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Title: The effects of adult progenitor cell transplantation on recipient cardiomyocyte excitation-contraction coupling
Author: Lee, Joon
ISNI:       0000 0001 3607 1079
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2008
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Cell transplantation is a promising strategy for treating heart failure but the mechanisms effecting functional improvements remain unknown. The hypothesis that cell transplantation influences the contractile properties and excitation-contraction (EC) coupling of recipient cardiomyocytes by paracrine mechanisms was tested. Adult rats underwent myocardial infarction and subsequently developed chronic heart failure. They then received intra-myocardial injections of either skeletal myoblasts or bone marrow mononuclear cells which were harvested from transgenic rats constitutively expressing green fluorescent protein. Four weeks after injection, both cell types increased ejection fraction and reduced cardiomyocyte size. Isolated cardiomyocytes emitted low levels of green fluorescence, indicating that they originated from the recipient heart. The cardiomyocytes were then studied using sarcomere length measurements, indo-1 fluorescence and whole-cell patch-clamping techniques. Injection of either bone marrow cells or skeletal myoblasts normalized the impaired contractile performance and the prolonged time-to-peak of the Ca2+ transients that were observed in failing cardiomyocytes. The smaller and slower L-type Ca2+ current observed in heart failure returned to normal values after skeletal myoblast, but not bone marrow mononuclear cell, transplantation. Analysis of Ca2+ sparks in isolated cardiomyocytes using confocal microscopy revealed that SR Ca2+ leak had increased in failing cardiomyocytes, but was normalized by skeletal myoblast transplantation. In order to test the hypothesis that these effects observed in vivo are mediated by paracrine substances secreted from the transplanted cells further experiments were performed. Cardiomyocytes were isolated from failing hearts and cultured for 48 hours. Co-culturing with either skeletal myoblasts or bone marrow mononuclear cells during this period improved cardiomyocyte contraction and Ca2+ handling. This effect was maintained even when the different cell populations were mechanically separated by means of a porous membrane, demonstrating that cell-to-cell contact was not required and that soluble substances mediated the effect. Analysis of the supernates obtained from these co-culture experiments identified four candidate substances as possible mediators, but confirmation of their importance requires further experimental investigation. In addition to the work described above, experiments were performed during the preparation of the whole-cell patch-clamping system. The system was tested by measuring the Na+/Ca2+ exchanger current densities in cardiomyocytes isolated from normal rat hearts. Using this system the acute effects of various -adrenergic agonists was assessed. The results obtained from this separate study are presented in Chapter 6.
Supervisor: Terracciano, Cesare Sponsor: Magdi Yacoub Institute ; Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral