Study On Regulatory Mechanism Of Protein S-Nitrosylation On Muscle Cell Apoptosis During Beef Postmortem Aging

As a biological signal transduction molecule,nitric oxide(NO)is catalyzed by nitric oxide synthase(NOS)and involved in the regulation of physiological processes such as muscle contraction,neurotransmission and apoptosis.NO can bind to sulfhydryl groups of protein cysteines,resulting in the occurrence of protein S-nitrosylation.NO and protein Snitrosylation can regulate various biochemical pathways during the conversion of muscle to meat,including energy metabolism and calcium homeostasis,thus affecting fresh meat quality.In the field of meat science,NO promoters and inhibitors are usually used to regulate the levels of NO and S-nitrosylation in animal muscles to investigate their effects on meat quality during postmortem aging.However,the regulatory mechanism of NO on postmortem beef muscle cell apoptosis has not been reported yet.Therefore,the main purpose of this paper was to study the effects and regulatory mechanism of protein Snitrosylation on muscle cell apoptosis during beef aging,which could provide a new perspective for explaining the mechanism of NO on fresh meat quality.The specific research contents and results are as follows:1.Activity and expression changes of nitric oxide synthase during beef agingThe beef semimembranous muscle(SM)was collected within 45 min after slaughter,and then vacuum-packed and aged at 4℃ for 0,1,3,7,and 14 d to investigate the activity,content and localization of nitric oxide synthase(NOS)in bovine SM during postmortem aging.The results showed that beef SM muscle retained NOS activity during the 14 d of postmortem aging,and the activity reached the highest(0.80 nmol/mg protein/min)at 0 d.Besides,NOS activity significantly decreased at 1,3 and 7 d(P<0.05).Moreover,neuronal NOS(nNOS)is considered a dominant isoform in skeletal muscle,and nNOS content decreased gradually over 7 d of aging(P<0.05),indicating that nNOS was degraded during beef aging.The reasons for the degradation of nNOS after slaughter were explored by treatments with calpain or caspase inhibitors.The results showed that both calpain and caspase were involved in the degradation of nNOS,but calpain could mainly contribute to nNOS degradation.Immunofluorescence results indicated that nNOS was distributed in sarcolemma,and the fluorescence intensity gradually weakened when aging time was extended.In addition,the level of protein S-nitrosylation in beef SM muscle was found to accumulate during postmortem aging,and it reached a maximum of 20.01 μM/mg protein at 14 d.The activity and expression of NOS in beef SM provide the possibility for NO formation and S-nitrosylation occurrence,which may be involved in muscle metabolism and play a potential role in the regulation of meat quality.2.Effect of protein S-nitrosylation on the proceeding of beef muscle cell apoptosis during postmortem agingThe bovine SM was taken within 45 min after slaughter,and then incubated with Snitrosoglutathione(GSNO,NO donor),Nω-nitro-L-arginine methyl ester hydrochloride(LNAME,NOS inhibitor)and NaCl(control group)at 4℃ for 24 h.The apoptosis-related indictors were measured at 1,6,12,24,72,and 168 h,respectively,to study the effect of protein S-nitrosylation on beef muscle cell apoptosis during postmortem aging.Compared to the control group,more chromatin condensation,apoptotic body formation,and mitochondrial swelling were observed in the L-NAME group,and these morphological changes were significantly inhibited by GSNO treatment.TdT-mediated dUTP nick-end labeling(TUNEL)results showed that the apoptotic rate increased with the aging time.When aging time was extended to 168 h,the apoptotic rate of the control group,GSNO group and L-NAME group reached 30.78%,20.51%and 45.68%,respectively.At 12,24,72,and 168 h,compared to the control,the apoptosis rate was significantly inhibited by GSNO treatment,while the apoptosis rate of L-NAME group was remarkably increased(P<0.05).In addition,GSNO treatment significantly decreased the activity and expression of caspase-3 and caspase-9 during the early postmortem period(P<0.05).The results suggest that protein S-nitrosylation plays a negative regulatory role in the proceeding of bovine SM apoptosis.3.Effects of protein S-nitrosylation on