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Obesity Preserves Myocardial Function During Blockade of the Glycolytic Pathway

Abstracts

Background:

Obesity is defined by excessive accumulation of body fat relative to lean tissue. Studies during the last few years indicate that cardiac function in obese animals may be preserved, increased or diminished.

Objective:

Study the energy balance of the myocardium with the hypothesis that the increase in fatty acid oxidation and reduced glucose leads to cardiac dysfunction in obesity.

Methods:

30-day-old male Wistar rats were fed standard and hypercaloric diet for 30 weeks. Cardiac function and morphology were assessed. In this paper was viewed the general characteristics and comorbities associated to obesity. The structure cardiac was determined by weights of the heart and left ventricle (LV). Myocardial function was evaluated by studying isolated papillary muscles from the LV, under the baseline condition and after inotropic and lusitropic maneuvers: myocardial stiffness; postrest contraction; increase in extracellular Ca2+ concentration; change in heart rate and inhibitor of glycolytic pathway.

Results:

Compared with control group, the obese rats had increased body fat and co-morbities associated with obesity. Functional assessment after blocking iodoacetate shows no difference in the linear regression of DT, however, the RT showed a statistically significant difference in behavior between the control and the obese group, most notable being the slope in group C.

Conclusion:

The energy imbalance on obesity did not cause cardiac dysfunction. On the contrary, the prioritization of fatty acids utilization provides protection to cardiac muscle during the inhibition of glycolysis, suggesting that this pathway is fewer used by obese cardiac muscle.

Obesity; Rats; Myocardial; Metabolism; Fatty Acid


Fundamento:

A obesidade é definida por um acúmulo excessivo do tecido adiposo em relação a massa magra tecidual. Estudos realizados nos últimos anos sugerem que a função cardíaca em animais obesos pode se encontrar preservada, aumentada ou reduzida.

Objetivo:

Estudar o balanço energético do miocárdio com a hipótese de que o aumento na oxidação de ácidos graxos e redução de glicose levam à disfunção cardíaca na obesidade.

Métodos:

Ratos Wistar machos com 30 dias de idade foram alimentados com uma dieta padrão ou hipercalórica durante 30 semanas. A função e morfologia cardíacas foram analisadas. Neste trabalho foram estudadas as características gerais e comorbidades associadas com a obesidade. A estrutura cardíaca foi determinada pelo peso do coração e do ventrículo esquerdo (VE). A função do miocárdio foi avaliada pela análise de músculos papilares isolados do VE, na condição basal e depois de manobras inotrópicas e lusitrópicas: rigidez do miocárdio, contração pós-pausa, aumento da concentração extracelular de Ca2+, mudança na frequência de estímulos e inibição da via glicolítica.

Resultados:

Os ratos obesos tiveram um aumento de tecido adiposo e comorbidades associadas à obesidade em relação aos ratos do grupo controle. A análise funcional após o bloqueio pelo iodoacetato não mostrou diferença na regressão linear da tensão desenvolvida (TD), entretanto, a tensão de repouso (TR) apresentou uma diferença estatística significativa entre o grupo controle e o grupo obeso, mais notadamente na inclinação da curva no grupo C.

Conclusão:

O desequilíbrio energético na obesidade não promoveu disfunção cardíaca. Pelo contrário, a priorização na utilização de ácidos graxos promoveu uma proteção do músculo cardíaco durante a inibição da glicólise, sugerindo que esta via é menos utilizada pelo músculo cardíaco obeso.

