| Long-term high-fat diet and sedentary lifestyle can lead to myocardial lipid deposition,induce a variety of heart diseases.Integration of sports and medicine is very necessary.The mechanism of myocardial lipid deposition is complex and the role of peroxisome proliferator activator receptor ?(PPAR?),a key transcription factor regulating the expression of glucolipid-related genes,is central.PPAR? is highly expressed in myocardial tissue and plays an important role in myocardial energy metabolism under physiological or pathological conditions.PPAR? is affected by a variety of molecules,but the specific mechanism has not been fully elucidated.Kruppel-like factor 5(KLF5)is widely expressed in different tissues of the body.As a basic transcription factor,KLF5 participates in the occurrence and development of cardiovascular diseases by regulating a variety of target genes.Some studies have found that PPAR? is one of the important target genes in cardiac tissue.Meanwhile,we used the bioinformatics software Target Scan to predict and combined with relevant research literature in recent years,found that miR-145-5p and miR-153-3p may target KLF5.Therefore,we speculated that miR-145-5p/miR-153-3p may be involved in myocardial lipid metabolism by regulating PPAR? expression through KFL5.Disturbance of miRNAs/KLF5/PPAR?signaling pathway may cause myocardial lipid deposition after long-term high-fat diet? The effectiveness of exercise on myocardial lipid metabolism depends on exercise load,different combinations of intensity and time may lead to different effects.Moderate intensity continuous training(MICT)and high intensity interval training(HIIT)have their own characteristics,could they prevent myocardial lipid deposition induced by long-term high fat diet? What are the differences and similarities between the effects? How does the myocardial miRNAs/KLF5/PPAR? signaling pathway change during this process? Are the differences in preventive effects related to the degree of activation of this signaling pathway? All the above problems need to be confirmed.Objectives:Human,animal and cell experiments were conducted to verify the signaling pathway of miRNAs/KLF5/PPAR? which participates in myocardial lipid metabolism,to explore the mechanism of miRNAs induced myocardial lipid deposition.The effects of MICT and HIIT on preventing myocardial lipid deposition and the activity of miRNAs/KLF5/PPAR?signaling pathway were compared in rats with high fat diet,the mechanism and the difference were analyzed,aims to provide a basis for selecting a better exercise program for preventing and controlling myocardial lipid deposition.Methods:1.Overweight and obese urban male residents aged 45 to 55 years old with university degree and with reduced cardiac function by echocardiography were selected as the control group(OB,BMI?24,n=10),and the age and sex matched group with normal cardiac function confirmed by echocardiography(N,18.5?BMI?23.9,n=10).Serum LDL,HDL,TC and TG were detected by microplate method.CTn T,a marker of myocardial injury,was detected by ELISA.Serum miR-145-5p and miR-153-3p levels were detected by q PCR,and the changes of serum miR-145-5p and miR-153-3p levels in overweight and obese patients with reduced cardiac function were analyzed.2.5-week-old clean male SD rats were randomly divided into normal diet group(NC,n=8)and high-fat diet group.After 12 weeks,the FC group were sorted according to their body weight from high to low,and the rats in the top eight for weight were selected as high-fat diet group(FC,n=8).Cardiac structure,systolic and diastolic function were examined by echocardiography.HE,oil red O staining and transmission electron microscopy were used to observe the structure and lipid deposition of rat myocardium.Serum lipid,myocardial injury markers and lipid content in myocardial tissue were measured by microplate method and ELISA.The expressions of miR-145-5p,miR-153-3p,KLF5 and PPAR? in myocardia were detected by q PCR,Western Blot and immunofluorescence.the possible role of the two miRNAs in the process of myocardial lipid deposition induced by high fat diet in obese rats were preliminarily analyzed and miR-145-5p was selected as the object of follow-up study based on the results of human experiments.3.H9C2 cell line was stimulated with palmitic acid(PA)at concentrations of 0,0.1,0.2,0.4 and 0.8mmol/L for 24 h and 48 h,respectively.Cell activity was detected by CCK8,and the appropriate concentration and time of PA stimulation were screened.H9C2 cell line were cultured in vitro and transfected into