Files in this Data Supplement:

• Supplemental Methods - (Word) (42 KB)
• Table I - (TIFF) (279 KB)
• Table II - (TIFF) (290 KB)
• Table III - (TIFF) (277 KB)
• Table IV - (TIFF) (281 KB)
• Table V - (TIFF) (264 KB)
• Table VI - (TIFF) (284 KB)
• Figure I - (TIFF) (1.93 MB) Characterization of ILK^S343D transgenic mice. a, Genomic DNA from ILK^S343D Tg and NTg littermates was analyzed by Southern blotting using a human ILK cDNA probe. b, ILK-specific RT-PCR of total RNA isolated from heart tissue with (upper panel) or without (control, middle panel) reverse transcriptase, and on skeletal muscle (bottom panel) with reverse transcriptase. This yields the expected product 1.46 kb in length, expressed in the hearts of Tg mice, but not in the hearts of NTg littermates or skeletal muscle of the Tg mice. The lane marked "P" is the PCR product obtained using α-MHC/ILK plasmid as template. This product is larger than 1.46 kb because the PCR primers encompass exons 1 and 2 of the α-MHC promoter. c, Western immunoblot analysis of ILK protein levels in ILK Tg and control (NTg) hearts. Signal densities normalized to that of GAPDH were 3-fold higher in ILK Tg hearts. d, ILK immune complex kinase assays of heart lysates from ILK^S343D Tg and NTg littermates. Purified myosin light chain II, 20 kDa regulatory subunit was added as exogenous substrate.
• Figure II - (TIFF) (7.83 MB) Increased cardiomyocyte size in ILK^S343D Tg mice. a, Gross morphology of hearts from ILK^S343D Tg mice and NTg littermates. Enlarged hearts of ILK^S343D mice exhibited concentric hypertrophy evident by an approximate 25% increase in heart weight to body weight ratios relative to that in NTg controls (controls for all comparisons are age- and sex-matched littermates, Table 1). Histological studies using Masson's trichrome and picrosirius red staining (not shown) indicated no conspicuous increase in collagen in the ILK^S343D Tg hearts. b, Mean values of cardiomyocyte areas based on approximately 500 cells per mouse with centrally positional nuclei. This analysis indicated a 20% to 25% increase in cardiomyocyte area, thereby accounting for the observed increase in LV mass. c, Representative echocardiograms showing details of dimensional measurements. At 15 months, ILK^S343D Tg mice exhibited significant increases in LV mass as well as LV cavitary dimensions at end-systole and end-diastole (P<0.05), and preserved LV function based on echocardiography (% fractional shortening, Table 2) and invasive hemodynamic measurements (Table I).
• Figure IIIa and IIIb - (TIFF) (2.13 MB) elective activation of hypertrophic signaling in ILK^WT, but not ILK^R211A transgenic hearts. Ventricular lysates from (a) ILK^WT and (b) ILK^R211A Tgmice were assayed for activation of Rac1, Cdc42 and RhoA, using specific immunoaffinity assays as described in Methods. In each panel, parallel assays of ventricular lysates from littermate NTg controls are shown.
• Figure IIIc and IIId - (TIFF) (2.46 MB) Ventricular lysates from (c) ILK^WT and (d) ILK^R211A Tgmice were resolved by SDS-PAGE and analyzed by western blotting for levels of the indicated total and phosphorylated proteins. GAPDH was analyzed in parallel as loading control. Controls were NTg littermates.
• Figure IIIe - (TIFF) (656 KB) Ventricular lysates from ILK^WT, ILK^S343D, and ILK^R211A mice were analyzed by western blotting for total ILK. GAPDH was the loading control (bottom panel).
• Figure IV - (TIFF) (4.77 MB) Selective activation of Rac1 and Cdc42 in ILK^S343D Tgmice. Affinity-based precipitation assays were conducted (see Methods) to determine the ratio of GTP-bound (activated) to total: a, Rac1; b, Cdc42; and c, RhoA GTPases in cardiac lysates of ILK^S343D Tgand non-Tglittermate mice. Histograms summarize data from 4 hearts of each genotype.
• Figure V - (TIFF) (933 KB) Activation of hypertrophic signaling in ILK^S343D Tg mice. Hearts from two ILK^S343D Tg and 2 NTg littermate controls were extracted and proteins resolved on 10% SDS-PAGE. Western blotting using antibodies against total and phosphorylated forms of the indicated protein kinases was performed to assess the relative activation levels of these pathways. For PKB and GSK3β determinations the ratio of densitometric signals of phosphorylated/total protein were determined for each sample, and are displayed under the panels. GAPDH was used as a loading control.