Identification of Ferroptosis from Severe Myocardial Infarction Induced in Neonatal Mice Based on Cellular Transcriptome Sequencing


Student thesis: Doctoral Thesis

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Award date1 Nov 2023


Cardiac diseases lead to millions of deaths yearly. This is because the adult heart is unable to fully recover after biophysical damage, which severely affects its function. However, during the first week of life, mammals can recover most of the injured heart tissue to some extent. Therefore, understanding which molecules are present after a cardiac injury will help to identify targetable therapeutics. In the present thesis, I prepared a myocardial infarction model in mice using cryoinjury techniques, genetic approaches, and inhibition of the novel cell death process known as ferroptosis.

Chapter I briefly describe the techniques I learned during the first two years of my PhD. I present pilot projects conducted when learning how to prepare the myocardial infarction model in adult and neonatal mice. From my cryoinjury trials in adult mice, I found that extensive collagen deposition could be observed along the non-resolved scar. I adjusted the protocol for performing cryoinjury in both adult and neonatal mouse hearts. The adult cryoinjury model was successfully adapted without requiring a mechanical ventilator. I adapted the neonatal cryoinjury protocol from different research groups. I used a different size of cryoprobe to induce the lesion over the neonatal ventricle, obtaining similar characteristics of an infarcted heart. Another adaptation from a published protocol was aimed at preventing cannibalism and increasing the survival rate after surgical intervention by adding surgical glue to the tail of the mother mouse. Overall, the cryoinjury technique was reliable with these modifications, and the characteristics of the injury mimicked the characteristics of MI.

Chapter II describes the principal studies of my doctorate project, where I investigated heart regeneration in neonates. Here, I describe how I generated two different sized injuries in neonates and how I performed RNA-sequencing, selecting candidate genes to induce cardiac regeneration. My initial hypothesis was that genes related to the extracellular matrix might be up-regulated the most. As expected, an extracellular matrix gene, Spp1 was the most up-regulated gene in both types of cryoinjured hearts. However, another set of genes was the main focus of interest since they were related to a novel type of cell death, called ferroptosis. Studying those candidate genes will provide an interesting area of research to study heart regeneration.

Chapter III describes the experiments I conducted to test the hypothesis based on the candidate genes identified in Chapter II. My hypotheses were focused on investigating the role of Ctss, a gene that encodes cathepsin S (CatS), as my first target of observation. I investigated if CatS might participate in the expansion of the severe cryoinjury. First, I targeted Ctss since it is an extracellular matrix gene and it is clustered among other genes related to ferroptosis. I used an in vitro approach testing the product of Ctss, CatS, in different cell types in the infarcted heart using histological methods of assessment. In addition, I used in vitro approaches to target CatS in different cell lines, especially in fibroblasts, in response to oxidative stress. I also observed the protein levels of the other candidate genes. This chapter contains the preliminary results of two principal candidate genes, Ctss and Hmox1.

Chapter IV presents results from experiments conducted to demonstrate the novel cell death process, ferroptosis, as a potential new target to prevent cell death using a severe myocardial infarction model using neonatal mice. To test the hypothesis that ferroptosis is an event that occurs after myocardial infarction, then I used the severe cryoinjury model and treated P1 mice with the anti-ferroptotic drug, Ferrostatin-1 (Fer-1). I induced a severe cryoinjury on the heart of neonatal mice and treated them with Fer-1 via intraperitoneal (IP) for three weeks after cryoinjury. Then, I extracted the hearts to analyze the expression of genes using RT-qPCR. I grouped the genes by different classifications including: candidate genes (from my previous RNA-seq experiments; see Chapter II), ferroptosis markers, cellular lineage markers, fatty acid markers, cardiac proliferative markers and autophagy markers. I also performed histological measurements to test the potential of Fer-1 to induce cell proliferation. Generally, my results showed that Fer-1 induced the increase of markers related to cell proliferation, peaking at 3 days post-severe cryoinjury in P1 mice. I also tested the potential of Fer-1 to induce regeneration in cryoinjured hearts by treating P1 mice daily with Fer-1 for three weeks (before or after performing severe cryoinjury), and prior to 120 days of birth (equivalent to days after severe cryoinjury), the treatment was induced for two weeks. In brief, female hearts who had Fer-1 injection 1 to 3 hours before surgery had complete heart healing after 120 days, different from those male or female injected 30 min or 1 h after cryoinjury. Lastly, a non-specific lesion related to a pathological condition was observed in mice treated with Fer-1, suggesting no chronic affection of vital organs. My results have an impact on continuing the study of ferroptosis as a hallmark to induce heart regeneration in a neonatal cryoinjury model.

Lastly, Chapter V presents the future directions of my study. With this project, I intend to contribute to research in the Cardiac Medicine field.