of mitonuclear communication in muscle degeneration is helpful to understand the role of mitochondrial dysfunction in the pathogenesis of sarcopenia.The expression of respiratory chain subunits is mainly regulated by mitochondrial biogenesis factor PGC-1?(peroxisome proliferator activated receptor-? coactivator-1?)and its downstream NRF-1(nuclear respiratory factor 1)and NRF-2.These factors also play an important role in the nuclear-mitochondrial cross-talk mechanism.However,the role of these factors in the skeletal muscle nucleus-mitochondrial imbalance has not been reported yet.Biological informatics provides convenience for related research.By combining with traditional laboratory methods,it will provide the possibility to further study the role of nuclear-mitochondrial dialogue mechanism in mitochondrial dysfunction and skeletal muscle degeneration.Htr A2/Omi is a nuclear encoding mitochondrial serine protease.Traditionally,most attention was paid to the role of Htr A2/Omi in the process of apoptosis.Htr A2/Omi can be released into the cytoplasm under the stimulation of apoptotic factors,and participate in apoptotic signal transduction by interacting with the caspase signal pathway.Recent studies have found that Htr A2/Omi can also participate in the degradation of unfolded proteins through its protease activity,hence promoting the mitochondrial protein homeostasis.Besides,Htr A2/Omi transducts signals involved in mitochondrial biogenesis,mitochondrial dynamics,and redox balance,which suggests that Htr A2/Omi may play an important role in the mitonuclear communication.mnd2 mice with Htr A2/Omi serine missense mutations at position 276 have neurodegeneration,abnormal movement,muscle atrophy,and premature aging death,suggesting a protective role of Htr A2/Omi protease activity in aging-related diseases.However,the correlation between Htr A2/Omi protease activity and skeletal muscle phenotype is still unknown.Therefore,combined with functional experiemnts,morphology analysis,genes expression,and bioinformatics analysis,we can further clarify the mechanism of mitonuclear communication in regulating mitochondrial function,and its role in sarcopenia.Method:1.This study used mnd2 mice to investigate the role of Htr A2/Omi protease activity in skeletal muscle degeneration,through muscle contractility tests,grip tests,suspension tests to evaluate muscle strength and movement phenotype of mnd2 mice.2.Evaluating the skeletal muscle degenerative phenotype using HE staining,Sirius red staining,other morphological techniques,and quantitative morphological analysis methods.3.Evaluating mitochondrial function,expression of respiratory chain complex subunits,and activation of mitochondrial biogenesis using q PCR,western blot and immunohistochemical staining,and other functional characterization experiments.4.In order to clarify the role of Htr A2/Omi in skeletal muscle degeneration,we employed sarcopenia expression chip data to perform bioinformatics analysis,and explored the biological function of Htr A2/Omi in sarcopenia.Result:1.The weight of mnd2 homozygous mice,gastrocnemius muscle weight,skeletal muscle body mass index(SMI)and physiological cross-sectional area were significantly lower than that of wild-type and heterozygous mice.Both the single twitch contraction and tonic contraction of gastrocnemius muscle in mnd2 homozygote were significantly reduced in gastrocnemius specific contractility test,and the significance was still maintained after weight correction.The results of movement phenotype experiments showed that the hanging time of mnd2 homozygous mice was significantly shortened,and the resting time in tail suspension test was significantly extended.Morphological experiment results showed that mnd2 homozygous gastrocnemius cross-section HE staining showed broken or segmented muscle fibers,eosinophilic homogeneous gelatinous staining indicating hyalinization,and fibrous interstitial fat filling.Some muscle fibers showed nuclear centralization and obvious multinuclearization.Sirius red staining showed that in mnd2 homozygous mice,gastrocnemius muscle fiber interstitials was widened and collagen was deposited.The morphological quantitative analysis results showed that mnd2 homozygous gastrocnemius muscle fibers cross-sectional area decreased and muscle fiber irregularity increased.The m RNA expression levels of muscle fiber typing marker genes Myh1,2,4,7 in the mnd2 homozygous gastrocnemius muscle were significantly reduced.The m RNA expression levels of denervation-sensitive genes Myo D and Myogenin in the homozygous gastrocnemius of mnd2 did not change significantly,while that of ACh R? and ACh R? decreased significantly.2.The protein expression level of mitochondrial marker gene VDAC1 in mnd2 homozygous gastrocnemius muscle was significantly reduced.Immunohistochemical staining of VDAC1 showed that mnd2 homozygous gastrocnemius muscle lost highly regular mitochondrial distribution.