Atg7 induces basal autophagy and rescues autophagic deficiency in CryABR120G cardiomyocytes

Abstract

Rationale: Increasing evidence suggests that misfolded proteins and intracellular aggregates contribute to cardiac disease and heart failure. Several cardiomyopathies, including the αB-crystallin R120G mutation (CryAB(R120G)) model of desmin-related cardiomyopathy, accumulate cytotoxic misfolded proteins in the form of preamyloid oligomers and aggresomes. Impaired autophagic function is a potential cause of misfolded protein accumulations, cytoplasmic aggregate loads, and cardiac disease. Atg7, a mediator of autophagosomal biogenesis, is a putative regulator of autophagic function.

Objective: To determine whether autophagic induction by Atg7 is sufficient to reduce misfolded protein and aggregate content in protein misfolding-stressed cardiomyocytes.

Methods and results: To define the gain and loss of function effects of Atg7 expression on CryAB(R120G) protein misfolding and aggregates, neonatal rat cardiomyocytes were infected with adenoviruses expressing either wild-type CryAB or CryAB(R120G) and coinfected with Atg7 adenovirus or with Atg7 silencing siRNAs to produce gain-of or loss-of Atg7 function. Atg7 overexpression effectively induced basal autophagy with no detrimental effects on cell survival, suggesting that Atg7 can activate autophagy with no apparent cytotoxic effects. Autophagic flux assays on CryAB(R120G)-expressing cardiomyocytes revealed reduced autophagic function, which probably contributed to the failure of misfolded proteins and aggregates to be cleared. Coexpression of Atg7 and CryAB(R120G) significantly reduced preamyloid oligomer staining, aggregate content, and cardiomyocyte cytotoxicity. Conversely, Atg7 silencing in the CryAB(R120G) background significantly inhibited the already reduced rate of autophagy and increased CryAB(R120G) aggregate content and cytotoxicity.

Conclusions: Atg7 induces basal autophagy, rescues the CryAB(R120G) autophagic deficiency, and attenuates the accumulation of misfolded proteins and aggregates in cardiomyocytes.

Figure 1. Atg7 induces basal autophagy

A, Representative immunoblots show a 7.8-fold increase in Atg7 protein levels in Ad Atg7 versus Ad LacZ, without induction of other autophagic proteins (n=4/treatment). B, Ultrastructural analyses confirmed an increase in autophagic structures with Atg7 expression. Amphisomes are denoted by black arrows. Multilamellar bodies are denoted by black asterisks. C, Autophagosomes are apparent as GFP-LC3-positive puncta (white arrows; n=4 wells/group). D, Autophagic flux assay shows a significant increase in GFP-LC3 puncta in Ad Atg7-infected cardiomyocytes in the presence of the lysosomal inhibitor BafA1. E, Autophagic flux assay shows increased LC3-II levels in representative immunoblots in Ad Atg7 versus Ad LacZ infected RNCs treated with BafA1 (n=6/treatment). F, LC3-II synthesis is significantly increased by Ad Atg7 expression with lysosomal inhibition. *P<0.05, significant difference Veh versus BafA1 treatment. †P<0.05, significant difference Ad Atg7 versus Ad LacZ infection.

Figure 2. Atg7 rescues autophagic inhibition of CryAB^R120G

A, Representative immunoblots of the autophagic flux assay shows reduced LC3-II levels with CryAB^R120G expression relative to CryAB^WT with and without lysosomal inhibitor BafA1 (n=4/treatment). B, LC3-II synthesis is significantly decreased with CryAB^R120G expression. *P<0.05, significant difference Veh versus BafA1. †P<0.05, significant difference CryAB^R120G versus CryAB^WT. C, A Representative immunoblots of the autophagic flux assay illustrating that Atg7 rescues the LC3-II deficiency of CryAB^R120G in the presence of BafA1 (n=6/treatment). D, LC3-II levels are elevated with CryAB^R120G and Atg7 co-expression. *P<0.05, significant difference between BafA1 versus Veh. †P<0.05, significant difference between LacZ+CryAB^R120G versus LacZ+CryAB^WT. ‡P<0.05, significant difference between Atg7+CryAB^R120G versus LacZ+CryAB^R120G. E, A representative immunoblot shows autophagic marker levels with CryAB^R120G and Atg7 co-expression (n=4/treatment).

