Katt C. Mattos

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.

Integrative Effect of Carvedilol and Aerobic Exercise Training Therapies on Improving Cardiac Contractility and Remodeling in Heart Failure Mice

The use of β-blockers is mandatory for counteracting heart failure (HF)-induced chronic sympathetic hyperactivity, cardiac dysfunction and remodeling. Importantly, aerobic exercise training, an efficient nonpharmacological therapy to HF, also counteracts sympathetic hyperactivity in HF and improves exercise tolerance and cardiac contractility; the latter associated with changes in cardiac Ca2+ handling. This study was undertaken to test whether combined β–blocker and aerobic exercise training would integrate the beneficial effects of isolated therapies on cardiac structure, contractility and cardiomyocyte Ca2+ handling in a genetic model of sympathetic hyperactivity-induced HF (α2A/α2C- adrenergic receptor knockout mice, KO). We used a cohort of 5–7 mo male wild-type (WT) and congenic mice (KO) with C57Bl6/J genetic background randomly assigned into 5 groups: control (WT), saline-treated KO (KOS), exercise trained KO (KOT), carvedilol-treated KO (KOC) and, combined carvedilol-treated and exercise-trained KO (KOCT). Isolated and combined therapies reduced mortality compared with KOS mice. Both KOT and KOCT groups had increased exercise tolerance, while groups receiving carvedilol had increased left ventricular fractional shortening and reduced cardiac collagen volume fraction compared with KOS group. Cellular data confirmed that cardiomyocytes from KOS mice displayed abnormal Ca2+ handling. KOT group had increased intracellular peak of Ca2+ transient and reduced diastolic Ca2+ decay compared with KOS group, while KOC had increased Ca2+ decay compared with KOS group. Notably, combined therapies re-established cardiomyocyte Ca2+ transient paralleled by increased SERCA2 expression and SERCA2:PLN ratio toward WT levels. Aerobic exercise trained increased the phosphorylation of PLN at Ser16 and Thr17 residues in both KOT and KOCT groups, but carvedilol treatment reduced lipid peroxidation in KOC and KOCT groups compared with KOS group. The present findings provide evidence that the combination of carvedilol and aerobic exercise training therapies lead to a better integrative outcome than carvedilol or exercise training used in isolation.

Lack of β2‐adrenoceptors aggravates heart failure‐induced skeletal muscle myopathy in mice

Skeletal myopathy is a hallmark of heart failure (HF) and has been associated with a poor prognosis. HF and other chronic degenerative diseases share a common feature of a stressed system: sympathetic hyperactivity. Although beneficial acutely, chronic sympathetic hyperactivity is one of the main triggers of skeletal myopathy in HF. Considering that β2‐adrenoceptors mediate the activity of sympathetic nervous system in skeletal muscle, we presently evaluated the contribution of β2‐adrenoceptors for the morphofunctional alterations in skeletal muscle and also for exercise intolerance induced by HF. Male WT and β2‐adrenoceptor knockout mice on a FVB genetic background (β2KO) were submitted to myocardial infarction (MI) or SHAM surgery. Ninety days after MI both WT and β2KO mice presented to cardiac dysfunction and remodelling accompanied by significantly increased norepinephrine and epinephrine plasma levels, exercise intolerance, changes towards more glycolytic fibres and vascular rarefaction in plantaris muscle. However, β2KO MI mice displayed more pronounced exercise intolerance and skeletal myopathy when compared to WT MI mice. Skeletal muscle atrophy of infarcted β2KO mice was paralleled by reduced levels of phosphorylated Akt at Ser 473 while increased levels of proteins related with the ubiquitin‐–proteasome system, and increased 26S proteasome activity. Taken together, our results suggest that lack of β2‐adrenoceptors worsen and/or anticipate the skeletal myopathy observed in HF.

Lack of β2-AR improves exercise capacity and skeletal muscle oxidative phenotype in mice

β2‐adrenergic receptor (β2‐AR) agonists have been used as ergogenics by athletes involved in training for strength and power in order to increase the muscle mass. Even though anabolic effects of β2‐AR activation are highly recognized, less is known about the impact of β2‐AR in endurance capacity. We presently used mice lacking β2‐AR [β2‐knockout (β2 KO)] to investigate the role of β2‐AR on exercise capacity and skeletal muscle metabolism and phenotype. β2 KO mice and their wild‐type controls (WT) were studied. Exercise tolerance, skeletal muscle fiber typing, capillary‐to‐fiber ratio, citrate synthase activity and glycogen content were evaluated. When compared with WT, β2 KO mice displayed increased exercise capacity (61%) associated with higher percentage of oxidative fibers (21% and 129% of increase in soleus and plantaris muscles, respectively) and capillarity (31% and 20% of increase in soleus and plantaris muscles, respectively). In addition, β2 KO mice presented increased skeletal muscle citrate synthase activity (10%) and succinate dehydrogenase staining. Likewise, glycogen content (53%) and periodic acid‐Schiff staining (glycogen staining) were also increased in β2 KO skeletal muscle. Altogether, these data provide evidence that disruption of β2‐AR improves oxidative metabolism in skeletal muscle of β2 KO mice and this is associated with increased exercise capacity.

Comparison betweent the Effects of Swimming and Treadmill-Based Aerobic Training Protocols in Diabetic Rats

Mailing Address: Elizabeth de Orleans Carvalho de Moura Universidade Federal de São Paulo Campus Baixada Santista Departamento de Biociências R. Silva Jardim, 136. Postal Code: 11015-020, Vl. Mathias, Santos, SP Brazil E-mail: elizabeth.c.moura@hotmail.com Comparison betweent the Effects of Swimming and Treadmill-Based Aerobic Training Protocols in Diabetic Rats Elizabeth de Orleans Carvalho de Moura,1 Kelvin Tanaka,1 Moisés Felipe Pereira Gomes,1 Evandro Nogueira,2 Ricardo Gomes,1 Debora Estadella,1 Katt Mattos,3 Patrícia Chakur Brum,3 Alessandra Medeiros1 Universidade Federal de São Paulo (UNIFESP),1 São Paulo Brazil Faculdade Integral Diferencial,2 Piauí Brazil Universidade de São Paulo (USP),3 São Paulo Brazil

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.