The Preliminary Studies On Effects Of LSDP5 On Lipid Droplets Metabolism In Mouse Cardiomyocytes

Metabolic syndrome(MS) seriously damaging human health consists of obesity,diabetes, hyperlipidemia and hypertension,all of wich are associated with the disorders of lipid metabolism. Lipid is an important form of storing energy. In cells,lipid is stored mainly in form of lipid droplets(LDs),which widely exist in all kinds of tissues and cells. The surface of LD is composed of a phospholipid monolayer,in which lipid droplet associated proteins embed. Lipid droplet associated proteins play key roles in lipid metabolism. PAT family,including perilipin,ADRP (Adipocyte Differentiation-Related Protein),TIP47,S3-12 and LSDP5 (Lipid Storage Droplet Protein 5),is an important constructive protein on the surface of LDs. LSDP5 is the newest member of PAT family,and expressed prominently in tissues that obtain energy mainly from fatty acid oxidation (heart,skeletal muscle,live,etc.),specially exhibits the highest level in heart. At present,the functional studies of LSDP5 have been mainly focused on COS-7 cell,live cell and other cells or tissues;however its roles on lipid metabolism in heart or cardiomyocytes have not been demonstrated,in order to define its effects on lipid metabolism in heart, the effects of LSDP5 on LDs in mouse cardiomyocytes were studied in this research.ObjectivesTo identify the changes of LSDP5 mRNA and protein expression levels caused by oleic acid stimulation in cardiomyocytes,and to define the effects of LSDP5 on LDs in cardiomyocytes by constructing recombinant adenovirus of LSDP5 overexpression and silence.Methods1. Suckling mouse cardiomyocytes were separated and then stimulated with different concentrations of oleic acid for 24 hours. Changes of LDs were observed through fluorescent staining with Bodipy 493/503;total mRNA and proteins in cells were extracted and their expression levels were detected by Real-time PCR and Western blot.2. Recombinant adenovirus,Ad-HA-LSDP5 and Ad-si-LSDP5 that can separately overexpress and silence LSDP5 were constructed using AdEasyTMXL adenovirus vector system. The plasmid pCMV5-HA and pSilencer-si-LSDP5 were cloned into adenovirus shuttle plasmid pShuttle. The linearized were transformed into competent cells BJ5183 for homologous recombination. The recombinant plasmid extracting from the positive clones were linearized and transfected into AD293 cells. The recombinant adenovirus were harvested and the recombinant adenovirus DNA were identified by PCR and Western blot. The recombinant adenovirus were amplified and their titer were detected. 3. Cardiomyocytes were infected respectively with Ad-HA-LSDP5 and Ad-si-LSDP5,while LSDP5 expression levels were detected by Western blot. At the same time,LDs in every group were induced by 100μM/L oleic acid and observed through fluorescent staining with Bodipy 493/503.Results1. The administration of oleic acid on seperated suckling mouse cardiomyocytes made LDs augment and increased the mRNA transcription and protein expression of LSDP5.2. The recombinant adenovirus,Ad-HA-LSDP5 and Ad-si-LSDP5 were identified by PCR,and the results showed the former could produce a 1400bp fragment,but the latter could not. AD293 cells were separately infected by the recombinant adenovirus and Western blot showed that Ad-HA-LSDP5 and Ad-si-LSDP5 made AD293 cells overexpress and silence LSDP5.3. The recombinant adenovirus could effectively overexpress or silence LSDP5 in the cardiomyocytes. After the cells were infected by the recombinant adenovirus and stimulated with 100μM/L oleic acid,the overexpression of LSDP5 resulted in LDs increasing in the number and enlargeing in the size. However,when LSDP5 was interfered by Ad-si-LSDP5,the number reduced and the volume minished.ConclusionsThe changes of LSDP5 expression during forming of LDs were preliminaryly identified in cardiomyocytes. The recombinant adenovirus,Ad-HA-LSDP5 and Ad-si-LSDP5 were constructed successfully and the overexpression of LSDP5 promoted LDs formation in cardiomyocytes. These works may lay a foundation for the further functional and mechanical researches of LSDP5 in LDs metabolism.