Biomedical Engineering Reference
In-Depth Information
First, we established a guineapig lung fibroblast cell line stably expressing HCN1
channels with a GFP reporter (HCN1-fibroblasts). These cells were fused with freshly
isolated guineapig ventricular myocytes using polyethylene glycol 1500 (PEG). The
PEG-induced membrane fusion events have served as a model system to create mouse
and human hybridomas [65], to study eukaryotic cell-cell fusion events [41], and to
deliver outward K
+
currents into myocytes [30]. In our experiments, PEG-induced
fusion occurred almost instantaneously in vitro since, within 3 min, the HCN1-
fibroblasts fused with ventricular myocytes as verified by the sudden introduction of
Calcein-AM fluorescence into the myocytes. The in vivo study was carried out by a
simple intracardiac, focal-injection of HCN1-fibroblasts suspended in 50% PEG into
the apex of guinea-pig hearts. Langendorff-isolation of ventricular myocytes from the
site of injection revealed GFP-positive myocytes. We also verified in vivo fusion
events by histology. HCN1-fibroblasts were first transduced with adenovirus
expressing cytoplasmic
ȕ
-galactosidase (Ad-
lacZ
). Immunohistochemistry against
ȕ
-galactosidase and myosin heavy chain (MHC) co-localized the two proteins in
regions of the myocardium, indicating fusion of cytoplasm from HCN1-fibroblasts
(expressing b
ȕ
-galactosidase) and cardiomyocytes.
These heterokaryons formed by in vivo fusion of myocytes and HCN1-fibroblasts
verified pacemaker function by displaying spontaneous action potentials with a slow
phase-4 depolarization. Biological pacemaker activities in vivo were also confirmed
by electrocardiography in guinea pigs injected with HCN1-fibroblasts in PEG.
Electrocardiograms recorded 1-22 days after the HCN1-fibroblast injection revealed
ectopic ventricular beats that were identical in polarity and similar in morphology to
those recorded during bipolar pace-mapping of the apex in the same animal (n = 5 of
13). Occasionally, junctional escape rhythms could be overtaken by ectopic
ventricular pacemaker activity. Such ectopic beats were not observed in animals
injected with control fibroblasts expressing GFP only (n =9).
An assumption of this study was that the fusion-induced generation of pacemaker
activity is independent of cell-cell coupling. Gap-junctional coupling between cardiac
fibroblasts and cardiomyocytes has been observed [37] and could provide an
alternative mechanism of pacemaker activity. To test the presence of cell-coupling in
our model, we loaded HCN1-fibroblasts with the membrane-impermeable dye,
Calcein-AM, and mixed them with non-loaded myocytes. The dye did not diffuse
from a loaded HCN1-fibroblast to the neighboring myocytes, indicating the absence
of cell-cell coupling. Thus, the data indicates that the
I
f
-mediated pacemaker activity
arises from fused heterokaryons rather than electrotonic coupling between myocytes
and fibroblasts.
Comparable to the hMSC approach [57], the syngeneic fibroblasts in our study
acted as a vehicle to deliver the pacemaker currents into ventricular cardiomyocytes.
However, our approach is independent of gap-junctional coupling between cells and
thus should be more stable in long term. Previous studies suggest that the in vivo
fusion-induced heterokaryons can maintain the nuclei from each fusion partner
separately and stably for at least several months [1, 22, 23, 76]. Our fusion-induced
biological pacemakers were stable for at least 3 weeks and functional in less than 1
day post-injection as revealed by the electrocardiography. Furthermore, straight
injection of hMSCs [57] or hESCs [36] into heart does not guarantee that the injected
cells will remain at the site of injection. The fusion approach implants the biological