Figure 3. Atg7 expression reduces CryAB^R120G aggregate content

A, Immunofluorescent staining shows decreased CryAB (green) aggregate content when Atg7 is co-expressed with CryAB^R120G. Cardiomyocytes counterstained by a TnI antibody (red). B, Aggregate area relative to the cardiomyocyte area (n=4/treatment). *P<0.05, significant difference between CryAB^R120G versus CryAB^WT. †P<0.05, significant difference CryAB^R120G+Atg7 versus CryAB^R120G+LacZ. C, The filter trap assay shows reduced insoluble CryAB and Ubiquitin with CryAB^R120G and Atg7 co-expression. D, Densitometry for insoluble CryAB and Ubiquitin (n=4/treatment). *P<0.05, significant difference between CryAB^R120G versus CryAB^WT. †P<0.001, significant difference between CryAB^R120G+Atg7 versus CryAB^R120G+LacZ.

Figure 4. Increasing Atg7 expression causes a dose-dependent reduction in aggregate content

A, RNC’s were infected with 100 MOI AdLacZ, or 10, 30, or 100 MOI AdAtg7 to show that Atg7 protein levels increase in a dose-dependent manner (n=4/group). B, RNCs were co-infected with 10 MOI of AdCryAB^R120G and 10, 30, or 100 MOI of AdLacZ or AdAtg7, respectively. Five days post-infection cells were fixed and immunostained with CryAB (green, aggregates), TnI (red, cardiomyocytes) and TO-PRO-3 (blue, nuclei). C, Aggregate area relative to the cardiomyocyte area (n=8/treatment). *P<0.001, significant difference between Atg7 versus LacZ. †P<0.1, significant difference between 10 versus 30 versus 100 MOI of Atg7. D, Autophagic flux was evaluated by co-infecting Ad GFP-LC3 with Ad LacZ (100MOI) or Ad Atg7 (10, 30 or 100 MOI) in the presence or absence of BafA1. E, Autophagic flux is significantly increased in a dose-dependent manner with increasing MOI of AdAtg7 (n=4 wells/group). *P<0.001, significant difference between BafA1 treated versus BafA1 treated with additional Atg7.

Figure 5. Atg7 reduces CryAB^R120G PAO content and cytotoxicity

A, Immunofluorescent staining shows decreased pre-amyloid oligomer PAO (green) content when Atg7 is co-expressed with CryAB^R120G. Cardiomyocytes counterstained by a TnI antibody (red). B, PAO area relative to the cardiomyocyte area (n=4 wells/treatment). *P<0.001, significant difference between CryAB^R120G versus CryAB^WT. †P<0.05, significant difference between CryAB^R120G+Atg7 versus CryAB^R120G+LacZ (P<0.001). C, CryAB^R120G adenylate kinase and lactate dehydrogenase release are reduced by Atg7 co-expression at 5 days post-infection (n=4/treatment). *P<0.001, significant difference between CryAB^R120G versus CryAB^WT. †P<0.001, significant difference between CryAB^R120G+Atg7 versus CryAB^R120G+LacZ.

Figure 6. Atg7 silencing inhibits autophagy and increases aggregate content of CryAB^R120G

A, Representative blot showing autophagic flux assay with reduced LC3-II levels from Atg7 knockdown with BafA1 (n=4/treatment). B, Decreased LC3-II protein levels with Atg7 siRNA. *P<0.05, significant difference between Veh versus BafA1. †P<0.05, significant difference Atg7 siRNA versus Ctrl siRNA. C, Increased CryAB aggregate content (green) when Atg7 siRNA is coupled with CryAB^R120G. Cardiomyocytes are counterstained with a TnI antibody (red). D, Aggregate area relative to the cardiomyocyte area (n=4/treatment) *P<0.001, significant difference CryAB^WT versus CryAB^R120G. †P<0.00 1, significant difference CryAB^R120G+Atg7 siRNA versus CryAB^R120G+Ctrl siRNA.

Figure 7. Atg7 siRNA increases CryAB^R120G PAO content and cytotoxicity

A, PAO content (green) is increased when Atg7 siRNA is co-expressed with CryAB^R120G. Cardiomyocytes are counterstained with a TnI antibody (red). B, PAO area relative to the cardiomyocyte area (n=4/treatment). *P<0.05, significant difference CryAB^WT versus CryAB^R120G. †P<0.05, significant difference between Atg7 siRNA versus Ctrl siRNA. C, CryAB^R120G adenylate kinase and lactate dehydrogenase release are increased by Atg7 siRNA co-expression at 5 days post-infection (n=4/treatment). *P<0.001, significant difference between CryAB^R120G versus CryAB^WT. †P<0.001, significant difference between CryAB^R120G+Atg7 siRNA versus CryAB^R120G+Ctrl siRNA.