Construction And Analysis Of Murf1 And MAFbx-knockout Pigs

As animal with significant agricultural ecnomic values,pigs are also used in basic science and biomedical studies as an important animal model.Muscle RING-Finger protein 1(MuRF1)and Muscle Atrophy F-box protein(MAFbx)are E3 ubiquitin ligases specifically expressed in striated muscles.They play important roles in the degradation of several factors and structural proteins in myofibers.It has been reported that Murf1 is a candidate gene related to human hypertrophic cardiomyopathy(HCM).HCM is charactarized as myocardial hypertrophy.A loss-of-function mutation of Murf1Q247*leads to overaccumulation of sarcromere proteins,with myocardial hypertrophy and disorder.According to the statistics,the morbidity of HCM is 0.2%,1%of which causes sudden cardiac death(SCD)every year.The typical clinical symptoms of HCM are dyspnea,chest pain,palpitation,syncope and SCD.SCD is the most serious symptom,and also the first symtom of some patients.HCM is a common autosomal dominant disease,mainly caused by mutations of genes encoding sarcomere proteins.But so far,the mechanism of sarcomere proteins abnormity to HCM remains unclear.Meanwhile,MuRF1 and MAFbx are the key factors in the process of muscle atrophy.Muscle spare in Murf1 and MAFbx null mice in response to several stimulus of atrophy.The triceps surae mass increases by 10-14%in Murf1 null mice vs WT mice.Therefore,the research about the regulation of muscle mass by MuRF1 and MAFbx may benefit the animal agricultural development.In the study of pig Murf1,we firstly identified its specific expression pattern in the striated muscles.Then,we modified the region of Murf1 exon1 by CRISPR/Cas9n system,which caused a homozygous insertion of 83 bp.Finally,we generated Murf1 deletion pig model with this mutation.The off-target effect was within an acceptable range.The Murf1 expression was significantly decreased on both mRNA and protein levels in the Murf1 deficient pig.Two of the F0 founders suffered SCD in 22 and 23 d with asymmetric hypertrophy in left ventricular(LV).However,the other F0 founders slaughtered in 11.5 mon were without hypertrophy in LV vs WT of same age.According to the Q-PCR results,the transcription of E3 ubiquitin ligases related to MuRF1 was decreased.The expressions of genes encoding sacromere proteins were also reduced on both mRNA and protein level.The level of myocardial enzymes in the serum of Murf1-/-pigs increased at beginning and reduced thereafter in 5 to 10 mon.This result indicated that dysfunction of Murf1-/-pig heart might appear in the early stage.By detection and analysis of the production traits in Murf1-/-pigs,we found no significant change in the lean muscle mass between mutants and WT.However,the cross sectional area of the longissimus dorsi in Murf1-/-pig increased significantly while the activation of the Akt-mTOR pathway was much higher,which suggested that the protein synthesis was enhanced.In the aspect of meat quality traits,most of the data of Murf1-/-pigs was in the regular base.The color 1 level was significantly reduced(P<0.05),whereas pH level was much higher(P<0.01)and drip loss was lower but not significantly vs WT.The traits showed that pigs with Murf1 deficiency had more firm and darker meat.In the study of pig MAFbx,we found that MAFbx expressed in various tissues in Duroc.We modified the exon2 of MAFbx gene by CRISPR/Cas9n,and screened a blended cell clone.After SCNT,we generated MAFbx knockout pigs.The influence of the deficiency of MAFbx in various tissues and organs needs further investigation.In summary,we generated Murf1 and MAFbx mutant pig models.Murf1-/-pig showed symptoms similar to human HCM and thicker skeletal muscle myofiber.It will be a great model in the study of HCM and the development of skeletal muscle,and it will also benefit the clinical diagnosis and treatment for HCM,providing effective tool for drug screening.