Obesidade; Ratos; Miocárdio; Metabolismo; Ácidos Graxos


Introduction

Obesity is a chronic metabolic disease defined by an excessive accumulation of body fat compared to lean tissue1World Health Organization (WHO). Obesity and overweight. Geneva;2011. [Access in 2014 May 03]. Available from: http://www.who.int/mediacentre/factsheets/fs311/en/
http://www.who.int/mediacentre/factsheet...
. According to estimates by the World Health Organization, in 2015, about 2.3 billion adults are overweight and of these, at least 700 million are considered obese (body mass index [BMI]> 30 g/m2)1World Health Organization (WHO). Obesity and overweight. Geneva;2011. [Access in 2014 May 03]. Available from: http://www.who.int/mediacentre/factsheets/fs311/en/
http://www.who.int/mediacentre/factsheet...
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Sumiyoshi M, Sakanaka M, Kimura Y. Chronic intake of high-fat and high-sucrose diets differentially affects glucose intolerance in mice. J Nutr. 2006;136(3):582-7.
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Sumiyoshi M, Sakanaka M, Kimura Y. Chronic intake of high-fat and high-sucrose diets differentially affects glucose intolerance in mice. J Nutr. 2006;136(3):582-7.
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Obesity is associated with increased risk of mortality and reduced life expectancy5Fontaine KR, Redden DT, Wang C, Westfall AO, Allison DB. Years of life lost due to obesity. JAMA. 2003;289(2):187-93.,6Olshansky SJ, Passaro DJ, Hershow RC, Layden J, Carnes BA, Brody J, et al. A potential decline in life expectancy in the United States in the 21st century. N Engl J Med. 2005;352(11):1138-45. and cardiovascular disease7Malnick SD, Knobler H. The medical complications of obesity. QJM. 2006;99(9):565-79.,8Poirier P, Giles TD, Bray GA, Hong Y, Stern JS, Pi-Sunyer FX, et al. Obesity and cardiovascular disease: pathophysiology, evaluation, and effect of weight loss. Arterioscler Thromb Vasc Biol. 2006;26(5):968-76.. Clinical studies or experimental show that obesity can lead to depression of cardiac function, the pathophysiological mechanisms responsible for this change are not entirely clear9Leopoldo AS, Lima-Leopoldo AP, Sugizaki MM, do Nascimento AF, de Campos DH, Luvizotto Rde A, et al. Involvement of L-type calcium channel and SERCA2a in myocardial dysfunction induced by obesity. J Cell Physiol. 2011:226(11):2934-42.. In normal aerobic conditions, approximately 70% of myocardial energy production derived from fatty acid metabolism, glycolytic contribution being about 30%1010 Stanley WC, Recchia FA, Lopaschuk GD. Myocardial substrate metabolism in the normal and failing heart. Physiol Rev. 2005;85(3):1093-129.

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15 Nuutila P, Koivisto VA, Knuuti J, Ruotsalainen U, Teras M, Haaparanta M, et al. Glucose-free fatty acid cycle operates in human heart and skeletal muscle in vivo. J Clin Invest. 1992;89(6):1767-74.
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18 Boudina S, Sena S, O'Neill BT, Tathireddy P, Young ME, Abel ED. Reduced mitochondrial oxidative capacity and increased mitochondrial unccoupling impair myocardial energetics in obesity. Circulation 2005;112(17):2686-95.

19 Aasum E, Belke DD, Severson DL, Riemersma RA, Cooper M, Andreassen M, et al. Cardiac function and metabolism in Type 2 diabetic mice after treatment with BM 17.0744, a novel PPAR-alpha activator. Am J Physiol Heart Circ Physiol. 2002;283(3):H949-57.

20 Belke DD, Larsen TS, Gibbs EM, Severson DL. Altered metabolism causes cardiac dysfunction in perfused hearts from diabetic (db/db) mice. Am J Physiol Endocrinol Metab. 2000;279(5):E1104-13.
-2121 Semeniuk LM, Kryski AJ, Severson DL. Echocardiographic assessment of cardiac function in diabetic db/db and transgenic db/db-hGLUT4 mice. Am J Physiol Heart Circ Physiol. 2002;283(3):H976-82.. In mice db/db obese and diabetic patients, normalization of energy metabolism, increased utilization of glucose, reversed the contractile dysfunction in these animals2020 Belke DD, Larsen TS, Gibbs EM, Severson DL. Altered metabolism causes cardiac dysfunction in perfused hearts from diabetic (db/db) mice. Am J Physiol Endocrinol Metab. 2000;279(5):E1104-13.,2121 Semeniuk LM, Kryski AJ, Severson DL. Echocardiographic assessment of cardiac function in diabetic db/db and transgenic db/db-hGLUT4 mice. Am J Physiol Heart Circ Physiol. 2002;283(3):H976-82.. This finding concurs with studies suggesting that ATP generated by the oxidation of glucose is used preferentially by the calcium pump (Ca2+ ) from the sarcoplasmic reticulum (SERCA2)1212 Okoshi K, Guimaraes JF, Di Muzio BP, Fernandes AA, Okoshi MP. Miocardiopatia diabética. Arq Bras Endocrinol Metabol. 2007;51(2):160-7.,1313 An D, Rodrigues B. Role of changes in cardiac metabolism in development of diabetic cardiomyopathy. Am J Physiol Heart Circ Physiol. 2006;291(4):H1489-506.,2222 Entman ML, Bornet EP, Van Winkle WB, Goldstein MA, Schwartz A. Association of glycogenolysis with cardiac sarcoplasmic reticulum: II. Effect of glycogen depletion, deoxycholate solubilization and cardiac ischemia: evidence for a phorphorylase kinase membrane complex. J Mol Cell Cardiol. 1977;9(7):515-28.

23 Weiss JN, Lamp ST. Glycolysis preferentially inhibits ATP-sensitive K+ channels in isolated guinea pig cardiac myocytes. Science. 1987;238(4823):67-9.
-2424 Weiss JN, Lamp ST. Cardiac ATP-sensitive K+ channels. Evidence for preferential regulation by glycolysis. J Gen Physiol. 1989;94(5):911-35., the protein responsible for reuptake of cytosolic calcium.

