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Aortic banding (AB). AB was performed on age-matched (~ 3 months) WT and KO
mice as described1. Both male and female were included in the experiments. After
anesthesia, the chest was opened, and a blunt dissection was performed to expose the
aortic arch. A 7-0 silk suture was placed around the aorta between the left common
carotid artery and brachiocephalic trunk. A 27-gauge needle was placed on top of the
exposed aortic arch. Tying a suture around the needle and its subsequent removal
produced stenosis. Sham animals underwent same surgery without banding. All the
measurements were made one month after AB. Systolic blood pressures of right carotid
artery in anesthetized animals were measured using Millar Mikro-tip catheter (Millar
Instruments).
Langendorff Preparation. LV function was measured using the isolated buffer-perfused
heart preparation as described2. The hearts were perfused retrogradely using KrebsHenseleit buffer at a constant perfusion pressure of 80 mmHg and paced at 7 Hz. A small
polyvinyl chloride fluid-filled balloon attached to polyethylene tubing was placed in the
LV via the left atrium and connected to a pressure transducer for determinations of LV
pressures. The balloon was then progressively filled in 5 μl increments to generate LV
filling and function curves.
Apoptosis. To detect apoptosis, terminal deoxynucleotidyl transferase-mediated dUTP
nick end-labeling (TUNEL) followed by propidium iodide staining was carried out in 4m thick sections from LV apex, mid cavity and base as described3. From each heart, the
total number of TUNEL-positive myocyte nuclei was counted and normalized to the total
number of nuclei.
Western Analysis. Tissue lysates were prepared in lysis buffer (1%Triton X-100, NaCl
150 mmol/L, Tris 10 mmol/L, pH 7.4, EDTA 1 mmol/L, EGTA 1mmol/L,
phenylmethylsulfonyl fluoride 0.2 mmol/l, sodium orthovanadate 0.2 mmol/L and 0.5%
nonidet P-40) using dounce homogenizer. Equal amounts of proteins (50 μg) were
analyzed by western blot as described4. Protein loading differences were normalized
using β-actin (Figure 1) and non-phospho-specific (Figures 4 and 5) antibodies.
References
1. Hill JA, Karimi M, Kutschke W, Davisson RL, Zimmerman K, Wang Z, Kerber RE,
Weiss RM. Cardiac hypertrophy is not a required compensatory response to shortterm pressure overload. Circulation. 2000;101:2863-2869.
2. Trueblood NA, Xie Z, Communal C, Sam F, Ngoy S, Liaw L, Jenkins AW, Wang J,
Sawyer DB, Bing OH, Apstein CS, Colucci WS, Singh K. Exaggerated left
ventricular dilation and reduced collagen deposition after myocardial infarction in
mice lacking osteopontin. Circ Res. 2001;88:1080-1087.
3. Sam F, Xie Z, Ooi H, Kerstetter DL, Colucci WS, Singh M, Singh K. Mice lacking
osteopontin exhibit increased left ventricular dilation and reduced fibrosis after
aldosterone infusion. Am J Hyper. 2004;17:188-193.
4. Xie Z, Singh M, Siwik DA, Joyner WL, Singh K. Osteopontin inhibits interleukin-1βstimulated increases in matrix metalloproteinase activity in adult rat cardiac
fibroblasts: role of protein kinase C-zeta. J Biol Chem. 2003;278:48546-48552.