Exercise and hypoxia unmask pulmonary vascular disease and right ventricular dysfunction in a 10- to 12-week-old swine model of neonatal oxidative injury

Abstract

Prematurely born young adults who experienced neonatal oxidative injury (NOI) of the lungs have increased incidence of cardiovascular disease. Here, we investigated the long-term effects of NOI on cardiopulmonary function in piglets at the age of 10-12 weeks. To induce NOI, term-born piglets (1.81 ± 0.06 kg) were exposed to hypoxia (10-12% $F_{{\mathrm{iO}}_2}$ ), within 2 days after birth, and maintained for 4 weeks or until symptoms of heart failure developed (range 16-28 days), while SHAM piglets were normoxia raised. Following recovery (>5 weeks), NOI piglets were surgically instrumented to measure haemodynamics during hypoxic challenge testing (HCT) and exercise with modulation of the nitric-oxide system. During exercise, NOI piglets showed a normal increase in cardiac index, but an exaggerated increase in pulmonary artery pressure and a blunted increase in left atrial pressure - suggesting left atrial under-filling - consistent with an elevated pulmonary vascular resistance (PVR), which correlated with the duration of hypoxia exposure. Moreover, hypoxia duration correlated inversely with stroke volume (SV) during exercise. Nitric oxide synthase inhibition and HCT resulted in an exaggerated increase in PVR, while the PVR reduction by phosphodiesterase-5 inhibition was enhanced in NOI compared to SHAM piglets. Finally, within the NOI piglet group, prolonged duration of hypoxia was associated with a better maintenance of SV during HCT, likely due to the increase in RV mass. In conclusion, duration of neonatal hypoxia appears an important determinant of alterations in cardiopulmonary function that persist further into life. These changes encompass both pulmonary vascular and cardiac responses to hypoxia and exercise. KEY POINTS: Children who suffered from neonatal oxidative injury, such as very preterm born infants, have increased risk of cardiopulmonary disease later in life. Risk stratification requires knowledge of the mechanistic underpinning and the time course of progression into cardiopulmonary disease. Exercise and hypoxic challenge testing showed that 10- to 12-week-old swine that previously experienced neonatal oxidative injury had increased pulmonary vascular resistance and nitric oxide dependency. Duration of neonatal oxidative injury was a determinant of structural and functional cardiopulmonary remodelling later in life. Remodelling of the right ventricle, as a result of prolonged neonatal oxidative injury, resulted in worse performance during exercise, but enabled better performance during the hypoxic challenge test. Increased nitric oxide dependency together with age- or comorbidity-related endothelial dysfunction may contribute to predisposition to pulmonary hypertension later in life.

Keywords: heart failure; neonatal; pulmonary vascular disease.

Figure 1. Schematic overview of the protocol

Neonatal oxidative injury (NOI) is induced by placing piglets in hypoxia (10–12% fraction of inspired oxygen) for a maximum of 28 days. After initial recovery (<7 days), swine underwent cardiovascular magnetic resonance (CMR) under anaesthesia. At the start of week 9, piglets were chronically instrumented. The exercise and hypoxic challenge experiments (photograph insets) took place from week 10 onward until the second CMR and sacrifice after week 12. SHAM operated piglets followed the same protocol, but were not subjected to hypoxia in the first 28 days. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2. Planning of the MRI

A and B, planning of the short axis plane parallel to the mitral valve in the 4‐(A) and 2‐chamber (B) long axis plane and perpendicular to the long‐axis of the left ventricle. C and D nine short axis cine slices of the ventricles in end‐diastole (C) and end‐systole (D). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3. Influence of duration of postnatal hypoxia exposure on right ventricular (RV) tissue measurements

Only NOI animals are included in the regression analysis. mRNA expression was normalized using housekeeping genes cyclophilin A and β‐actin. Data are shown as means ± SD and as individual data points as a function of duration of hypoxia; n = 8 for SHAM and n = 10 for NOI.

Figure 4. Influence of duration of hypoxia on pulmonary vascular resistance (PVR), stroke volume and wall‐to‐lumen ratio of pulmonary arterioles

Only NOI animals are included in the regression analysis. Data are shown as means ± SD and as individual data points plotted as a function of duration of hypoxia; n = 6–8 for SHAM and n = 10–11 for NOI.

Figure 5. Pulmonary vascular resistance (PVR) and stroke volume during hypoxic challenge testing and exercise in 10‐ to 12‐week‐old, chronically instrumented piglets

Only NOI animals are included in the regression analysis. Data are shown as means ± SD and as individual data points plotted as a function of duration of hypoxia; n = 6–7 for SHAM and n = 8 for NOI. Mean Δ: Average change during hypoxic challenge test from measurements at 2, 5, 10, 15 and 20 min compared to baseline value. $\mathrm{B}{\dot{V}}_{{\mathrm{O}}_2}$, body oxygen consumption. *Difference between groups. †Difference over time or $\mathrm{B}{\dot{V}}_{{\mathrm{O}}_2}$. ‡Interaction group × time/$\mathrm{B}{\dot{V}}_{{\mathrm{O}}_2}$.

Figure 6. Inhibition of nitric oxide synthase (NOS‐i) during exercise in 10‐ to 12‐week‐old, chronically instrumented piglets

NOI, neonatal oxidative injury piglets; PVR, pulmonary vascular resistance. Means ± SD are shown, n = 6 for SHAM and n = 8 for NOI at rest; in both groups one animal did not exercise. *NOS‐i within group. †$\mathrm{B}{\dot{V}}_{{\mathrm{O}}_2}$ within group. ‡Interaction NOS‐i × $\mathrm{B}{\dot{V}}_{{\mathrm{O}}_2}$ within groups. §NOS‐i between groups. §Interaction NOS‐i × $\mathrm{B}{\dot{V}}_{{\mathrm{O}}_2}$ between groups.

Figure 7. Total endothelial nitric oxide synthase (eNOS) protein normalized to GAPDH and phosphorylated eNOS (p‐eNOS)/eNOS ratio in lung tissue in 10‐ to 12‐week‐old, chronically instrumented swine

NOI, neonatal oxidative injury piglets. Shown are individual data points (n = 9 for SHAM and n = 8 for NOI). Mean values are denoted by the horizontal lines. Original Western blots are shown in the supplementary data.

Figure 8. Inhibition of phosphodiesterase 5 (PDE5‐i) at rest and during exercise in 10‐ to 12‐week‐old, chronically instrumented piglets

NOI, neonatal oxidative injury piglets; PVR, pulmonary vascular resistance. Mean ± SD are shown, n = 6 for SHAM and n = 8 for NOI. *PDE5‐i within group. †$\mathrm{B}{\dot{V}}_{{\mathrm{O}}_2}$ within group. ‡Interaction PDE5‐i × $\mathrm{B}{\dot{V}}_{{\mathrm{O}}_2}$ within groups. §PDE5‐i between groups. §Interaction PDE5‐i × $\mathrm{B}{\dot{V}}_{{\mathrm{O}}_2}$ between groups.