Final Report Summary - HES-CARDIO (Cardiac myosin light chain kinase function in human pluripotent stem cell derived cardiomyocytes)
Diseases of the heart and circulatory system are the main cause of death in Europe, accounting for over 4 million deaths each year. Cells of the human heart are principally non-regenerative and are difficult to obtain for research or transplantation. However, cardiac cells produced from human stem cells represent an accessible and renewable source of heart cells. These human cardiac cells, termed cardiomyocytes, have immense potential value to the pharmaceutical industry, in particular as model systems for drug toxicity screening. Further, the recent derivation of other cardiovascular cell types, including smooth muscle and epicardial cells represents further sources of cells for therapy and drug toxicity screening.
The main hurdle to the effective use of human embryonic stem cell (hESC) and human induced pluripotent stem cell (hIPSC) derived cardiomyocytes (hESC-CMs and hIPSC-CMs) in drug toxicology screening and transplantation therapy is their embryonic nature. It is recognised that hESC-CM and hIPSC-CM are immature and undergo limited maturation in the laboratory. This creates problems as the cells may not display appropriate responses to target drugs in toxicology screening and may not be suitable for transplantation into the damaged heart.
The key objective of this project was to derive and characterise a reproducible source of mature human cardiomyocytes suitable for drug toxicology screening and human cardiac cell biology studies. The impact of the research conducted would be to illustrate the potential use of hESC-CM and hIPSC-CM as cell based models for drug screening, with the aim of reducing drug attrition rates, improving the accuracy of predicting drug cardiotoxicity and reducing animal testing.
The hypothesis to induce maturation of hESC-CM was to overexpress cardiac myosin light chain kinase (MLCK). MLCKs phosphorylate myosin light chain proteins, the principle components of thick filaments that are required for cardiac motor activity. Expression of cardiac MLCK is limited to the heart, and initial studies in animal models suggested that cardiac MLCK is expressed specifically in the heart of neonates and adults, is required for normal cardiac development and cardiomyocyte contractility, and regulates sarcomere assembly. Further work has shown that increased cardiac MLCK levels improve cardiac output post-myocardial infarction in rats. Therefore, cardiac MLCK is an important myofilament regulatory protein with a potential cardioprotective capacity that represents an ideal candidate gene for study.
During the course of the project a number of publications and expert opinion indicated that overexpression of cardiac specific genes alone would be unlikely to induce maturation of hESC-CM. Thus, the main goals of the project changed slightly. Research focused primarily on studying the role of cardiac transcription factors, particularly NKX2-5, in the early stages of cardiac development and their influence on the maturation of cardiomyocytes. Gene editing technology was used to disrupt expression of NKX2-5 during cardiomyocyte differentiation from hESCs. This resulted in immature cardiac phenotypes indicating that NKX2-5 is critical for the correct development and maturation of cardiomyocytes. This research is currently under review for publication.
Ultimately, publication of this work will shed light on the development of the human cardiovascular system and will provide new leads for efforts to improve the maturation of hESC-CM. It will therefore aid efforts to generate suitable cellular material for transplantation therapy as a cure for heart disease.
The main hurdle to the effective use of human embryonic stem cell (hESC) and human induced pluripotent stem cell (hIPSC) derived cardiomyocytes (hESC-CMs and hIPSC-CMs) in drug toxicology screening and transplantation therapy is their embryonic nature. It is recognised that hESC-CM and hIPSC-CM are immature and undergo limited maturation in the laboratory. This creates problems as the cells may not display appropriate responses to target drugs in toxicology screening and may not be suitable for transplantation into the damaged heart.
The key objective of this project was to derive and characterise a reproducible source of mature human cardiomyocytes suitable for drug toxicology screening and human cardiac cell biology studies. The impact of the research conducted would be to illustrate the potential use of hESC-CM and hIPSC-CM as cell based models for drug screening, with the aim of reducing drug attrition rates, improving the accuracy of predicting drug cardiotoxicity and reducing animal testing.
The hypothesis to induce maturation of hESC-CM was to overexpress cardiac myosin light chain kinase (MLCK). MLCKs phosphorylate myosin light chain proteins, the principle components of thick filaments that are required for cardiac motor activity. Expression of cardiac MLCK is limited to the heart, and initial studies in animal models suggested that cardiac MLCK is expressed specifically in the heart of neonates and adults, is required for normal cardiac development and cardiomyocyte contractility, and regulates sarcomere assembly. Further work has shown that increased cardiac MLCK levels improve cardiac output post-myocardial infarction in rats. Therefore, cardiac MLCK is an important myofilament regulatory protein with a potential cardioprotective capacity that represents an ideal candidate gene for study.
During the course of the project a number of publications and expert opinion indicated that overexpression of cardiac specific genes alone would be unlikely to induce maturation of hESC-CM. Thus, the main goals of the project changed slightly. Research focused primarily on studying the role of cardiac transcription factors, particularly NKX2-5, in the early stages of cardiac development and their influence on the maturation of cardiomyocytes. Gene editing technology was used to disrupt expression of NKX2-5 during cardiomyocyte differentiation from hESCs. This resulted in immature cardiac phenotypes indicating that NKX2-5 is critical for the correct development and maturation of cardiomyocytes. This research is currently under review for publication.
Ultimately, publication of this work will shed light on the development of the human cardiovascular system and will provide new leads for efforts to improve the maturation of hESC-CM. It will therefore aid efforts to generate suitable cellular material for transplantation therapy as a cure for heart disease.