The Effect of PDK4 Knockdown Delayed on D-gal Induced Cardiomyocyte Senescence by Improving Mitochondria-associated Endoplasmic Reticulum Membranes
ZHENG Yang
HUANG Yubing
HUANG Wenxia
LI Haitao
Abstract：Objective:To explore the effect of knockdown pyruvate dehydrogenase kinase 4(PDK4)on D-galactose(D-gal)-induced cardiomyocyte senescence and its regulatory effect on mitochondria-associated endoplasmic reticulum membranes(MAMs).Methods:si-PDK4 recombinant plasmid and negative control si-NC recombinant plasmid were transfected into rat cardiomyocytes H9c2 to knock down the expression of PDK4.Real-time quantitative fluorescent polymerase chain reaction(qRT-PCR)and Western Blot were used to determine the transfection effect.H9c2 cells were divided into control group,D-gal group,si-NC+D-gal group,and si-PDK4+D-gal group.The control group was cultured normally,the D-gal group was given 10 g/L D-gal induction for 48 h,and the si-NC+D-gal group and the si-PDK4+D-gal group were transfected with si-NC and si-PDK4 respectively,and then given 10 g/L D-gal for 48 h.H9c2 cells were collected from each group after completion.Cell activity assay(CCK-8)was used to detect cell activity in each group.Cell senescence status was determined by β-galactosidase activity(S Aβ-gal)staining.The levels of reactive oxygen species(ROS)were detected by 2',7'-dichlorofluorescein yellow diacetate(DCFH-DA)fluorescence probe.Cell fluorescence staining was used to detect MAMs in each group.The expression level of mitochondrial fusion protein-2(MFN2)protein in each group was determined by Western Blot.Fluo-3 AM fluorescence probe was used to detect calcium ion(Ca2+)levels in each group.Results:The relative expression of PDK4 mRNA and protein in H9c2 cells transfected with si-PDK4 was significantly lower than that in H9c2 cells transfected with si-NC and normal H9c2 cells(P＜0.05),indicating that PDK4 expression was successfully knocked down.Compared with the control group,the survival rate of H9c2 cells in D-gal group significantly decreased,the proportion of SAβ-gal labeled senile cells significantly increased,ROS level significantly increased,and MAMs co-localization significantly decreased,the relative expression of MFN2 protein significantly down-regulated,and the intracellular Ca2+level significantly increased(P＜0.05).Compared with D-gal group and si-NC+D-gal group,the survival rate of H9c2 cells in si-PDK4+D-gal group significantly increased,the proportion of senile cells labeled with SA β-gal significantly decreased,and the ROS level significantly decreased,MAMs colocalization significantly increased,MFN2 protein relative expression significantly up-regulated,and intracellular Ca2+level significantly decreased(P＜0.05).Conclusion:Knockdown of PDK4 can delay D-gal induced cardiomyocyte senescence,and its mechanism may be related to increasing the formation of MAMs to promote intracellular Ca2+operation.
Keywords：cardiomyocyteagingpyruvate dehydrogenase kinase 4mitochondria-associated endoplasmic reticulum membranesD-galactose
Publication Date：2025-01-15
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：9( 61-69 )