six groups: H9C2 cell control group(C),H9C2 cell +PA group(CP),H9C2 cell +PA control group(CIN),H9C2 cell +PA+ miR-145-5p inhibitor group(IN),H9C2 cell +PA control mimics group(CMI)and H9C2 cells +PA+ miR-145-5P mimics group(MI).The contents of FFA and TG in supernatant of culture medium were detected by microplate method.The mRNA and protein expressions of KLF5 and PPAR?were detected by q PCR and Western Blot,and the role of miR-145-5p/KLF5/PPAR?signaling pathway was analyzed.4.Target Scan software was used to predict that miR-145-5p has bases corresponding to KLF5 3,-UTR region.miR-145-5p corresponding target gene KLF5 3,-UTR target sites and their adjacent sequences were constructed into pmir Glo vector as wild type,miR-145-5p corresponding target gene KLF5 3,-UTR after base mutation at target site were constructed into Pmir Glo vector as mutant type,and transfected into human renal epithelial cells for 48 h.Dual luciferase kit was used for detection.The relative luciferase activity = firefly luciferase activity value/sea kidney luciferase activity value was calculated to verify the relationship between miR-145-5p and KLF5.5.5-week-old clean grade male SD rats were randomly divided into high-fat diet quiet group(F),high-fat diet MICT group(M)and high-fat diet HIIT group(H),n=8.After 12 weeks,cardiac structure,systolic and diastolic function of rats were detected by echocardiography.HE,oil red O staining and transmission electron microscopy were used to observe the structure and lipid deposition in myocardium.Serum lipid,myocardial injury markers and lipid content in myocardial tissue were measured by microplate method and ELISA.The expressions of miR-145-5p,KLF5 and PPAR? were detected by q PCR,Western Blot and immunofluorescence double-label method.Effects of MICT and HIIT on myocardial lipid deposition and the role of miR-145-5p/KLF5/PPAR? in high-fat diet rats were analyzed to explore the mechanism of action and difference between the two exercise programs.Results:1.Serum miR-145-5p content increased in overweight and obese patients with reduced cardiac function,while miR-153-3p content did not change significantly.(1)There was no significant difference in age and height between the two groups.(2)The body weight and BMI of OB group were higher than those of N group(P<0.01).(3)Compared with N group,IVS and IVRT in OB group were increased(P<0.05),LVSV,LVCO,LVEF,LVFS and E/A were decreased(P<0.05).(4)Compared with N group,the contents of LDL,TG and TC in OB group were increased(P<0.05),while the contents of HDL and cTnT were not significantly different.miR-145-5p content increased(P<0.01),and miR-153-3p had no significant.2.The expression levels of miR-145-5p and miR-153-3p in myocardium of obese rats were increased after high-fat diet.(1)Before the experiment,there was no significant difference in the initial body weight between the two groups.During the 12-week feeding period,the body weight of the two groups increased steadily with good growth and development.After 12 weeks,compared with NC group,body weight,heart weight and Lee's index of FC group increased(P<0.05);Compared with NC group,FC group had no significant difference in total intake of food,and total intake of calories increased(P<0.05).(2)Compared with NC group,LVDs,LVDd and LVCO in FC group were increased(P<0.05),LVEF,LVFS and E/A were decreased(P<0.05).(3)Longitudinal HE staining results of left ventricular axis brachycardia of rats showed that myocardial fibers in NC group were in normal direction,arranged in parallel with each other,bifurcated,closely connected and anastomosed into a network.In FC group,the direction of myocardial fibers was basically normal,but myocardial fibers were obviously damaged,broken more,the gap between each other was significantly increased,the connection was loose,and the whole showed a trend of atrophy.The results of oil-red O staining in longitudinal section of left ventricular axis muscle of rats showed that compared with NC group,lipid deposition in myocardial tissue of rats in FC group were significantly increased.Transmission electron microscopy of ultra-thin sections of rat myocardium showed that the myofilaments of myocardium of rats in NC group were arranged neatly and in a normal direction,the distribution of sarcomere was regular,the bands,regions and lines were clearly visible,the mitochondria were developed and arranged in a linear manner,the structure was regular,the mitochondrial crest was dense,no swelling or vacuolation changes,and the myocardial interstitium was not abnormal.In the FC group,the myofilament arrangement and alignment of rat cardiomyocytes were basically normal,but the Z-line of some myofilament disappeared and appeared dissolution phenomenon,with disordered arrangement,the number of mitochondria decreased,the regular linear arrangement was damaged,some mitochondria showed dissolution vacuoles,the mitochondrial crists decreased,and a large number of lipid droplets accumulated near mitochondria.