Ang 2

Hearts were excised into ice-cold modified Krebs-Henseleit perfusion buffer. The aorta was perfused at 80 mmHg with perfusion buffer equilibrated with 95% O₂ and 5% CO₂ at 37°C and pH 7.4. A fluid-filled balloon was inserted via the mitral valve, and inflated to a diastolic pressure of ∼5 mmHg. Hearts were maintained at 37±0.1°C in a water-jacketed bath. Experiments, performed in separate groups, were: 1) perfusion with successively increasing concentrations of A61603 (0.1 nM−1.0 µM; Sigma, Australia), a selective α_1A-AR agonist; 2) before and after perfusion with angiotensin II (AngII, 100 nM, 10 min, MP Biomedicals, Australia) or with one of two selective α_1A-AR antagonists, RS100329 (50 nM, 10 min, Sigma, Australia) or KMD3213 dihydrobromide (100 nM, 10 min, Kissei Pharmaceutical Co. Matsumoto, Japan); 3) for contractile protein measurements, perfusion with saline or RS100329 (50 nM, 8 min), then snap-frozen (liquid nitrogen); 4) for RhoA/ROCK pathway, perfusion with saline or Y-27632, a selective ROCK inhibitor (1 µM, 5 min, Merck Millipore, MA), then snap-frozen; 5) for RhoA/ROCK signaling in agonist-induced responses, A61603 (0.1 nM−1.0 µM) in absence or presence of Y-27632.