The Role And Associated Mechanisms Of TANK-Binding Kinase 1 In Pathological Cardiac Hypertrophy

Background:Heart failure(HF)is a group of syndromes with clinical manifestations of congestion in the pulmonary and(or)systemic circulation and insufficient blood perfusion in organs and tissues.It is caused by various cardiac structural or functional diseases leading to impaired ventricular filling and(or)ejection function,because of which the cardiac output can't meet the body's metabolic needs.HF is one of the leading causes of death worldwide,with high morbidity and high mortality.According to an epidemiological survey,of the total population,there are 3 to 5 people of the average 100 people having heart failure with varying degrees.Therefore,heart failure is a serious medical problem.Under the stresses of extrinsic factors(such as pressure or capacity overload caused by hypertension or valvular diseases)and intrinsic factors(such as cardiac remodeling caused by hypertrophic cardiomyopathy or myocardial ischemia),the heart will occur compensatory hypertrophy to ensure sufficient left ventricular output to meet the body's needs.However,since chronic stresses and diseases exist all the time,the heart will go from the stage of compensation to the stage of decompensation over time.Then,heart failure occurs to the heart causing various clinical manifestations because the heart can't supply enough blood to meet the body's needs at that time.Therefore,as the compensatory phase of HF,it will have great X significance in the treatment of heart failure if a therapeutic target can be found to delay or even reverse pathological cardiac hypertrophy in a timely and effective manner.Autophagy is a self-clearing process involving lysosomes in cells.It is very conservative in evolution and mainly degrades long-lived proteins,macromolecules and some damaged organelles.There are three main types of autophagy: macroautophagy,microautophagy and chaperone-mediated autophagy.Then,macroautophagy can be divided into nonselective autophagy and selective autophagy represented by mitochondrial autophagy.Although more and more studies demonstrate that autophagy plays a critical role in the development of pathological cardiac hypertrophy,the underlying regulatory mechanisms have not been fully elucidated.TANK-Binding Kinase 1(TBK1)is a serine/threonine kinase in the IKK-kinase family.It is known for inducing type I interferons(IFN-Is)production in innate immunity.TBK1 is ubiquitously expressed in the body,such as: hematopoietic tissues,hearts,lungs,kidneys and so on.Previous studies exhibited that AMPK was an upstream factor of TBK1 and could phosphorylate and activate TBK1.Besides,other studies reported that TBK1 also played an important role in autophagy,in addition to its important role in innate immunity.TBK1 can phosphorylate autophagy receptor proteins,like p62,OPTN and NDP52,enhancing their abilities to link LC3 and ubiquitinated cargos to promote degradation of these cargos through autophagy.P62 is one of the downstream factors of TBK1.And it has been reported that TBK1 can phosphorylate p62 at Ser403 to promote engulfment and degradation of damaged mitochondria through mitophagy.Previous studies have demonstrated that TBK1 was implicated in pathological cardiac hypertrophy,but whether TBK1-mediated autophagy plays a role in pathological cardiac hypertrophy is unknown.If does,then whether it is regulated by AMPK-TBK1-p62 pathway is also unknown.Therefore,we use TBK1 inhibitors or overexpressing viruses to inhibit or overexpress TBK1 respectively to observe changes of autophagy in vivo and in vitro models of pathological cardiac hypertrophy.Then,we try to clarify the specific mechanisms of TBK1-mediated autophagy in pathological cardiac hypertrophy,and provide a new therapeutic target and strategy for clinical treatment of pathological cardiac hypertrophy and heart failure.Objectives:The objectives of this study were to clarify the role of TBK1 in pathological cardiac hypertrophy and explore its underlying mechanisms,in order to provide a new therapeutic target and strategy for subsequently clinical research.Methods: 1.Construct a TAC-induced cardiac hypertrophy model and constrict aortic arch.2.In vivo experiments of TBK1 inhibition: administer TBK1 inhibitor amlexanox(30mg/kg/day)for four weeks by intragastric administration after TAC surgery.3.In vivo experiments of TBK1 overexpression: mice were transfected through an intramyocardial injection of AAV9 of TBK1 after TAC surgery.4.Detect the cardiac function by ultrasound after