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.