Calorie Restriction Initiate In Different Age Have Distinct Effects On Cardiac Function

Caloric restriction(CR)that reduces calorie intake by 20-50%without causing malnutrition is a classical anti-aging intervention.Abundant studies show that short-term CR has a cardioprotective effect.Short-term CR started in old could improve age-related ventricular remodeling,decline of cardiac function,mitochondrial dysfunction,oxidative stress and apoptosis.Short-term CR started in middle-age could not only reduce mitochondrial oxidative stress and oxidative damage,but also improve ischemic tolerance.Although lots of studies find that short-term CR started in young could improve ischemic tolerance and impaird cardiac structure and function of subjects with metabolic disorders.However,these protection are based on myocardial ischemia reperfusion or metabolic disorders such as obesity or diabetes.In addition,sometimes it was found that the benefits of short-term CR on mitochondria and cardiac function couldn’t been observed in young heathy heart.Some studies found short-term young-onset CR even induced accumulation of myocardial triglycerides and suppressed left ventricular diastolic function in otherwise healthy subjects.Thus,with respect to cardioprotective properties of short-term CR,the experimental data are not uniformly in accord.The age when CR is started,the severity of restriction,and physical states of heart determine the effect of CR.Due to the different state of heart during aging,it is important to investigate whether a similar degree of short-term CR could also have a similar cardioprotective effect.Therefore,our study investigated the change of cardiac structure and function during aging,as well as the effect of short-term CR on heart of mice at different age,so as to find the opportunity at which time short-term CR could have cardioprotection.We then focused on the molecular mechanisms of short-term CR started at different age in regulation on cardiac aging.It would provide a new strategy for preventing and treating of cardiac aging in clinic.Our research was divided into two parts.Part Ⅰ:Distinct effects of short-term calorie restriction initiate in different age on cardiacfunction.Part Ⅱ:Molecular mechanisms of short-term calorie restriction initiate in different age in regulating cardiac function.Part Ⅰ:Distinct effects of short-term calorie restriction initiate in different age on cardiac functionObjective.Caloric restriction is a classical intervention that could delay the aging of heart.Short-term CR started in old could improve age-related ventricular remodeling,decline of cardiac function,mitochondrial dysfunction,oxidative stress and apoptosis.Short-term CR started in middle-age could not only reduce mitochondrial oxidative stress and oxidative damage,but also improve ischemic tolerance.Short-term CR started in young could improve ischemic tolerance,impaird cardiac structure and function of subjects with metabolic disorders.However,sometimes it was found that the benefits of short-term CR on mitochondria and cardiac function couldn’t been observed in young heathy heart.Some studies found short-term young-onset CR even suppressed left ventricular diastolic function in otherwise healthy subjects.Thus,the age when CR is started,the severity of restriction,and physical states of heart determine the effect of CR.Therefore,our study investigated the effect of short-term CR with similar degree on cardiac structure and function of mice at different age.Methods.We placed C57BL/6 mice on a calorically restricted diet(60%of the control diet)for 3 months initiated in different age(3 months old,12 months old and 19 month old).To assess the changes of heart,we measured the body weight and heart weight of mice.We evaluated the cardiac systolic function by echocardiography.Myocardium cell membrane stained with the wheatgerm agglutinin and mRNAlevels of the hypertrophic markers measured by real-time PCR were used for assessing cardiomyocyte hypertrophy.In addition,Masson’strichrome staining of heart tissue sections,mRNA and protein levels of the fibrosis markersmeasured by real-time PCR and Western Blot respectively were all used for assessing cardiac fibrosis.Real-time PCR and Western Blot were used for detecting cardiac inflammation.Furthermore,using electron microscopy,we studied not onlythe mitochondrial morphology and number,but also the lipiddroplets in cardiomyocyte.Real-time PCR was used to detect the telomere length and senescence markers.Senescence markers were also measured by Western Blot in protein level.Results.The data from this study showed