The Effect Of Lysosomal Dysfunction In The Ischemic/Hypoxic Injuries Of Cardiomyocytes And Its Mechanisms

BackgroundIschemia/hypoxia is one of the most common pathological factors for the myocardium.In the early stages of patients with severe burn,cardiac tissues also undergo ischemic/hypoxic injuries.Autophagy is activated with ischemic injuries despite that its effect on the myocardium is still under debate.Now it has been widely accepted that the impaired autophagic flux caused by the dysfunction of the autophagic-lysosomal pathway(ALP)could be detrimental for cells.But the underlying mechanisms still remain to be determined.The blockade of autophagic flux can cause the accumulation of autophagic substrates and the enlargement of the lysosomes,which make the lysosomes more vulnerable.Lysosomal membrane injury,usually termed as lysosomal membrane permeabilization(LMP),characterized by the leakage of lysosomal contents including protons and mutiple hydrolases into the cytoplasm,often causes the acidification of the cytoplasm and even various types of cell death.Previous studies have suggested that myocardial ischemia can cause the abnormal distribution and decreased activity of the lysosomal hydrolases.However,it still remains uncertain about the occurrence of LMP and its regulatory role towards ischemic cardiac tissues.Lysosomal dysfunction is an important factor for the blockade of the autophagic flux and has been found to be pathogenic in many diseases.In recent years,the studies of the isoproterenol induced myocardial infarcted rats report that ischemia results in the dysfunction of myocardial lysosomes.Proteins that can regulate the function of lysosomes include lysosomal hydrolases,lysosomal-associated membrane proteins(LAMPs)and those participating in the sorting,transport and posttranslational modifications of the lysosomal contents.However,the molecular mechanisms of lysosomal dysfunction caused by myocardial ischemic injuries are still under mist.Lysosomal-associated membrane protein 2(Lamp2)is an important lysosomal membrane protein.The accumulation of undigested autophagic structures and substrates has been found in multiple tissues of the Lamp2 mutant mice.Previous studies have suggested that it helps to maintain the autophagic flux by promoting the fusion between autophagosomes and lysosomes.Lamp2 deficiency in hepatocytes causes a significant downregulation of the activities of lysosomal hydrolases.Meanwhile,Lamp2 can be protective against various pathogenic conditions,including oxidative stresses,chronic acidic environment of tumor cells.But it remains to be discovered whether Lamp2 is also protective for ischemic cardiomyocytes and its underlying mechanisms.Cation-independent mannose 6-phosphate receptor(Cl-MPR)is an important protein tag for the target transport of lysosomal contents and is vital for the function of lysosomes.Loss of Cl-MPR can cause the abnormal distribution and maturation of lysosmal hydrolases and the consequent dysfunction of lysosomes.The transport of Cl-MPR from the endosomes to the Golgi apparatus or the cell membranes is called retrograde transport,which is an important mechanism for the maintainance of its protein level and to avoid the degradation by the lysosomal hydrolases.However,the impacts of ischemic injuries on the function of the celluar retrograde transport haven't been investigated.The retrograde transport of Cl-MPR is mainly regulated by the retromer complex,although the mechanisms are still under debate.Studies regarding the retrograde transport of Cl-MPR in cardiomyocytes haven't been reported yet.So far,many other proteins including Rab7,SNX3,the cytoskeletal system and its motor proteins have been reported to participate in this process apart from the retromer complex.The active Rab7 recruits the retromer complex to the endosomal membranes,which promotes the progression of the retrograde transport.TBC1D5 is a relatively specific GTP ase-activating protein(GAP)for Rab7,which can promote its inactivation and dissociation from endosomal membranes.It is still controversial about the regulatory role of TBC1D5 over the retrograde transport and Cl-MPR and the underlying mechanisms are unclear.In this study,we first detected the changes of lysosomes and autophgy in cardiomyocytes with ischemia/hypoxia treatment and revealed the correlations among the impaired autophagic flux,LMP and cell injuries.We further studied the specific blocked period of the autophagic flux and its possible mechanisms,aiming to find out the proteins participating in the regulation of lysosomal function with ischemia/hypoxia stress.Therefore,the effects of Lamp2 on the ischemic