A Study Of The Pathogenesis Of Cardiac Toxicity Induced By Amyloidogenic Light Chains

Background and ObjectivesLight chain(AL)amyloidosis is a lethal plasma cell disorder.In AL amyloidosis,mismatched immunoglobulin light chains deposit in various organs,leading to organ dysfunction.The prognosis of patients with AL amyloidosis is mainly determined by the severity of cardiac involvement.It is recognized that the deposition of fibrils of abnormal light chains(LC)in heart as well as the direct cytotoxicity of AL LCs both deteriorate the cardiac function.However,the molecular mechanisms of developing AL amyloidosis are yet unknown.This study aims to figure out the way of internalization and subcellular localization of AL LCs,and to study the manifestations of AL LC-induced cardiac cytotoxicity and its related molecular mechanisms.MethodsThe AL LC protein was expressed using a eukaryotic cell protein expression system.The H9C2 cell line was used in this study.Fluorescent confocal microscope was used to study cellular internalization of AL LCs with different incubation time,and to determine the effect of different inhibitors on AL LC's cellular internalization.Lysotracker and Mitotracker were used to mark lysosomes and mitochondria.The subcellular localization of AL LCs as well as the effect of AL LCs on subcellular structures were identified using a confocal microscope and a transmission electron microscope(TEM).A TUNEL assay and a CCK-8 test were used to study the apoptotic and toxic effect of AL LCs on cells.DCFH-DA was chosen to detect the cellular ROS level through flow cytometry.Immunofluorenscence assay,Western blot,and TEM were used to detect autophagy flux and molecular changes on autophagy pathway.Finally,transcriptome sequencing and time series analysis were used to investigate the molecular mechanisms of AL LC-induced cytotoxicity at different incubation time(3,6,12,and 24 hours).ResultsFor AL LC internalization and subcellular localization:AL LC was found inside H9C2 cells after 3 hours' incubation.Besides,the amount of internalized AL LC was increased as incubation time passed by,which was far more than that of multiple myeloma(MM)LC.Both Dynasore(P = 0.042)and Genistein(P = 0.007)significantly reduced the internalization of AL LCs into H9C2 cells.After internalized into cells,AL LCs mainly presented a parinuclear location.Though AL LCs didn't colocalize with mitochondria,the mitochondria significantly changed into shortened round shape,which was swollen and vacuolated,with widened cristae(P = 0.03).AL LCs can largely colocalize with lysosome.For the cardiac cytotoxicity of AL LCs:Cellular apoptosis rate wasn't affected when treated with less than 120 ?g/ml AL LC,neither was cell viability affected when treated with less than 240 ?g/ml AL LC.The ROS levels were remarkably increased in H9C2 cells treated with 240 ?g/ml AL LC for 3 hours(P = 0.01)and 24 hours(P = 0.004).To note,cells treated with AL LC had an increase in autophagy flux,which presented as increased number of autophagosomes,increased expression of Beclin-1,and decreased expression of p62.Meanwhile,the expression of p-PDK1,Akt,p-Akt,and p-mTOR were all decreased,and p-p70s6k remained the same.For transcriptome sequencing:There were 1211 differently expressed genes identified in total,of which 73 genes were differentially expressed at all time points.Compared to other groups,AL LCs treated for 3 hours changed the most in gene expression profiling.Differentially expressed genes were classified to four groups under cluster analysis.The biological processes related to differential genes include cell death,oxidative stress,endoplasmic reticulum stress,cell signal transduction,molecular transport,and damaged DNA repair.The related pathways include p38 MAPK,FoxO,IGF-1,PI3K/Akt,Jak/Stat,Erk/MAPK etc.ConclusionsH9C2 cells can internalize AL LCs even when incubated together for as short as 3 hours;and such internalization is mainly carried out by receptor-mediated endocytosis.After internalized into cells,AL LCs present a parinuclear distribution without colocalization with mitochondria,while mitochondria exhibit round and shortened swollen shapes,with widened cristae.AL LCs can colocalize with lysosomes.AL LCs can increase ROS levels in cardiac cells.Autophagy is also activated in AL LC treated cells,probably through a non-p70s6k PI3K/AKT/mTOR pathway.AL LCs treated for 3 hours changed the most in gene expression profiling.Important pathways may include p38 MAPK,FoxO,IGF-1,PI3K/Akt,Jak/Stat,Erk/MAPK etc.