Impaired pulmonary vasomotor control in exercising swine with multiple comorbidities

Figures

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  Fig. 1. Pulmonary hemodynamic and function. Heart rates at rest and during exercise were similar between groups (A), cardiac index was lower in DM + HC + CKD compared to Healthy swine (B) for any given level of body oxygen consumption (BVO2). Pulmonary function was maintained as oxygen saturation (SaO2, C) was similar between groups. Left atrial pressures were similar between groups, suggesting no left ventricular backward failure (D). Mean pulmonary arterial (PA) pressure was similar for any given level of BVO2 (E), but was increased for any given level of filling (F). Transpulmonary gradient (TPG) was also increased for any level of cardiac index (G), resulting in a higher pulmonary vascular resistance (PVR) in DM + HC + CKD compared to Healthy (H). Systemic vascular resistance (SVR) was higher in DM + HC + CKD compared to Healthy (I). PVRi n = 11, rest of parameters n = 12 for both groups. Values are mean ± SEM. *P ≤ 0.05, (*)P ≤ 0.1 for Healthy versus DM + HC + CKD, †P ≤ 0.05 for interaction DM + HC + CKD × BVO2

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  Fig. 2. Pulmonary vessel structure of DM + HC + CKD and Healthy swine. Typical examples of pulmonary elastin stained (A), lectin and smooth muscle stained (C), Endothelin B (ETB) receptor stained (E) and hematoxylin–eosin stained (G) sections. Wall-to-lumen ratio of pulmonary arterioles (B) and muscularization of small (10–30 µm) pulmonary vessels (0—non-muscularized; 1, 2, 3—partially muscularized and 4—fully muscularized) (D) was not different between groups. Number of quadrants stained for the endothelin B (ETB) receptor in the pulmonary endothelium of arterioles (20–60 µm) were lower (F) and perivascular inflammation score tended to be lower (H) in DM + HC + CKD compared to Healthy (H). Black arrows show scored quadrants in panel C and E, white arrows represent unscored quadrants. Values are mean ± SEM (solid line for panel B, D and F), or median ± IQR (dashed line for panel H). *P ≤ 0.05, (*)P ≤ 0.10 for Healthy versus DM + HC + CKD

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  Fig. 3. Role of the endothelin-pathway in pulmonary and systemic vasomotor control in DM + HC + CKD and Healthy swine. Transpulmonary gradient (TPG) as a function of cardiac index during graded exercise showed no significant change in response to endothelin receptor A and B blockade (ETA + Bi) in Healthy (A) but did decrease slightly in DM + HC + CKD (D) swine particularly at rest. Pulmonary vascular resistance (PVR) was unchanged in response to ETA + Bi in Healthy (B), while DM + HC + CKD showed pulmonary vasodilation in response to ETA + Bi (E). ETA + Bi resulted in similar reduction in systemic vascular resistance (SVR) in Healthy and DM + HC + CKD (C, F). Healthy n = 6, DM + HC + CKD n = 6. Values are mean ± SEM. *P ≤ 0.05, (*)P ≤ 0.1 for effect ETA + Bi within group, †P ≤ 0.05, (†)P ≤ 0.10 for interaction ETA+Bi × cardiac index (C) or ETA + Bi × BVO2 (D) within groups, ‡P ≤ 0.05, (‡)P ≤ 0.10 for interaction ETA+Bi × group

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  Fig. 4. Nitric oxide in pulmonary and systemic vasomotor control in DM + HC + CKD and Healthy swine. Transpulmonary gradient (TPG) as a function of cardiac index markedly increased as a result of endothelial nitric oxide synthase inhibition (NOSi) in both Healthy (A) and DM + HC + CKD (D) both at rest and during exercise. NOSi resulted in a similar increase in pulmonary vasoconstriction, as evidenced by the increase in pulmonary vascular resistance (PVR), in Healthy (B) and DM + HC + CKD swine (E). Nitric oxide synthase inhibition (NOSi) has a more pronounced effect on systemic vascular resistance (SVR) in DM + HC + CKD (F) than in Healthy swine (C). Healthy n = 7, DM + HC + CKD n = 7 at rest and n = 5 during exercise. Values are mean ± SEM. *P ≤ 0.05 for effect NOSi within group, †P ≤ 0.05 for interaction NOSi × cardiac index (D) or NOSi × BVO2 (E) within group. ‡P ≤ 0.05 for interaction NOSi × group (F)

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  Fig. 5. Endothelial nitric oxide (eNOS) pathway activity patterns in bulk lung tissue of DM + HC + CKD and Healthy, detected by Western blots. Total eNOS protein (A), eNOS monomer/dimer ratios (B), phosphorylated eNOS/eNOS ratios (C) and phosphorylated VASP/VASP ratios (D) were unchanged in DM + HC + CKD compared to Healthy. Values are mean ± SEM (solid lines, panel A and D) or median ± IQR (dashed lines, panel B and C). Uncut Western blots are provided in the supplemental data file