The objective of this study was to test the hypothesis that the myocardial energy balance, increased fatty acid oxidation and decreased glucose in obese animals leads to cardiac dysfunction resulting ATP deficit for SERCA2. In order to test this premise, the glycolytic pathway was inhibited by myocardial administration of iodoacetate (IAA), a drug that blocks the activity of the enzyme glyceraldehyde 3-phosphate dehydrogenase2525 Kusuoka H, Marban E. Mechanism of the diastolic dysfunction induced by glycolytic inhibition. Does adenosine triphosphate derived from glycolysis play a favored role in cellular Ca2+ homeostasis in ferret myocardium? J Clin Invest. 1994;93(3):1216-23.. The inhibition of glycolysis enhance cardiac dysfunction in obese mice.

Methods

Animal Model and Experimental Protocol

Thirty-day-old male Wistar rats were randomly assigned to one of two groups: control (C, n=12) and obese (Ob, n=12). The control group was fed a standard rat chow containing 11.2% fat, 55.5% carbohydrate, and 33.3% protein; whereas the obese animals received a high-fat diet containing 45.2% kcal fat, 28.6% carbohydrate, and 26.2% protein. Each group was fed the diet for 30 consecutive weeks. High fat diet was designed in our laboratory and contained powdered commercial Agroceres rat chow (Agroceres®, Rio Claro, SP, Brazil), industrialized feed, protein supplement, vitamins and minerals. The high-fat diet was calorically rich (high-fat diet = 3.65 kcal/g versus standard diet = 2.95 kcal/g) due to the higher fat composition, made with saturated (20.2%) and unsaturated fatty acid (79.8%). All rats were housed in individual cages in an environmentally-controlled clean-air room at 23 ± 3ºC with a 12 h light/dark cycle and 60 ± 5 % relative humidity. Food consumption was measured daily and water ad libitun. Initial and final body weights (IBW and FBW, respectively) were recorded. Weekly caloric intake was calculated as the average weekly food consumption x caloric value of each diet. Feed efficiency, the ability to translate calories consumed into body weight, was also evaluated.

All experiments and procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals, published by the U.S. National Institutes of Health2626 Medei E, Lima-Leopoldo AP, Pereira-Junior PP, Leopoldo AS, Campos DH, Raimundo JM, Sudo RT, et al. Could a high-fat diet rich in unsaturated fatty acids impair the cardiovascular system? Can J Cardiol. 2010:26(10):542-8., and were approved by the Botucatu Medical School Ethics Committee (UNESP, Botucatu, SP, Brazil).

Composition of Experimental Diets

The experimental diets provided sufficient amounts of protein, vitamins, and minerals according to the Nutrient Requirements of Laboratory Animals (1995). The standard and the four high-fat diets used in the study were formulated by Agroceres (Rio Claro, SP, Brazil). The ingredients were first ground and then mixed with vitamins and minerals. The mixture was made into pellets, dried in a ventilated drying oven at 55 ± 5ºC, and stored at -20ºC. The standard diet (RC Focus 1765) contained soybean oil, whole corn, wheat bran, soybean bran, dicalcium phosphate, sodium chloride, fish and meat flour, antioxidant additive, and a vitamins and minerals mixture. Meanwhile, the dietary ingredients used to prepare the high-fat diets were sodium chloride, casein, powdered milk, soybean protein concentrate, whole corn, cracker flour, dicalcium phosphate, Ca2+ carbonate, additives emulsifier, antioxidants and flavoring (cheese, vanilla, chocolate, and bacon), and a vitamins and minerals mixture. The composition of the high-fat diet consisted of saturated and unsaturated fatty acids, which provided 20% and 80% of the fat-derived calories, respectively9Leopoldo AS, Lima-Leopoldo AP, Sugizaki MM, do Nascimento AF, de Campos DH, Luvizotto Rde A, et al. Involvement of L-type calcium channel and SERCA2a in myocardial dysfunction induced by obesity. J Cell Physiol. 2011:226(11):2934-42.,2626 Medei E, Lima-Leopoldo AP, Pereira-Junior PP, Leopoldo AS, Campos DH, Raimundo JM, Sudo RT, et al. Could a high-fat diet rich in unsaturated fatty acids impair the cardiovascular system? Can J Cardiol. 2010:26(10):542-8..

Determination of Obesity

A criterion based on the adiposity index was used to determine obesity according to several authors9Leopoldo AS, Lima-Leopoldo AP, Sugizaki MM, do Nascimento AF, de Campos DH, Luvizotto Rde A, et al. Involvement of L-type calcium channel and SERCA2a in myocardial dysfunction induced by obesity. J Cell Physiol. 2011:226(11):2934-42.,2626 Medei E, Lima-Leopoldo AP, Pereira-Junior PP, Leopoldo AS, Campos DH, Raimundo JM, Sudo RT, et al. Could a high-fat diet rich in unsaturated fatty acids impair the cardiovascular system? Can J Cardiol. 2010:26(10):542-8.

