Progressive aortic stiffness in aging C57Bl/6 mice displays altered contractile behaviour and extracellular matrix changes

Figures

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  Fig. 1. Age-related aortic stiffening in C57Bl/6 mice. Aortic stiffness was measured in vivo as aPWV (a) and ex vivo in the isolated thoracic aorta at isobaric conditions of 80–120 mmHg as Peterson modulus (Ep, b). Wall thickness was determined on orcein-stained aortic tissue sections (c) and total medial cell number was counted on haematoxylin-stained aortic tissue sections (d). Orcein staining was used to quantify elastin positive area (e) and counting of elastin breaks (f). Each symbol represents an individual biological repeat, with n > 8 (a, b) and n = 5 (c, f). Statistical analysis using one-way ANOVA. Overall significance (bottom of graph) and post hoc significance vs. 4-month value (in graph) are listed. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 2. More pronounced aortic stiffening at high distending pressure corresponds to increased levels of types I and III collagen. Peterson modulus expressed in function of age for increasing distending pressure from 60–100, 80–120, 100–140, to 120–160 mmHg (a). Values of slope from linear regression analysis are listed on the right for each pressure. Histological assessment of area positivity for type I and II collagen (b, c). Data are listed as mean ± SEM (n > 8) (a) or each symbol represents an individual biological repeat (n = 5) (b, c). Statistical analysis using two-way ANOVA (a) or one-way ANOVA (b, c). Overall significance (bottom of graph) and post hoc significance vs. 4-month value (in graph) are listed. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 3. Increased PE-contractility and VGCC alterations. Maximal PE contraction in the absence (circles) and presence (squares) of L-NAME (a). Basal NO levels, quantified as the relative increase in PE contraction after addition of L-NAME (b). Contraction-dependent aortic stiffening by addition of 2 µM PE + 300 µm L-NAME at isobaric 80–120 mmHg pressure (c). Maximal PE contractions, as obtained from non-linear regression of the PE concentration-response curves, in the absence (circles) and presence (squares) of 30 nM BAY-K8644 (d), and calculated absolute BAY-K8644 effect (circles, e). Contraction-inhibition by diltiazem in the absence (circles) or presence (squares) of BAY-8644 (f). Statistical analysis using two-way ANOVA (a, d, f), one-way ANOVA (b, c) or one-sample t-test vs. 0 (e). Data are listed as mean ± SEM (a, d–f, n > 8) or each symbol represents an individual biological repeat (b, c, n > 8). Overall significance (bottom of graph) and post hoc significance vs. 4-month value (in graph) are listed. *p < 0.05, **p < 0.01, ***p < 0.001. In d, post hoc comparison for BAY-K8644 effect is listed as #p < 0.05, ##p < 0.01.

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  Fig. 4. Functional and molecular VGCC phenotyping. Potassium concentration-response curves of 5- and 26-month-old mice were plotted in the absence (circles) and presence (squares) of L-NAME (a). Non-linear regression analysis was used to calculate the maximal effect (b) and EC50 value (c). 50 mM potassium-induced contraction-dependent change in Peterson modulus at calculated isobaric 80–120 mmHg distending pressure was measured in the absence and presence of 300 µM L-NAME (d). Splice variation was assessed for CaV1.2 exon 9* and exon 33, and quantification of the % mRNA with inclusion of exon 9* or exon 33 is shown (e). The fraction nuclear/total myocardin in the media was assessed using immunohistochemical staining, as a marker of VSMC phenotype regulation (f). Data are listed as mean ± SEM (a, e, n > 5) or each symbol represents an individual biological repeat (b–d, f, n > 5). Statistical analysis using three-way ANOVA (a), two-way ANOVA (b–d), or one-way ANOVA (e, f). Overall significance (bottom of graph) and post hoc significance vs. 4-month value (in graph) are listed (post hoc significance was not added in a). **p < 0.01, ***p < 0.001. In c, d, post hoc comparison for L-NAME…

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  Fig. 5. Ageing induces slight endothelial dysfunction and increased basal VSMC tone. ACh concentration-response curves of PE-precontracted aortic rings (a), with logIC50 value (b) and maximal effect (c) from non-linear regression analysis. Statistical analysis using two-way ANOVA (a) or one-way ANOVA (b, c). DEANO concentration-response curves of PE + L-NAME-precontracted aortic rings (d), with a calculated maximal effect (e) from non-linear regression analysis. Effect of replacing normal Krebs solution with 0Ca Krebs on isometric force (f). Data are listed as mean ± SEM (a, d, n > 8). Statistical analysis using two-way ANOVA (a, d), one-way ANOVA (b, c), or repeated one-sample t-test vs 100% (e) or 0 mN (f). Overall ANOVA significance (bottom of graph) and post hoc significance as indicated (above brackets) are listed (post hoc significance was not added in a, d). *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 6. Altered transient SR-mediated contractions. A tracing of the transient SR-mediated contraction (a) was fitted with a bi-exponential regression equation encompassing the amplitude of the upward (Aon) and downward (Aoff) phase (b), as well as the time constant of the upward (τon) and downward (τoff) phase (c). Statistical analysis using two-way ANOVA (a) or one-way ANOVA (b, c). Data are listed as mean ± SEM (n > 8). Overall significance (bottom of graph) and post hoc significance vs. 4-month value (in graph) are listed (post hoc significance was not added in a). *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 7. Arterial-stiffness precedes cardiac disease and peripheral blood pressure alterations. Echocardiograms were used to assess interventricular septum (IVS) thickness (a) and left ventricular internal diameter (LVID, b). Cardiomyocyte cross-sectional area was studied using immunohistochemical staining for laminin (c). Peripheral systolic blood pressure (SBP, d), diastolic blood pressure (DBP, e) and pulse pressure (PP, f) were measured. Each symbol represents an individual biological repeat (n > 8). Statistical analysis using one-way ANOVA. Overall significance (bottom of graph) and post hoc significance vs. 4-month value (in graph) are listed. ***p < 0.001.