“Alopecia areata” of VDAC1 staining in mnd2 homozygous gastrocnemius muscle are observed.Consistently,the copy number of mitochondrial gene(mt DNA)decreased significantly in mnd2 homozygotes.When corrected for total protein content and mt DNA copy number,the levels of ATP production and mitochondrial respiratory chain complex I activity were significantly reduced in the mnd2 homozygous gastrocnemius muscle.The level of ROS corrected for total protein content(total ROS)was significantly reduced in the mnd2 homozygous gastrocnemius muscle,but the level of ROS corrected for mt DNA was significantly increased.3.In the results of GSEA analysis,the gene sets enriched in the Htr A2/Omi low expression group were mitochondrial complexes or oxidative phosphorylation.The expression data of structural subunits and chaperone molecules in five complexes of the mitochondrial respiratory chain were extracted,and the correlation analysis was performed.The results showed that there was a strong negative correlation(? <-0.7)among Htr A2/Omi and subunits which are encoded by nuclear genes(n DNA),while mt DNA-encoded subunits have no correlation or strong positive correlation with Htr A2/Omi(?> 0.7).The study further verified the above results biologically in mnd2 mice.The results showed that compared to wild type,m RNA expression levels of most n DNA-encoding subunits were significantly down-regulated in mnd2 homozygous gastrocnemius muscles,while NDUFS7,NDUFV2,SDHC,SDHD,COX6A1 and COX8 A had no significant changes,only SDHA and UQCRB and UQCRFS1 showed a significant increase.In contrast,the mt DNA coding subunits showed no significant change or significant upregulation.4.Analysis of sarcopenia expression chip data shows that Htr A2/Omi has a strong negative correlation with NRF-1 and a strong positive correlation with NRF-2.ETC subunits(eg,NDUFA1,NDUFB4,NDUFS1,NDUPV1,UQCRH,CYCS,COX5 A,COX6C,ATP5A1,ATP5 B,etc.)which have negative correlation with Htr A2/Omi are all NRF-1 targeted genes.ETC subunits(such as COX6A1,COX8 A,SDHD and UQCRFS1,and mt DNA coding subunits)that are positively correlated or unrelated to Htr A2/Omi are all NRF-2 targeted genes.The biological verification results showed that the protein and m RNA expression of PGC-1? in mnd2 homozygote gastrocnemius muscle decreased,and the expression of its target genes SOD1/2,UCP2,GPX1,etc.were down-regulated.In addition,the expression of NRF-1 and NRF-2 showed opposite trends.5.The study used sarcopenia expression chip data to screen key differentially expressed genes(DEGs),and to construct a protein interaction network.The results showed that 8 key DEGs were interacted with Htr A2/Omi,among which Akt1 has been reported to post-translationally modify PGC-1? in skeletal muscle.Further analysis showed that Akt1 has a strong negative correlation with PGC-1?,NRF-1 and its ETC target genes,and a strong positive correlation with NRF-2 and its ETC target genes.Biological verification results showed that: compared with the wild type,the m RNA expression level of Akt1 in the homozygous gastrocnemius of mnd2 was significantly up-regulated.Conclusion:1.Compared with wild type,the muscle weight,SMI,contractility and motor performance of mnd2 homozygous mice were significantly reduced,indicating that loss of Htr A2/Omi enzyme activity will lead to significantly decreased muscle mass,muscle strength and motor function.2.Compared with wild type,mnd2 homozygous mice had lower mitochondrial content,less ATP and total ROS generation,and ETC complex I activity,suggesting that lack of Htr A2/Omi enzyme activity lead to mitochondrial dysfunction in skeletal muscle.3.Most of the ETC subunits encoded by n DNA and mt DNA in the gastrocnemius muscle of mnd2 homozygous mice showed differential expression,namely the mitonuclear imbalance among oxidative phosphorylation genes,suggesting that the lack of Htr A2/Omi protease activity caused oxidative phosphorylation disorders in skeletal muscle.In addition,the differential expression patterns of these ETC subunits are consistent with the differential expression patterns of their transcription factors NRF-1 and NRF-2,suggesting that mitonuclear imbalance caused by biogenesis disorders may be responsible for skeletal muscle degeneration and sarcopenia.4.Analysis of sarcopenia gene chip data and biological verification in mnd2 mice showed that the upregulation of Akt1 expression may be one of the mechanisms underlying mitochondrial biogenesis and oxidative phosphorylation disorders in mnd2 homozygous muscles.In conclusion,this study may provide new direction for the study of skeletal muscle degeneration and sarcopenia,and provide new targets for its prevention and treatment.