Effect of exercise training and food restriction in the cardiac function and insulin resistance in obese rats

INTRODUÇÃO: A obesidade está associada com alterações na função cardíaca e no metabolismo hepático de gordura. Por outro lado, o treinamento físico e a restrição alimentar são conhecidos por reverter às alterações metabólicas decorrentes da obesidade e melhorar o prognóstico em pacientes obesos. No entanto, se estas intervenções melhoram a função cardíaca e os seus mecanismos moleculares associados ao transiente de Ca2+ e a concentração hepática de gordura ainda são pouco conhecidos. Os objetivos deste estudo foram avaliar os efeitos do treinamento físico e da restrição alimentar: 1) na função cardíaca e no perfil molecular das proteínas responsáveis pelo transiente de Ca2+em ratos obesos; 2) na esteatose em ratos obesos. Além disso, se essas duas intervenções associadas tinham um efeito sinérgico nessas respostas. MÉTODOS: Ratos Wistar machos foram alimentados com dieta normocalórica ou dieta hipercalórica durante 25 semanas. Após este período, os ratos com dieta de cafeteria foram randomizados em 4 grupos e acompanhados por 10 semanas: 1) dieta hipercalórica (GO); 2) dieta hipercalórica e treinamento físico (60 % do VO2pico, GOTF); 3) restrição alimentar (-20% da ingestão diária, GORA); 4) treinamento físico e restrição alimentar (GOTFRA). Os ratos do grupo controle continuaram recebendo a dieta normocalórica (GM). O controle hepático glicêmico foi determinado pelo índice HOMA-IR (avaliação do modelo de homeostasia), a esteatose pela concentração hepática de triglicérides, a função cardíaca foi determinada pela ecocardiografia, Modo M e Doppler tecidual e a expressão das proteínas responsáveis pelo transiente de Ca2+ por Western blotting. RESULTADOS: Os ratos do GO apresentaram maior peso corporal, índice de adiposidade, HOMA-IR, concentração de glicose, leptina, adrenalina e noradrenalina e menor fração de encurtamento (39±1 vs 44±1%, P<0,05), fosforilação do receptor de rianodina (P-RyR-Ser2808/RyR , 52±7 vs 100±16%, P<0,01) e da fosfolambam (PPLB- Tre17/PLB, 76±6 vs 100±6%, P<0,05), concentração de nitrato e razão glutationa reduzida e oxidada quando comparados com os ratos do GM. Treinamento físico, restrição alimentar e a associação das dua intervenções diminuiram o índice de adiposidade, a concentração de leptina, adrenalina e noradrenalina e aumentaram a fração de encurtamento (41±1, 42±1 e 43±1%, respectivamente), a fosforilação do receptor de rianodina (80±9, 79±7 e 66±12 %, respectivamente) a da fosfolambam (107±9, 109±4 e 122±11 %, respectivamente), concentração de nitrato e razão glutationa reduzida e oxidada. Apenas o treinamento físico aumentou o consumo de oxigênio de pico. As intervenções envolvendo a restrição alimentar diminuíram o peso corporal e o conteúdo de triglicerídeo hepático (GORA= -52 e GORATF= - 63%). Nenhuma das intervenções normalizou o HOMA-IR e a concentração de glicose. CONCLUSÕES: A perda de peso por treinamento físico ou restrição alimentar por 10 semanas previne a alteração na função sistólica do ventrículo esquerdo relacionada ao perfil molecular das proteínas responsáveis pelo transiente de Ca2+ em ratos obesos. A perda de peso por essas intervenções diminui a esteatose, apesar de não normalizar o HOMA-IR. A associação do treinamento físico e da restrição alimentar não tem efeito sinérgico na função cardíaca, na expressão das proteínas responsáveis pelo transiente de Ca2+ e no conteúdo hepático de gordura.BACKGROUND. Obesity is associated with cardiac function and hepatic fat metabolism abnormalities. On the other hand, exercise training and food restriction are known to restore obesity metabolic disorders and improve prognosis in obese individuals. However, whether these interventions improve cardiac function and its molecular mechanism associated with Ca2+ handling proteins are little unknown. The aims of this study were to investigate the effects of exercise training and food restriction on: 1) cardiac function and molecular net Ca2+ handling proteins in obese rats; 2) liver fat content in obese rats. In addition, we investigated whether the association of these two interventions had an additive effect on those responses. METHODS: Male Wistar rats (30 days-old) were fed with standard chow or cafeteria diet with high-fat for 25 weeks. At 25th week, the cafeteria diet rats were randomly assigned into 4 groups followed by 10 weeks: high-fat-chow (GO); high-fat-chow submitted to running exercise training (60% VO2peak, GOTF); food restriction (20% less intake of standard chow, GORA); and exercise training and food restriction (GOTFRA). Control rats continued fed with standard chow (GM). Hepatic insulin resistance was evaluated by HOMA-IR index (Homeostastic Metabolic Assessement- Insulin Resistance), liver fat content by liver triglyceride level, cardiac function was evaluated by echocardiography, M Modus and Tissue Doppler, and protein expression by Western blotting. RESULTS: Obese rats had increased body weight, adiposity index, HOMA-IR, glucose, leptin, epinephrine and norepinephrine levels and decreased left ventricular fractional shortening (39±1 vs 44±1%, P<0.05), ryanodine receptor phosphorylation (P-RyR-Ser2808/RyR , 52±7 vs 100±16%, P<0.01), phospholamban phosphorylation (P-PLB-Tre17/PLB, 76±6 vs 100±6%, P<0.05), nitrate level and reduced/oxidized glutathione ratio when compared with lean rats. Exercise training, food restriction or both decreased adiposity index, leptin, epinephrine and norepinephrine levels and increased left ventricular fractional shortening (41±1, 42±1 e 43±1%, respectively), ryanodine receptor phosphorylation (80±9, 79±7 and 66±12 %, respectively), phospholamban phosphorylation (107±9, 109±4 e 122±11%, respectively), nitrate level and reduced /oxidized glutathione ratio. Exercise training increased peak oxygen uptake. Food restriction alone or associated with exercise training decreased body weight and liver triglyceride level (GORA= -52 e GORATF= -63%). Neither exercise training nor food restriction restored HOMA-IR or glucose level. COCLUSIONS: Body weight reduction by exercise training or food restriction during 10 weeks prevents abnormalities in left ventricular systolic function associated with cardiac Ca2+ handling proteins expression in obese rats. Body weight reduction by these two interventions decreases steatosis without normalized HOMA-IR and glucose levels. Exercise training associated with food restriction does not have synergic effects on cardiac function and molecular net Ca2+ handling proteins, and liver fat content

