Proceedings of The Physiological Society

Physiology 2014 (London, UK) (2014) Proc Physiol Soc 31, C64

Oral Communications

Flicker-assisted localisation microscopy reveals altered mitochondrial architecture in vascular smooth muscle in hypertension

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

1. Strathclyde Institute of Pharmacy & Biomedical Systems, University of Strathclyde, Glasgow, United Kingdom. 2. Department of Physics, Durham University, Durham, United Kingdom.

Mitochondria vary from being small puncta to forming large interconnected networks. Mitochondrial structure is easily visualised in cultured cells; however the precise structure of the organelle in native cells is poorly understood due to the cells' complex organisation and to imaging resolution limitations. We have made use of the electrical changes during transient de- and re-polarisations of membrane potential (‘flickers') of single mitochondria within live, native vascular smooth muscle cells (visualised by epifluorescence imaging of the membrane-potential sensitive indicator TMRE, 62.5 nM) to determine the organelles' dimensions, position and relationship with Ca2+ signals. Custom image-analysis procedures written in Python software measure the spatial and temporal covariance of changes in fluorescence in areas around each image pixel, to functionally resolve organelle boundaries in clusters of visually inseparable mitochondria. We show that the apparently wide range of mitochondrial sizes in native cells (1-10 μm) is explained by varying numbers of multiple, tightly-packed small organelles. Alterations in mitochondrial positioning and architecture are associated with the proliferative state of vascular smooth muscle (1, 2) and so this analysis was applied to single smooth muscle cells freshly-isolated from resistance arteries of spontaneously hypertensive rats (SHR) and normotensive (WKY) control. Remarkably, the size of individual mitochondria increased in SHR (geometric mean area±SEM 0.826±1.97 μm2, n=48, compared to 0.347±3.083 μm2, n=20, in WKY controls; significantly different at the 0.01 level, Mann-Whitney 2 independent samples, U=1.19E6). Despite this mitochondria in SHR have a slightly higher average number of neighbouring mitochondria (mean ±SEM 4.67±0.02) than those in control (WKY; 4.48±0.03; p<0.01, one-way ANOVA). Those mitochondria in SHR are more tightly packed (mean separation±SEM 1.91±0.011 μm apart compared to 2.21±0.016 μm in WKY; p<0.01, one-way ANOVA). These two aspects combine to increase the extent of mitochondrial clustering within the vascular smooth muscle of SHR compared to WKY. Resistance artery smooth muscle cells were then co-loaded with the Ca2+-indicator fluo4AM (10 μM) plus mitochondrial TMRE and patch-clamped in the whole cell configuration. Transient plasma-membrane depolarisation (-70 to 10 mV, 1 s) activated voltage-gated Ca2+ channels and elevated [Ca2+]c. Pixel-by-pixel analysis reveals that those pixels that are closest to mitochondria show the highest [Ca2+]c changes. In summary, this work has developed a novel tool for increased resolution of mitochondrial architecture in live cells. This tool revealed a previously unrecognised change in mitochondrial structure that may contribute to changes in Ca2+ signalling and smooth muscle function characteristic of hypertension.

Where applicable, experiments conform with Society ethical requirements