Abstract 1388: Heat-Shock Protein 60 is Required for Cardiac Regeneration in Zebrafish
[Introduction] It is generally believed that the mammalian cardiomyocytes could not regenerate themselves after ischemic injury. In contrast, zebrafish can regenerate not only fins but also hearts after 20% ventricular resection. Regeneration of the heart in zebrafish occurs through proliferation of cardiomyocytes localized at the leading epicardial edge of the new myocardium. The purpose of this study is to clarify what gene is involved in this process and character its gene in this process.
[Methods and Results] (1) We used a chemical mutagenesis screen to identify a zebrafish mutant with a regeneration defect of fins and heart. The obtained mutant shows no blastema (regeneration bud) formation, and named it no blastema (nbl). (2) Positional cloning revealed that heat shock protein 60 (hsp60) is critically involved in this process. (3) Mutational analyses revealed that nbl results from a Val324Glu missense mutation of this protein. (4) We surgically removed 20% of the ventricular myocardium from 1-year-old adults, and performed histological analysis of the regenerating hearts. Wild-type fish formed fibrin clots by 7 days post amputation, and cardiac myofibers penetrated the clot and constructed a new muscle around the wound by 17 days. (5) The restoration of cardiac muscle resulted from cardiomyocyte proliferation. In contrast, nbl failed to fill in the wound area with fibrin clots or collagen scar. (6) BrdU-uptake studies showed no evidence of myocyte or nonmyocyte proliferation around the wound at 12 days post amputation. (7) We injected mRNA encoding wild type hsp60 and mutant Val324Glu hsp60 into nbl embryos, and score the ability to swim and pericardial effusion. Genetic rescue experiment revealed that the wild type hsp60 significantly improved these scores, while Val324Glu hsp60 did not. (8) We injected morpholino oligonucleotide for hsp60 into wild type embryo. Most of these embryo showed pericardial effusion and showed nbl phenotype, indicating that hsp60 is the cause of nbl mutation.
[Conclusions] The heart of zebrafish with a mutation in hsp60 failed to regenerate cardiac muscle. These findings indicate that zebrafish will be useful for genetically dissecting the molecular mechanisms of cardiac regeneration.