A De Novo Mutation In CHKB Induces Serious Dilated Cardiomyopathy By Disturbing Phospholipids Homeostasis

Background and AimHeart failure affects 40 million people worldwide and is associated with significant clinical burden and economic cost worldwide.Dilated cardiomyopathy(DCM)is the major common cause of heart failure,accounting for approximately 36%of all heart failure cases and incidence in 1/250 of the general population¹.DCM defined as the presence of both left ventricular enlargement and systolic dysfunction,excluding hypertension,heart valvular disease and other causes,is easier to diagnose whereas the cause of the disease is far from clear^2,3.DCM is now commonly defined as genetically heterogeneous and inheritable heart disease.At present,more than 100 genes related to the pathogenesis of DCM have been studied by many laboratories,and studies have shown that mutations in genes encoding cytoskeleton,sarcomere,mitochondria,desmosome,nuclear membrane and RNA-binding proteins are all related to the occurrence of DCM⁴.However the DCM related gene is not sufficient to explain all of the genetic causes of DCM,in view of the genetic mechanism of DCM,the clinical use of whole exome sequencing for the genetic mechanism of dilated cardiomyopathy is increasing,and information about the presence of variations in undefined genes can be obtained.The identification and validation of rare genetic variants in DCM,as well as advances in response to disease progression and treatment,will eventually lead to precision medicine for DCM and hopefully improve outcomes for patients and their families.Finding and correcting the function of molecular abnormalities in DCM is an urgent problem⁵.A case of severe DCM attracted our attention.The patient had early onset and severe DCM.The prognosis is poor and he still died of again heart enlargement after transplantation.The patient did not have any other risk factors for DCM,in view of the possible genetic mechanism of DCM,we analyzed the whole exome sequencing data and found that the patient carried no genetic variants known to contribute to the development of DCM whereas a new mutation in the CHKB gene.The CHKB gene encodes the first key enzyme in the phospholipid synthesis pathway,a pathway in mammalian cells that makes the main phosphatidylcholine(PC)and phosphatidylethanolamine(PE),essential for life⁶.Abnormal phospholipid metabolism is involved in the occurrence and development of many diseases.Recent studies have found changes in phospholipid metabolism in the pathological process of heart,and the underlying mechanism remains unclear ^7-10.CHKB knockout mice can spontaneously develop juvenile bone deformities and hindlimb muscular dystrophy.The study firstly demonstrated that decreased CHKB activity and tissue PC level can lead to muscular dystrophy^11,12.CHKB catalyzes the first reaction in the de novo synthesis pathway of PC and accounts for 95%of the heart's choline kinase activity¹³,but its role in the heart has not been reported.The study aimed to study the role and the underlying mechanism of the de novo mutation in DCM,and explore the potential therapeutic targets of DCM.Methods and Results1.The de novo mutation of CHKB gene leads to the abnormal CHKB splicing and expressionIn this study,we found a case of severe DCM patient.For the possible genetic mechanism of patients with DCM,we conducted the whole exon sequencing of the patient and his parents.Through candidate gene strategy and family analysis,we found that a new mutation in the CHKB gene carried by the patient may be the pathogenic cause of DCM.We then validated the mutation by Sanger sequencing and found that the mutation at the splicing site.Using c DNA amplification and bioinformatics prediction,we found that the mutation resulted in abnormal splicing and expression of CHKB.HE staining revealed that the myocardial fibers were denatured,swollen and partially compensatory hypertrophy,vacuoles appeared in the cytoplasm.Moreover,Sirius Red staining showed severe fibrosis.Further fluorescence staining showed that the expression of CHKB in heart was decreased and mitochondrial distribution was abnormal.2.CHKB knock in CMs displayed similar morphology signatures and phospholipids metabolism alterations to that of CHKB knockout CMsIn order to further examine the effect of the de novo mutation on the function of CHKB and the effect of CHKB deficiency on the function of cardiomyocytes,we used the CRISPR/Cas9 system to generate CHKB de novo mutation knock-in and CHKB knockout CMs lines.Phalloidine staining revealed that CHKB^KI and CHKB^KO CMs were round and the cell area was increased compared with CHKB^WTCMs.Lipidomic analysis showed that the lack of CHKB interfered with the balance of