Systemic maternal inflammation and neonatal hyperoxia induces remodeling and left ventricular dysfunction in mice

Abstract

Aims: The impact of the neonatal environment on the development of adult cardiovascular disease is poorly understood. Systemic maternal inflammation is linked to growth retardation, preterm birth, and maturation deficits in the developing fetus. Often preterm or small-for-gestational age infants require medical interventions such as oxygen therapy. The long-term pathological consequences of medical interventions on an immature physiology remain unknown. In the present study, we hypothesized that systemic maternal inflammation and neonatal hyperoxia exposure compromise cardiac structure, resulting in LV dysfunction during adulthood.

Methods and results: Pregnant C3H/HeN mice were injected on embryonic day 16 (E16) with LPS (80 µg/kg; i.p.) or saline. Offspring were placed in room air (RA) or 85% O(2) for 14 days and subsequently maintained in RA. Cardiac echocardiography, cardiomyocyte contractility, and molecular analyses were performed. Echocardiography revealed persistent lower left ventricular fractional shortening with greater left ventricular end systolic diameter at 8 weeks in LPS/O(2) than in saline/RA mice. Isolated cardiomyocytes from LPS/O(2) mice had slower rates of contraction and relaxation, and a slower return to baseline length than cardiomyocytes isolated from saline/RA controls. α-/β-MHC ratio was increased and Connexin-43 levels decreased in LPS/O(2) mice at 8 weeks. Nox4 was reduced between day 3 and 14 and capillary density was lower at 8 weeks of life in LPS/O(2) mice.

Conclusion: These results demonstrate that systemic maternal inflammation combined with neonatal hyperoxia exposure induces alterations in cardiac structure and function leading to cardiac failure in adulthood and supports the importance of the intrauterine and neonatal milieu on adult health.

Figure 1. Functional and morphological parameters achieved by M-mode echocardiography of mice exposed to maternal saline or LPS on E16 and 14 days of neonatal RA or O₂ at 2 and 8 weeks of age.

Data were analyzed using two-way ANOVA and Bonferroni post hoc, n = 8–9 mice per group, p<0.05 compared to saline/RA (*), saline/O₂ (†), or LPS/RA (‡) exposed mice.

Figure 2. In vitro cardiomyocyte function in saline/RA and LPS/O₂ exposed mice at 8 weeks of age.

(A) % Peak shortening (% PS) was increased in the LPS/RA exposed mice. (B) Shortening velocity (Dep) and relengthening velocity (Rel) was significantly increased in cardiomyocytes isolated from LPS/RA exposed mice and decreased in LPS/O₂-exposed mice compared to saline/RA controls. (C) Time-to-90% shortening (TPS 90) was significantly increased in cardiomyocytes isolated from saline/O₂ and LPS/O₂-exposed mice, indicating systolic dysfunction at the cellular level. (D) Time-to-90% relengthening (TR 90) was significantly increased in myocytes from saline/O₂ and LPS/O₂-exposed mice, indicating significant diastolic dysfunction at the cellular level. Data were analyzed using one-way ANOVA and Bonferroni post hoc, *p<0.05, n = 20 cells per mouse and three to five mice per group. p<0.05 compared to saline/RA (*), saline/O₂ (†), or LPS/RA (‡) exposed mice.

Figure 3. MHC protein contents in LV tissues at 8 weeks of age.

Representative Western blots and quantified data indicating changes in α-MHC and β-MHC protein contents due to LPS, O₂ or combined treatments. Data were analyzed using one-way ANOVA and Bonferroni post hoc, n = 5 mice per group, p<0.05 compared to saline/RA (*) exposed mice.

Figure 4. Connexin-43 proteins in LV tissues at 8 weeks of age.

Representative confocal images showing reduced numbers of connexin-43 positive gap junctions and CX-43 lateralization in saline/O₂, LPS/RA, and LPS/O₂ compared to saline/RA-exposed mice (Figure 6A). Representative Western blots and quantified data indicating significantly reduced connexin-43 content in LPS/O₂ compared to saline/RA exposed mice (Figure 6B). Data were analyzed using one-way ANOVA and Bonferroni post hoc, n = 5 mice per group, p<0.05 compared to saline/RA (*) exposed mice.

Figure 5. Western blot assessments of Nox4 and VEGFA protein levels.

Representative Western blots and quantified data indicating changes in Nox4 and VEGFA protein contents due to LPS treatment. Data were analyzed using one-way ANOVA and Bonferroni post hoc, n = 5 mice per group, p<0.05 compared to saline/RA (*), saline/O₂ (†), or LPS/RA (‡) exposed mice.

Figure 6. Capillary density was assessed in LV tissues by immunohistochemistry.

Images of tissue sections were immuno-stained for CD31 (Figure 6A). Capillary numbers were counted in 5 high power fields (HPF) per slide and n = 3 mice per group (HPF = 22,000 µm²). Data were analyzed using one-way ANOVA and Bonferroni post hoc, p<0.05 compared to saline/RA (*), saline/O₂ (†), or LPS/RA (‡) exposed mice.