The Role of Autophagy in Zebrafish Heart Regeneration


Student thesis: Doctoral Thesis

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Award date27 May 2019


Myocardial infarction (MI) is an acute heart disease causing morbidity and mortality worldwide. In humans and mice, the regenerative capacity of cardiomyocytes is very low, which is an ineffective way to heal the injured myocardium and recover cardiac function. In contrast to mammals, zebrafish possess a highly efficient regenerative capacity in adult heart after injury, and is a fascinating model organism to study heart regeneration. Understanding the healing processes not only can elucidate their underlying cellular and molecular mechanisms, but also can offer a promising therapeutic strategy for heart failure in humans.

Autophagy is an evolutionarily conserved catabolic cellular process that controls protein and organelle degradation. It occurs ubiquitously in all eukaryotic cells, and has essential roles in energy supply, cellular homeostasis, development and immunity. Zebrafish heart regeneration is a dynamic process, and a multitude of signals and factors are recruited to regulate the process. My data indicate that autophagy is also required for zebrafish heart regeneration. Following cryoinjury, autophagy is up-regulated during zebrafish heart regeneration. Interestingly, it is found that autophagy is induced not only in injured area, but also in bulbous arteriosus. Furthermore, autophagy is blocked following treatment with chloroquine, an inhibitor of autophagy, which neutralizes the lysosomal pH. Chloroquine treatment decreases cell proliferation, and delays myocardium, epicardium, endocardium and vascular endothelium regeneration and
deposition of collagen, resulting in impairment of zebrafish heart regeneration. Conversely, Autophagy is promoted following treatment with Torin 1, an inducer of autophagy, which inhibits mTOR signaling. Torin 1 treatment increases cell proliferation, and accelerates myocardium, epicardium, endocardium and vascular endothelium regeneration and deposition of collagen, leading to acceleration of zebrafish heart regeneration.

Metformin is a first-line drug for treatment of type 2 diabetes. It is well known that through activation of AMPK and inhibition of mTORC1, metformin can directly or indirectly activate several core autophagy-related proteins, therefore promoting autophagy. In my studies, autophagy was enhanced following treatment with metformin at therapeutic dose range (i.e., 50 µM). Similar to Torin 1, metformin treatment accelerates zebrafish heart regeneration after cryoinjury. Moreover, the changes of myocardial function were non-invasively evaluated by echocardiography. Metformin treatment transiently promotes ventricular systolic function for the compensation of blood supply during heart regeneration. Altogether, chronic metformin treatment can therapeutically accelerate adult zebrafish heart regeneration via induction of autophagy. These evidences suggest that metformin may have clinical value for the prevention and amelioration of myocardial infarction by enhancing cardiac autophagic activity.

The extracellular matrix (ECM) is composed of a complex set of proteins and macromolecules that provide structural scaffold and actively direct cell behavior, form and function. ECM controls cell survival, development, proliferation, cell size and shape, as well as cells interactions and migration. In zebrafish, transient fibrosis is beneficial for for cardiomyocyte proliferation and heart regeneration. My results reveal that activation of autophagy is required for transient fibrosis during zebrafish heart regeneration. Furthermore, it has been found that inhibition of TGF-β signaling with SB431542, a specific inhibitor of TGF-β type I receptors Alk5/4, not only impairs collagen deposition, but also reduces the activity of autophagy after cryoinjury. In summary, TGF-β activates autophagy for collagen deposition in injured zebrafish heart, and autophagy is a novel aspect of biological effects of TGF-β signaling. Zebrafish heart regeneration is a dynamic biological process that multiple signals and factors are activated for hemostasis, inflammation, proliferation and maturation. I used RNA sequencing (RNA-Seq) to carry out genome-wide transcriptional profiling of the regenerating hearts at 4 dpc and 7 dpc. After RNA-Seq and bioinformatics analysis, differentially expressed genes (DEGs), including 134 DEGs at 4 dpc and 64 DEGs at 7 dpc, were identified and were classified into different functional categories. The data indicate that ECM components including collagen, fibronectin and thrombospondin iv stimulate integrin receptors which transduce the activities of Rap1 and PI3K/AKT in regenerating zebrafish heart. Activation of Rap1 further regulates cell migration by modulating actin cytoskeleton, cell adhesion and aggregation. Concomitantly, PI3K/AKT signaling also interacts with FoxO, HIF-1, and Hippo signaling pathways for promoting cell survival, proliferation, growth and metabolism, thereby contributing to zebrafish heart regeneration. Moreover, it is possible that PPAR pathway is triggered for response to adipocyte differentiation, lipid and glucose metabolism, and inflammation.These results highlight the power of high-throughput RNA-Seq to identify and provide some insight into molecules and pathways whose roles in regulating zebrafish heart
regeneration after cryoinjury