age-related changes in heart including increase in heart weight,decline in systolic function,induced cardiac hypertrophy,fibrosis,inflammation and mitochondrial damage,as well as shorten of telomere and increased senescence-associated proteins.Our results suggested that short-term CR in middle-aged and old mice effectively induce regression of age-related decline in systolic function while has adverse effect in young mice.Consistent with this,short-term CR in middle-aged and old mice reduced aging-induced cardiac hypertrophy,cardiac fibrosis and cardiac inflammation,but which all aggravated in young mice.In addition,aging-induced cardiac mitochondrial damage and infiltration of lipid droplets were reduced by short-term CR in middle-aged and old mice,but both were increased by short-term CR in young mice.We also found that short-term CR in middle-aged and old mice both reduced aging-induced senescence markers and increased telomere length,but did oppositely in young mice.Conclusions.These data indicated that short-term CR started in different age have distinct effects on heart.Short-term CR retard cardiac aging in middle-aged and old mice wherase accelerate the decline of cardiac function and ventricular remodeling in young mice.Part II:Molecular mechanisms of short-term calorie restriction initiate in different age in regulating cardiac functionObjective.Evidence from animal models and preliminary studies in humans indicates that CR delays cardiac aging and prevents cardiovascular disease.These effects are mediated by a wide spectrum of biochemical and cellular adaptations,including redox homeostasis,mitochondrial function,inflammation,apoptosis,and autophagy.lt is well known that a number of nutrient-sensitive proteins have been identified in the health and longevity effects of CR,including the sirtuins,forkhead box transcription factors(FOXOs),mammalian target of rapamycin(mTOR)and Adenosine Monophosphate Activated Protein Kinase(AMPK).Activation or repression of these nutrient-sensitive signal pathways could regulate redox homeostasis,mitochondrial function,inflammation,apoptosis,and autophagy,so as to protect the heart.MiRNAs play important regulatory roles in a variety of biological processes including cardiac aging and cardiac disease.Report about the regulation role of miRNA on CR in heart is very few so far.The first part of this study suggested that short-term CR started in different age have distinct effects on heart.Therefore,we further studied the molecular mechanisms of short-term CR inregulating the cardiac aging.Methods.To elucidate the possible molecular pathways underlying the observed different effect of short-term CR on cardiac aging,we performed whole genome and miRNA microarray in myocardial tissue of CR models.Differentially expressed(DE)genes or miRNAs were identified using a threshold of fold-change≥1.5.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were used for mapping DE genes to Biocartapathways.Then we filtrated the pathway which changed in the opposite between young group and older group(middle-aged and old group).Real-time PCR and Western Blot were used for detecting key genes and proteins in the pathway.Results.Our microarray dataanalysis demonstrated that FOXO signaling pathway was down-regulated by short-term CR initiate in young mice,while up-regulated when initiate in middle-aged and old mice,suggesting FOXO signaling pathway may be involved in the different regulation of CR.In addition,the downstream of FOXO,autophagy was inhibited by short-term CR initiate in young,while promoted when initiate in middle-age and old,both confirmed by microarray and Western Blot.Since AMPK cound modulate FOXO transcription factor expression,which leads to expression of autophagy-associated genes.Then we further assessed AMPK activity and found that AMPK phosphorylation was down-regulated by CR in young mice.To the contrary,AMPK phosphorylation was up-regulated by CR in both middle-aged and old mice,suggesting that CR activated AMPK.Our microarray data analysis demonstrated that miRNAs are modulated differentially by CR beginning in different age.Several miRNAs which were down-regulated by CR in young mice but were up-regulated in middle-aged and old mice have been proved to inhibit inflammation and fibrosis.Conclusions.In summary,our data showed that AMPK activation may be a major contributor to various regulation of FOXO Signaling Pathway by short-term CR initiate in different age,thus lead to different regulation of autophagy and cardiac aging.It was also suggested that miRNAs might also involved in the regulation of cardiac aging by CR.