Rats, Inbred BN

The rat macrophages were isolated from inbred male Brown Norway rats (Harlan, Indianapolis, IN) and cultured as described in an MRI cell phantom study (21 (link)). Phantoms with different cell densities (0.8 x 10⁷, 2 x 10⁷, 4 x 10⁷, and 8 x 10⁷, cells/mL) were prepared by diluting cells in different volumes of 2% agarose gel in shortened 5-mm NMR tubes (Fig. 1a). 4 cell phantoms and one control phantom (2% agarose gel with no cell) were scanned using a Bruker BioSpec 11.7-T scanner (Bruker, Billerica MA). The diffusion acquisition was conducted using a 2D stimulated echo sequence. The b-table was a grid sampling scheme with 101 diffusion encoding gradient directions (22 (link)). The b-table is publicly available on the DSI Studio website (http://dsi-studio.labsolver.org). The maximum b-value was 6,000 mm²/sec, diffusion time Δ = 80 ms (i.e., diffusion gradient separation), and the diffusion gradient pulse duration δ = 1 ms. The diffusion weighted images were acquired with TE = 10 ms, TM = 75 ms, TR = 2600 ms. The FOV was 1.26 cm with an in-plane resolution of 0.2 mm and slices thickness of 0.35 mm. ρ(L) and δ(L) were calculated by Equations 3 and 4, respectively, and correlated with the cell density. The data were analyzed using DSI Studio (http://dsi-studio.labsolver.org).

The animals used in this study-- all obtained from the National Institutes of Aging colony (Harlan Sprague-Dawley, Inc., Indianapolis, IN)-- were adult (six months-old) and elderly (31 months-old) Fischer 344 X Brown Norway hybrid rats and adult (six months-old) and elderly (24 months-old) Fischer 344 rats. The animal studies were approved by the Institutional Animal Care and Use Committees of the School of Medicine, Case Western Reserve University, and of the Louis Stokes Cleveland Department of Veterans Affairs Medical Center. The rats were allowed food and water ad libitum, and were maintained on a 12 hour light:dark cycle. They were killed by decapitation and specimens of left ventricle removed. Samples of the left ventricle were immediately fixed by immersion in the triple aldehyde-DMSO mixture of Kalt and Tandler (1971) (link). After rinsing in distilled water, the tissue blocks were postfixed in ferrocyanide-reduced osmium tetroxide (Karnovsky, 1971 ). After again rinsing in distilled water, they were soaked overnight in acidified ranyl acetate (Tandler, 1990 (link)). Another rinse in distilled water was followed by dehydration in ascending concentrations of ethanol, passage through propylene oxide, and embedment in Poly/Bed resin. Thin sections were sequentially stained with acidified uranyl acetate (Tandler, 1990 (link)) followed by Sato’s triple lead stain as modified byHanaichi et al. (1986) and examined in a JEOL 1200EX electron microscope.
Pellets of isolated subsarcolemmal (SSM) and of interfibrillar mitochondria (IFM) were prepared as described previously byPalmer et al. (1977) (link) and by Chen et al. (2007). These initially were fixed in eighth-strength Karnovsky’s fixative (1965) ; from this point on, the pellets were treated in the same fashion as the solid tissues, as described above.

All experiments were approved by the Institutional Animal Care and Use Committee of The Ohio State University and are in compliance with the laws of The United States of America and conform to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health. Male Brown Norway rats (approximately 2–4 months old, ~200–250 grams) were intraperitoneally injected with euthasol (~400 mg/kg pentobarbital sodium and ~50 mg/kg phenytoin sodium). The chest cavity was exposed by bilateral thoracotomy and the heart was quickly removed after it was injected with 1000 units of heparin. The heart was perfused via the aorta in a retrograde fashion with a modified Krebs-Henseleit solution containing (in mM) 137 NaCl, 5 KCl, 20 NaHCO₃, 1.2 NaH₂PO₄, 1.2 MgSO₄, 10 glucose, 0.25 CaCl_2, and 20 2,3-butanedione monoxime (BDM) at room temperature. This solution was bubbled with 95% O₂/5% CO₂ where the buffering system results in a pH of 7.4. Trabeculae were isolated from the right ventricle and mounted horizontally in a custom made setup as previously described(Milani-Nejad et al. 2013 (link)). Muscle were stimulated at 20% over theshhold voltage, using 2 ms bipolar pulses, typically ~5 V. The perfusion solution during experiments was changed to a modified Krebs-Henseleit solution without (BDM) and containing 2 mM CaCl₂. This solution was also continuously bubbled with 95% O₂/5% CO₂ resulting in a pH of 7.4. Muscles were gradually stretched until reaching optimal length as previously described (Milani-Nejad et al. 2013 (link)) and all of the experiments were performed at this length. Muscles for force-frequency relationship and post-rest potentiation experiments were stabilized at a baseline frequency of 4 Hz and 37 °C. Muscles used for rapid cooling contracture experiments were stabilized at 1 Hz and 27 °C, the latter temperature was chosen to allow for a rapid cooling; cooling from 37 °C in this particular set-up led to incomplete cooling of the muscles. Muscle chamber size was ~400 μl, with a flow rate that refreshed the bath once every ~3 seconds. Due to space restrictions of the small bath (needed for rapid cooling), The temperature probe as situated after the muscle, leading to a small lag time in recording of the actual temperature at the muscle level. Confocal imaging experiments were stabilized at 1 Hz and room temperature (~23 °C) to avoid actifacts from solution flow vibrations. All force recordings in all protocols were normalized to cross-sectional area of the muscles.