ABC | Volume 115, Nº1, July 2020

Original Article Silva-Bertani et al. Mechanism of decreased heart collagen I in obesity Arq Bras Cardiol. 2020; 115(1):61-70 rabbits fed a high-fat diet for 12 weeks, a previous study by our group, Silva et al., 13 found decreased cardiac collagen type I in obese rats fed an unsaturated high-fat diet for 30 weeks. 13 The mechanisms responsible for the decreased myocardial collagen type I, however, were not studied. One of the possible mechanisms involved in myocardial type I collagen regulation is the increased leptin hormone. 5,14-16 Supporting this hypothesis, most studies in vitro have shown that leptin increases MMP-2 activity, 5,15,16 which is involved in the degradation of collagen type I. Therefore, the purpose of this study was to test the hypothesis that the reduction in myocardial collagen type I, associated with increased activity of MMP-2, is linked to elevation of leptin in obese rats. Materials and methods Animals and Experimental Protocol After one week for acclimatization, 30-day-old male Wistar rats were randomly assigned, by lottery, to one of two groups: control (n = 20) and obese (n = 21). The sample size used in this study was based on the literature and on our previous studies. 13,17-19 The control group was fed standard rat chow (RC Focus 1765, Agroceres ® , Rio Claro, SP, Brazil) containing 12.3% of kilocalories from fat, 57.9% from carbohydrates, and 29.8% from protein, whereas the obese group was fed one of four alternating high-fat diets (RC Focus 2413, 2414, 2415, and 2416, Agroceres ® , Rio Claro, SP, Brazil) containing 49.2% of kilocalories from fat, 28.9% from carbohydrates, and 21.9% from protein. The four high-fat diets had the same nutritional composition, except flavoring additives, namely, cheese, bacon, chocolate, or vanilla. Each diet was changed daily, and the rats were maintained on their respective diets for 34 consecutive weeks. The high-fat diet was calorically rich compared to the standard diet (3.65 kcal/g vs. 2.95 kcal/g) due to the higher fat composition. The high-fat diet consisted of saturated and unsaturated fatty acids, which provided 20% and 80% of the fat-derived calories, respectively. Rats were housed in individual cages in an environmentally controlled clean-air room at 23 (± 3)ºC with a 12-hour light/ dark cycle and 60% (± 5%) relative humidity. All experiments and procedures were conducted according to the Guide for the Care and Use of Laboratory Animals, published by the National Research Council (1996), 20 and they received approval from the Botucatu Medical School Ethics Committee (UNESP, Botucatu, SP, Brazil, Protocol: 861-2011). Animal general characteristics and metabolic and endocrine profiles Animal general characteristics and metabolic and endocrine profiles were evaluated according to the following parameters: body weight, body fat (BF), adiposity index (AI), food consumption, caloric intake, feed efficiency, glucose tolerance, insulin resistance, serum lipid profile, and serum leptin and insulin concentrations. A criterion based on the AI was used to determine obesity. The AI is an easy and consistent method used by several authors to evaluate the amount of BF in rodents. 21-23 The animals’ food intake and body weight were measured weekly. Caloric intake was determined by multiplying the energy value of each diet (g × kcal) and weekly food intake. To analyze the animals’ capacity to convert consumed food energy to body weight, feed efficiency was calculated, dividing the total body weight gain (g) by total energy intake (Kcal). Glucose tolerance was evaluated by the oral glucose tolerance test one week before euthanasia. After a 6-hour fast, blood samples were collected by puncture from the tail tip at baseline and after intraperitoneal administration of 30% glucose solution (Sigma-Aldrich®, St Louis, MO, USA), equivalent to 2.0 g/kg body weight. Blood glucose concentrations were analyzed at 0 minutes (baseline) and after 15, 30, 60, 90, and 120 minutes of glucose infusion, using a handheld glucometer (Accu-chek Advantage; Roche Diagnostics Co., Indianapolis, IN, USA). Glucose intolerance was assessed by the area under the curve (AUC) for glucose. At the end of the experimental protocol, after fasting for 12 hours, animals were anesthetized (sodium pentobarbital 50 mg/kg, intraperitoneal injection), decapitated, and thoracotomized; the adipose tissue fat pads were dissected and weighed. BF was calculated as the sum the weight of the individual fat pads as follows: BF = epididymal fat + retroperitoneal fat + visceral fat. AI was calculated by the following formula: AI = (BF/final body weight) × 100. Blood samples were collected in heparinized tubes, centrifuged at 3,000 × g for 10 minutes at 4°C, and stored at −80°C for later analysis. Triacylglycerol, total cholesterol, and high- (HDL) and low-density lipoprotein (LDL) concentrations were determined using specific kits (BIOCLIN®, Belo Horizonte, MG, Brazil). Hormone levels of leptin and insulin were determined by enzyme-linked immunosorbent assay (ELISA), using commercially available kits (EMDMillipore Corporation, Billerica, MA, USA). The homeostatic model assessment of insulin resistance (HOMA-IR) was used as an insulin resistance index, calculated according to the formula: HOMA-IR = [fasting glucose (mmol/L) × fasting insulin (μU/mL)]/22.5. 24 Cardiovascular profile The cardiovascular profile of the animals was also assessed according to the following parameters: systolic blood pressure (SBP); cardiac tissue morphology; myocardial protein expression of collagen type I, TIMP-1, TIMP-2, and leptin; and MMP-2 activity. Systolic blood pressure At the end of the experiment, one week before euthanasia, SBP was measured in conscious rats using the non-invasive tail-cuff method with an electro- sphygmomanometer, Narco BioSystems ® (International Biomedical, Austin, TX, USA). 25 Arterial pulsations were recorded in a computerized data acquisition system (Biopac Systems Inc., CA, USA). The average of two readings was recorded for each measurement. 62