27 Carroll JF, Zenebe WJ, Strange T. Cardiovascular function in rat model of diet-induced obesity. Hypertension. 2006;48(1):65-72.
-2828 Boustany-Kari CM, Gong M, Akers WS, Guo Z, Cassis LA. Enhanced vascular contractility and diminished coronary artery flow in rats made hypertensive from diet-induced obesity. Int J Obes. 2007;31(11):1652-9.. After animals had been anesthetized (sodium pentobarbital 50 mg/kg intraperitoneal [i.p.]), decapitated, and thoracotomized, the fat pads of adipose tissue were dissected and weighed. The adiposity index was calculated by the following formula: adiposity index = (total body fat (BF)/final body weight) × 100. BF was measured from the sum of the individual fat pad weights as follows: BF = epididymal fat + retroperitoneal fat + visceral fat 9Leopoldo AS, Lima-Leopoldo AP, Sugizaki MM, do Nascimento AF, de Campos DH, Luvizotto Rde A, et al. Involvement of L-type calcium channel and SERCA2a in myocardial dysfunction induced by obesity. J Cell Physiol. 2011:226(11):2934-42.,2626 Medei E, Lima-Leopoldo AP, Pereira-Junior PP, Leopoldo AS, Campos DH, Raimundo JM, Sudo RT, et al. Could a high-fat diet rich in unsaturated fatty acids impair the cardiovascular system? Can J Cardiol. 2010:26(10):542-8..

Comorbidities Associated with Obesity

As the rat models of diet-induced obesity may develop some of the characteristics of human obesity, such as hypertension, glucose intolerance, insulin resistance, dyslipidemia, hyperinsulinemia, and hyperleptinemia, the following evaluations were performed in all groups. For biochemical analysis, ten animals of each group were used.

Systolic Blood Pressure

At the conclusion of the experiments, the systolic blood pressure was assessed by using the non-invasive tail-cuff method2727 Carroll JF, Zenebe WJ, Strange T. Cardiovascular function in rat model of diet-induced obesity. Hypertension. 2006;48(1):65-72. with a Narco BioSystems® Electro-Sphygmomanometer (International Biomedical, Austin, TX, USA). The average of two pressure readings was recorded for each animal.

Oral Glucose Tolerance Test

At the end of the 30-week feeding period, an oral glucose tolerance test was performed. Rats fasted overnight (12-15 h) and blood samples were drawn from the tip of the tail. Blood glucose was collected under basal conditions and after intraperitoneal [i.p.] administration of 2 g/kg glucose load9Leopoldo AS, Lima-Leopoldo AP, Sugizaki MM, do Nascimento AF, de Campos DH, Luvizotto Rde A, et al. Involvement of L-type calcium channel and SERCA2a in myocardial dysfunction induced by obesity. J Cell Physiol. 2011:226(11):2934-42.. Blood samples were collected at 0, 15, 30, 60, 90 and 120 minutes, and analyzed using a glucometer (Accu-Check Go Kit; Roche Diagnostic Brazil Ltda, SP, Brazil).

Plasma Analysis of Hormones

At the end of treatment, animals were subjected to 12-15 h fast, anaesthetized with sodium pentobarbital (50 mg/kg intraperitoneal) and euthanized by decapitation. Blood was collected in heparinized tubes, centrifuged at 3000 × g for 15 minutes at 4ºC, and then stored at -80ºC. Plasma leptin and insulin concentrations were determined by ELISA9Leopoldo AS, Lima-Leopoldo AP, Sugizaki MM, do Nascimento AF, de Campos DH, Luvizotto Rde A, et al. Involvement of L-type calcium channel and SERCA2a in myocardial dysfunction induced by obesity. J Cell Physiol. 2011:226(11):2934-42. using specific commercial kits (Linco Research Inc., St. Louis, MO, USA).

Cholesterol, Triacylglycerol, non-esterified Tatty Acid, Insulin, and Leptin

At the end of the experimental period, animals were submitted to 12-15 h of fasting, anesthetized with sodium pentobarbital (50 mg/kg i.p.), and euthanized by decapitation. Blood samples were collected in heparinized tubes, and the serum was separated by centrifugation at 3000 × g for 15 minutes at 4ºC and stored at -80ºC until further analysis. Serum was analyzed for levels of GL, triglycerides (TG), total cholesterol (T-Chol), non-esterified fatty acid (NEFA), and hormones (insulin and leptin). Serum concentrations of GL, TG, and T-Chol were measured with an automatic enzymatic analyzer system (Technicon, RA-XTTM System, Global Medical Instrumentation, Minnesota, USA). NEFA levels were determined by method of Johnson and Peters (1993) by using colorimetric kits (WAKO NEFA-C, Wako Pure Chemical Industries, Osaka, Japan). Leptin and insulin levels were determined by ELISA using specific commercial kits (Linco Research Inc., St. Louis, MO, USA).