Abstract 104: Sex-specific Impact Of S-nitrosoglutathione Reductase (gsnor) On Ventricular Remodeling And Function Following Myocardial Infarction In Mice

Introduction: Female hearts are less susceptible to myocardial injury following myocardial infarction (MI) and estrogen working through the nitric oxide (NO) pathway is thought to play a role. S-nitrosylation of cysteine thiols is a major signaling pathway through which NO exerts its action and mice with targeted deletion of S-nitrosoglutathione reductase (GSNOR), a denitrosylase that regulates S-nitrosylation, show increased levels of nitrosylated proteins. Male GSNOR -/- mice show a more favorable outcome after MI as compared to wild-type (WT), including reduced myocardial infarct size, improved ejection fraction and preserved left ventricular volumes. Whether female GSNOR -/- mice show gender-related cardiac protection following MI was not known and thus investigated. Methods & Results: MI was induced in male and female GSNOR -/- mice and their respective controls, C57Bl/6J, at 3-5 months via left anterior descending coronary artery occlusion. Serial echocardiography was performed prior to MI and after 1- and 4-weeks post-MI to assess ejection fraction (EF) and left ventricular volume both at diastole (end-diastolic volume, EDV) and systole (end-systolic volume, ESV). Compared to WT, GSNOR -/- males showed less dilation in both EDV (98.6 ± 9.3 mm vs. 140.6 ± 7.4 mm in WT, P<0.001) and ESV (73.1 ± 9.2 vs. 112.7 ± 7.3 mm in WT, P<0.05) at 4 weeks post-MI. Whereas, GSNOR -/- females showed greater dilation in both EDV (162.2 ± 13 vs. 83.8 ± 12 mm in WT, P<0.001) and ESV (141.8 ± 13 vs. 65.6 ± 12 mm in WT, P<0.001) as compared to WT females. EF decreased (P<0.001) in all groups post-MI, but at 4 weeks post-MI, it was significantly worse in GSNOR -/- females compared to GSNOR -/- males (14 ± 4% vs. 28 ± 3%, P<0.05). Conclusion: GSNOR -/- females exhibit significantly lower EF and greater dilation of left ventricular volumes both at diastole and systole following MI than any of the other groups suggesting that S-nitrosylation plays an important role in gender-related cardiac protection following myocardial injury. These findings suggest the importance of taking gender into account when exploring novel therapeutic treatments for myocardial injury

