Exploring Therapeutic Strategies Targeting Cardiomyocytes for Heart Regeneration Open Access
Cho, Kyuwon (Spring 2022)
Abstract
Heart failure is the leading cause of morbidity and mortality in industrialized countries and is caused by dysfunction or deficiency of cardiomyocytes (CMs). No effective treatment is available due to limited regenerative capacity of the adult heart after ischemic injury. Among therapeutic strategies to generate CMs for heart regeneration, the current study explored two promising options: transplantation of pluripotent stem cell-derived CMs (PSC-CMs) and activation of pre-existing cardiomyocyte (CM) proliferation. One of the most critical hurdles for the use of PSC-CMs is their heterogeneity that leads to ventricular arrhythmias after transplantation. Isolation of ventricular cardiomyocytes (vCMs) has been challenging due to the lack of specific surface markers. We showed that vCMs can be purified from differentiating mouse embryonic stem cells (mESCs) using molecular beacons (MBs) targeting specific intracellular mRNAs. We found that, of the cells isolated, ~98% displayed vCM-like action potentials by electrophysiological analyses. These MB-purified vCMs continuously maintained their CM characteristics as verified by spontaneous beating, Ca2+ transient, and expression of vCM-specific proteins. Our study shows the feasibility of isolating pure vCMs via cell sorting without modifying host genes. The homogeneous and functional vCMs generated via the MB-based method can be useful for disease investigation, drug discovery, and cell-based therapies. As for the second strategy, we aimed to define regulatory mechanism how CMs lose proliferative capacity because limited proliferative capacity of adult mammalian CMs restricts heart regeneration. Polycomb group proteins are epigenetic regulators of the cell cycle, but their role in CM proliferation is unclear. Gene expression profiling revealed that CBX7, a subunit of Polycomb Repressive Complex 1, was upregulated in the postnatal heart. To determine whether CBX7 inhibits CM proliferation, we generated double mutant mouse with haplodeficiency of CBX7 in CMs. The mutant mice exhibited perinatal lethality with cardiomegaly. Histological analyses showed increased proliferative capacity of perinatal CMs by the targeted inhibition of CBX7. Gene expression profiling revealed that mitosis-related genes were derepressed whereas cardiac maturation genes were downregulated by targeted inhibition of CBX7. Gain of function analyses using adenoviral particles showed repressed proliferative capacity and promoted multinucleation of neonatal CMs by CBX7 overexpression in vitro. These results indicate that CBX7 is a critical repressor of perinatal CM proliferation and an attractive therapeutic target for inducing CM proliferation and ultimately for heart regeneration.
Table of Contents
Chapter 1: General introduction …1
1.1 Therapeutic strategies to generate cardiomyocytes for heart regeneration…2
1.2 Pluripotent stem cells (PSCs) as a source for CMs and clinical hurdles…5
1.3 Molecular regulation of cardiomyocyte proliferation...7
1.4 Epigenetic regulation of cardiac development…10
1.5 Polycomb group proteins and their roles in cardiac development…13
1.6 Expression, structure, and function of Polycomb group protein CBX7…13
1.7 Aim and scope of the dissertation...20
Chapter 2: Non-genetic purification of ventricular cardiomyocytes from differentiating embryonic stem cells through molecular beacons targeting IRX-4…21
2.1 Introduction…22
2.2 Results…24
2.2.1 Ventricular cardiomyocyte-specific gene selection and generation of IRX4 MBs...24
2.2.2 Delivery of MBs into different cell types...28
2.2.3 Selection of optimal IRX4 MB for isolating ventricular-like cardiomyocytes...29
2.2.4 Generation of ventricular cardiomyocytes from mESCs31
2.2.5 Purification of mESC-derived ventricular cardiomyocytes through IRX4-2 MBs...34
2.2.6 Electrophysiological characteristics of IRX4 MB+ ventricular-like cardiomyocytes…35
2.2.7 Cellular characterization of FACS-sorted ventricular-like cardiomyocytes…39
2.3 Discussion…41
2.4 Materials and Methods...44
Chapter 3: Expression profiling of Polycomb group proteins during cardiac development…48
3.1 Introduction…49
3.2 Results…50
3.2.1 Polycomb group subunits are differentially expressed in neonatal and adult mouse cardiomyocytes…50
3.2.2 CBX family proteins are dynamically regulated during cardiac development...52
3.2.3 Expression of CBX7 gene is induced in the perinatal mouse hearts…54
3.2.4 CBX7 protein is expressed in the adult mouse cardiomyocytes…56
3.2.5 Cbx7 is downregulated in the border zone of infarcted heart…58
3.2.6 Downstream chromatin modifications of CBX7 are detected in the adult mouse cardiomyocytes...60
3.3 Discussion…62
3.4 Materials and Methods...64
Chapter 4: Loss-of-function analyses of CBX7 in mice in vivo…68
4.1 Introduction…69
4.2 Results…70
4.2.1 Generation of mutant mice with cardiac haploinsufficiency of CBX7...70
4.2.2 Cardiac haploinsufficiency of CBX7 causes cardiomegaly and cardiac remodeling during perinatal period...72
4.2.3 Cardiac haploinsufficiency of CBX7 sustains proliferative capacity of perinatal cardiomyocytes...74
4.2.4 Cardiac haploinsufficiency of CBX7 causes reduced cell size of perinatal cardiomyocytes...79
4.2.5 Cardiac haploinsufficiency of CBX7 causes upregulation of mitotic signaling and downregulation of cardiac maturation-related genes...81
4.2.6 Cardiac haploinsufficiency of CBX7 results in increased number of Cyclin B1+ cardiomyocytes...83
4.2.7 Cardiac haploinsufficiency of CBX7 decreases cardiac connexin 43…86
4.2.8 CBX7 plays minimal roles in the regulation of senescence-related genes in the neonatal mouse heart...88
4.3 Discussion…90
4.4 Materials and Methods…93
Chapter 5: Gain-of-function analyses of CBX7 using neonatal mouse cardiomyocytes in vitro…96
5.1 Introduction…97
5.2 Results…99
5.2.1 CBX7 overexpression decreases proliferative capacity and promotes binucleation of neonatal mouse cardiomyocytes…99
5.2.2 CBX7 overexpression increases DNA content of neonatal mouse cardiomyocytes…101
5.2.3 CBX7 overexpression represses proliferation of cardiac fibroblasts…103
5.3 Discussion…105
5.4 Materials and Methods…107
Chapter 6: Conclusion, Discussion, and Future Directions...112
Chapter 7: References...124
About this Dissertation
School | |
---|---|
Department | |
Subfield / Discipline | |
Degree | |
Submission | |
Language |
|
Research Field | |
Keyword | |
Committee Chair / Thesis Advisor | |
Committee Members |
Primary PDF
Thumbnail | Title | Date Uploaded | Actions |
---|---|---|---|
|
Exploring Therapeutic Strategies Targeting Cardiomyocytes for Heart Regeneration () | 2024-04-24 13:01:58 -0400 |
|
Supplemental Files
Thumbnail | Title | Date Uploaded | Actions |
---|