cardiomyocytes and its underlying mechanisms were determined.In addition,we also detected the changes of the retrograde transport in cardiomyocytes with ischemia/hypoxia treatment and investigated the molecular mechanisms.Methods1.We constructed an in vitro cellular ischemic/hypoxic model using primary neonatal cardiomyocytes.The acridine orange(AO)release experiment,galectin 3(Gal3)immunofluorescence(IF)and the detection of the activity of cytosolic cathepsin B(Cat B)were used to determine the occurrence of LMP with ischemia/hypoxia treatment.The cell counting kit 8(CCK8)and the lactate dehydrogenase(LDH)release experiments were used to assess the cell viability and cytotoxicity respectively with the application of the inhibitors or the siRNA of Cat D.The changes of the myocardial autophagic flux caused by ischemia/hypoxia treatment were detected by western blot(WB)and the fluorescence of mCherry-GFP-LC3.Last,we determined the cell viability and cytotoxicity with drug intervention and gene regulation to alleviate and exacerbate the autophagic flux blockade respectively.2.The Cat B kit was used to detect the change of its activity caused by ischemia/hypoxia treatment.The distribution of cathepsins in the lysosomes was assessed by the immunofluorescence co-localization analysis.We also determined the protein levels of the two important tags,Cl-MPR and CD-MPR by the WB assay.These results reflected the changes of lysosomal function with ischemia/hypoxia treatment and might sugguested possilble mechanisms.3.We first explored the changes of the protein levels of the LAMPs by WB and IF assays.Then we constructed the Lamp2 overexpression cardiomyocyte model using the adenovirus carrying full-length Lamp2.Next the protein levels of Cl-MPR and CD-MPR and the localization of lysosomal cathepsins were detected with Lamp2 overexpression.Then we determined the changes of the autophagic flux,LMP and cell injury of cardiomyocytes with Lamp2 overexpression and aimed to explore the effect of Lamp2 overexression on ischemic/hypoxic injuries and its underlying mechanisms.4.To uncover the effect of the ischemia/hypoxia treatment on the myocardial retrograde transport,the changes of the distributions of Cl-MPR and glucose transporter 4(GLUT4)were assessed by the immunofluorescence co-localization analysis.With the regulation of the activities of lysosomal hydrolases,the activation of autophagy and the protein level of the retromer,the protein level of Cl-MPR was determined to evaluate the regulation of lysosomal degradation,autophagy activation and the function of the retromer complex,which further reflected the effect of ischemia/hypoxia on the function of the retrograde transport.Then,the changes of the protein levels of the retromer complex and its recruitment to the late endosomes/lysosomes were assessed with ischemia/hypoxia treatment.We next undertook IF and the fluorescence recovery after photobleaching(FRAP)experiments of Rab7 to determine its activity and membrane turnover with ischemia/hypoxia treatment.We also tested the protein level of its GAP,TBC1D5.Then the TBC1D5 siRNAs and adenovirus carrying full-length TBC1D5 were applied in the normal and ischemic/hypoxic group respectively and the changes of Cl-MPR were detected.To explore whether TBC1D5 regulates Cl-MPR through its effect on the retromer complex,we used VPS29 siRNAs in the ischemic/hypoxic group with TBC1D5 overexpression and detected the protein level of Cl-MPR.To further explore the mechanisms of the regulatory role of TBC1D5 over the retromer complex,we determined the protein levels of the retromer complex and the membrane turnover of Rab7 with the genetic means to regulate the protein level of TBC1D5 in both the normal and ischemic/hypoxic conditions.We next assessed the incorporation of the retromer to the microtubule and the motor protein with immunoprecipitation(IP)and immunofluorescence co-localization analysis after treating normal cardiomyocytes with TBC1D5 siRNAs.We also studied the recruitment of the retromer to the endosomal membranes with the immunofluorescence co-localization analysis.Last,we aimed to find out whether TBC1D5 regulated the distribution of lysosomal cathepsins.The immunofluorescence co-localization analysis of cathepsins and lysosomes was used with genetic regulation of TBC1D5 in both normal and ischemic/hypoxic conditions.Results1.Ischemia/hypoxia treatment caused the injury of the myocardial lysosomal membranes and the leakage of Cat B to the cytoplasm,which suggested the occurrence of LMP.The applications of the inhibitors of Cat B or Cat D and Cat D siRNA greatly attenuated cell injury.Ischemia/hypoxia treatment also impaired the