Marcele A. Coelho

Intracellular mechanisms of specific β-adrenoceptor antagonists involved in improved cardiac function and survival in a genetic model of heart failure

beta-blockers, as class, improve cardiac function and survival in heart failure (HF). However, the molecular mechanisms underlying these beneficial effects remain elusive. In the present study, metoprolol and carvedilol were used in doses that display comparable heart rate reduction to assess their beneficial effects in a genetic model of sympathetic hyperactivity-induced HF (alpha(2A)/alpha(2C)-ARKO mice). Five month-old HF mice were randomly assigned to receive either saline, metoprolol or carvedilol for 8 weeks and age-matched wild-type mice (WT) were used as controls. HF mice displayed baseline tachycardia, systolic dysfunction evaluated by echocardiography, 50% mortality rate, increased cardiac myocyte width (50%) and ventricular fibrosis (3-fold) compared with WT. All these responses were significantly improved by both treatments. Cardiomyocytes from HF mice showed reduced peak [Ca(2+)](i) transient (13%) using confocal microscopy imaging. Interestingly, while metoprolol improved [Ca(2+)](i) transient, carvedilol had no effect on peak [Ca(2+)](i) transient but also increased [Ca(2+)] transient decay dynamics. We then examined the influence of carvedilol in cardiac oxidative stress as an alternative target to explain its beneficial effects. Indeed, HF mice showed 10-fold decrease in cardiac reduced/oxidized glutathione ratio compared with WT, which was significantly improved only by carvedilol treatment. Taken together, we provide direct evidence that the beneficial effects of metoprolol were mainly associated with improved cardiac Ca(2+) transients and the net balance of cardiac Ca(2+) handling proteins while carvedilol preferentially improved cardiac redox state.

Angiotensin receptor blockade improves the net balance of cardiac Ca2+ handling-related proteins in sympathetic hyperactivity-induced heart failure

AIMS The clinical benefits of angiotensin II type 1 (AT1) receptor blockers (ARB) in heart failure (HF) include cardiac anti-remodeling and improved ventricular function. However, the cellular mechanisms underlying the benefits of ARB on ventricular function need to be better clarified. In the present manuscript, we evaluated the effects of AT1 receptor blockade on the net balance of Ca(2+) handling proteins in hearts of mice lacking α(2A) and α(2C) adrenoceptors (α(2A)/α(2C)ARKO), which develop sympathetic hyperactivity (SH) induced-HF. MAIN METHODS A cohort of male wild-type (WT) and congenic α(2A)/α(2C)ARKO mice in a C57BL6/J genetic background (5-7mo of age) was randomly assigned to receive either placebo or ARB (Losartan, 10mg/kg for 8wks). Ventricular function (VF) was assessed by echocardiography, and cardiac myocyte width and ventricular fibrosis by a computer-assisted morphometric system. Sarcoplasmic reticulum Ca(2+) ATPase (SERCA2), phospholamban (PLN), phospho-Ser(16)-PLN, phospho-Thr(17)-PLN, phosphatase 1 (PP1), Na(+)-Ca(2+) exchanger (NCX), Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and phospho-Thr(286)-CaMKII were analyzed by Western blot. KEY FINDINGS α(2A)/α(2C)ARKO mice displayed ventricular dysfunction, cardiomyocyte hypertrophy and cardiac fibrosis paralleled by decreased SERCA2 and increased phospho-Thr(17)-PLN, CaMKII, phospho-Thr(286)-CaMKII and NCX levels. ARB induced anti-cardiac remodeling effect and improved VF in α(2A)/α(2C)ARKO associated with increased SERCA2 and phospho-Ser(16)-PLN levels, and SERCA2:NCX ratio. Additionally, ARB decreased phospho-Thr(17)-PLN levels as well as reestablished NCX, CaMKII and phospho-Thr(286)-CaMKII toward WT levels. SIGNIFICANCE Altogether, these data provide new insights on intracellular Ca(2+) regulatory mechanisms underlying improved ventricular function by ARB therapy in HF.

