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Cardiac side population cells (cSPCs) were the first group of
progenitor cells identified in the heart; however, their progenitor
cell properties have only been established in cell culture and after
transplantation. To determine whether cSPCs possess progenitor cell
properties in vivo, we generated an Abcg2-driven, lineage-tracing
mouse model. In this model, 75.8 ± 10.87% of cSPCs are labeled
with GFP. Over a four-week chase period, there is a five-fold increase
in cardiomyocyte-labeling from 0.17 ± 0.15% to 0.84 ± 0.24%.
Surprisingly, 90.7 ± 2.66% of labeled cardiomyocytes arise from
fusion events and not direct differentiation from cSPCs based on
labeling in Abgc2MCM/+ R26mTmG/+ mice. Given the extensive
labeling of bone marrow and endothelial cells in our model, we
hypothesized that the increase in cardiomyocyte labeling over
the four-week chase period arises from fusion of unlabeled cardio-
myocytes with GFP-labeled bone marrow cells or endothelial cells.
To test this hypothesis, we irradiated Myh7Cre/+ R26tdTomato/+ mice,
transplanted them with hematopoietic stem (LSK) cells isolated
from Abgc2MCM/+ R26GFP/+ and pulsed them with tamoxifen. At
the end of a four-week chase period, a similar percentage of bone
marrow side population cells, LSK cells and differentiated lineages
were labeled with GFP. More importantly, only 0.02 ± 0.03% of
cardiomyocytes were labeled with both GFP and tdTomato. No
cardiomyocytes were labeled with only GFP. Next, we evaluated
whether fusion with endothelial cells could account for the increase
in cardiomyocyte labeling we observed. Using an endothelial-
specific, lineage-tracing mouse model, Cdh5CreER/+ R26mTmG/+, we
found no cardiomyocytes labeled at the end of a four-week chase
period. Our results demonstrate that the primary mechanism of
cardiomyocyte labeling in our Abcg2-driven, lineage-tracing mouse
model arises from fusion of cardiomyocytes with lineage-traced
cells, but not with bone marrow or endothelial cells. These results
indicate that fusion in the heart may occur at much higher rates than
previously assumed, and may play important roles in homeostatic
maintenance of cardiac function.
doi:10.1016/j.yjmcc.2017.07.025
013
Cardiac hypertrophy suppresses glucose oxidation in newborns
with congenital heart defects
Sonia Rawata, Arata Fukushimaa,b, Liyan Zhanga, Alda Huqia,c,
Tariq Altamimia, Cory Wagga, Lisa Hornbergera, Paul Kantora,
Ivan Rebeykaa, Gary Lopaschuka
a
Cardiovascular Research Centre, University of Alberta, Edmonton,
Alberta, Canada
b
Department of Cardiovascular Medicine, Hokkaido University Graduate
School of Medicine, Sapporo, Hokkaido, Japan
c
Cardio Thoracic and Vascular Department, University of Pisa, Pisa,
Tuscany, Italy
Introduction: Cardiac fatty acid oxidation in the newborn dramat-
ically increases shortly after birth, while glucose oxidation remains low
until weaning. In newborn rabbit hearts, volume overload hypertrophy
delays this increase in fatty acid oxidation, while keeping glucose
oxidation low. This results in a decreased capacity to produce energy
in the heart and increases the susceptibility to ischemic injury. The
presence of cardiac hypertrophy in patients with congenital heart
defects also decreases the maturation of fatty acid oxidation, but it is not
clear what happens to glucose oxidation. We therefore determined
what happens to the control of glucose oxidation in hypertrophied
human newborn hearts. Method: Human right ventricular biopsy
samples were collected during corrective heart surgery from neonates
aged 101-200 days (an age group with increased fatty acid oxidation
Abstracts
enzyme activity compared to 0-100 day old hearts). Samples were
grouped based on the presence or absence of right ventricular hyper-
trophy, as assessed by echocardiography. Results: Pyruvate dehydro-
genase (PDH), the rate-limiting enzyme of glucose oxidation, had
significantly increased phosphorylation in hypertrophied vs. non-
hypertrophied hearts (1.08±0.09 vs. 0.81±0.06 arbitrary units,
respectively, n=6, pb0.05), resulting in inactivation of PDH. Con-
comitantly, expression of the PDH kinases, PDK2 and PDK4, were
significantly increased in hypertrophied hearts. Transcription factors
PPARα and ERRα, which are known to regulate PDH kinases, showed no
changes between groups, while E2F1, a transcriptional regulator of
PDK2 and PDK4, was increased. Components of the E2F1 pathway were
also upregulated in the hypertrophied group, namely P-cyclin D1 and
CDK4. Conclusion: Hypertrophy of the newborn heart upregulates
E2F1-mediated transcription of PDH kinases, thereby phosphorylating
and inhibiting PDH and reducing glucose oxidation rates. This may
contribute to compromised energetics in the newborn heart.
doi:10.1016/j.yjmcc.2017.07.026
014
Cardiac Progenitor Cell Lineage Tracing During
Embryonic Cardiomyogenesis
Bingyan Wang, Alvin Muliono, Roberto AlvarezJr., Mark Sussman
San Diego State University, San Diego, CA, USA
Background: Stem cell therapy represents great promises to
myocardium regeneration. Multipotent c-Kitpos cardiac progenitor
cells (CPCs) are able to differentiate into endothelial cells, smooth
muscle cells, and cardiomyocytes. However, fundamental knowledge
of CPC biology remains incomplete. Studies in rodent myocardial
infarction model revealed that CPCs have poor long-term survival
and engraftment after adoptive transfer, perhaps due to severely
damaged host environment. Therefore, it is critical to understand
how CPCs interface with recipient environment following transfer in
order to enhance their true regenerative potentials. Hypothesis:
Adoptively transferred stem cells are thought to survive and engraft
best in an environment closely resembling their original habitat.
Thus, we hypothesized that embryonic environment provides opti-
mal spatiotemporal conditions to promote CPCs engraftment and
commitment to cardiac fate. Methods: CPCs isolated from adult
mouse hearts were expanded, fluorescence-tagged, and injected into
blastocysts at E3.75 and in utero at E15.5. Embryos were analyzed
following cardiogenesis by immunofluorescence for presence of CPC-
derived tissues. Additionally, CPCs were injected intramyocardially at
various stages from P0 to P7, to follow long-term adoptive transfer
and assess CPCs lineage commitment. Results and Conclusions: At
48 hours post injection, donor CPCs were found anchoring in
blastocoel and trophoblasts at E5.5, and were detected within host
myocardium at E17.5 predominantly at perivascular regions (n =4).
Interestingly, CPCs also integrated into aminochorionic sac, indicat-
ing a novel non-cardiogenic fate of CPCs (n= 5). CPCs injected at
P3 stably engrafted into left ventricular myocardium by 14 days post
injection (n=4), sharing gap junction proteins (ZO-1, Connexin-43)
with neighboring cells. In conclusion, this study provides vivid
evidence for the first time of CPC engraftment and survival in vivo
under homeostasis during cardiogenesis. Future studies will assess
permissive environmental conditions and cardiogenic potential of
CPCs, which may optimize their use in therapeutic applications and
provide fundamental insights on CPCs biology.
doi:10.1016/j.yjmcc.2017.07.027