Body Fat Analysis

After animals had been anesthetized (sodium pentobarbital 50 mg/kg i.p.), decapitated and thoracotomized, the fat pads of adipose tissue were dissected and weighed. The total body fat was measured from the sum of the individual fat pad weights: epididymal fat + retroperitoneal fat + visceral fat. The adiposity index was calculated from: (body total fat/final body weight)*100 9Leopoldo AS, Lima-Leopoldo AP, Sugizaki MM, do Nascimento AF, de Campos DH, Luvizotto Rde A, et al. Involvement of L-type calcium channel and SERCA2a in myocardial dysfunction induced by obesity. J Cell Physiol. 2011:226(11):2934-42.,2626 Medei E, Lima-Leopoldo AP, Pereira-Junior PP, Leopoldo AS, Campos DH, Raimundo JM, Sudo RT, et al. Could a high-fat diet rich in unsaturated fatty acids impair the cardiovascular system? Can J Cardiol. 2010:26(10):542-8..

Cardiac Structure and Function

The heart and left ventricle (LV) weights were determined as indices of cardiac structure in absolute values and after normalization with the length of the shin-bone (SB).

Myocardial function was evaluated by studying isolated papillary muscles from the LV. This procedure has been utilized by various authors9Leopoldo AS, Lima-Leopoldo AP, Sugizaki MM, do Nascimento AF, de Campos DH, Luvizotto Rde A, et al. Involvement of L-type calcium channel and SERCA2a in myocardial dysfunction induced by obesity. J Cell Physiol. 2011:226(11):2934-42.,1717 Leopoldo AS, Sugizaki MM, Lima-Leopoldo AP, do Nascimento AF, Luvizotto Rde A, de Campos DH, et al. Cardiac remodeling in a rat model of diet-induced obesity. Can J Cardiol. 2010;26(8):423-9.,2929 Bruder-Nascimento T, Campos DHS, Leopoldo AS, Lima-Leopoldo AP, Okoshi K, Cordellini S, et al. Chronic stress improves the myocardial function without altering L-type Ca+2 channel activity in rats. Arq Bras Cardiol. 2012;99(4):907-14.. This preparation allows us to measure the capacity of cardiac muscle to shorten and develop force independently of influences that can modify in vivo mechanical performance of the myocardium, such as heart rate, preload, and afterload. Briefly, at the time of investigation, rats were anesthetized with sodium pentobarbital (50 mg/kg IP) and sacrificed by decapitation. The hearts were quickly removed and placed in oxygenated Krebs-Henseleit solution at 28ºC. LV papillary muscles from C (n = 12) and Ob rats (n = 12) were dissected, mounted between two spring clips, and placed vertically in a chamber containing Krebs-Henseleit solution (118.5 mM NaCl; 4.69 mM KCl; 2.5 mM CaCl2; 1.16 mM MgSO4; 1.18 mM KH2PO4; 5.50 mM GL, and 24.88 mM NaCO3) maintained at 28ºC with a thermostatic water circulator. The bathing solution was bubbled with 95% oxygen and 5% carbon dioxide, with a pH of 7.4. The lower spring clip was attached to a 120T-20B force transducer (Kyowa, Tokyo, Japan) by a thin steel wire (1/15,000 inch), which passed through the mercury seal at the bottom of the chamber. The upper spring clip was connected by a thin steel wire to a rigid lever arm, above which a micrometer stop was mounted for adjusting the muscle length. The muscle preparation was placed between two parallel platinum electrodes (Grass E8, GRASS Technologies, An Astro-Med, Inc. Product Group, West Warwick, RI, USA) and stimulated at a frequency of 0.2 Hz (12 pulses/min) by using square-wave pulses of 5 ms in duration. Voltage was set to a value 10% greater than the minimum required to produce a maximal mechanical response.

The muscles were contracted isotonically with light loads for 60 min and then loaded (50 g) to contract isometrically and stretched to the maximum of their length-tension curves. After a 5-min period during which preparations underwent isotonic contractions, muscles were again placed under isometric conditions, and the peak of the length-tension curve (Lmax) was carefully determined. A 15-min period of stable isometric contraction was imposed prior to the experimental period, during 10 which one isometric contraction was then recorded.

The following parameters were measured from the isometric contraction at Lmax: peak developed tension (DT [g/mm2]), resting tension (RT [g/mm2]), maximum rate of tension development (+dT/dt [g/mm2/s]) and maximum rate of tension decline (-dT/dt [g/mm2/s]) normalized per cross-sectional area (CSA). The myocardial stiffness was determined by the ratio between the muscle length variation and resting tension. Resting tension was analyzed in muscle length corresponding to 90, 92, 94, 96, 98, and 100 % of the Lmax. Resting tension-length curves were plotted using exponential regression analysis: log (RT) = -51.1118 + 25.5425 log(Lmax) for the C group and log (RT) = -58.1992 + 29.1455 log(Lmax) for the Ob group.

