Adverse effects of fetal exposure of electronic-cigarettes and high-fat diet on male neonatal hearts

Figures

• 

  Fig. 1.. In situ detection of ventricular CM apoptosis detected by TUNEL. Incidence of CM apoptosis is minimal in controls at PND1, PND3, and PND14. In contrast, gestational exposure of e-cigarettes and HFD triggers a marked increase in the incidence of neonatal CM apoptosis at PND3 and PND14(A). For quantitation of CM apoptosis, apoptotic rate was expressed as the percentage of TUNEL-positive nuclei per total nuclei (apoptotic plus non-apoptotic nuclei) counted in a unit reference area (B,C,D). Values are means ± SEM (n = 10). * Significantly different (P <0.05) from respective controls. (Bar 25 μm).

• 

  Fig. 2.. Control CMs from pups born to mothers exposed to saline and HFD exhibited normal myofibrillar architecture and sarcomeric pattern with normal nuclei and abundant mitochondria. In contrast representative TEM images of ventricles at PND14 following in utero exposure of e-cigarettes and HFD show varying degrees of nuclear and myofibrillar abnormalities in CMs. The most predominant nuclear abnormalities (yellow arrow) included shrunken nuclei with chromatin condensation and fragmentation (characteristic of apoptosis) and nuclear malformation with extensively convoluted nuclear membrane. The most pronounced cytoplasmic abnormalities (yellow asterisk) were myofibrillar thinning and derangement. Representative higher magnified TEM images of ventricles from pups at PND14 following in utero exposure of e-cigarettes and HFD show disappearance of cristae (black asterisks) crystolysis when compared with controls (bottom panel). Bar 2 μm (upper and middle panels). Bar 0.5 μm (bottom panel).

• 

  Fig. 3.. Visualization of AMPK inactivation (dephosphorylation) by immunohistochemistry. Compared with controls, in utero exposure of e-cigarettes and HFD results in a decrease of phospho-ventricular AMPK immunoreactivity in PND14 pups (A). In utero exposure of e-cigarette and HFD causes a greater oxidative stress, as evidenced by elevated ventricular 4-HNE levels, relative to pups born to mothers exposed to saline and HFD (B). Bar 25 μm. Quantitation of ventricular staining intensities of p-AMPK (C) and 4-HNE (D). Values are means ± SEM (n = 10). *Significantly different (P <0.05) from respective controls.

• 

  Fig. 4.. Temporal changes in the expression of ventricular BAX, BCL-2, and active Caspase 3 detected by immunohistochemistry in pups at different postnatal days following in utero exposure of e-cigarettes and HFD (A). Note a significant increase in BAX expression (upper panel) and a decrease in BCL-2 expression (middle panel) after gestational exposure of e-cigarettes and HFD at PND 14. Perturbation of the ventricular BAX/BCL-2 ratio at PND14 following in utero exposure of e-cigarettes and HFD was further associated with activation of caspase 3 in ventricles, as evidenced by an increase in the immunostaining of active caspase 3 (bottom panel). Quantitation of ventricular staining intensities of BAX (B), BCL-2 (C), and active caspase 3 (D). Values are means ± SEM (n = 6). *Significantly different (P < 0.05) from respective controls. Bar 25 μm.