['3b.', 'digitizing', 'tablet', 'for', 'six', 'consecutive', 'beats', 'during', 'normal', 'sinus', 'rhythm.', 'Left', 'ventricular', 'length', 'is', 'determined', 'by', "Simpson's", 'rule', 'and', 'mass', 'calculated', 'by', 'subtracting', 'epicardial', 'volumes', 'from', 'endocardial', 'volumes', 'and', 'multiplying', 'the', 'results', 'by', 'the', 'specific', 'gravity', 'of', 'myocardial', 'muscle.', 'The', 'accuracy', 'of', 'the', 'echo', 'measurement', 'of', 'canine', 'left', 'ventricular', 'mass', 'has', 'been', 'previously', 'reported', 'with', 'postmortem', 'validation', '(10).', 'The', 'average', 'wall', 'thickness', 'is', 'obtained', 'as', 'the', 'mean', 'end-diastolic', 'wall', 'thickness', 'over', '22.5', 'degree', 'radial', 'intervals', 'in', 'the', 'midpapillary', 'short-axis', 'view.', 'The', 'relative', 'wall', 'thickness', 'is', 'calculated', 'as', 'the', 'average', 'wall', 'thickness/left', 'ventricular', 'end-diastolic', 'dimension.', 'A', 'no.', '8', 'Goodale-Lubin', 'catheter', 'is', 'placed', 'in', 'the', 'proximal', 'aorta', 'from', 'the', 'carotid', 'artery', 'under', 'sterile', 'conditions.', 'A', 'Statham', 'transducer', 'is', 'used', 'for', 'the', 'measurement', 'of', 'aortic', 'pressures,', 'which', 'are', 'recorded', 'on', 'an', 'amplifier-recorder', 'system', '(Honeywell).', 'The', 'micromanometer', 'tipped', 'catheter', 'will', 'be', 'placed', 'in', 'the', 'ventricle,', 'zeroed', 'and', 'balanced', 'against', 'the', 'saline', 'filled', 'catheter.', 'The', 'maximal', 'rate', 'of', 'rise', 'of', 'left', 'ventricular', 'pressure', '(dP/dt)', 'is', 'obtained', 'using', '@', 'resistance', 'capacitance', 'differentiating', 'circuit.', 'The', 'system', 'has', 'a', 'linear', 'frequency', 'response', 'from', '0-30', 'cycles/s.', 'Systolic', 'ejection', 'period', 'is', 'measured', 'from', 'superimposed', 'aortic', 'and', 'left', 'ventricular', 'pressure', 'pulse', 'tracings.', 'End', 'diastolic', 'pressure', 'is', 'measured', 'over', 'at', 'least', 'three', 'respiratory', 'cycles', 'and', 'averaged.', 'As', 'an', 'index', 'of', 'myocardial', 'contractility,', 'we', 'use', 'the', 'velocity', 'of', 'contractile', 'elements', '(Vce)', 'at', 'peak', 'dP/dt,', 'calculated', 'as', 'the', 'ratio', 'of', 'maximal', 'dP/dt,', 'related', 'to', 'the', 'simultaneous', 'left', 'ventricular', 'pressure.', 'Simultaneous', 'measures', 'of', 'left', 'ventricular', 'pressures', 'and', 'volumes', 'by', 'ECHO', 'will', 'be', 'made', 'during', 'pacing', 'and', 'volume', 'loading.', 'For', 'the', 'latter', '10%', 'dextran-40', '(3ml/kg/min', 'over', '4', 'min)', 'will', 'be', 'infused', 'to', 'observe', 'graded', 'increases', 'of', 'end-diastolic', 'pressure', 'and', 'volume', 'and', 'to', 'calculate', 'chamber', 'stiffness', '(7).', 'Myocardial', 'Composition', 'After', 'the', 'hemodynamic', 'study', 'and', 'a', 'thoracotomy,', 'the', 'heart', 'will', 'be', 'cold', 'arrested', 'with', 'iced', "Ringer's", 'solution.', 'Appropriate', 'sized', 'samples', 'are', 'taken', 'from', 'the', 'mid-left', 'and', 'ventricle.', 'For', 'mRNA', 