Abstract 7: S-nitrosylation Affects Differentiation of Mesenchymal Stem Cells

Introduction: Bone marrow-derived mesenchymal stem cells (BMMSCs) appear to play an important role in the formation of atherosclerotic lesions. Circulating BMMSCs are attracted to arterial wall where they undergo adipogenic or osteogenic differentiation to form ectopic aggregates of fat and bone. The factors that determine the direction of BMMSC differentiation are poorly understood. We assessed the role of nitric oxide (NO), an inhibitor of lipid formation and vascular calcification, in BMMSC differentiation. Hypothesis: S-nitrosylation mediated-NO signaling controls the balance between adipogenic and osteogenic differentiation of BMMSCs. Methods: We isolated BMMSCs from wild type mice (WT) and mice deficient in S-nitrosoglutathione reductase (GSNOR -/- ), an enzyme that governs levels of S-nitrosylation by promoting protein denitrosylation. Cells were cultured in either adipogenic or osteogenic differentiation media followed by functional and gene expression assays. Results: MSCs derived from GSNOR -/- mice had impaired fat droplet formation and lower expression of the adipogenic markers PPARγ (1329±415.3-fold increase in WT vs. 158±65.61-fold in GSNOR -/- , P<0.05) and FABP4 (11.06±3.29-fold in WT vs. 4.06±0.62-fold in GSNOR -/- , P<0.05). Conversely, GSNOR -/- MSCs exhibited enhanced osteogenic differentiation as indicated by greater calcium deposition and higher expression of the osteogenic marker Osteopontin (1.48±0.17-fold in WT vs. 16.18±5.26-fold in GSNOR -/- , P<0.05). Interestingly, GSNOR -/- cells had higher baseline expression of Osteopontin, Osteocalcin and Runx2. Treatment of GSNOR -/- MSCs with L-NAME, a nitric oxide synthase (NOS) inhibitor, resulted in decreased osteogenic differentiation without affecting adipogenic differentiation. Conclusion: Our results indicate that GSNOR enhances adipogenic differentiation and impairs osteogenic differentiation of BMMSCs in vitro and suggest that interaction of GSNOR- and NOS-mediated processes regulates the balance between adipogenic and osteogenic differentiation of BMMSCs

Abstract 175: Genetic Ablation Of S-nitrosoglutathione Reductase In Mice Enhances Proliferative Expansion Of Adult Heart Progenitors And Myocytes Post Myocardial Infarction

