The Role of Cardiac Energy Metabolism in Congenital Diaphragmatic Hernia-associated Ventricular Dysfunction

Background: Ventricular dysfunction is associated with mortality in congenital diaphragmatic hernia (CDH). However, the myocardial cellular pathology underlying this phenomenon remains unknown.

Methods: Using the Sprague-Dawley nitrofen model of CDH, ventricular tissue was harvested from newborn control and CDH pups. Wet and dry weights were collected. Gross, histologic, and transmission electron microscopic (TEM) images were captured. Differential gene expression and mitochondrial yield were assessed. Hypoxyprobe was used to measure tissue hypoxia.

Results: CDH hearts displayed a rightward anatomical shift, RV hypertrophy (38% increased RV thickness vs. controls, p=0.008, n=3), and decreased LV volume, though this did not reach statistical significance (29% decreased LV volume; p=0.463, n=3) (Figure 1A-B). Ventricular mass analysis showed that CDH pups displayed increased LV edema (Figure 1C). CDH cardiac tissue was 1.5-fold more hypoxic than controls (p=0.01, n=3) (Figure 1D). qRT-PCR and western blotting analyses revealed a slight upregulation of Hypoxia-inducible factor-1α (HIF-1α) and HIF-2α mRNA levels, however mRNA and protein levels of Bcl-2 interacting protein 3 (BNIP3), a downstream regulator of HIF-1α, was significantly upregulated in CDH cardiac tissues compared to control (Figure 1E&F). On RNA sequencing analysis, 46 genes relating to mitochondrial and fatty acid biogenesis were downregulated in CDH cardiac tissue (Figure 1G). Mitochondrial protein yield was decreased in both ventricles in CDH rodents (Figure 1H). Western blotting of RV tissue demonstrates decreased expression of mitochondrial DNA-encoded protein, cytochrome B (CytB) in CDH (Figure 1I). On TEM analysis, CDH-ventricular mitochondria had a more swollen shape compared to controls (Figure 1J). Additionally, CDH mitochondria displayed disorganization, cristae loss, and a less dense mitochondrial matrix. Cristae in the CDH mitochondria displayed fusion and vacuolation, and a majority (71% RV, 73% LV) of CDH ventricular mitochondria had abnormal ultrastructure (both p<0.001).

Conclusion: Mitochondrial diminution, particularly in the left ventricle and in association with hypoxia, may play a critical role in the pathophysiology of ventricular dysfunction in CDH.