The parameters used to characterize papillary muscle were length (mm), weight (mg), and CSA (mm2). After the end of each experiment, the muscle length at Lmax was measured with a cathetometer (Gartner Scientific Corporation, Chicago, IL, USA) and the muscle between the two clips was blotted dry and weighed. The cross-sectional area was calculated from the muscle weight and length by assuming uniformity and a specific gravity of 1.0. All force data were normalized for the muscle cross-sectional area.

To determine the mechanism by which obesity induces negative inotropic effects on contractile function, the papillary muscles were evaluated under the baseline condition of 2.5 mmol/L Ca2+ and after inotropic and lusitropic maneuvers: postrest contraction of 10, 30 and 60 seconds; increase in extracellular Ca2+ concentration from 0,5 to 2.5 mmol/L; change in heart rate from 0.1 to 2.0 Hz; addition of the acid iodo acetic; the evaluation of glycolytic pathway was performed using a specific inhibitor, acid iodo acetic, 10-3 mM/L. The myocardial function was recorded 5, 10, 15, 20, 25, 30, 35, 40 and 45 min after the addition of acid iodo acetic at the solution nutrient. The behavior of the TD and TR after blocker administration of the glycolytic pathway, was carried through the construction of the linear regression model.

Statistical Analysis

General, nutritional, hormonal and morphologic characteristics, and cardiac function evaluation were reported as means ± standard deviation and the comparison between groups was analyzed using Student's t-test for independent samples. The glucose profile of the groups was compared by ANOVA for repeated measures. When significant differences were found (p < 0.05), the Bonferroni test post hoc for multiple comparisons was carried out. The regression curves of the blockade of the glycolytic pathway and myocardial stiffness were compared by the angular coefficient test and linear regression. The program used for statistical analysis was the Sigma Plot 3.5 for Windows (Systat Software Inc., San Jose, CA, USA). The normality of variables was analyzed by the Kolmogorov-Smirnov test. The level of significance considered was 5 %.

Results

General characteristics and comorbities associated to obesity

The general characteristics and comorbities associated to obesity are shown in Table 1. The final body weight, total body fat, adiposity index and glucose, leptin and insulin levels were higher in Ob than in C. There was no significant difference between groups in protein and lipids levels. The test results of glucose tolerance are shown in Figure 1. Glucose levels were similar at baseline between groups. After intraperitoneal administration of glucose, blood glucose was higher in Ob group at all times evaluated.

Table 1
General characteristics and comorbities associated to obesity
Figure 1
Oral glucose tolerance test in control animals (C, n = 12) and obese (Ob, n = 12). Values expressed as mean ± standart deviation. ANOVA and Bonferroni test. * p < 0.05 vs C.

Cardiac structure and function

The macroscopic cardiac structure is presented in Table 2; there was no significant difference between the two groups. Basal papillary myocardial function is presented in the Table 3; there were no significant differences in mechanical data. Moreover both groups, control and obese, presented same behavior with inotropic stimulations. The influence of variation of muscle length on the resting tension of the papillary muscle is shown in Figure 2; there were no significant differences between the two groups. This result shows that obesity did not increase the stiffness of the myocardium. Functional evaluation after blockade by iodoacetate is shown in Figure 3A and 3B; while there was no difference between linear regression of DT (Figure 3A), the RT showed a statistically significant difference in behavior between control and obese group, being the slope of the more remarkable in group C (Figure 3B).

Table 2
Macroscopic cardiac structure
Table 3
Basal isometric contraction
Figure 2
The influence of variation of muscle length on the resting tension in control animals (C, n = 12) and obese (Ob, n = 12). Linear regression.
Figure 3
Effects of blockade of glyceraldehyde 3-phosphate dehydrogenase with iodoacetate in the myocardium of rats by linear regression model in control animals (C, n = 12) and obese (Ob, n = 12). A: DT: peak developed tension. B: RT: resting tension. Linear regression. * p < 0,05 vs Ob.