Objectives: The molecular pathways underlying the proliferative activity of adult cardiac myocytes and stem/progenitors in response to heart damage remain elusive. Since perturbing nitroso-redox balance in mice by genetic deletion of GSNOR (GSNOR -/- ) confers resilience to experimental myocardial infarction (MI), we investigated whether GSNOR knockout influences the proliferative activity of the post-MI heart. Hypothesis: Knockout of GSNOR in mice enhances the proliferative expansion of CMs and cKit + cardiac stem cells (CSCs) in response to MI. Methods: Wild-type (WT) and GSNOR -/- mice (n=5/ group) underwent experimental MI. To assess proliferative activity, animals received intraperitoneal injections of 5-bromodeoxyuridine (BrdU) at selected time-points during the first 2 weeks post-MI. Immunnohistochemical evaluation was performed 1 month post-MI. Results: Confocal immunofluorescence revealed that GSNOR -/- hearts exhibited higher rates of BrdU incorporation in CSCs after MI (10.9%±4.99% of WT CSCs compared to 15.9%±3% of GSNOR -/- CSCs, p =0.02). Similarly, there were ~3-fold more BrdU + /Tropomyosin + cardiomyocytes in the infarct zone of GSNOR -/- mice compared to WT ( p <0.05). Immunohistochemical evaluation of cardiac troponin-T + cardiomyocytes co-expressing the mitotic marker ser-10 phosphorylated histone H3 (H3P) showed further that cardiomyocyte mitosis was 2.4-fold greater in GSNOR -/- compared to WT mice ( p <0.05), whereas the presence of aurora-b kinase in the cleavage furrow of GSNOR -/- cardiomyocytes substantiated their competence for mitosis after MI. The rate of cardiomyocyte apoptosis after MI, was not different between GSNOR -/- and WT mice, as shown by activated cleaved caspase-3 immunofluorescence. Conclusions: Collectively, our findings suggest that protein S-nitrosothiol turnover by GSNOR regulates proliferation of cardiomyocytes and CSCs in the adult heart in response to damage. These findings have therapeutic implications for the treatment of heart disease since they reveal novel pathways by which nitroso-redox balance influences cardiac repair

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BACKGROUND: Mammalian heart regenerative activity is lost before adulthood but increases after cardiac injury. Cardiac repair mechanisms, which involve both endogenous cardiac stem cells (CSCs) and cardiomyocyte cell-cycle reentry, are inadequate to achieve full recovery after myocardial infarction (MI). Mice deficient in S-nitrosoglutathione reductase (GSNOR(−⁄−)), an enzyme regulating S-nitrosothiol turnover, have preserved cardiac function after MI. Here, we tested the hypothesis that GSNOR activity modulates cardiac cell proliferation in the post-MI adult heart. METHODS AND RESULTS: GSNOR(−⁄−) and C57Bl6/J (wild-type [WT]) mice were subjected to sham operation (n=3 GSNOR(−⁄−); n=3 WT) or MI (n=41 GSNOR(−⁄−); n=65 WT). Compared with WT,GSNOR(−⁄−) mice exhibited improved survival, cardiac performance, and architecture after MI, as demonstrated by higher ejection fraction (P<0.05), lower endocardial volumes (P<0.001), and diminished scar size (P<0.05). In addition, cardiomyocytes from post-MI GSNOR(−⁄−) hearts exhibited faster calcium decay and sarcomeric relaxation times (P<0.001). Immunophenotypic analysis illustrated that post-MI GSNOR(−⁄−) hearts demonstrated enhanced neovascularization (P<0.001), c-kit(+) CSC abundance (P=0.013), and a ≈3-fold increase in proliferation of adult cardiomyocytes and c-kit(+)/CD45(−) CSCs (P<0.0001 and P=0.023, respectively) as measured by using 5-bromodeoxyuridine. CONCLUSIONS: Loss of GSNOR confers enhanced post-MI cardiac regenerative activity, characterized by enhanced turnover of cardiomyocytes and CSCs. Endogenous denitrosylases exert an inhibitory effect over cardiac repair mechanisms and therefore represents a potential novel therapeutic target

Discrete effects of A57G-myosin essential light chain mutation associated with familial hypertrophic cardiomyopathy

The functional consequences of the familial hypertrophic cardiomyopathy A57G (alanine-to-glycine) mutation in the myosin ventricular essential light chain (ELC) were assessed in vitro and in vivo using previously generated transgenic (Tg) mice expressing A57G-ELC mutant vs. wild-type (WT) of human cardiac ELC and in recombinant A57G- or WT-protein-exchanged porcine cardiac muscle strips. Compared with the Tg-WT, there was a significant increase in the Ca(2+) sensitivity of force (ΔpCa(50) ≅ 0.1) and an ∼1.3-fold decrease in maximal force per cross section of muscle observed in the mutant preparations. In addition, a significant increase in passive tension in response to stretch was monitored in Tg-A57G vs. Tg-WT strips indicating a mutation-induced myocardial stiffness. Consistently, the hearts of Tg-A57G mice demonstrated a high level of fibrosis and hypertrophy manifested by increased heart weight-to-body weight ratios and a decreased number of nuclei indicating an increase in the two-dimensional size of Tg-A57G vs. Tg-WT myocytes. Echocardiography examination showed a phenotype of eccentric hypertrophy in Tg-A57G mice, enhanced left ventricular (LV) cavity dimension without changes in LV posterior/anterior wall thickness. Invasive hemodynamics data revealed significantly increased end-systolic elastance, defined by the slope of the pressure-volume relationship, indicating a mutation-induced increase in cardiac contractility. Our results suggest that the A57G allele causes disease by means of a discrete modulation of myofilament function, increased Ca(2+) sensitivity, and decreased maximal tension followed by compensatory hypertrophy and enhanced contractility. These and other contributing factors such as increased myocardial stiffness and fibrosis most likely activate cardiomyopathic signaling pathways leading to pathologic cardiac remodeling

