Proceedings of The Physiological Society

Ageing and Degeneration (Edinburgh, UK) (2015) Proc Physiol Soc 33, PC11

Poster Communications

Age-associated alterations in mitochondrial architecture and motility in vascular smooth muscle

S. Chalmers1, C. Saunter2, J. M. Girkin2, J. G. McCarron1

1. SIPBS, University of Strathclyde, Glasgow, Please Select, United Kingdom. 2. Physics, Durham University, Durham, United Kingdom.

The motility and architecture of mitochondria is central to cell function. Mitochondrial function declines with age, and may underlie the development of a diverse range of common diseases. Changes in mitochondrial structure and motility may also contribute to changes in function with age but are ill-defined due to challenges of imaging in native cells and tissues. We have developed methods to determine the structure and motility of mitochondria in live functional native cells based upon epifluorescence microscopy. To determine the spatial extent of individual, electrically-distinct mitochondria (i.e. their functional boundaries), stochastic transient "flickers" of mitochondrial membrane potential were induced and measured as fluctuations in cationic fluorophore intensity (Flicker-assisted Localization Microscopy, FaLM). Custom image analysis, written in Python, defined mitochondrial boundaries as having a strong positive spatio-temporal co-variance of fluorescence intensity changes, measured on a pixel-by-pixel basis around each mitochondrial centre. Single native smooth muscle cells isolated from cerebral resistance arteries from young (3 month) and aged (18 month) rats (humanely sacrificed in accord with UK legislation) were loaded with tetramethylrhodamine-ethyl ester (62.5 nM) and imaged at 37°C.FaLM revealed a range of mitochondrial sizes in smooth muscle cells from young and aged rats. In young animals the majority of mitochondria were very small (most common size: mode area of 0.051 µm2; geometric mean±s.d. of 0.35±3.08; n=19 cells/1259 mitochondria) when compared to older animals (mode 0.76 µm2; geometric mean±s.d. of 1.38±2.44; n=12 cells/805 mitochondria; significantly different at the .05 α level by two-sample t-test with Welch correction for unequal variance). Interestingly, smooth muscle from older animals contained many more long mitochondria than from young (5.4% of mitochondria in young were > 1 µm vs. 39.5% in older; two-sample t-test between proportions significant at the .05 α level, p<0.01). Smooth muscle from older animals also contained a subpopulation (4.2%) of highly-elongated, electrically-contiguous mitochondria (length:width ratio>3) not observed in younger animals (mean±s.d. length:width of 5% most elongated was 1.93±0.54 µm; two-sample t-test between proportions significant at the .05 α level, p<0.01). Additionally, mitochondria in smooth muscle from older animals were not observed to move. In the younger animals, both directed and Brownian-like mitochondrial motility occurred.In conclusion, vascular smooth muscle from young animals contained small, motile mitochondria. In aged animals the mitochondria were larger, sometimes highly-elongated and were immobile. These age-associated alterations in mitochondrial behaviour may present a barrier for re-entry into the cell cycle and hence inhibit smooth muscle proliferation.

Where applicable, experiments conform with Society ethical requirements