Long-Term Strenuous Exercise Promotes Vascular Injury by Selectively Damaging the Tunica Media: Experimental Evidence

Abstract

Moderate exercise has well-founded benefits in cardiovascular health. However, increasing, yet controversial, evidence suggests that extremely trained athletes may not be protected from cardiovascular events as much as moderately trained individuals. In our rodent model, intensive but not moderate training promoted aorta and carotid stiffening and elastic lamina ruptures, tunica media thickening of intramyocardial arteries, and an imbalance between vasoconstrictor and relaxation agents. An up-regulation of angiotensin-converter enzyme, miR-212, miR-132, and miR-146b might account for this deleterious remodeling. Most changes remained after a 4-week detraining. In conclusion, our results suggest that intensive training blunts the benefits of moderate exercise.

Keywords: CACS, coronary artery calcium score; CAD, coronary artery disease; CV, cardiovascular; MMP9, matrix metalloproteinase 9; NO, nitric oxide; Phe, phenylephrine; VSMC, vascular smooth muscle cell; atherosclerosis; coronary artery disease; endurance exercise; extreme sport; vascular stiffening.

Graphical abstract

Figure 1

Aortic Morphological Changes (A) Representative microphotographs of hematoxylin/eosin-stained thoracic aorta sections from sedentary (SED) (n = 10), moderate (MOD) (n = 6), and intensive (INT) (n = 6) training groups. (B to D) Quantification (mean ± SEM) of tunica media thickness (B), lumen area (C), and wall thickness to lumen diameter ratio (D). (E) Representative echocardiographic images of the root (blue arrow) and ascending thoracic aorta (yellow arrow). (F and G) Echocardiographic measurements (mean ± SEM) of ascending aorta (F) and aortic root (G) diameters (SED, n = 16; MOD, n = 17; INT, n = 16). All analyses were performed with a 1-way analysis of variance; the omnibus test was only significant for G at the P < 0.05 level. ∗P < 0.05.

Figure 2

Aortic Tunica Media Structural Remodeling (A) Representative Picrosirius-stained pictures of thoracic aorta sections. (B) Quantification of collagen content (mean ± SEM) in the tunica media (SED, n = 7; MOD, n = 9; INT, n = 9). (C) Representative images of elastic laminae revealed by autofluorescence. (D) Density of elastic lamina ruptures (white arrow in right panel of C) (mean ± SEM). (E) Assessment of elastic lamina elongation (mean ± SEM) (SED, n = 9; MOD, n = 7; INT, n = 7). All analyses were performed with a 1-way analysis of variance; omnibus tests were significant at the P < 0.01 (B and D) and the P < 0.05 (E) levels. ∗P < 0.05, ∗∗P < 0.01. Abbreviations as in Figure 1.

Figure 3

Intramyocardial Vessels Remodeling (A) Representative Picrosirius-stained pictures of intramyocardial arteries. (B to D) Area (mean ± SEM) of tunica media (B), lumen (C), and perivascular fibrosis (D) adjusted for total vessel area (SED, n = 9; MOD, n = 9; INT, n = 9). Analyses were performed with a linear mixed-effects model (vessel nested into ventricle, nested into rat). Omnibus tests were significant at the P < 0.05 level (B and C). ∗P < 0.05. Abbreviations as in Figure 1.

Figure 4

Aortic Stiffness Assessment (A) Representative invasive blood pressure recordings in all groups. Both the electrocardiogram (top) and the central arterial pressure (bottom) are shown. (B) Quantification (mean ± SEM) of pulse pressure (PP), obtained from central pressure recordings in all groups (SED, n = 15; MOD, n = 13; INT, n = 11). (C) Representative echocardiographic images (M-mode) of ascending aorta in all groups. The maximum aortic systolic (AoS) (purple arrow) and aortic diastolic (AoD) (cyan arrow) diameters are shown. (D and E) Quantification (mean ± SEM) of aortic root (D) and ascending aorta (E) pulsatility obtained from echocardiographic data (SED, n = 16; MOD, n = 19; INT, n = 17). (F) Correlation between hemodynamic and echocardiographic data (Ao-P). (G and H) Estimation (mean ± SEM) of arterial stiffness by the β-index (G) and elastic modulus (Ep) (H) (SED, n = 9; MOD, n = 13; INT, n = 8). Most analyses (B, D, E, G, H) were performed with a 1-way analysis of variance; omnibus tests were significant at the P < 0.001 (D and E); P < 0.01 (B) and P < 0.05 (G and H) levels. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviations as in Figure 1.

Figure 5

VSMC Stiffness Measurement By Atomic Force Microscopy (A) Cultured vascular smooth muscle cells (VSMCs) with the AFM cantilever (left). (B) VSMC stiffness measurements (mean ± SEM) in all groups (right). Analyses were performed with a linear mixed-effects model (each measurement nested into cell, and each cell nested into rat). The omnibus test was significant at the P < 0.05 level. ∗P < 0.05; ∗∗P < 0.01.

Figure 6

Vascular Reactivity of the Thoracic Aorta (A and B) Dose-response vasorelaxation curves to Carbachol (CCh) in intact endothelium conditions (A) (SED, n = 19; MOD, n = 21; INT, n = 11) and nitric oxide (NO)-independent conditions after LNMMA incubation (B) (SED, n = 20; MOD, n = 19; INT, n = 15). (C and D) Dose-response vasoconstriction curves to Phenylephrine (Phe) in intact endothelium conditions (C) (SED, n = 9; MOD, n = 12; INT, n = 8) and NO-independent conditions after LNMMA incubation (D). Shaded areas represent 95% CI. Comparisons performed with sum-of-squares F tests by comparing fitted logEC50 and maximum effect in 3-parameter equations. ∗∗P < 0.01. Abbreviations as in Figure 1.

Figure 7

Mechanisms of Exercise-Induced Vascular Remodeling (A) Venn diagram showing the 21 unique miRNAs that were deregulated between the different comparisons in the miRNA microarray analysis of thoracic aorta. (B) Relative expression (mean ± SEM) by RT-PCR of the miRNA that were deregulated in the INT group in comparison to both SED and MOD groups in the miRNA array. (C) Ingenuity analysis of the validated targets of the 3 selected miRNAs. Analyses point to MMP9 as a central component of the largest network including validated targets of miR-132-3p, miR-212-3p, and miR-146b-5p. (D) Representative ACE-1 blots and Ponceau-stained membranes (top), and relative quantification (mean ± SEM) of ACE-1 protein in all groups (bottom). (SED, n = 6; MOD, n = 7; INT, n = 6). Analyses were performed with 1-way analysis of variance; omnibus tests were significant at the P < 0.05 level. ∗P < 0.05.