autophagic flux of cardiomyocytes,characterized by the accumulated autophagosomes and autolysosomes.The extent of LMP and cell injury could be reversed by the methods to improve the autophagic flux and further aggravated by the exacerbation of the autophagic flux blockade.2.The activity of myocardial lysosomal Cat B significantly decreased and the distributions of Cat B and Cat D greatly reduced in the lysosomes with ischemia/hypoxia treatment.Correspondingly,the protein levels of Cl-MPR and CD-MPR were also down-regulated in the ischemic/hypoxic group.These results suggested the impairment of lysosomal function with ischemia/hypoxia treatment,which was possibly caused by the decrease of Cl-MPR and CD-MPR.3.The protein level of Lamp1 increased while that of Lamp2 decreased with ischemia/hypoxia treatment.Consequently,we infected cardiomyocytes with adenovirus carrying full-length Lamp2 to restore its protein level.Overexpression of Lamp2 significantly up-regulated the protein level of CD-MPR and increased the distribution of cathepsins inside the lysosomes with no significant effect on the protein level of Cl-MPR.The impaired autophagic flux was also improved represented by the reduction of the autophagosomes and autolysosomes with the restoration of Lamp2.Overexpression of Lamp2 remarkably reversed the leakage of Cat B and the injury of the lysosomal membranes caused by ischemia/hypoxia treatment,thus improved the cell viability and reduced the injury of cardiomyocytes.4.The distribution of Cl-MPR robustly changed with ischemia/hypoxia treatment,with more localization in the endosome/lysosome system and less localizaiton in the Golgi apparatus.Consistently,more GLUT4 was transported to lysosomes.Inhibition of lysosomal hydrolases or downregulation of the activity of autophagy significantly reversed the decrease of Cl-MPR caused by ischemia/hypoxia treatment.Loss of VPS29 in the normal group caused a remarkable reduction of Cl-MPR while the reduction in the ischemic/hypoxic group was less prominent with VPS29 siRNAs.These results suggested that the insufficient retrograde transport of cardiomyocytes of with ischemia/hypoxia treatment caused the decrease of Cl-MPR.Ischemia/hypoxia treatment didn't decrease the protein levels of the retromer complex and its recruitment to the endosomal membranes.However,Rab7 activity was shifted towards the active state,with a striking loss of Rab7 mobility/membrane turnover in cardiomyocytes with ischemia/hypoxia treatment.In accordance,the protein level of TBC1D5 also decreased,which could explain the impairment of Rab7 membrane turnover.Overexpession of TBC1D5 remarkably promoted the membrane turnover of Rab7 and increased the protein level of Cl-MPR with ischemia/hypoxia treatment while TBC1D5 siRNA transfection in the normal group caused similar results as the ischemic/hypoxic group.The increase of Cl-MPR caused by the overexpression of TBC1D5 could be inhibited by the application of VPS29 siRNAs.These results suggested that the reduction of TBC1D5 caused by ischemia/hypoxia treatment impaired the function of the retrograde transport,resulting in the decrease of Cl-MPR.Loss of TBC1D5 decreased the corporation of the retromer to the microtubule and the motor protein and inhibited its dissociation from endosomal membranes.Overexpression of TBC1D5 in the ischemic/hypoxic group significantly increased the distribution of cathepsins to the lysosomes and TBC1D5 knockdown in the normal group decreased this distribution.Conclusions1.Ischemia/hypoxia treatment caused the occurrence of LMP through impeding the autophagic flux,which could result in the cell injury of cardiomyocytes.2.Ischemia/hypoxia treatment caused the dysfunction of myocardial lysosomes,represented by the impairment of the transport and maturation of lysosomal cathepsins and the reduction of their activities.3.The protein level of Lamp2 decreased with ischemia/hypoxia stress.Overexpression of Lamp2 improved the transport defect of lysosomal cathepsins,decreased the blockade of the autophagic flux and the occurrence of LMP,which could be protective for the cardiomyocytes with ischemia/hypoxia treatment.4.The decrease of myocardial TBC1D5 with ischemia/hypoxia treatment caused the impairment of Rab7 membrane turnover and therefore the defect of the retrograde transport,which increased the degradation of Cl-MPR in the lysosomes and reduced its protein level.Deletion of TBC1D5 impeded the corporation of the retromer to the microtubule and the motor protein and thus caused the retarded dissociation of the retromer from the endosome membranes.TBC1D5 is of great significance for the proper distribution of lysosomal cathepsins.