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                                MATERIALS AND METHODS


      Methylarginine hydrochloride (L-NMMA) was a gift from Glaxo Wellcome.

Dobutamine was obtained from Lilly. BH4 was obtained from Alexis Corp. (San

Diego, CA). L-[14C]-arginine was obtained from New England Nuclear (Boston,

MA). NOS3, NOS2, caveolin-1 and caveolin-3 antibodies were obtained from

Transduction laboratories (Lexington, KY).

Chronic preparation, hemodynamic analysis, and pacing protocol:

      Twenty-two mongrel dogs of either sex (20-25 kg) were chronically

instrumented to measure LV pressure and dimensions, and paced to heart failure

as previously described.1 Studies were performed in conscious animals resting

comfortably in a sling. Pressure-dimension signals were digitized at 250 Hz using

a pentium microprocessor and custom software. Signal-averaged data from 10

to 20 consecutive beats were used to derive steady-state parameters, and data

measured during transient IVC occlusion were used to assess pressure-

dimension relations over a wide range of preload pressure and dimensions.

Measurements were obtained with heart rate maintained constant at either 160

or 170 bpm by atrial pacing.

      Isovolumic contraction was indexed by the peak rate of LV pressure rise

(+dP/dt). Myocardial contractility was also assessed by the relatively load-

independent indices obtained from pressure-dimension relationships during IVC

occlusion: Ventricular elastance (Ees) calculated as the slope of the end-systolic

pressure-dimension relation, and dP/dt-EDD calculated as the slope of the
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relation between dP/dt and EDD. Short axis dimension has been validated an

index of LV volume.1, 2 Afterload was indexed by arterial elastance (Ea), the ratio

of LV systolic pressure to stroke dimension,3 and preload was directly measured

as the end-diastolic dimension.

Two photon microscopy:

   Isolated myocytes obtained from control and HF dogs (n=2 each) were

attached to coverslips with laminin, fixed in 50% methanol/50% acetone, and

incubated overnight with monoclonal antibodies to caveolin-3 and NOS3.

Secondary incubation was performed at room temperature for 1 hour using anti-

mouse rhodamine (Jackson Immunoresearch, West Grove PA) and anti-rabbit

Alexa 488 (Molecular Probes, Eugene OR). Imaging was performed on a Nikon

E600FN upright physiological fluorescence microscope with a Bio-Rad MRC-

1024/2-P multiphoton imaging system attachment (Bio-Rad, Hercules, CA). Two

photon excitation at 780 nm was provided by a Tsunami mode-locked Ti:Sa

pulsed laser pumped by a 10W Millenia X solid state laser (Spectra Physics,

Mountain View, CA). The pulse duration was < 60 femtoseconds. Image

colocalization analysis was accomplished using LaserSharp MRC-1024 Laser

Scanning Confocal Imaging System software.4 The extent of antibody

colocalization was determined by plotting 5the red fluorescence intensity as a

function of the green intensity for each pixel location after subtracting off

background fluorescence for each component and generating an image overlay

representing colocalized regions.5
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Data analysis:

      Data are presented as meanSEM. Comparisons between baseline

variables in control and heart failure dogs were performed using Student’s t-tests.

Comparison within individual experiments for a shift in the ESPDR were made

using multiple linear regression of pressure-dimension data from individual IVC

occlusions with an interaction term for drug effect (either L-NMMA or

dobutamine). To assess population responses to L-NMMA and the L-NMMA

effect on dobutamine response, a two-way ANOVA was applied with a

categorical term identifying each experiment.6 Concentration-effects to L-NMMA

were assessed using an interaction term (concentration x effect). Post hoc

testing employed the Student-Newman Keuls test. Comparisons of

concentration-effect relationships to dobutamine before and after L-NMMA were

made using repeated measures ANOVA.6 Statistical calculations were

performed using Systat and SAS software.

                                  Reference List

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     oxygen consumption and increases mechanical efficiency in dogs with

     pacing-induced heart failure. Circ Res. 1999;85:437-445.
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 2. Little WC, Freeman GL, O'Rourke AR. Simultaneous determination of left

     ventricular end-systolic pressure-volume and pressure-dimension

     relationships in closed-chest dogs. Circulation. 1985;71:1301-1308.

 3. Kelly RP, Ting C-T, Yang T-M, Liu C-P, Maughan WL, Chang M-S, Kass

     DA. Effective arterial elastance as index of arterial vascular load in humans.

     Circulation. 1992;86:513-521.

 4. Xu C, Zipfel W, Shear JB, Williams RM, Webb WW. Multiphoton

     fluorescence excitation: new spectral windows for biological nonlinear

     microscopy. Proc Natl Acad Sci U.S.A. 1996;93:10763-10768.

 5. Soeller C, Cannell MB. Examination of the transverse tubular system in

     living cardiac rat myocytes by 2-photon microscopy and digital image-

     processing techniques. Circ Res. 1999;84:266-275.

 6. Wallenstein S, Zucker C, Fleiss J. Some statistical methods useful in

     Circulation Research. Circ.Res. 1980;47:1-9.

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