Discussion

In the study period the diet was able to develop obesity because the adiposity index was 39.2% higher than the control. Data from this study are consistent with work done in our laboratory using the same methodology9Leopoldo AS, Lima-Leopoldo AP, Sugizaki MM, do Nascimento AF, de Campos DH, Luvizotto Rde A, et al. Involvement of L-type calcium channel and SERCA2a in myocardial dysfunction induced by obesity. J Cell Physiol. 2011:226(11):2934-42.,1717 Leopoldo AS, Sugizaki MM, Lima-Leopoldo AP, do Nascimento AF, Luvizotto Rde A, de Campos DH, et al. Cardiac remodeling in a rat model of diet-induced obesity. Can J Cardiol. 2010;26(8):423-9.,2626 Medei E, Lima-Leopoldo AP, Pereira-Junior PP, Leopoldo AS, Campos DH, Raimundo JM, Sudo RT, et al. Could a high-fat diet rich in unsaturated fatty acids impair the cardiovascular system? Can J Cardiol. 2010:26(10):542-8. . In this paper was viewed some comorbidities associated with obesity experimental as glucose intolerance, hyperinsulinemia and hyperleptinemia. These results reveal that our model promotes changes in metabolic and hormonal parameters and are in agreement with the findings of several researches9Leopoldo AS, Lima-Leopoldo AP, Sugizaki MM, do Nascimento AF, de Campos DH, Luvizotto Rde A, et al. Involvement of L-type calcium channel and SERCA2a in myocardial dysfunction induced by obesity. J Cell Physiol. 2011:226(11):2934-42.,3030 Li L, Yang G, Li Q, Tang Y, Li K. High-fat- and Lipid-induced insulin resistance in rats: the comparison of glucose metabolism, plasm resistin and adiponectin levels. Ann Nutr Metab. 2006;50(6):499-505.,3131 Nivoit P, Morens C, Van Assche FA, Jansen E, Poston L, Remacle C, et al. Established diet-induced obesity in female rats leads to offspring hyperphagia, adiposity and insulin resistance. Diabetologia. 2009;52(6):1133-42..

Obesity did not cause structural remodeling in heart rate and systolic blood pressure. The data differ from studies that showed that obesity induced by high-fat diet promoted cardiac hypertrophy9Leopoldo AS, Lima-Leopoldo AP, Sugizaki MM, do Nascimento AF, de Campos DH, Luvizotto Rde A, et al. Involvement of L-type calcium channel and SERCA2a in myocardial dysfunction induced by obesity. J Cell Physiol. 2011:226(11):2934-42.,3232 Smith AD, Brands MW, Wang MH, Dorrance AM. Obesity-induced hypertension develops in young rats independently of the renin-angiotensin-aldosterone system. Exp Biol Med. 2006;231(3):282-7.,3333 Fitzgerald SM, Henegar JR, Brands MW, Henegar LK, Hall JE. Cardiovascular and renal responses to a high-fat diet in Osborne-Mendel rats. Am J Physiol Regul Integr Comp Physiol. 2001;281(2):R547-52.,2626 Medei E, Lima-Leopoldo AP, Pereira-Junior PP, Leopoldo AS, Campos DH, Raimundo JM, Sudo RT, et al. Could a high-fat diet rich in unsaturated fatty acids impair the cardiovascular system? Can J Cardiol. 2010:26(10):542-8. and increased blood pressure3434 du Toit EF, Nabben M, Lochner A. A potential role for angiotensin II in obesity induced cardiac hypertrophy and ischaemic/reperfusion injury. Basic Res Cardiol. 2005;100(4):346-54.. The change in these variables do not suggest that obesity did not cause significant change neuro hormone capable of producing cell proliferation, vasoconstriction, and hydro-saline retention. In this study, myocardial function was assessed in vitro using isolated LV papillary muscles. Papillary muscle preparations permit the measurement of cardiac muscle force ability to develop and to shorten, independent of changes in cardiac load and heart rate that might modify mechanical performance of the myocardium in vivo. Inotropic stimulation allows the identification of alterations in contraction and relaxation phases observed that can not be read help under basal conditions and in the under-standing of the mechanisms involved alterations in myocardial function. The animals used in both groups had similar cross-sectional area and this allows to avoid the homogenizing influence of cross-sectional area on the results3535 Bing OH, Wiegner AW, Brooks WW, Fishbein MC, Pfeffer JM. Papillary muscle structure-function relations in the aging spontaneously hypertensive rat. Clin Exp Hipertens A. 1988;10(1):37-58.. The results show that obesity did not cause deterioration of cardiac function and myocardial stiffness in basal and with inotropic stimulation. This behavior is similar to that obtained by other authors, who used isolated hearts of obese rabbits by 12 weeks36 36 Carroll JF, Summers RL, Dzielak DJ, Cockrell K, Montani JP, Mizelle HL. Diastolic compliance is reduced in obese rabbits. Hypertension. 1999;33(3):811-5.and isolated myocytes of obese mice for 14 weeks3737 Ricci E, Smallwood S, Chouabe C, Mertani HC, Raccurt M, Morel G, et al. Electrophysiological characterization of left ventricular myocytes from obese Sprague-Dawley rat. Obesity. 2006;14(5):778-86.. However, researchers differ from that observed depression of mechanical function in isolated myocytes of obese mice for 12 weeks3838 Ouwens DM, Boer C, Fodor M, de Galan P, Heine RJ, Maassen JA, et al. Cardiac dysfunction induced by high-fat diet is associated with altered myocardial insulin signalling in rats. Diabetologia. 2005;48(6):1229-37. and the authors found that elevation of basal contractile performance of papillary muscle in obese mice by 7 weeks. Leopoldo et al9Leopoldo AS, Lima-Leopoldo AP, Sugizaki MM, do Nascimento AF, de Campos DH, Luvizotto Rde A, et al. Involvement of L-type calcium channel and SERCA2a in myocardial dysfunction induced by obesity. J Cell Physiol. 2011:226(11):2934-42. did not observe change in basal myocardial function in obeses rats by 15 weeks; however, these authors found differences between obese and control groups after L-type Ca2+ channel and SERCA2 blockers.

