Abstract
Key points Age is proposed to be associated with altered structure and function of mitochondria; however, in fully‐differentiated cells, determining the structure of more than a few mitochondria at a time is challenging. In the present study, the structures of the entire mitochondrial complements of cells were resolved from a pixel‐by‐pixel covariance analysis of fluctuations in potentiometric fluorophore intensity during ‘flickers’ of mitochondrial membrane potential.Mitochondria are larger in vascular myocytes from aged rats compared to those in younger adult rats.A subpopulation of mitochondria in myocytes from aged, but not younger, animals is highly‐elongated.Some mitochondria in myocytes from younger, but not aged, animals are highly‐motile.Mitochondria that are motile are located more peripherally in the cell than non‐motile mitochondria. Mitochondrial function, motility and architecture are each central to cell function. Age‐associated mitochondrial dysfunction may contribute to vascular disease. However, mitochondrial changes in ageing remain ill‐defined because of the challenges of imaging in native cells. We determined the structure of mitochondria in live native cells, demarcating boundaries of individual organelles by inducing stochastic ‘flickers’ of membrane potential, recorded as fluctuations in potentiometric fluorophore intensity (flicker‐assisted localization microscopy; FaLM). In freshly‐isolated myocytes from rat cerebral resistance arteries, FaLM showed a range of mitochondrial X‐Y areas in both young adult (3 months; 0.05–6.58 μm2) and aged rats (18 months; 0.05–13.4 μm2). In cells from young animals, most mitochondria were small (mode area 0.051 μm2) compared to aged animals (0.710 μm2). Cells from older animals contained a subpopulation of highly‐elongated mitochondria (5.3% were >2 μm long, 4.2% had a length:width ratio >3) that was rare in younger animals (0.15% of mitochondria >2 μm long, 0.4% had length:width ratio >3). The extent of mitochondrial motility also varied. 1/811 mitochondria observed moved slightly (∼0.5 μm) in myocytes from older animals, whereas, in the younger animals, directed and Brownian‐like motility occurred regularly (215 of 1135 mitochondria moved within 10 min, up to distance of 12 μm). Mitochondria positioned closer to the cell periphery showed a greater tendency to move. In conclusion, cerebral vascular myocytes from young rats contained small, motile mitochondria. In aged rats, mitochondria were larger, immobile and could be highly‐elongated. These age‐associated alterations in mitochondrial behaviour may contribute to alterations in cell signalling, energy supply or the onset of proliferation.
Highlights
Vascular function declines with age as a result of a range of morphological and molecular alterations, such as vessel wall intima-media thickening, smooth muscle cell hyperplasia, endothelial cell dysfunction, increased oxidative stress and decreased nitric oxide bioavailability
Mitochondrial architecture was examined in vascular smooth muscle cells from young adult and aged adult rats (3 and 18 months, respectively) using flicker-assisted localization microscopy (FaLM)
Mitochondria from young animals were dispersed throughout the cell (Fig. 2Aa), largely presenting as spheres or short rods
Summary
Vascular function declines with age as a result of a range of morphological and molecular alterations, such as vessel wall intima-media thickening, smooth muscle cell hyperplasia, endothelial cell dysfunction, increased oxidative stress and decreased nitric oxide bioavailability. Vascular smooth muscle cells have an increased production of oxidative species with age, both via the activity of plasma membrane NADPH oxidase and the mitochondria; mitochondrial superoxide production may precede, and induce, NADPH superoxide production (Mistry et al 2013) Attenuating this mitochondrial oxidant production inhibits vascular smooth muscle cell switching to a proliferative and migratory phenotype and, reducing mitochondrial anti-oxidants promotes proliferation (Wang et al 2012). The extent of mitochondrial motility in fullydifferentiated, post-mitotic cells, is less clear (Chalmers et al 2012; Song & Dorn, 2015) and it remains to be seen whether alterations in mitochondrial motility are associated with vascular smooth muscle ageing
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