(4)Compared with NC group,the contents of LDL,TG and FFA in serum of FC group were increased(P<0.05),while the HDL content was decreased(P<0.05);The contents of TG and FA in myocardial tissue of FC group were increased(P<0.01).There was no significant difference in serum cTnI and cTnT in FC group.(5)Compared with NC group,the expressions of miR-145-5p and miR-153-3p in myocardium of FC group were increased(P<0.05),and the mRNA and protein expressions of KLF5 and PPAR? in myocardium of FC group were decreased(P<0.05).(6)Compared with NC group,the number of KLF5 positive cells,PPAR? positive cells and KLF5/PPAR? co-expression positive cells in myocardium of FC group was decreased(P<0.01).3.miR-145-5p/KLF5/PPAR? signaling pathway regulates lipid metabolism in H9C2 cell line.(1)the cell activity was 46.33% at 0.2mmol/L PA for 48 h,24.33% and 20.67% at 0.4 and0.8mmol/L PA for 48 h,respectively.Low cell activity is not conducive to the development of subsequent experiments,so the concentration of PA stimulation was set at 0.2mmol/L and the stimulation time was set at 48 h in this study.(2)Compared with C group,the expression of miR-145-5p in CP group was increased(P<0.05);After cell transfection with miR-145-5p inhibitor,compared with CIN group,the expression of miR-145-5p in the IN group was decreased(P<0.01),down regulated by about 5 times;After cell transfection with miR-145-5p mimics,the expression of miR-145-5p in the MI group was increased(P<0.01)compared with that in the CMI group,which was up regulated by about 127 times.(3)Compared with C group,the contents of FFA and TG in supernatant of cell culture medium in CP group were increased(P<0.01);Compared with IN group,the contents of FFA and TG in supernatant of cell culture medium IN CP and CIN groups were increased(P<0.05);Compared with MI group,FFA content in supernatant of cell culture medium in CP group was not significantly changed,but TG content was decreased(P<0.01),while FFA and TG contents in supernatant of cell culture medium in CMI group were decreased(P<0.05).(4)Compared with C group, the mRNA and protein expression levels of KLF5 and PPAR? in CP group were decreased(P<0.01);Compared with IN group,the mRNA and protein expression levels of KLF5 and PPAR? in CP and CIN groups were decreased(P<0.01).Compared with MI group,the mRNA and protein expressions of KLF5 and PPAR? in CP and CMI groups were increased(P<0.05).4.miR-145-5p can inhibit its expression at the post-transcriptional level by binding to KLF5 3,-UTR.(1)Comparing the sequencing results with the target gene sequence,the results showed that the inserted fragment was completely consistent with the design sequence of KLF5-wt/mut,and the insertion direction was correct.(2)The luciferase expression of firefly and sea kidney was stable,and the luciferase activity of KLF5 3,-UTR pmir Glo vector and miR-145-5p mimics was decreased compared with that of mimics NC group(P<0.05).Compared with the mimics NC group,there was no significant change in lucifase activity of after transfection of mutant KLF5 3,-UTR pmir Glo vector and miR-145-5p mimics.5.MICT and HIIT prevents myocardial lipid deposition in high-fat diet rats by affecting the activity of miR-145-5p/KLF5.(1)Before the experiment,there was no significant difference in the initial body weight of rats in each group.During the 12-week feeding period,the body weight of rats in each group increased steadily with good growth and development.After 12 weeks,compared with F group,body weight and Lee's index of rats in M and H groups decreased(P<0.01),heart weight of rats in H group decreased(P<0.05),and heart weight of rats in M group had no significant change;Compared with group M,there were no significant differences in body weight,heart weight and Lee's index in group H.During the12-week feeding period,the average daily intake and caloric intake of rats in group F were relatively stable,while the average daily intake and caloric intake of rats in group M and H showed a decreasing trend at the beginning,especially in group H,and then gradually increased and tended to be stable.Compared with group F,total food intake and calorie intake were decreased in groups M and H(P<0.01);Compared with group M,there were no significant differences in total food intake and calories in group H.