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. 2018 Aug;17(4):e12773.
doi: 10.1111/acel.12773. Epub 2018 May 9.

Restoring Mitochondrial DNA Copy Number Preserves Mitochondrial Function and Delays Vascular Aging in Mice

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Free PMC article

Restoring Mitochondrial DNA Copy Number Preserves Mitochondrial Function and Delays Vascular Aging in Mice

Kirsty Foote et al. Aging Cell. .
Free PMC article

Abstract

Aging is the largest risk factor for cardiovascular disease, yet the molecular mechanisms underlying vascular aging remain unclear. Mitochondrial DNA (mtDNA) damage is linked to aging, but whether mtDNA damage or mitochondrial dysfunction is present and directly promotes vascular aging is unknown. Furthermore, mechanistic studies in mice are severely hampered by long study times and lack of sensitive, repeatable and reproducible parameters of arterial aging at standardized early time points. We examined the time course of multiple invasive and noninvasive arterial physiological parameters and structural changes of arterial aging in mice, how aging affects vessel mitochondrial function, and the effects of gain or loss of mitochondrial function on vascular aging. Vascular aging was first detected by 44 weeks (wk) of age, with reduced carotid compliance and distensibility, increased β-stiffness index and increased aortic pulse wave velocity (PWV). Aortic collagen content and elastin breaks also increased at 44 wk. Arterial mtDNA copy number (mtCN) and the mtCN-regulatory proteins TFAM, PGC1α and Twinkle were reduced by 44 wk, associated with reduced mitochondrial respiration. Overexpression of the mitochondrial helicase Twinkle (Tw+ ) increased mtCN and improved mitochondrial respiration in arteries, and delayed physiological and structural aging in all parameters studied. Conversely, mice with defective mitochondrial polymerase-gamma (PolG) and reduced mtDNA integrity demonstrated accelerated vascular aging. Our study identifies multiple early and reproducible parameters for assessing vascular aging in mice. Arterial mitochondrial respiration reduces markedly with age, and reduced mtDNA integrity and mitochondrial function directly promote vascular aging.

Keywords: aging; mitochondria; mitochondrial DNA; vascular stiffness.

Figures

Figure 1
Figure 1
Functional evidence that mice develop vascular stiffness by 44 wk of age. (a–f) Pulse pressure, heart rate, aortic pulse wave velocity (PWV), carotid artery compliance, carotid artery distensibility and carotid artery β‐stiffness index in WT mice aged 8–72 wk. Data are means ± SEM. *p < .05 using ANOVA with Tukey post‐test (n = 10–17)
Figure 2
Figure 2
Structural evidence that mice develop increased collagen, elastin breaks, reduced cellularity and vessel thickening by 44 wk of age. (a) Representative images of aorta sections stained with Sirius Red, Verhoeff–van Gieson (VVG) or haematoxylin and eosin (H&E). Examples of elastin breaks are indicated by the white arrows. Scale bars = 25 μm. (b) Collagen quantification using Sirius Red‐positive staining, (c) number of elastin breaks quantified by VVG staining, (d) medial cellularity and (e) aortic wall thickness. Data are means ± SEM. *p < .05 using ANOVA with Tukey post‐test (n = 4–9 mice)
Figure 3
Figure 3
Reductions in vascular mitochondrial DNA copy number, mitochondrial protein expression and mitochondrial respiration with age in mice. (a) Relative aortic mitochondrial copy number (mtCN) (n = 4–6 mice), (b) relative aortic mtDNA lesions, (c–g) Western blots showing protein levels of the major mtCN regulators Twinkle, PGC1α or TFAM with their respective quantifications (relative to β‐actin), (h) relative protein expression levels of mitochondrial complexes (I–V relative to citrate synthase levels) quantified from Western blot, (i) Seahorse oxygen consumption rate (OCR) in intact aortas indicating injection points (grey dotted lines) of oligomycin (O), FCCP (F), antimycin A and rotenone (A/R) and (j) quantified baseline OCR (n = 3 aortas per group). Data are means ± SEM. *p < .05 using ANOVA with Tukey post‐test
Figure 4
Figure 4
Tw+ mice have increased vascular mtCN and enhanced mitochondrial respiration. (a) qPCR for Twinkle mRNA (n = 3), (b–c) Western blot for Twinkle protein in aortas of WT and Tw+ mice at 44 wk (n = 3–4), (d) mitochondrial copy number (mtCN) in aorta in WT and Tw+ mice at 44 and 72 wk (n = 5–6), (e–f) representative Western blot of mitochondrial complexes (I–V) in aortas from 72‐wk‐old WT and Tw+ mice with quantified levels relative to citrate synthase (CS) (n = 3–5), (g) Seahorse profile of WT and Tw+ mouse aortas at 44 wk indicating injection of the compounds (grey dotted lines) oligomycin (O), FCCP (F) and antimycin A and rotenone (A/R), with (h) Quantification of OCR at baseline and after addition of the uncoupler FCCP (n = 2 aortas). WT (grey labels); Tw+ (blue labels). Data are means ± SEM. *p < .05 using ANOVA with Tukey post‐test
Figure 5
Figure 5
Tw+ mice have delayed vascular aging. (a–f) Pulse pressure, heart rate, aortic pulse wave velocity (PWV), and carotid artery compliance, carotid artery distensibility and carotid β‐stiffness index in WT and Tw+ mice aged 22–72 wk (n = 6–9), (g) representative images of aorta from 44‐wk‐old WT and Tw+ mice stained with Sirius Red and quantification of the percentage positive staining (scale bar = 50 μm) and (h) representative images of aorta from 44‐wk‐old WT and Tw+ mice stained with Verhoeff–van Gieson (VVG) showing elastin breaks (white arrows) and quantification of the number of elastin breaks. Scale bar = 20 μm (n = 3–7). WT (grey labels); Tw+ (blue labels). Data are means ± SEM. *p < .05 using ANOVA with Tukey post‐test
Figure 6
Figure 6
PolG mice have accelerated vascular aging. (a–f) Pulse pressure, heart rate, aortic pulse wave velocity (PWV) and carotid artery compliance, carotid distensibility and carotid β‐stiffness index in WT and PolG mice aged 8–32 wk relative to 8‐wk‐old mice (n = 5–9), (g) representative images of aortas from 22‐wk‐old WT and PolG mice stained with Sirius Red and quantification of positive staining (scale bar = 50 μm) and (h) representative images of aortas from 8‐wk‐old WT and PolG mice stained with Verhoeff–van Gieson (VVG) for measuring elastin breaks (scale bar = 20 μm) (n = 4–6). WT (grey labels); PolG (red labels). Data are means ± SEM. *p < .05 using ANOVA with Tukey post‐test
Figure 7
Figure 7
Changes in WT, Tw+ and PolG mice during vascular aging. Summary of the age at which changes are first observed over 8–72 wk of age in WT, Tw+ and PolG mice. mtDNA, mitochondrial DNA; mtCN, mitochondrial copy number; PWV, pulse wave velocity

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