Pathophysiological Mechanisms Of Cardiac Damage And Metabolic Abnormalities Caused By Lmna P.R349W Mutation

Study Objective:The Lmna gene encodes Lamin A/C,an essential component of the nuclear lamina,playing a crucial role in maintaining nuclear shape stability,cell cohesion,regulating signal transduction,and gene expression.Lmna gene mutations can cause various diseases,collectively referred to as laminopathies,involving multiple tissues and organs.To date,more than 500 mutation types of the Lmna gene have been discovered.This study focuses on the c.1045C>T/R349W mutation site of the Lmna gene,which has a strong genotype-phenotype correlation.So far,21 cases have been reported,with the most common clinical manifestations including cardiac and metabolic abnormalities.The pathogenic mechanisms of Lmna mutations mainly include mechanical hypothesis and gene expression regulation hypothesis.It has been reported that Lmna mutations inhibit autophagy levels;when autophagy is activated by inhibiting mTOR(mammalian target of rapamycin)phosphorylation with rapamycin,it can improve the cardiomyopathy caused by the mutation and ameliorate the lipodystrophy phenotype.However,the pathogenic mechanisms of Lmna gene mutations have not been fully elucidated and require further research.Moreover,there is currently no specific treatment for Lmna mutations;the main treatment strategies are still based on the treatment of complications and comorbidities.Notably,there have been no reports on the pathogenic mechanism of Lmna R349W domestically and abroad.Therefore,this study aims to establish an Lmna R349W mouse model and perform cardiac and metabolic phenotype identification to explore possible pathogenic mechanisms,fill the research gap,and lay a theoretical foundation for drug research and development.The results of this study are expected to provide new ideas and methods for the treatment of Lmna mutation-related diseases,thereby helping to improve patients’ prognosis and quality of life.Study Methods:In this study,the Lmna R349W gene was successfully knocked-in in C57BL6/J background mice using the CRISPR/Cas9 method,establishing a homozygous mutant mouse model.Phenotypic identification was performed through various methods,including body weight,appearance changes,heart function assessment,glucose metabolism phenotype assessment,organ weight,biochemical indicators,and myocardial tissue changes.Autophagy levels and mitochondrial function in mouse cardiomyocytes were detected in vivo and in vitro using techniques such as western blotting,immunofluorescence,and transmission electron microscopy.In addition,a 2-month intervention with hypoglycemic drug pioglitazone and autophagy activator rapamycin was conducted at 6 months of age,evaluating the intervention’s effect.Autophagy and mitochondrial conditions were detected at the cellular level in HEK293 cells overexpressing Lmna R349W and primary cardiomyocytes using western blotting,immunofluorescence,and transmission electron microscopy methods,with pioglitazone and rapamycin intervention.A lipidomic study was performed on the liver and white adipose tissue to evaluate the impact of the Lmna R349W mutation on metabolism.Study Results:1.A novel Lmna R349W homozygous mutant mouse model was established,effectively simulating human Lmna R349W mutation-induced manifestations,such as cardiomyocyte hypertrophy,decreased left ventricular function,myocardial fibrosis,and glucose and lipid metabolic disorders.2.The Lmna R349W mutation leads to an imbalance in mitochondrial homeostasis and impaired autophagy flux in cardiomyocytes.3.Mutant Lamin A/C may bind to the mitochondrial fission protein Fis1,affecting mitochondrial division and fusion,leading to mitochondrial homeostasis imbalance.4.This study found that pioglitazone and rapamycin effectively improved cardiac function and metabolic phenotype in mutant mice,possibly by inhibiting mTOR phosphorylation levels,activating the autophagy pathway,promoting the clearance of damaged mitochondria,and restoring cell activity and mitochondrial membrane potential.5.This study analyzed the lipidomic characteristics of the liver in Lmna R349W homozygous mutant mice,revealing several lipid metabolism abnormalities.TAG(Triacylglycerol)and DGDG(Digalactosyldiacylglycerol)levels were significantly increased,while PC(Phosphatidylcholine)and Cer(Ceramide)levels were significantly decreased,possibly related to non-alcoholic fatty liver disease.Both pioglitazone and rapamycin improved some lipid abnormalities in the liver.6.This study also conducted a systematic lipidomic analysis of white adipose tissue,revealing that the Lmna R349W mutation affects lipid metabolism in white adipose tissue.Study Conclusion:This study established an Lmna R349W homozygous mutant mouse model,simulating various manifestations induced by the mutation in cardiomyocytes.The mutation leads to an imbalance in mitochondrial homeostasis in cardiomyocytes,which may be related to Lamin A/C binding to the mitochondrial fission protein Fis1 and impaired autophagy.Pioglitazone and rapamycin can activate the autophagy pathway by inhibiting mTOR phosphorylation levels,restoring mitochondrial function,and improving cardiac function and metabolic phenotype.This study also found that the Lmna R349W mutation affects lipid metabolism in the liver and white adipose tissue,with pioglitazone and rapamycin improving some liver lipid abnormalities.