Studies On CNPase In Regulating Mitochondrial Calcium Overload Through Mitochondria Associated Endoplasmic Reticulum Membrane

Heart failure is the end stage of cardiovascular diseases,which is characterized by inadequate cardiac output and insufficient blood supply to tissues and organs.1.3% of the population over 35 years old in China suffers from heart failure(HF).There are four stages of heart failure ranging from being at risk of heart failure to having advanced heart failure.With the increasing incidence of hypertension,diabetes and coronary heart disease,the HF incidence in China is rising.The state-of-art HF treatment is challenged with poor long-term prognosis,and novel therapeutics is dewanding.The cardiac energy metabolism hypothesis is a way to explain HF pathogenesis,which is based on the data in clinical practice.Mitochondrion is the main organelle of ATP production and the dysfunction contributes to the onset and progression of heart failure.Calcium plays a fundamental role in maintaining mitochondria energy homeostasis and myocardial contraction.2’,3’-Cyclic nucleotide 3’-phosphodiesterase(CNPase),an atypical phosphodiesterase hydrolyzing 2’,3’-cyclic nucleotides,is involved in regulating mitochondria function.The mitchondria associated endoplasmic reticulum membrane(MAM),a physical junction structure associating endoplasmic reticulum(ER)and mitochondria,is essential for the exchange of ER-mitochondria calcium,lipids and metabolites.Myocardial hypertrophy is an early stage of heart failure.This study aims to articulate that CNPase regulates the mitochondria calcium and energy homeostasis through MAM.The thesis consists of following chaptors,(1)the establishment of CNPase-deficient myocardial cell line.The CRISPR/Cas9 system is a new efficient and convenient gene editing tool.A dual sg RNA targeting the first exon sequence of CNPase was designed,and the CRISPR/Cas9 recombinant vectors were constructed.H9C2 cells were transfected and screened with puromycin to eliminate the untransfected cells.Thirty cells were seeded in a96-well plate to produce monoclonal cell lines genotyped by Sanger sequencing and immunoblotting.(2)Regulation of mitochondrial energy homeostasis by CNPase.CNPase knockout reduced mitochondria calcium overload,the depolarization of mitochondria membrane potential,ROS accumulation,and apoptosis susceptibility.By using the Seahorse XFe96 energy analyzer,CNPase deficiency was found to preserve the mitochondria respiration and ATP production.(3)CNPase is a constituent of MAM and regulates calcium overload.The cellular components,including endoplasmic reticulum,crude and purified mitochondria,and MAM,were separated by ultracentrifugation to explore the sub-location of CNPase.The scanning electronic microscopy demonstrates that CNPase deficiency significantly reduced the ER-mitochondria contact and the calcium deposition in cytoplasm,mitochondria,and ER.The protein-protein interaction between CNPase and IP3 R,VDAC,Camk II was identified by immunoprecipitation,indicating that CNPase is involved in the ER-mitochondria calcium exchange.This study reveals the sub-localization of CNPase in MAM and explores how CNPase maintains the mitochondria energy and calcium ovrload,providing an evaluation of CNPase as a therapeutic target in cardiac hypertrophy.