(2)Related parameters of left ventricular structure: Compared with group F,LV Mass AW,LVDs and LVAWd were decreased in group M and H(P<0.05);Compared with M group,LV Mass AW was increased(P<0.05)and LVDs was decreased(P<0.01)in H group.Compared with F group,LVFS increased in M group(P<0.05),LVCO,LVEF and LVFS increased in H group(P<0.05),LVVs decreased in H group(P<0.01).Compared with M group,LVEF and LVFS in H group increased(P<0.05),LVVs decreased(P<0.01);Parameters related to left ventricular diastolic function: Compared with group F,MPI of rats in M and H groups was decreased(P<0.05),while E/A value was increased(P<0.05);There was no significant difference in the parameters of group M and group H.(3)HE staining of longitudinal section of left ventricular short axis muscle of rats showed that the direction of myocardial fibers in group F was basically normal,but there were obvious damage and fracture of myocardial fibers,the gap between each other increased significantly,the connection was loose,and the whole showed a tendency of atrophy.The myocardial fibers in groups M and H were in a normal direction,arranged in parallel,closely connected,basically without breaking disorder,relatively closely connected,and there was no significant difference between the two groups of rats.Oil red O staining in longitudinal section of left ventricular axis axis muscle of rats showed that compared with group F,oil red O staining in myocardial tissue of rats in groups M and H was decreased and lipid deposition was significantly decreased.Compared with group M,the myocardial tissue of rats in group H had no obvious oil red O staining mass and less lipid deposition.Rat myocardial ultrathin slices tem showed that muscle group F rat myocardial cell arrangement,to the basic normal,but some muscle Z line disappear,appear to dissolve phenomenon,disordered arrangement,the decrease in the number of mitochondria,the rules are linear damaged,some mitochondria dissolved cavity,mitochondrial cristae decreased,a large amount of lipid droplets are located near the accumulation.There was no significant difference in myocardial cell structure between groups M and H,but both groups were significantly better than group F,with orderly and compact arrangement of myofilaments,less dissolution,more mitochondria,relatively regular structure,fewer mitochondria vacuoles,and no obvious accumulation of lipid droplets near mitochondria.(4)Compared with F group,the contents of LDL and FFA in M and H groups were decreased(P<0.01),while the contents of HDL and TG in M group were not significantly different,while the contents of HDL in H group were increased(P<0.01)and decreased(P<0.01).Compared with group M,the serum HDL content of group H was increased(P<0.01),while the contents of LDL,TG and FFA were not significantly different.Compared with F group,the contents of TG and FA in myocardial tissue of rats in M and H groups were decreased(P<0.01).Compared with group M,the content of TG in group H was decreased(P<0.01),while the content of FA was not significantly different.Compared with F group,there was no significant difference in the contents of cTnI and cTnT in M and H groups.Compared with M group,there was no significant difference in serum cTnI and cTnT contents in H group.(5)Compared with F group,the expression of miR-145-5p was decreased in M and H groups(P<0.01),and the mRNA and protein expression levels of KLF5 and PPAR? were increased(P<0.05).Compared with group M,the mRNA and protein expression levels of miR-145-5p,KLF5 and PPAR? in group H showed no significant difference.(6)Compared with F group,KLF5 and PPAR? positive cells and KLF5/PPAR? co-positive cells were significantly increased in M and H groups(P<0.01).Compared with M group,the ratio of KLF5,PPAR? positive cells,KLF5/PPAR? co-positive cells and KLF5 and PPAR? positive cells in H group was significantly increased(P<0.01).Conclusion:1.miR-145-5p/KLF5/PPAR? is a signaling pathway of myocardial lipid deposition induced by long-term high-fat diet.2.Both MICT and HIIT can prevent myocardial lipid deposition and protect myocardial structure and function in high-fat diet rats by affecting miR-145-5p/KLF5/PPAR? signaling pathway.Compared with MICT,HIIT is more effective,which may be related to the activation level of myocardial miR-145-5p/KLF5/PPAR? signaling pathway. |
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