The blockade of the glycolytic pathway with iodoacetate on myocardial function showed that the slope of developed tension in obese animals was similar to control group; however, this slope of the RT in obese rats was significantly lower than controls. This finding suggests, contrary to expectations, that the control rats showed myocardial stiffness higher than the obese. This behavior could be related to the highest elevation of cytosolic calcium, possibly resulting from a lower recapture by SERCA2 and/or greater affinity between calcium and troponin C. The smallest contracture observed in obese rats suggests that prioritization of fatty acids in the myocardium at the expense of glucose increased energy availability for SERCA2 provided by fatty acids led to myocardial protection during the blockade of the glycolytic pathway. This suggests that in situations that occur in increased concentrations of fatty acids, the SERCA2 also uses energy from the beta-oxidation. Therefore, in this experiment, obesity promoted metabolic changes that resulted in myocardial protection.

The difference in behavior between the TR and TD, the TD being equal between the groups could be related to the ratio between the amount of calcium that diffuses and is removed from the cytosol during a cardiac cycle. The reason, according to Katz3939 Katz AM. Physiology of the heart. 4th ed. Philadelphia: Lipponcott Williams&Wilkins; 2006. p. 162-99., approximately 150 shows that the amount of calcium that is removed is less than the spread to the interior of the cell3939 Katz AM. Physiology of the heart. 4th ed. Philadelphia: Lipponcott Williams&Wilkins; 2006. p. 162-99.. Since SERCA2, largely responsible for the reuptake of calcium ions in rodents4040 Opie LH, Bers DM. Excitation-contraction coupling and calcium. In: Opie LH. Heart physiology: from cell to circulation. 4th ed. Philadelphia: Lipponcott Williams&Wilkins;2004.p. 159-85., uses ATP to its basic function, an energy deficit for this organelle would have severe deleterious consequences for diastole than in systole4040 Opie LH, Bers DM. Excitation-contraction coupling and calcium. In: Opie LH. Heart physiology: from cell to circulation. 4th ed. Philadelphia: Lipponcott Williams&Wilkins;2004.p. 159-85..

Conclusion

In conclusion, the energy imbalance in obesity does not cause cardiac dysfunction. Rather, the prioritization of the use of fatty acids provides protection to the heart muscle during the blockade of glycolysis, suggesting that this pathway is lesser utilized by myocyte in obese animals.

The results of this study suggest that the use of fatty acids by the myocardium, in certain situations, may have beneficial effects; thus, could do his job in models of cardiac remodeling as a therapy of low cost and easy implementation.

  • Author contributions
    Conception and design of the research: Campos DHS; Nascimento AF; Cicogna AC. Acquisition of data: Campos DHS; Leopoldo AS; Lima-Leopoldo AP; Nascimento AF; Oliveira-Júnior AS; SILVA DCT. Analysis and interpretation of the data: Campos DHS; Leopoldo AS; Lima-Leopoldo AP; Oliveira-Júnior AS; SUGIZAKI MM; Cicogna AC. Statistical analysis: Campos DHS; Leopoldo AS; Padovani CR. Obtaining fundung: Campos DHS; Cicogna AC. Writing of the manuscript: Campos DHS; Cicogna AC. Critical revision of the maniscript for intelectual contente: Campos DHS; Sugizaki MM; Cicogna AC.
  • Sources of Funding
    This study was funded by FAPESP - process nº 07/59747-7 e 07/46595-0.
  • Study Association
    This article is part of the thesis of master submitted by Dijon Henrique Salomé de Campos, from Faculdade de Medicina de Botucatu - UNESP.

Acknowledgments

We are grateful to José C. Georgette, Mário B. Bruno, Sandra A. Fabio, Camila R. C. Camacho and Corina J. Correa for their technical assistance. This manuscript has been proofread and edited by native English speakers with a related biomedical background in BioMed Proofreading (Cleveland, Ohio, USA).

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Publication Dates

  • Publication in this collection
    12 Sept 2014
  • Date of issue
    Oct 2014

History

  • Received
    06 Dec 2013
  • Reviewed
    14 Mar 2014
  • Accepted
    19 May 2014
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