four weeks,and observe the overall changes of heart and measure the ratio of heart weight to body weight and the ratio of heart weight to tibia length.5.HE and WGA staining were used to observe the cross-sectional area of the heart; Masson and picrosirius red(PSR)staining were used to observe the myocardial fibrosis.6.Neonatal rat cardiomyocytes were isolated and treated with TBK1 inhibitor amlexanox(50?M)or TBK1 overexpression adenovirus(MOI=50),followed by 48h of Ang ?.7.Neonatal rat cardiomyocytes were isolated and treated with compound C or AICAR, followed by 48 h of Ang ?.8.TRITC Phalloidin staining was used to measure the cardiomyocyte surface area.9.JC-1 staining was used to detect the mitochondrial membrane potential of cardiomyocytes.10.qPCR was used to detect the BNP m RNA and ?-MHC m RNA in cardiomyocytes.11.A laser confocal microscope was used to analyzed the colocalizations of COXIV and LC3,COXIV and p-p62,COXIV and TBK1 as well as TBK1 and p-p62.12.Western Blot was used to detect the levels of AMPK,p-AMPK,TBK1,p-TBK1, LC3,p62,p-p62(Ser403)and so on. Results:Part One: TBK1 inhibition improves TAC or Ang ?-induced cardiac hypertrophy phenotype through promoting autophagy 1.Cardiac function indexes LVEF and LVFS in mice were significantly reduced (p<0.05)while LVEDd and LVESd were significantly increased(p<0.05)after TAC surgery compared with Sham group;Based on those results,LVEF and LVFS were increased(p<0.05)while LVEDd and LVESd were decreased(p<0.05)after being treated with TBK1 inhibitor Amx compared with TAC group.2.The whole heart of mice was enlarged after TAC surgery compared with Sham group.Then,it was reduced after being treated with TBK1 inhibitor Amx compared with TAC group.3.Heart weight/body weight(HW/BW)and heart weight/tibial length(HW/TL)ratios were significantly increased(p<0.05)in mice after TAC surgery compared with Sham group;Then,they were reduced(p<0.05)after being administrated with TBK1 inhibitor Amx compared with TAC group.4.According to the HE and WGA staining,the cross-sectional area of heart was significantly increased(p<0.05)after TAC surgery compared with Sham group; Then,it was reduced(p<0.05)after being administrated with TBK1 inhibitor Amx compared with TAC group.5.According to the Masson and PSR staining,myocardial fibrosis was increased (p<0.05)after TAC surgery compared with Sham group.Then,it was lessened(p<0.05)after being administrated with TBK1 inhibitor Amx compared with TAC group.6.Cardiomyocyte surface area was significantly increased(p<0.05)after Ang ? stimuli compared with CON-DMSO group.Then,it was reduced(p<0.05)after being treated with TBK1 inhibitor Amx compared with Ang ?-DMSO group.7.The mitochondrial membrane potential of cardiomyocytes was significantly downregulated(p<0.05)after Ang ? stimuli compared with CON-DMSO group.Then,it was improved(p<0.05)after being treated with TBK1 inhibitor Amx compared with Ang ?-DMSO group.8.According to the qPCR results,the hypertrophic markers such as BNP and ?-MHC were significantly increased(p<0.05)after Ang ? stimuli compared with CON-DMSO group.Then,they were reduced(p<0.05)after being treated with TBK1 inhibitor Amx compared with Ang ?-DMSO group.9.According to the Western Blot results,p-TBK1 and p62 were significantly upregulated(p<0.05),and LC3 was significantly reduced(p<0.05)while TBK1 had no change after TAC surgery compared with Sham group;Based on those results, p-TBK1 and p62 were significantly reduced(p<0.05)while LC3 and TBK1 had no change after being administrated with TBK1 inhibitor Amx compared with TAC group.So were the results in vitro model induced by Ang ? stimuli.Part Two: TBK1 overexpression exacerbated TAC or Ang ?-induced cardiac hypertrophy phenotype through inhibiting autophagy 1.Cardiac function indexes LVEF and LVFS in mice were significantly reduced (p<0.05)while LVEDd and LVESd were significantly increased(p<0.05)after TAC surgery compared with Sham-NC group;Based on those results,LVEF and LVFS were further downregulated(p<0.05)while LVEDd and LVESd were further increased(p<0.05)after being transfected with AAV9 of TBK1 compared with TAC-NC group.2.The whole heart of mice was enlarged after TAC surgery compared with Sham-NC group.Then,it was further enlarged after being transfected with AAV9 of TBK1 compared with TAC-NC group.3.Heart weight/body weight and heart weight/tibial length ratios were significantly increased(p<0.05)in mice after TAC surgery compared with Sham-NC group.Then, they were further increased(p<0.05)after being transfected with AAV9 of TBK1 compared with