Abstract 16703: p62 is Required for Pre-Amyloid Oligomer Formation in CryABR120G Hearts.
Introduction: Increasing evidence suggests misfolded proteins can cause cardiac disease and heart failure. Several cardiomyopathies, including the CryABR120G model of desmin-related cardiomyopathy, accumulate cytotoxic misfolded proteins, in the form of pre-amyloid oligomers (PAOs) and aggregates. The mechanisms responsible for promoting PAO and aggregate formation are largely unknown. Previous publications suggest the ubiquitin- and LC3-binding protein “p62” regulates the formation of misfolded proteins and aggregates. p62 functions as a cargo receptor for autophagy by binding to ubiquitinated, misfolded proteins and aggregates.
Hypothesis: Loss of p62 will reduce PAO and aggregate content in CryABR120G expressing cardiomyocytes and hearts.
Methods: Rat neonatal cardiomyocytes were infected with adenoviruses to overexpress wild-type CryAB or CryABR120G and were transfected with negative control or p62 silencing siRNAs. CryABR120G transgenic mice were crossed with p62-null mice to study the necessity of p62 in forming misfolded protein intermediates in vivo.
Results: Previously, autophagic deficiency has been shown to induce protein misfolding and aggregate formation in a p62-dependent manner. Autophagic flux assays show that CryABR120G expressing cardiomyocytes have an autophagic deficiency. CryABR120G expression induces p62 protein levels in vitro and in vivo 7.5-fold, respectively. Immunohistochemistry shows that p62 localizes in CryABR120G aggregates. Silencing p62 expression in CryABR120G expressing cardiomyocytes results in decreased PAO and aggregate content, with a concomitant reduction in cytotoxicity. Loss of p62 in CryABR120G expressing hearts causes an overt loss of PAO staining. However, p62 deficiency has no effect on CryABR120G aggregate content in vivo. The mechanism by which loss of p62 prevents PAO formation in vivo remains to be defined. Autophagic flux assays show that the loss of p62 does not effect autophagic function in vitro, suggesting that p62-dependent PAO formation occurs by an autophagy-independent mechanism.
Conclusions: p62 is necessary for PAO formation in the CryABR120G model of heart failure.
- © 2010 by American Heart Association, Inc.