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
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
Data are presented as meanSEM. 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.
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