Moderate exercise training promotes adaptations in coronary blood flow and adenosine production in normotensive rats

OBJECTIVES: Aerobic exercise training prevents cardiovascular risks. Regular exercise promotes functional and structural adaptations that are associated with several cardiovascular benefits. The aim of this study is to investigate the effects of swimming training on coronary blood flow, adenosine production and cardiac capillaries in normotensive rats. METHODS: Wistar rats were randomly divided into two groups: control (C) and trained (T). An exercise protocol was performed for 10 weeks and 60 min/day with a tail overload of 5% bodyweight. Coronary blood flow was quantified with a color microsphere technique, and cardiac capillaries were quantified using light microscopy. Adenine nucleotide hydrolysis was evaluated by enzymatic activity, and protein expression was evaluated by western blot. The results are presented as the means ± SEMs (p<0.05). RESULTS: Exercise training increased the coronary blood flow and the myocardial capillary-to-fiber ratio. Moreover, the circulating and cardiac extracellular adenine nucleotide hydrolysis was higher in the trained rats than in the sedentary rats due to the increased activity and protein expression of enzymes, such as E-NTPDase and 5′-nucleotidase. CONCLUSIONS: Swimming training increases coronary blood flow, number of cardiac capillaries, and adenine nucleotide hydrolysis. Increased adenosine production may be an important contributor to the enhanced coronary blood flow and angiogenesis that were observed in the exercise-trained rats; collectively, these results suggest improved myocardial perfusion.

Akt/mTOR pathway contributes to skeletal muscle anti-atrophic effect of aerobic exercise training in heart failure mice.

BACKGROUND Exercise intolerance is one of the main clinical symptoms of heart failure (HF) and is associated with skeletal muscle wasting due to an imbalance between proteolysis and protein synthesis. In this study, we tested whether aerobic exercise training (AET) would counteract skeletal muscle atrophy by activating IGF-I/Akt/mTOR pathway in HF mice. METHODS Sympathetic hyperactivity induced HF mice were assigned into 8-week moderate intensity AET. Untrained wild type and HF mice were used as control. Soleus cross sectional area was evaluated by histochemistry and motor performance by rotarod. 26S proteasome activity was assessed by fluorimetric assay, and components of IGF-I/Akt/mTOR pathway or myostatin pathway by qRT-PCR or immunoblotting. A different subset of mice was used to evaluate the relative contribution of mTOR inhibition (rapamycin) or activation (leucine) on AET-induced changes in muscle mass regulation. RESULTS AET prevented exercise intolerance and impaired motor performance in HF mice. These effects were associated with attenuation of soleus atrophy. Rapamycin treatment precluded AET effects on soleus mass in HF mice suggesting the involvement of IGF signaling pathway in this response. In fact, AET increased IGF-I Ea and IGF-I Pan mRNA levels, while it reduced myostatin and Smad2 mRNA levels in HF mice. At protein levels, AET prevented reduced expression levels of IGF-I, pAkt (at basal state), as well as, p4E-BP1 and pP70(S6K) (leucine-stimulated state) in HF mice. Additionally, AET prevented 26S proteasome hyperactivity in HF mice. CONCLUSIONS Taken together, our data provide evidence for AET-induced activation of IGF-I/Akt/mTOR signaling pathway counteracting HF-induced muscle wasting.

Lactate Up-Regulates the Expression of Lactate Oxidation Complex-Related Genes in Left Ventricular Cardiac Tissue of Rats

Background Besides its role as a fuel source in intermediary metabolism, lactate has been considered a signaling molecule modulating lactate-sensitive genes involved in the regulation of skeletal muscle metabolism. Even though the flux of lactate is significantly high in the heart, its role on regulation of cardiac genes regulating lactate oxidation has not been clarified yet. We tested the hypothesis that lactate would increase cardiac levels of reactive oxygen species and up-regulate the expression of genes related to lactate oxidation complex. Methods/Principal Findings Isolated hearts from male adult Wistar rats were perfused with control, lactate or acetate (20mM) added Krebs-Henseleit solution during 120 min in modified Langendorff apparatus. Reactive oxygen species (O2 ●-/H2O2) levels, and NADH and NADPH oxidase activities (in enriched microsomal or plasmatic membranes, respectively) were evaluated by fluorimetry while SOD and catalase activities were evaluated by spectrophotometry. mRNA levels of lactate oxidation complex and energetic enzymes MCT1, MCT4, HK, LDH, PDH, CS, PGC1α and COXIV were quantified by real time RT-PCR. Mitochondrial DNA levels were also evaluated. Hemodynamic parameters were acquired during the experiment. The key findings of this work were that lactate elevated cardiac NADH oxidase activity but not NADPH activity. This response was associated with increased cardiac O2 ●-/H2O2 levels and up-regulation of MCT1, MCT4, LDH and PGC1α with no changes in HK, PDH, CS, COXIV mRNA levels and mitochondrial DNA levels. Lactate increased NRF-2 nuclear expression and SOD activity probably as counter-regulatory responses to increased O2 ●-/H2O2. Conclusions Our results provide evidence for lactate-induced up-regulation of lactate oxidation complex associated with increased NADH oxidase activity and cardiac O2 ●-/H2O2 driving to an anti-oxidant response. These results unveil lactate as an important signaling molecule regulating components of the lactate oxidation complex in cardiac muscle.