'analysis,', 'sterile', 'myocardium', 'will', 'be', 'frozen', 'in', 'liquid', 'N,', 'and', 'stored', 'at', '-70°', 'C.', 'Other', 'samples', 'will', 'be', 'stored', 'at', '-20°C.', 'Transmural', 'sections,', 'trimmed', 'of', 'epicardial', 'adipose', 'tissue', 'and', 'vasculature,', 'are', 'taken', 'from', 'the', 'free', 'wall', 'of', 'the', 'left', 'ventricle,', 'placed', 'in', 'formalin', 'for', 'histochemical', 'analysis', 'or', 'lyophilized', 'for', 'hydroxyproline', 'analysis.', 'To', 'determine', 'collagen-linked', 'fluorescence', 'as', 'measure', 'of', 'advanced', 'glycosylation', 'products', '(18),', 'samples', 'of', 'left', 'ventricular', 'myocardium', 'will', 'be', 'minced,', 'dilipidated', 'and', 'washed', '3', 'times', 'in', 'phosphate', 'buffered', 'saline.', 'The', 'tissue', 'is', 'subjected', 'to', 'sequential', 'extraction', 'steps', 'with', 'acetic', 'acid,', 'pepsin,', 'protease', 'K,', 'and', 'collagenase.', 'The', 'first', 'extract', 'is', 'in', '2', 'ml', 'of', '0.5', 'M', 'acetic', 'acid', 'overnight', 'at', '4', 'degrees.', 'This', 'is', 'retreated', 'twice', 'before', 'centrifuging.', 'The', 'undigested', 'material', 'is', 'treated', 'with', 'a', '1%', 'proteinase', 'K', 'solution', 'in', '0.1%', 'sodium', 'dodecyl', 'sulfate', '(wt/vol)', 'at', '370°', 'C', 'overnight', 'with', 'shaking.', 'The', 'remaining', 'tissue', 'is', 'digested', 'with', 'collagenase', 'as', 'described', 'above.', 'A', 'small', 'pellet', 'remains', 'undigested.', 'Portions', 'of', 'each', 'fraction', 'are', 'acid', 'hydrolyzed', 'with', '6N', 'HCL', 'at', '110', 'degrees', 'for', '24', 'hours', 'for', 'quantitation', 'of', 'hydroxyproline', 'content.', 'Fluorescence', 'is', 'also', 'assessed', 'in', 'the', 'soluble', 'fractions.', 'Five', 'hundred', 'microliters', 'of', 'a', '5%', '(wt/wt)', 'collagenase', 'solution', '(type', 'VII,', 'Sigma,', 'St', 'Louis,', 'MO)', 'is', 'added,', 'and', 'samples', 'are', 'incubated', 'at', '37°', 'for', '24', 'hours.', 'After', 'centrifugation', 'for', '3', 'minutes', 'with', 'a', 'microphuge,', 'fluorescence', 'measurements', 'are', 'taken', 'at', 'an', 'excitation', 'wavelength', 'of', '350', 'nm', 'and', 'an', 'emission', 'wavelength', 'of', '410', 'nm', 'with', 'an', 'Perkin-Elmer', 'spectrophotofluorometer.', 'Northem', 'Hybridization', 'Analysis', 'of', 'Total', 'RNA', 'Total', 'RNA', 'is', 'extracted', 'by', 'single', 'step', 'method', 'from', 'the', 'confluent', 'myocardial', 'fibroblast', 'cultures', 'by', 'scraping', 'the', 'cells', 'off', 'the', 'dishes', 'in', '4', 'M', 'GIT', 'and', 'homogenizing', 'in', 'the', 'same', 'solution', 'as', 'described', 'by', 'Chomezynski', 'and', 'Sacci', '(19).', 'Total', 'RNA', 'is', 'fractionated', 'by', 'electrophoresis', 'in', '0.8%', 'formaldehyde/agarose', 'gels', 'and', 'blotted', 'onto', 'nitrocellulose', 'filters', 'using', 'standard', 'methods', '(20,21).', 'The', 'nitrocellulose', 'filters', 'are', 'baked,', 'prehybridized', 'and', 'hybridized', 'at', '42', 'degrees', 'C', 'for', '16', 'hours', 'to', 'specific', 'cDNAs', 'which', 'had', 'been', ' ']