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The Study of Mechanisms for Neonatal Mouse Heart Regeneration

Author: WangZhenHua
Tutor: HuShengShou
School: Beijing Union Medical College
Course: Surgery
Keywords: Heart regeneration transcription factor c-kit stem cell cardiomyocyte
CLC: R654.2
Type: PhD thesis
Year: 2013
Downloads: 62
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Abstract


Rational:With the development of social economy and improvements of people’s living standard, the expectancy of human’s life is also greatly elongated. Following that, the morbidity of heart disease such as coronary artery disease is also raising up. Great damage of heart muscle brought by heart attack posts a great threaten to people’s lives around the world. Cardiomyocyte regeneration seems to be a promising method to cure the patients suffering from heart attack. Huge amount of research labs are after this subject. As we all know, some animals have amazing ability in regeneration. The adult zebrafish and newt retain a remarkable capacity for cardiac regeneration throughout life, can regenerate their hearts in a very short time after up to20%surgical resection of their ventricle apex. The mammalian heart may also retain regenerative capacity because a number of studies showed that new cardiomyocytes can not only be detected in mouse heart but also in human heart. However the ability to regenerate is not sufficient enough to restore contractile function after substantial cardiac injury. To our knowledge, there is no good solution to regenerate heart muscle quickly and efficiently. A recent study showed us that1-day-old neonatal mice can fully regenerate cardiomyocytes after partial surgical resection in3weeks, characterized by cardiomyocyte proliferation with minimal hypertrophy or fibrosis. This regeneration capacity is lost by7days of age. Genetic fate mapping indicated that the majority of cardiomyocytes within the regenerated tissue originated from preexisting cardiomyocytes.Heart development is governed by a core set of evolutionarily conserved transcription factors (NK2, MEF2, GATA, Tbx, and Hand) that controls cardiac cells fates, the expression of genes encoding contractile proteins, and the morphogenesis of cardiac structurs. The cascade of these transcription factors network play an important role in serving to stabilize and reinforce the cardiac gene program and they also regulate each other’s expression. During the development of mammalian heart, cardiomyocytes originate from two cell sources, the first heart field and the second heart field. Nkx2.5is possibly the earliest marker for progenitors, which regulate the development of the first heart field in combination with transcription factors Tbx5, Tbx2and Handl. The right ventricular chamber and outflow tract are later evolutionary advancements, formed primarily from the secondary or anterior heart field. The secondary heart field seems to be very special. During evolution, the heart evolved from a single-layered tube to a more efficient and powerful pump with thick muscular chambers dedicated to receiving and pumping blood. The transition from and aquatic to a terrestrial environment required several additional adaptations of the heart to separate oxygenated and deoxygenated blood. The hearts of fish only contain a single atrial chamber which is connected to a ventricle. Amphibians have two atria separated by a septum and a single ventricle. Mammalian hearts have two atrial and two ventricles which is a more efficient four-chambered heart. Transcription factors highly expressed in second heart field include but not limited to Nkx2.5、Isll、Foxhl、Tbx20、Gata4、 Mef2c、Bop and Hand2. Transcription factor Isll is of extremely importance to second heart field. The second heart field is absent in Isll mutants. These regulating transcription factors for embryogenesis can also play important role in adult heart regeneration. Study in zebrafish indicated that Gata4is strongly expressed in surgery site. New muscle come from pre-existing cardiomyocytes expressing transcription factor Gata4.Endogenous regeneration is present in organs including the skin, liver, kidney, bone marrow and skeletal muscle. The aging and dying cells are substituted by the proliferation and differentiation of stem cell. There are some well-established markers for stem cell, such as c-kit (tyrosine protein kinase) and SCA1(stem cell antigen1). There is one c-kit+CPC for every approximately30,000cells in human heart. These CPCs regulate myocyte turnover and repair after injury. Study in adult mouse showed that these precursor cells might participate in the formation of new cardiomyocytes after injury but do not contribute significantly to the formation of new cardiomyocytes during normal