mitochondrial apoptosis during beef postmortem agingAt 45 min after slaughter,bovine SM was incubated with GSNO,L-NAME,and NaCl(control group)for 24 h at 4℃.After incubation,beef SM was aged continuously and collected at 1,6,12,24,72,and 168 h,respectively,for indicator measurement to explore the pathway of protein S-nitrosylation regulating beef muscle cell apoptosis.Results showed that GSNO significantly increased the expression of anti-apoptotic protein Bcl-2 at 6 h postmortem(P<0.05).In addition,the mitochondrial membrane potential of the GNSO group was significantly higher than the control and L-NAME groups,indicating that GSNO delayed the decrease of mitochondrial membrane potential.Besides,GSNO significantly inhibited the translocation of cytochrome c from mitochondria to cytoplasm(P<0.05).In addition,protein S-nitrosylation was involved in the mitochondria-dependent downregulation of caspase-9-caspase-3 cascade.Besides,S-nitrosylation of caspase-3 and-9 resulted in the decrease in their activities(P<0.05),which further confirmed the inhibition of beef SM apoptosis by S-nitrosylation.Therefore,it can be proposed that protein Snitrosylation regulates beef SM apoptosis through the intrinsic mitochondrial apoptotic pathway.4.Effect of protein S-nitrosylation on caspase-3 activity and its proteolytic ability on beef myofibrillar protein in vitroDifferent concentration gradients of NO donor(GSNO)were set up,and then incubated with recombinant caspase-3 in vitro.The activity and the S-nitrosylation degree of caspase-3 were determined,and S-nitrosylation sites of caspase-3 were identified by mass spectrometry.In addition,beef myofibrillar protein was extracted and incubated with S-nitrosylated caspase-3,and the degradation of desmin and troponin-T was detected by western blotting.The results showed that caspase-3 was S-nitrosylated at Cys 116,170,184,220 and 264.After GSNO treatment,the activity of caspase-3 was significantly reduced(P<0.05)in a dose-dependent manner,and the activity of caspase-3 in the 400 μM GSNO group was the lowest.However,the S-nitrosylation degree of caspase-3 was improved with the increasing concentration of GSNO(P<0.05).The results indicated that S-nitrosylation of the cysteine sites of caspase-3 might be an intrinsic mechanism regulating its activity.Compared to 0 μM GSNO group,the addition of GSNO to caspase-3 resulted in the content of intact desmin significantly increased(P<0.05),and the 400 μM GSNO group showed the highest intensity of intact desmin band.Meanwhile,200 μM and 400 μM GSNO groups had significantly lower troponin-T degradation fragment than 0 μM and 100 μM GSNO treatment groups(P<0.05).It was concluded that in vitro S-nitrosylation modification of caspase-3 inhibited the degradation of desmin and troponin-T.5.Identification and analysis of S-nitrosylated proteins in postmortem beef SM during agingThe bovine SM was incubated with GSNO or L-NAME for 24 h to exogenously regulate the levels of protein S-nitrosylation in beef SM.The differential S-nitrosylated proteins in different treatment groups were identified by quantitative proteomics of TMT labeling.Bioinformatics analysis was conducted to explore the pathways of protein Snitrosylation in postmortem muscle metabolism.The results showed that 95 differential Snitrosylated proteins were identified,among which the main proteins involved in postmortem aging were sarcoplasmic reticulum calcium ATPase,ryanodine receptor,yruvate kinase,glyceraldehyde-3-phosphate dehydrogenase,lactate dehydrogenase,malate dehydrogenase,actin,nebulin,type Ⅱ myosin,plectin,cytochrome b-c1 complex,and ubiquitin-conjugating enzyme.It was suggested that protein S-nitrosylation could regulate calcium release,energy metabolism,protein degradation,and apoptosis to affect the quality of beef.Besides,it was found that enzyme was dominant in the classification of differential S-nitrosylated proteins,and most of the differential S-nitrosylated proteins had only one cysteine modification site,which accounted for 93%.GO analysis and KEGG enrichment analysis showed that glycolysis was a typical metabolic pathway regulated by Snitrosylation.The above results indicate that protein S-nitrosylation might cause different biochemical metabolism level of postmortem beef,which led to the differences in beef quality.