Abstract 13585: Up Regulation of Autophagy in Hsp60 Mutant Heart is an Adaptive Response to Increased Oxidative Stress and Causes Cardiomyopathy

Introduction: Despite the recent advance of genetic studies, genetic causes of hereditary dilated cardiomyopathy (DCM) are still unknown in most cases. Heat shock protein 60 (Hsp60) is a well-known chaperonin, responsible for correct folding and transportation of cytoplasmic protein to mitochondria. This study is aimed to investigate whether dysfunction of Hsp60 leads to cardiomyopathy in a fish model. Methods: We previously developed a zebrafish mutant, nbl, which has a missense mutation in hsp60, leading to the loss of function. To evaluate the phenotype of cardiomyopathy in nbl, we performed RT-PCR, western blot and immunohistochemistry of the hearts. Results: Homozygous nbl embryos showed lower survival rate (65%), compared to 81% in wild-type (WT) embryos, when subjected to 33°C (stress condition). We observed pericardial edema in 92% of nbl homozygous mutants. Also, nbl homozygotes showed sudden death at around 8 months post fertilization (mpf), when grown in non-stress condition. At 8 mpf, nbl mutants showed dilated heart and high expression of reactive oxygen species (ROS). Both mRNA and protein levels of Hsp60 were similar in nbl homozygotes and WT, at 3 mpf but, much higher expression of Hsp60 in nbl homozygotes was observed at 6 mpf, beginning of death of nbl homozygous mutants. Electron microscopy analysis showed dark mitochondria, disrupted sarcomeric structure and higher number of autophagosomes in nbl homozygote hearts at 8 mpf. We, then, analyzed autophagy related genes and found that atg5, atg3 and gabarap mRNAs were increased in nbl homozygotes, suggesting the increased autophagy might underlie the pathogenesis of DCM. Furthermore, analysis of genetically unrelated patients with familial DCM, who had no mutations in the known DCM-causing genes, identified an hsp60 mutation in one DCM family in which two of four mutation prone individuals died suddenly. Over expression of nbl mutation or DCM-associated hsp60 mutation, but not normal hsp60, increased autophagosomes in Hela cells carrying GFP-LC3. Conclusions: Functional loss of Hsp60 increased oxidative stress in the heart, which leads to increased autophagy and confer the susceptibility to cardiomyopathy.