aging. The latest investigation reported that c-kit+progenitor cells are responsible for heart regeneration after myocardial infarction in neonatal mouse heart.Aims:1. If different parts of neonatal mouse heart injury could lead to cardiomyocytes regeneration.2. Investigate the expression of development related transcription factors during cardiomyocytes regeneration. Clarify the differences of transcription factors’ expression between left ventricular apex resection and right ventricle injury.3. Investigate the activation of c-kit+progenitor cells during neonatal mouse heart regeneration.Methods and results:Part1:Different part of neonatal mouse heart injury leads to cardiomyocytes regeneration.We performed the apex resection or right ventricle injury surgery on12-h-old neonatal mice, then evaluated hearts from operated and sham-operated group at1,4,7and21day after surgery. Masson and HE stain indicated that neonatal mouse can fully regenerate their heart in a very brief time. Ultrasonic cardiogram was performed to evaluate the heart function. Data indicated that the function of left or right ventricle was not impaired by surgery. The expression was kept at a high level in the first week after birth. The number of Ki67+cells per field in control group was3.2±1, while it was5.5±1.1(p<0.001) in apex resection group. The expression of pH3also rose up among cells in apex resection group. At day4, the number of pH3+cardiomyocytes was1.65±0.12/field in apex resection group and it was1.18±0.15/field (P<0.01) in control group. Cardiomyocytes with dissembled sarcomeres were widely distributed in the whole heart. All this indicated that new cardiomyocytes may come from proliferation of preexisting cardiomyocytes.Part2:Specific expression of development related transcription factors.We tested the mRNA expression of transcription factors Nkx2.5, Isll, Foxhl, Tbx20, Gata4, Mef2c, Hand2and c-Myc in different time points following surgery. Their expression level at day1after birth served as control. Expression of these8transcription factors was kept at low level during normal development. After apex resection, gene Mef2c was highly activated, which was confirmed by immunohistochemistry and western blot. These data indicated that Mef2c might be associated with apex regeneration. The expression of transcription factors Foxhl and Tbx20was raised up in right ventricle injury group, demonstrated by qRT-PCR, western and immunohistochemistry. High level expression of Foxhl and Tbx20was not seen in apex resection group. Our study showed that gene cascades were different after injury of right or left ventricle. Different mechanisms of regeneration may underlie neonatal mouse cardiomyocytes regeneration.Part3:Activation of c-kit+progenitor cells after neonatal mouse apex resection.Although new cardiomyocytes may arise from pre-existing cardiomyocytes, cardiac progenitor cells still might be considered as a potential cell resource. In this study, we tested whether heart injury in neonatal mouse induced the proliferation of c-kit+cardiac progenitor cells during the process of regeneration. For quantification of c-kit+stem cells, it was counted and expressed as number per mm2. The expression of c-kit was tested by quantitative PCR and western blot. Apex resection in neonatal mouse leads to complete regeneration of heart and brisk increase of cardiac progenitor cell. Gene c-kit expression and synthesis of Kit protein were also significantly elevated shortly after injury. The number of c-kit+progenitor cells significantly increased at day1after surgery (sham operated:3.71/mm2; operated8.38/mm2, p<0.05). c-kit mRNA and protein expression levels also increased significantly after amputation in a time dependent manner. Some c-kit+cells were marked with cTnT, which is particularly expressed by cardiomyocytes. Apex resection in neonatal mouse leads to complete regeneration of heart and significant up regulation of c-kit+progenitor cell population. c-kit+cardiac progenitor cells may contribute to the apex regeneration in neonatal mouse heart.Conclusions:Left or right ventricle of neonatal mouse heart injury leads to cardiomyocytes regeneration.The expression of transcription factors is different in neonatal mouse heart after apex resection or right ventricle injury. Therefore, the mechanisms of heart regeneration may be different for right ventricle and left ventricle. c-kit+cardiac progenitor cells may contribute to the heart regeneration in neonatal mouse heart. These findings have implications for promoting regeneration of the injured human heart.

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CLC: > Medicine, health > Surgery > Of surgery > Cardiovascular and lymphatic system surgery > Heart
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