CLINICS 2012;67(5):489-496 stimulated with isolated rectangular pulses (10 to 15 V, 12- ms duration) using a pair of platinum electrodes placed along the entire length of the muscle. The standard stimulation rate was 0.5 Hz (steady-state). Recording was begun after 60 minutes to allow for the beating preparation to adapt to the new environmental conditions. The bathing solution was a modified Krebs solution with the following composition (in mM): 120 NaCl, 5.4 KCl, 1.2 MgCl2 6H2O, 1.25 CaCl2 2H2O, 2.0 NaH2PO4H2O, 1.2 Na2SO4, 27 NaHCO3, and 11 glucose, gassed with 95% O2 and 5% CO2, pH 7.4. The developed force (F) was measured with an isometric force transducer (Nihon-Kohden, TB 612T, Tokyo), which was recorded on a chart recorder (Nihon-Kohden, RM-6200, Tokyo) and normalized to the muscle cross-sectional area (g/mm2). Considering the papillary muscle as a cylinder and the tissue density as 1, the cross-sectional area was calculated by dividing the muscle length at Lmax by its weight. To avoid the possibility of a hypoxic core, we performed the experiments at low temperatures (29¡1˚C) as previously described (20). The influence on the contractile machinery was studied in another group of papillary muscles during tetanic stimula- tion. Tetanic tension was obtained after treatment with 5 mM caffeine (for 20 minutes, at a rate of 10 Hz, for 15 s) as previously described (20). The tetanic contractions devel- oped a fast upstroke (tetanic peak force) followed by a slow decay (tetanic plateau force). Tension was measured at the peak and in the middle of the plateau. The muscles remained under steady-state stimulation for at least 10 min between stimuli. This protocol was repeated twice. Studies of myosin ATPase activity To evaluate myosin ATPase activity, the left ventricle was dissected and assayed as described previously (11). Briefly, myosin was prepared from minced and homogenized left ventricles, which were extracted briefly with KCl phosphate buffer (0.3 M KCl and 0.2 M phosphate buffer, pH 6.5). Myosin ATPase activity was assayed by measuring Pi liberation from 1 mM ATP in the presence of 50 mM HEPES, pH 7, 0.6 M KCl, and 5 mM CaCl2 or 10 mM EGTA in a final volume of 200 ml. The level of myosin activity was measured without Mg2+ in the medium. There is no significant Ca2+-ATPase activity in the sarcoplasmic reticulum membrane, which requires high Mg2+ and low Ca2+ concentrations. Controls containing the enzyme pre- paration and trichloroacetic acid were used to correct for non-enzymatic hydrolysis of the substrate. All samples were prepared in duplicate. The level of enzyme activity was calculated as the difference between the levels observed in the presence of Ca2+ and in the presence of 10 mM EGTA. The level of inorganic phosphate was determined according to the method proposed by Chan et al. (21). Specific activity is reported as nmol Pi released per min per mg of protein unless otherwise stated. Protein was measured using the Coomassie Blue method according to Bradford (22) using bovine serum albumin as a standard. Drugs used Pentobarbital sodium was obtained from Cristalia Produtos Qu´ımicos Farmaceˆuticos Ltda. (Sa˜o Paulo, SP, Brazil). Heparin was obtained from Roche Q.F.S.A. (Rio de Janeiro, RJ, Brazil) and Sigma. Bovine serum albumin, Na2ATP, and anhydrous caffeine were purchased from 491 Tension cost and contractility changes Moreira CM et al. Sigma Chemical Co. (St. Louis, MO, U.S.A.). All of the other reagents used were of analytical grade and from Sigma, E. Merck (Darmstadt, Germany), or Reagen (Rio de Janeiro, RJ, Brazil). Statistical analysis The results are presented as the means ¡ SEM with n indicating the number of observations. The values were analyzed using ANOVA (one- and two-way) and unpaired Student t-tests. When ANOVA revealed a significant difference, the Tukey test was applied. Linear regression was used for correlation analysis. p,0.05 was considered to be significant. Data analysis and figure preparation were performed using GraphPad PrismTM (version 2.0, GraphPad Software, San Diego, CA, USA) and GB-STAT (version 4.0, Dynamic Microsystem Inc., Silver Spring, MD). RESULTS Four weeks after myocardial infarction, the isometric force development (Figure 1A) and peak and plateau tetanic tension (Figure 2A, Table 3) were reduced. The level of myosin ATPase activity was also reduced compared to that observed in sham-operated rats (Figure 3A). SHRs were hypertensive (systolic pressure; SHR = 213¡ 7.5 mmHg vs. WKY = 124¡1.3 mmHg; p,0.01, unpaired Student’s t-test) and showed left ventricular hypertrophy (Table 2). Force development and peak and plateau tetanic tension were similar between SHRs and WKY rats (Figure 1B and 2B). For SHRs and WKY rats, this similarity in terms of tetanic tension was paralleled by similar levels of myosin ATPase activity (Figure 3B). Thirty days after streptozotocin administration, the diabetic group showed an increased blood glucose level (DB = 488¡ 20.5 mg/dL and Control = 82¡1.8 mg/dL, p,0.01, unpaired Student’s t-test) and a reduction in body weight (Table 2) when compared to baseline (Control: 270¡3.16 vs Diabetic: 280¡3.63, p.0.05). The papillary muscles from diabetic rats developed less isometric tension than those from controls (Figure 1C); the same trend was observed for peak and plateau tetanic tension (Figure 2C, Table 3). The level of myosin ATPase activity was reduced (Figure 3C). In the DOCA-salt group, only the DOCA salt-treated rats were hypertensive. The other three subgroups (nephrectomized, salt treated, and DOCA treated) were normotensive (DOCA salt = 201¡3.4 mmHg vs. Nefrec = 119¡1.1 mmHg; Salt = 120¡2.1 mmHg and DOCA = 135¡2.0 mmHg: p,0.01, one- way randomized ANOVA). DOCA-salt rats also presented reduced body weight compared to the other three subgroups and displayed significant LV hypertrophy at the end of the treatment (Table 2). Isometric tension (Figure 1D) and peak and plateau tetanic tension (Figure 2D, Table 3) were reduced in the DOCA-salt group compared with the other groups. Myosin ATPase activity was also reduced in the DOCA-salt group (Figure 3D). We used L-NAME treatment for 30 days to create a model with hypertension but without LV hypertrophy. Arterial systolic pressure increased (Control = 119¡0.8 mmHg and L- NAME = 196¡4.4 mmHg; p,0.01, unpaired Student t-test), but no hypertrophy developed (Table 2). L-NAME-treated rats developed increased isometric force (Figure 1E), but peak and plateau tetanic tension presented no significant differences (Figure 2E, Table 3). Myosin ATPase activity increased in the L-NAME group (Figure 3E).