TAC-NC group.4.According to the HE and WGA staining,the cross-sectional area of heart was significantly increased(p<0.05)after TAC surgery compared with Sham-NC group.Then,it was further increased(p<0.05)after being transfected with AAV9 of TBK1 compared with TAC-NC group.5.According to the Masson and PSR staining,myocardial fibrosis was increased XIV (p<0.05)after TAC surgery compared with Sham-NC group.Then,it was further increased(p<0.05)after being transfected with AAV9 of TBK1 compared with TAC-NC group.6.Cardiomyocyte surface area was significantly increased(p<0.05)after Ang ? stimuli compared with CON-NC group.Then,it was further increased(p<0.05)after being transfected with Ad TBK1 compared with Ang ?-NC group.7.The mitochondrial membrane potential of cardiomyocytes was significantly downregulated(p<0.05)after Ang ? stimuli compared with CON-NC group.Then,it was further downregulated(p<0.05)after being transfected with Ad TBK1 compared with Ang ?-NC group.8.According to the qPCR results,the hypertrophic marker BNP was significantly increased(p<0.05)after Ang ? stimuli compared with CON-NC group.Then,it was further increased(p<0.05)after being transfected with Ad TBK1 compared with Ang ?-NC group.9.According to the Western Blot results,p-TBK1 and p62 were significantly upregulated(p<0.05),and LC3 was significantly reduced(p<0.05)while TBK1 had no change after Ang ? stimuli compared with CON-NC group;Based on those results,p-TBK1 and p62 were further upregulated(p<0.05)and TBK1 was overexpressed successfully(p<0.05)while LC3 had no change after being transfected with Ad TBK1 compared with Ang ?-NC group.Part Three: Mitophagy triggered by TBK1 through phosphorylating p62(Ser403)unlikely played a role in pathological cardiac hypertrophy 1.P62 was increased(p<0.05)while p-p62 was reduced(p<0.05)after Ang ? stimuli compared with CON-NC group;Based on those results,p62 was further increased(p<0.05)and p-p62 was also increased(p<0.05)after TBK1 overexpression compared with Ang ?-NC group.2.The colocalization indexes such as Pearson's co-efficient and Overlap co-efficient of COXIV and LC3 were both downregulated(p<0.05)after Ang ? stimuli compared with CON-NC group;Based on those results,they were upregulated(p<0.05)after TBK1 overexpression compared with Ang ?-NC group.3.The colocalization indexes such as Pearson's co-efficient and Overlap co-efficient of COXIV and p-p62 were both downregulated(p<0.05)after Ang ? stimuli compared with CON-NC group;Based on those results,they were upregulated(p<0.05)after TBK1 overexpression compared with Ang ?-NC group.4.The colocalization indexes such as Pearson's co-efficient and Overlap co-efficient of COXIV and TBK1 were both downregulated(p<0.05)after Ang ? stimuli compared with CON-NC group;Based on those results,they were upregulated(p<0.05)after TBK1 overexpression compared with Ang ?-NC group.5.The colocalization indexes such as Pearson's co-efficient and Overlap co-efficient of TBK1 and p-p62 were both downregulated(p<0.05)after Ang ? stimuli compared with CON-NC group;Based on those results,they were upregulated(p<0.05)after TBK1 overexpression compared with Ang ?-NC group.Part Four:TBK1 regulated Ang ?-induced cardiomyocyte hypertrophy through AMPK-TBK1-p62 axis.1.P-AMPK?,p-TBK1 and p62 were significantly increased(p<0.05)while LC3 was reduced(p<0.05)after Ang ? stimuli compared with CON group;Based on those results,p-AMPK?,TBK1,p-TBK1 and p62 were significantly downregulated(p<0.05)while LC3 was increased(p<0.05)after being treated with AMPK inhibitor compound C compared with Ang ? group.2.P-AMPK?,p-TBK1 and p62 were significantly increased(p<0.05)while LC3 was reduced(p<0.05)after Ang ? stimuli compared with CON-DMSO group;Based on those results,p-AMPK?,TBK1,p-TBK1 and p62 were further increased(p<0.05) while LC3 had no change after being treated with AMPK activator AICAR compared with Ang ?-DMSO group.3.The mitochondrial membrane potential of cardiomyocytes was significantly downregulated(p<0.05)after Ang ? stimuli compared with CON group.Then,it was improved(p<0.05)after being treated with AMPK inhibitor compound C compared with Ang ? group.4.The mitochondrial membrane potential of cardiomyocytes was significantly downregulated(p<0.05)after Ang ? stimuli compared with CON-DMSO group.Then,it was further reduced(p<0.05)after being treated with AMPK activator AICAR compared with Ang ?-DMSO group.Conclusions: TBK1 is a negative regulator of pathological cardiac hypertrophy and regulating macroautophagy through AMPK-TBK1-p62 pathway may be its underlying mechanisms.