Short communication Rapid telomere attrition in cardiac tissue of the ageing Wistar rat Richard Hastings * , Nai-Chang Li, Peter S. Lacy, Hasmukh Patel, Karl E. Herbert, Adrian G. Stanley, Bryan Williams Department of Cardiovascular Sciences, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, UK Received 5 November 2003; received in revised form 28 January 2004; accepted 17 February 2004 Abstract Many studies show an association between ageing and mean telomere length in DNA isolated from peripheral blood mononuclear cells, few studies have examined less accessible tissues. This study has two objectives: (i) to define the best method to prepare rodent DNA for telomere length measurement by Southern blotting and (ii) to determine whether there are differential rates of telomere attrition in different rodent tissues. We found that the use of agarose plugs for DNA isolation was essential for the accurate measurement of rodent telomere length. Tissue was collected from neonatal (3 days) or aged (18 – 24 months) male Wistar rats and terminal restriction fragment (TRF) length was measured by Southern blotting. Cardiac tissue from aged rats showed a 38% loss of TRF length compared with newborn animals (p , 0:001; n ¼ 13), this contrasts with much smaller reductions in brain (1.6%), liver (14.2%), kidney (8.9%) and lung (9.7%). This study demonstrates that the methods of DNA preparation are critical for accurate measurement of telomeres in rodent tissues. Moreover, we show differential rates of telomere attrition in rat tissues, the heart being most susceptible to telomere loss. These observations could have important implications for the study of age-specific changes in tissue function. q 2004 Elsevier Inc. All rights reserved. Keywords: Telomere; Molecular ageing; Senescence; Wistar rat; Cardiovascular system 1. Introduction The progressive loss of the protective cap structures, telomeres, at the ends of chromosomes is one of several molecular changes that characterise ageing (reviewed in McEachern et al., 2000; Campisi et al., 2001). In humans, telomeres are approximately 15 kb at birth, but shorten to less than 10 kb with ageing. Oxidative damage to telomeric DNA repeats has been proposed as the major cause of telomere attrition (von Zglinicki, 2002). When telomeres become critically shortened, the cell enters a crisis that in most cases promotes cell senescence. This process is characterised by an inability to enter cell division and by a phenotypic shift and deleterious changes in cell function. The age-associated accumulation of senescent cells is likely to have profound effects on the function of many organs. Rodent telomeres are generally much longer than those of humans (Kipling and Cooke, 1990), and have been technically difficult to study. Consequently, tissue-specific telomere loss in rodent models of ageing is poorly understood. This study has two objectives: (i) to determine the most accurate method for measuring rodent telomere lengths by Southern blotting and (ii) to study tissue-specific changes in the telomere length of ageing Wistar rats. 2. Materials and methods 2.1. Measurement of rat telomeres Tissues were taken from male neonatal (3 days old) and adult male (18–24 months old) Wistar rats. Lung, liver, heart, kidney and brain were taken from each animal and were rapidly frozen or used immediately. Samples were minced then homogenised in buffer L (0.1 M EDTA, 10 mM Tris–HCl, 20 mM NaCl, pH7.5). Tissue homogen- ates were centrifuged and resuspended in an appropriate volume of buffer L (determined by experiment to provide approximately equal DNA concentrations). An equal 0531-5565/$ - see front matter q 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2004.02.003 Experimental Gerontology 39 (2004) 855–857 www.elsevier.com/locate/expgero * Corresponding author. Tel.: þ44-116-252-5837; fax: þ 44-116-252- 5847. E-mail address: rah22@le.ac.uk (R. Hastings).