Angiogenic genes and proteins are differentially expressed in left and right ventricular tissue in perinatal sheep hearts

Abstract

Birth places a great stress on the fetal heart due to increased cardiac workload, following a disconnection from the low-resistance placenta and closure of fetal shunts. The mechanisms that regulate myocardial microvascular growth and development during the perinatal period are largely unknown. We hypothesized that birth stimulates an increase in angiogenic gene and protein expression in a ventricular dependent manner to reflect the changing roles for the left and right ventricles (LV, RV). Fetal (135 d gestation, dGA; term=147 dGA) and neonatal (1 and 5 postnatal days, PD) sheep LV and RV were snap frozen. Gene expression of key angiogenic regulators ( VEGFA, VEGFR1, VEGFR2, ANGPT1, ANGPT2, PGC1A), endothelial cell growth ( DDL4, NOTCH4, SPP1, PECAM1, NCL), nitric oxide synthesis ( NOS2, NOS3), and extracellular matrix (ECM) remodeling ( MMP2, MMP9, THBS1) were quantified using real-time qPCR (n=12/age group). Protein expression of VEGFA and VEGF-R2 were measured by Western blot (n=5/age group). Data were analyzed by two-way ANOVA with Tukey’s post-hoc test to determine effects of age, ventricle, and interaction. Significance was set at P<0.05. At 135 dGA, all angiogenic, nitric oxide synthesis, ECM, DDL4, SPP1, and PECAM1 mRNA expression levels were higher in the RV vs. LV. At PD1, there were no differences between LV and RV. At PD5, VEGFA, VEGFR1, ANGPT1, ANGPT2, PGC1A, DDL4, SPP1, PECAM1, NCL, MMP2, and THBS1 mRNA expression levels were again higher in the RV vs. LV. At 135 dGA, VEGFA and VEGF-R2 protein expression levels were 76% and 72% lower, respectively, in the RV vs. LV. Within each ventricle, age related changes to mRNA expression patterns were unique. In the LV, mRNA expression of NOTCH4 and NCL increased stepwise from 135 dGA to PD5; however, in the RV, both were upregulated at PD1 but returned to fetal levels by PD5. In contrast, all angiogenic, ECM, DDL4, SPP1, PECAM1, and NOS2 genes were downregulated in the RV at PD1 but all returned to fetal levels by PD5. In the LV, VEGF-R2 protein expression was 56% and 73% lower at PD 1 and 5, respectively, vs. 135 dGA. In summary, gene and protein expression patterns involved in angiogenesis and vascular remodeling change over the perinatal period and are differentially expressed in the ventricles. Greater angiogenic gene expression levels in the RV vs. the LV at 135 dGA might be due to the RV serving as the main systemic pump in utero, thus requiring relatively increased microcirculation. However, reduced VEGFA and VEGF-R2 protein levels at 135 dGA contradict this hypothesis; histological studies to determine vascularity are ongoing. Likewise, the subsequent downregulation of angiogenic genes in the RV at PD1 might be due to the RV’s sudden decrease in cardiac output as the LV becomes the main pump. The underlying reasons for why LV angiogenic genes are not significantly upregulated in the early postnatal period, given its new role as the main systemic pump, are yet to be elucidated. NIH R21-HL152112 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.