S‐Nitrosoglutathione Reductase Deficiency Causes Aberrant Placental S‐Nitrosylation and Preeclampsia

Background Preeclampsia, a leading cause of maternal and fetal mortality and morbidity, is characterized by an increase in S‐nitrosylated proteins and reactive oxygen species, suggesting a pathophysiologic role for dysregulation in nitrosylation and nitrosative stress. Methods and Results Here, we show that mice lacking S‐nitrosoglutathione reductase (GSNOR−⁄−), a denitrosylase regulating protein S‐nitrosylation, exhibit a preeclampsia phenotype, including hypertension, proteinuria, renal pathology, cardiac concentric hypertrophy, decreased placental vascularization, and fetal growth retardation. Reactive oxygen species, NO, and peroxynitrite levels are elevated. Importantly, mass spectrometry reveals elevated placental S‐nitrosylated amino acid residues in GSNOR−⁄− mice. Ascorbate reverses the phenotype except for fetal weight, reduces the difference in the S‐nitrosoproteome, and identifies a unique set of S‐nitrosylated proteins in GSNOR−⁄− mice. Importantly, human preeclamptic placentas exhibit decreased GSNOR activity and increased nitrosative stress. Conclusions Therefore, deficiency of GSNOR creates dysregulation of placental S‐nitrosylation and preeclampsia in mice, which can be rescued by ascorbate. Coupled with similar findings in human placentas, these findings offer valuable insights and therapeutic implications for preeclampsia

Exercise Training and Caloric Restriction Prevent Reduction in Cardiac Ca2+-Handling Protein Profile in Obese Rats

Previous studies show that exercise training and caloric restriction improve cardiac function in obesity. However, the molecular mechanisms underlying this effect on cardiac function remain unknown. Thus, we studied the effect of exercise training and/or caloric restriction on cardiac function and Ca2+ handling protein expression in obese rats. To accomplish this goal, male rats fed with a high-fat and sucrose diet for 25 weeks were randomly assigned into 4 groups: high-fat and sucrose diet, high-fat and sucrose diet and exercise training, caloric restriction, and exercise training and caloric restriction. An additional lean group was studied. The study was conducted for 10 weeks. Cardiac function was evaluated by echocardiography and Ca2+ handling protein expression by Western blotting. Our results showed that visceral fat mass, circulating leptin, epinephrine, and norepinephrine levels were higher in rats on the high-fat and sucrose diet compared with the lean rats. Cardiac nitrate levels, reduced/oxidized glutathione, left ventricular fractional shortening, and protein expression of phosphorylated Ser(2808)-ryanodine receptor and Thr(17-)phospholamban were lower in rats on the high-fat and sucrose diet compared with lean rats. Exercise training and/or caloric restriction prevented increases in visceral fat mass, circulating leptin, epinephrine, and norepinephrine levels and prevented reduction in cardiac nitrate levels and reduced: oxidized glutathione ratio. Exercise training and/or caloric restriction prevented reduction in left ventricular fractional shortening and in phosphorylation of the Ser(2808)-ryanodine receptor and Thr(17)-phospholamban. These findings show that exercise training and/or caloric restriction prevent cardiac dysfunction in high-fat and sucrose diet rats, which seems to be attributed to decreased circulating neurohormone levels. In addition, this nonpharmacological paradigm prevents a reduction in the Ser(2808)-ryanodine receptor and Thr(17-)phospholamban phosphorylation and redox status. (Hypertension. 2010;56:629-635.)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP