ABC | Volume 115, Nº1, July 2020

Original Article Oliveira-Junior et al Effects of losartan in obesity Arq Bras Cardiol. 2020; 115(1):17-28 General characterization and in vitro analysis of myocardial performance Caloric intake was assessed daily. 6-8 Feeding efficiency was obtained from the relationship between body weight variation and total energy intake. 6-8 Body weight was measured weekly, while weight gain was obtained from the difference between initial and final body weight values. Adipose tissue from the retroperitoneal, epididymal, and visceral regions was used to determine body fat content. 6-12 Myocardial performance was assessed by in vitro study with papillary muscle isolated from LV. 7,16,18,29 After 20 weeks, animals were submitted to intraperitoneal anesthesia with ketamine hydrochloride (80 mg/kg), xylazine (5 mg/kg), and euthanasia. After median thoracotomy, the heart was removed and dissected. Atria, right ventricle (RVW) as well as left ventricle (LVW) were weighted for macroscopic morphological analysis. Dissected LV papillary muscles were placed between two stainless steel rings and positioned vertically within a glass chamber containing Krebs-Henseleit solution at 28°C, continuously oxygenated with O 2 (95%) and CO 2 (5%). The Krebs solution composition was the following: 118.5 mM NaCl; 4.69 mM KCl; 2.50 mM CaCl 2 ; 1.16 mM MgSO 4 ; 1.18 mM KH2PO 4 ; 5.50 mM glucose; and 24.88mMNaCO 3 . The lower end of the inferior ringwas coupled to a 120T-20B force transducer (Kyowa, Tokyo, Japan) by a steel wire (1/15,000) running through a mercury-filled slot in the glass chamber floor. 7,16,18,29 The muscles were kept on isotonic contraction against a light loading for 60 minutes; afterwards, they were then kept on isometric contraction and gradually stretched until the maximum developed tension (DT) was achieved. After 5 minutes under isotonic contraction, the muscles were placed back in isometric contraction to determine the tension-length curve (L max ) peak. The papillary muscles behavior was assessed at baseline with a 2.50 mM calcium concentration ([Ca 2+ ] o ) and after the following inotropic maneuvers: post-rest potentiation (PPP), extracellular [Ca 2+ ] elevation since 0.5 until 2.5 mM, and during beta-adrenergic stimulation with 0.1 and 1.0 mM isoproterenol. Post-pause potentiation was studied in extracellular [Ca 2+ ] equal to 1.50 mM, where the stimulus was stopped for 30 and 60 seconds before it restarted. 7,30 After PPP, the papillary muscle response was assessed after extracellular [Ca 2+ ] o maneuver. 31 Isometric contractile parameters were recorded after 10 minutes with progressive calcium addition (0.5 to 2.5mM) in the extracellular solution. The beta-adrenergic system stimulation has also been studied to test beta-adrenergic complex integrity, troponin C sensitivity, and calcium absorption by the sarcoplasmic reticulum. 7,31 Beta adrenergic receptor stimulation was induced using cumulative isoproterenol concentrations (0.1 to 1.0 mM) in the presence of 1.0 mM [Ca 2+ ] o . Mechanical variables Conventional mechanical responses at L max were obtained in isometric contraction: maximum developed tension normalized by the transverse sectional area of the papillary muscle (DT [g/mm 2 ]) and maximum positive variation velocities (+dT/dt [g/mm 2 /s]); and maximum negative variation velocity (-dT/dt [g/mm 2 /s]) of maximum developed tension (DT), normalized by the transverse sectional area of the papillary muscle. The measures used to characterize papillary muscle size included length (mm), muscle weight (mg) and transverse sectional area (TSA [mm 2 ]). At the end of each experiment, L max was measured with the Gaertner catheter (Gaertner Scientific Corporation, Chicago, IL, USA), and the muscle portion between the steel rings was cut and weighed. TSA was obtained from a ratio between muscle weight and length, assuming uniformity and a specific 1.0 gravity. Statistical analysis A Sigma-Stat version 3.5 software was used for data analysis. Firstly, the results were subjected to normality analysis by the Kolmogorov-Smirnov test. Since the variables had parametric distribution, measures were presented as mean and standard-deviation. Nutritional results, body composition, cardiac morphology and functional performance of the papillary muscle were analyzed using the two-way analysis of variance ( Two-Way ANOVA) and the Tukey’s multiple comparisons test. SBP and echocardiogram measurements were analyzed by Two-Way ANOVA in the repeated measures (RM) model, and Bonferroni multiple comparison test. The level of significance was set at 5%. Results Results of nutritional profile, body composition and cardiac macroscopic morphology are shown in Table 1. Although caloric intake was unchanged, OB and OL showed higher fat intake and feed efficiency than CO and CL, respectively. Obesity was characterized by higher measures of body weight and adiposity. OB presented higher values of atria weight and respective relationships between atrial weight and LV weight with tibia length compared to CO regarding cardiac morphology. Losartan promoted lower atrial and LV measurements comparing OL with OB in absolute values and when normalized by tibia length, as shown in Table 1. Table 2 presents SBP results, structure and performance of the heart, assessed by echocardiography. After 16 weeks, obesity was associated with higher SBP; losartan led to SBP reduction in CL and OL at the end of the experiment. The ratio between left ventricular diastolic diameter (LVDD) and tibia length was similar among groups and between the moments. At the end of the experiment, obesity culminated in a higher posterior wall shortening velocity (PWSV), as observed in OB and OL. Considering the diastolic performance, OL presented lower E/A ratio than CL at the 20 th week. Tissue Doppler of late diastolic mitral valve annular velocity (A’ average) was lower in CL than CO; S average and E’ average were increased from week 16 to week 20 in OL. The functional performance of the papillary muscles is shown in Figures 1 to 4. Under basal conditions, the DT and +dT/dt indexes were similar among groups (Figure 1A and 1B), while the -dT/dt was lower in CL than CO (Figure 1C). The effects of diverse calcium concentrations on papillary muscle performance are shown in Figure 2. Increasing [Ca 2+ ] o from 1.0 to 2.5 mM resulted in higher DT, +dT/dt and -dT/dt values in all groups. OL showed higher DT, +dT/dt and -dT/ dt values compared to CL at calcium concentrations of 1.5, 19