| Background and Object:Long-term high fat diet（HFD）leads to myocardial lipid deposition,lipid metabolites accumulation,and myocardial injury.Mitochondria is the main carriers of energy metabolism,plays a key role in HFD-induced myocardial injury.Timely removal of damaged mitochondira by mitophagy is beneficial for cardiac energy metabolism and physiological function.However,over-activated mitophagy not only removes damaged mitochondria,but also initiates the removal mechanism of normal mitochondria,even programmed cell death,resulting in cardiac energy failure.Aparting from non-canonical autophagy pathways that independent on ATG5 and LC3,Rab9 dependent non-canonical autophagy has been identified recently.Beclin1 is a key regulatory protein of autophagy,which is involved in the regulation of ATG5/LC3 depemdent canonical autophagy and Rab9-depedent non-canonical autophagy.However,the role of Beclin 1 and Rab9-mediated noncanonical autophagy in long-term HFD-induced cardiomyopathy remains unclear.We set to explore the role of Rab9-mediated non-canonical autophagy and Beclin 1 heterozygous knockout(Beclin 1+/-)in HFD induced cardiomyopathy.Methods:Wide type（WT）and Beclin 1+/-mice were fed with HFD（60%fat）or normal diet（10%fat）for 24 weeks,respectively.Changes in food intake and body weight were recorded,and glucose tolerance test was performed.Western blot was used to detect the protein expressions of autophagy/mitophagy pathway and mitochondrial biosynthesis pathway.Single cardiomyocytes were isolated to detect the systolic and diastolic function of cardiomyocytes.Single cardiomyocytes isolated from WT and Beclin 1+/-mice were further stimulated with non-classical autophagy inhibitor（Brefeldin）or autophagy inhibitor（Bafilomycin A1）with or without palmitic acid（PA）to measure the diastolic and systolic function of cardiomyocytes.The mitochondria and cytoplasm of cardiomyocytes were also isolated to detect the changes of autophagy indicators.In vitro,ATG5 and Rab9 knockout lentivirus were constructed and transfected into H9C2 cardiomyocytes.After PA stimulation,mitochondrial morphology was observed under electron microscope.Mito-keima transfection combined with laser confocal microscopy was used to observe mitophagy levels.And western blot was used to detect autophagy proteins expressions in cardiomyocytes mitochondrial and cytoplasmic proteins.Results:1.HFD significantly impaired glucose tolerance and cardiomyocyte contraction in WT mice,the effects of which were rescued in Beclin 1+/-mice.2.Immunoblot results revealed that HFD activated non-canonical autophagy and inhibited conventional autophagy.Beclin 1haploinsufficiency exhibited little effect on conventional autophagy pathway（ATG5,LC3）although it further upregulated Rab9 expression,marker of non-canonical autophagy,in the face of HFD challenge.These results suggested that Beclin 1-mediated cardioprotection might be through Rab9-dependent non-classical autophagy pathway.3.Non-canonical autophagy inhibition or Beclin 1 haploinsufficiency abolished PA-induced cardiomyocyte contractile anomalies,indicating the cardioprotection role of non-canonical autophagy and Beclin 1+/-in HFD-induced cardiomyopathy.4.Although PA inhibited conventional autophagy but increased Rab 9-dependent non-canonical autophagy.Furthermore,PA-induced mitophagy led to increased mitochondira damage,increased mitophagy,and lower mitochondrial content in H9C2and ATG5 knockout H9C2 cells（conventional autophagy defficiency）.However,PA related decreased mitochondrial content was partly abolished in non-canonical autophagy deficiency cells（Rab 9 knockout）.5.HFD inhibited mitochondrial biosynthesis（PGC1 and p AMPK）.However,Beclin 1 haploinsufficiency rescued HFD-induced mitochondrial biogenesis impairment.In addition,Beclin 1+/-prevented HFD-induced cardiac dysfunction through inhibition of Rab9translocation to mitochondria,thus inhibiting Rab9-mediated mitophagy over-activation.Conclusions:These results demonstrated that non-canonical autophagy is crucial for chronic HFD/PA-induced cardiac dysfunction,and Beclin 1+/-prevented HFD/PA induced cardiac dysfunction through inhibiting Rab9 dependent non-canonical autophagy,thus attenuate non-canonical mitophagy induced mitochondria over-degradation,and rescued HFD-induced mitochondrial biogenesis impairment in metabolic cardiomyopathy. |