Esteróide anabolizante inibe a angiogênese induzida pelo treinamento físico de natação em músculo sóleo de ratos normotensos

Androgenic anabolic steroids (EAA) are synthetic derivatives of testosterone, used in therapeutic dosages in medical practice and in high doses by physically active people that could be health damaging. To study the effects of EAA on the cardiovascular system, Wistar rats were randomized into Sedentary Control (SC), Sedentary Steroid (SA), Trained Control (TC) and Trained Steroid (TA) groups. We evaluated the effects of swimming training (60min/day, 5x/week during 10 week) and AAS (nandrolone decanoate - 5 mg/kg sc, 2x/week) on cardiac output, basal blood flow (Qb, DC basal) and after injection of a vasodilator to observe the endothelium dependent vasodilatation (acetylcholine - Q Ach)(Q Ach, DC Ach), capillary to fiber ratio (r c/f) and vascular-endothelial growth factor expression (VEGF) in soleus muscle (oxidative fibers). Serum testosterone increased in SA and TA. Exercise training significantly decreased resting heart rate. Qb was not different among groups, and QAch was higher in TC group, however in TA group this beneficial effect of swimming exercise training was lost by association with EAA. Rc/f and VEGF were higher only in TC group. These results suggest that swimming training associated with EAA inhibit angiogenesis and arteriogenesis observed as effects of aerobic training, and impairs the red skeletal muscle blood flow which predispose physically active AAS users to vascular diseases.

Resposta taquicárdica e controle autonômico no exercício físico em modelo genético de insuficiência cardíaca

Increase of sympathetic nervous activity and tachycardia at rest or during physical exertions are associated with increase of morbimortality, even in the absence of clinical signs of cardiac disease. Considering the importance of the α2A/α2C-adrenergic receptors in the modulation of the nervous activity and heart rate (HR), the present study uses a genetic model of cardiomyopathy induced by excess of circulating catecholamine in the gene inactivation of the α2A/α2 -adrenergic receptors in mice (α2A/α2CKO) to verify the HR response to physical exercise (PE), as well as the sympathetic-vagal control of the HR to PE. The hypothesis is that there would be exacerbated tachycardic response during PE in α2A/α2CKO mice even when the cardiac function was still preserved at rest, being the α2A-adrenergic receptor the main reason for this response. Male mice of the C57Bl6J lineage, control (CO) and with gene inactivation for the a2A (α2AKO), α2C α2CKO) and α2A/α2CKO receptors were submitted to tolerance to a physical exercise test. Two other groups of mice, CO and α2A/α2CKO, were submitted to pharmacological blocking of the muscarinic and β-adrenergic receptors as well as to progressive PE to assess the sympathetic-vagal contribution to PE tachycardia. Intolerance to physical exercise (1.220 ± 18 and 1.460 ± 34 vs. 2.630 ± 42m, respectively) and higher tachycardia to PE (765 ± 16 e 792 ± 13 vs. 603 ± 18 bpm, respectively) in the α2AKO and α2A/α2CKO vs. CO mice was observed. Moreover, the autonomic balance was altered in the α2A/α2CKO mice by the sympathetic hyperactivity and lower cardiac vagal effect. These outcomes demonstrated the importance of the α2A/α2C-adrenergic receptors in autonomic control not only at rest, but also during PE, being theα2A-adrenergic receptor responsible for the sympathetic hyperactivity and lower vagal effect observed. This exacerbated tachycardic response in α2A/α2CKO mice is present even when cardiac dysfunction is not observed.