The Role And Mechanism Of Resveratrol In The Prevention And Therapy Of Metabolic Cardiovascular Diseases

BackgroundNowadays,approximately 290 million patients are suffering from cardiovascular disease?CVD?,which is the leading cause of prevalence and mortality,bringing a heavy burden to the economy of national development.Therefore,it is quite essential to search for novel targets as well as preventional methods for CVDs.The change in lifestyle and dietary patterns have raised the mortality of CVDs.Particularly,high-fat-diet?HFD?is the leading cause of dyslipidemia and obesity,which are major risk factors for the development of CVDs.In the liver,when fat gets overloaded by HFD,the serum lipid level will go up,leading to the inflammation and oxidative stress of endothelial cells,which is the first step of AS development.AS is the core pathological process of CVD,with coronary artery thrombosis formation,causing the irreversible ischemic injury to cardiomyocytes,resulting in heart failure or death.Optimizing dietary pattern is an economical and effective approach to preventing metabolic-related diseases.Resveratrol?RSV?is a polyphenol compound abundantly existing in different fruits and vegetables.Several studies have reported the cardiac protective function of RSV.Our group previously found that RSV could protect the HFD-induced non-alcoholic fatty liver disease?NAFLD?and AS through regulating protein kinase A?PKA?signaling pathway.However,since the subunits of PKA genes locate in different loci of chromosomes,it is hard to construct PKA global knockout animal model through gene editing,make it difficult to perform the in vivo PKA loss-of-function study of lipid metabolism in the liver.On the other hand,endothelial oxidative stress induced by HFD is closely related to the mitochondrial dynamics and redox metastasis.We found RSV could protect endothelial cells?ECs?through regulating redox homeostasis,but whether it is through the classic RSV down-stream target,Tyrosyl-tRNA synthetase?TyrRS?-Poly?ADP-ribose?polymerase 1?PARP1?and mitochondrial dynamics mechanisms remains unknown.Meanwhile,to increase the cardiomyocytes'resistance to ischemic injury and stress,it is necessary to understand the mechanism of cardiomyocytes apoptosis.Evidence showed that in the ventricle of the adult heart,there are two kinds of ventricular myocytes:mononucleated ventricular myocytes?MVMs?and binucleated ventricular myocytes?BVMs?.Compared to BVMs,MVMs are less matured and have a potential of proliferation.Since it is difficult to isolate and culture BVMs and MVMs respectively,the reaction of BVMs and MVMs under stress remains largely unknown.Identifying and illustrating the basic difference between these two subpopulations of cardiomyocytes will hopefully help us to find novel methods to protect the heart from injury.Objectives1.To construct a genetic mouse model to inhibit the activity of PKA in the liver to study the function of PKA in liver lipid metabolism in vivo by detecting the NAFLD phenotype under HFD with PKA inhibited and perform global transcriptome analysis.2.To illustrate the mechanism behind the RSV protecting ECs through regulating mitochondrial dynamics and redox metastasis,and to find preventative methods from the view of dietary pattern.3.To study the physiological reaction of MVMs and BVMs under basal condition or stress.To illustrate the gene signatures of MVMs and BVMs by single-cell transcriptome analysis.To test whether RSV could bring a protective effect to the two types of cells.Methods1.Constructing the loxP-PKAinhibitor-GFP-loxP mice,and then crossing the mice with Alb-Cre mice to have the liver tissue-specific PKA inhibited?PKAi mice?.Using WB to validate the expression of PKAi;using PKA activity assay kit to measure the PKA activity in the liver.Feeding the PKAi mice and WT mice with chow diet or HFD,and test the body weight,liver index,triglyceride in the liver.Using H&E staining to test the morphology of liver,oil red O staining to test the hepatic lipid droplet accumulation.Finally,using RNA-seq to test the transcriptome alterations in the liver of WT and PKAi mice with or without HFD treatment to explain the underlying mechanism.2.Using palmitic acid?PA?to induce oxidative stress on human umbilical vein endothelial cell?HUVEC?,then test the viability with cell counting kit 8?CCK8?and optimize the treatment condition.Using flow cytometry and JC-1 probe to test the membrane potential of mitochondria.Using confocal and electron microscope