Introduction:
Gestational diabetes mellitus (GDM) is associated with a five-fold increase in congenital heart defects. It is critical to determine the biological effects of diabetes mellitus (DM)
in vivo
and high glucose
in vitro
on neonatal cardiomyocyte progenitor cells (nCPCs) to maximize their regenerative potential. In the present study we seek to investigate the roles of Mir-195 and its hypothesized target gene, enhancer of zeste homolog 2 (Ezh2), in GDM.
Hypothesis:
We predict that high glucose is associated with decreased cellular proliferation, viability and increased senescence through oxidative stress. We also hypothesize that expression of Mir-195 will be higher in DM-nCPCs and inhibit cell proliferation via Ezh2 silencing.
Methods:
We subjected nCPCs
in vitro
to increasing glucose concentrations; cellular proliferation, migration, reactive oxygen species generation, and apoptosis were assessed using cell counting kit-8, wound healing, dihydroethidium, and annexin assays respectively. Our
in vivo
experiments involved injecting four-week old female mice with streptozocin. After pairing diabetic mice with non-diabetic male mice, timed embryos at E14.5 were evaluated for viability, proliferation and characterization. mRNA expression levels of Mir-195 and Ezh2 protein levels were detected using RT-qPCR and western blot analysis respectively. Lipofectamine transfection, with siRNA inhibiting Mir-195, was performed on c-kit+ cardiac stem cells obtained from diabetic mothers.
Results:
We found that subjecting nCPCs
in vitro
to increased glucose concentration led to increased % cell death, decreased proliferation and expression of paracrine factors indicating poorer secretome quality from these cells. Our
in vivo
models showed that maternal diabetes impedes prenatal development as decreased expression of c-kit+/Lin- cells and ISL1+ cells and increased DHE positivity were seen in DM-nCPCs at E14.5. Expression of Mir-195 was higher in DM-nCPCs but Ezh2 mRNA and protein expression levels were significantly decreased. siRNA inhibition of Mir-195 revealed higher EZH2 expression in c-kit+ cells and concomitant increase in regenerative capacity.
Conclusion:
In conclusion, the viability of DM-nCPCs both
in vivo
and
in vitro
is decreased compared to NDM-nCPCs suggesting decreased postnatal regenerative capacity. Mir-195 is associated with increased apoptosis and decreased proliferation of nCPCs via abrogation of the protective effects of EZH2.
Anti‐Diabetic, Anti‐Inflammatory, and Anti‐Oxidant Effects of Naringenin in an In Vitro Human Model and an In Vivo Murine Model of Gestational Diabetes Mellitus