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  Fig. 6. Role of scavenging of reactive oxygen species (ROSi) in pulmonary and systemic vasomotor control in DM + HC + CKD and Healthy swine. Transpulmonary gradient (TPG) as a function of cardiac index did not result in significant change after ROSi in both Healthy (A) and in DM + HC + CKD (D). Pulmonary vascular resistance (PVR) showed no response to ROSi in Healthy (B), and in DM + HC + CKD (E). In the systemic vasculature, no effect of ROSi was observed in Healthy (C), but resulted in vasodilatation in DM + HC + CKD (F) at rest, while this effect was lost during exercise. Healthy n = 8, DM + HC + CKD n = 8. Values are mean ± SEM. *P ≤ 0.05 for effect ROSi within group, (†)P ≤ 0.10 for interaction ROSi × BVO2 within group

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  Fig. 7. Role of Endothelin 1 after eNOS inhibition in pulmonary and systemic vasomotor control in DM + HC + CKD and Healthy swine. Transpulmonary gradient (TPG) as a function of cardiac index decreased significantly after endothelin receptor A and B inhibition (ETA + Bi) in the presence of NOS inhibition (NOSi) in Healthy (A) but not in DM + HC + CKD (D). Pulmonary vascular resistance (PVR) decreased significantly in response to ETA + Bi after NOSi in Healthy (B), while in DM + HC + CKD (E) ETA + Bi additional to NOSi did not result in pulmonary vasodilation. In the systemic vasculature, effect of ETA + Bi during NOSi was only observed at rest in Healthy (C), but resulted in vasodilatation at rest and during exercise in DM + HC + CKD (F). Healthy n = 6, DM + HC + CKD n = 4. Values are mean ± SEM. *P ≤ 0.05 for effect ETA + Bi within group, †P ≤ 0.05, (†)P ≤ 0.10 for interaction ETA+Bi × group

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  Fig. 8. Phosphodiesterase 5 (PDE5) in pulmonary and systemic vasomotor control in DM + HC + CKD and Healthy swine. The relation between transpulmonary gradient (TPG) and cardiac index shifted downward as a result of PDE5 inhibition (PDE5i) in DM + HC + CKD (C) but not in Healthy (A). PDE5i resulted in pulmonary vasodilation in DM + HC + CKD (D) but not in Healthy swine (B) as evidenced by the decrease in pulmonary vascular resistance (PVR). The reduction in systemic vascular resistance (SVR) with administration of PDE5i was similar between both groups. Healthy n = 6, DM + HC + CKD n = 7. Values are mean ± SEM. *P ≤ 0.05, (*)P ≤ 0.1 for effect PDE5i within group, (†)P ≤ 0.1 for interaction PDE5i*group

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  Fig. 9. Right ventricular function at rest, during exercise and during cardiovascular magnetic resonance imaging in DM + HC + CKD and Healthy swine. Right ventricular afterload at rest and during exercise was similar as the maximal right ventricular pressure (RVPmax, A) and pulmonary arterial elastance (Ea, B) were similar between groups for any given level of body oxygen consumption (BVO2). Systolic (dP/dtmax; maximum of the first derivate of the systolic RV pressure increase, E) and diastolic function (dP/dtmin; minimum of first derivate of the RV diastolic pressure decrease, F) were preserved in DM + HC + CKD swine for any level of heart rate. n = 11 for Healthy and n = 10 for DM + HC + CKD. Right ventricle cardiovascular magnetic resonance imaging parameters were measured under general anesthesia. End-diastolic (EDV, C) and end-systolic volume (ESV, D) ejection fraction (EF, G) and global longitudinal strain (GLS, H) were unaltered in DM + HC + CKD (n = 7) compared to Healthy swine (n = 6). Values are mean ± SEM

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  Fig. 10. Right ventricular structure of DM + HC + CKD and Healthy swine typical examples of right ventricular Gomori stained (A), Lectin stained (D) and Picrosirius red stained (G) sections. The cross-sectional area of the RV cardiomyocytes was decreased in DM + HC + CKD swine (B), RV weight was also lower in DM + HC + CKD swine (C). Capillary density was similar between groups (E) but capillary-to-fiber ratio was lower in DM + HC + CKD (F). Total interstitial collagen content (H) was unaltered in DM + HC + CKD swine, but there was a shift the composition of the specific collagen fibers in DM + HC + CKD (I). Values are mean ± SEM. *P ≤ 0.05 for Healthy versus DM + HC + CKD, scale bars are 100 µm

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  Fig. 11. Proposed mechanisms of pulmonary vascular dysfunction in DM + HC + CKD. Upper cells represent endothelial cells, lower cells the smooth muscle cells. Significant differences in DM + HC + CKD compared to Healthy are shown as highlighted numbers in the figure. AKT protein kinase B, Big-ET-1 big endothelin 1, Ca2+ calcium, cGMP cyclic guanosine monophosphate, CM calmodulin, ECE endothelin converting enzyme, ET-1 endothelin 1, ETA endothelin receptor A, ETB endothelin receptor B, GTP guanosine triphosphate, Gq G alpha subunit, IP3 inositol triphosphate, L-Arg l-arginine, M-A myosin–actin, MLCk myosin light chain kinase, MLCp myosin light chain phosphatase, NO nitric oxide, NOS nitric oxide synthase, PDE5 phosphodiesterase 5, PIP3 phosphatidylinositol triphosphate, PKG protein kinase G, PLC phospholipase C, sGc soluble guanylate cyclase, SMC smooth muscle cell, VASP vasodilatation stimulating proteins. This figure is produced by adapting images from Servier Medical Art by Servier (https://smart.servier.com/), licensed under a Creative Commons attribution 3.0 Unported Licence