measure the morphology change of mitochondria.Using WB to detect the expression of mitochondrial fusion proteins.Using siRNA knockdown TyrRS and PARP1 in HUVEC,to test the function of TyrRS-PARP1 pathway in mitochondrial dynamics and oxidative stress.3.Using a switching mechanism at 5'end of RNA template-sequencing?SMART-seq?to test the transcriptome of manually selected mouse adult MVMs and BVMs under basal condition or with isoproterenol?ISO?treatment.Using TdT-mediated dUTP Nick-End Labeling?TUNEL?to test the apoptosis rates of MVMs and BVMs with or without ISO treatment.Using JC-1,DCFH-DA to label mitochondrial membrane potential and ROS respectively and measure them with confocal microscopy.Using DCFH-DA to label the intracellular ROS,Rhod-2 to label the Ca²⁺in mitochondria,and then measure them with confocal microscope in time-lipase scanning mode.Finally,using Trypan blue staining MVMs and BVMs pre-treated with RSV to detect the viability,and confocal microscope measuring ROS labeled by DCFH-DA.Results1.Tissue-specific PKA inhibition increased the HFD induced triglyceride accumulation in the liver.1.1 Using the loxP/Cre system,we constructed a liver-specific PKA inhibited mouse model,with highly PKAi-GFP expression in liver and reduced 61%of PKA activity.1.2 Compared to WT mice,under chow diet,PKAi mice showed no difference in body weight,liver index,or triglyceride in the liver.When fed HFD for 8 weeks,compared to WT mice,PKAi mice'liver index increased and showed more severe lipid accumulation.1.3 We analyzed the transcriptome in the liver of WT and PKAi mice with or without HFD treatment and found under chow diet the differentially expressed genes between WT and PKAi mice are mainly enriched in lipid metabolism pathway.HFD influenced the Hot Shock Proteins to stress responding pathway in PKAi mice,while influenced immune response in WT mice.2.RSV protects the PA-induced ECs oxidative stress through activating TyrRS-PARP1pathway.1.1 Treating HUVEC with 200?M PA for 24h decreased viability by 47%compared to non-PA treatment group.PA treatment decreased the viability through inducing ROS,oxidation of lipid,and decreasing SOD activity,which can be reversed by RSV.1.2 In HUVEC oxidative stress model,mitochondrial membrane potential decreased with the fission of mitochondria along with the decrease of the fusion protein?OPA1,MFN1,MFN2?expression,which can be reversed by RSV.However,when knockdown of TyrRS or PARP1,the protective function of RSV lost.3.RSV protects BVMs from ISO induced apoptosis through decreasing oxidative stress.3.1 The transcriptome of MVMs and BVMs under basal condition showed a significant difference.Highly expressed genes of MVMs mainly enriched in RNA biogenesis and anti-apoptosis,while in BVMs highly expressed genes are enriched in protein degradation and P53 mediated apoptosis.3.2 When treated with ISO,BVMs are more prone to apoptosis than MVMs,because of a higher ROS,dysfunction of mitochondrial membrane potential regulation and faster in Ca2+overload.RSV could protect BVMs from ISO induced apoptosis through reducing the production of ROS,while does not influence MVMs.3.3 The transcriptome of MVMs and BVMs under ISO treatment condition showed a significant difference.Highly expressed genes of MVMs mainly enriched in autophagy and metabolism arrest to maintain basic energy consumption,while in BVMs highly expressed genes are enriched in anabolism to maintain the cell function which will lead to apoptosis.Conclusions1.We successfully constructed a liver-specific PKA inhibition mice model with loxP/Cre system.When treated with HFD,PKAi mice showed a more severe lipid accumulation with a change in the liver transcriptome.2.RSV protect ECs from PA-induced oxidative stress through activating TyrRS-PARP1 and regulating mitochondrial dynamics.3.MVMs and BNMs are two sub-population of cardiomyocytes with a distinct difference in transcriptome and physiological functions.Under ISO treatment,MVMs are more prone to survive than BVMs,resulting in a different outcome to stress.RSV could protect BVMs from oxidative stress.In summary,our study provided a mechanism of CVD protective methods from a dietary pattern view.The PKAi mouse model study and MVMs-BVMs studies brought the therapeutic potential of RSV in the translational study.We successfully constructed a liver specific PKA inhibition mice model with loxP/Cre system.When treated with HFD,PKAi mice showed a more severe lipid accumulation with a change in the liver transcriptome.