phospholipid metabolism in the heart,leading to a significant decrease in the main phospholipids in cardiomyocytes.Further analysis of individual phospholipids showed that the changes of phospholipid species in CHKB^KI CMs lines were similar to those in the CHKB^KO CMs lines and that the changes of phospholipid fatty acid chain induced by CHKB deficiency were consistent with the changes of phospholipid fatty acid chain during heart failure,suggesting that the disorder of phospholipid metabolism caused by CHKB deficiency may be involved in the occurrence and development of heart failure.3.Cardiomyocyte-specific CHKB knockdown mice exhibited DCMIn attempt to mimic the CHKB knockdown status in the patient,CHKB cardiomyocyte-specific knockdown mice were generated by administrating r AAV-Tn T-sh CHKB.Echocardiography was then used to conduct regular detection of cardiac function in mice and we found that at the age of 20 weeks,r AAV-Tn T-sh CHKB mice developed LV dilatation and reduced ejection fraction with normal heart ventricular wall thickness.The r AAV-Tn T-sh CHKB mice exhibited decreased myocardial contraction force by using cardiac catheterization and single myocyte contractile force measurement also showed the same result.Histological staining showed that CHKB knockdown resulted in myocardial fibrosis,denatured myocardial fibers,swollen and partially compensatory hypertrophy,vacuoles in the cytoplasm.4.CHKB knockdown mice displayed abnormal phospholipids metabolism and mitochondrial defectsTo further explore the mechanisms underlying cardiac dysfunction in the absence of CHKB,RNA sequencing of mice heart was then conducted.The transcriptome analysis indicated that the top enriched KEGG pathway terms of changed genes were mostly involved in mitochondrial energy metabolism.The content of PC and PE in the whole heart and mitochondria were both dropped significantly,especially in the mitochondria.Further,transmission electron microscopy and western blot also showed significant morphology and function detects in mitochondrial,which was consistent with the severely reduced contractile force of the r AAV-Tn T-sh CHKB mice.5.CDP-choline supplement improved cardiac function in CHKB knockdown miceCDP-choline is an important metabolic substrate in phospholipid synthesis pathway.Studies have found that CDP-choline can increase the level of PC.In recent years,studies have also found that CDP-choline has a protective effect on CVD.We then fed with 500?M CDP-choline every other day in r AAV-Tn T-sh CHKB mice to detect the function of CDP-choline in hearts.The echocardiography revealed that CDP choline improved cardiac function in the CHKB knockdown mice,and hemodynamics and cardiomyocyte contractile tests showed that CDP-choline saved myocardial contractile function in the CHKB knockdown mice.Through histomorphology,we observed the damaged myocardial fiber and cardiac fibrosis caused by CHKB knockdown was ameliorated by CDP-choline.6.CDP-choline supplement promoted phospholipids homeostasis and mitochondrial energy balanceFinally,through quantitative detection of choline metabolism pathway products and phospholipids,we found that CDP-choline increased the levels of phosphocholine and the major phospholipids PC and PE.Through transmission electron microscopy,mitochondrial stress test and western blot,we found that CDP-choline improved mitochondrial defects by increasing mitochondrial matrix density and cristae and improving mitochondrial respiratory function and energy production.ConclusionConsidering the genetic mechanism of DCM in the patient,we found that the de novo mutation in the CHKB gene lead to abnormal splicing and decreased expression of CHKB.The downregulated CHKB expression resulted in a decrease in the level of major phospholipids in the heart and mitochondria.Mice with cardiac-specific CHKB knockdown showed spontaneous cardiac dilation and reduced contractile force.Lipid metabonomics showed that CHKB knockdown disturbed the phospholipid metabolism in the heart tissues and mitochondria and and transcriptome analysis showed that CHKB knockdown led to significant changes in the mitochondrial energy metabolism pathways.CHKB knockdown resulted in mitochondrial structure defects and mitochondrial respiratory chain damages.CDP-choline supplement can increase the content of PC and PE and improve the cardiac function in r AAV-Tn T-sh CHKB.We also demonstrated that CDP-choline improved mitochondrial defects by increasing mitochondrial matrix density and cristae and improving mitochondrial respiratory function.This provides us with a new strategy for the treatment of heart failure,that is,to supplement the intermediate metabolites to correct the metabolic disorders caused by the gene function defects.